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Pharmacological regulation of ovarian function in dairy cows to improve reproductive performance

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Title:
Pharmacological regulation of ovarian function in dairy cows to improve reproductive performance
Creator:
Bartolome, Julian Alberto, 1965-
Publication Date:
Language:
English
Physical Description:
xviii, 268 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Cattle ( jstor )
Dairy cattle ( jstor )
Estrus ( jstor )
Estrus cycle ( jstor )
Heifers ( jstor )
Insemination ( jstor )
Ovulation ( jstor )
Plasmas ( jstor )
Pregnancy ( jstor )
Pregnancy rate ( jstor )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 2004.
Bibliography:
Includes bibliographical references.
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Julian A. Bartolome.

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University of Florida
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The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (dissertations@uflib.ufl.edu) with any additional information they can provide.
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880637476 ( OCLC )
ocn880637476

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PHARMACOLOGICAL REGULATION OF OVARIAN FUNCTION IN DAIRY
COWS TO IMPROVE REPRODUCTIVE PERFORMANCE












By

JULIAN ALBERTO BARTOLOME



















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

2004






























Copyright

By

Julidn Alberto Bartolome













To Milagros and Santiago













ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my advisers Dr. Louis F. Archbald

and Dr. William W. Thatcher. The knowledge, support, encouragement, and confidence

they provide me were endless and I will be grateful for the rest of my days. From Dr.

Archbald I received not only a great deal of knowledge in reproduction, but also a lot

support, enthusiasm and friendship. From Dr. Thatcher I also received his support and

confidence, his passion for teaching and learning, and his knowledge in reproductive

physiology. We had a very productive interaction, and it reflected in my progress as

professional and as human being. They gave me an outstanding education and friendship.

I am very grateful to all the members of my committee. The insights and

discussion with Dr. Jorge Hernandez about experimental design and statistics plus what I

learned in his class will be knowledge and expertise extremely useful for my future

career. My sincere gratitude goes to Dr. Carlos Risco, not only for sharing with me his

skills and enthusiasm in the area of bovine reproduction, but also for creating a nice

environment of discussion plus all the fun during meetings and seminars. I want to

express my appreciation to Dr. Lokenga Badinga for his scientific contributions, his

teaching in the area of nutrition, and why not, for sharing the passion of soccer.











iv








I would like to express my gratitude to Dr. Pedro Melendez for his friendship and

for his scientific and field work contributions. Also, to his family-Maria Ester, Elisa,

Diego and Ignacio-are now part of my family. I will remember my friends Dr. Antonio

Landaeta and Dr. Martin Giangreco for the time we spent together in graduate school.

Marie Joelle Thatcher provided excellent, very kind, and continuous technical

support in the laboratory and with computer techniques. All of the members of Dr.

Thatcher Laboratory-Oscar Hernandez, Dr. Aydin Gulezoglu, Dr. Ricardo Mattos, Dr.

Frederico Moreira, Dr. Metin Pancarci, Todd Bilby, Dr. Ana Meikle-helped me with

discussions and comments about my research. Drs. Alvaro Arteche, Alessandro Sozzi,

and Shun Kamimura gave me invaluable help and joy during my field work at DRU and

North Florida Holstein.

I am very grateful to the faculty, staff, students and residents in Large Animal

Clinical Sciences and to Delores Foreman, for creating a nice environment to study and

for their support, discussions,'and friendship. I really appreciated all the help from Dr.

Charles Courtney, Sally O'Connell and the Phi Zeta Organization. They were always

ready to help me and solve every problem I had.

There are not enough words to express all the fun, friendship and knowledge I

received during my six years of work at North Florida Holstein. I owe special gratitude

to Mr. Don Bennink, Dr. David Kelbert, and the entire staff of North Florida Holstein and

the invaluable help from Dr. Jennifer MacHale and John Karanja. I am also grateful for

the help and friendship of all the guys at NFH: Gabriel, Gonzalo, Raul, Panfilo, Jorge,

Burt and Concepcion.






v








I want to extend my thanks to my home university for providing the leave during

years away and to the faculty members who were in charge of my classes. My friends in

Argentina, Julio y Silvana, provided unconditional support and help.

Finally, I want to express my love and gratitude to my family (parents, brothers,

sister, brother-in-law, sisters-in-law, nieces and nephews) for their continuous support,

and to my son and daughter, Santiago and Milagros, for their unconditional love.









































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TABLE OF CONTENTS

Page

ACKNOW LEDGEM ENTS ..................................................... .............. iv

LIST OF TABLES.......................................................................x

LIST OF FIGURES............ ................ .. .... ...... ...... .. ......... .. ........xiv

AB STRA CT ............................................ ................................. xvii

CHAPTER

1 INTRODUCTION .......................................... .................. ................

2 REVIEW OF LITERATURE ...................................................................................5

The Estrous Cycle and Follicular Dynamics.............. .......... ...............5
Drugs Used for the Pharmacologycal Control of the Estrous Cycle.................... 18
Methods for Synchronization of Estrus in Cattle ......................... ...............24
Induction of Ovulation and Timed Insemination................ ... ................29
Function of the Corpus Luteum.................... ...... ....... ................ 44

3 ADMINISTRATION OF HUMAN CHORIONIC GONADOTROPIN (hCG)
DURING ESTRUS IN NONLACTATING DAIRY COWS AND ITS
EFFECT ON CORPUS LUTEUM SIZE AND PROGESTERONE
PRODUCTION .............................. .............. 54

Introduction .................... .. .......................... .... .... 54
M aterials and M ethods.................. ..................... .............. ............55
R esults .............................................. ........ ........... .......... 56
Discussion................... ....................... .. .................... .. ......... 57

4 INDUCTION OF OVULATION IN NONLACTATING DAIRY COWS AND
HEIFERS USING DIFFERENT DOSES OF A DESLORELIN IMPLANT...........62


Introduction ............................................. ................... ... .......... 62
M aterials and M ethods ................. .................. ...... .......... ........ ... 63
R esults .......................................... ....................................... 66
D iscussion ............................................. ................... ... ........69





vii








5 EFFECT OF A DESLORELIN IMPLANT IN A TIMED ARTIFICIAL
INSEMINATION PROTOCOL ON FOLLICLE DEVELOPMENT,
LUTEAL FUNCTION AND REPRODUCTIVE PERFORMANCE
OF LACTATING DAIRY COWS......................................... ................82

Introduction ................. .............. ... ......................... ......... 82
M aterials and M ethods............ ................. ................. ..... ....... ...84
R esults......... .................................... .. .............. ............ 89
Discussion ............................................. .. ......... .... ..........92

6 STRATEGIC USE OF GONADOTROPHIN-RELEASING HORMONE
(GnRH) TO INCREASE PREGNANCY RATE AND REDUCE
PREGNANCY LOSS IN LACTATING DAIRY COWS SUBJECTED
TO SYNCHRONIZATION OF OVULATION AND TIMED
INSEM INATION ..................................... ... ........... .............. 106

Introduction........................................................... . ............. .. 106
M aterials and M ethods............... ........... ................ ................. 108
R esu lts................... ................... ..... .................. ..... .. ....... .. 111
D iscussion................... ................ ...................... ............ 113

7 THE USE OF A GNRH AGONIST (DESLORELIN IMPLANT) DURING
LATE EMBRYONIC PERIOD TO ENHANCE EMBRYO
SURVIVAL.................................... .......................... 124

Introdu ction .............. .................................... ... .. ...... ..........124
Materials and Methods ..... ......................... ...... .... ....................126
R esults................................................................................ ......... 129
Discussion........................................... .................. ........... 130

8 RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION
(TAI) IN LACTATING DAIRY COWS, I: USE OF THE OVSYNCH AND
HEATSYNCH PROTOCOLS AFTER NONPREGNANCY DIAGNOSIS
BY ULTRASONOGRAPHY..........................................................141

Introdu ction ........................................................................... 14 1
M aterials and M ethods .................................... .... .................142
R esults................................................................................ ......... 146
D iscussion................... ................ ................. .... ......... 148

9 RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION
(TAI) IN LACTATING DAIRY COWS, II: ASSIGNING PROTOCOLS
ACCORDING TO STAGES AND TWO COMMON ABNORMALITIES
OF THE ESTROUS CYCLE.............................................. .............159





viii








Intro du ction ................... ............... ................ ..... ....... ......... 159
Materials and Methods .............. ........... ........... .......... 160
Results ....................................................................... 164
D iscussion ............................................. 169

10 RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION
(TAI) IN LACTATING DAIRY COWS, III: ADMINISTRATION OF
GNRH 23 DAYS POST AI AND ULTRASONOGRAPHY FOR
NONPREGNANCY DIAGNOSIS ON DAY 30...................................191

Intro du ction .................................. ................................... .....19 1
M aterials and M ethods................................................. .... ........ 192
R esults............................................. .................. .. ......... 197
D iscussion.............................................................. .............. 201

11 SUMMARY AND CONCLUSIONS................... ..................................226

REFERENCE LIST...... .................................. .......... ..... ... ... ..... 232

BIOGRAPHICAL SKETCH......... ...................................... ................ 267
































ix














LIST OF TABLES

Table Page

5-1 Effect of a deslorelin implant in a timed AI protocol on fertility of lactating
dairy cow s................. ............................... ...... ....... 103

5-2 Effect of a deslorelin implant in a timed AI on ovarian activity of dairy cows
diagnosed nonpregnant on Day 27 after AI..........................................104

5-3 Effect of a deslorelin implant in a timed AI on ovarian activity 7 d after
resynchronization with GnRH on Day 27 after the initial timed insemination..... 105

6-1 Baseline comparison for parity, season, and inseminator.........................119

6-2 Pregnancy rates on Days 27 and 55 and pregnancy losses (PL) between
Days 27 and 55 for cows in all groups.................................................120

6-3 Pregnancy rates on Day 27, and the adjusted odd ratios (AOR), 95 %
confidence interval (CI), and level of significance for the risk of
nonpregnancy in cows in all groups.................. .............. .............. .121

6-4 Pregnancy rates on Day 55, adjusted odd ratios (AOR), 95% confidence
interval (CI), and level of significance for the risk of nonpregnancy in
cows in all groups........................... ........................122

6-5 Percentages, adjusted odd ratios (AOR), 95 % confidence interval (CI),
and level of significance for the risk of pregnancy loss between
Day 27 and Day 55.................... .............. .................. ....... ..123

7.1 Distribution of cows by parity, previous services and type of artificial
insemination in both groups............... ........................................133

7.2 Pregnancy loss in lactating dairy cows after administration of a 2.1 mg
deslorelin implant on Day 27 of pregnancy................ ..... ............... 134

7.3 Number of cows, percentages, adjusted odd ratios and 95% confidence interval
for the risk of pregnancy loss between Day 27 and Day 45 for cows with
accessory CL in the both groups.................................................... 135





x








8-1 Criteria for determination of the stage and two common abnormalities
of the estrous cycle based on ultrasonography and rectal palpation of the
genital tract (adapted from Zemjanis 1962 and Pierson and
Ginther 1984a, 1987).....................................................152

8-2 Distribution of cows by parity, season and DIM for both groups .................. 153

8-3 Distribution of cows by inseminator, and stages/abnormalities of the
estrous cycle for both groups ................. .................. .... ...................154

8-4 Pregnancy rate on Days 27, 45, and 90 and pregnancy losses between
Days 27-45 an 45-90 for cows in the Ovsynch and Heatsynch
groups ...... ........... ............................... ...........155

8-5 Pregnancy rate on Day 27, odd ratios (OR), 95% confidence interval
(CI) and level of significance for the risk of nonpregnancy for cows in
both groups and at different stages and two common abnormalities
of the estrous cycle....................... .............. ........ .. ........ ..... 156

8-6 Pregnancy rate on Day 45, adjusted odd ratios (AOR), 95% confidence
interval (CI) and level of significance for the risk of nonpregnancy for
cows in both groups and at different stages and two common
abnormalities of the estrous cycle ............. ......................... 157

8-7 Pregnancy rate on Day 90, adjusted odd ratios (AOR), 95% confidence
interval (CI) and level of significance for the risk of nonpregnancy for cows
in both groups and at different stages and two common abnormalities
of the estrous cycle............... .. ............ ........................... 158

9-1 Number of cows evaluated for different stages and two common
abnormalities of the estrous cycle and protocols at Days 30, 55 and 90
after resynchronized insemination.......................... ......... ... .............. 178

9-2 Distribution of cows by parity, season, DIM and inseminator for cows
in diestrus from January to May for both groups...................................... 179

9-3 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in diestrus for the Ovsynch and
Quicksynch Groups from January to M ay ............................. ................180

9-4 Distribution of cows by parity, season, DIM, and inseminator for in
diestrus from June to December for both groups .................. ...............181

9-5 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in diestrus for the Ovsynch and Modified
Quicksynch groups from June to December................... ..................182



xi









9-6 Distribution of cows by parity, season, DIM, and inseminator for in
metestrus from January to December ................ ........... ............... 183

9-7 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in metestrus for the Ovsynch, Heatsynch,
and GnRH+Ovsynch groups from January to December........................184

9-8 Distribution of cows by parity, season, DIM, and inseminator for in
proestrus from January to December ........................................185

9-9 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in proestrus for the Ovsynch, and
GnRH+Ovsynch groups from January to December ............ .................186

9-10 Distribution of cows by parity, season, DIM, and inseminator for with
ovarian cysts January to December.............. ................ ................ 187

9-11 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows with ovarian cysts subjected to the
Ovsynch and GnRH+Ovsynch groups from January to December ....................188

9-12 Plasma P4 concentration in cows with ovarian cysts diagnosed using
a clinical method combining per rectum examination and
ultrasonography of the genital tract............... ................. ............... 189

9-13 Pregnancy rate at Days 30, 55 and 90 for Ovsynch and GnRH+Ovsynch
groups in cows with ovarian cysts classified according type of cyst and
plasm a P4 concentration............. ... ..... ... .............. ... ................ 190

10-1 Distribution of cows by parity, DIM, inseminator and lot for cows
in diestrus subjected to the GnRH and ECP groups...............................209

10-2 Pregnancy rate and losses for cows in diestrus and subjected to the
GnRH or ECP groups .............................................. ...............210

10-3 Distribution of cows by parity, DIM, inseminator and housing lot
for cows without a CL on Day 0 ............. .................. ...... ............211

10-4 Distribution of cows by BCS and stages/abnormalities of the estrous
cycle for cows without CL on Day 0...................................................212

10-5 Ovarian responses on Days 7 and 17 for cows in different stages/
abnormalities of the estrous cycle in all 4 groups ................................... 213





xii








10-6 Number of cows, adjusted odd ratios (AOR), 95% confidence interval
(CI) and levels of significance for the risk of presence of a CL on Day 7
in cows in proestrus and with ovarian cysts treated with GnRH on
Day 0 adjusting for BCS and plasma P4 concentration on Day 0...................214

10-7 Number of cows, adjusted odd ratios (AOR), 95% confidence interval
(CI) and levels of significance for the risk of presence of a CL on Day 17
in cows in proestrus and with ovarian cysts treated with GnRH on
Day 0 adjusting for BCS............................................................215

10-8 LSM and SEM for plasma P4 concentration on Day 0, 7, 10, and 17 for
cows in proestrus and with ovarian cysts for all 4 groups........................216

10-9 Pregnancy rates on Day 30, 55, and 90 for cows in different stages/
abnormalities of the estrous cycle in all 4 groups ............... ...............217

10-10 Number of cows, adjusted odd ratios (AOR), 95% confidence
interval (CI) and levels of significance for the risk of pregnancy on
Day 30 in cows in proestrus and with ovarian cysts treated with
CIDR on Day 0 adjusting for BCS.................................. ...............218

10-11 Number of cows, adjusted odd ratios (AOR), 95% confidence
interval (CI) and levels of significance for the risk of pregnancy on
Day 55 in cows in proestrus and with ovarian cysts treated with
CIDR on Day 0 adjusting for BCS ................ ................... .............. 219

10-12 Number of cows, adjusted odd ratios (AOR), 95% confidence
interval (CI) and levels of significance for the risk of pregnancy on
Day 90 in cows in proestrus and with ovarian cysts treated with
CIDR on Day 0 adjusting for BCS.................................. ..............220




















xiii













LIST OF FIGURES

Figure Page

3-1 Experim ental design ...... .............. ...... .......................... ............. 59

3-2 Plasma P4 concentrations in nonlactating dairy cows after induction of
ovulation with human chorionic gonadotropin (hCG) .................................60

3-3 Size of the CL in nonlactating dairy cows treated with human chorionic
gonadotropin (hCG) 8 to 10 hours after onset of estrus................................61

4-1 Least squares means and SE for plasma P4 concentration between Day 0
and Day 16 for Control (m), DESLORELIN 750 (e), DESLORELIN 1000
(A) groups in Experiment 1. DESLORELIN 1000 vs Control on Day 11
(P<0.05), DESLORELIN 750 vs. Control on Day 12 (P<0.05)........................73

4-2 Least squares means and SE for the first-wave largest follicles from Day 0
to Day 16 for Control (m), DESLORELIN 750 (*) and DESLORELIN 1000
(A)groups (P<0.02 Control vs. DESLORELIN 1000) and second-wave
largest follicle for Control (c), DESLORELIN 750 (0), and DESLORELIN
1000 (A) groups (P<0.01) in Experiment 1............................ .................74

4-3 A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (m), DESLORELIN 750
(*) and DESLORELIN 1000 (A) groups in Experiment 1 (P50.01).
B. Least squares means and SE for the number of class III follicles after
treatment for from Day 0 to Day 16 for Control (m), DESLORELIN 750
(e) and DESLORELIN 1000 (A) groups in Experiment 1 (P<0.01)............... 75

4-4 Least squares means and SE for the largest follicle after PGF2a on Day 16
for cows in Control (m), DESLORELIN 750 (e) and DESLORELIN 1000
(A) groups in Experiment 1 (P<0.001)............... ............ ...............76

4-5 Least squares means and SE for plasma P4 concentration between Day 0
and Day 16 for Control (m) and DESLORELIN 450 (e) groups in
Experiment 2. DESLORELIN 450 vs. Control on Day 9 (P50.10), and
DESLORELIN 450 vs. Control on Day 10 (P50.05)...............................77





xiv








4-6 Least squares means and SE for the first-wave largest follicles from Day 0
to Day 16 for Control (m) and DESLORELIN 450 (*) groups (P50.01),
and second-wave largest follicle for control (o), DESLORELIN 450 (0)
groups (P 0.001) in Experiment 2................ ............... ................ 78

4-7 A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (w), DESLORELIN 450
(*) groups (P<0.01) in Experiment 2. B. LSM and SE for the number of
Class III follicles after treatment for from Day 0 to Day 16 for Control (m)
and DESLORELIN 450 (e) groups (P<0.01) in Experiment 2.......................79

4-8 Least squares means and SE for the largest follicle after PGF2a on Day 16
for cows in Control (m) and DESLORELIN 450 (*) groups (P<0.001)
in Experim ent 2................. ................. ............... ... ......80

4-9 Least squares means and SE for plasma P4 concentration on Days 7, Day 13
and Day 16 for Control (m) and DESLORELIN 750 (*) groups (P50.07)
in Experiment 3 ................................................... 81

5-1 Diagram of activities during the study ............. ......... .............101

5-2 Relationship between body condition score (BCS) at first postpartum
artificial insemination (AI) and frequency of anovulatory dairy cows.............02

6-1 Interaction between administration of GnRH on Day 5 and Day 15 on
pregnancy rate at Day 27 (P<0.10)................ ................ ................ 118
/
7-1 Number of class 2 follicles on Day 27 and Day 45 of pregnancy in lactating
dairy cows after treatment with 2.1 mg deslorelin implant (P<0.01) .............136

7-2 Number of class 3 follicles on Day 27 and Day 45 of pregnancy in lactating
dairy cows after treatment with 2.1 mg deslorelin implant (P<0.01) ..............137

7-3 Number of CL on Day 27 and Day 45 of pregnancy in lactating dairy cows
after treatment with 2.1 mg deslorelin implant (P<0.01).............................. 138

7-4 Plasma P4 concentrations on Days 27 and 45 of pregnancy in lactating dairy
cows after treatment with 2.1 mg of deslorelin implant (P<0.10)...................139

7-5 Interaction between group and presence of accessory CL at Day 45 on
pregnancy losses between Day 27 and Day 45 (P<0.03)............................ 140

9-1 Protocols used for resynchronization depending upon stage of the estrous
cycle at the time of nonpregnancy diagnosis at Day 30 after insemination.......... 176





xv








9-2 Distribution ofinterestrus intervals in 359 lactating dairy cows diagnosed
nonpregnant based on expression of estrus and insemination prior to
ultrasonography for pregnancy diagnosis .................. ........................177

10-1 Experimental design for cows in diestrus, metestrus, or without a CL at
the time of a nonregnancy diagnosis (experimental Day 0) ............ ......221

10-2 The effect of GnRH treatment on Day 0 on the proportion of cows with
a corpus luteum at PGF2a treatment on Day 7 (P<0.11).............................222

10-3 The effect of GnRH treatment on Day 0 on the proportion of cows with
a corpus luteum at on Day 17 (i.e., ovulation rate; P<0.12)........................223

10-4 The effect of GnRH treatment on Day 0 on the rise of progesterone from
Day 10 to Day 17 for cows in proestrus and with ovarian cysts
(Treatment* Stage*Day P<0.06)............. ................... .. ............... 224

10-5 Pregnancy rates on Day 30, 55 and 90 in cows without a corpus luteum
on Day 0 (proestrus and ovarian cysts) and treated with a CIDR insert
(CID R Stage P<0.05)....................................................................225































xvi













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

PHARMACOLOGICAL REGULATION OF OVARIAN FUNCTION IN DAIRY
COWS TO IMPROVE REPRODUCTIVE PERFORMANCE

By

Julian A. Bartolome

May 2004

Chairman: Dr. Louis F. Archbald
Cochairman: Dr. William W. Thatcher
Major Department: Veterinary Medical Sciences



Dairy herds experience low pregnancy rates and high embryonic mortality. The

objective was to evaluate pharmacological strategies to improve reproductive

performance. Nonlactating dairy cows (n=17) received human chorionic gonadotropin

(hCG) at estrus or no treatment. Nonlactating dairy cows (n=20 and n=14) and dairy

heifers (n=44) received a deslorelin implant or gonadotropin releasing homone (GnRH)

for induction of ovulation. Lactating dairy cows (n=593) received a deslorelin implant or

GnRH for induction of ovulation and timed artificial insemination (TAI). Lactating dairy

cows (n=831) received no treatment or GnRH on Day 5, Day 15, or both after TAI.

Lactating dairy cows (n=179) received no treatment or a deslorelin implant at Day 27 of

pregnancy. Lactating dairy cows (n=332) were assigned to the Ovsynch or Heatsynch

protocols after nonpregnancy diagnosis. Lactating dairy cows (n=1083) received different

protocols for resynchronization. Lactating dairy cows (n=624) received GnRH on Day 23


xvii








after previous AI and were assigned to different protocols for resynchronization.

Administration of hCG at estrus impaired CL function. Induction of ovulation with a

deslorelin implant suppressed follicular growth, and slightly improved CL function in

nonlactating cows and heifers and tended to reduce late embryonic losses in lactating

cows. Administration of GnRH on Days 5 and 15 after TAI did not increase pregnancy

rate. A deslorelin implant administered at 27 days of pregnancy suppressed follicular

growth, induce accessory corpora lutea, increased progesterone (P4) production, an

reduce late embryonic mortality in cows that formed an accessory CL. Forty to 45% of

the cows detected not pregnant after AI presented a CL and the stage of the estrous cycle

influenced the response to different resynchronization protocols. Administration of

GnRH 23 days after AI resulted in 75% of cows with a CL at nonpregnancy diagnosis

and these cows obtained similar pregnancy rate after resynchronization of ovulation with

either GnRH or estradiol cypionate (ECP). For cows without a CL, administration of a P4

insert for resynchronization resulted in increased pregnancy rate in cows with ovarian

cysts. In summary, treatments affected ovarian function and indicated that may be

possible to increase pregnancy rate and reduce embryo mortality. Resynchronization

protocols assigned based on stages of the estrous cycle increased pregnancy rate.
















xviii













CHAPTER 1
INTRODUCTION


Reproductive efficiency is one of the most important factors affecting dairy herd

productivity (Louca and Legates, 1968; Pellisier, 1976; Oltenacu et al., 1981; Galligan, 1999).

Fertility has been reduced in the last 50 years due to genetic selection for milk production and

intensive management, which are characteristics of modem dairy herds (Butler and Smith,

1989; Stevenson, 2001; Lucy, 2001).

Reproductive efficiency is based on high estrous detection/service rate, high

conception rate, enhanced embryo and fetal survival, and efficient detection and re-

insemination of cows that did not conceive or lost their embryos after first and subsequent

services. Therefore, enhancing reproductive efficiency using pharmacological and

management strategies should focus at three different levels. First, by increasing pregnancy

rate (either by increasing estrous detection or conception rate); second, by enhancing embryo

and fetal survival; and third, by detecting and rapidly re-inseminating cows that did not

conceive or failed to maintain pregnancy.

Pregnancy rate for 21-day periods is the product of estrous detection and conception

rates, and is the most commonly used index to evaluate reproductive performance (Ferguson

and Galligan, 1993). Estrous detection rate is defined as the number of animals detected in

estrus in a 21-day period divided by the number of animals available. Conception rate is

defined as the number of pregnant animals divided by the number of inseminated animals




1





2

(Fetrow et al., 1990). When estrous detection rates of 35 %, 55 % and 75%, and conception

rates of 42 %, 50 % and 58% were used to model reproductive efficiency, the estrous

detection rate had a major impact (Oltenacu et al., 1981).

Pregnancy rate can be increased by either increasing conception rate or estrous

detection rate. Estrous detection and conception are affected by several factors. Some ofthem

include level of production, management, nutrition, health problems, climate, and genetic

selection. Even though conception rate is slightly reduced in intensively-managed dairy herds,

estrous detection has been the most affected variable (Nebel et al., 1987). The length and

intensity of estrus have been reduced throughout the years because of genetic selection, level

of production and intensive management (Washburn et al., 2002). Consequently, estrous

detection is a difficult task that usually becomes compromised. Estrous detection rate by

visual observation is very low (-50%) in both intensively-managed dairy herds (King et al.,

1976) and in small herds on pasture-based systems (Williamson et al., 1972). This low estrous

detection rate can have a devastating effect on reproductive performance (Heersche and

Nebel, 1994). In addition, estrous detection could be inaccurate, and results in the

insemination of cows that are not in estrus, which further contributes to reduced conception

rate (Heersche and Nebel, 1994).

Even though estrous synchronization programs were developed to concentrate

expression of estrus in 3 to 4 days to make detection of estrus more efficient, estrous

detection rates continue to be less than 50% in dairy herds, and 5 % to 30% of the cows are

not in estrus at time of insemination (Senger, 1994). Synchronization of ovulation in a group

of cows allows insemination by appointment (timed artificial insemination, TAI) and

eliminates the need for estrous detection. Several studies provided information regarding the





3

intervals between onset ofestrus, luteinizing hormone (LH) surge and time of ovulation after

estrous synchronization protocols which led to development of TAI programs. Timed

artificial insemination increase pregnancy rate despite a slight decrease in conception rate

since 100% of the cows are inseminated (Stevenson, 2001).

After fertilization is achieved, increasing reproductive efficiency depends on embryo

and fetal survival. Several factors (genetics, endocrine and metabolic changes, infectious

diseases, climate) may be responsible for embryo and fetal mortality (Ayalon, 1978). Different

strategies to increase embryo and fetal survival include administration of hCG, GnRH or

GnRH agonists during diestrous and early pregnancy. The aim of these strategies is to

increase plasma P4 concentration, decrease follicular growth, estrogen production, and inhibit

PGF2a release and the luteolytic cascade (Hansel and Seifart, 1967; Schmitt et al., 1996).

The third step to maintain high reproductive efficiency is to detect and rapidly re-

inseminate cows that did not conceive or failed to maintain a pregnancy (Stevenson et al.,

2003). Different methods such as nonreturn to estrus, plasma P4 concentrations, detection of

early pregnancy factors, ultrasonograpy and per rectum palpation of the genital tract have

been used to detect nonpregnant cows. At the time of pregnancy diagnosis, when cows are

diagnosed not pregnant, they should be re-inseminated as soon as possible. However, since

estrous detection is not very efficient, protocols for resynchronization and TAI need to be

applied. The clinical findings of the reproductive tract at the time ofnonpregnancy diagnosis

allow classifying cows in different stages and abnormalities of the estrus cycle (Zemjanis,

1962; Pierson and Ginther, 1984b; 1987). This information can be used to implement different

resynchronization protocols because the stage of the estrous cycle affects the response to

hormonal treatments.





4




Interactions between hormones secreted by the hypothalamus, anterior pituitary,

ovaries, uterus, the influence ofneurotransmitters in the central nervous system and growth

factors are responsible for regulation the reproductive cycle in cattle. These interactions are

complex and a thorough understanding of the reproductive physiology is important in order to

implement different strategies for the pharmacological regulation of ovarian function and

reproductive management to optimize reproductive efficiency in lactating dairy cows.

The hypothesis of this study was that pharmacological manipulation of ovarian

function in lactating dairy cows will contribute to increased pregnancy rate, reduced embryo

mortality and allow for resynchronization of nonpregnant cows. The objective was to use

different strategies to enhance CL function and reduce follicular growth to increase pregnancy

rate and reduce embryo mortality, and to regulate follicular dynamics and ovulation to

resynchronize and time-inseminate nonpregnant cows.













CHAPTER 2
LITERATURE REVIEW

The Estrous Cycle and Follicular Dynamics

The bovine female experiences the first estrous cycle after puberty, and

subsequent ovulations occur every 17 to 24 days (Roberts, 1956). The estrous cycle may

be interrupted physiologically (pregnancy) or pathologically (anestrus, cystic ovarian

degeneration, pyometra) and then reinitiated during the postpartum period or after the

pathological conditions are resolved (Roberts, 1956). The estrous cycle is controlled by

endocrine changes in the central nervous system (CNS) and the hypothalamic-pituitary-

gonadal axis which cause physiological and clinical changes in the genital tract which are

divided in 4 stages as follows: estrus, metestrus, diestrus, and proestrus (Rathbone et al.,

2001). The onset of estrus coincides with a peak of follicle stimulating hormone (FSH),

maximum follicular growth, peak of estrogen levels and the gonadotropin releasing

hormone (GnRH) and luteinizing hormone (LH) surge. The LH surge lasts for 7 to 10

hours, estrus for 16.9 hours and ovulation occurs 25 hours after the LH surge (Day 0).

The corpus luteum forms from the ovulated follicle, P4 levels increase slowly from Day

1 to 4 during formation of the corpus luteum (metestrus), and then rapidly from Day 4 to

10 to reach a plateau and decrease to basal concentrations on Day 17 (diestrus). During

diestrus, FSH shows a wave-like pattern with peaks on Day 4, 8, and 12 parallel with

follicular growth. Between Days 16 and 17, the endometrium releases PGF2a, which






5





6

causes regression of the corpus luteum. Subsequently, FSH and LH increase, and

follicular growth is stimulated (proestrus), and this result in a subsequent estrous period

and ovulation (Schams et al., 1977).

Estrus

The onset of estrus coincides with the LH surge and lasts until the end of the

estrus behavior (receptivity to be mounted; 12-20 h). During estrus, P4 levels are low,

secretion of gonadotropins (FSH and LH) by the anterior pituitary increases, LH pulse

amplitude and frequency are high (Rahe et al., 1980; Stumpf et al., 1989), follicular

growth is accelerated and dominant preovulatory follicles reach 14 to 16 mm in cows

with two or three follicular waves, respectively (Ginther et al., 1989). During estrus,

estradiol concentration reaches a maximum (6-7 pg/ml; Glencross et al., 1973) and under

low P4 levels (Kesner et al., 1981; Schallenberger et al., 1982), induces the GnRH/LH

surge (Ireland and Roche, 1982a). The hypothalamic neurons responsible for the GnRH

surge (preoptic area and anterior hypothalamic nuclei) do not contain estrogen receptors.

However, estrogen binds to its receptor in the central nervous system, and the effect on

GnRH release is mediated by neurotransmitters (Kalra et al., 1993). Estradiol stimulates

GnRH release and increases sensitivity of the pituitary to GnRH (Reeves et al., 1971 in

sheep; Kaltenbach et al., 1974 in cows) by increasing GnRH receptors in the anterior

pituitary gland (Schoenemann et al., 1985). In addition, estrogen increases the expression

of its own receptors in the anterior pituitary gland (Clarke et al., 1981). The onset of

estrus occurs at the time of the LH surge and last for 16.9 hours. Then, estradiol levels

drop after the LH surge and ovulation occurs approximately 24 hours after the LH surge

(Schams et al., 1977).





7

Duration and intensity (number of mounts) of estrus is influenced by several

factors and have been reported to be reduced in the last 20 years in dairy cows.

Nonlactating dairy cows show estrus for a period of 11.4 hours and receive an average of

19 mounts per estrous period (Dalton et al., 2001). However, intensively-managed high

producing dairy cows express estrus for 7.1 hours with an average of 8.5 mounts per

estrous period (Dransfield et al., 1998). Several factors affect the duration and intensity of

estrus in dairy cows. Heat stress reduced the length of estrus in dairy heifers (Thatcher

and Chenault, 1975) and the levels of estrogen in lactating dairy cows (Gwazdauskas,

1985) which have been shown to be lower than that observed in nonlactating dairy cows

(De la Sota et al., 1993). These factors contribute to lower estrus detection rates.

Follicular growth after CL regression (preovulatory follicles) is faster in June (2.00.6

mm) compared to April (1.10.6 mm), August (10.0+0.6 mm) and November (1.20.6

mm) and larger follicles and corpora lutea detected in November (Badinga et al., 1994).

Overall, the time a cows stands to be mounted is around 1% of the entire duration

of the estrous cycle, and this makes estrus detection by visual observation very difficult

(Senger, 1994). Protocols for synchronization of estrus and ovulation that include

estrogen may enhance estrus behavior. A TAI protocol which included estradiol

cypionate (ECP, 1 mg) induced estrus with a length of 12.5 hours and 20.3 mounts per

estrous period (Pancarci et al., 2002). Therefore, this protocol may result in an increased

estrus detection rate.

Clinical signs associated with estrus are swollen vulva, reddened vestibulum,

discharge of copious quantities of elastic clear mucous, increased walking activity

(restlessness), mounting and standing to be mounted, interrupted grazing, and decrease in





8

milk production (Hammond, 1927). Receptivity to be mounted is the most precise sign

of estrus and is used by different methods for estrus detection such as visual observation

(Trimbeger, 1943), rump-mounted detectors (Stevenson and Britt, 1977) and Heatwatch

system (Dohi et al., 1993). The pedometer detects 2.75 time-increase in walking activity

during estrus (Kiddy, 1977), and used in combination with reduced milk production can

estimate the onset of estrus. An increase of 100% in walking activity may indicate estrus

and there was a positive correlation with fertility up to an increase of 400-500% (Yaniz et

al., 2003). Plasma P4 concentrations measured by radioimmunoassay or milk P4 levels

evaluated by an on-farm ELISA test can be used to monitor the accuracy of estrus

detection methods (Nebel et al., 1987).

The palpable characteristics of the genital tract associated with estrus are a

dominant ovarian follicle, absence of a corpus luteum or presence of a regressing corpus

luteum, and increased uterine tonicity (Zemjanis, 1962). On ultrasonography,

preovulatory dominant ovarian follicles are observed (usually from 12 to 17 mm), the

uterine horns display a very heteregenous pattern, and a hypoechogenic image indicating

the presence of mucus can sometimes be observed in the uterine lumen (Pierson and

Ginther, 1984a; 1987).

Metestrus

The stage of metestrus is considered to begin either after the end of estrus (10 to h

after LH surge) or at ovulation (24-30 h after LH surge; Day 0) and ends after the corpus

luteum (CL) is fully matured (Day 5). Considering that metestrus has to begin at the end

of estrus, ovulation occurs during metestrus. The LH surge triggers inflammatory-like

changes that allow for rupture of epithelia, albuginea, theca, basal membrane and





9

granulosa cells and the release of the oocyte (Espey, 1994). After the oocyte is released,

theca interna and granulosa cells form the small and large cells of the corpus luteum

during a period of rapid mitosis and vascularization under LH stimulation (Hansel and

Dowd, 1986). A second peak of LH and a rise in follicle-stimulating hormone (FSH) at

the beginning of metestrus stimulate the first follicular wave (Adams et al., 1992). The

tonic releases of LH and FSH during the luteal phase are controlled by the arcuate

nucleus, ventromedial nucleus and median eminence in the hypothalamus. Tonic and

pulsatile secretion of LH from the anterior pituitary gland stimulate CL development and

P4 secretion gradually rises (Rahe et al., 1980). Progesterone exerts a negative feedback

on the anterior pituitary gland and inhibits gonadotropin secretion. However, during

metestrus, P4 concentrations are still low and LH pulsatility is greater compared to mid-

luteal phase (Rahe et al., 1980; Peters et al., 1994).

During metestrus, the CL is refractory to a single luteolytic dosage of

PGF2a (Lauderdale et al., 1972; Rowson et al., 1972). The mechanism responsible for this

is not completely understood. One possible explanation involves a mechanism that

contributes to luteolysis by uterine and exogenous PGF2, involving stimulation of

prostaglandin secretion by the large luteal cells (Tsai and Wiltbank, 1997). However, this

mechanism may not be functional in the early luteal phase. Nevertheless, the magnitude

of the luteolytic effect of intraluteal prostaglandin still needs to be demonstrated (Tsai

and Wiltbank, 1998). An alternative mechanism involves the role of endothelin-1 in

luteolysis. Injecting PGF2a in early luteal phase (Day 4) did not induce the expression of

ET-1, its receptor type A (ETA-R), and COX-2 compared with cows injected in midcycle

(Day 10; Levy et al., 2000). In adittion, P4 levels, StaR protein mRNA, and cytochrome





10


P450 mRNA were only affected when PGF2a was administered in mid-luteal phase (Day

10) but not early in the estrous cycle (Levy et al., 2000).

A clinical sign that may be observed during the first day of metestrus, especially

in heifers and rarely in cows, is a bloody mucous discharge caused by the increased blood

supply as an effect of estrogen during estrus. The increased blood supply results in

uterine edema during metestrus which is one the clinical characteristics at per rectum

examination of the genital tract (Zemjanis, 1962). Another palpable characteristic is the

absence of significant structures in the ovaries and sometimes the presence of an

ovulation depression (Zemjanis, 1962). At ultrasonography, carry-over follicles (did not

ovulate at estrus) may be present, but usually nonsignificant structures are observed. The

uterine horns appear very heteregoneous and a hypoechogenic line can be observed in the

vascular layer of the submucosa reflecting the edema of the uterus (Pierson and Ginther,

1984a; 1987).

Diestrus

The diestrus stage begins on Day 5 (mature CL) and ends with CL regression

between Days 16 and 18. It is characterized by high levels of plasma P4 concentration

secreted by the CL (Schams et al., 1977). Corpus luteum development and differentiation

are supported by tonic secretion of LH from the anterior pituitatry gland (Hansel and

Seifart, 1967). The CL secretes P4 to support embryo development and at the same time

P4 inhibits the amplitude and frequency of LH pulses in a negative feed back mechanism

(Roberson et al., 1989) influencing follicular growth. Follicles grow in a wave pattern

during diestrus (Rajakoski, 1960). Using ultrasonography, two and three follicular waves

have been described (Savio et al., 1988; Sirois ad Fortune., 1988), and oestradiol-17p





11

produced by the follicles peak on Days 6 to 7 of the estrous cycle and rise again on Day

16 around luteolysis (Glencross et al., 1973). Secretion of estrogen in the presence of

high levels of P4, also inhibits gonadotropin release from the anterior pituitary gland

(Kesner et al., 1981; Schallemberger et al., 1982). High levels of P4 during diestrus are

associated with a more rapid follicular turnover (three instead of two follicular waves),

and high fertility in next estrus (Savio et al., 1993; Wehrman et al., 1993; Ahmad et al.,

1997).

Progesterone concentration during diestrus can be affected by several factors such

as parity (heifers and cows), lactational stage, metabolic diseases, season, and size of the

follicle at the time of ovulation. Plasma P4 is lower during summer time in lactating dairy

cows (Rosemberg et al., 1977; De la Sota et al., 1993). In addition, lactating dairy cows

have an increased feed intake that results in increased liver blood flow and steroid

metabolism (Sangsritavong et al., 2002) that may result in lower P4 concentrations.

Induction of ovulation commonly used in lactating dairy cows may result in ovulation of

immature follicles and formation of a corpus luteum with reduced production of P4

(Vasconcelos et al., 1999, 2001; Peters and Pursley, 2003).

Clinical characteristics of diestrus at per rectum examination of the genital tract

are the presence of a corpus luteum determined by distortion in the shape of the ovary

and a line of demarcation between the corpus luteum and the rest of the ovarian stroma,

at least one graffian follicle, and moderate tonicity of the uterus (Zemjanis, 1962).

Specificity of per rectum examination of the ovaries to determine the presence of a

corpus luteum has been reported to be low (Ott et al., 1986). However, the positive

predictive value obtained by evaluating the proportion of cows in estrus after





12


administering PGF2 in cows with CL at clinical examination has been reported to be

80% (Archbald et al., 1994). Using plasma P4 concentration as the gold standard, positive

predictive value was 87 % for rectal palpation (> 1 ng/ml; Archbald et al., 1992), and

90% for rectal palpation combined with ultrasonography (> 2 ng/ml; Bartolome et al.,

2002). At ultrasonography, a corpus luteum and one graffian follicle are observed, and

the echogenicity of the uterus is moderate (Pierson and Ginther, 1984a; 1987). Follicles

during the estrous cycle can be classified in class I (3-5 mm), class II (6-9 mm) and class

III (> 9 mm) by ultrasonography of the ovaries (Macmillan and Thatcher, 1991).

Proestrus

The stage of proestrus begins at the time of CL regression (Days 16-18) and ends

with the LH surge at the onset of estrus (Day 21). The length of proestrus is affected by

the size of the dominant follicle at the time of luteolysis (Sirois and Fortune, 1988).

Around Day 16 of the estrous cycle, uterine PGF2a is collected in the uterine-ovarian vein

and is transported to the ovarian artery through a counter-current transfer mechanism. It

then passes directly into the ovary to cause luteolysis (McCracken et al., 1972) which

results in decline of P4 and initiation of a new follicular phase (Kindahl et al., 1976).

Functional luteolysis includes the decline in P4 which occurs 30 minutes after the

levels of PGF2a increase (Thatcher and Chenault, 1975; Behrman and Hitches, 1976), and

structural luteolysis takes approximately 48 hours to be completed (Pate, 1994).

Luteolysis is carried out by a positive feed-back loop between PGF2a from the

endometrium and oxytocin from the CL in pulses every 6 to 8 h (Flint and Sheldrick,

1982; 1983). Prostaglandin F2a stimulates the release of oxytocin using the

phosphoinositol-Inositol 3-phosphate/diacilglycerol/Ca2-protein kinase second-





13

messenger system (Stormshak et al., 1995) and the same mechanism is used by oxytocin

to stimulate PGF2a release by the endometrium (Silvia and Homanics, 1988). Other

substances such as endothelin-1 and angiotensin II seem to mediate the effect of PGF2z in

the corpus luteum.

Endothelin-1 (ET-1) is a potent vasoconstrictor secreted by endothelial cells and

expressed in the corpus luteum when stimulated by PGF2a (Girsh et al. 1996a, 1996b;

Miyamoto et al., 1997). It suppresses P4 production in luteal-derived cells in the presence

ofPGF2a (Girsh, et al., 1996a). Endothelin-1 is known to play a role in luteolysis, and by

using an Endothelin-A receptor antagonist, it was shown that ET-1 has a potent

contractile effect on human myometrial cells (Dallot et al., 2003). Prostaglandin F2a has

been shown to stimulate angiotensin II in vitro, and angiotensin II, ET-1, and oxytocin

may interact to inhibit P4 secretion by causing functional luteolysis, and through

vasoconstriction may initiate structural regression (Hayashi and Miyamoto, 1999).

Structural luteolysis involves apoptosis and is characterized by expression of class II

MHC antigens, an autoimmune-like response with invasion of immune cells, and

secretion of TNF-at and other cytokines (Benyo et al., 1991; Benyo and Pate, 1992).

After P4 drops, the mean concentration of LH (Chenault et al., 1975), frequency

(Ireland and Roche, 1982b) and amplitude ofLH pulses (Stumpft et al., 1989) stimulate

follicular growth and consequently a rise in estrogen levels (Schallemberger et al., 1984)

and a new estrous cycle begins.

Clinical signs characteristic of proestrus are increasing walking activity,

involment in sexual active groups and mounting behavior. Clinical characteristics at

rectal palpation are increase in uterine tonicity due to the effect of oxytocin during luteal





14

regression and a growing follicle and regressing CL in the ovaries (Zemjanis, 1962). At

ultrasonography, the uterine horn will appear highly echogenic and in the ovaries a

dominant follicle should be present (Pierson and Ginther 1984a; 1987). The CL may still

be observed since structural regression takes 48 h to be completed (Kastelic et al.,

1990b).

Follicular Dynamics

Ovarian follicles grow from the primordial stage (1"t generation of granulosa

cells) to the preovulatory stage during a period of 180 days. A pool of follicles grow

slowly and with low rate of atresia until 7 to 8th generations of granulosa cells under the

influence of basal levels of gonadotropins (FSH and LH), activin, inhibin, follistatin,

growth factors (IGF-1, EGF, FGF, TGFa and b, TNFa), cytokines (IL-1, Interferon-y and

endothelin) and metabolic hormones (Findlay et al., 1996). At the 9th granulosa cells'

generation and at 0.5 mm in diameter, a group of preantral follicles express FSH

receptors and under FSH stimulation grow rapidly and at higher rate of atresia (Findlay et

al., 1996) than occurs by apoptosis (Kaipia and Hsueh, 1997).

The occurrence of follicular waves in the ovaries during the estrous cycle was

reported using postmortem specimens of the reproductive tracts of heifers slaughtered at

different stages after estrus (Rajakoski, 1960). This was subsequently confirmed by

ultrasonography (Pierson and Ginther, 1984a). Studies using daily ultrasonography

described two or three follicular waves in the ovaries during the estrous cycle in cattle

(Savio et al., 1988; Sirois and Fortune, 1988, Knopf et al., 1989). Follicular waves in

primates were described by having a phase of recruitment, selection and dominance

(Goodman and Hodgen, 1983). These waves consisted of a group of 3 to 6 follicles that





15

grow larger than 5 mm and then one (dominance) or two (co-dominance) become

dominant and grow larger than 10 mm and the others become subordinate and regress

(Savio et al., 1988; Sirois and Fortune, 1988). Follicular waves were evaluated in 10

heifers after estrus (Day 0) and two animals presented two follicular waves (starting on

Days 2 and 11), seven animals presented three follicular waves (starting on Days 2, 9 and

16) and one animal presented four follicular waves (starting on Days 2, 8, 14 and 17;

Sirois and Fortune, 1988). The dominant follicle of the first wave reaches maximum

diameter (15-18 mm) on Day 6 after ovulation, goes through a plateau phase from Days 6

to 10 and was undetectable on Day 15 (Savio et al., 1988). The dominant follicle of the

second wave reached maximum diameter on Day 16 (or Day 19 in cows with two

follicular waves) and was undetectable by Day 19 and the dominant follicle of the third

follicular wave reached maximum size on Day 21 and became the ovulatory follicle

(Savio et al., 1988).

The differences between estrous cycle of two and three waves are the length of

the inter-ovulatory interval (20.4 vs 22.8 d), the mean day of luteal regression (16.5 vs

19.2), the interval from emergence of the follicle to ovulation (10.9 vs 6.8) and the

diameter of the follicle the day before ovulation (16.5 vs 13.9; Ginther et al., 1989b).

Follicles acquire ovulatory capacity at an approximate size of 12 mm with 80% of

ovulation in follicles 10 mm in diameter (Sartori et al., 2001). Follicular waves are

characterized by three phases called recruitment, selection and dominance (Ginther et al.,

1989a). The dominant follicle grows 1.8 mm a day until it reaches 16 mm, remains static

for 6 days and regresses linearly at 1 mm a day (Ginther et al., 1989a). The subordinate

follicle ceases to grow at 4.4 days after emergence (Ginther et al., 1989a). Basal





16

concentration of FSH and LH are parallel during most part of the luteal phase, and before

ovulation there is a coincident FSH and LH surge (Rahe et al., 1980). However, there are

two instances during the estrous cycle in which FSH and LH diverge (Turzillo and

Fortune, 1990). Firstly, after luteolysis LH pulsatility increases and FSH decreases (Rahe

et al., 1980), and secondly, early in the luteal phase there is an increase in FSH but no rise

in LH (Dobson, 1978). This post-ovulatory surge of FSH precedes the first follicular

wave (Turzillo and Fortune, 1990) and an increase in FSH precedes the emergence of

each follicular wave by 1 to 2 days (Adams, 1992).

Follicular deviation in cattle occurs during the selection phase when the dominant

follicle is approximately 8.5 mm in diameter (Ginther et al., 2000). The mechanism by

which one or two follicles are selected to be dominant and the rest become subordinate is

not clearly understood and has been the subject of several studies. Intravenous

administration of the proteinaceous fraction of the follicular fluid inhibited follicular

growth (Kastelic et al., 1990a). After deviation (Ginther et al., 2001), the dominant

follicles become dependent on LH pulsatility (Gong et al., 1996), secrete inhibin and

estrogen, and both suppress FSH secretion which may be the mechanism to inhibit

development of subordinate follicles (Badinga et al., 1992; Ginther et al., 2000).

Dominant follicles have a greater aromatase activity and estrogen production than

subordinate follicles with a high ratio of P4/estrogen indicating the beginning of atresia

(Badinga et al., 1992). Follicular dominance or terminal follicular development is

characterized by decreased intrafollicular concentrations of IGF- binding proteins

(IGFBP 2, 4 and 5) that result from a decreased synthesis and increased proteolysis

(Monniaux et al., 1997). In contrast, atresia is associated with an increased concentration





17

of IGFBs and reduced availability of IGF (Monniaux et al., 1997). The increased

availability of IGF within the ovarian follicles amplifies the action of gonadotropins in

follicular cells (Monniaux et al., 1997, Ginther et al., 2001). After a period of dominance

and under high concentration of P4 characteristic of the luteal phase, LH pulsatility

decreases (Rahe et al., 1980) and the dominant follicle regresses and a new wave is

recruited. During an estrous cycle with three waves the dominant follicle of the second

wave grows to a smaller size (Sirois and Fortune, 1988); because of high levels of P4 and

lower LH pulsatility during mid-cycle it is unable to maintain follicular growth (Lucy et

al., 1992; Stock and Fortune, 1993). The decrease in LH support results in reduced

androgen availability through the steroidogenic pathway causing a decrease in estradiol

production and triggering atresia (Lucy et al., 1992).

Energy balance and lactation affect follicular growth which could result in lower

fertility of lactating dairy'cows compared with dairy heifers (Lucy et al., 1992). Negative

energy balance reduces LH secretion and follicular growth (Lucy et al., 1992). Lactating

dairy cows have lower levels of P4 compare to nonlactaing dairy cows and therefore have

larger follicles and a longer period of dominance (De la Sota et al., 1993). Heat stress

also affects follicular dynamics increasing the number of large follicles and accelerating

the regression of the first-wave dominant follicle and the emergence of the second

follicular wave by two days (Wolfenson et al., 1995). Heat stress also appears to reduce

follicular dominance in nonlactating dairy cows (Guzeloglu et al., 2001). In cows treated

with bovine somatotrofine (bST), the growth of the dominant follicle of the first follicular

wave was reduced, the growth of subordinate follicle was increased, and the subordinate

follicle of the second follicular wave was larger (de la Sota et al., 1993). Administration





18

of bST may contribute to a negative energy balance that may result from increased milk

production which could affect growth of the dominant follicle indirectly, and a direct

effect of bST stimulating subordinate follicles and also caused by the increase in IGF-I

secretion (Lucy et al., 1992). Administration of bST in cattle increases the population of

small ovarian follicles (Gong et al., 2002) and in combination with FSH increases the

superovulatory response of dairy heifers (Kuehner e al., 1993).

Drugs Used for the Pharmacological Control of the Estrous Cycle

Prostaglandin F2a

Prostaglandins (PGs) were first detected in human semen and sheep seminal

plasma. They were named PGs since it was believed that they were produced in the

prostate gland. They were shown to cause smooth muscle contraction and changes in

blood pressure. They are a derivative of the prostanoid acid which is form by five

cyclopentane rings (Zubay et al., 1995). They were reported as luteolytic in pregnant rats

(Pharris and Wingarden, 1969), sheep (McCracken et al., 1972) and consequently either

natural (Lauderdale et al., 1972; Rowson et al., 1972) or synthetic PGF2a (Tervit et al.,

1973) were used to induce luteolysis in cattle. The half-life of prostaglandins is very short

and that is useful for the organism in order to remove the hormonal action when it is no

longer required (Zubay et al., 1995). The half-life of the primary PGF2a is 1 minute and

the half-life of PGFM is 8 minutes (Kindahl et al., 1976). Natural PGF2a (25 mg i.m.,

dinoprost tromethamine; Lutalyse, Pharmacia) and synthetic PGF2a (500 utg i.m.,

cloprostenol sodium, Estrumate, Schering Plough) can be used to induce luteolysis in

cattle.





19

The route, dose, and frequency of administration for different types (natural and

synthetic) of PGF2a were evaluated in several studies. Prostaglandins are usually

administered intramuscular. However, attempts to delay absorption or metabolism and

reduce the minimal dose have been made by testing other routes of administration such as

uterine wall (Inskeep, 1973), uterine lumen (Tervit et al., 1973, Louis et al., 1974), or the

antrum of the ovarian follicle (Inskeep et al., 1974). Reduced doses of cloprostenol

administered intra-vulvosubmucosally or subcutaneously resulted in incomplete

luteolysis and prolonged intervals from treatment to ovulation compared with a standard

dose used intramuscularly (Dhaliwal et al., 1991). A reduced dose (10 mg) or standard

dose (25 mg) of dinoprost tromethamine (10 mg) administered into the isquio-rectal fossa

caused luteolysis in cattle with the same effectiveness as did 25 mg injected

intramuscular (Colazo et al., 2002b). Cloprostenol sodium (500 ug) administered

subcutaneously (Colazo et al., 2002a) or intravenously (Stevens et al., 1995) was also

effective in causing luteolysis in heifers.

Administration of 5, 10, 25 and 30 mg of PGF2a (dinoprost tromethamine) during

early (Days 5-9), mid (Days 10-14) or late (Days 15-19) luteal phase showed that 5 mg

were not effective and 25 to 30 mg caused luteolysis in 56% of beef heifers in early luteal

phase and 100% of the animals in mid or late luteal phase (Berardinelli and Adair, 1989).

Similarly results were obtained using either 25 mg of PGF2a (Watts and Fuquay, 1985) or

500 p.g of cloprostenol sodium (Jackson et al., 1979) with increased effectiveness as the

stage of the estrous cycle advanced. Frequency of administration can also influence the

luteolytic effect. Two doses of PGF2a 8 h apart may be more effective in causing

luteolysis than a single or double injection 24 h apart (Archbald et al., 1993, 1994).





20

Progesterone and Progestins

Oral administration of progestins inhibits estrus and ovulation in cattle, and can

be used for estrus synchronization (Hansel et al., 1961). Natural P4 (Lamond, 1964) or

synthetic progestins (Wiltbank, 1968) have been used for estrus synchronization in cattle.

Based on the ability of progestins to synchronize estrus various long-term delivery

intravaginal devices such as PRID (Roche, 1976) and CIDR (Macmillan et al., 1991) or

ear implant such as Synchro Mate B (Burrel et al., 1972) were developed. These devices

together with oral administration of melengestrol acetate (MGA) are the most common

approaches using P4 administration for estrus synchronization.

Melengestrol acetate (MGA; 6a-methyl-6-dehydro-16-methylene-17a-acetoxy-

pregn-4,6-diene-3-20-dione) is an oral product with progestin activity and is

recommended at a dose of 0.5 mg/day (Zimbelman and Smith, 1966). Other oral

progestins are 16-alpha-17 dihydroxyprogesterone acetophenomide (DHPA) and 6-

chloro-A6-17 cetoxyprogesterone (CAP). They are not commonly used. Oral

administration of 400 mg/head/day of DHPA for 9 days combined with 5 mg of estradiol

benzoate on Day 2 (Wiltbank et al., 1968) or 10 mg/heifer/day of CAP for 14 d (Wagner

et al., 1973) have been used to synchronize estrus in beef cattle.

Norgestomet (SC21009;17a-acetoxy-11 1-methyl-19-nor-preg-4-ene-3, 20-dione)

is a potent synthetic progestin that suppress estrus and ovulation at a daily i.m. dose of

0.14 mg (Wishart, 1972). It was incorporated in a polymer as a ear implant (Burrel et al.,

1972). An implant containing 3 or 6 mg of norgestomet implanted for 9 d and combined

with an i.m. injection of 3 mg of norgestomet and 5 mg of estradiol valerate at the time of

implant insertion (Wishart and Young, 1974) was named Synchro-Mate-B.





21

An intravaginal insert consisting of a stainless steel spiral coated with silicone

rubber and impregnated with P4 (Progesterone Releasing Intravaginal Device, PRID,

CEVA, Paris, France) was developed (Mauer et al., 1975) and shown to suppress estrus

and ovulation in heifers and cows (Roche et al., 1976). The CIDR intravaginal insert is

produced by coating a nylon spine with silicon-based elastomer containing 1.9 g (10%

w/w) of P4 (Macmillan et al., 1991). The retention rates of the CIDR insert are 99% in

heifers when used for 4-15 day periods and 98% in cows when used for a 4-7 day period

(Macmillan et al., 1991). Currently in the USA, the CIDR insert contains 1.38 g of P4

(CIDR-B, Pharmacia, Kalamazoo, MI, 49001) and is approved for use in lactating dairy

cows for resynchronization of estrus administered from Day 14 to Day 21 after

insemination.

GnRH and GnRH Agonists

Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted by the

hypothalamus into hypophyseal portal system and stimulates secretion of gonadotropins

from the anterior pituitary gland (Goodman, 1988). The half-life of GnRH is very short

and is cleared from the circulation in 4 to 7 minutes (Redding et al., 1972). GnRH and

GnRH analogue have been used to stimulate follicular growth, induce follicular turnover,

or ovulation in cattle according to the stage of follicular development at the time of

treatment (Macmillan and Thatcher, 1991, Thatcher et al., 1993). GnRH (native GnRH)

includes gonadorelin diacetate tetrahydrate (Cystorelin; Fertagyl) and gonadorelin

hydrochloride (Factrel). GnRH analogues and agonists include buserelin (D-

Ser(tBu)6,Pro9 Net; Receptal), fertirelin acetate (Ovulyse) and deslorelin ((pGlu-His-

Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-NEHt; Ovuplant). The chemical modification of





22

GnRH agonists makes them more stable and increases their affinity to plasma proteins,

membranes and the GnRH receptor (Conn and Crowley, 1991). Fertirelin acetate is 4 to

10 times and buserelin 50 times more potent than gonadorelin (Chenault et al., 1990).

The recommended or commonly used doses are 10-20 pg for buserelin, 25-50 gg for

fertirelin acetate and 100-200 pg for gonadorelin acetate (Chenault et al., 1990). In a

protocol for synchronization of ovulation and timed insemination (TAI; Ovsynch) the

use of either 100 or 50 jtg of gonadorelin acetate was equally effective (Fricke et al.,

1998). Gonadorelin diacetate was more effective in inducing the LH surge in dairy cows

than gonadorelin hydrochloride, but no differences were detected in beef heifers and

difference were observed between commercial preparations of the same analogue

(Martinez et al., 2002). Deslorelin (GnRH analogue, 2,100 ug, Ovuplant, Peptech

Animal Health, North Ryde, Sydney, Australia 2113) is a nonapeptide administered as a

subcutaneous implant and approved for induction of ovulation in horses.

Human Chorionic Gonadotropin (hCG)

Human chorionic gonadotropin is a glycoprotein secreted by the placenta of the

pregnant woman and excreted in the urine. The administration ofhCG during diestrus has

been used to stimulate CL function and enhance embryo survival (Wiltbank et al., 1961).

Intravenous injection of hCG (5,000 IU) on Days 5, 10, 15 and 20 of the estrous cycle

had variable results in terms of P4 increase, induced ovulations and prolonged the estrous

cycle (Schomberg et al., 1967). The administration of 1,000 and 2,000 IU of hCG from

Days 1 to 7 of the estrous cycle were also able to stimulate CL function in beef heifers

(Veenhuizen et al., 1972). The administration of 1,000 to 2,000 IU ofhCG at estrus or 3

days later has been used to increase pregnancy rate in synchronized beef heifers with





23

variable results (Wagner et al., 1973). Currently, hCG (i.m., 10,000 IU, Chorulon,

Intervet, Millsboro, DE 19966) is available for the treatment of cystic ovaries.

Estrogens

The naturally occurring estrogens in the cow are estradiol and estrone. Estradiol is

produced by the granulosa cells of mature follicles. The half-life of estradiol is around 5

minutes and metabolites are excreted in feces and urine. Synthetic estrogens are

metabolized more slowly in the liver and half-life is extended (Wright and Malmo, 1992).

Estradiol has been used to induce regression of the CL (Wiltbank et al., 1968), induce

atresia of antral follicles and synchronize ovarian follicular waves (Bo et al., 1995) and

induce ovulation (Smith and Zimbelman, 1968a, Welch et al., 1975). Different salts of

estradiol (estradiol valerate, estradiol cypionate, estradiol benzoate), and estradiol-170

can be used in cattle, but they are not approved for use in domestic farm animals. Doses

from 0.5 to 10 mg have been administered at different stages of the estrous cycle either

by intramuscular injections or in intravaginal capsules to synchronize follicular waves.

Estradiol-173 (Bo et al., 1994), estradiol benzoate (Macmillan and Burke, 1996),

estradiol valerate (Bo et al., 1993) and estradiol cypionate (Thundatil et al., 1998) have

been used alone or in combination with P4, to synchronize follicular waves in cattle.

Estradiol benzoate (10 mg) in a gelatin capsule placed in the groove of a CIDR device

and administered intravaginally had a variable effect on synchronization of estrus

(Macmillan and Burke, 1996). Estradiol-17P and estradiol benzoate have been the most

effective to synchronize the follicular wave in cattle in doses from 2 to 5 mg (Bo et al.,

1995). Estradiol cypionate (Pancarci et al., 2002) and estradiol benzoate 1 mg i.m. (Day,

1998) have been administered 48 h after PGF2, to synchronize ovulation in cattle.





24

Methods for Synchronization of Estrus in Cattle

The ability to freeze bovine semen while maintaining acceptable fertility has

allowed artificial insemination to be a reproductive technology of tremendous impact in

cattle genetics and productivity (Foote, 1996). Artificial insemination is a commonly

used technique in dairy and beef herds. It requires estrus detection since cows need to be

inseminated at a specific time after onset of estrus. Timing of insemination was the

objective of many studies that determined that cows need to be inseminated 12 hours

after onset of estrus (AM-PM rule; Trimberger and Davis, 1943) or within 24 hours after

onset estrus when using morning insemination only (Barret and Casida, 1946, Robbins et

al., 1978). An interval of 12 hours between onset of estrus and insemination seems to be

ideal since it increases fertilization rate and accessory sperm per embryo without

reducing embryo quality (Dalton et al., 2001). In order to eliminate much of the time

required for estrus detection and be able to inseminate most cows in a short period of

time, estrous synchronization protocols were developed.

Estrus occurs after P4 levels drop resulting in an increase in LH pulsatility which

stimulates follicular growth and estrogen production. Estrus can be induced either by

administration and subsequent removal of P4 supplementation (Lamond et al., 1964),

inducing luteolysis with PGF2a(Lauderdale et al., 1974), or by a combination of both

approaches.

Progesterone and Progestins

The use of P4 supplementation for extended periods of time (14 to 21 days)

creates an artificial luteal phase, provides enough time for any CL to regress, and induces

estrus in 2 to 5 days after P4 removal ( Hansel and Malven, 1960; Zimbelman and Smith,





25

1966). However, P4 supplementation for an extended period of time in beef heifers

reduced pregnancy rate (Hansel, 1967, Roche 1974a). Long term (14-21 days) treatment

with P4 for estrus synchronization results in low levels of P4 which increase LH

pulsatility and extends the growth of the dominant follicle which suppress growth of

other follicle for period of more than 20 days (Savio et al., 1993). Persistent follicles are

associated with follicular and oocyte degeneration (Mihn et al., 1994) and reduced

fertility (Savio et al 1993b). Reduction in the length of the treatment was a strategy to

improve fertility, and an oral progestin (Wiltbank 1968), a silastic subcutaneous implant

of P4 (Roche 1974b, 1974c) or a norgestomet implant (Wiltbank et al., 1971) were

combined with estrogen at initiation of treatment to induce premature regression of the

CL.

However, the dose of P4 will influence gonadotropins control (Begfeld et

al.,1996) and treatments with MGA or Syncro-Mate-B generated levels of P4 lower than

mid-luteal phase resulting in greater LH pulsatility, persistent follicles and greater

production of estrogen (Kojima et al., 1992). Low levels (-2 ng/ml) compared with high

levels (-6 ng/ml) of P4 created with PRID inserts generated an acute (6-24 hours)

increase in LH pulsatility and 171-estradiol concentrations (Bergfeld et al., 1996). The

use of 2 compared with 1 PRID insert during a 9 day period and administration of PGF2a

on Days -9 and -3 to lyse the natural CL, reduce estradiol concentrations and increased

pregnancy rate in the cow (Wehrman et al., 1993). Similarly, norgestomet treated cows

had lower levels of estradiol during treatment and higher conception rate to synchronized

estrus when a CL was present during the period of treatment (Sanchez et al., 1993).





26

Syncro-Mate-B combines a norgestomet ear implant for 9 days and estrogen at

initiation of the treatment and after implant insertion. Therefore, estradiol increases in the

first 24 hours, and remains higher than controls until the end of the treatment, P4

decreases, LH pulses seem to be slightly increased but mean concentrations decrease,

FSH levels are reduced and LH and FSH increase 48 hours after implant removal

(Rathbone et al., 2001). Synchronization of estrus with Syncro-Mate-B resulted in

reduced estrus expression (Roche 1974b, 1974c), when treatment was initiated in cows in

proestrus or metestrus (Roche 1974c). This indicated that many cows did not regress the

CL or the estrogen injection induced ovulation and the formation of CL that did not

regressed after implant removal (Smith and Zimbelman, 1968b). Consequently,

pregnancy rates were variable (Hansel, 1965; Beal, 1996). In addition, fertility after P4

implant (Roche 1974b) or a norgestomet implant (Wiltbank et al., 1971) is variable due to

variation in LH surge and behavioral estrus after cessation of treatment.

Prostaglandin F2a

In 1972, PGF2a was identified as luteolytic in sheep (McCraken et al., 1972) and

used to synchronize estrus in cattle (Lauderdale 1972, Rowson et al., 1972) allowing for

certain control of time of ovulation (Lauderdale et al., 1974). After PGF2a administration,

plasma P4 declines 50% within 7 hours and to less than 0.5 ng/ml within 24 hours,

estradiol increases abruptly 1 to 9 hous after PGF2a treatment and remains high until the

LH surge occurs and then decreases (Thatcher and Chenault 1975). Animals treated with

PGF2a usually express estrus in 64 hours with an average of 36 to 96 hours (Dalton et al

2001) and fertility is similar to cows inseminated at spontaneous estrus (Lauderdale 1972,

Rowson et al., 1972).





27

Some of the limitations need to be considered regarding the use of PGF2a to

induce estrus: 1) PGF2a is not effective during the first five days of the estrous cycle

(Lauderdale et al., 1972; Rowson, 1972); 2) the interval from treatment to estrus varies

according to the stage of the estrous cycle at the time of PGF2 treatment (King et al.,

1982; Stevenson et al., 1984; Kastelic et al., 1990); 3) even tough conception rates after

PGF2z-induced estrus are high (Beal, 1996). Reduced or inconsistent conception rates

after PGF2a-induced estrus have been reported in lactating dairy cows on a few occasions

(Macmillan et al., 1977; 1983); and 4) PGF2 will not induce estrus in noncycling cows.

Until 1982 it was believed that all cows will respond to PGF2a after Day 4 of the

estrous cycle (Beal, 1996). However, it was demonstrated that there was an interaction

between dose and stage of the estrous cycle, with high doses being effective in early

stages of the estrus cycle (5-9) and lower doses being also effective in late stages (9-17)

of the estrous cycle (Berardinelli and Adair, 1989). The bovine CL becomes more

sensitive as the cycle progresses, and after Day 4 maximum responsive can be achieved

with a 25 mg dose or multiple administrations with equal doses given 6 to 24 hours apart

(Beal, 1996). Since PGF2a is not effective during the first 4 days of the estrous cycle, a

schedule including 2 doses 11 days apart (heifers) or 14 d apart (cows) will synchronize

the majority of the animals (Beal, 1996).

The stage of the estrous cycle also affects the interval from treatment to

expression of estrus (Beal, 1996). Administration of PGF2a between Days 5 to 8 of the

estrous cycle synchronizes estrus within 48 to 59 h (King et al., 1982) but the average

was 52 to 72 h when administered between Days 12 and 15 of the estrous cycle. The

response depends on the stage of the follicular wave at the time of treatment (Beal, 1996).





28

Fertility to PGF2a-synchronized estrus is high compared to cows inseminated after

spontaneous estrus breeding 12 hours after onset of estrus for both cases (Moody and

Lauderdale, 1974). The levels of P4 exposure may have an effect on fertility since P4

values 2 days before the LH surge was higher in cows that conceived compared to cows

that failed to conceive (Erb et al., 1976), cows with higher P4 during the 12 days prior to

estrus had higher conception rate (Fonseca et al., 1983), and cows treated with PGF2 in

late luteal phase had higher fertility (Tanabe et al., 1984; Xu et al., 1997). However, other

reports did not find any relationship between the stage of the estrous cycle at the time of

treatment with PGF2a and fertility (Stevenson et al., 1984).

Combinations of Progesterone or Progestins and PGF2a

Progesterone supplementation was combined with PGF2a in order to reduce the

length of treatment from 7 to 9 days (Beal, 1996), and elevate P4 concentrations before

PGF2a administration (Xu et al 1997).

A combination of PGF2a and MGA is commonly used in beef and dairy heifers to

synchronize estrus. It includes administration of MGA for 14 days and heifers express

estrus within 2 to 6 days after MGA removal. Then, heifers are treated with PGF2, 17

days after MGA removal when heifers are in late luteal phase obtaining high fertility after

insemination at detected estrus (Patterson et al., 1989). Several studies using a

combination of progestins and PGF2a showed increased (Xu et al., 1997), acceptable

(Roche,1976; Folman et al., 1990, Ryan et al., 1995; Whittier et al., 1986; Beal and Good

1986) or reduced fertility (Xu et al., 1997).

The use of MGA for 7 days combined with a dose of PGF2a on Day 7 was applied

in order to reduce the length of the treatment (Beal and Good 1986). However, when this





29

protocol was used during diestrus pregnancy rates were reduced (Beal, 1996). The

reduced fertility was probably due to the low level of P4 which was not able to inhibit LH

pulsatility like a natural CL, and, therefore, persistent follicles developed (Beal, 1996).

Even though fertility may have been improved after 9-day P4 treatments in combination

with PGF2a at P4 removal, the distribution of estrus and the interval to ovulation were not

synchronized enough to allow for TAI.

The understanding of follicular waves led to the use of GnRH to synchronize the

wave and increase the synchrony of induction of estrus (Thatcher et al., 1989;

Twagiramungu et al., 1992; Wolfenson et al., 1994). When GnRH was administered on

Day 12 of the estrous cycle, synchronization of estrus was tighter compared to cows

receiving PGF2a, and the dominant follicle was more estrogenic and dominant

(Wolfenson et al., 1994). The use of GnRH 7 days before PGF2a treatment avoids the

problem of persistent follicles that occur when estrus is synchronized with progestins or

any other method (Savio et al., 1993). Similarly, estrogen administration induces

follicular atresia and synchronizes the follicular wave allowing for a more control timing

of ovulation (Bo et al., 1995).

Induction of Ovulation and TAI

Induction of ovulation allows for TAI and eliminates the need for estrus detection.

In order for TAI to be successful, an LH surge must be induced, and such factors as

interval from LH surge to ovulation, insemination time, sperm transport and viability,

ovum viability, and number of accessory sperm per embryo need to be considered to

optimize fertilization rate and embryo quality (Saacke et al., 2000). The time from LH

surge to ovulation is 24 hours in dairy heifers (Thatcher and Chenault, 1975), sperm





30

transport takes 6 to 12 hours (Hawk, 1987), sperm viability is 24 to 32 hours, ovum

viability is 8 to 12 hours (Thibault, 1967) and at least 10 accessory sperm per embryo are

necessary to achieve satisfactory embryo quality (Saacke et al., 2000). Based on the

dynamics during periovulatory period, insemination is recommended at 12 hours after the

LH surge (Trimberger and Davis, 1943; Dalton et al., 2001).

Ovulation can be induced by administering GnRH to directly stimulate the

anterior lobe of the pituitary gland to release LH (Reeves et al., 1972; Kaltenbach et al.,

1974), hCG to directly induce the follicle in the ovaries to ovulate (Graves and Dziuk,

1968; Schmitt et al., 1996), or estradiol to stimulate GnRH release from the

hypothalamus and indirectly LH release from the pituitary gland (Hobson and Hansel,

1972; Inskeep, 1973; Roche, 1974). In order to obtain better synchronization of estrus,

two doses of PGF2a could be administered 12 days apart. Although estrus is induced in

the majority of the cows, the estrus periods were still spread from Days 2 to 4 after the

last PGF2a treatment (Day 2=23%; Day 3=54%; Day 4=15%; Lauderdale et al., 1974).

After the two doses of PGF2a, TAI without induction of ovulation (Lauderdale et al.,

1974; Stevenson et al., 1987) or after administering GnRH at 48 hours after the last dose

of PGF2a (Lucy and Stevenson, 1986), or GnRH 48 hours after PGF2a (Thatcher and

Chenault, 1975) resulted in reduced fertility, but not when it was combined with estrus

detection. Cows in diestrus treated with PGF2a and inseminated at detected estrus for 72

hours, and cows not detected in estrus treated with GnRH and time-inseminated at 72-80

hours had pregnancy rates similar to cows inseminated after detected spontaneous or

PGF2a-induced estrus (Archbald et al., 1992).





31

The description of follicular waves by macroscopic observation of reproductive

tracts from heifers at different stages of the estrous cycle (Rajakoski, 1960) or by

ultrasonography (Pierson and Ginther, 1984) and a detailed description of follicular

waves during diestrus using ultrasonography (Savio et al., 1988; Sirois and Fortune,

1988) made researchers realize that follicular dynamics during diestrus should be

controlled in order to improve fertility (Thatcher et al., 1989). Through different

mechanisms, GnRH, estradiol, and P4 will induce follicular turnover in cows with ovarian

follicles at different stages. Estradiol suppresses FSH secretion and induces atresia of

follicles that are in the recruitment phase (2-8 mm; Burke et al., 2000). However, P4 will

suppress LH pulsatility and induce atresia in follicles that are larger than 10 mm

(Anderson and Day, 1994), and GnRH will induce LH and FSH release with ovulation of

follicles larger than 10 mm and recruitment of a new follicular wave (Thatcher et al.,

1989). A new follicular wave is recruited approximately 2 days after GnRH

administration (Thatcher et al., 1989), 2 days after hCG (Rajamahendran and

Sianangama, 1992; Niasari-Naslaji et al., 1996) 3-4 days after P4 (Anderson and Day,

1994), and 4-5 days after estrogen administration (Bo et al., 1995).

The Use of GnRH and PGF2a to Synchronize Estrus

Administration of a GnRH agonist (buserelin) induces an immediate release of

FSH and LH from the pituitary, a rise in estrogen, ovulation, luteinization or atresia of

follicles larger than 10 mm, and recruitment of a new follicular wave within 2-3 days

(Thatcher et al., 1989). Administration of PGF2a 7 days after GnRH (Select Synch;

Thatcher et al., 1989) will induce a more synchronized incresase in estradiol (Wolfenson

et al., 1994) and estrus within 36-72 hours after luteolysis. The Select Synch protocol in





32


comparison to administering PGF2a alone, increases estrus synchronization rate, and may

induce estrus in some noncyclic animals and may increase fertility. An extra benefit can

be obtained by including a P4 supplement (CIDR Insert) between the GnRH and the

PGF2a in the Select Synch protocol. This will avoid the occurrence of estrus before

PGF2a treatment (Stevenson et al., 1997) and induce estrus in some noncyclic animals.

Induction of Ovulation and TAI Using GnRH and PGF2a (Ovsynch Protocol)

The administration of GnRH induced an LH surge and ovulation in the cow

(Fernandez Limia et al., 1977; Graves et al., 1974), and therefore it was administered at

estrus or at AI in normal and repeat-breeding cows in order to increase conception rate

(Mee et al., 1990). The results were inconsistent since stage of follicular development at

the time of GnRH treatment was not considered. However, a second dose of GnRH

administered 48 hours after PGF2a injection in the Select Synch protocol will

synchronize ovulation and allow for TAI (Pursley et al., 1995; Burke et al., 1996). Once

the follicular wave is controlled, synchronization of ovulation for TAI is possible by

inducing an LH surge since there is a constant interval between LH surge and time of

ovulation (Cumming et al., 1973).

Administration of GnRH at random stages of the estrous cycle induced ovulation

in approximately 70 % of lactating dairy cows (Vasconcelos et al., 1999), and treatment

with PGF2a 7 days later caused luteolysis in 90 to 100% of the cows (Pursley et al., 1995;

Vasconcelos et al., 1999). Injection of GnRH 48 hours later (Ovsynch protocol)

synchronized ovulation within 24 to 32 hours later (Pursley et al., 1995) and TAI is

conducted 16 hours after the second GnRH treatment (Burke et al., 1996). GnRH

administered after PGF2a induces ovulation, but cows usually do not express estrus





33

(Rodriguez et al., 1975; Lucy and Stevenson, 1986) since the preovulatory follicle does

not have time to produce enough estrogen (Lucy and Stevenson, 1986). Despite a slight

reduction in conception rates, the Ovsynch protocol maintains or increases pregnancy

rates when compared to different reproductive strategies because 100% of the cows are

inseminated without the need for estrus detection (Schmitt et al., 1996; Burke et al., 1996;

Pursley et al., 1997a; Pursley et al., 1997b; Fricke et al., 1998 Momcilovic et al., 1998;

Britt and Gaska., 1998; Stevenson et al., 1999).

Different variations of the Ovsynch protocol have been used. It is important to

maintain a 7 day interval from the first GnRH injection to PGF2 treatment because a 6

day interval may not be enough for the GnRH-induced CL to be responsive to the

luteolytic effect of PGF2a. On the other hand, an 8 day interval will allow time for more

cows to express estrus before synchronization of ovulation. Other modifications include

administration of the second GnRH 30 to 56 hours after PGF2a as well as TAI at different

times (0, 16, 24 or 32 hours) after the second GnRH. If the interval from PGF2a to

second GnRH injection is less than 48 hours, a smaller, less mature dominant follicle is

ovulated with reduced fertility. Nonlactating cows and heifers that received GnRH for

induction of ovulation 24 hours after PGF2a formed a CL that regressed earlier (Schmitt

et al., 1996), and this short luteal phase will compromise fertility (Peters and Pursley,

2002).

An interval from PGF2a to second GnRH of more than 48 hours may allow for a

higher proportion of cows to show estrus before GnRH injection and these cows need to

be inseminated at detected estrus in order to maintain fertility. In addition, GnRH

administration after the spontaneous LH surge may compromise corpus luteum





34

development and decrease P4 levels during diestrus (Lucy and Stevenson, 1986).

Intervals from second GnRH to TAI of 0, 8, 16, 24, and 32 hours, generated pregnancy

rates at 30 days of 37, 41, 45, 41 and 32%, respectively. Furthermore, pregnancy losses

were higher when cows were timed inseminated at 32 hours (Pursley et al., 1998).

Therefore, TAI 16 hours after the second GnRH seems ideal, but at 0 and 24 hours

pregnancy rates were acceptable. Therefore, it is very important to maintain the timing of

injections for the Ovsynch protocol to avoid reductions in pregnancy rate.

In several studies, the Ovsynch protocol increased reproductive efficiency in

dairy herds. The Ovsynch protocol for TAI reduced days open and eliminated the need

for estrus detection in lactating dairy cows compared to a reproductive program based on

PGF2a and AI using the AM-PM rule (60 and 100 d postpartum pregnancy rates were 37

and 53% for Ovsynch and Control respectively; Pursley et al., 1997a). The Ovsynch

protocol was also more efficient when compared to a program involving PGF2a injections

every 14 days and AI at detected estrus or TAI 72-80 hours after the third treatment

(Pursley et al., 1997b). In addition, the Ovsynch protocol in lactating dairy cows

achieved a similar pregnancy rate (30%) compared to cows that were inseminated at

detected estrus after injection of GnRH and PGF2a given 7 days apart, estrus detection

rate in the latter group was 70% (Burke et al., 1996).

In a similar study but with a lower estrus detection rate, higher pregnancy rates

were obtained with the Ovsynch protocol compared with AI at detected estrus after a

single dose or double dose ofPGF2 given 14 days apart or after GnRH and PGF2a given

7 days apart (Stevenson et al., 1999). Higher pregnancy rate and a reduced interval from

calving to conception was obtained using the Ovsynch protocol at Day 50 postpartum in





35

lactating dairy cows compared with AI to a spontaneously detected estrus or after one

dose of PGF2a on Day 57 or two doses on Days 33 and 47 postpartum (Momcilovic et al.,

1998). Following a Presynch protocol, a Co-Synch protocol involving GnRH and TAI at

72 h after PGF2a, increased pregnancy rate in lactating dairy cows compared to Ovsynch

(GnRH at 48 h and TAI 24 h later) or Co-Synch (GnRH and TAI at 48 h; Portaluppi and

Stevenson, 2003).

Heatsynch Protocol

Administration of estradiol during proestrus induced an LH surge and

synchronized ovulation in cattle (Inskeep, 1973). The second treatment with GnRH of the

Ovsynch protocol was replaced by estradiol cypionate (ECP) administered 24 hours

after PGF2a and induced ovulation 60 hours later in dairy heifers. The protocol includes

100 utg of GnRH given on Day 0, 25 mg of PGF2a on Day 7, 1 mg of ECP on Day 8 and

a TAI 48 hours later (Lopez et al., 2000). This program was named Heatsynch because of

the synchronized occurrence of estrus. In lactating dairy cows, ECP induced ovulation 55

hours later, estrus occurred 29 hours after ECP and the interval from onset of estrus to

ovulation was 27 hours. However, 20% of the cows showed estrus by 24 hours after ECP

and ovulated before 48 hours (i.e. time of AI)). Therefore, it is recommended that the

Heatsynch protocol include inseminating cows detected in estrus within 24 hours after

ECP injection (Pancarci et al., 2002). Seventy five to 85% of the cows subjected to the

Heatsynch protocol show estrus, and AI technicians report greater uterine tonicity and

ease of Al compared to the Ovsynch protocol, in which only 30% of the cows showed

estrus after the second GnRH injection. (Pancarci et al., 2002).





36

Factors Affecting the Efficacy of GnRH to Synchronize Follicular Waves

Several factors such as stage of the estrous cycle, parity, days in milk, heat stress,

bovine somatotropin (bST) treatments, and ovarian abnormalities (anestrus and cystic

ovarian degeneration) may affect pregnancy rate to protocols for TAI using GnRH and

PGF2a. The key for success of estrous synchronization and induction of ovulation

protocols using GnRH and PGF2a lies in the effectiveness of the GnRH treatment to

ovulate a follicle present in the ovaries and to recruit a new follicular wave which will

produce a healthy follicle which will ovulate and form a strong CL after PGF2a treatment.

Stage of the estrous cycle affects the response to an injection of a GnRH analogue

or agonist to synchronize follicular wave emergence. Induction of follicular turnover

depends on the presence of a dominant follicle of at least 10 mm in diameter, and the

ability of GnRH to induce ovulation of that follicle (Moreira et al., 2000). The Ovsynch

protocol was more efficient when initiated during mid-cycle (Day 5 to 9) since most of

the cows ovulated to the first GnRH injection (Moreira et al., 2000). When Ovsynch

was initiated on Days 1-4, 5-9, 10-16 and 17-21, ovulation rate to first GnRH was 23, 96,

54 and 77 %, respectively. The ovulation rate to the second GnRH was 94, 89, 85 and

81% when size of the follicle at second GnRH (i.e. 48 h after PGF2a) was 19, 17, 18 and

20 mm, respectively. Overall, 93% of the animals regressed their CL after PGF2a

(Vasconcelos et al., 1999). When cows ovulated to the first GnRH, 92% of them ovulated

to the second GnRH injection, but only 79% ovulated if they failed to ovulate to the first

GnRH injection. When the protocol is initiated in late diestrus, 6% of cows ovulated

before the second GnRH, and 7% failed to ovulate at 48 hours after the second dose of

GnRH (Vasconcelos et al., 1999). The Ovsynch' protocol initiated early in the luteal





37

phase (Day 2) fails to synchronize follicular recruitment since the dominant follicle of the

first wave is too small to turn over in response to GnRH. The follicle will continue to

grow and be an aged follicle at the time of the second GnRH for induction of ovulation

(Moreira et al., 2000). Extended periods of follicular dominance are associated will low

fertility (Mihn et al., 1994) and higher pregnancy losses (Ahmad et al., 1995). Similarly,

if the Ovsynch protocol is initiated late in the luteal phase (e.g. Day 15) the dominant

follicle of the second wave may be too small and PGF2a will be spontaneously released

approximately 2 days later. This will cause the CL to regress and estrus will be induced

well before the TAI resulting in low fertility (Moreira et al., 2000).

Based on this information, a protocol including two doses of PGF2a given 14 d

apart (Presynch) was developed that allowed initiation of the Ovsynch' protocol 12 days

after the second PGF2a injection. Following this protocol, cyclic cows will be between

Days 5 and 10 of the estrous cycle when the Ovsynch program is initiated (Moreira et

al., 2001). The Presynch-Ovsynch protocol increased pregnancy rate by 10% in cyclic

cows compared with the Ovsynch protocol with no presynchronization (Moreira et al.,

2001). There was no difference in pregnancy rate between Presynch-Ovsynch and

Presynch-Heatsynch protocols in lactating dairy cows (Pancarci et al., 2002). The

Presynch-Ovsynch protocol is 36 to 38 days long (depending if the Ovsynch is initiated

either 12 or 14 days after the second PGF2a injection) and can be initiated any time after

calving according to the designed voluntary waiting period and first insemination.

Initiating the Ovsynch at 14 days after the second PGF injection has been designated as

a modified Presynch-Ovsynch protocol and increases pregnancy rate compared to

Ovsynch with no presynchronization (Fricke et al., 2004). Insemination at detected





38

estruses can be considered after any of the PGF2a injections according to management

decisions. Since administration of PGF2a 12 and 26 days after calving in cows with

abnormal parturition improved conception rate (Risco et al., 1994), the Presynch protocol

inititated early in postpartum may have an additional benefit on cows with uterine

infections.

Another practical alternative of using a presynchronization protocol is to assign

Ovsynch or Heatsynch protocol at the right stage of the estrous cycle based on the

findings at rectal palpation or ultrasonography. Cows with a corpus luteum could be

treated with PGF2a followed by insemination at detected estrus for 14 days and cows not

detected in estrus could be subjected to the Ovsynch protocol. Cows without a corpus

luteum and signs of estrus (follicles and high uterine tonicity) could be subjected to the

Ovsynch or Heatsynch protocol and cows without corpus luteum that are in metestrus,

anestrus or have ovarian cysts could receive GnRH to induce formation of a corpus

luteum and then submitted to the Ovsynch protocols 8 days later. Using this approach,

insemination at detected estrus is combined with TAI and reduces some costs and time

for resynchronization and targets protocols for TAI that are most appropriate for various

stages of the estrous cycle (Bartolome et al., 2002).

The Ovsynch protocol was effective in lactating dairy cows but not dairy heifers

(Pursley et al., 1995; 1997b). However, Schmitt et al., 1996 used a modified Ovsynch

protocol with dairy heifers (Ovsynch protocol with heifers detected in heat at < 39 hours

after PGF2a being inseminated) reported a reduced conception rate, but similar pregnancy

rate compared to a Select Synch protocol involving insemination at detected estrus. The

greater rate of follicular turnover in heifers results in premature expression of estrus.





39


Reproductive responses of heifers treated with PGF2a, Select Synch and Ovsynch

protocols are described in Table 2 (Stevenson et al., 2000).

Days in milk are known to affect the response to TAI protocols. Pregnancy rates

were optimized when the Ovsynch protocol was initiated after 75 days postpartum

(Pursley et al., 1997b). Anestrus may be one of the factors reducing pregnancy rate to the

Ovsynch protocol early in lactation. In fact, pregnancy rate to a TAI protocol was

reduced in cows with BCS less than 2.5 (1-5 scale) and reducing the proportion of cows

with BCS < 2.5 may have an economical benefit (Moreira et al., 2000). The effect of

anestrus can be even more pronounced with use of the Heatsynch protocol that include an

estrogen to induce ovulation. Cows with BCS less than 3.0 had a higher pregnancy rate to

the Presynch-Ovsynch protocol compared to the Presynch-Heatsynch protocol (Pancarci

et al., 2002). Inclusion of P4 supplementation from the first GnRH to the PGF2a in the the

Ovsynch" protocol will likely avoid premature occurrence of estrus and enhance cyclic

responses in anestrous animals (Thatcher et al., 2001).

Heat stress is another factor affecting fertility in dairy herds. During a heat stress

period estradiol production by preovulatory follicles is reduced and estrus expression is

compromised. In addition, heat stress increases embryo mortality. The Ovsynch

protocol increased cumulative pregnancy rate (Arechiga et al., 1998) and increased

pregnancy rate, reduced days open and increased net revenue per cow due to the

elimination of estrus detection during the summer time (De la Sota et al., 1998).

Administration of bST is a common practice in dairy herds in USA to increase

milk production. The effect of bST on reproductive efficiency has been reported to be

negative due to a reduction in estrus expression. However, administration of bST at the





40

time of the first GnRH of the Ovsynch protocol or at TAI increased pregnancy rate

(Moreira et al., 2001). Both bST and IGF-I stimulate in vitro development of embryos

(Moreira et al., 2002). Effects ofbST on the embryo and intrauterine environment likely

contribute to the increase in pregnancy rate in lactating dairy cows.

Anestrus and cystic ovarian degeneration are abnormalities of ovulation and may

occur in nonpregnant cows at any time but are more common during the postpartum

period. Anestrus and a high proportion of cows with ovarian cystic degeneration are

characterized by lack of expression of estrus. Approximately 25% of the cows are not

cyclic (anestrus and ovarian cystic degeneration) by Day 63 postpartum (Moreira et al.,

2001). For cows to have a LH surge, ovulation and a normal luteal phase of P4 need to

rise either by a progressive luteinization of follicles or by treatment with exogenous P4

(Inskeep, 1995). A treatment with P4 for 7 days and 1 mg of estradiol benzoate 24 hours

later induced estrus, and cows can be inseminated within 4 days with acceptable fertility

(Macmillan and Burke, 1996). Such a protocol could be used to presynchronize cows that

may be at high risk of anestrus before a TAI protocol is applied. Cows with ovarian cysts

had a similar pregnancy rate to either the Ovsynch protocol (24%) or Select Synch

protocol (18%). Although conception rate for Select Synch was 50%, the estrus detection

rate was only 35% (Bartolome et al., 2000).

Select Synch, Ovsynch and Cosynch in Beef Heifers and Cows

The Select Synch, Ovsynch and modifications of Ovsynch (e.g. Cosynch) have

been used in beef animals (cows and heifers). Similar to dairy heifers, beef heifers may

be prepuberal and noncyclic and may fail to respond to the initial GnRH. They may also

express estrus (10%) before the PGF2,.injection given 7 d after the GnRH. Beef cows





41


may be in postpartum anestrus and GnRH and PGF2a may not be effective. However,

GnRH based protocols are more effective than PGF2a alone since the GnRH injection

may induce cyclicity in a small proportion of anestrus cows (-15-25%; Day, 1998). An

extra benefit can be obtained by including a P4 supplement (e.g. CIDR Insert) between

the GnRH and PGF2a of the Select Synch protocol which collectively avoid estrus

expression before PGF2a, increases estrous synchronization by 10%, and induces estrus in

an additional 20% of anestrus cows (Day, 1998).

However, if noncyclic status of prepuberal heifers and postpartum beef cows are

very pronounced, presynchronization with p P4 may be necessary. Administration of

GnRH induced ovulation in 75% (Thomson et al., 1999) to 83% (Troxel and Kesler,

1984) of the anestrous beef cows between Days 17 to 44 and 21 to 30 postpartum,

respectively. When a progestin was administered for 8 days, GnRH treatment on Day 8

induced a greater LH surge, induced ovulation in 100% of the cows, and reduced the

incidence of short luteal phases (Troxel and Kesler, 1984). A progestin between GnRH

and PGF2a given 7 days apart increased the proportion of cows that ovulated and had a

normal luteal phase to a GnRH given 48 hours after the PGF2a (Thompson et al., 1999).

A traditional protocol for estrus synchronization in beef heifers in the USA was to

feed MGA for 14 days and administer PGF2a 19 days after MGA removal (Patterson

et al., 1989). Administration of GnRH 7 days before PGF2a in the MGA protocol

increased synchronization of estrus and is called MGA Select (Patterson et al., 2003).

Reduction in the length of the treatment was achieved by using the 7-11 Synch protocol

(MGA Day 0 to 7, PGF2a Day 7, GnRH Day 11, PGF2a Day 18 and insemination at

detected estrus). The 7-11 Synch program increased estrus synchrony compared to the





42

Select Synch protocol (Kojima et al., 2000). Replacing MGA with a CIDR-B in the 7-11

Synch protocol seems to even increase estrus synchronization and pregnancy rate in beef

animals (Patterson and Kojima, 2003).

Timed insemination is also an alternative to increase the proportion of beef herds

that can adopt artificial insemination to improve genetic selection and productivity. After

the MGA Select or 7-11 Synch protocols, a second GnRH treatment at 60-72 hours plus

TAI at 60-72 hours after PGF2a generated acceptable pregnancy rates (60%; Patterson

and Kojima, 2003). In a study with postpartum beef cows, pregnancy rate was higher for

the Ovsynch protocol combined with 48-hour calf removal beginning at PGF2a injection

(54%) compared to a Syncro-Mate-B protocol alone (42%) or with calf removal for 48

hours after implant removal (42%; Geary et al., 1998). The lower fertility with Syncro-

Mate-B may be related to estradiol valerate not being as consistent in synchronizing the

follicular wave and that PGF2a was not used at implant removal (Geary et al., 1998). In

order to reduce handling the Ovsynch protocol was modified such that TAI was done at

the second GnRH injection and is referred to as the Cosynch protocol. A high pregnancy

rate was obtained using the Cosynch protocol with calf removal for 48 hours from PGF2a

injection until the GnRH injection and TAI (Geary et al., 2000). In another study,

pregnancy rate was 52% for Ovsynch, 54% for Co-Synch, 61% for Ovsynch plus calf

removal and 63% for Co-Synch plus calf removal (Stevenson et al., 1997). An alternative

protocol that may work in heifers and cows that effectively deals with premature

expression of estrus, prepuberal stages and anestrus is the Cosynch protocol including

insertion of a CIDR device during the first 7 days and with the second GnRH injection

and TAI completed at 70-84 hours (Geary, 2003). A P4 or progestin supplement between





43


GnRH and PGF2a will induce estrus in noncyclic cows and prepuberal heifers. Protocols

combining GnRH, PGF2a and P4 to synchronize estrus and ovulation in comparison with

those utilizing estrogens and P4 resulted in similar pregnancy rates (Martinez et al.,

2002).

The Use of Estrogen to Synchronize the Follicular Wave

Different salts of estradiols such as estradiol benzoate (Burke et al., 2000),

estradiol valerate (Bo et al., 1991), estradiol cypionate (Thundatil et al., 1998) and

estradiol-173 (Bo et al., 1995) have been used alone or in combination with P4 to

synchronize follicular wave in cattle.

Estradiol valerate administered at different stages of the estrous cycle induced

follicular atresia but follicular wave emergence was variable and inconsistent (Bo et al.,

1993). In contrast, estradiol-170 suppressed the dominant follicle and emergence of a

new follicular wave occurred in 4.3 days (Bo et al., 1995).

Estradiol benzoate induces atresia of the dominant follicle in 36 hours (Burke et

al., 2001) and delays FSH surge in a dose-dependent manner (Burke et al., 2003). After

ablation of the dominant follicle, FSH peaks in 29 hours, but in cows receiving 1, 2 or 4

mg of estradiol benzoate FSH peaks at 53, 80 and 90 hours, respectively, and a new

follicular wave was recruited 15 hours after the FSH peak (Burke et al., 2003).

Administration of 0.5 or 1 mg of ECP with 100 mg of P4 on Day 3 of the estrous

cycle was effective, but 1 mg of ECP with 50 mg of P4 in cows at random stages of the

estrous cycle and treated with a CIDR insert failed to synchronize the follicular wave

(Thundathil et al., 1997).





44

Progesterone administered at the time of follicular deviance inhibits LH secretion

and the largest follicle had reductions in diameter and concentration of estradiol and IGF-

1 in the follicular fluid (Ginther et al., 2001). Progesterone in high doses is able to induce

atresia of the dominant follicle and induce a new follicular wave (Anderson and Day,

1994). However, administration of 200 mg of P4 at the time of insertion of an intravaginal

P4 device to synchronize estrus at random stages of the estrous cycle was not as effective

as estradiol benzoate in inducing follicular turnover (Cavalieri et al., 2003).

Since estradiol can be used to synchronize the emergence of a new follicular wave

(Bo et al., 1995) and at the same time can induce GnRH release, increase GnRH receptors

in the pituitary and induce ovulation when administered during proestrus (Sumpf et al.,

1989; Kinder et al., 1991), protocols involving estradiol on Day 0 and then 24 hours after

P4 withdrawal have been used to synchronize ovulation for TAI. The advantage of

estradiol lies in the low cost of the treatment and the increased expression of behavioral

estrus. Estrus and ovulation synchronization with estradiol benzoate (1 mg) 24 hours after

or GnRH (gonadorelin, 0.25 mg) 34 hours after CIDR removal resulted in 97.5% of the

cows that received estradiol and 36.2% of the cows that received GnRH expressing

estrus (Cavalieri and Macmillan, 2002).

Function of the Corpus Luteum

Corpus Luteum Development and Differentiation

The LH surge secreted by the anterior pituitary gland induces ovulation and under

LH stimulation the ovarian follicle becomes the corpus luteum which secretes P4

(Niswender and Nett, 1994). Luteinizing hormone supports CL development,

differentiation and P4 production during the luteal phase (Hansel and Seifart, 1967). After





45

the LH surge, there is a hypertrophy of the granulosa cells and nuclear activation, the

basement membrane breaks down and blood vessels from the theca interna invade the

cavity of the rupture follicle (Niswender and Nett, 1994). During CL development, the

vascularized theca cells and the avascular granulosa cells of the ovarian follicle become

the small and large luteal cells, respectively (Donaldson and Hansel, 1965; Alila and

Hansel, 1984), and during advanced stages of luteal phase some of the small luteal cells

are transformed to large luteal cells (Alila and Hansel, 1984).

The amount of large luteal cells in the CL is consistent with the number of

granulosa cells in the preovulatory follicle (Rodgers et al., 1984). Mitosis in the granulosa

cells ends with the LH surge by inhibition of cyclin D and upregulation of p27M'P and

p21cipl which are inhibitors of cyclin D complexes (Robker and Richards, 1998). Other

cells that form the CL are fibroblasts, vascular smooth muscle, pericytes and endothelial

cells (Niswender and Nett, 1994). Matrix metalloproteinases 2 (MMP2) which are

endopeptidases that cleave extracellular proteins (lattice network of basement membranes

that support epithelial cells and endothelium) are mediators of ovulation and luteinization

facilitating connective tissue projections that provide a framework for cellular migration

and angiogenesis (Gottsch et al., 2002).

The LH surge changes the steroidogenic pathway from the two-cell two-

gonadotropin secretion of estrogen by the follicle to secretion of P4 by small and large

luteal cells (Niswender et al., 2000). Cholesterol from high density lipoproteins (HDL)

and low density lipoproteins (LDL) enters the cell and is transformed to pregnelonone in

the mitochondria (enzyme P450cc) and then to P4 in the endoplasmic reticulum (enzyme

3b-HSD) and P4 diffuses from the cells and the amount stored is very low (Niswender et





46

al., 2000). Cholesterol is transported into the mitochondria by the steroidogenic acute

regulatory protein (StAR), and StAR protein (Pescador et al., 1996) and P450cc (Rao et

al., 1978) are expressed in the CL after luteinization. The small luteal cells are

responsible for increasing secretion of P4 in response to LH and the large luteal cells

contribute to high basal levels of P4 (Koos and Hansel, 1981) producing around 80% of

total P4 (Niswender et al., 1985). Small luteal cells are 8-22 um in diameter and respond

to LH with a 6-12 fold increase in steroid production (Fitz et al., 1982). The small luteal

cells express LH receptors in large amounts but also receptors are expressed in large

luteal cells (Chegini et al., 1991) and both cells receptors for prostaglandin (Niswender

and Nett, 1994). Therefore, also luteal cells respond to stimulatory effect of prostaglandin

1-2 (Fitz et al., 1984) and E-2 (Chegini et al., 1991). Large luteal cells are greater than 22

um in diameter and do no respond to LH stimulation (Rodgers et al., 1983). Large luteal

cells express growth hormone (GH) and IGF-I receptors and therefore GH and IGF-I may

have a role in basal secretion of P4 (Niswender et al., 2000). In fact, IGF-I accelerates the

appearance of StAR during in vitro luteinization and augments gonadotropin-induced

StAR expression (Pescador et al., 1999).

The pulsatile secretion of LH is important for CL maintenance and development,

especially during the first 12 days of the luteal phase (Peters et al., 1994). Luteinizing

hormone binds its receptor (seven-transmembrane G-protein receptor) in the plasma

membrane of the large luteal cells and stimulate steroidogenesis through the cAMP-

protein kinase second messenger system (Niswender and Nett, 1994). This protein kinase

system may be constituvely activated in the large luteal cells (Hoyer et al., 1984).

Luteinizing hormone facilitates the transport of cholesterol through the cell and to the





47

P450cc enzyme (Wiltbank et al., 1993). Administration of large doses of LH or hCG may

result in loss of LH receptors due to either inactivation within the plasma membrane or

internalization of the hormone-receptor complex by endocytic vesicles with posterior

degradation by lysosomal enzymes (Niswender and Nett, 1994). The luteal phase in the

cow last for 16 days approximately and then if a viable embryo is not present in the

uterus at Day 16 post ovulation the uterus secretes PGF2a and the CL regresses

(McCracken et al 1972). The role of the uterus in luteolysis was clear after CL lifespan

was prolonged following hysterectomy (Wiltbank and Casida 1956). If a viable embryo is

present in the uterus, this embryo will secrete IFN-T, inhibit PGF2a secretion which

results in CL maintenance and pregnancy recognition (Bazer et al., 1991).

The Role of Progesterone on Embryo Survival

Production of P4 by the CL supports embryo development, pregnancy

maintenance and embryo survival (Staples and Hansel, 1961). Production of P4 by the CL

is affected by the process of development and differentiation. The rate of growth of the

CL is self-limiting and related to the body size and numbers of corpora lutea (Reynolds et

al., 1994). Mitosis, cell turnover and luteal differentiation are rapid, and the early CL has

the same ability to produce P4 than a mature CL, but it produces less due to the small size

(Reynolds et al., 1994). In fact, concentrations of P4 in blood will depend on the amount

of steroidogenic tissue, blood flow and capacity of the steroidogenic tissue to produce P4

(Niswender et al., 2000). The rate of mitosis and cell turnover is stimulated by

proliferation of endothelial cells and the blood flow that supports luteinization (Reynolds,

1986). Fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) ad

angiogenic factors present in the CL contribute to luteinization. Granulosa cells produce





48

FGF-2 and platelet-derived growth factor (PDGF) which stimulate pericyte migration and

proliferation. The pericytes produce VEGF which stimulates thecal-derived endothelial

cells (Reynolds and Redmer, 1999). The CL increases in size by hypertrophy of large

luteal cells and increase in number of small luteal cells, fibroblasts and endothelial cells

(Farin et al., 1986).

Cows that become pregnant after insemination had higher plasma P4

concentrations during the luteal phase compared with nonpregnant cows (Lukesewska

and Hansel 1980). In addition, luteal insufficiency has been observed in subfertile cows

compared with heifers (Shelton et al., 1990). The role of P4 on CL maintenance and

embryo survival is carried out by: 1) stimulating embryo development and consequently

stimulating the antiluteolytic signal of interferon-z (Mann and Lamming, 1995); 2)

inhibiting LH pulsatility, follicular growth and estrogen production which contribute to

suppression of oxytocin receptors and the luteolytic cascade involving estrogen, oxytocin

and prostaglandin (Vallet and Lamming, 1991); and 3) inhibiting apoptosis of the luteal

cells by a local mechanism (Rueda et al., 2000).

Progesterone production by the CL stimulates secretion of the endometrial glands

necessary for embryo development (Geisert et al., 1992). Insufficient amount ofP4 may

result in compromised embryo development, reduction in interferon-T secretion, failure to

inhibit the luteolytic cascade and embryo loss (Mann and Lamming 1995). High levels of

P4 and low levels of estrogen during the luteal phase contribute to attenuation of the

luteolytic cascade (Lamming and Mann, 1995). Progesterone is able to inhibit oxytocin

receptors (Vallet and Lamming, 1991), and cows with low plasma P4 concentrations had

a greater PGF2, release in response to oxytocin administration compared with cows with





49

high plasma P4 concentrations (Mann and Lamming, 1995). Excessive follicular

development and high estradiol during the luteal phase may be detrimental to embryo

survival (Inskeep, 1995). Destruction of ovarian follicles during diestrus prolonged the

lifespan of the corpus luteum in cows indicating a contribution of estradiol to the

luteolytic cascade (Hughes et al., 1987).

Progesterone also contributes by a local mechanism mediated by P4 receptors

located in small and large luteal cells to repress the onset of apoptosis in the corpus

luteum (Rueda et al., 2000). Also mediated by the same mechanisms, P4 may be involved

in autocrine/paracrine regulation of follicular function (Rae et al., 1998). In addition, P4

downregulates estrogen receptors and P4 receptor antagonist inhibits luteinization in

bovine granulosa cells (Niswender et al., 2000).

Embryo Mortality

The evaluation of fertilization rate and different methods to monitor embryo

development (oviductal and uterine flushings) as well as methods for diagnosis of

pregnancy (biochemical, immunological, hormonal, and clinical) allow for determination

of embryo and fetal losses. After service, fertilization occurs in approximately 95%

(Ayalon, 1978; Diskin and Sreenan, 1980) but calving rates are around 50% (Humblot,

2001). Therefore, around 45% of pregnancy losses seem to occur which reduce

reproductive efficiency. Pregnancy losses can be classified in early embryonic mortality

(from fertilization to Day 16 with return to estrus before Day 25), late embryonic

mortality (from Day 17 to Day 45) and fetal loss (from Day 45 to calving; Humblot,

2001).





50

Based on artificial insemination and slaughter at different stages of pregnancy it

was estimated in Hereford beef heifers that pregnancy losses were 9 % from Day 8 to

Day 14, and 15 % between Day 14 and Day 18, and there seemed to be no losses between

Day 18 and Day 28 (Roche et al 1981). In dairy cows, early embryo loss reflected by

return to estrus within 25 days is around 28 % (Lamming et al., 1989) and most losses

have already occurred by Day 7 (Ayalon, 1978; Gustafsson et al., 1985) or by Day 5

(Weibold, 1988) in dairy cows, or by Day 14 in beef heifers (Dunne et al., 2000). More

recents experiments indicate that fertilization rates can be as low as 80% in cattle (Saake

et al., 2000) and even lower in lactating dairy cows during the summer (Sartori et al.,

2002a) and it needs to be considered when calculating early embryonic mortality. Fetal

loss calculated between 31 and 260 day of gestation was estimated to be 10.8% in dairy

herds (Forar et al., 1996).

Embryo and fetal losses are caused by multiple factors including infectious

agents, endocrine disorders, chromosomal abnormalities and environmental factors. Early

reports did not show a clear difference in the levels of P4 and estrogen between cows that

become pregnant or those that did not become pregnant after breeding (Ayalon, 1978).

Lactating dairy cows, however, are at a higher risk for endocrine disorders for three

reasons: 1) they have lower levels of P4 during diestrus to support embryo development

(Sangsritavong et al., 2002; Sartori et al., 2002b); 2) they are commonly subjected to

protocols of synchronization of ovulation that may alter follicular development, ovulation

and CL formation with a potential detrimental effect for the embryo (Peters and Pursley,

2003); and 3) environmental factors such as heat stress have also been involved in

increasing early embryonic mortality in lactating dairy cows (Ryan et al., 1993).





51

The detrimental effect of low P4 and high estrogen concentrations on early

embryo development may be exacerbated in high-producing dairy cows subjected to

synchronization of ovulation. Comparing lactating dairy cows with nonlacting dairy cows

(de la Sota et al., 1993) or with heifers (Sartori et al., 2002b) it was concluded that

lactating dairy cows have a reduced level of circulating P4. Lactating dairy cows have an

increased feed intake that results in increased liver blood flow and steroid metabolism

(Sangsritavong et al., 2002) that may result in low P4 concentrations. Low levels of P4

during diestrus increase LH pulses (Ireland and Roche, 1982b) which stimulate follicular

growth (Savio et al., 1993; Stock and Fortune, 1993) resulting in elevated concentration

of estradiol. In fact, follicular development is more pronounced in lactating than in

nonlactating dairy cows (Lucy et al., 1992) and it may be detrimental for embryo survival

since elimination of follicles during diestrus is known to extend the lifespan of the CL in

cattle (Fogwell et al., 1985), and follicular development is attenuated on the CL-bearing

ovary ipsilateral to the pregnant uterine horn (Guibault et al., 1986). Lactating dairy cows

inseminated at detected estrus with high fertility semen had more nonviable embryos

compared to nonlactating dairy cows (Sartori et al., 2002a).

The pulsatile secretion of LH and follicular development during proestrus are

important for differentiation and development of the CL and P4 production (Quintal-

Franco et al., 1999). In protocols for TAI, the timing between GnRH administration and

follicular development may be altered and could result in ovulation and formation of a

corpus luteum with reduced production ofP4 (Vasconcelos et al., 1999; 2001; Peters and

Pursley, 2003), and it may result in increased embryo mortality. Timing of insemination

and protocols for synchronization of ovulation may affect CL formation and indirectly





52

embryo development. In nonlactating dairy cows, the fertilization rate was 67, 79 and

82% when cows were inseminated either 0, 12 or 24 hours after onset ofestrus, and 98%

after natural breeding (Dalton et al., 2001). Using Ovsynch, pregnancy losses between

Day 28 and Day 98 were 24 % (PR on Day 28 of 32% and on Day 98 of 25%) for cows

with a large follicle (> 15 mm at PGF2a and >18mm at second GnRH) and 14% (PR on

Day 28 of 42% and at Day 98 of 36 %) for cows with smaller follicle (<15 mm at PGF2a,

and <18 mm at GnRH (Vasconcelos et al., 1999). Administration of GnRH 72 hours after

PGF2a will induce a shorter and less pronounced LH surge compared to control cows but

some cows will have a spontaneous LH surge before GnRH administration (Lucy and

Stevenson 1986). This may be explained by the fact that the estrogen produced by the

preovulatory follicles induces GnRH receptors in the anterior lobe of the pituitary gland

(Reeves et al., 1971) which indicates the importance of estrogen levels to induce a potent

LH surge (Zolman et al., 1974).

There is a clear effect of climate on reproductive efficiency through out the year

with reduced pregnancy rates during the summer (Badinga et al., 1985; Al-Katanani et

al., 1999). This effect is observed mainly in lactating dairy cows (Badinga et al., 1985).

Heat stress increased embryo mortality in lactating dairy cows (Ryan et al., 1993). In

addition, heat stress reduced luteal tissue and conceptus weight in beef cattle (Biggers et

al., 1987). In lactating dairy cows, fertilization rate evaluated on Day 5 (-56%) was

reduced compared to dairy heifers during the summer (Sartori et al., 2002). Temperatures

that exceed normal body temperature are detrimental to both in vitro and in vivo embryo

at the two-cell stage causing disruption of the cytoskeleton and damage of the

mitochondria (Rivera et al., 2003). In addition, heat stress alters follicular dynamics.





53


Therefore, cows develop more large follicles during the summer (Wolfenson et al., 1995)

that can be detrimental for embryo survival.













CHAPTER 3
ADMINISTRATION OF HUMAN CHORIONIC GONADOTROPIN (hCG) DURING
ESTRUS IN NONLACTATING DAIRY COWS AND ITS EFFECT ON CORPUS
LUTEUM SIZE AND PROGESTERONE PRODUCTION

Introduction

The preovulatory rise in luteinizing hormone (LH) induces functional and

morphological changes in theca and granulosa cells and they are transformed into luteal cells

and begin to secrete P4 (Baird, 1992). Progesterone production by the corpus luteum supports

establishment and maintenance of pregnancy in cattle (Mann and Lamming, 1995). Cows

diagnosed pregnant after breeding had higher levels of circulating P4 during early luteal phase

compared to cows diagnosed not pregnant (Lee et al., 1985).

Luteinizing hormone is the major factor involved in stimulating CL development and

P4 production (Donaldson and Hansel, 1965, Hansel and Seifart., 1967). Human Chorionic

Gonadotropin has LH-like activity, binds LH-receptors in the luteal tissue and has been

demonstrated to be luteotrophic in cattle (Donaldson et al., 1965; Lee and Ryan, 1973). In

sheep, the steroidogenic response to hCG is longer than the one observed with ovine LH

(Bourdage et al., 1984). Therefore, daily (Wiltbank et al., 1961) or systemic (Schomberg et

al., 1967) administration of hCG during the luteal phase increased P4 concentrations and

prolonged the lifespan of the CL in cattle. In addition, the importance of basal levels and

pulsatility of LH during early stages of CL development have been demonstrated using a

GnRH antagonist (Peters et al., 1994).




54





55

The objective of the present experiment was to evaluate the ability ofhCG to enhance

CL development and increase P4 production when injected during estrus to extend the natural

LH surge.

Materials and Methods

Study Population

The experiment was conducted at the Dairy Research Unit of the University of

Florida. A group of twenty three multiparous nonlactating cycling dairy cows were kept in

free-stalls with access to a dry lot and fed 80 % sorghum silage and 20% lactating cow ration.

Study Design

All cows had a body condition score (BCS) of 3.5 to 4.5, and were treated with

GnRH (100 ug, im, Cystorelin) and PGF2a (25 mg, im, 2 doses 8 h apart, Lutalyse@) 7 days

later. Estrus detection was conducted using the HeatWatch system considering cows in

estrus if they had a at least 3 mounts of at least 2 seconds duration within a 4 hours period.

Seventeen cows that showed estrus between 2 and 3 days after PGF2a were assigned

randomly to receive either hCG (3,000 IU, im, Chorulon, Intervet, Millsboro, DE 19966;

n=9) or saline (n=8) 8 to 10 hours after the onset of estrus (Figure 3.1).

Plasma P4 Concentrations

Blood samples were collected from 12 hours after treatment and every 12 hours

thereafter to determine plasma P4 concentration. Plasma P4 concentration was determined

with a single antibody radioimmunoassay procedure (Knickerbocker et al., 1986). Inter- and

intra-assay coefficients of variation were 8.5 and 23.5 % respectively. Sensitivity of the assay

was 0.15 ng/mL.





56

Ultrasonography of the Ovaries

Ovarian ultrasound examination was done every 3 days using a real-time ultrasound

scanner (Aloka 500, 7.5 Mhz linear transrectal probe) to evaluate number of follicular

waves and CL size during 23 days after treatment. To evaluate CL size two cross-sectional

observations were done and the three greatest radii were used to estimate the volume using

the formula: 3/*7*rl *r2*r3. Cavities of corpora lutea were subtracted from measurements in

order to considered only luteal tissue.

Statistical Analysis

The response variables were plasma P4 concentration, CL size and number offollicular

waves. Data for plasma P4 concentration were analyzed using repeated measures ANOVA for

mixed models and the homogeneity of regression procedure. The CL size was evaluated using

repeated measures ANOVA for mixed models. The number of follicular waves was compared

using Fisher exact test. Treatment differences with P < 0.05 were considered significant.

Results

There was no difference in the number of follicular waves between cows treated with

hCG (78% of cows with 2 waves; 7/9) and control cows (75% of cows with 2 waves; 6/8).

Plasma P4 concentration was lower in cows treated with hCG (6.46 ng/ml 0.41) than

in control cows (7.89 ng/ml 0.43; P<0.05), with major differences detected between Day 14

and Day 16 after treatment (Figure 3.2). Plasma P4 concentration was also higher for control

than for hCG cows (cubic effect; P<0.01) when evaluated by homogeneity of regression

procedures. Evaluating differences on specific days using the Proc MIXED, plasma P4

concentrations were higher for control group on Day 14 (P<0.003), Day 16 (P<0.02), Day 20

(P<0.01) and Day 20.5 (P<0.03)





57

In addition, on Day 16, CL tended to be smaller (P<0.08) in cows treated with hCG

(6.96 cm3 0.71) compared to control cows (8.82 cm3 0.76; Figure 3.3).

Discussion

Administration of hCG 10 hours after onset of estrus in nonlactating dairy cows

resulted in a reduction in the size of the CL and plasma P4 concentration. Even though basal

levels of LH can support the development of the CL, LH pulsatility is required for

differentiation and development of the CL during early stages of the luteal phase (Peters et al.,

1994). In the present study, hCG administered during or right after the spontaneous LH surge

may have produced an excess of LH and LH-like activity and negatively affected mitosis and

differentiation of small luteal cells and loss of response to the luteotrophic stimulus of LH. In

fact, administration of hCG in rats induced loss of LH receptors and desensitization of

adenylate cyclase in the ovary (Conti et al., 1976).

A previous study reported that administration of hCG (1,000 and 2,000 IU) 12 hours

after estrus increases CL size, plasma P4 concentrations. However, the increment in CL size in

cows treated 12 hours after onset of estrus was less than cows that were treated on Day 3

(Veenhuizen et al., 1972). Using the same treatment, Wagner et al. (1973) reported an

increased in pregnancy rate but a decrease when hCG was administered on Day 3 after first

service. Cows submitted to estrus synchronization using 2 doses ofPGF2a 14 days apart and

then treated with GnRH 72 hours after the second dose ofPGF2a had reduced P4 levels during

the luteal phase (Lucy and Stevenson, 1986). It was concluded that the administration ofhCG

during or right after the spontaneous LH surge with a high LH and LH-like exposure in the

peri-ovulatory period did not appear to cause a hyper-stimulation of CL function.





58

Gonadotropin releasing hormone administered either 0 to 5 hours before or at the time

of insemination increased conception rate in some studies but not in others, and the largest

response was observed when conception rate in the control group was under 50 %

(Rosenberg et al., 1991). In subfertile cows, administration of GnRH before or at the time of

insemination increased conception rate (Rosenberg et al., 1991). In contrast, there was a

decrease in pregnancy rate in lactating dairy cows inseminated and treated with GnRH 12 to

16 hours after onset of estrus (Mee et al 1990). Treatment of GnRH on Day 2 after estrus

resulted in reduced concentration of P4 during late luteal phase in heifers (Rodger and

Stormshak, 1984). Administration of GnRH on Day 2 of the estrous cycle may attenuate

corpus luteum function by down-regulating luteal LH receptors (Rodger and Stormshak

1984).

In conclusion, administration of hCG 10 hours after onset of estrus in nonlactating

dairy cows decreased the size of the CL and reduced P4 concentrations during the luteal phase

and is not recommended to enhance luteal function in nonlactating cattle.





59






Treatment Group (n=9)
hCG 3,000 IU
GnRH PG 8-10 h after estrus
I RHEstrus Bleeding every 12 h
Detection U/S every 3 d
Day 0 Day 7 Day 30
U/S U/S

Control Group (n=8)
Saline
GnRH PGF2a Bleeding every 12 h
S Estrus U/S every 3 d
Detection
Day 0 Day 7 Day 30
U/S U/S

Figure 3.1. Experimental Design





60






20 -

15 P<0.05

o 10



0

1 3 5 7 9 11 13 15 17 19 21 23
Day after onset of estrus

--"*hCG -*Control

Figure 3.2. Plasma P4 concentrations in nonlactating dairy cows after
induction of ovulation with human chorionic gonadotropin (hCG).





61






12
10
P<0.08
8
.N 6


2-


4 7 10 13 16 19
Days after onset of estrus

-- Control '-" -hCG

Figure 3.3. Size of CL in nonlactating dairy cows treated with human
chorionic gonadotropin (hCG) 8 to 10 h after onset of estrus.













CHAPTER 4
INDUCTION OF OVULATION IN NONLACTATING DAIRY COWS AND HEIFERS
USING DIFFERENT DOSES OF A DESLORELIN IMPLANT

Introduction

Timed-artificial insemination is a common practice in dairy herds to improve

reproductive efficiency since all cows are inseminated independent of estrus detection

(Pursley et al., 1997a; Momcilovic et al., 1998). Timed-insemination requires

synchronization of follicular development, regression of the corpus luteum (CL), and

synchronous induction of ovulation (Pursley et al., 1995).

Human chorionic gonadototrophin (hCG; Schmitt et al., 1996), gonadotropin

releasing hormone (GnRH; Pursley et al., 1995), GnRH agonists (Schmitt et al., 1996),

and estradiol (Dailey et al., 19986) all have been used to induce ovulation. A synthetic

GnRH-agonist (deslorelin) implant was administered to synchronize ovulation, enhance

CL function, and delay the first-wave dominant follicle (Ambrose et al., 1998). Enhanced

CL function is associated with high blood concentrations of P4 early in the estrous cycle;

this stimulates embryo development, increases interferon-r production (Mann and

Lamming, 2001), and may contribute to improved fertility (Butler et al., 1996). In

addition, suppression of follicular development also may be beneficial to extend CL

lifespan (Fogwell et al., 1985).

The hypothesis of this study was that induction of ovulation with a deslorelin

implant would increase plasma P4 concentrations (due to enhanced development of the

CL) and suppress follicular development, without extending the interestrus interval. The


62





63

objective was to evaluate ovarian function after inducing ovulation with different doses

(450, 750 and 1000 pag) of a deslorelin implant (GnRH agonist) in comparison to

gonadorelin diacetate (GnRH, 100 plg) in nonlactating dairy cows and heifers.

Materials and Methods

Experiment 1

Nonlactating dairy cows (n=20) were treated on Day 9 with 100 g.g im GnRH

(CystorelinTM; Merial Ltd, Iselin, NJ), and with two doses of 25 mg im PGF2a

(LutalyseTM; Pharmacia, Kalamazoo, MI,) 8 h apart on Day 2. On Day 0, cows were

assigned randomly to three treatment groups and received either 100 ljg im GnRH

(Control, n=6), a subcutaneous (sc) biodegradable implant (Peptech Animal Health,

North Ryde, Australia) containing 750 jlg deslorelin (D-Trp6-Pro6-des-Glylo-LHRH

ethylamide, DESLORELIN 750; n=7) or a sc implant containing 1000 lpg deslorelin

(DESLORELIN 1000; n=7). On Day 16, cows were treated with PGF2a (two, 25-mg

doses given 8 h apart) to induce luteolysis. Ovaries were evaluated by ultrasonography

daily from Day 0 to ovulation and every other to Day 28. Daily blood samples were

collected from Day 0 until PGF2, administration on Day 16 to determine plasma P4

concentrations. The Heat Watch system (DDx Inc., Denver, CO 80221) was used to

monitor estrus. After Day 16, cows that did not display signs of estrus and ovulate

received 100 4ig im GnRH when the largest follicle reached 20 mm in diameter, 25 mg

im PGF2a 7 d later, and 100 jig im GnRH 2 d after PGF2a (Ovsynch protocol; 3). The

outcome variables were plasma P4 concentrations (ng/mL), first-wave largest follicle

(mm; the term largest will be used since deslorelin-implanted cows did not have a

dominant follicle), second-wave largest follicle until Day 16 (mm), number of Class II





64

(5-9 mm) and Class III (> 10 mm) follicles (Macmillan and Thatcher, 1991) between Day

0 and Day 16, size of the largest follicle between Day 16 and Day 20 (mm), rate of

growth for largest follicle (mm/day) between Day 16 and Day 28 for DESLORELIN 750

and DESLORELIN 1000 groups, expression of estrus after Day 16 (yes/no), number of

Class II and Class III follicles between Day 16 and Day 28 for DESLORELIN 750 and

DESLORELIN 1000 groups, and response to Ovsynch for cows that did not ovulate after

Day 16.

Experiment 2

Fourteen nonlactating dairy cows were synchronized using GnRH on Day 9 and

two doses of PGF2a 12 h apart on Day 2. On Day 0, cows received either 100 jig of

GnRH (Control, n=8) or a 450 4g deslorelin implant (DESLORELIN 450, n=6). On Day

16, all cows received PGF2,. Ovaries were evaluated by ultrasonography daily from Day

0 to ovulation and every other day to Day 28. Daily blood samples (to determine plasma

P4 concentrations) were collected from Day 0 until PGF2a administration on Day 16. The

Heat Watch@ system was used to monitor estrus. After Day 16, cows that did not show

estrus and did not ovulate (i.e., based upon ultrasonography) received 100 jtg imGnRH at

the time the largest follicle reached 10 mm in diameter, 25 mg im PGF2a 7 d later, and

100 jtg im GnRH 2 d after PGF2a (Ovsynch protocol). The outcome variables were

plasma P4 concentrations (ng/mL), first-wave largest follicle (mm), second-wave largest

follicle to Day 16 (mm), number of Class II and Class III follicles between Day 0 and

Day 16, size of the largest follicle (mm) between Day 16 and Day 28, expression of

estrus after Day 16 (yes/no), and response to Ovsynch for cows that did not ovulate after

Day 16.





65

Experiment 3

Forty-four dairy heifers were synchronized using GnRH on Day 9 and PGF2a on Day 2.

On Day 0, heifers received either 100 4g of CystorelinM (Control, n=22) or a 750 jg

deslorelin implant (DESLORELIN 750, n=22). On Day 16, all heifers received PGF2a

and estrus was monitored for 6 d. Heifers that had not displayed signs of estrus within 10

d after PGF2a (i.e., Day 26) were evaluated by ultrasonography to determine ovarian

structures. Blood samples were collected on Days 7, 13, and 16 after either GnRH

injection or insertion of the deslorelin implant (i.e., Day 0) to evaluate plasma P4

concentrations. Outcome variables were plasma P4 concentration (ng/mL) and estrus

expression after PGF2a on Day 16 (yes/no).

Collection of Blood Samples and Radioimmunoassay

Blood samples for P4 analysis were collected by puncture of the coccygeal vein or

artery into evacuated tubes containing ethylene diamine tetra acetic acid (EDTA) as an

anticoagulant (10.5 mg, Monoject, Sherwood Medical, St. Louis, MO). Plasma P4

concentration was determined with a single antibody radioimmunoassay procedure

(Knickerbocker et al., 19986). Inter- and intra-assay coefficients of variation were 6.7 and

10.3 %, respectively. Sensitivity of the assay was 0.15 ng/mL.

Statistical Analyses

Plasma P4 concentrations were analyzed using the repeated measures procedure

(Proc MIXED, SAS) for Mixed Models (Littell et al., 1997) adjusting for the number of

CL as a covariate. Differences between means for specific days were examined by the

PDIFF option. Follicular growth was analyzed using the repeated measures procedure

and by the homogeneity of regression procedure (Proc GLM, SAS, Wilcox et al., 1990).





66

Number of Class II and III follicles was compared using the repeated measures procedure

(Litell et al., 1997) and the repeated procedure for counted data (Proc GENMOD, SAS;

Agresti, 1996). The expression of estrus was analyzed by using Chi Square (Proc FREQ,

SAS). Differences were considered significant when P < 0.05.

Results

Experiment 1

All cows ovulated and formed a CL after treatments on Day 0. Average plasma

P4 concentrations from Day 0 to Day 16 were not different among cows in the Control

(5.9 0.6 ng/mL), DESLORELIN 750 (6.4 0.6 ng/mL), and DESLORELIN 1000 (6.3

0.6 ng/mL) groups. However, sporadic elevations in plasma P4 concentrations were

detected on Day 11 for cows in the DESLORELIN 1000 (11.1 1.0 ng/mL) compared to

cows in the Control (8.2 1.1 ng/mL; P<0.05) groups, and on Day 12 for cows in the

DESLORELIN 750 (12.4 1.0 ng/mL) compared to cows in the Control (9.0 1.1

ng/mL, P<0.05; Figure 4.1) groups. Overall a deslorelin-induced increase in plasma

concentrations of progesterone was minimal.

The first-wave largest follicle was larger (P<0.02) for cows in the Control Group

(9.48 + 1.1 mm) compared to cows in DESLORELIN 1000 Group (4.92 + 1.0 mm), and

not different from that of cows in DESLORELIN 750 Group (7.82 + 1.0 mm). The

second-wave largest follicle evaluated until Day 16 was larger (P<0.01) for cows in

Control Group (7.63 + 0.6 mm) compared to cows in DESLORELIN 750 (5.25 + 0.6

mm) and DESLORELIN 1000 (4.05 + 0.6 mm; Figure 4.2) Groups.

The number of Class II follicles from Day 0 to Day 16 was higher (P<0.01) for

cows in Control Group (2.1 + 0.3) compared to cows in DESLORELIN 750 Group (1.2 +





67

0.3) and DESLORELIN 1000 Group (0.5+ 0.3; Figure 4.3A). The number of Class III

follicles from Day 0 to Day 16 was higher (P<0.05) for cows in Control Group (1.1 +

0.2) compared to cows in DESLORELIN 1000 Group (0.7 + 0.2; Figure 4.3B).

From Day 16 to Day 20, the average diameter of the largest follicle was greater

(P<0.001) for cows in Control Group (16.1 + 0.9 mm) compared to cows in

DESLORELIN 750 Group (6.3 + 0.8 mm) and DESLORELIN 1000 Group (4.7 + 0.8

mm, Figure 4.4). The number of cows expressing estrus and ovulating after PGF2a on

Day 16 until Day 20 was higher (P<0.001) in Control Group (6/6) compared to cows in

DESLORELIN 750 Group (1/7) and DESLORELIN 1000 Group (0/7). The rate of

follicular growth from Day 16 to Day 28 was greater in cows in DESLORELIN 750

Group (0.67 mm/d) than in cows in DESLORELIN 1000 Group (0.4 mm/d; P<0.01,

Figure 4.4). Twelve of 14 (86 %) cows in DESLORELIN 750 and DESLORELIN 1000

groups expressed estrus between Day 24 to Day 30 (1 in estrus on Day 19 and 1 did not

show estrus) but failed to ovulate. The 12 cows that failed to ovulate were subjected to

the Ovsynch protocol, and 9/12 (75 %) ovulated synchronously in response to the second

GnRH injection.

The number of Class II follicles from Day 16 to Day 28 was not different

(P=0.36) between cows in DESLORELIN 750 Group (1.2 + 0.4) compared to those in

DESLORELIN 1000 Group (0.6 + 0.3). Likewise, the number of Class III follicles from

Day 16 to Day 28 was not different (P=0.57) between cows in DESLORELIN 750 Group

(0.5 + 0.2) compared to cows in DESLORELIN 1000 Group (0.4 + 0.2).





68

Experiment 2

Overall plasma P4 concentration was not different among cows in the Control

Group (5.9 + 0.80 ng/mL) and DESLORELIN 450 Group (7.4 + 1.0 ng/mL). However,

plasma P4 concentration tended to be higher (P<0.10) on Day 9 for cows in the

DESLORELIN 450 Group (11.4 + 1.5 ng/mL) compared to cows in Control Group (8.0 +

1.2 ng/mL), and was higher (P<0.05) on Day 10 for cows in DESLORELIN 450 Group

(13.0 + 1.5 ng/mL) compared to cows in Control Group (8.2 + 1.2 ng/mL; Figure 4.5).

The first-wave largest follicle was larger (P<0.01) for cows in Control Group

(11.4 + 1.0 mm) compared to that for cows in DESLORELIN 450 Group (6.0 + 1.1 mm).

The second-wave largest follicle monitored until Day 16 was larger (P<0.001) for cows

in Control Group (8.5 + 0.4 mm) compared to cows in DESLORELIN 450 Group (3.3 +

0.5 mm; Figure 4.6).

The number of Class II follicles from Day 0 to Day 16 was higher (P<0.01) for

cows in Control Group (2.5 + 0.2) compared to cows in DESLORELIN 450 Group (0.9+

0.2, Figure 4.7A). The number of Class III follicles from Day 0 to Day 16 was higher

(P<0.01) for cows in Control Group (1.3 + 0.1) compared to cows in DESLORELIN 450

Group (0.4 + 0.1; Figure 4.7B).

The average diameter of the largest follicles from Day 16 to Day 28 was larger

(P<0.001) in cows in Control Group (16.5 + 1.85 mm) compared to cows in

DESLORELIN 450 Group (9.7 + 1.85 mm; Figure 4.8). The number of cows expressing

estrus and ovulating after PGF2a on Day 16 until Day 20 was higher (P< 0.001) in

Control Group (8/8) compared to cows in DESLORELIN 450 Group (1/6). One cow in

DESLORELIN 450 Group showed estrus on Day 28, but failed to ovulate. Therefore,





69

five cows in DESLORELIN 450 Group where subjected to the Ovsynch protocol and

three of them ovulated in response to the second GnRH.

Experiment 3

Overall plasma P4 concentration tended (P<0.07) to be higher for heifers in

DESLORELIN 750 Group (12.9 + 0.7 ng/mL) compared to that of heifers in the Control

Group (11.1 + 0.7 ng/mL; Figure 4.9). Within the 6-d period after PGF2a, estrus

expression was higher (P<0.01) for heifers in Control group (22/22) compared to that for

heifers in DESLORELIN 750 Group (12/22). Among the 12 heifers that did not show

estrus, 11 of them did not have follicles larger than 10 mm in diameter.

Discussion

In the present study, ovulation was synchronized with administration of either

gonadorelin diacetate (GnRH) or a deslorelin implant (GnRH agonist) in nonlactating

dairy cows (Experiments 1 and 2) and dairy heifers (Experiment 3). Clearly, deslorelin

implants did not suppress plasma P4 concentrations; rather it increased P4 concentrations

on certain days in late diestrus. However, these increases were sporadic and of minimal

magnitude for all three experiments. The results are in agreement with a previous report

using a 700 ptg deslorelin implant to induce ovulation in nonlactating dairy cows

(Ambrose et al., 1998). Size of the CL was not evaluated and there was no effect of

treatment on the number of CL. In a previous study, it was suggested that a deslorelin

implant may increase differentiation and development of the CL, since the number and

size of the CL had no effect on plasma P4 concentration (Ambrose et al., 1998).

Luteinizing hormone (LH) surge and pulsatile secretion from the pituitary gland

are responsible for ovulation, CL development and CL differentiation (Peters et al.,





70

1994). Pulsatile secretion of LH is necessary to maintain luteal activity between days 2

and 12, but not between days 12 and 17 (Peters et al., 1994). Administration ofdeslorelin

induces a normal LH surge and elevates basal LH during the following 2 (Rajamahendran

et al., 1998) or 7 (D'Occhio et al., 1996) days. High basal levels of LH during early

stages of the estrous cycle may stimulate CL development and differentiation in cows

induced to ovulate with a deslorelin implant.

Follicular development was decreased after induction of ovulation with a

deslorelin implant. The first-wave largest follicle was inhibited by the deslorelin implant;

however there was variability among doses in the degree of inhibition. In addition,

deslorelin implants reduced the number of Class II and III follicles from Days 0 to 16, but

again variability existed among deslorelin implant doses. The differences could be

explained due to cow-to-cow sensitivity to different doses of the implant. Substantial

variation among cattle has been detected previously using deslorelin implants (Ambrose

et al., 1998, D'Occhio et al., 1996). Second-wave largest follicle was clearly suppressed

using 1000, 750 or 450 .g deslorelin implants.

Follicular growth and return to estrus after PGF2a on day 16 were clearly delayed

in cows and heifers induced to ovulate with a deslorelin implant (1000, 750 or 450 ig)

and the rate of follicular growth was decreased in a dose-dependent manner. In cows that

failed to ovulate, the Ovsynch protocol had to be used in order to induce ovulation. It is

clear that cows that experience follicular suppression and delayed return to estrus will

grow follicles in the absence of P4 and therefore they will need to be resynchronized to

avoid the development of anovulatory follicles. The deslorelin implants used in the

present series of experiments are biodegradable and therefore not removed. Although the





71

release pattern of deslorelin in vitro is over a 4-day period (Mattos et al., 2001), there

appears to be a greater period of deslorelin release in vivo or an extended period of

pituitary desensitization to GnRH that was induced by deslorelin.

Continuous administration of GnRH (Gong et al., 1995; Gong et al., 1996;

Lamming and McLeod, 1988) or a single administration of a deslorelin implant (Mattos

et al., 2001) desensitizes the pituitary gland to the stimulatory effect of GnRH. Chronic

administration of 10 jig of GnRH and 5 4ig of buserelin (GnRH agonist) twice a day for

21 d in beef heifers suppressed pulsatile secretion of LH, but not FSH, and inhibited the

growth of follicles greater than 9 mm (Gong et al., 1995). A more prolonged buserelin

administration (using a minipump) suppressed LH and FSH concentrations, and follicles

did not grow more than 5 mm in diameter (Gong et al., 1996). Administration of different

doses of a deslorelin implant during the postpartum period and at various stages of the

estrous cycle suppressed follicular development and delayed first estrus or extended the

interestrus interval (Rajamahendran et al., 1998; D'Occhio et al., 1996; Mattos et al.,

2001). Induction of ovulation with a 700 jlg deslorelin implant reduced the number of

Class III follicles, did not extend CL lifespan, but some cows failed to ovulate by Day 36

(Ambrose et al., 1998).

The luteolytic effect of free and conjugated estrogens in cattle has been reported

previously (Greenstein et al, 1958; Wiltbank, 1966; Eley et al., 1979). Administration of

estradiol-17j on Day 13 (Thatcher et al., 1986) or Day 17 (Knickerbocker et al., 1986) of

the estrous cycle induces PGF2, secretion that was associated with luteolysis. In addition,

follicular development and estradiol levels have a negative effect on CL lifespan

(Fogwell et al., 1985) and fertility (Maurer and Echternkamp, 1982; Randel et al., 1971).





72

Electrocauterization and x-radiation of ovarian follicles on day 10 of the cycle reduced

estradiol levels and extended CL lifespan (Fogwell et al., 1985). Pregnant heifers had a

lower estradiol/progesterone ratio compared to nonpregnant heifers (Maurer and

Echternkamp, 1982). High levels of estradiol on days 14 to 17 of the cycle may have a

negative effect on fertility (Pritchard et al., 1994). Interestingly, higher conception rate

was reported in cows with three follicular waves than those with two follicular waves

(Ahmad et al., 1997). In addition, cows with follicles smaller than 15 mm in diameter

between days 12 and 14 of the cycle had higher conception rate compared with cows

having follicles larger than 15 mm (Hernandez Ceron et al., 2001). The present

experiment indicates that the administration of a deslorelin implant, as part of a timed

insemination protocol, reduced follicular development during this critical period when

the CL is maintained for pregnancy. Indeed a deslorelin implant appears to reduce

pregnancy losses from day 27 to day 45 compared to GnRH when used to induce

ovulation in the Ovsynch protocol. However, pregnancy rates at day 27 and day 45 were

not different (Santos et al., 2002).

In conclusion, deslorelin implants induced ovulation, stimulated development of a

normal CL, and delayed follicular growth during the subsequent diestrus period. The

effect of a slight increase in plasma concentrations of P4 and the attenuation of follicular

development on embryo survival warrant further investigation. However, the dose will

have to be adjusted and nonpregnant cows will have to be resynchronized (e.g., at the

time of pregnancy diagnosis) to minimize the abnormal return to estrus and ovulation.





73


16

14 -

12-

10












1 2 3 4 5 6 7 8 9 10111213141516
Interval after treatment (d)


Figure 4.1. Least squares means and SE for plasma P4 concentration between Day 0 and
Day 16 for Control (m), DESLORELIN 750 (*) and DESLORELIN 1000 (A) groups in
Experiment 1. DESLORELIN 1000 vs. Control on Day 11 (P<0.05), DESLORELIN 750
vs. Control on Day 12 (P0.05
1 2 3 4 5 6 7 8 9 1011 1213141516
Interval after treatment (d)


Figure 4.1. Least squares means and SE for plasma P4 concentration between Day 0 and
Day 16 for Control (m), DESLORELIN 750 (e) and DESLORELIN 1000 (A) groups in
Experiment 1. DESLORELIN 1000 vs. Control on Day 11 (P<0.05), DESLORELIN 750
vs. Control on Day 12 (P<0.05).





74


16-

14

__ 12 .

10
8-


6-
2,
_-
4-

2-

0- I------------------r
0 1 2 3 5 7 9 12 14 16
Interval after treatment (d)


Figure 4.2. Least squares means and SE for the first-wave largest follicles from Day 0 to
Day 16 for Control (m), DESLORELIN 750 (e) and DESLORELIN 1000 (A) groups
(P<0.02 Control vs. DESLORELIN 1000) and second-wave largest follicle for Control
(t), DESLORELIN 750 (O),'and DESLORELIN 1000 (A) groups (P<0.01) in
Experiment 1.






75



Figure 3.A.

6











o 1 2 3 5 7 9 12 14 16
Interval after treatment (d)




Figure 3.B.

2.5
4-


o3

S1-





















0.5



0 1 2 3 5 7 9 12 14 16
Interval after treatment (d)


Figure 4.3. A. Least squares means and SE for the number of Class II follicles after
2-

















treatment for from Day 0 to Day 16 for Control (i), DESLORELIN 750 (.) and
DESLORELIN 1000 (A) groups in Experiment 1 (P<0.01). B. Least squares means and
SE for the number of class III follicles after treatment for from Day 0 to Day 16 for
Control (m), DESLORELIN 750 (e) and DESLORELIN 1000 (A) groups in Experiment
1 (P<0.01).





76



25


20


| 15


10


5-


0-
16 18 20 22 24 26 28
Interval after treatment (d)






Figure 4.4. Least squares means and SE for the largest follicle after PGF2a on Day 16 for
cows in Control (m), DESLORELIN 750 (*) and DESLORELIN 1000 (A) groups in
Experiment 1 (P<0.001).





77







16

14
















0 1 2 3 4 5 6 7 8 9 101112131415
oDa 10(




0
D4-

2-


0 1 2 3 4 5 6 7 8 9 101112131415
Interval after treatment (d)


Figure 4.5. Least squares means and SE for plasma P4 concentration between Day 0 and
Day 16 for Control (m) and DESLORELIN 450 (*) groups in Experiment 2.
DESLORELIN 450 vs. Control on Day 9 (P50.10), and DESLORELIN 450 vs. Control
on Day 10 (P<0.05).





78






18-

16-

14-

12-



10

4-

2-


0 1 2 3 5 7 9 12 14 16
Days after treatment

Figure 4.6. Least squares means and SE for the first-wave largest follicles from Day 0 to
Day 16 for Control (m) and DESLORELIN 450 (e) groups (P50.01), and second-wave
largest follicle for control (o), DESLORELIN 450 (0) groups (P<0.001) in Experiment
2.





79


Figure 7.A.

6-
5 -
a4
3
0 2
z



0 1 2 3 5 7 9 12 14 16
Interval after treatment (d)



Figure 7.B.

2.5
CD 2
.2 1.5
4- 1
Z 0.5

0 1 2 3 5 7 9 12 14 16
Interval after treatment (d)


Figure 4.7. A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (m), DESLORELIN 450 (e) groups
(P<0.01) in Experiment 2. B. LSM and SE for the number of Class III follicles after
treatment for from Day 0 to Day 16 for Control (m) and DESLORELIN 450 (*) groups
(P<0.01) in Experiment 2.





80








25

E 20 -

I 15



_J
0 ------
0


16 18 20 22 24 26 28
Interval after treatment (d)



Figure 4.8. Least squares means and SE for the largest follicle after PGF2a on Day 16 for
cows in Control (m) and DESLORELIN 450 (e) groups (P<0.001) in Experiment 2.





81










18
2 16 -
E14
5 12
S10
S8-
S6-
4
2
1 2-
0 T
7 13 16
Interval after treatment (d)



Figure 4.9. Least squares means and SE for plasma P4 concentration on Days 7, Day 13
and Day 16 for Control (i) and DESLORELIN 750 (e) groups (P<0.07) in Experiment 3.













CHAPTER 5
EFFECT OF A DESLORELIN IMPLANT IN A TIMED ARTIFICIAL
INSEMINATION PROTOCOL ON FOLLICLE DEVELOPMENT, LUTEAL
FUNCTION AND REPRODUCTIVE PERFORMANCE OF LACTATING DAIRY
COWS

Introduction

Reproductive efficiency is a major component of economic success in dairy herds.

Pharmacological control of the estrous cycle involves synchronization of follicular

development, control of corpus luteum (CL) regression, and synchronization of ovulation

to improve conception and pregnancy rates (PR). Such pharmacological controls have

allowed development of a successful timed artificial insemination (TAI) protocol with

adequate PR (Pursley et al., r997a). Systematic breeding programs that involve TAI have

become an integral part of reproductive management in high-producing dairy herds

(Stevenson, 2001). However, TAI protocols have not increased conception rates or

altered pregnancy losses in lactating dairy cows (Santos et al., 2003).

Recently, manipulation of the estrous cycle prior to initiation of the TAI protocol

resulted in increased PR in lactating cyclic cows (Moreira et al., 2001). Optimizing

fertility in TAI protocols should result in higher PR and greater adoption of such

protocols to improve reproductive performance in dairy herds.

Embryo mortality in lactating dairy cows has been reported to be as high as 21%

between 27 and 45 d after TAI (Moreira et al., 2001). Losses of pregnancy in lactating

dairy cows have been similar when insemination is performed at detected estrus or after

the Ovsynch/TAI protocol (Santos et al., 2003). Furthermore, conception rate in cows


82




Full Text
Copyright
By
Julin Alberto Bartolom


5 EFFECT OF A DESLORELIN IMPLANT IN A TIMED ARTIFICIAL
INSEMINATION PROTOCOL ON FOLLICLE DEVELOPMENT,
LUTEAL FUNCTION AND REPRODUCTIVE PERFORMANCE
OF LACTATING DAIRY COWS 82
Introduction 82
Materials and Methods 84
Results 89
Discussion 92
6 STRATEGIC USE OF GONADOTROPHIN-RELEASING HORMONE
(GnRH) TO INCREASE PREGNANCY RATE AND REDUCE
PREGNANCY LOSS IN LACTATING DAIRY COWS SUBJECTED
TO SYNCHRONIZATION OF OVULATION AND TIMED
INSEMINATION 106
Introduction 106
Materials and Methods 108
Results Ill
Discussion 113
7 THE USE OF A GNRH AGONIST (DESLORELIN IMPLANT) DURING
LATE EMBRYONIC PERIOD TO ENHANCE EMBRYO
SURVIVAL 124
Introduction 124
Materials and Methods 126
Results 129
Discussion 130
8 RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION
(TAI) IN LACTATING DAIRY COWS, I: USE OF THE OVSYNCH AND
HEATSYNCH PROTOCOLS AFTER NONPREGNANCY DIAGNOSIS
BY ULTRASONOGRAPHY 141
Introduction 141
Materials and Methods 142
Results 146
Discussion 148
9 RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION
(TAI) IN LACTATING DAIRY COWS, II: ASSIGNING PROTOCOLS
ACCORDING TO STAGES AND TWO COMMON ABNORMALITIES
OF THE ESTROUS CYCLE. 159
viii


CHAPTER 7
THE USE OF A GNRH AGONIST (DESLORELIN IMPLANT) DURING LATE
EMBRYONIC PERIOD TO ENHANCE EMBRYO SURVIVAL
Introduction
Early and late embryonic mortality is an important factor affecting reproductive
efficiency in dairy herds (Silke et al., 2002; Thatcher et al., 2003). Early embryonic
mortality is considered when embryos die between fertilization and maternal recognition
of pregnancy and cows return to estrus within 25 days after insemination (Humblot,
2002). Late embryonic mortality occurs between Days 25 and 45 after insemination, and
cows return to estrus or are diagnosed nonpregnant between Days 25 to 45 (Humblot,
2002).
Several experiments have been conducted to understand and reduce early
pregnancy loss. However, studies evaluating strategies to reduce late embryonic mortality
are limited. Late embryonic mortality has been reported to be 7.2% between Day 28 and
Day 84 of pregnancy (Silke et al., 2001), 8-17.5% after Day 24 of pregnancy (Humblot,
2002) and 10 to 12 % between Day 25 and Day 60 of pregnancy (Van Cleef et al., 1991)
in cattle inseminated at detected estrus, and 14.6% between Day 34 and Day 74 of
pregnancy in cows subjected to timed insemination (Moreira et al., 2001). Several factors
such as infectious agents (bacteria, virus, protozoa), environmental stress, physiological
124


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iv
LIST OF TABLES x
LIST OF FIGURES xiv
ABSTRACT xvii
CHAPTER
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 5
The Estrous Cycle and Follicular Dynamics 5
Drugs Used for the Pharmacologycal Control of the Estrous Cycle 18
Methods for Synchronization of Estrus in Cattle 24
Induction of Ovulation and Timed Insemination 29
Function of the Corpus Luteum 44
3 ADMINISTRATION OF HUMAN CHORIONIC GONADOTROPIN (hCG)
DURING ESTRUS IN NONLACTATING DAIRY COWS AND ITS
EFFECT ON CORPUS LUTEUM SIZE AND PROGESTERONE
PRODUCTION 54
Introduction 54
Materials and Methods 55
Results 56
Discussion 57
4 INDUCTION OF OVULATION IN NONLACTATING DAIRY COWS AND
HEIFERS USING DIFFERENT DOSES OF A DESLORELIN IMPLANT 62
Introduction 62
Materials and Methods 63
Results 66
Discussion 69
vii


239
de la Sota RL, Lucy MC, Staples CR, Thatcher WW. Effects of recombinant bovine
somatotropin (sometribove) on ovarian function in lactating and nonlactating dairy cows.
JDairySci 1993;76:1002-1013.
de la Sota, RL, Risco CA, Moreira F, Burke JM, Thatcher WW. Efficacy of a timed
insemination program in lactating dairy cows during summer heat stress. Theriogenology
1998;49:761-770.
De Silva M, Reeves JJ. Hypothalamic-pituitary function in chronically cystic and
regularly cycling dairy cows. Biol Reprod 1988;38:264-269.
Dhaliwal GS, Sharma RD, Prabhakar S. Ovarian changes in buffaloes following PGF 2-
alpha administration using two routes. Buffalo Bulletin 1991; 10:32-37.
Diaz T, Schmitt EJ-P, de la Sota RL, Thatcher M-J, Thatcher WW. Human chorionic
gonadotropin-induced alterations in ovarian follicular dynamics during the estrous cycle
of heifers. J Anim Sci 1998;76:1929-1936.
Diskin MG, Sreenan JM. Fertilization and embryonic mortality rates in beef heifers after
artificial insemination. J Reprod Fert 1980;59:463-468.
Dobson H. Plasma gonadotrophins and oestradiol during oestrus cycle in the cow. J
Reprod Fert 1978;52:51-53. .
Dobson H, Alam MGS. Preliminary investigations into the endocrine systems of
subfertile cattle: location of a common lesion (rate-limiting step). J Endocrinol
1987;113:167-171.
Dobson H, Rankin JEF, Ward WR. Bovine cystic ovarian disease; plasma progesterone
concentrations and treatment. VetRec 1997;101:459-461.
Dohi H, Yamada A, Tsuda S, Sumikawa T, Entsu S. Technical note: a pressure-sensitive
sensor for measuring the characteristics of standing mounts of cattle. J Anim Sci
1993;71:369-72.
Donaldson LE, Hansel W. Prolongation of the lifespan of the bovine corpus luteum by
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intravaginal device. Vet Rec 2000;147:355-359.


12
administering PGF2ain cows with CL at clinical examination has been reported to be
80% (Archbald et al., 1994). Using plasma P4 concentration as the gold standard, positive
predictive value was 87 % for rectal palpation (> 1 ng/ml; Archbald et al., 1992), and
90% for rectal palpation combined with ultrasonography (> 2 ng/ml; Bartolom et al.,
2002). At ultrasonography, a corpus luteum and one graffian follicle are observed, and
the echogenicity of the uterus is moderate (Pierson and Ginther, 1984a; 1987). Follicles
during the estrous cycle can be classified in class I (3-5 mm), class II (6-9 mm) and class
III (> 9 mm) by ultrasonography of the ovaries (Macmillan and Thatcher, 1991).
Proestrus
The stage of proestrus begins at the time of CL regression (Days 16-18) and ends
with the LH surge at the onset of estrus (Day 21). The length of proestrus is affected by
the size of the dominant follicle at the time of luteolysis (Sirois and Fortune, 1988).
Around Day 16 of the estrous cycle, uterine PGF2a is collected in the uterine-ovarian vein
and is transported to the ovarian artery through a counter-current transfer mechanism. It
then passes directly into the ovary to cause luteolysis (McCracken et al., 1972) which
results in decline of P4 and initiation of a new follicular phase (Kindahl et al., 1976).
Functional luteolysis includes the decline in P4 which occurs 30 minutes after the
levels of PGF2 increase (Thatcher and Chenault, 1975; Behrman and Hitches, 1976), and
structural luteolysis takes approximately 48 hours to be completed (Pate, 1994).
Luteolysis is carried out by a positive feed-back loop between PGF2a from the
endometrium and oxytocin from the CL in pulses every 6 to 8 h (Flint and Sheldrick,
1982; 1983). Prostaglandin F2a stimulates the release of oxytocin using the
phosphoinositol-Inositol 3-phosphate/diacilglycerol/Ca2+-protein kinase second-


178
Table 9.1. Number of cows evaluated for different stages and two common abnormalities
of the estrous cycle and protocols at Day 30, 55 and 90 after resynchronized
insemination.
Stage/Abn
P
Day 30
Day 55
Day 90
NU
QP
D
T
C
D
T
C
D
T
Diestrus
0
2
2
92
96
-
96
96
- '
96
96
(Jan-Mar)
Q
-
4
92
96
-
96
96
-
96
96
Diestrus
0
28
-
128
156
2
154
156
1
153
154
(Jun-Dec)
M
20
4
118
142
1
141
142
1
140
141
0
9
1
58
68
_
68
68
_
68
68
Metestrus
H
8
54
62
-
62
62
1
61
62
G
14
2
48
64
3
61
64
2
59
61
Proestrus
0
15
1
73
89
1
88
89
1
87
61
G
11
1
75
87
1
86
87
2
84
88
Cystic
0
15
3
79
97
-
97
97
2
95
97
G
19
1
89
109
-
109
109
-
109
109
Anestrus
0
3
-
5
8
-
8
8
-
8
8
G
1
-
8
9
-
9
9
-
9
9
Total
145
19
919
1083
8
1075
1083
10
1065
1075
0=0vsynch; Q=Quicksynch; M=Modified Quicksynch; H=Heatsynch;
G=GnRH+Ovsynch; NU=not ultrasounded; QP=questionable pregnant;
D=diagnosed;C=culled;T=total


50
Based on artificial insemination and slaughter at different stages of pregnancy it
was estimated in Hereford beef heifers that pregnancy losses were 9 % from Day 8 to
Day 14, and 15 % between Day 14 and Day 18, and there seemed to be no losses between
Day 18 and Day 28 (Roche et al 1981). In dairy cows, early embryo loss reflected by
return to estrus within 25 days is around 28 % (Lamming et al., 1989) and most losses
have already occurred by Day 7 (Ayalon, 1978; Gustafsson et al., 1985) or by Day 5
(Weibold, 1988) in dairy cows, or by Day 14 in beef heifers (Dunne et al., 2000). More
recents experiments indicate that fertilization rates can be as low as 80% in cattle (Saake
et al., 2000) and even lower in lactating dairy cows during the summer (Sartori et al.,
2002a) and it needs to be considered when calculating early embryonic mortality. Fetal
loss calculated between 31 and 260 day of gestation was estimated to be 10.8% in dairy
herds (Forar et al., 1996).
/
Embryo and fetal losses are caused by multiple factors including infectious
agents, endocrine disorders, chromosomal abnormalities and environmental factors. Early
reports did not show a clear difference in the levels of P4 and estrogen between cows that
become pregnant or those that did not become pregnant after breeding (Ayalon, 1978).
Lactating dairy cows, however, are at a higher risk for endocrine disorders for three
reasons: 1) they have lower levels of P4 during diestrus to support embryo development
(Sangsritavong et al., 2002; Sartori et al., 2002b); 2) they are commonly subjected to
protocols of synchronization of ovulation that may alter follicular development, ovulation
and CL formation with a potential detrimental effect for the embryo (Peters and Pursley,
2003); and 3) environmental factors such as heat stress have also been involved in
increasing early embryonic mortality in lactating dairy cows (Ryan et al., 1993).


217
Table 10.9. Pregnancy rates on Day 30, 55 and 90 for cows in different
stages/abnormalities of the estrous cycle in all 4 groups.
Group* stage
Pregnancy Rates
Day 30
Day 55
Day 90
%
N
%
N
%
N
GnRH
Proestrus
50.0
6/12
38.5
5/13
38.5
5/13
Ovarian cysts
16.7
2/12
8.3
1/12
8.3
1/12
CIDR
Proestrus
5.9
1/17
5.9
1/17
5.9
1/17
Ovarian cysts
27.3
3/11
27.3
3/11
27.3
3/11
GnRH+CIDR
Proestrus
21.4
3/14
20.0
3/15
20.0
3/15
Ovarian cysts
37.5
3/8
37.5
3/8
37.5
3/8
Control
Proestrus
35.7
5/14
35.7
5/14
35.7
5/14
Ovarian cysts
27.3
3/11
27.3
3/11
18.2
2/11
For pregnancy rates on Days 30, 55 and 90: CIDR*Stage P<0.05


4
Interactions between hormones secreted by the hypothalamus, anterior pituitary,
ovaries, uterus, the influence of neurotransmitters in the central nervous system and growth
factors are responsible for regulation the reproductive cycle in cattle. These interactions are
complex and a thorough understanding of the reproductive physiology is important in order to
implement different strategies for the pharmacological regulation of ovarian function and
reproductive management to optimize reproductive efficiency in lactating dairy cows.
The hypothesis of this study was that pharmacological manipulation of ovarian
function in lactating dairy cows will contribute to increased pregnancy rate, reduced embryo
mortality and allow for resynchronization of nonpregnant cows. The objective was to use
different strategies to enhance CL function and reduce follicular growth to increase pregnancy
rate and reduce embryo mortality, and to regulate follicular dynamics and ovulation to
resynchronize and time-inseminate nonpregnant cows.


8-1 Criteria for determination of the stage and two common abnormalities
of the estrous cycle based on ultrasonography and rectal palpation of the
genital tract (adapted from Zemjanis 1962 and Pierson and
Ginther 1984a, 1987) 152
8-2 Distribution of cows by parity, season and DIM for both groups 153
8-3 Distribution of cows by inseminator, and stages/abnormalities of the
estrous cycle for both groups 154
8-4 Pregnancy rate on Days 27, 45, and 90 and pregnancy losses between
Days 27-45 an 45-90 for cows in the Ovsynch and Heatsynch
groups 155
8-5 Pregnancy rate on Day 27, odd ratios (OR), 95% confidence interval
(Cl) and level of significance for the risk of nonpregnancy for cows in
both groups and at different stages and two common abnormalities
of the estrous cycle 156
8-6 Pregnancy rate on Day 45, adjusted odd ratios (AOR), 95% confidence
interval (Cl) and level of significance for the risk of nonpregnancy for
cows in both groups and at different stages and two common
abnormalities of the estrous cycle 157
8-7 Pregnancy rate on Day 90, adjusted odd ratios (AOR), 95% confidence
interval (Cl) and level of significance for the risk of nonpregnancy for cows
in both groups and at different stages and two common abnormalities
of the estrous cycle 158
9-1 Number of cows evaluated for different stages and two common
abnormalities of the estrous cycle and protocols at Days 30, 55 and 90
after resynchronized insemination 178
9-2 Distribution of cows by parity, season, DIM and inseminator for cows
in diestrus from January to May for both groups 179
9-3 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in diestrus for the Ovsynch and
Quicksynch Groups from January to May 180
9-4 Distribution of cows by parity, season, DIM, and inseminator for in
diestrus from June to December for both groups 181
9-5 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in diestrus for the Ovsynch and Modified
Quicksynch groups from June to December 182
xi


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ME, Clopton DT, Kinder JE.Corpus luteum development and function in cattle with
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phases of the estrous cycle. Biol Reprod 1999;61:921-926.
Rae MT, Menzies GS, Bramley TA. Bovine ovarian non-genomic progesterone binding
sites: presence in follicular and luteal cell membranes. J Endocrinol 1998;159:413-27.
Rahe CH, Owens RE, Fleeger JL, Newton HJ, Harms PG. Pattern of plasma luteinizing
hormone in the cyclic cow: dependence upon the period of the cycle. Endocrinology
1980, 107:498-503.
Rajakoski E. The ovarian follicular system in sexually mature heifers with special
reference to seasonal, cyclical, and left-right variations. Acta Endocrinol 1960;34(Suppl
52):7-68.
Rajamahendran R, Ambrose JD, Schmitt EJP, Thatcher MJ, Thatcher WW. Effects of
buserelin injection and deslorelin (GnRH agonist) implants on plasma progesterone, LH,
accessory CL formation and corpus luteum dynamics in Holstein cows. Theriogenology
1998;50:1141-1155.


58
Gonadotropin releasing hormone administered either 0 to 5 hours before or at the time
of insemination increased conception rate in some studies but not in others, and the largest
response was observed when conception rate in the control group was under 50 %
(Rosenberg et al., 1991). In subfertile cows, administration of GnRH before or at the time of
insemination increased conception rate (Rosenberg et al., 1991). In contrast, there was a
decrease in pregnancy rate in lactating dairy cows inseminated and treated with GnRH 12 to
16 hours after onset of estrus (Mee et al 1990). Treatment of GnRH on Day 2 after estrus
resulted in reduced concentration of P4 during late luteal phase in heifers (Rodger and
Stormshak, 1984). Administration of GnRH on Day 2 of the estrous cycle may attenuate
corpus luteum function by down-regulating luteal LH receptors (Rodger and Stormshak
1984).
In conclusion, administration of hCG 10 hours after onset of estrus in nonlactating
/
dairy cows decreased the size of the CL and reduced P4 concentrations during the luteal phase
and is not recommended to enhance luteal function in nonlactating cattle.


143
requirements for lactating cows (NRC, 2001). Cows were milked three times a day, and
had a rolling herd average for milk production of 10,700 kg. Beginning at 60 d
postpartum, cows received a bST treatment (Posilac, 500 mg sometribove zinc,
subcutaneously; Monsanto, St Louis, Missouri) every 14 d during the entire lactation.
Reproductive management consisted of a voluntary waiting period of 75 d that
incorporated a Presynch-Ovsynch program (Moreira et al., 2001) for first service, and
estrus detection using visual observation and a computerized pedometer system
(Afimilk, S.A.E. Afikim, Kibbutz Afikim, 15148, Israel) for subsequent services. The
study included 332 lactating dairy cows detected nonpregnant by ultrasonography at Day
27 (experimental Day 0) after artificial insemination. Cows with reproductive
abnormalities (i.e. metritis, pyometra, uterine-ovarian adhesions, granulosa cell tumor)
were excluded.
Experimental Design
On Day 0, nonpregnant cows (n=332) were divided randomly into two groups.
Cows in the Ovsynch Group (n=166) received 100 pg (i.m.) GnRH (Cystorelin, Merial
Limited, Iselin, NJ, 08830) on Day 0, 25 mg (i.m.) PGF2a (Lutalyse, Pharmacia,
Kalamazoo, MI, 49001) on Day 7, 100 pg (i.m.) GnRH on Day 9, and TAI 16 h later
(Day 10). Cows in the Heatsynch Group (n=166) received 100 pg (i.m.) GnRH on Day 0,
25 mg (i.m.) PGF2a on Day 7, 1 mg (i.m.) ECP on Day 8, and TAI 48 h later (Day 10).
Cows detected in estrus on Days 8 and 9 in both groups were inseminated and included in
the study. On Day 0, information for parity, time of the year (season), days in milk
(DIM), and inseminator were recorded.


148
(OR=4.9; 95% CI=1.1-36.9; P=0.03), and tended to decrease for cows in metestrus
subjected to the Heatsynch Group (OR=0.4; 95% 0=0.1-1.1; P=0.08).
Discussion
The objectives of this study were to compare pregnancy rates and pregnancy
losses in lactating dairy cows resynchronized using the Ovsynch and Heasynch
protocols, and to evaluate the effect of the stages/abnormalities of the estrous cycle.
There was no difference in overall pregnancy rate and pregnancy losses in cows
resynchronized using either the Ovsynch or the Heatsynch protocols initiated after
diagnosis of nonpregnancy by ultrasonography on Day 27 post-insemination. However,
pregnancy rate was affected by the stages/abnormalities of the estrous cycle that either
Ovsynch or Heatsynch was initiated. Pregnancy rates increased when cows in metestrus
were subjected to the Heatsynch protocol and cows with ovarian cysts received the
Ovsynch protocol.
The importance of reducing the interval between first and second insemination
has stimulated the development of different strategies to resynchronize return to estrus
(Chebel et al., 2003b; Macmillan et al., 1997; 1999). Administration of 1 mg of estradiol
benzoate on Day 13 after estrus synchronizes the ovarian follicular wave and subsequent
estrus (Burke et al., 2000). Such a treatment in combination with a medroxyprogesterone
acetate impregnated sponge increased the number of cows re-inseminated between Days
18 and 25 after insemination (Cavestany et al., 2003). Insertion of the CIDR
(progesterone intravaginal insert) by itself (Chenault et al., 2003) or in combination with
an estrogen injection at CIDR insertion, at CIDR removal, or 24 h after removal, can
resynchronize the return to estrus following a prior insemination (Macmillan et al., 1999;


179
Table 9.2. Distribution of cows by parity, season, DIM and inseminator for cows in
diestrus from January to May for both groups.
Variable
Ovsynch
Quicksynch
P value
%
N
%
N
Parity
1
20.8
20/96
32.3
31/96
0.18
2
32.3
31/96
30.2
29/96
3+
46.9
45/96
37.5
36/96
Season
Jan-Mar
49.0
47/96
58.4
56/96
0.19
Apr-May
51.0
49/96
41.6
40/96
DIM
90-145
21.9
21/96
28.1
27/96
0.26
146-178
19.8
19/96
27.1
26/96
179-233
31.2
30/96
20.8
20/96
234-685
27.1
26/96
24.0
23/96
Inseminator
A
25.0
24/96
31.2
30/96
0.48
B
7.3
7/96
9.4
9/96
C
67.7
65/96
59.4
57/96


93
The incidence of anovulatory cows during the first 58 DIM was similar between
treatments and the higher incidence in primiparous than multiparous cows has been
reported previously (Stevenson, 2001; Santos et al., 2003; Moreira et al., 2001).
Incidence of anovulatory cows during the first 58 DIM is variable and is probably
affected by herd, season of the year, level of milk production, nutrition, and BCS in the
early postpartum period, among other factors. Clearly, a relationship between BCS at 68
DIM and frequency of anovulatory cows in the first 2 mo postpartum has been
established. Moreira et al. (2001) and Santos et al. (2003) also indicated that frequency of
anovulatory cows decreased as BCS increased prior to the first postpartum Al. Body
condition score reflects the energy reserves of dairy cows (Ferguson et al., 1994); cows in
better energy status have earlier resumption of ovarian function and higher fertility.
Agonists of GnRH have been synthesized to have increased binding affinity to the
/
GnRH receptors and to have a longer half-life (Karten and Rivier, 1986). The response of
cattle to treatment with deslorelin involves an acute and chronic phase (dOcchio et al.,
2000). The acute phase lasts for several days, which increases the secretion of
gonadotropins. When compared to buserelin, deslorelin induces a greater secretion of LH
(Mann and Lamming, 2001), which might improve CL formation resulting in higher mid-
luteal phase progesterone concentrations when compared with a Buserelin injection
(Ambrose et al., 1998).
Different concentrations of deslorelin have been used successfully to induce
ovulation in non-lactating dairy cows and dairy heifers when incorporated into the
Ovsynch protocol (Ambrose et al., 1998; Bartolom et al., 2004). Treatment with
deslorelin at doses of 450 to 750 pg increased plasma progesterone concentrations after


171
nonpregnant at ultrasonography on Day 28 (1) post insemination subjected to a
shortened Ovsynch protocol (PGF2a Day 0, GnRH on Day 2 and TAI 16-20 h later) had a
similar pregnancy rate to cows inseminated at detected estrus or TAI at 72-80 h after
PGF2a. However, protocol responses were not compared to a protocol that synchronizes
follicular waves (Stevenson et al, 2003). Administration of GnRH at 7 d before a
nonpregnancy diagnosis may be an alternative to synchronize the follicular wave prior to
administration of PGF2a at the nonpregnancy diagnosis and to ensure presence of a
functional CL and a preovulatory follicle. Such a program would allow for a
synchronized ovulation and permit a rapid TAI (Moreira et al., 2000; Chebel et al., 2003).
However, time of GnRH injection needs to be optimized based upon dynamics of
interestrus intervals.
Since pregnancy rate for cows in the Quicksynch Group was acceptable for cows
that showed estrus activity based on pedometers, the protocol was modified, and the
Modified Quicksynch Group included insemination at estruses detected by pedometers
during the first four days and a TAI protocol (Ovsynch) assigned on Day 4 for cows that
had not expressed estrus. There was no difference in pregnancy rates for cows in the
Modified Quicksynch and Ovsynch protocols. In the Modified Quicksynch protocol, a
high proportion (72%) of cows were inseminated at estrus within 4 d after PGF2a and
ECP administration. Therefore, this protocol may be an option to combine estrus
detection and TAI to maintain pregnancy rates, and reduce the cost for resynchronization.
For cows in metestrus, there was a reduction in pregnancy rates for cows in the
Heatsynch Group. In a previous study, the Heatsynch protocol increased pregnancy rates
in cows in metestrus since a high proportion (41%) of cows were inseminated at estrus


152
Table 8.1. Criteria for determination of the stage and two common abnormalities of the
estrous cycle based on ultrasonography and rectal palpation of the genital tract (adapted
from Zemjanis 1962 and Pierson and Ginther 1984a, 1987)
Clinical Findings
Stages/abnormalities of the
Estrous Cycle
Ovaries
Uterus
Diestrus
Functional CL
Follicle >10 mm
Slight tonus
Metestrus
Corpus hemorrhagicum
Edema
Follicle < 10 mm
Moderate tonus
Proestrus/Estrus
Follicle ~ 18 mm
Regressing CL
High tonus
Ovarian Cysts
Multiple follicles > 18 mm
Absence of CL
Flaccid
Anestrus
Follicle < 18 mm
Flaccid


This dissertation was submitted to the Graduate Faculty of the College of
Veterinary Medicine and to the Graduate School and was accepted as partial fulfillment
of the requirements for the degree of Doctor of Philosophy.
May 2004


146
Proc GENMOD, SAS (Agresti, 1996). Treatment differences with P<0.05 were
considered significant.
Results
The distribution of cows for the different variables between treatment groups was
not different (Tables 8.2 and 8.3) other than the distribution of cows among inseminators
(P<0.01; Table 8.3). The number of cows in different stages/abnormalities of the estrous
cycle on Day 0 was as follow: diestrus (46.1%, 153/332); metestrus (14.8%, 49/332);
proestrus (22.0%, 73/332); ovarian cysts (14.4%, 48/332); anestrus (2.7%, 9/332).
The number of cows that were bred at estrus on Days 8-9 were lower (P<0.01) in
the Ovsynch Group [15.1%, 25/166 (10/81 diestrus, 1/22 metestrus, 8/35 proestrus, 6/23
ovarian cyst and 0/5 anestrus)] than in the Heasynch Group [36.1%, 60/166 (25/72
diestrus, 11/27 metestrus, 12/38 proestrus, 11/25 ovarian cyst and 1/4 anestrus)].
On Day 27 after the resynchronized TAI, 259 (127 in Ovsynch and 132 in
Heatsynch) cows were classified as pregnant or nonpregnant, four cows were classified
as a questionable pregnant (two in each group) and 69 cows were not presented for
ultrasonography diagnosis. On Day 45, 332 cows (166 in Ovysnch and 166 in Heatsynch)
were evaluated for pregnancy diagnosis. On Day 90, five cows that were pregnant on Day
45 were culled from the herd and 327 cows (165 in Ovsynch and 162 in Heatsynch) were
evaluated for pregnancy diagnosis. Pregnancy rates on Days 27, 45 and 90 and pregnancy
losses between Days 27 and 45 and 45 and 90 for cows in the Ovsynch and Heatsynch
groups were not different (Table 8.4).
In the multivariate analysis for pregnancy rates, there was an interaction (P<0.01)
between group and stages/abnormalities of the estrous cycle. Therefore, pregnancy rates


162
plasma was stored at -20C until assayed for P4 using a solid-phase, no-extraction RIA
(Coat-a-Count Progesterone, DPC, Diagnostic Products Corporation, Los Angeles,
California). The standard curve dilution consisted of 4 plane tubes for total counts and
coated tubes for nonspecific binding and 100 ul of increasing concentrations of P4 (0.1,
0.25, 0.5, 2, 5, 10, 20 and 40 ng/ml). Serum from ovariectomized and estrus cows and
from cows in diestrus (low and high) were used as references. A duplicate was included
every 6th sample. Sensitivity of the assay was 0.1 ng/ml. The duplicates were categorized
into high (>3 ng/ml), medium (1-3 ng/ml) and low (<1 ng/ml) concentrations of P4. The
intra-assay coefficients of variation for the high, medium and low duplicates samples
were 5.5, 4.8 and 9.3 % respectively. Intra and inter-assay coefficient of variation for the
references were 12.5 and 10.5 % respectively.
Plasma P4 concentration was used as the reference to determine the positive
predictive value of per rectum examination and ultrasonography of the genital tract to
determine stages/abnormalities of the estrous cycle. The limit values for plasma P4
concentration for different stages/abnormalities of the estrous cycle were established as
follow: diestrus > 2 ng/ml, metestrus < 2 ng/ml, proestrus <1 ng/ml, and anestrus < 1
ng/ml. For cows with ovarian cysts: follicular cysts < 1 ng/ml and luteal cysts > 1 ng/ml
(Kesler and Garverick, 1982). Plasma P4 concentration also was used to determine
sensitivity, specificity, and positive predicted value of the clinical diagnosis (per rectum
examination and ultrasonography of the genital tract) to differentiate follicular and luteal
cysts (Martin et al., 1987; Farin et al., 1990).


46
al., 2000). Cholesterol is transported into the mitochondria by the steroidogenic acute
regulatory protein (StAR), and StAR protein (Pescador et al., 1996) and P450cc (Rao et
al., 1978) are expressed in the CL after luteinization. The small luteal cells are
responsible for increasing secretion of P4 in response to LH and the large luteal cells
contribute to high basal levels of P4 (Koos and Hansel, 1981) producing around 80% of
total P4 (Niswender et al., 1985). Small luteal cells are 8-22 urn in diameter and respond
to LH with a 6-12 fold increase in steroid production (Fitz et al., 1982). The small luteal
cells express LH receptors in large amounts but also receptors are expressed in large
luteal cells (Chegini et al., 1991) and both cells receptors for prostaglandin (Niswender
and Nett, 1994). Therefore, also luteal cells respond to stimulatory effect of prostaglandin
1-2 (Fitz et al., 1984) and E-2 (Chegini et al., 1991). Large luteal cells are greater than 22
um in diameter and do no respond to LH stimulation (Rodgers et al., 1983). Large luteal
/
cells express growth hormone (GH) and IGF-I receptors and therefore GH and IGF-I may
have a role in basal secretion of P4 (Niswender et al., 2000). In fact, IGF-I accelerates the
appearance of StAR during in vitro luteinization and augments gonadotropin-induced
StAR expression (Pescador et al., 1999).
The pulsatile secretion of LH is important for CL maintenance and development,
especially during the first 12 days of the luteal phase (Peters et al., 1994). Luteinizing
hormone binds its receptor (seven-transmembrane G-protein receptor) in the plasma
membrane of the large luteal cells and stimulate steroidogenesis through the cAMP-
protein kinase second messenger system (Niswender and Nett, 1994). This protein kinase
system may be constituvely activated in the large luteal cells (Hoyer et al., 1984).
Luteinizing hormone facilitates the transport of cholesterol through the cell and to the


Pregnancy
£ 40 -
30 -
20 -
10 -
0 -
GnRH on Day 5
No GnRH on Day 5
No Yes
GnRH on Day 15
Figure 6.1. Interaction between administration of GnRH on Day 5
and Day 15 on pregnancy rate at Day 27 (P<0.10).


165
In the Quicksynch Group, 78% (72/92) of cows showed pedometer activity 1 d
from TAI, and pregnancy rate on Day 30 was higher (P<0.01) for the cows with
pedometer activity (27.8%, 20/72) compared to cows without activity (0.0%, 0/20).
Plasma P4 concentrations on Day 0 were not different for cows in the Ovsynch
(6.070.32 ng/ml) and Quicksynch (6.40.30 ng/ml) groups. There was no difference in
the proportion of cows with plasma P4 concentration greater than 2 ng/ml between
Ovsynch (87.5%, 84/96) and Quicksynch (92.5%, 87/94) groups. The positive predictive
value for per rectum examination and ultrasonography of the genital tract to detect cows
in diestrus was 90% (171/190).
Cows in Diestrus (June to December)
The distribution of cows by the different variables between groups was not
different (P>0.15) except among inseminators (PO.01, Table 9.4). Pregnancy rates on
Days 30, 55, and 90 were evaluated by logistic regression adjusting for inseminator.
Pregnancy rates on Days 30, 55, and 90 for cows in the Ovsynch (34.0%, 24%, and
23.6%) and Modified Quicksynch (27.1%, 26.2%, and 21.6%) groups were not different.
Pregnancy losses between Days 30-55 were higher (P<0.05) for cows in the Ovsynch
Group (23.2%) compared to cows in the Modified Quicksynch Group (6.4%). Pregnancy
losses between Days 55-90 for cows in the Ovsynch (5.7%) and Modified Quicksynch
(8.3%) groups were not different (Table 9.5).
In the Modified Quicksynch Group, 72.0% (85/118) of the cows were
inseminated based on pedometer activity and 28.0% (33/118) were TAI. Pregnancy rate
on Day 30 for cows inseminated at detected estrus (29.0%; 25/85) and for cows TAI
(21.1%; 7/33) within the Modified Quicksynch Group were not different.


102
Figure 5.2. Relationship between body condition score (BCS) at first postpartum artificial
insemination (AI) and frequency of anovulatory dairy cows. Frequency of anovulatory
cows = 52.2393 12.5 x BCS at AI (P < 0.002).


Treatment Group (n=9)
GnRH
l
PGF
2a
hCG 3,000 IU
8-10 h after estrus
i Bleeding every 12 h
U/S every 3 d
Estrus
Detection
Day 0
U/S
Day 7
U/S
Day 30
Control Group (n=8)
GnRH
i
I
PGF2a
i
1
Saline
Esms i
Detection
Day 0 Day 7
U/S u/S
Bleeding every 12 h
U/S every 3 d
Day 30
Figure 3.1. Experimental Design


Thatcher WW, Binelli M, Burke J, Staples CR, Ambrose JD, Coelho S. Antiluteolytic
signals between the conceptus and endometrium. Theriogenology 1997;47:131-140.
262
Thatcher WW, Chenault JR. Reproductive physiological responses of cattle to exogenous
prostaglandin F2a. J Dairy Sci 1975, 59:1366-1375.
Thatcher WW Driancourt MA, Terqui M, Badinga L. Dynamics of ovarian follicular
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Thatcher WW, Drost M, Savio JD, Macmillan KL, Entwistle KW, Schmitt EJ, de la Sota
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1993,33:27-49.
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Norgestomet, and Prostaglandin F2alpha.J Anim Sci 1999;77:1823-1832.
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125
stage (days in milk, parity; Humblot, 2002), nutritional status (lost in body condition
score; Silke et al., 2001), mastitis (Risco et al., 1999), and endocrine disorders (Thatcher
et al., 2003) may be responsible for late embryonic mortality. Vasconcelos et al. (1999)
reported a lower late embryo mortality (Days 28 to 98) in cows subjected to the Ovsynch
protocol that ovulated a large versus a small follicle (14 vs 24%).
Corpus luteum regression can precede embryo mortality in heifers (Kastelic et al.,
1991) and supplementation with norgestomet at 36 hours after PGF2a-induced luteolysis
on Day 15 after insemination was able to maintain pregnancy in cows (Lulai et al., 1994).
However, administration of a norgestomet implant on Days 15-16 after insemination did
not increase pregnancy rate in heifers (Lulai et al., 1994).
Excessive follicular development and high levels of estrogen during the luteal
phase can be detrimental for the embryo (Pritchard et al., 1994; Inskeep, 1995; Bridges et
/
al., 2000). Several factors including heat stress and high milk production are known to
affect follicular dominance and may allow for a greater follicular development during
early pregnancy (de la Sota et al., 1993). The effect of estrogenic follicles on conceptus
survival and luteolysis is not clearly understood, but there seems to be a positive
association between higher levels of estrogen and early pregnancy losses (Inskeep, 2000;
Bridges et al., 2000). Interestingly, follicular growth is suppressed in the ovary ipsilateral
to the pregnant uterine horn during the embryonic stage (Guibault et al., 1986; Thatcher
et al., 1986, 1991).
A deslorelin implant suppresses follicular growth without affecting corpus luteum
function (Ambrose et al., 1998; Bartolom et al., 2003). Treatment with a GnRH agonist
implant to induce accessory corpora ltea, to reduce follicular growth and to increase P4


166
Plasma P4 concentration on Day 0 for cows in the Ovsynch (4.770.20 ng/ml) and
Modified Quicksynch (4.770.19 ng/ml) groups was not different. There was no
difference in the proportion of cows with plasma P4 concentration greater than 2 ng/ml
between the Ovsynch (136/152, 89.5%) and Modified Quicksynch (124/140, 92.5%)
groups. The positive predictive value for per rectum examination and ultrasonography of
the genital tract to detect cows in diestrus was 85.6% (250/292).
Cows in Metestrus
The distribution of cows by the different variables among groups was not
different (P>0.15) except by DIM (P<0.01, Table 9.6). Pregnancy rates on Days 30, 55
and 90 were evaluated by logistic regression adjusting for DIM. Pregnancy rate on Day
30 tended (P<0.13) to be higher for cows in the GnRH+Ovsynch Group (33.3%)
compared to cows in the Ovsynch (24.1%) and Heatsynch (20.3%) groups. Pregnancy
rate on Days 55 and 90 tended (P<0.10) to be higher for cows in the GnRH+Ovsynch
(24.5 and 20.3%) and Ovsynch (25.3 and 23.8%) groups compared to cows in the
Heatysnch Group (12.9 and 9.8%). Pregnancy losses between Days 30-55 and Days 55-
90 for cows in the Ovsynch (7.1 and 5.9%), Heatsynch (36.3 and 14.3%), and
GnRH+Ovsynch (13.3 and 7.8%) groups were not different (Table 9.7).
In the Heatsynch Group, 16.0 % (10/62) of the cows were inseminated at detected
estrus and 40.0% (4/10) were pregnant at Day 30. In the Ovsynch Group, 10.4% (7/67)
cows were inseminated at detected estrus and 28.6% (2/7) were pregnant at Day 30. In
the GnRH+Ovsynch Group only 1 cow of 59 (1.7%) was inseminated at detected estrus
and was diagnosed pregnant at Day 30.


230
insert (i.e. CIDR device) on Day 14 after AI with removal at the time GnRH injection is
administered on Day 23 for resynchronization may control follicular growth such that the
administration of GnRH on Day 23 will be more efficient causing follicular turnover and
increase follicular synchronization at Day 30. In Figure 11.1 different strategies to
increase pregnancy rate, reduce embryonic loss and rapidly resynchronize nonpregnant
cows are described. In addition, a positive effect on the progesterone insert on Day 14 in
combination with a GnRH dose on Day 23 on a potentially established pregnancy
warrants further investigation.
>
Presynch
Ovsynch
GnRH+TAl
Induction
accessory
CL
:;Z;XvX Induction
accessory
CL
Days from Breeding to Estrus
This strategy for resynchronization includes early pregnancy diagnosis with
ultrasonography. However, the concepts for manipulating follicular growth and CL
formation before a nonpregnancy diagnosis apply for different reproductive
managements. Even though the number of cows in different stages of the estrous cycle,


206
Since cows with ovarian cysts also have follicular waves (Cook et al., 1990; Hamilton et
al., 1995; Yoshioka et al., 1996), the beneficial effect of GnRH is due to ovulation of a
dominant follicle that may be present at the time of treatment and recruitment of a new
potential ovulatory follicle rather than luteinization or regression of the cysts (Kesler et
al., 1981). In the present study, GnRH on Day 0 in cows with ovarian cysts increased the
proportion of corpora ltea on Day 7, and this effect was not observed in cows at
proestrus. It indicates that cows detected with multiple follicles, absence of a CL and lack
of uterine tonicity (ovarian cysts) are less likely to ovulate spontaneously and form a CL
compared with cows in proestrus with a dominant follicle and tonicity in the uterus.
Temporary P4 exposure has been used previously to induce estrus, ovulation and
CL formation in lactating dairy cows with ovarian cyst degeneration (Johnson and
Ulberg, 1967; Archbald and Thatcher, 1992; Douthwaite et al., 2000; Wiltbank et al.,
2002; Zulu et al., 2003). Progesterone treatment seems to restore the positive feed-back
of estradiol on GnRH release (Nanda et al., 1991) that is compromised in cows with
ovarian cysts (Dobson and Alam, 1987; Refsal et al., 1988; De Silva and Reeves, 1988;
Kaneko et al., 2002). However, the majority of these protocols were not intended to
synchronize the follicular waves in order to synchronize ovulation and evaluate fertility
to a TAI. Previous research indicated that induction of estrus in cows with ovarian cysts
has to be combined either with high estrus detection (Archbald et al., 1991; Archbald and
Thatcher, 1992) or TAI in order to increase pregnancy rate (Bartolom et al., 2001).
A treatment that combines GnRH on Day 0, PGF201 on Day 7 and a CIDR insert
from Day 0 to 7 was used to induce follicular turnover, elevate P4 during follicular
growth and then induce an LH surge after CIDR removal and PGF2a administration


74
Figure 4.2. Least squares means and SE for the first-wave largest follicles from Day 0 to
Day 16 for Control (), DESLORELIN 750 () and DESLORELIN 1000 (A) groups
(P<0.02 Control vs. DESLORELIN 1000) and second-wave largest follicle for Control
(), DESLORELIN 750 (0)/and DESLORELIN 1000 (A) groups (P<0.01) in
Experiment 1.


70
1994). Pulsatile secretion of LH is necessary to maintain luteal activity between days 2
and 12, but not between days 12 and 17 (Peters et al., 1994). Administration of deslorelin
induces a normal LH surge and elevates basal LH during the following 2 (Rajamahendran
et al., 1998) or 7 (DOcchio et al., 1996) days. High basal levels of LH during early
stages of the estrous cycle may stimulate CL development and differentiation in cows
induced to ovulate with a deslorelin implant.
Follicular development was decreased after induction of ovulation with a
deslorelin implant. The first-wave largest follicle was inhibited by the deslorelin implant;
however there was variability among doses in the degree of inhibition. In addition,
deslorelin implants reduced the number of Class II and III follicles from Days 0 to 16, but
again variability existed among deslorelin implant doses. The differences could be
explained due to cow-to-cow sensitivity to different doses of the implant. Substantial
s
variation among cattle has been detected previously using deslorelin implants (Ambrose
et al., 1998, DOcchio et al., 1996). Second-wave largest follicle was clearly suppressed
using 1000, 750 or 450 p.g deslorelin implants.
Follicular growth and return to estrus after PGF2 on day 16 were clearly delayed
in cows and heifers induced to ovulate with a deslorelin implant (1000, 750 or 450 p.g)
and the rate of follicular growth was decreased in a dose-dependent manner. In cows that
failed to ovulate, the Ovsynch protocol had to be used in order to induce ovulation. It is
clear that cows that experience follicular suppression and delayed return to estrus will
grow follicles in the absence of P4 and therefore they will need to be resynchronized to
avoid the development of anovulatory follicles. The deslorelin implants used in the
present series of experiments are biodegradable and therefore not removed. Although the


88
305-d mature equivalent milk yield (multiparous), and randomly assigned to one of the
two treatments. Cows in the deslorelin treatment were randomly assigned to receive
either a 450 or a 750 pg implant. Plasma progesterone concentration on Day 11 after TAI
was analyzed by the GLM procedure of the SAS program (SAS, 1999), with the effects
of treatment, parity, cyclicity, body condition score at AI, and higher-order interactions.
Follicular populations and interval between the first and second postpartum AI were
analyzed by the GLM procedure of the SAS program (SAS, 1999), with the effects of
treatment, parity, cyclicity, body condition score at the first AI, plasma progesterone
concentration on Day 11, and higher-order interactions. Orthogonal contrasts were
performed to determine the effect of type of GnRH (control vs DESLORELIN 450 and
DESLORELIN 750) and the effect of dose of deslorelin (DESLORELIN 450 vs
DESLORELIN 750) on the outcome variables evaluated.
/
Binomially distributed data were analyzed by logistic regression (Allison, 1999),
using the LOGISTIC procedure of the SAS program (SAS, 1999), with a model that
included the effects of treatment, parity, cyclicity, body condition score at AI, BCS
change from 30 DIM to AI, plasma progesterone concentration on Day 11, and higher-
order interactions. Contrasts were performed to determine the effect of type of GnRH
(control vs DESLORELIN 450 and DESLORELIN 750) and the effect of dose of
deslorelin (DESLORELIN 450 vs DESLORELIN 750) on the outcome variables
evaluated as described previously.
Regression analyses were performed to determine the best-fitted line between
BCS at AI and the frequency of anovulatory/anestrous cows prior to the first AI. Linear,
quadratic and cubic effects were tested. Cows were grouped based on their BCS at AI,


229
a CIDR insert). The distribution of estrus in cows not pregnant based on expression of
estrus and re-insemination indicated that the majority of the cows return to estrus around
Day 22 after previous insemination. However, only 40 to 45% of nonpregnant cows had
corpora ltea at the time of nonpregnancy diagnosis by ultrasonography on Days 28 to
30. Administration of GnRH on Day 23 after previous insemination resulted in 75% of
the cows with corpora ltea, and these diestrus cows obtained similar pregnancy rate to a
resynchronized timed insemination after induction of luteolysis with PGF2 and
synchronization of ovulation with either GnRH or ECP. In cows without a CL, GnRH
treatment was beneficial, and a P4 insert during the 7 days between injection of GnRH
and PGF201 improved pregnancy rate in cows with ovarian cysts, but not in proestrus
cows. In cows detected nonpregnant after previous service, protocols for
resynchronization of ovulation and timed insemination that considered the clinical
findings of the reproductive tract at the nonpregnancy diagnosis increased pregnancy rate
to a resynchronized service.
Previous research indicated that presynchronization combined with
synchronization of ovulation and timed insemination increased pregnancy rate to first
service in lactating dairy cows (Moreira et al., 2001). Administration of hCG on Day 5
after insemination induce accessory CL and increased pregnancy rate (Santos et al,
2001). These strategies can optimize pregnancy rate at Day 28-30. Administration of a
deslorelin implant (450 (ig, or short-term release deslorelin) or hCG may induce
accessory CL and reduce late embryonic loss and optimize pregnancy rates at Day 45-55.
For cows that did not conceive to first service or loss the embryo before Day 28-30, the
distribution of estruses after insemination suggest that the inclusion of a progesterone


130
Follicular suppression was higher (P<0.01) for cows in the Implant Group
(23.3%, 20/86) than for cows in the Control Group (1.1%, 1/89). Accessory CL formation
was also higher (P<0.01) for cows in the Implant Group (45.4%, 39/86) than for cows in
the Control Group (10.1%, 9/89).
There was an interaction between treatment and accessory CL formation on
pregnancy loss (Figure 7.5). In the Implant Group, cows that formed an accessory CL had
a reduced pregnancy loss between Day 27 and Day 45 compared to cows that did not
form an accessory CL (5.1% vs 34.0%; Table 7.3). In contrast, pregnancy loss was
greater in control cows with an accessory CL (33% versus 14%). In addition, among all
experimental cows, cows with follicular suppression had an increased embryonic loss
(42.9 vs 14.9%; Table 3).
There was also an interaction between treatment and accessory CL formation on
/
plasma P4 concentrations on Day 45 (P<0.01). In the Implant Group, cows that formed an
accessory CL had increased plasma P4 concentration on Day 45 compared to cows that
did not form an accessory CL (10.7 ng/ml versus 5.5 ng/ml). In contrast, plasma P4
concentrations were not different for control cows with or without an accessory CL (7.8
ng/ml versus 6.3 ng/ml).
Discussion
Administration of a deslorelin implant (2.1 mg) on Day 27 of pregnancy in
lactating dairy cows increased the number of corpora ltea, increased plasma P4
concentration, reduced follicular growth but did not reduce pregnancy loss as a treatment
main effect. However, the deslorelin treatment did affect pregnancy loss dependably
upon presence of an accessory corpus luteum. Cows treated with the deslorelin implant


183
Table 9.6. Distribution of cows by parity, season, DIM and inseminator for cows in
metestrus from January to December for all groups.
Variable
Ovsynch
Heatsynch
GnRH+Ovsynch
P value
%
N
%
N
%
N
Parity
1
20.6
14/68
29.0
18/62
31.2
20/64
P=0.67
2
36.8
25/68
35.5
22/62
31.2
20/64
3+
42.6
29/68
35.5
22/62
37.5
24/64
Season
Mar-Sep
50.0
34/68
50.0
31/62
51.6
33/64
P=0.97
Oct-Feb
50.0
34/68
50.0
31/62
48.4
31/64
DIM
90-162
29.4
20/68
17.7
11/62
25.0
16/64
P=0.04
163-197
14.7
10/68
29.0
18/62
35.9
23/64
198-263
32.3
22/68
25.8
16/62
14.1
9/64
264-783
23.5
16/68
27.4
17/62
25.0
16/64
Inseminator
A
47.1
32/68
51.6
32/62
46.9
30/64
P=0.80
B
10.3
7/68
14.5
9/62
15.6
10/64
C
42.6
29/68
33.9
21/62
37.5
24/64


211
Table 10.3. Distribution of cows by parity, DIM, inseminator, and housing lot for cows
without CL on Day 0.
Variable
Group
P
GnRH
CIDR
GnRH+CIDR
Control
%
N
%
N
%
N
%
N
Parity
1
46.4
13/28
41.2
14/34
50.0
16/32
42.9
12/28
0.78
2
28.6
8/28
17.6
6/34
18.7
6/32
28.6
8/28
3+
28.6
8/28
41.2
14/34
31.2
10/32
28.6
8/28
DIM
95-150
32.1
9/28
23.5
8/34
15.6
5/32
25.0
7/28
0.20
151-170
14.3
4/28
26.5
9/34
18.7
6/32
10.7
3/28
171-227
10.7
3/28
23.5
8/34
12.5
4/32
32.1
9/28
228-899
42.9
12/28
26.5
8/34
53.1
17/32
32.1
9/28
Ins.
A
21.4
6/28
20.6
7/34
21.9
7/32
35.7
10/28
0.27
B
50.0
14/28
26.5
9/34
37.5
12/32
25.0
7/28
C
28.6
8/28
52.9
18/34
40.6
13/32
39.3
11/28
Lot
A
28.6
8/28
20.6
7/34
37.5
12/32
39.3
11/28
0.04
B
25.0
7/28
26.5
9/34
12.5
4/32
42.9
12/28
C
46.4
13/28
52.9
18/34
50.0
16/32
17.9
5/28
Ins.=inseminator


95
pregnancy losses than cows treated with gonadorelin (P = 0.13). Plasma progesterone
concentrations during mid-diestrus did not differ between DESLORELIN 450 and control
cows, and progesterone concentrations on Day 11 did not affect pregnancy losses
between Days 27 and 41 after TAI (P = 0.28). However, treatment with deslorelin
attenuated follicular growth and the trend of reduced pregnancy losses for the
DESLORELIN 450 supported the concept that follicular development during the period
of early embryo development may adversely affect embryo survival.
During diestrus, the stimulation caused by the rise in progesterone re-establishes
the endometrial lipid pool. In the absence of viable embryo during the period of maternal
recognition of pregnancy, the uterine endometrium under stimulation of estradiol and
oxytocin produces PGF2a, which is responsible for the lysis of the CL and termination of
pregnancy or re-initiation of a new estrous cycle (Thatcher et al., 2003). We speculate
that suppressed follicular development as caused by deslorelin (Bartolom et al., 2004) is
expected to result in a lower estrogenic environment during the early stages of pregnancy,
which might reduce the ability of the endometrium to secrete PGF2a and improve embryo
survival.
When pregnant cows on Day 27 after TAI were either left as controls (n = 90) or
treated with a deslorelin implant (n = 89), Bartolom et al. (2002) observed that the
number of CL and the concentration of progesterone on Day 45 was increased, at the
same time that follicular population was reduced, but no effects on losses of pregnancy
between Days 27 and 45 and between Days 27 and 90 after insemination were observed.
Thus, late treatment with deslorelin does not alter pregnancy losses, but when cows are
exposed to a low dose of deslorelin during the early stages of embryonic development


Table 10.10. Number of cows, adjusted odd ratios (AOR), 95% confidence interval (Cl)
and levels of significance for the risk of pregnancy on Day 30 in cows in proestrus and
with ovarian cysts treated with CIDR on Day 0 adjusting for BCS.
PR on Day 30
Variable
%
N
AOR
95% Cl
P value
Treatment* stage
CIDR Proestrus
12.9
4/31
Referent
Referent
No CIDR Proestrus
42.3
11/26
4.9
1.3-19.3
0.02
CIDR Ovarian cysts
31.6
6/19
4.0
0.9-18.0
0.06
No CIDR Ovarian cysts
BCS
21.7
5/23
1.8
0.4-7.9
0.41
<3.0
10.0
2/20
Referent
Referent
-
>3.0
30.3
24/79
4.5
0.9-22.2
0.06
PR=Pregnancy rate


13
messenger system (Stormshak et al., 1995) and the same mechanism is used by oxytocin
to stimulate PGF201 release by the endometrium (Silvia and Homanics, 1988). Other
substances such as endothelin-1 and angiotensin II seem to mediate the effect of PGF2a in
the corpus luteum.
Endothelin-1 (ET-1) is a potent vasoconstrictor secreted by endothelial cells and
expressed in the corpus luteum when stimulated by PGF2 (Girsh et al. 1996a, 1996b;
Miyamoto et al., 1997). It suppresses P4 production in luteal-derived cells in the presence
of PGF2a (Girsh, et al., 1996a). Endothelin-1 is known to play a role in luteolysis, and by
using an Endothelin-A receptor antagonist, it was shown that ET-1 has a potent
contractile effect on human myometrial cells (Dallot et al., 2003). Prostaglandin F2 has
been shown to stimulate angiotensin II in vitro, and angiotensin II, ET-1, and oxytocin
may interact to inhibit P4 secretion by causing functional luteolysis, and through
/
vasoconstriction may initiate structural regression (Hayashi and Miyamoto, 1999).
Structural luteolysis involves apoptosis and is characterized by expression of class II
MHC antigens, an autoimmune-like response with invasion of immune cells, and
secretion of TNF-a and other cytokines (Benyo et al., 1991; Benyo and Pate, 1992).
After P4 drops, the mean concentration of LH (Chenault et al., 1975), frequency
(Ireland and Roche, 1982b) and amplitude of LH pulses (Stumpft et al., 1989) stimulate
follicular growth and consequently a rise in estrogen levels (Schallemberger et al., 1984)
and a new estrous cycle begins.
Clinical signs characteristic of proestrus are increasing walking activity,
involment in sexual active groups and mounting behavior. Clinical characteristics at
rectal palpation are increase in uterine tonicity due to the effect of oxytocin during luteal


8
milk production (Hammond, 1927). Receptivity to be mounted is the most precise sign
of estrus and is used by different methods for estrus detection such as visual observation
(Trimbeger, 1943), rump-mounted detectors (Stevenson and Britt, 1977) and Heatwatch
system (Dohi et al., 1993). The pedometer detects 2.75 time-increase in walking activity
during estrus (Kiddy, 1977), and used in combination with reduced milk production can
estimate the onset of estrus. An increase of 100% in walking activity may indicate estrus
and there was a positive correlation with fertility up to an increase of 400-500% (Yaniz et
al., 2003). Plasma P4 concentrations measured by radioimmunoassay or milk P4 levels
evaluated by an on-farm ELISA test can be used to monitor the accuracy of estrus
detection methods (Nebel et al., 1987).
The palpable characteristics of the genital tract associated with estrus are a
dominant ovarian follicle, absence of a corpus luteum or presence of a regressing corpus
luteum, and increased uterine tonicity (Zemjanis, 1962). On ultrasonography,
preovulatory dominant ovarian follicles are observed (usually from 12 to 17 mm), the
uterine horns display a very heteregenous pattern, and a hypoechogenic image indicating
the presence of mucus can sometimes be observed in the uterine lumen (Pierson and
Ginther, 1984a; 1987).
Metestrus
The stage of metestrus is considered to begin either after the end of estrus (10 to h
after LH surge) or at ovulation (24-30 h after LH surge; Day 0) and ends after the corpus
luteum (CL) is fully matured (Day 5). Considering that metestrus has to begin at the end
of estrus, ovulation occurs during metestrus. The LH surge triggers inflammatory-like
changes that allow for rupture of epithelia, albuginea, theca, basal membrane and


CHAPTER 8
RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION (TAI) IN
LACTATING DAIRY COWS, I: USE OF THE OVSYNCH AND HEATSYNCH
PROTOCOLS AFTER NONPREGNANCY DIAGNOSIS BY ULTRASONOGRAPHY
Introduction
Reproductive efficiency in dairy herds increases by inseminating all the cows at
the end of the voluntary waiting period, obtaining high pregnancy rate to first service,
enhancing embryonic and fetal survival, and detecting and re-inseminating nonpregnant
cows. Ultrasonography allows for early pregnancy diagnosis and detection of
nonpregnant cows (Pierson and Ginther, 1984a; Kastelic et al., 1988), which need to be
subjected to resynchronization of ovulation and TAI in order to minimize the problem of
/
poor estrus detection (Nebel et al., 1987).
Protocols for synchronization of ovulation and TAI, such as Ovsynch (Pursley et
al., 1995; Pursley et al., 1997a) and Heatsynch (Pancarci et al., 2002), ensure that all
cows are inseminated, and generate acceptable pregnancy rates in lactating dairy cows.
Since the stage of the estrous cycle is known to affect the response to these protocols
(Vasconcelos et al., 1999; Moreira et al., 2000), a Presynch program was developed to
initiate Ovsynch in the early to mid-luteal phase improving pregnancy rate to first
service in cyclic cows (Moreira et al., 2001).
Protocols for resynchronization that considered the stage of the estrous cycle have
been applied after detection of nonpregnant cows by rectal palpation and assignment of
protocols based on the presence or absence of a CL in the ovaries (Bartolom et al.,
141


214
Table 10.6. Number of cows, adjusted odd ratios (AOR), 95% confidence interval (Cl)
and levels of significance for the risk of presence of a CL on Day 7 in cows in proestrus
and with ovarian cysts treated with GnRH on Day 0 adjusting for BCS and plasma P4
concentration on Day 0.
Variable
CL on Day 7
% N
AOR
95% Cl .
P value
Treatment* Stage
GnRH Ovarian cysts
77.3
17/22
Referent
Referent
-
No GnRH Ovarian cysts
40.9
9/22
0.1
0.02-0.5
0.005
GnRH Proestrus
75.9
22/29
1.6
0.1-2.6
0.50
No GnRH Proestrus
66.7
22/33
1.2
0.3-4.6
0.82
BCS
<3.0
50.0
12/24
Referent
Referent
-
>3.0
69.5
57/82
3.6
1.2-10.5
0.01
P4 on Day 0
< 1 ng/ml
59.4
41/69
Referent
Referent
-
> 1 ng/ml
78.4
29/37
6.2
1.7-22.4
0.005


pgf2o
GnRH
TAI A
| 16h
DO
D2
D3
PGFto
ECP
TAI
36h
DO
D2
D3 >
GnRH
DO
Cows in diestras
PGF^ ECP TAI
D7 D8 D9
^j>- Cows in metestras
DO D7
B/US B/US
B=blood sample; US=ultrasonography
D9 DIO
B
D17
B/US
Cows without
a CL
Figure 10.1. Experimental design for cows in diestrus, metestrus or
without a CL at the time of a nonpregnancy diagnosis (exp. Day 0).


Pregnancy Rate Pregnancy Rate
Pregnancy RateonDay55
Pregnancy Rate on Day 30
No CIDR CIDR
Proestrus ili Ovarian Cysts
Pregnancy Rate on Day 90
No CIDR
Proestrus ;;
50
40
30
20
10
0
No CIDR CBR
Proestrus is- Ovarian Cysts
CIDR
S Ovarian Cysts
Figure 10.5. Pregnancy rate on Days 30, 55, and 90 in cows
without a corpus luteum on Day 0 (proestrus and ovarian cysts)
and treated with a CIDR insert (CIDR*Stage P<0.05).


260
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169
There was an interaction (P<0.05) between group and type of ovarian cysts.
Pregnancy rates on Day 30, 55 and 90 in cows with follicular cysts as determined by
clinical diagnosis was lower for the Ovsynch Group (18.6, 17.2 and 12.9%) compared to
the GnRH+Ovsynch Group (31.1, 27.0 and 23.0%) but there was no difference in cows
with luteal cysts (Table 9.13). However, even though pregnancy rate was numerically
higher for the GnRH+Ovsynch Group, there was no difference between Ovsynch and
GnRH+Ovsynch in cows with either low (<1 ng/ml) or high (>1 ng/ml) plasma P4
concentration (Table 9.13).
Cows in Anestrus
Pregnancy at Days 30, 55 and 90 for cows in the Ovsynch (0/5; 1/8; 0/8) and
GnRH+Ovsynch (1/8; 1/9; 1/9) groups were not statistically evaluated due to small
sample size. Plasma P4 concentration on Day 0 for cows in the Ovsynch (0.25+0.10
ng/ml) and GnRH+Ovsynch (0.55+0.29 ng/ml) groups was not different. There was no
difference in the proportion of cows with plasma P4 concentration lower than 1 ng/ml in
the Ovsynch (8/8, 100%) and GnRH+Ovsynch (6/8, 75.0%) groups. The positive
predictive value for per rectum examination and ultrasonography of the genital tract to
detect cows in anestrus was 87.5% (14/16).
Discussion
Objective of the present study was to compare pregnancy rates and pregnancy
losses for different protocols of resynchronization of ovulation and TAI of nonpregnant
cows assigned to protocols based on their stages/abnormalities of the estrous cycle.
Pregnancy rates for cows in different stages and two common abnormalities (ovarian


175
protocol may be the alternative to increase pregnancy rate. For cows with ovarian cysts a
double administration of GnRH (GnRH+Ovsynch) increased pregnancy rate.
Finally, the distribution of interestrus intervals and stages of the estrous cycle
indicated that different strategies (GnRH, P4 inserts, estradiol) could be used to
synchronize follicular growth before ultrasonographic diagnosis of nonpregnancy. Such
strategies may increase the percentage of cows in diestrus that will respond to PGF2 and
have a fresh dominant follicle for rapid reinsemination.


52
embryo development. In nonlactating dairy cows, the fertilization rate was 67, 79 and
82% when cows were inseminated either 0, 12 or 24 hours after onset of estrus, and 98%
after natural breeding (Dalton et al., 2001). Using Ovsynch, pregnancy losses between
Day 28 and Day 98 were 24 % (PR on Day 28 of 32% and on Day 98 of 25%) for cows
with a large follicle (> 15 mm at PGF2 and >18mm at second GnRH) and 14% (PR on
Day 28 of 42% and at Day 98 of 36 %) for cows with smaller follicle (<15 mm at PGF2a,
and <18 mm at GnRH (Vasconcelos et al., 1999). Administration of GnRH 72 hours after
PGF2 will induce a shorter and less pronounced LH surge compared to control cows but
some cows will have a spontaneous LH surge before GnRH administration (Lucy and
Stevenson 1986). This may be explained by the fact that the estrogen produced by the
preovulatory follicles induces GnRH receptors in the anterior lobe of the pituitary gland
(Reeves et al., 1971) which indicates the importance of estrogen levels to induce a potent
LH surge (Zolman et al., 1974).
There is a clear effect of climate on reproductive efficiency through out the year
with reduced pregnancy rates during the summer (Badinga et al., 1985; Al-Katanani et
al., 1999). This effect is observed mainly in lactating dairy cows (Badinga et al., 1985).
Heat stress increased embryo mortality in lactating dairy cows (Ryan et al., 1993). In
addition, heat stress reduced luteal tissue and conceptus weight in beef cattle (Biggers et
al., 1987). In lactating dairy cows, fertilization rate evaluated on Day 5 (-56%) was
reduced compared to dairy heifers during the summer (Sartori et al., 2002). Temperatures
that exceed normal body temperature are detrimental to both in vitro and in vivo embryo
at the two-cell stage causing disruption of the cytoskeleton and damage of the
mitochondria (Rivera et al., 2003). In addition, heat stress alters follicular dynamics.


186
Table 9.9. Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between Days
30-55 and 55-90 for cows in proestrus for the Ovsynch and GnRH+Ovsynch groups
from January to December.
Group
Variable
Ovsynch
% (N)
GnRH+Ovsynch
% (N)
Pregnancy rate
Day 30
30.1 (22/73)
28.0 (21/75)
Day 55
23.9 (21/88)
23.2 (20/86)
Day 90
21.8 (19/87)
20.2(17/84)
Pregnancy losses
Day 30-55
19.0(4/21)
15.0(3/20)
Day 55-90
5.2(1/19)
5.9(1/17)


109
sometribove zinc, subcutaneously; Monsanto, St Louis, Missouri) every 14 d during the
entire period of the study. The reproductive management employed in this herd consisted
of a voluntary waiting period of 100 d for multiparous cows, and 140 d for primiparous
cows. All cows were subjected to Presynch-Ovsynch program (Moreira et al., 2001) for
first service.
The methods of estrus detection used in this study included a computerized
system which used increased walking activity and an accompanying decrease in milk
production (Afimilk, S.A.E. Afikim, Kibbutz Afikim, 15148, Israel), as well as daily
visual observations of the cows. A specific individual was assigned the duty of observing
these cows for estrus during each 8-h shift. Thus, the cows were continuously observed
for estrus during an 8-h period of time for 24 h.
Study Design
/
A total of 831 reproductively normal cows was used in this study. Multiparous
cows were treated with one luteolytic dosage of PGF2Q (25 mg, im; Lutalyse, Pharmacia
& Upjohn, Kalamazoo, MI, 49001) on Day 64 and Day 78 PP, GnRH (100 pg im;
Cystorelin, Merial Limited, Iselin, NJ, 08830) on Day 90 PP, PGF2a on Day 97 PP,
GnRH on Day 99 PP, and time inseminated 16 h later (Day 100 PP; Experimental Day 0).
Primiparous cows were treated with PGF201 (25 mg, im) on Day 104 PP and Day 118 PP,
GnRH (100 pg im) on Day 130 PP, PGF2a on Day 137 PP, GnRH on Day 139 PP, and
time inseminated 16 h later (Day 140 PP; Experimental Day 0).
On Day 0, 831 primiparous and multiparous cows were randomly assigned to
receive either GnRH on Day 5 or Day 15 after TAI in a 2x2 factorial design. Sample size
was calculated based on a expected difference in pregnancy rate of 5% for the main


151
The use of per rectum examination in combination with ultrasonography of the genital
tract to determine stages/abnormalities of the estrous cycle may be a valuable tool for
selective resynchronization of nonpregnant cows at the time of pregnancy diagnosis.


39
Reproductive responses of heifers treated with PGF20C, Select Synch and Ovsynch
protocols are described in Table 2 (Stevenson et al., 2000).
Days in milk are known to affect the response to TAI protocols. Pregnancy rates
were optimized when the Ovsynch protocol was initiated after 75 days postpartum
(Pursley et al., 1997b). Anestrus may be one of the factors reducing pregnancy rate to the
Ovsynch protocol early in lactation. In fact, pregnancy rate to a TAI protocol was
reduced in cows with BCS less than 2.5 (1-5 scale) and reducing the proportion of cows
with BCS < 2.5 may have an economical benefit (Moreira et al., 2000). The effect of
anestrus can be even more pronounced with use of the Heatsynch protocol that include an
estrogen to induce ovulation. Cows with BCS less than 3.0 had a higher pregnancy rate to
the Presynch-Ovsynch protocol compared to the Presynch-Heatsynch protocol (Pancarci
et al., 2002). Inclusion of P4 supplementation from the first GnRH to the PGF2a in the the
/
Ovsynch protocol will likely avoid premature occurrence of estrus and enhance cyclic
responses in anestrous animals (Thatcher et al., 2001).
Heat stress is another factor affecting fertility in dairy herds. During a heat stress
period estradiol production by preovulatory follicles is reduced and estrus expression is
compromised. In addition, heat stress increases embryo mortality. The Ovsynch
protocol increased cumulative pregnancy rate (Arechiga et al., 1998) and increased
pregnancy rate, reduced days open and increased net revenue per cow due to the
elimination of estrus detection during the summer time (De la Sota et al., 1998).
Administration of bST is a common practice in dairy herds in USA to increase
milk production. The effect of bST on reproductive efficiency has been reported to be
negative due to a reduction in estrus expression. However, administration of bST at the


19
The route, dose, and frequency of administration for different types (natural and
synthetic) of PGF2a were evaluated in several studies. Prostaglandins are usually
administered intramuscular. However, attempts to delay absorption or metabolism and
reduce the minimal dose have been made by testing other routes of administration such as
uterine wall (Inskeep, 1973), uterine lumen (Tervit et al., 1973, Louis et al., 1974), or the
antrum of the ovarian follicle (Inskeep et al., 1974). Reduced doses of cloprostenol
administered intra-vulvosubmucosally or subcutaneously resulted in incomplete
luteolysis and prolonged intervals from treatment to ovulation compared with a standard
dose used intramuscularly (Dhaliwal et al., 1991). A reduced dose (10 mg) or standard
dose (25 mg) of dinoprost tromethamine (10 mg) administered into the isquio-rectal fossa
caused luteolysis in cattle with the same effectiveness as did 25 mg injected
intramuscular (Colazo et al., 2002b). Cloprostenol sodium (500 ug) administered
subcutaneously (Colazo et al., 2002a) or intravenously (Stevens et al., 1995) was also
effective in causing luteolysis in heifers.
Administration of 5, 10, 25 and 30 mg of PGF2a (dinoprost tromethamine) during
early (Days 5-9), mid (Days 10-14) or late (Days 15-19) luteal phase showed that 5 mg
were not effective and 25 to 30 mg caused luteolysis in 56% of beef heifers in early luteal
phase and 100% of the animals in mid or late luteal phase (Berardinelli and Adair, 1989).
Similarly results were obtained using either 25 mg of PGF2a (Watts and Fuquay, 1985) or
500 p.g of cloprostenol sodium (Jackson et al., 1979) with increased effectiveness as the
stage of the estrous cycle advanced. Frequency of administration can also influence the
luteolytic effect. Two doses of PGF2a 8 h apart may be more effective in causing
luteolysis than a single or double injection 24 h apart (Archbald et al., 1993, 1994).


CHAPTER 3
ADMINISTRATION OF HUMAN CHORIONIC GONADOTROPIN (hCG) DURING
ESTRUS IN NONLACTATING DAIRY COWS AND ITS EFFECT ON CORPUS
LUTEUM SIZE AND PROGESTERONE PRODUCTION
Introduction
The preovulatory rise in luteinizing hormone (LH) induces functional and
morphological changes in theca and granulosa cells and they are transformed into luteal cells
and begin to secrete P4 (Baird, 1992). Progesterone production by the corpus luteum supports
establishment and maintenance of pregnancy in cattle (Mann and Lamming, 1995). Cows
diagnosed pregnant after breeding had higher levels of circulating P4 during early luteal phase
compared to cows diagnosed not pregnant (Lee et al., 1985).
/
Luteinizing hormone is the major factor involved in stimulating CL development and
P4 production (Donaldson and Hansel, 1965, Hansel and Seifart., 1967). Human Chorionic
Gonadotropin has LH-like activity, binds LH-receptors in the luteal tissue and has been
demonstrated to be luteotrophic in cattle (Donaldson et al., 1965; Lee and Ryan, 1973). In
sheep, the steroidogenic response to hCG is longer than the one observed with ovine LH
(Bourdage et al., 1984). Therefore, daily (Wiltbank et al., 1961) or systemic (Schomberg et
al., 1967) administration of hCG during the luteal phase increased P4 concentrations and
prolonged the lifespan of the CL in cattle. In addition, the importance of basal levels and
pulsatility of LH during early stages of CL development have been demonstrated using a
GnRH antagonist (Peters et al., 1994).
54


49
high plasma P4 concentrations (Mann and Lamming, 1995). Excessive follicular
development and high estradiol during the luteal phase may be detrimental to embryo
survival (Inskeep, 1995). Destruction of ovarian follicles during diestrus prolonged the
lifespan of the corpus luteum in cows indicating a contribution of estradiol to the
luteolytic cascade (Hughes et al., 1987).
Progesterone also contributes by a local mechanism mediated by P4 receptors
located in small and large luteal cells to repress the onset of apoptosis in the corpus
luteum (Rueda et al., 2000). Also mediated by the same mechanisms, P4 may be involved
in autocrine/paracrine regulation of follicular function (Rae et al., 1998). In addition, P4
downregulates estrogen receptors and P4 receptor antagonist inhibits luteinization in
bovine granulosa cells (Niswender et al., 2000).
Embryo Mortality
/
The evaluation of fertilization rate and different methods to monitor embryo
development (oviductal and uterine flushings) as well as methods for diagnosis of
pregnancy (biochemical, immunological, hormonal, and clinical) allow for determination
of embryo and fetal losses. After service, fertilization occurs in approximately 95%
(Ayalon, 1978; Diskin and Sreenan, 1980) but calving rates are around 50% (Humblot,
2001). Therefore, around 45% of pregnancy losses seem to occur which reduce
reproductive efficiency. Pregnancy losses can be classified in early embryonic mortality
(from fertilization to Day 16 with return to estrus before Day 25), late embryonic
mortality (from Day 17 to Day 45) and fetal loss (from Day 45 to calving; Humblot,
2001).


164
or high P4 using the Chi-square test (Proc FREQ, SAS system). Treatment differences
with P<0.05 were considered significant.
Results
The numbers of cows evaluated at Days 30, 55 and 90 are described in Table 9.1.
Overall pregnancy rates were 28% (257/919) on Day 30, 23.1% (248/1075) on Day 55,
and 20.3% (216/1065) on Day 90. Among the 72% (662/919) of cows detected
nonpregnant on Day 30 after resynchronized insemination, 54% (359/662) were
diagnosed not pregnant based on expression of estrus and insemination before
ultrasonography, and 46% (303/662) based on the absence of pregnancy at
ultrasonography. The median interestrus interval for cows diagnosed not pregnant based
on expression of estrus and insemination before ultrasonography was 22 d, and the
distribution of interestrus intervals is described in Figure 9.2.
Cows in Diestrus (January to May)
The distributions of cows by parity, season, DIM and inseminator were not
different between groups (P>0.15; Table 9.2). Pregnancy rates on Days 30 and 55 were
higher (P<0.05) for cows in the Ovsynch Group (35.9 and 29.2%) compared to cows in
the Quicksynch Group (21.7 and 16.7%). Pregnancy rate on Day 90 tended (P<0.07) to
be higher for cows in the Ovsynch Group (26.0%) compared to cows in the Quicksynch
Group (15.6%). There was no difference in pregnancy losses between Days 30-55 or 55-
90 for cows in the Ovsynch (21.2 and 10.7%) and Quicksynch (20.0 and 6.2%) groups
(Table 9.3).


219
Table 10.11. Number of cows, adjusted odd ratios (AOR), 95% confidence interval (Cl)
and levels of significance for the risk of pregnancy on Day 55 in cows in proestrus and
with ovarian cysts treated with CIDR on Day 0 adjusting for BCS.
Variable
PRe on Day 55
% N
AOR
95% Cl
P value
Treatment*stage
CIDR Proestrus
12.5
4/32
Referent
Referent
-
No CIDR Proestrus
37.0
10/27
4.6
1.2-19.3
0.02
CIDR Ovarian cysts
31.6
6/19
4.0
0.9-18.0
0.06
No CIDR Ovarian cysts
17.4
4/23
1.4
0.3-6.4
0.67
BCS
<3.0
9.5
2/21
Referent
Referent
-
>3.0
37.5
22/80
4.3
0.9-21.1
0.07
PR=Pregnancy rate


170
cysts and anestrus) of the estrous cycle were optimized after assignment to various
protocols for resynchronization of ovulation and TAI.
In a previous study, ultrasonography for pregnancy diagnosis was conducted at 28
d after insemination, and the proportion of cows in diestrus was 46% (Bartolom et al.,
2004). Based on an expected interestrus interval of 17-24 d in cows previously
inseminated, ultrasonography was conducted at 30 d after insemination in this study to
increase the number of cows with a CL at the time of ultrasonography. However, the
proportion of cows in diestrus was still 45%. Fifty five percent of the cows in the present
study were detected in metestrus, proestrus, with ovarian cysts and anestrus indicating
that these cows had an estrous cycle of abnormal length probably due to various causes
such as failure of previous synchronization, early regression of the CL, embryo loss with
delayed return to estrus, or ovulatory failure of a return estrus. A treatment with GnRH at
7 d before nonpregnancy diagnosis on Day 30 could be an alternative to reduce the
number of cows without CL at the time a resynchronization program is initiated.
For cows in diestrus, there was a decrease in pregnancy rates for cows in the
Quicksynch protocol compared to cows in the Ovsynch protocol. The lack of follicular
wave synchronization reflected by the distribution of interestrus intervals in nonpregnant
cows (Figure 9.2) may explain this decreased pregnancy rate. In another study, there was
no difference in pregnancy rate for cows subjected to Quicksynch and Heatsynch
protocols for resynchronization after nonpregnancy by ultrasonography (Chebel et al.,
2003). In the present study, even though 78% of the cows in the Quicksynch group
showed estrus activity 1 d of TAI, timing of insemination may differ from studies using
insemination at or 12 h after visual observation of estrus (Chebel et al., 2003). Cows


98
size to express LH receptors in the granulosa cells (Ginther et al., 2001 and to respond to
an injection of LH (Sartori et al., 2001). The proportion of cows with a follicle greater
than 10 mm at the moment of GnRH injection on Day 27 after TAI was reduced for
deslorelin compared to control, which would limit the ability of any LH surge to induce
ovulation. Obviously, re-synchronization at Day 27 after TAI was not successful for
those treated with deslorelin, based on the proportion of cows that responded to the first
GnRH of the Ovsynch and the proportion of cows with a large follicle at the moment of
the PGFia injection. All these factors should impair the ability of the last GnRH of the
Ovsynch protocol to induce a synchronous ovulation of a healthy follicle.
The attenuation in follicular development and the lack of response to gonadorelin
treatment during the re-synchronization period reduced PR to the second Al. Lack of
ovulation and ovulation of a small incompetent follicle are some of the possible reasons
for the reduced fertility during the re-synchronization period.
Vasconcelos et al. (2001) demonstrated that reduction in the size of the ovulatory
follicle compromised the ability of the resulting CL to sustain high concentrations of
plasma progesterone and reduced PR. Although the means by which follicles did not
reach adequate ovulatory size were different between the current study and the study
reported by Vasconcelos et al. (2001), perhaps part of the effect of deslorelin on reduced
fertility during the second postpartum Al was due to a induced ovulation of a smaller
ovulatory follicle that would compromise subsequent luteal function.
After cows underwent the Ovsynch protocol between 58 and 67 DIM, 94.0% of
them had plasma progesterone concentrations >1.5 ng/mL. This indicates that a majority
of the lactating cows in this study had progesterone concentrations compatible with the


65
Experiment 3
Forty-four dairy heifers were synchronized using GnRH on Day 9 and PGF2a on Day 2.
On Day 0, heifers received either 100 of Cystorelin (Control, n=22) or a 750 jag
deslorelin implant (DESLORELIN 750, n=22). On Day 16, all heifers received PGF2a
and estrus was monitored for 6 d. Heifers that had not displayed signs of estrus within 10
d after PGF2a (i.e., Day 26) were evaluated by ultrasonography to determine ovarian
structures. Blood samples were collected on Days 7, 13, and 16 after either GnRH
injection or insertion of the deslorelin implant (i.e., Day 0) to evaluate plasma P4
concentrations. Outcome variables were plasma P4 concentration (ng/mL) and estrus
expression after PGF2aon Day 16 (yes/no).
Collection of Blood Samples and Radioimmunoassay
Blood samples for P4 analysis were collected by puncture of the coccygeal vein or
/
artery into evacuated tubes containing ethylene diamine tetra acetic acid (EDTA) as an
anticoagulant (10.5 mg, Monoject, Sherwood Medical, St. Louis, MO). Plasma P4
concentration was determined with a single antibody radioimmunoassay procedure
(Knickerbocker et al., 19986). Inter- and intra-assay coefficients of variation were 6.7 and
10.3 %, respectively. Sensitivity of the assay was 0.15 ng/mL.
Statistical Analyses
Plasma P4 concentrations were analyzed using the repeated measures procedure
(Proc MIXED, SAS) for Mixed Models (Littell et al., 1997) adjusting for the number of
CL as a covariate. Differences between means for specific days were examined by the
PDIFF option. Follicular growth was analyzed using the repeated measures procedure
and by the homogeneity of regression procedure (Proc GLM, SAS, Wilcox et al., 1990).


188
Table 9.11. Pregnancy rates at Days 30, 55 and 90, and PL between Days 30-55 and 55-
90 for cows with ovarian cysts subjected to the Ovsynch and GnRH+Ovsynch groups
from January to December.
Group
Variable
Ovsynch
% (N)
GnRH+Ovsynch
% (N)
Pregnancy rate
Day 30
20.2 (16/79)a
30.3 (27/89)b
Day 55
18.5 (18/97)a
26.6 (29/109)b
Day 90
14.7 (14/95)a
22.9 (25/109)b
Pregnancy losses
Day 30-55
12.5 (2/16)
11.1 (3/27)
Day 55-90
12.5 (2/16)
13.8 (4/29)
a-b PO.05


189
Table 9.12. Plasma P4 concentration in cow with ovarian cysts diagnosed using a clinical
method combining per rectum examination and ultrasonography of the genital tract
Plasma P4
concentration
Total
Clinical Diagnosis
< 1 ng/ml
> 1 ng/ml
Follicular
144
37
181
Luteal
2
17
19
Total
146
54
200


15
grow larger than 5 mm and then one (dominance) or two (co-dominance) become
dominant and grow larger than 10 mm and the others become subordinate and regress
(Savio et al., 1988; Sirois and Fortune, 1988). Follicular waves were evaluated in 10
heifers after estrus (Day 0) and two animals presented two follicular waves (starting on
Days 2 and 11), seven animals presented three follicular waves (starting on Days 2, 9 and
16) and one animal presented four follicular waves (starting on Days 2, 8, 14 and 17;
Sirois and Fortune, 1988). The dominant follicle of the first wave reaches maximum
diameter (15-18 mm) on Day 6 after ovulation, goes through a plateau phase from Days 6
to 10 and was undetectable on Day 15 (Savio et al., 1988). The dominant follicle of the
second wave reached maximum diameter on Day 16 (or Day 19 in cows with two
follicular waves) and was undetectable by Day 19 and the dominant follicle of the third
follicular wave reached maximum size on Day 21 and became the ovulatory follicle
(Savio et al., 1988).
The differences between estrous cycle of two and three waves are the length of
the inter-ovulatory interval (20.4 vs 22.8 d), the mean day of luteal regression (16.5 vs
19.2), the interval from emergence of the follicle to ovulation (10.9 vs 6.8) and the
diameter of the follicle the day before ovulation (16.5 vs 13.9; Ginther et al., 1989b).
Follicles acquire ovulatory capacity at an approximate size of 12 mm with 80% of
ovulation in follicles 10 mm in diameter (Sartori et al., 2001). Follicular waves are
characterized by three phases called recruitment, selection and dominance (Ginther et al.,
1989a). The dominant follicle grows 1.8 mm a day until it reaches 16 mm, remains static
for 6 days and regresses linearly at 1 mm a day (Ginther et al., 1989a). The subordinate
follicle ceases to grow at 4.4 days after emergence (Ginther et al., 1989a). Basal


212
Table 10.4. Distribution of cows by BCS and stages/abnormalities of the estrous cycle
status for cows without CL on Day 0.
Variable
Group
P
GnRH
CIDR
GnRH+CIDR
Control
%
N
%
N
%
N
%
N
BCS
>3.0
75.0
21/28
76.5
26/34
62.5
20/32
75.0
21/28
0.31
<3.0
25.0
7/28
23.5
8/34
37.5
12/32
25.0
7/28
Stage
Proestrus
53.6
15/28
55.9
19/34
56.2
18/32
50.0
14/28
0.63
Ov. Cysts
46.4
13/28
32.3
11/34
34.4
11/32
42.9
12/28
Anestrus
0.0
0/28
11.8
4/34
9.4
3/32
7.1
2/28
Ov. Cysts=ovarian cysts


40
time of the first GnRH of the Ovsynch protocol or at TAI increased pregnancy rate
(Moreira et al., 2001). Both bST and IGF-I stimulate in vitro development of embryos
(Moreira et al., 2002). Effects of bST on the embryo and intrauterine environment likely
contribute to the increase in pregnancy rate in lactating dairy cows.
Anestrus and cystic ovarian degeneration are abnormalities of ovulation and may
occur in nonpregnant cows at any time but are more common during the postpartum
period. Anestrus and a high proportion of cows with ovarian cystic degeneration are
characterized by lack of expression of estrus. Approximately 25% of the cows are not
cyclic (anestrus and ovarian cystic degeneration) by Day 63 postpartum (Moreira et al.,
2001). For cows to have a LH surge, ovulation and a normal luteal phase of P4 need to
rise either by a progressive luteinization of follicles or by treatment with exogenous P4
(Inskeep, 1995). A treatment with P4 for 7 days and 1 mg of estradiol benzoate 24 hours
later induced estrus, and cows can be inseminated within 4 days with acceptable fertility
(Macmillan and Burke, 1996). Such a protocol could be used to presynchronize cows that
may be at high risk of anestrus before a TAI protocol is applied. Cows with ovarian cysts
had a similar pregnancy rate to either the Ovsynch protocol (24%) or Select Synch
protocol (18%). Although conception rate for Select Synch was 50%, the estrus detection
rate was only 35% (Bartolom et al., 2000).
Select Synch, Ovsynch and Cosynch in Beef Heifers and Cows
The Select Synch, Ovsynch and modifications of Ovsynch (e.g. Cosynch) have
been used in beef animals (cows and heifers). Similar to dairy heifers, beef heifers may
be prepuberal and noncyclic and may fail to respond to the initial GnRH. They may also
express estrus (10%) before the PGF2a.injection given 7 d after the GnRH. Beef cows


86
TAI, cows were observed for signs of estrus once daily in the morning by tail chalking
(Macmillan et al., 1988) using paintsticks (All-weather Paintstick, LA-CO Industries,
Chicago, IL). Cows detected in estrus were inseminated in the same morning.
Pregnancy was diagnosed by ultrasonography 27 d after AI. Observation of
embryonic fluid, appearance of the embryo, and embryonic heartbeat were used as
determinants of pregnancy. A real-time ultrasound scanner (Aloka SSD 500 V, Aloka Co.
Ltd., Tokyo, Japan) equipped with a 7.5 MHz rectal probe was used. Cows diagnosed
pregnant on Day 27 had their pregnancies reconfirmed by palpation per rectum of an
embryonic vesicle 14 d later.
Ovaries of non-pregnant cows that had not been re-inseminated were examined on
Day 27 to determine the presence of CL and follicles, and the number of follicles in the
different class sizes: Class I < 5.0 mm; Class II = 6 to 9 mm; and Class III = 10 to 19
mm; and class IV > 20 mm. Non-pregnant cows 27 d after AI that had not been
reinseminated were resynchronized with the Ovsynch protocol (Pursley et al., 1997a).
Ultrasound examination of the ovaries was performed again, 7 d later, at the moment of
the PGF2a injection during the re-synchronization protocol to determine the presence of
CL and the size of the largest follicle. Cows resynchronized were submitted to TAI 16 to
18 h after the last GnRH injection of the Ovsynch protocol. Pregnancy during the re
synchronization period was determined by palpation per rectum of an embryonic vesicle
at 38 3 d after the second postpartum AI (Figure 5.1).
The interval between the first and second postpartum AI for those cows
reinseminated prior to pregnancy diagnosis on Day 27 after the initial TAI was evaluated.


17
of IGFBs and reduced availability of IGF (Monniaux et al., 1997). The increased
availability of IGF within the ovarian follicles amplifies the action of gonadotropins in
follicular cells (Monniaux et al., 1997, Ginther et al., 2001). After a period of dominance
and under high concentration of P4 characteristic of the luteal phase, LH pulsatility
decreases (Rahe et al., 1980) and the dominant follicle regresses and a new wave is
recruited. During an estrous cycle with three waves the dominant follicle of the second
wave grows to a smaller size (Sirois and Fortune, 1988); because of high levels of P4 and
lower LH pulsatility during mid-cycle it is unable to maintain follicular growth (Lucy et
al., 1992; Stock and Fortune, 1993). The decrease in LH support results in reduced
androgen availability through the steroidogenic pathway causing a decrease in estradiol
production and triggering atresia (Lucy et al., 1992).
Energy balance and lactation affect follicular growth which could result in lower
fertility of lactating dairy cows compared with dairy heifers (Lucy et al., 1992). Negative
energy balance reduces LH secretion and follicular growth (Lucy et al., 1992). Lactating
dairy cows have lowfer levels of P4 compare to nonlactaing dairy cows and therefore have
larger follicles and a longer period of dominance (De la Sota et al., 1993). Heat stress
also affects follicular dynamics increasing the number of large follicles and accelerating
the regression of the first-wave dominant follicle and the emergence of the second
follicular wave by two days (Wolfenson et al., 1995). Heat stress also appears to reduce
follicular dominance in nonlactating dairy cows (Guzeloglu et al., 2001). In cows treated
with bovine somatotrofine (bST), the growth of the dominant follicle of the first follicular
wave was reduced, the growth of subordinate follicle was increased, and the subordinate
follicle of the second follicular wave w as larger (de la Sota et al.. 1993). Administration


63
objective was to evaluate ovarian function after inducing ovulation with different doses
(450, 750 and 1000 gig) of a deslorelin implant (GnRH agonist) in comparison to
gonadorelin diacetate (GnRH, 100 pg) in nonlactating dairy cows and heifers.
Materials and Methods
Experiment 1
Nonlactating dairy cows (n=20) were treated on Day 9 with 100 pg im GnRH
(Cystorelin; Merial Ltd, Iselin, NJ), and with two doses of 25 mg im PGF2a
(Lutalyse; Pharmacia, Kalamazoo, MI,) 8 h apart on Day 2. On Day 0, cows were
assigned randomly to three treatment groups and received either 100 pg im GnRH
(Control, n=6), a subcutaneous (sc) biodegradable implant (Peptech Animal Health,
North Ryde, Australia) containing 750 pg deslorelin (D-Trp6-Pro6-des-Gly10-LHRH
ethylamide, DESLORELIN 750; n=7) or a sc implant containing 1000 pg deslorelin
(DESLORELIN 1000; n=7). On Day 16, cows were treated with PGF2ct (two, 25-mg
doses given 8 h apart) to induce luteolysis. Ovaries were evaluated by ultrasonography
daily from Day 0 to ovulation and every other to Day 28. Daily blood samples were
collected from Day 0 until PGF2a administration on Day 16 to determine plasma P4
concentrations. The Heat Watch system (DDx Inc., Denver, CO 80221) was used to
monitor estrus. After Day 16, cows that did not display signs of estrus and ovulate
received 100 pg im GnRH when the largest follicle reached 20 mm in diameter, 25 mg
im PGF2a 7 d later, and 100 pg im GnRH 2 d after PGF2a (Ovsynch protocol; 3). The
outcome variables were plasma P4 concentrations (ng/mL), first-wave largest follicle
(mm; the term largest will be used since deslorelin-implanted cows did not have a
dominant follicle), second-wave largest follicle until Day 16 (mm), number of Class II


156
Table 8.5. Pregnancy rate on Day 27, odd ratios (OR), 95 % confidence interval (Cl) and
levels of significance for the risk of nonpregnancy for cows in both groups and at
different stages and two common abnormalities of the estrous cycle.
Variable
Pregnancy Rate
% N
OR
95 % Cl
P value
Group
Ovsynch
Stage/Abn.
Diestrus
30.2
19/63
Referent
Referent
Heatsynch
Diestrus
24.1
14/58
1.3
0.6-3.0
0.45
Ovsynch
Metestrus
15.4
2/13
2.4
0.5-11.8
0.29
Heatsynch
Metestrus
52.2
12/23
0.4
0.1-1.0
0.06
Ovsynch
Proestrus
19.2
5/26
1.8
0.6-5.5
0.29
Heatsynch
Proestrus
24.1
7/29
1.4
0.5-3.7
0.55
Ovsynch
Ovarian Cyst
30.0
6/20
1.0
0.3-3.0
0.98
Heatsynch
Ovarian Cyst
5.0
1/20
8.2
1.0-65.8
0.04


Fricke PM, Caraviello DZ, Weigel KA, Welle ML. Fertility of dairy cows after
resynchronization of ovulation at three intervals following first timed insemination. J
Dairy Sci 2003;86:3941-3950.
242
Fricke PM, Guenther JN, Wiltbank MC. Efficacy of decreasing the dose of GnRH used in
a protocol for synchronization of ovulation and timed AI in lactating dairy cows.
Theriogenology 1998;50:1275-1284.
Fricke PM, Reynolds LP, Redmer DA. Effect of human chorionic gonadotropin
administered early in the estrous cycle on ovulation and subsequent luteal function in
cows. J Anim Sci 1993;71:1242-1246.
Galligan DT. The economics of optimal health and productivity in the commercial dairy.
Rev Sci Tech 1999;18:512-519.
Geary T. Estrus synchronization in beef cows and heifers. Select Sires Think Tank,
Columbus, OH, September 21-22, 2003.
Geary TW, Whittier JC, Downing ER, LeFever DG, Silcox RW, Holland MD, Nett TM,
Niswender GD. Pregnancy rates of postpartum beef cows that were synchronized using
Syncro-Mate-B or the Ovsynch protocol. J Anin Sci 1998, 76:1523-1527.
Geary TW, Whittier JC, Hallford Dm, MacNeil MD. Calf removal improves conception
rates to the Ovsynch and CO-Synch protocols. J Anim Sci 2001, 79:1-4.
Geisert RD, Morgan GL, Short EC, Zavey MT. Endocrine events associated with
endometrial function and conceptus development in cattle. Rep Fert Dev 1992;4:301-305.
Ginther OJ, Beg MA, Bergfelt DR, Donadeu FX, Kot F. Follicle selection in monovular
species. Biol Reprod 2001; 65:638-647.
Ginther OJ, Bergfelt DR, Beg MA, and Kot K. Follicle selection in cattle: role of
luteinizing hormone. Biol Reprod 2001;64:197-205.
Ginther OJ, Bergfelt DR, Kulik LJ, and Kot K. Selection of the dominant follicle in
cattle: role of estradiol. Biol Reprod 2000;63:383-389.
Ginther OJ, Kastelic JP, Knopf L. Composition and characteristics of follicular waves
during the bovine estrous cycle. Anim Rep Sci 1989a;20:187-200.
Ginther OJ, Knopf L, Kastelic JP. Temporal association among ovarian events in cattle
during oestrus cycles with two and three follicular waves. J Reprod Fert 1989b;87:223-
230.


66
Number of Class II and III follicles was compared using the repeated measures procedure
(Litell et al, 1997) and the repeated procedure for counted data (Proc GENMOD, SAS;
Agresti, 1996). The expression of estrus was analyzed by using Chi Square (Proc FREQ,
SAS). Differences were considered significant when P < 0.05.
Results
Experiment 1
All cows ovulated and formed a CL after treatments on Day 0. Average plasma
P4 concentrations from Day 0 to Day 16 were not different among cows in the Control
(5.9 0.6 ng/mL), DESLORELIN 750 (6.4 0.6 ng/mL), and DESLORELIN 1000 (6.3
0.6 ng/mL) groups. However, sporadic elevations in plasma P4 concentrations were
detected on Day 11 for cows in the DESLORELIN 1000 (11.1 1.0 ng/mL) compared to
cows in the Control (8.2 1.1 ng/mL; P<0.05) groups, and on Day 12 for cows in the
s
DESLORELIN 750 (12.4 1.0 ng/mL) compared to cows in the Control (9.0 1.1
ng/mL, P<0.05; Figure 4.1) groups. Overall a deslorelin-induced increase in plasma
concentrations of progesterone was minimal.
The first-wave largest follicle was larger (P<0.02) for cows in the Control Group
(9.48 1.1 mm) compared to cows in DESLORELIN 1000 Group (4.92 1.0 mm), and
not different from that of cows in DESLORELIN 750 Group (7.82 1.0 mm). The
second-wave largest follicle evaluated until Day 16 was larger (P<0.01) for cows in
Control Group (7.63 + 0.6 mm) compared to cows in DESLORELIN 750 (5.25 0.6
mm) and DESLORELIN 1000 (4.05 + 0.6 mm; Figure 4.2) Groups.
The number of Class II follicles from Day 0 to Day 16 was higher (PO.01) for
cows in Control Group (2.1 0.3) compared to cows in DESLORELIN 750 Group (1.2 +


123
Table 6.5. Percentages, adjusted odd ratios (AOR), 95 % confidence interval (Cl), and
level of significance for the risk of pregnancy loss between Day 27 and Day 55.
Variable
Pregnancy Loss
AOR
95 % Cl
P value
%
N
Group
1
19.6
20/102
1.0
0.5-2.0
0.74
2
13.2
12/91
0.6
0.3-1.4
0.11
3
23.0
18/78
1.2
0.6-2.6
0.25
4
Season
19.5
17/87
Referent
Referent
NA
November-February
13.9
30/215
Referent
Referent
NA
March/April
25.9
37/143
2.2
1.3-3.7
0.005
NA= not appplicable


192
1999; Moreira et al., 2000). Cows in proestrus obtained acceptable pregnancy rate to the
Ovsynch protocol (Moreira et al., 2000), and the distribution of interestrus intervals
after prior insemination indicated that around Days 22-23 most of the nonpregnant cows
will be in the peri-estrus period (Bartolom et al., 2004). Therefore, administration of
GnRH on Days 22-23 after previous insemination should induce ovulation in the majority
of the cows resulting in formation of a CL and synchronize follicular wave development.
In this study, GnRH was administered on Day 23 after previous insemination and
pregnancy diagnosis by ultrasononography was conducted on Day 30. Nonpregnant cows
were resynchronized using different protocols according to ovarian and uterine
characteristics. The objective was to compare pregnancy rate in cows in diestrus after
synchronization of ovulation with either GnRH or ECP, and compare ovarian function
and pregnancy rate in cows without a CL after synchronization of ovulation and TAI in a
protocol including either GnRH or a progesterone (P4) intravaginal device (CIDR).
Materials and Methods
Study Population
The study was conducted from January to March 2003 in a large commercial
dairy with 3,200 milking cows. The herd is in north central Florida and is divided into 14
lots according to levels of production and stage of lactation, and cows from all lots were
included in the study. Cows were housed in free stall barns and dry lots and fed a total
mixed ration (TMR) three times daily. The TMR was formulated to meet or exceed
requirements for lactation (NRC, 2001). Cows were milked three times a day with a
rolling herd average for milk production of 10,700 kg. Beginning at 60 d postpartum,
cows received a bST treatment (Posilac, 500 mg sometribove zinc, subcutaneously;


34
development and decrease P4 levels during diestrus (Lucy and Stevenson, 1986).
Intervals from second GnRH to TAI of 0, 8, 16, 24, and 32 hours, generated pregnancy
rates at 30 days of 37, 41, 45, 41 and 32%, respectively. Furthermore, pregnancy losses
were higher when cows were timed inseminated at 32 hours (Pursley et al., 1998).
Therefore, TAI 16 hours after the second GnRH seems ideal, but at 0 and 24 hours
pregnancy rates were acceptable. Therefore, it is very important to maintain the timing of
injections for the Ovsynch protocol to avoid reductions in pregnancy rate.
In several studies, the Ovsynch protocol increased reproductive efficiency in
dairy herds. The Ovsynch protocol for TAJ reduced days open and eliminated the need
for estrus detection in lactating dairy cows compared to a reproductive program based on
PGF2a and AI using the AM-PM rule (60 and 100 d postpartum pregnancy rates were 37
and 53% for Ovsynch and Control respectively; Pursley et al., 1997a). The Ovsynch
s
protocol was also more efficient when compared to a program involving PGF201 injections
every 14 days and AI at detected estrus or TAI 72-80 hours after the third treatment
(Pursley et al., 1997b). In addition, the Ovsynch protocol in lactating dairy cows
achieved a similar pregnancy rate (30%) compared to cows that were inseminated at
detected estrus after injection of GnRH and PGF201 given 7 days apart, estrus detection
rate in the latter group was 70% (Burke et al., 1996).
In a similar study but with a lower estrus detection rate, higher pregnancy rates
were obtained with the Ovsynch protocol compared with AI at detected estrus after a
single dose or double dose of PGF2 given 14 days apart or after GnRH and PGF2a given
7 days apart (Stevenson et al., 1999). Higher pregnancy rate and a reduced interval from
calving to conception was obtained using the Ovsynch protocol at Day 50 postpartum in


35
lactating dairy cows compared with AI to a spontaneously detected estrus or after one
dose of PGF2a on Day 57 or two doses on Days 33 and 47 postpartum (Momcilovic et al.,
1998). Following a Presynch protocol, a Co-Synch protocol involving GnRH and TAI at
72 h after PGF2a, increased pregnancy rate in lactating dairy cows compared to Ovsynch
(GnRH at 48 h and TAI 24 h later) or Co-Synch (GnRH and TAI at 48 h; Portaluppi and
Stevenson, 2003).
Heatsynch Protocol
Administration of estradiol during proestrus induced an LH surge and
synchronized ovulation in cattle (Inskeep, 1973). The second treatment with GnRH of the
Ovsynch protocol was replaced by estradiol cypionate (ECP) administered 24 hours
after PGF2a and induced ovulation 60 hours later in dairy heifers. The protocol includes
100 gg of GnRH given on Day 0, 25 mg of PGF2a on Day 7, 1 mg of ECP on Day 8 and
s
a TAI 48 hours later (Lopez et al., 2000). This program was named Heatsynch because of
the synchronized occurrence of estrus. In lactating dairy cows, ECP induced ovulation 55
hours later, estrus occurred 29 hours after ECP and the interval from onset of estrus to
ovulation was 27 hours. However, 20% of the cows showed estrus by 24 hours after ECP
and ovulated before 48 hours (i.e. time of AI)). Therefore, it is recommended that the
Heatsynch protocol include inseminating cows detected in estrus within 24 hours after
ECP injection (Pancarci et al., 2002). Seventy five to 85% of the cows subjected to the
Heatsynch protocol show estrus, and AI technicians report greater uterine tonicity and
ease of AI compared to the Ovsynch protocol, in which only 30% of the cows showed
estrus after the second GnRH injection. (Pancarci et al., 2002).


56
Ultrasonography of the Ovaries
Ovarian ultrasound examination was done every 3 days using a real-time ultrasound
scanner (Aloka 500, 7.5 Mhz linear transrectal probe) to evaluate number of follicular
waves and CL size during 23 days after treatment. To evaluate CL size two cross-sectional
observations were done and the three greatest radii were used to estimate the volume using
the formula: 3A*n*rl *r2*r3. Cavities of corpora ltea were subtracted from measurements in
order to considered only luteal tissue.
Statistical Analysis
The response variables were plasma P4 concentration, CL size and number of follicular
waves. Data for plasma P4 concentration were analyzed using repeated measures ANOVAfor
mixed models and the homogeneity of regression procedure. The CL size was evaluated using
repeated measures ANOVA for mixed models. The number of follicular waves was compared
/
using Fisher exact test. Treatment differences with P < 0.05 were considered significant.
Results
There was no difference in the number of follicular waves between cows treated with
hCG (78% of cows with 2 waves; 7/9) and control cows (75% of cows with 2 waves; 6/8).
Plasma P4 concentration was lower in cows treated with hCG (6.46 ng/ml 0.41) than
in control cows (7.89 ng/ml 0.43; P<0.05), with major differences detected between Day 14
and Day 16 after treatment (Figure 3.2). Plasma P4 concentration was also higher for control
than for hCG cows (cubic effect; P<0.01) when evaluated by homogeneity of regression
procedures. Evaluating differences on specific days using the Proc MIXED, plasma P4
concentrations were higher for control group on Day 14 (P<0.003), Day 16 (P<0.02), Day 20
(P<0.01) and Day 20.5 (P<0.03)


181
Table 9.4. Distribution of cows by parity, season, DIM and inseminator for cows in
diestrus from June to December for both groups.
Variable
Ovsynch
Modified Quicksynch
P value
%
N
%
N
Parity
1
25.0
39/156
19.7
28/142
0.53
2
29.5
46/156
30.3
43/142
3+
45.5
71/156
50.0
71/142
Season
Jun-Sep
52.6
92/156
52.1
74/142
0.93
Oct-Dec
47.4
74/156
47.9
68/142
DIM
90-145
22.4
35/156
28.2
40/142
0.07
146-178
24.4
38/156
23.9
34/142
179-233
26.9
42/156
23.9
34/142
234-685
26.3
41/156
23.9
34/142
Inseminator
A
22.5
35/156
22.5
32/142
<0.01
B
17.9
28/156
33.8
48/142
C
59.6
93/156
43.7
62/142


9-6 Distribution of cows by parity, season, DIM, and inseminator for in
metestrus from January to December 183
9-7 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in metestrus for the Ovsynch, Heatsynch,
and GnRH+Ovsynch groups from January to December 184
9-8 Distribution of cows by parity, season, DIM, and inseminator for in
proestrus from January to December 185
9-9 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows in proestrus for the Ovsynch, and
GnRH+Ovsynch groups from January to December 186
9-10 Distribution of cows by parity, season, DIM, and inseminator for with
ovarian cysts January to December 187
9-11 Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between
Days 30-55 and 55-90 for cows with ovarian cysts subjected to the
Ovsynch and GnRH+Ovsynch groups from January to December 188
9-12 Plasma P4 concentration in cows with ovarian cysts diagnosed using
a clinical method combining per rectum examination and
ultrasonography of the genital tract 189
9-13 Pregnancy rate at Days 30, 55 and 90 for Ovsynch and GnRH+Ovsynch
groups in cows with ovarian cysts classified according type of cyst and
plasma P4 concentration 190
10-1 Distribution of cows by parity, DIM, inseminator and lot for cows
in diestrus subjected to the GnRH and ECP groups 209
10-2 Pregnancy rate and losses for cows in diestrus and subjected to the
GnRH or ECP groups 210
10-3 Distribution of cows by parity, DIM, inseminator and housing lot
for cows without a CL on Day 0 211
10-4 Distribution of cows by BCS and stages/abnormalities of the estrous
cycle for cows without CL on Day 0 212
10-5 Ovarian responses on Days 7 and 17 for cows in different stages/
abnormalities of the estrous cycle in all 4 groups 213
xii


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42
Select Synch protocol (Kojima et al., 2000). Replacing MGA with a CIDR-B in the 7-11
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s
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184
Table 9.7. Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between Days
30-55 and 55-90 for cows in metestrus for the Ovsynch, Heatsynch, and
GnRH+Ovsynch groups from January to December.
Group
Variable
Ovsynch
% (N)
Heatsynch
% (N)
GnRH+Ovsynch
% (N)
Pregnancy rate
Day 30
24.1 (14/58)
20.3 (11/54)
33.3 (16/48)a
Day 55
25.0 (17/68)a
12.9 (8/62)b
24.5 (12/59)a
Day 90
23.5 (16/68)a
9.8 (6/6l)b
20.3 (12/59)3
Pregnancy losses
Day 30-55
7.1 (7/14)
36.3 (4/11)
13.3 (2/15)
Day 55-90
5.9(1/17)
14.3 (1/7)
7.8 (1/13)
a-b P<0.13


24
Methods for Synchronization of Estrus in Cattle
The ability to freeze bovine semen while maintaining acceptable fertility has
allowed artificial insemination to be a reproductive technology of tremendous impact in
cattle genetics and productivity (Foote, 1996). Artificial insemination is a commonly
used technique in dairy and beef herds. It requires estrus detection since cows need to be
inseminated at a specific time after onset of estrus. Timing of insemination was the
objective of many studies that determined that cows need to be inseminated 12 hours
after onset of estrus (AM-PM rule; Trimberger and Davis, 1943) or within 24 hours after
onset estrus when using morning insemination only (Barret and Casida, 1946, Robbins et
al., 1978). An interval of 12 hours between onset of estrus and insemination seems to be
ideal since it increases fertilization rate and accessory sperm per embryo without
reducing embryo quality (Dalton et al., 2001). In order to eliminate much of the time
/
required for estrus detection and be able to inseminate most cows in a short period of
time, estrous synchronization protocols were developed.
Estrus occurs after P4 levels drop resulting in an increase in LH pulsatility which
stimulates follicular growth and estrogen production. Estrus can be induced either by
administration and subsequent removal of P4 supplementation (Lamond et al., 1964),
inducing luteolysis with PGF2a (Lauderdale et al., 1974), or by a combination of both
approaches.
Progesterone and Progestins
The use of P4 supplementation for extended periods of time (14 to 21 days)
creates an artificial luteal phase, provides enough time for any CL to regress, and induces
estrus in 2 to 5 days after P4 removal ( Hansel and Malven, 1960; Zimbelman and Smith,


261
Stevenson JS, Kobayashi Y, Thompson KE. Reproductive performance of dairy cows in
various programmed breeding systems including Ovsynch and combinations of
gonadotropin-releasing hormone and prostaglandin F2a. J Dairy Sci 1999;99:506-515.
Stevenson JS, Lucy MC, Call EP. Failure of timed inseminations and associated luteal
function in dairy cattle after two injections of prostaglandin F2a. Theriogenology 1987,
28:937-946.
Stevenson JS, Phatak AP, Rettmer I, Stewart RE. Postinsemination administration of
receptakfollicular dynamics, duration of cycle, hormonal responses and pregnancy rate. J
Dairy Sci 1993;76:2536-2547.
Stevenson JS, Schmidt MK, Call EP. Stage of estrous cycle, time of insemination, and
seasonal effects on estrus and fertiity of Holstein heifers after prostaglandin F2ct. J Dairy
Sci 1984, 67:1798-1805.
Stock AE, Fortune JE. Ovarian follicular dominance in cattle: relationship between
prolonged growth of the ovulatory follicle and endocrine parameters. Endocrinology
1993;132:1108-1114.
Stormshak F, Orwig KE, Bertrand JE. Dynamics of molecular mechanisms underlying
ovarian oxytocin secretion. J. Reprod. Fert 1995; Suppl. 49:379-390.
Stumpf TT, Day ML, Wolfe MW, Clutter AC, Stotts JA, Wolfe PL, Kittok RJ, Kinder
JE.Effect of estradiol on secretion of luteinizing hormone during the follicular phase of
the bovine estrous cycle. Biol Reprod 1989;41:91-97.
Tanabe TY, Hahn RC. Synchronized estrus and subsequent conception in dairy heifers
treated with prostaglandin F2a I. Influence of stage of cycle at treatment. J Anim Sci
1984;58:805-811.
Taponen J, Katila T, Rodriguez-Martinez H. Induction of ovulation with gonadotropin
releasing hormone during proestrus in cattle: influence on subsequent follicular growth
and luteal function. Anim Rep Sci 1999, 55:91-105.
Tefera M, Chaffaux S, Thibier M, Humblot P. A short note: lack of effect of post-AI hCG
or GnRH treatment on embryonic mortality in dairy cattle. Livestock Prod Sci
2001;71:277-281.
Tervit HR, Rowson LEA, Brand A. Synchronization of oestrus in cattle using a
prostaglandin F2a analogue (ICI-80996). J Reprod Fert 1973;34:179-181.
Thatcher WW, Bazer FW, Sharp DC, Roberts RM. Inter-relationships between uterus and
conceptus to maintain corpus luteum function in early pregnancy: sheep, cattle, pigs,
horses. J Anim Sci 1986;62(Suppl.2):25-46.


153
Table 8.2. Distribution of cows by parity, season and DIM for both groups.
Variable
Ovsynch
% (N)
Heatsynch
N (%)
P value
Parity
1
43.4 (72/166)
39.8 (66/166)
0.56
2
19.9 (33/166)
24.7 (41/166)
3+
36.7 (61/166)
35.5 (59/166))
Season
Aug-Sept
60.8 (101/166)
60.2(100/166)
0.91
Oct-Dec
39.2 (65/166)
39.8 (66/166)
DIM
72-160
25.3 (42/166)
22.9 (38/166)
0.83
161-225
23.5 (39/166)
27.1 (45/166)
226-315
25.9 (43/166)
23.5 (39/166)
316-724
25.3 (42/166)
26.5 (44/166)


CHAPTER 9
RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION (TAI) IN
LACTATING DAIRY COWS, II: ASSIGNING PROTOCOLS ACCORDING TO
STAGES AND TWO COMMON ABNORMALITIES OF THE ESTROUS CYCLE
Introduction
Synchronization of ovulation and TAI increases the number of cows inseminated
after the voluntary waiting period and consequently increases pregnancy rate (Burke et
al., 1996; Pursley et al., 1997a; 1997b; De La Sota et al., 1998; Risco et al., 1998;
Momcilovic et al., 1998; Stevenson et al., 1999). Protocols for TAI are more efficient
when initiated at a particular stage of the estrous cycle (Vasconcelos et al., 1999; Moreira
et al., 2000). After first services, nonpregnant cows need to be reinseminated in a timely
manner to maintain reproductive efficiency (Stevenson et al., 2003). Therefore, protocols
for resynchronization need to be short, but still consider the stage of the estrous cycle.
Per rectum examination and ultrasonography of the genital tract was used to
determine stages and two common abnormalities (ovarian cysts and anestrus) of the
estrous cycle which influenced the response to the Ovsynch and Heatsynch protocols for
resynchronization (Bartolom et al., 2004).Therefore, pregnancy rates could be optimized
by assigning different resynchronization of ovulation protocols according
stages/abnormalities of the estrous cycle.
The objective of this study was to compare pregnancy rates at Days 30, 55 and 90
and pregnancy losses between Days 30-55 and 55-90 in lactating dairy cows subjected to
different protocols of resynchronization for TAI. These protocols were assigned based on
159


BIOGRAPHICAL SKETCH
I was born in Bahia Blanca, Buenos Aires Province, Argentina, on May 13 of
1965. During my first two years of life I lived in El Recreo a dairy farm owned my
grandfather Don Julian Bartolom. Thereafter I went to elementary school in Coronel
Granada and Florentino Ameghino and finally I moved to General Pico, La Pampa,
where my father attended the Veterinary School and I completed my bachelors degree.
In 1983 I initiated my studies at the College of Veterinary Medicine, University of La
Pampa, and graduated with the DVM degree in 1988. This program is oriented towards
bovine production medicine and specifically strong in beef production system since the
University is located in the pampas of Argentina where beef production is the principal
economic activity. I completed a two-year (1989-90) residency program in reproductive
physiopathology and herd health management in cattle and small ruminants at the
National Institute of Agricultural Technology (INTA), Balcarce, Argentina. In 1991, I
participated in the 19th International Course in Animal Reproduction at the College of
Veterinary Medicine of Uppsala, Sweden, and I was awarded the Fellowship of the Royal
Veterinary College of Sweden (FRVCS). This nine-month course involved lectures and
laboratories in reproduction in farm animals covering all aspects of reproductive
physiology and pathology with a strong component on breeding soundness evaluation in
bulls, artificial insemination programs and reproductive management. From 1992 to 1997
I was appointed as a lecturer at the College of Veterinary Medicine, University of La
267


LIST OF TABLES
Table Page
5-1 Effect of a deslorelin implant in a timed AI protocol on fertility of lactating
dairy cows 103
5-2 Effect of a deslorelin implant in a timed AI on ovarian activity of dairy cows
diagnosed nonpregnant on Day 27 after AI 104
5-3 Effect of a deslorelin implant in a timed AI on ovarian activity 7 d after
resynchronization with GnRH on Day 27 after the initial timed insemination 105
6-1 Baseline comparison for parity, season, and inseminator 119
6-2 Pregnancy rates on Days 27 and 55 and pregnancy losses (PL) between
Days 27 and 55 for cows in all groups 120
6-3 Pregnancy rates on Day 27, and the adjusted odd ratios (AOR), 95 %
confidence interval (Cl), and level of significance for the risk of
nonpregnancy in cows in all groups 121
6-4 Pregnancy rates on Day 55, adjusted odd ratios (AOR), 95% confidence
interval (Cl), and level of significance for the risk of nonpregnancy in
cows in all groups 122
6-5 Percentages, adjusted odd ratios (AOR), 95 % confidence interval (Cl),
and level of significance for the risk of pregnancy loss between
Day 27 and Day 55 123
7.1 Distribution of cows by parity, previous services and type of artificial
insemination in both groups 133
7.2 Pregnancy loss in lactating dairy cows after administration of a 2.1 mg
deslorelin implant on Day 27 of pregnancy 134
7.3 Number of cows, percentages, adjusted odd ratios and 95% confidence interval
for the risk of pregnancy loss between Day 27 and Day 45 for cows with
accessory CL in the both groups 135
x


257
Roche JF. Fertility in cows after treatment with a prostaglandin analogue with or without
progesterone. JReprod Fert 1976a;46:341-345.
Roche JF. Retention rate in cows and heifers of intravaginal silastic coils impregnated
with progesterone. J Reprod Fert 1976b, 46:253-255.
Roche JF, Boland MP and McGeady TA. Reproductive wastage following artificial
insemination of heifers. Vet Rec 1981, 109: 401-404.
Rodgers RJ, OShea JD, Bruce NW. Morphometric analysis of the ovine corpus luteum. J
Anat 1984;138:757-769.
Rodgers RJ, OShea JD, Findlay JK. Progesterone production in vitro by small and large
ovine luteal cells. J Reprod Fert 1983;69:113-124.
Rodriguez TR, Fields MJ, Bums WC, Franke DE, Hentges JF, Thatcher WW, Warnick
AC. Breeding at a predetermined time in the bovine following PGF2a and GnRH. J Anim
Sci 1975, 40:188 (Abstr.).
Rosenberg M, Chun SY, Kaim M, Herz Z, Folman Y. The effect of GnRH administered
to dairy cows during oestrus on plasma LH and conception in relation to the time of
treatment and insemination. Anim Rep Sci 1991, 24:13-24.
Rosenberg M, Herz Z, Davidson M, Folman Y. Seasonal variations in postpartum plasma
progesterone levels and conception in primiparous and multiparous dairy cows. J Reprod
Fert 1977, 51:363-367.
Rowson LEA, Teruit R, Brand A. The use of prostaglandins for synchronization of
oestrus in cattle. J Reprod Fert 1972, 29:145 (abst.).
Rueda BR, Hendry IR, Hendry III WJ, Stormshak F, Slayden OD, Davis JS. Decreased
progesterone levels and progesterone receptor antagonists promote apoptotic cell death in
bovine luteal cells. Biol Reprod 2000;62:269-76.
Ryan DP, Prichard JF, Kopel E, Godke RA. Comparing early embryo mortality in dairy
cows during hot and cool seasons of the year. Theriogenology 1993;39:719-737.
Ryan DP, Snijders S, Yaakub H, OFarrell KJ. An evaluation of estrus synchronization
programs in reproductive management of dairy herds. J Anim Sci 1995;73:3687-3695.
Saacke RG, Dalton JC, Nadir S, Nebel RL, Bame JH. Relationship of seminal traits and
insemination time to fertilization rate and embryo quality. Anim Rep Sci 2000, 60-
61,663-677.
Sanchez T, Wehrman ME, Bergfeld EG, Peters KE, Kojima FN, Cupps AS, Mariscal V,
Kittok RJ, Rasby RJ, Kinder JE. Pregnancy rate is greater when the corpus luteum is


94
Day 10 of the estrous cycle and suppressed follicle development for variable intervals
(Ambrose et al., 1998; Bartolom et al., 2004). Plasma progesterone concentrations
during mid-diestrus were not affected by treatment and there was no evidence for a
deslorelin -induced decrease in luteal phase progesterone concentrations.
Pregnancy rates determined on Days 27 and 41 after TAI did not differ between
cows in the control and DESLORELIN 450 groups, but it was lower for cows receiving
DESLORELIN 750. It is not clear why the higher dose deslorelin suppressed fertility in
lactating dairy cows. When Bartolom et al. (2001) treated non-lactating dairy cows with
a deslorelin containing either 750 or 1000 pg, all cows ovulated and formed a CL after
treatment, and their CL supported similar, or even higher, plasma concentrations of
progesterone when compared to an injection of 100 pg of gonadorelin. Therefore, timing
of ovulation and progesterone support are not expected to be the cause for the lower PR
s
in cows treated with DESLORELIN 750. Higher concentrations of deslorelin are
expected to prolong the chronic phase of the GnRH agonist effect on the gonadotrophs,
which should result in a longer period of suppressed follicular development (D;Occhio et
al., 2000). Perhaps more prolonged suppression of gonadotropin support from the
pituitary when higher doses of deslorelin are used affects establishment of pregnancy,
although no clear mechanism is known. Previously, non-lactating dairy cows were treated
with either buserelin or deslorelin and more deslorelin cows became pregnant, although
the number of animals was extremely limited for assessment of pregnancy data (Ambrose
et al., 1998).
Pregnancy losses between 27 and 41 d after TAI were similar for control and
deslorelin cows, but cows in the DESLORELIN 450 treatment tended to have lower


10-6 Number of cows, adjusted odd ratios (AOR), 95% confidence interval
(Cl) and levels of significance for the risk of presence of a CL on Day 7
in cows in proestrus and with ovarian cysts treated with GnRH on
Day 0 adjusting for BCS and plasma P4 concentration on Day 0 214
10-7 Number of cows, adjusted odd ratios (AOR), 95% confidence interval
(Cl) and levels of significance for the risk of presence of a CL on Day 17
in cows in proestrus and with ovarian cysts treated with GnRH on
Day 0 adjusting for BCS 215
10-8 LSM and SEM for plasma P4 concentration on Day 0, 7, 10, and 17 for
cows in proestrus and with ovarian cysts for all 4 groups 216
10-9 Pregnancy rates on Day 30, 55, and 90 for cows in different stages/
abnormalities of the estrous cycle in all 4 groups 217
10-10 Number of cows, adjusted odd ratios (AOR), 95% confidence
interval (Cl) and levels of significance for the risk of pregnancy on
Day 30 in cows in proestrus and with ovarian cysts treated with
CIDR on Day 0 adjusting for BCS 218
10-11 Number of cows, adjusted odd ratios (AOR), 95% confidence
interval (Cl) and levels of significance for the risk of pregnancy on
Day 55 in cows in proestrus and with ovarian cysts treated with
CIDR on Day 0 adjusting for BCS 219
10-12 Number of cows, adjusted odd ratios (AOR), 95% confidence
interval (Cl) and levels of significance for the risk of pregnancy on
Day 90 in cows in proestrus and with ovarian cysts treated with
CIDR on Day 0 adjusting for BCS 220
xiii


CHAPTER 2
LITERATURE REVIEW
The Estrous Cycle and Follicular Dynamics
The bovine female experiences the first estrous cycle after puberty, and
subsequent ovulations occur every 17 to 24 days (Roberts, 1956). The estrous cycle may
be interrupted physiologically (pregnancy) or pathologically (anestrus, cystic ovarian
degeneration, pyometra) and then reinitiated during the postpartum period or after the
pathological conditions are resolved (Roberts, 1956). The estrous cycle is controlled by
endocrine changes in the central nervous system (CNS) and the hypothalamic-pituitary-
gonadal axis which cause physiological and clinical changes in the genital tract which are
divided in 4 stages as follows: estrus, metestrus, diestrus, and proestrus (Rathbone et al.,
2001). The onset of estrus coincides with a peak of follicle stimulating hormone (FSH),
maximum follicular growth, peak of estrogen levels and the gonadotropin releasing
hormone (GnRH) and luteinizing hormone (LH) surge. The LH surge lasts for 7 to 10
hours, estrus for 16.9 hours and ovulation occurs 25 hours after the LH surge (Day 0).
The corpus luteum forms from the ovulated follicle, P4 levels increase slowly from Day
1 to 4 during formation of the corpus luteum (metestrus), and then rapidly from Day 4 to
10 to reach a plateau and decrease to basal concentrations on Day 17 (diestrus). During
diestrus, FSH shows a wave-like pattern with peaks on Day 4, 8, and 12 parallel with
follicular growth. Between Days 16 and 17, the endometrium releases PGF2a, which
5


240
Dransfield MBG, Nebel RL, Pearson RE, Warnick LD. Timing of insemination for dairy
cows identified in estrus by a radiotelemetric estrus detection system. J Dairy Sci
1998;81:1874-1882.
Driancourt MA, Thatcher WW, Terqui M, Andrieu D. Dynamics of ovarian follicular
development in cattle during the estrous cycle, early pregnancy and in response
to PMSG. Domest Anim Endocrinol 1991;8:209-221.
Dunne LD, Diskin MG, Sreenan JM. Embryo and foetal loss in beef heifers between day
14 of gestation and full term. Anim Rep Sci 2000;58:39-44.
Eley RM, Thatcher WW, Bazer FW. Luteolytic effect of oestrone sulphate on cyclic beef
heifers. J Reprod Fert 1979;55:191-193.
El-Zarkouny SZ, Cartmill JA, Richardson AM, Medina Britos MA, Hensley BA,
Stevenson JS. Presynchronization of estrous cycle in lactating dairy cows with
Ovsynch+CIDR and resynchronization of repeat estms using the CIDR. J Anim Sci
2001;79,l(Suppl):249(abst.).
El-Zarkouny SZ, Hensley BA, Stevenson JS. Estms, ovarian and hormonal responses
after resynchronization with progesterone (P4) and estrogen in lactating dairy cows of
unknown pregnancy status. J Anim Sci 2002;80,l(Suppl):390(abst.).
Erb RE, Garverick HA, Randel RD, Brown BL, Callahan CJ. Profiles of reproductive
hormones associated with fertile and nonfertile inseminations of dairy cows.
Theriogenology 1976;5:227-242.
Espey LL. Current status of the hypothesis that mammalian ovulation is comparable to an
inflammatory reaction. Biol Reprod 1994;50:233-238.
Farin CE, Moeller CL, Sawyer HR, Gamboni F, Niswender GD. Morphometric analysis
of cells types in the ovine corpus luteum throughout the estrous cycle. Biol Reprod
1986;35:1299-1308.
Farin PW, Youngquist RS, Parfet JR, Garveick HA. Diagnosis of luteal and follicular
ovarian cysts in dairy cows by sector scan ultrasonography. Theriogenology
1990;34:633-642.
Farin PW, Youngquist RS, Parfet JR, Garverick HA. Diagnosis of luteal and follicular
ovarian cysts by palpation per rectum and linear-array ultrasonography in dairy cows. J
Am Vet Med Assoc 1992;200:1085-1089.
Ferguson JD, Galligan DT. Reproductive programs in dairy herds. Proc Centr Vet Conf
1993:161-178.


204
time of a nonpregnancy diagnosis at Day 30. This may further optimize fertility at the
TAI and warrants investigation.
Pregnancy losses for cows subjected to synchronization of ovulation and TAI
were 14.6% between Days 32 and 74 (Moreira et al., 2001). In a previous
resynchronization experiment (Bartolom et al., 2004), pregnancy losses between
ultrasonography on Day 30 and rectal palpation on Day 55 for cows in diestrus and TAI
were around 20%. In the present study, pregnancy losses between Days 30 and 55 for
cows in diestrus were 7.0 and 9.8%. All resynchronized cows also received a GnRH
treatment at 23 d after the previous service since they were resynchronized again if
detected nonpregnant at ultrasonography on Day 30. However, since all the cows were
treated, it was not possible to evaluate the effect of a GnRH treatment at 23 d after
previous service on pregnancy losses of cows that conceived to the resynchronized
service.
Cow detected in metestrus (5.6%) were subjected to a modified Heatsynch
protocol and pregnancy rates were lower than expected. Pregnancy rate was high when
cows in metestrus were inseminated at detected estrus on Day 9 after the Heatsynch
protocol (Bartolom et al., 2004). In addition, pregnancy rate was low for cows in
metestrus subjected to the Heatsynch protocol when a low proportion of cows were
inseminated at detected estrus on Day 9 (Bartolom et al., 2004). However, in the present
study, when the cows were TAI on Day 9, pregnancy rate was lower than expected. Cows
that were inseminated at 36 h after ECP (i.e., Day 9.5) achieved satisfactory pregnancy
rate, but the number of cows was insufficient to compare 24 vs 36 h or to compare the
overall pregnancy rate of cows in metestrus with cows in diestrus. The use of GnRH to


11
produced by the follicles peak on Days 6 to 7 of the estrous cycle and rise again on Day
16 around luteolysis (Glencross et al., 1973). Secretion of estrogen in the presence of
high levels of P4, also inhibits gonadotropin release from the anterior pituitary gland
(Kesner et al., 1981; Schallemberger et al., 1982). High levels of P4 during diestrus are
associated with a more rapid follicular turnover (three instead of two follicular waves),
and high fertility in next estrus (Savio et al., 1993; Wehrman et al., 1993; Ahmad et al.,
1997).
Progesterone concentration during diestrus can be affected by several factors such
as parity (heifers and cows), lactational stage, metabolic diseases, season, and size of the
follicle at the time of ovulation. Plasma P4 is lower during summer time in lactating dairy
cows (Rosemberg et al., 1977; De la Sota et al., 1993). In addition, lactating dairy cows
have an increased feed intake that results in increased liver blood flow and steroid
metabolism (Sangsritavong et al., 2002) that may result in lower P4 concentrations.
Induction of ovulation commonly used in lactating dairy cows may result in ovulation of
immature follicles and formation of a corpus luteum with reduced production of P4
(Vasconcelos et al., 1999, 2001; Peters and Pursley, 2003).
Clinical characteristics of diestrus at per rectum examination of the genital tract
are the presence of a corpus luteum determined by distortion in the shape of the ovary
and a line of demarcation between the corpus luteum and the rest of the ovarian stroma,
at least one graffian follicle, and moderate tonicity of the uterus (Zemjanis, 1962).
Specificity of per rectum examination of the ovaries to determine the presence of a
corpus luteum has been reported to be low (Ott et al., 1986). However, the positive
predictive value obtained by evaluating the proportion of cows in estrus after


4-6 Least squares means and SE for the first-wave largest follicles from Day 0
to Day 16 for Control () and DESLORELIN 450 () groups (P<0.01),
and second-wave largest follicle for control (), DESLORELES 450 (O)
groups (P<0.001) in Experiment 2 78
4-7 A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (), DESLORELIN 450
() groups (P<0.01) in Experiment 2. B. LSM and SE for the number of
Class III follicles after treatment for from Day 0 to Day 16 for Control ()
and DESLORELIN 450 () groups (P<0.01) in Experiment 2 79
4-8 Least squares means and SE for the largest follicle after PGF2a on Day 16
for cows in Control () and DESLORELIN 450 () groups (P<0.001)
in Experiment 2 80
4-9 Least squares means and SE for plasma P4 concentration on Days 7, Day 13
and Day 16 for Control () and DESLORELIN 750 () groups (P<0.07)
in Experiment 3 81
5-1 Diagram of activities during the study 101
5-2 Relationship between body condition score (BCS) at first postpartum
artificial insemination (AI) and frequency of anovulatory dairy cows 102
6-1 Interaction between administration of GnRH on Day 5 and Day 15 on
pregnancy rate at Day 27 (P<0.10) 118
7-1 Number of class 2 follicles on Day 27 and Day 45 of pregnancy in lactating
dairy cows after treatment with 2.1 mg deslorelin implant (P<0.01) 136
7-2 Number of class 3 follicles on Day 27 and Day 45 of pregnancy in lactating
dairy cows after treatment with 2.1 mg deslorelin implant (P<0.01) 137
7-3 Number of CL on Day 27 and Day 45 of pregnancy in lactating dairy cows
after treatment with 2.1 mg deslorelin implant (P<0.01) 138
7-4 Plasma P4 concentrations on Days 27 and 45 of pregnancy in lactating dairy
cows after treatment with 2.1 mg of deslorelin implant (P<0.10) 139
7-5 Interaction between group and presence of accessory CL at Day 45 on
pregnancy losses between Day 27 and Day 45 (P<0.03) 140
9-1 Protocols used for resynchronization depending upon stage of the estrous
cycle at the time of nonpregnancy diagnosis at Day 30 after insemination 176
xv


33
(Rodriguez et al., 1975; Lucy and Stevenson, 1986) since the preovulatory follicle does
not have time to produce enough estrogen (Lucy and Stevenson, 1986). Despite a slight
reduction in conception rates, the Ovsynch protocol maintains or increases pregnancy
rates when compared to different reproductive strategies because 100% of the cows are
inseminated without the need for estrus detection (Schmitt et al., 1996; Burke et al., 1996;
Pursley et al., 1997a; Pursley et al., 1997b; Fricke et al., 1998 Momcilovic et al., 1998;
Britt and Gaska., 1998; Stevenson et al., 1999).
Different variations of the Ovsynch protocol have been used. It is important to
maintain a 7 day interval from the first GnRH injection to PGF2oitreatment because a 6
day interval may not be enough for the GnRH-induced CL to be responsive to the
luteolytic effect of PGF2oc. On the other hand, an 8 day interval will allow time for more
cows to express estrus before synchronization of ovulation. Other modifications include
administration of the second GnRH 30 to 56 hours after PGF2a as well as TAI at different
times (0, 16, 24 or 32 hours) after the second GnRH. If the interval from PGF2a to
second GnRH injection is less than 48 hours, a smaller, less mature dominant follicle is
ovulated with reduced fertility. Nonlactating cows and heifers that received GnRH for
induction of ovulation 24 hours after PGF2a formed a CL that regressed earlier (Schmitt
et al., 1996), and this short luteal phase will compromise fertility (Peters and Pursley,
2002).
An interval from PGF2a to second GnRH of more than 48 hours may allow for a
higher proportion of cows to show estrus before GnRH injection and these cows need to
be inseminated at detected estrus in order to maintain fertility. In addition, GnRH
administration after the spontaneous LH surge may compromise corpus luteum


195
Corporation, Los Angeles, California) with an standard curve, references and duplicates
as previously described (Bartolom et al., 2004). The sensitivity of the assay was 0.1
ng/ml. The intra-assay coefficients of variation for the high, medium and low duplicate
samples were 5.7, 7.1 and 10.6% respectively. Intra and inter-assay coefficients of
variation for a luteal phase reference sample (7.14 0.69 ng/ml) were 9.6 and 10.4%,
respectively.
Ovarian Responses
For cows without a CL on Day 0, per rectum examination and ultrasonography
(linear scanner, 5 MHz transrectal probe, Aloka 500V, Wallingford, CT 06492) of the
genital tract were conducted on Days 0, 7, and 17 to determine the presence of CL on the
ovaries.
Pregnancy Diagnosis to Resynchronized Insemination
Pregnancy diagnosis by ultrasonography using a 5 MHz transrectal probe was
carried out on Day 30 1 after the resynchronized insemination. Cows were classified as
1) pregnant: embryo proper with heartbeats and surrounded by a fluid-filled cavity
representing the allantoic cavity as detected in the uterine lumen (Pierson and Ginther,
19984; Kastelic et al., 1988); 2) questionable pregnant: fluid in the uterine lumen and CL
but no embryo observed, or 3) not pregnant: cows that were inseminated at detected
estrus before ultrasonography or nonpregnant at ultrasonography. On Days 55 and 90,
pregnancy was reconfirmed by per rectum examination of the genital tract based on
previous described criteria (Zemjanis, 1962).


47
P450cc enzyme (Wiltbank et al., 1993). Administration of large doses of LH or hCG may
result in loss of LH receptors due to either inactivation within the plasma membrane or
internalization of the hormone-receptor complex by endocytic vesicles with posterior
degradation by lysosomal enzymes (Niswender and Nett, 1994). The luteal phase in the
cow last for 16 days approximately and then if a viable embryo is not present in the
uterus at Day 16 post ovulation the uterus secretes PGF2a and the CL regresses
(McCracken et al 1972). The role of the uterus in luteolysis was clear after CL lifespan
was prolonged following hysterectomy (Wiltbank and Casida 1956). If a viable embryo is
present in the uterus, this embryo will secrete IFN-t, inhibit PGF2 secretion which
results in CL maintenance and pregnancy recognition (Bazer et al., 1991).
The Role of Progesterone on Embryo Survival
Production of P4 by the CL supports embryo development, pregnancy
s
maintenance and embryo survival (Staples and Hansel, 1961). Production of P4 by the CL
is affected by the process of development and differentiation. The rate of growth of the
CL is self-limiting and related to the body size and numbers of corpora ltea (Reynolds et
al., 1994). Mitosis, cell turnover and luteal differentiation are rapid, and the early CL has
the same ability to produce P4 than a mature CL, but it produces less due to the small size
(Reynolds et al., 1994). In fact, concentrations of P4 in blood will depend on the amount
of steroidogenic tissue, blood flow and capacity of the steroidogenic tissue to produce P4
(Niswender et al., 2000). The rate of mitosis and cell turnover is stimulated by
proliferation of endothelial cells and the blood flow that supports luteinization (Reynolds,
1986). Fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) ad
angiogenic factors present in the CL contribute to luteinization. Granulosa cells produce


83
inseminated at detected estrus is higher than that in cows inseminated following TAI
(Santos et al., 2003). Therefore, TAI protocols such as the Ovsynch have improved
service rates, but have not increased conception rates or improved embryo survival in
cattle.
During the late stages of the estrous cycle, there is an upregulation of estradiol
receptors, oxytocin mRNA and prostaglandin F synthase mRNA in cyclic non-lactating
dairy cows (Thatcher et al., 2003). This is thought to be coordinated partially by ovarian
function, because it coincides with development of the second wave dominant follicle
and is associated with a high estrogenic environment. Thus, follicle development during
the period of maternal recognition of pregnancy may contribute to the development of
luteolytic signals that compromise embryo survival. In fact, high levels of estradiol
during late diestrus may have a negative impact on fertility (Pritchard et al., 1994).
/
Therefore, it is possible that attenuation of follicle development during this period might
reduce early pregnancy losses.
The GnRH agonist deslorelin induced ovulation in dairy cows when used as the
last GnRH injection in the Ovsynch protocol (Ambrose et al., 1998) or on Day 5 of the
estrous cycle (Rajamahendran et al., 1998). It induces a greater secretion of LH when
compared with Buserelin (Rajamahendran et al., 1998), which might improve CL
differentiation, resulting in higher mid luteal phase progesterone concentrations when
compared with a Buserelin injection (Ambrose et al., 1998). Cows with higher plasma
progesterone have more developed embryos that secrete more interferon-x (Mann and
Lamming, 2001). Moreover, chronic GnRH stimulation caused by deslorelin on the
gonadotrophs down-regulates GnRH receptors and creates a period of GnRH insensitivity


after previous AI and were assigned to different protocols for resynchronization.
Administration of hCG at estrus impaired CL function. Induction of ovulation with a
deslorelin implant suppressed follicular growth, and slightly improved CL function in
nonlactating cows and heifers and tended to reduce late embryonic losses in lactating
cows. Administration of GnRH on Days 5 and 15 after TAJ did not increase pregnancy
rate. A deslorelin implant administered at 27 days of pregnancy suppressed follicular
growth, induce accessory corpora ltea, increased progesterone (P4) production, an
reduce late embryonic mortality in cows that formed an accessory CL. Forty to 45% of
the cows detected not pregnant after AI presented a CL and the stage of the estrous cycle
influenced the response to different resynchronization protocols. Administration of
GnRH 23 days after AI resulted in 75% of cows with a CL at nonpregnancy diagnosis
and these cows obtained similar pregnancy rate after resynchronization of ovulation with
either GnRH or estradiol cypionate (ECP). For cows without a CL, administration of a P4
insert for resynchronization resulted in increased pregnancy rate in cows with ovarian
cysts. In summary, treatments affected ovarian function and indicated that may be
possible to increase pregnancy rate and reduce embryo mortality. Resynchronization
protocols assigned based on stages of the estrous cycle increased pregnancy rate.
xviii


100
the chronic stimulation by the GnRH agonist on suppressed fertility during the
subsequent insemination.


CHAPTER 10
RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION (TAI) IN
LACTATING DAIRY COWS, III: ADMINISTRATION OF GNRH 23 DAYS POST AI
AND ULTRASONOGRAPHY FOR NONPREGNANCY DIAGNOSIS ON DAY 30
Introduction
Systematic breeding programs including synchronization of ovulation and TAI
enhance reproductive efficiency and consequently increase the profitability of dairy herds
(Galligan, 1999). After first service, nonpregnant cows need to be detected as early as
possible, and then resynchronized and inseminated in a timely manner to maintain high
reproductive performance (Stevenson et al., 2003).
Different strategies for resynchronization of ovulation and TAJ include
synchronizing the follicular wave in advance by initiating the Ovsynch protocol 7 d
before nonpregnancy diagnosis (Moreira et al., 2000; Chebel et al., 2003; Fricke et al.,
2003). An alternative was not to induce follicular wave synchronization after previous
insemination but administer PGF2a to induce luteolysis at the time of nonpregnancy
diagnosis and then GnRH (Stevenson et al., 2003) or ECP (Chebel et al., 2003;
Bartolom et al., 2004) to induce ovulation for a TAI.
Synchronization of ovulation for TAI is more effective when the first dose of
GnRH in the Ovsynch protocol is able to induce follicular turnover (Vasconcelos et al.,
191


29
protocol was used during diestrus pregnancy rates were reduced (Beal, 1996). The
reduced fertility was probably due to the low level of P4 which was not able to inhibit LH
pulsatility like a natural CL, and, therefore, persistent follicles developed (Beal, 1996).
Even though fertility may have been improved after 9-day P4 treatments in combination
with PGF201 at P4 removal, the distribution of estrus and the interval to ovulation were not
synchronized enough to allow for TAI.
The understanding of follicular waves led to the use of GnRH to synchronize the
wave and increase the synchrony of induction of estrus (Thatcher et al., 1989;
Twagiramungu et al., 1992; Wolfenson et al., 1994). When GnRH was administered on
Day 12 of the estrous cycle, synchronization of estrus was tighter compared to cows
receiving PGF2a, and the dominant follicle was more estrogenic and dominant
(Wolfenson et al., 1994). The use of GnRH 7 days before PGF2 treatment avoids the
/
problem of persistent follicles that occur when estrus is synchronized with progestins or
any other method (Savio et al., 1993). Similarly, estrogen administration induces
follicular atresia and synchronizes the follicular wave allowing for a more control timing
of ovulation (Bo et al., 1995).
Induction of Ovulation and TAI
Induction of ovulation allows for TAI and eliminates the need for estrus detection.
In order for TAI to be successful, an LH surge must be induced, and such factors as
interval from LH surge to ovulation, insemination time, sperm transport and viability,
ovum viability, and number of accessory sperm per embryo need to be considered to
optimize fertilization rate and embryo quality (Saacke et al., 2000). The time from LH
surge to ovulation is 24 hours in dairy heifers (Thatcher and Chenault, 1975), sperm


Ovarian cysts
Proestrus
Figure 10.4. The effect of a GnRH treatment on Day 0 on the rise
of progesterone from Day 10 to Day 17 for cows in proestrus and
with ovarians cysts (Treatment*Stage*Day P<0.06)


128
The standard curve was composed of four tubes for total counts and coated tubes for
nonspecific binding and 100 p.1 of increasing concentrations of P4 (0.1, 0.25, 0.5, 2, 5, 10,
20 and 40 ng/ml). Serum from ovariectomized and estrus cows and from cows in diestrus
(low and high) were used as references. Every sixth sample was duplicated. Sensitivity of
the assay was 0.1 ng/ml. Intra and inter-assay coefficients of variation for the luteal phase
reference were 12.7 and 4.5 % respectively.
Statistic Analysis
Outcome (dependent) variables were pregnancy loss at Day 45 and Day 90
(yes/no), number of class 2 and class 3 follicles at Day 27 and Day 45, plasma P4
concentrations (ng/ml) at Day 27 and Day 45, follicular suppression at Day 45 (absence
of class 3 follicles) and accessory CL formation (Yes= number of CL at Day 45 greater
than the number of CL at Day 27). Explanatory (independent) variables were treatment

(deslorelin implant), parity (1,2, 3+), month (June, July and August), number of previous
services (0, 1, 2, 3+) and insemination type (standing estrus, pedometer response or timed
insemination). Baseline comparison for parity, previous service, insemination type and
month was conducted using Chi-squared (Proc FREQ, SAS). Data for the number of
follicles and CL were analyzed by the repeated measures (Proc MIXED, SAS) and
counted data procedures (Proc GENMOD, SAS), and the presence of follicular
suppression and accessory CL formation using Chi-square (Proc FREQ, SAS). Plasma P4
concentration was analyzed by analysis of variance (Proc GLM, SAS) controlling for
parity, season and previous services and insemination type. The backward elimination
procedure of Logistic Regression (Proc GENMOD SAS) was use to evaluate the effect of
treatment, follicular suppression, accessory CL formation, parity, previous services,


237
Cartmill JA, El-Zarkouny SZ, Hensley BA, Rozell TG, Smith JF, Stevenson JS. An
alternative AI breeding protocol for dairy cows exposed to elevated ambient temperatures
before or after calving or both. J Dairy Sci 2001;84:799-806.
Cavalieri J, Hepworth G, Parker KI, wright PJ, Macmillan KL. Effect of treatment with
progesterone and oestradiol when starting treatment with an intravaginal progesterone
releasing insert on ovarian follicular development and hormonal concentrations in
Holstein cows. Anim Rep Sci 2003;76:177-193.
Cavalieri J, Macmillan KL. Synchronization of oestrus and reproductive performance of
dairy cows following administration of oestradiol benzoate or gonadotrophin releasing
hormone during synchronized proestrus. Aust Vet J 2002;8:486-493.
Cavestany D, Cibils J, Freire A, Sastre A, Stevenson JS. Evaluation of two different
oestrus-synchronisation methods with timed artificial insemination and resynchronisation
of returns to oestrus in lactating Holsteins cows. Anim Reprod Sci 2003;77:141-155.
Cerri RLA, Galvao KN, Juchem SO, Chebel RC, Santos JEP. Timed AI (TAI) with
estradiol cypionate (ECP) or inseminated at detected estrus in lactating dairy cows. J
Dairy Sci 2003;86,l(Suppl):181(abst.).
Chebel RC, Cerri RLA, Galvao KN, Juchem SO, Santos JEP. Effect of rapid
resynchronization of nonpregnant cows with estradiol cypionate (ECP) and PGF2a on
pregnancy rates (PR) and pregnancy losses (PL) in lactating dairy cows. J Anim Sci
2003b; 81,1 (Suppl):182 (abst.).
Chebel R, Santos JEP, Cerri RLA, Juchem S, Galvao KN, Thatcher WW. Effect of
resynchronization with GnRH on Day 21 after artificial insemination on pregnancy rate
and pregnancy loss in lactating dairy cows. Theriogenology 2003a;60:1389-1399.
Chegini N, Lei ZM, Rao CV, Hansel W. Cellular distribution and cycle phase
dependance of gonadotropin and eicosanoid binding sites in bovine corpora. Biol Reprod
1991;45:506-513.
Chenault JR, Boucher JF, Dame KJ, Meyer JA, Wood-Follis SL. Intravaginal
progesterone insert to synchronize return to estrus of previously inseminated dairy cows.
J Dairy Sci 2003;86:2039-2049.
Chenault JR, Kratzer DD, Rzepkowski RA, Googwin MC. LH and FSH response of
Holstein heifers to fertirelin acetate, gonadorelin and buserelin. Theriogenology
1990;34:81-98.
Chenault JR, Thatcher WW, Kalra PS, Abrams RM, Wilcox CJ. Transitory changes in
plasma progestins, estradiol and luteinizing hormone approaching ovulation in the
bovine. J Dairy Sci 1975;58-70-77.


CHAPTER 4
INDUCTION OF OVULATION IN NONLACTATING DAIRY COWS AND HEIFERS
USING DIFFERENT DOSES OF A DESLORELIN IMPLANT
Introduction
Timed-artificial insemination is a common practice in dairy herds to improve
reproductive efficiency since all cows are inseminated independent of estrus detection
(Pursley et al., 1997a; Momcilovic et al., 1998). Timed-insemination requires
synchronization of follicular development, regression of the corpus luteum (CL), and
synchronous induction of ovulation (Pursley et al., 1995).
Human chorionic gonadototrophin (hCG; Schmitt et al., 1996), gonadotropin
releasing hormone (GnRH; Pursley et al., 1995), GnRH agonists (Schmitt et al., 1996),
and estradiol (Dailey et al., 19986) all have been used to induce ovulation. A synthetic
GnRH-agonist (deslorelin) implant was administered to synchronize ovulation, enhance
CL function, and delay the first-wave dominant follicle (Ambrose et al., 1998). Enhanced
CL function is associated with high blood concentrations of P4 early in the estrous cycle;
this stimulates embryo development, increases interferon-x production (Mann and
Lamming, 2001), and may contribute to improved fertility (Butler et al., 1996). In
addition, suppression of follicular development also may be beneficial to extend CL
lifespan (Fogwell et al., 1985).
The hypothesis of this study was that induction of ovulation with a deslorelin
implant would increase plasma P4 concentrations (due to enhanced development of the
CL) and suppress follicular development, without extending the interestrus interval. The
62


9-2 Distribution of interestrus intervals in 359 lactating dairy cows diagnosed
nonpregnant based on expression of estrus and insemination prior to
ultrasonography for pregnancy diagnosis 177
10-1 Experimental design for cows in diestrus, metestrus, or without a CL at
the time of a nonregnancy diagnosis (experimental Day 0) 221
10-2 The effect of GnRH treatment on Day 0 on the proportion of cows with
a corpus luteum at PGF2a treatment on Day 7 (P<0.11) 222
10-3 The effect of GnRH treatment on Day 0 on the proportion of cows with
a corpus luteum at on Day 17 (i.e., ovulation rate; P<0.12) 223
10-4 The effect of GnRH treatment on Day 0 on the rise of progesterone from
Day 10 to Day 17 for cows in proestrus and with ovarian cysts
(Treatment* Stage*Day P<0.06) 224
10-5 Pregnancy rates on Day 30, 55 and 90 in cows without a corpus luteum
on Day 0 (proestrus and ovarian cysts) and treated with a CIDR insert
(CIDR* Stage P<0.05) 225
xvi


104
Table 5.2. Effect of a deslorelin implant in a timed AI on ovarian activity of dairy
cows diagnosed non-pregnant on Day 27 after AI.
Treatment
p<2
Parameter3
GnRH
Deslorelin 450
Deslorelin 750
C1
C2
Follicles
(X SEM)
8.13a
11.08b
10.62b
0.001
0.68
5 to 9 mm
(0.77)
2.10a
( 1.06)
0.89b
( 0.74)
0.94b
0.001
0.83
10 to 19 mm
(0.18)
1.27a
( 0.24)
0.58b
(0.17)
0.58b
0.001
0.97
> 20 mm
(0.12)
0.21d
(0.16)
0.15
\
O H-
b P
0.12
0.10
Follicle > 10
(0.07)
83.1a
( 0.09)
60.2bf
( 0.06)
45.9bg
0.001
0.09
mm (%)
CL
72.9a
60.2e
49.5bf
0.001
0.10
(%)
Ovarian activity
92.2a
88.0C
73 4bd
0.002
0.03
(%)
Reinseminated
50.6a
27.3b
11.6C
0.001
0.001
<27d%
Reinsemination
22.2a
26.8b
25.9b
0.001
0.60
interval (d)
( 0.9)
(1.2)
(1.5)
a,b,c Superscripts within the same row differ (P < 0.01).
d,e Superscripts within the same row differ (P < 0.05).
f,s Superscripts within the same row tend to differ (P < 0.15).
1 GnRH = 100 |ug of gonadorelin; DESLORELIN 450 = biodegradable implant
containing 450 jig of deslorelin; DESLORELIN 750 = biodegradable implant containing
750 p.g of deslorelin.
2 Cl = control vs deslorelin; C2 = DESLORELIN 450 vs DESLORELIN 750.
3 Ovarian activity = presence of a CL or a follicle greater than 10 mm; Re-insemination
interval = interval between the first and second AI for those cows re-inseminated prior to
pregnancy diagnosis on Day 27 after the initial AI.


134
Table 7.2. Pregnancy loss between Days 27-45 and Days 45-90 for cows in both groups.
Response
Control Group
Implant Group
P value
%
N
%
N
Pregnancy Loss at Day 45
15.2
14/92
20.2
18/89
0.37
Pregnancy Loss at Day 90
11.0
8/77
7.1
5/70
0.48
Pregnancy Loss Total
23.9
22/92
25.8
23/89
0.76
Note: one cow in each group was culled between Day 45 and 90.


55
The objective of the present experiment was to evaluate the ability of hCG to enhance
CL development and increase P4 production when injected during estrus to extend the natural
LH surge.
Materials and Methods
Study Population
The experiment was conducted at the Dairy Research Unit of the University of
Florida. A group of twenty three multiparous nonlactating cycling dairy cows were kept in
free-stalls with access to a dry lot and fed 80 % sorghum silage and 20% lactating cow ration.
Study Design
All cows had a body condition score (BCS) of 3.5 to 4.5, and were treated with
GnRH (100 ug, im, Cystorelin) and PGF2a (25 mg, im, 2 doses 8 h apart, Lutalyse) 7 days
later. Estrus detection was conducted using the HeatWatch system considering cows in
estrus if they had a at least 3 mounts of at least 2 seconds duration within a 4 hours period.
Seventeen cows that showed estrus between 2 and 3 days after PGF2a were assigned
randomly to receive either hCG (3,000 IU, im, Chorulon, Intervet, Millsboro, DE 19966;
n=9) or saline (n=8) 8 to 10 hours after the onset of estrus (Figure 3.1).
Plasma P4 Concentrations
Blood samples were collected from 12 hours after treatment and every 12 hours
thereafter to determine plasma P4 concentration. Plasma P4 concentration was determined
with a single antibody radioimmunoassay procedure (Knickerbocker et al., 1986). Inter- and
intra-assay coefficients of variation were 8.5 and 23.5 % respectively. Sensitivity of the assay
was 0.15 ng/mL.


90
induced to ovulate with DESLORELIN 750 compared with DESLORELIN 450 (P <
0.01) and with controls (P < 0.05). On Day 41 after TAI, PR for control and deslorelin
were similar (32.4%; P = 0.49), but cows treated with DESLORELIN 750 had lower PR
than DESLORELIN 450 (P < 0.01) and controls (P < 0.05). Primiparous cows had higher
PR than multiparous cows on Days 27 (43.0 vs 30.3; P = 0.08) and 41 (39.4 vs 25.5%; P
= 0.04) after TAI. Interestingly, cyclicity prior to TAI had no effect on PR of dairy cows
(P > 0.50). At Day 27 (36.6 vs 37.3%) and Day 41 (32.4 vs 32.9%) cycling and
anovulatory cows had similar PR.
Although pregnancy loss between Days 27 and 41 after TAI almost doubled for
control cows (12.7%) compared to cows receiving deslorelin (6.9%), incorporation of a
deslorelin implant to induce ovulation in the Ovsynch protocol did not affect pregnancy
loss (P = 0.18) during the respective period. However, pregnancy loss tended to decrease
for cows receiving the DESLORELIN 450 compared to cows in the control group (P =
0.13).
Plasma P4 concentrations during mid cycle after TAI did not differ for control and
deslorelin cows (7.08 vs 7.44 ng/mL; P < 0.34). Although a similar proportion of cycling
and anovulatory cows had plasma P4 on Day 11 after TAI above 1.5 ng/mL, anovulatory
cows prior to TAI had higher concentrations of plasma P4 than cycling cows (7.86 vs
6.67 ng/mL; P = 0.02).
In addition to cyclicity, BCS at TAI also affected mid-cycle concentrations of
plasma P4. Cows in higher BCS also had higher plasma progesterone on Day 11, with a
steady increase as BCS changed from low to medium to high (6.40 vs 7.59 vs 7.84
ng/mL; P = 0.03). Plasma progesterone concentrations on Day 11 after TAI affected PR


113
Cows in Group 3 (GnRH treatment on both Day 5 and Day 15) had a lower
proportion of cows diagnosed not pregnant based on expression of estrus and
insemination before ultrasonography on Day 27, and these cows had an extended
interestrus interval compared with cows in Group 4 (untreated controls). The proportion
of cows diagnosed not pregnant based on expression of estrus and insemination before
ultrasonography on Day 27 was 46.2% (49/106) for Group 1, 42.3% (48/113) for Group
2, 25.6% (33/129) for Group 3, and 52.9% (55/104) for Group 4 (P<0.01). The interval
(days) between first insemination and insemination before ultrasonography on Day 27
was 22.5 (0.41) d for cows in Group 1, 22.7 d (0.47) for cows in Group 2, 23.4 d
(0.39) for cows in Group 3, and 21.5 d (0.41) for cows in Group 4 (P=0.05).
Discussion
In the present experiment, GnRH administration on either Day 5 or Day 15 did
not increase pregnancy rate on Day 27 or Day 55, and did not reduce pregnancy loss
between Day 27 and Day 55. Moreover, administration of GnRH on both Day 5 and Day
15 reduced pregnancy rate on Day 27 and Day 55.
Previous research showed that administration of GnRH, GnRH agonist or hCG
after insemination at detected estrus and at specific times coincident with the presence of
the dominant follicle/s of the first and second follicular waves may stimulate CL
function, induce accessory CL formation, increase P4 and reduce estrogen production
with a consequent positive effect on embryo survival (Thatcher et al., 2003).
Human chorionic gonadotropin administered on Day 7 after insemination induced
\
accessory CL, increase P4 concentration and reduce embryo mortality compared to cows
treated on Day 0, Day 14 or nontreated controls (Rajamahendran and Sianangama, 1992).


147
were evaluated for both protocols in cows at different stages/abnormalities of the estrous
cycle using logistic regression considering in the model group, stage, parity, season,
DIM, and inseminator. Cows in anestrus were not considered in the statistical analysis
due to the low number in this category.
For pregnancy rate on Day 27, parity, season, DIM and inseminator were not
included in the final model (P>0.15). Pregnancy rates, odd ratios, 95 % Cl (confidence
interval), and levels of significance for the risk of nonpregnancy on Day 27 are shown in
Table 8.5. The risk of nonpregnancy on Day 27 was higher for cows with ovarian cysts
subjected to the Heatsynch Group (OR=8.2, 95% Cl 1.0-65.8, P=0.04) and the risk of
nonpregnancy on Day 27 tended to decrease for cows in metestrus subjected to the
Heatsynch Group (OR=0.4, 95% Cl 0.1-1.0, P=0.06).
For pregnancy rate on Day 45, season and inseminator (P<0.15) were considered
in the final model. Pregnancy rates, adjusted odd ratios (AOR), 95 % Cl, and levels of
significance for the risk of nonpregnancy on Day 45 are shown in Table 8.6. The risk of
nonpregnancy on Day 45 tended to increase in cows with ovarian cysts subjected to the
Heatsynch Group (AOR=4.2, 95% 0=0.8-20.1, P=0.07) and decreased for cows in
metestrus subjected to the Heatsynch Group (AOR=0.3, 95% 0=0.1-0.8, P=0.01).
For pregnancy rate on Day 90, DIM was included in the final model (P<0.15).
Pregnancy rates, AOR, 95 % Cl, and levels of significance for the risk of nonpregnancy
on Days 90 are shown in Table 8.7. The risk of nonpregnancy on Day 90 tended to
increase for cows with ovarian cysts subjected to the Heatsynch Group (OR= 6.0; 95%
0=0.7-44.7, P=0.09), increase for cows in metestrus subjected to Ovsynch Group


223
Figure 10.3. The effect of a GnRH treatment on Day 0 on the
proportion of cows with a corpus luteum on Day 17 (i.e.,
ovulation rate; P<0.12)


238
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ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my advisers Dr. Louis F. Archbald
and Dr. William W. Thatcher. The knowledge, support, encouragement, and confidence
they provide me were endless and I will be grateful for the rest of my days. From Dr.
Archbald I received not only a great deal of knowledge in reproduction, but also a lot
support, enthusiasm and friendship. From Dr. Thatcher I also received his support and
confidence, his passion for teaching and learning, and his knowledge in reproductive
physiology. We had a very productive interaction, and it reflected in my progress as
professional and as human being. They gave me an outstanding education and friendship.
I am very grateful to all the members of my committee. The insights and
discussion with Dr. Jorge Hernandez about experimental design and statistics plus what I
learned in his class will be knowledge and expertise extremely useful for my future
career. My sincere gratitude goes to Dr. Carlos Risco, not only for sharing with me his
skills and enthusiasm in the area of bovine reproduction, but also for creating a nice
environment of discussion plus all the fun during meetings and seminars. I want to
express my appreciation to Dr. Lokenga Badinga for his scientific contributions, his
teaching in the area of nutrition, and why not, for sharing the passion of soccer.
IV


14
regression and a growing follicle and regressing CL in the ovaries (Zemjanis, 1962). At
ultrasonography, the uterine horn will appear highly echogenic and in the ovaries a
dominant follicle should be present (Pierson and Ginther 1984a; 1987). The CL may still
be observed since structural regression takes 48 h to be completed (Kastelic et al.,
1990b).
Follicular Dynamics
Ovarian follicles grow from the primordial stage (1th generation of granulosa
cells) to the preovulatory stage during a period of 180 days. A pool of follicles grow
slowly and with low rate of atresia until 7 to 8 generations of granulosa cells under the
influence of basal levels of gonadotropins (FSH and LH), activin, inhibin, follistatin,
growth factors (IGF-1, EGF, FGF, TGFa and b, TNFa), cytokines (EL-1, Interferon-y and
endothelin) and metabolic hormones (Findlay et al., 1996). At the 9th granulosa cells
generation and at 0.5 mm in diameter, a group of preantral follicles express FSH
receptors and under FSH stimulation grow rapidly and at higher rate of atresia (Findlay et
al., 1996) than occurs by apoptosis (Kaipia and Hsueh, 1997).
The occurrence of follicular waves in the ovaries during the estrous cycle was
reported using postmortem specimens of the reproductive tracts of heifers slaughtered at
different stages after estrus (Rajakoski, 1960). This was subsequently confirmed by
ultrasonography (Pierson and Ginther, 1984a). Studies using daily ultrasonography
described two or three follicular waves in the ovaries during the estrous cycle in cattle
(Savio et al., 1988; Sirois and Fortune, 1988, Knopf et al., 1989). Follicular waves in
primates were described by having a phase of recruitment, selection and dominance
(Goodman and Hodgen, 1983). These waves consisted of a group of 3 to 6 follicles that


79
Figure 7.A.
Figure 7.B.
Interval after treatment (d)
Figure 4.7. A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (), DESLORELIN 450 () groups
(P<0.01) in Experiment 2. B. LSM and SE for the number of Class III follicles after
treatment for from Day 0 to Day 16 for Control () and DESLORELIN 450 () groups
(P<0.01) in Experiment 2.


246
Jackson PS, Johnson CT, Furr BJ, Beattie JF. Influence of oestrus cycle on time of
oestrus following cloprostenol treatment in the bovine. Theriogenology 1979;12:153-167.
Kaipia A, Hsueh JW. Regulation of ovarian follicle atresia. Annu Rev Physiol
1997;59:349-363.
Kaltenbach CC, Dunn TG, Kiser TE, Corah LR, Akbar AM, Niswender GD. Release of
FSH and LH in beef heifers by synthetic gonadotropin releasing-hormone. J Anim Sci
1974;38:357-362.
Kaneko H, Todoroki J, Noguchi J, Kikuchi K, Mizoshita K, Kubota C, Yamakuchi H.
Perturbation of estradiol-feedback control of luteinizing hormone secretion by
immunoneutralization induces development of follicular cysts in cattle. Biol Reprod
2002;67:1840-1845.
Karten MJ, Rivier JE. Gonadotropin-releasing hormone analog design. Structure-function
studies towards the development of agonists and antagonists: rationale perspectives.
Endocr Rev 1986;7:44-66.
Kastelic JP, Curran S, Pierson RA, Ginther OJ. Ultrasonic evaluation of the bovine
conceptus. Theriogenology 1988;29:39-54.
Kastelic JP, Knopf L, Ginther OJ. Effect of day of prostaglandin F2a treatment on
selection and development of the ovulatory follicle in heifer. Anim Rep Sci 1990,
23:169-180.
Kastelic JP, Ko JCH, Ginther OJ. Suppression of dominant and subordinate ovarian
follicles by a proteinaceous fraction of follicular fluid in heifers. Theriogenology
1990a;34:499-509.
Kastelic JP, Northey DL, Ginther OJ. Spontaneous embryonic death on days 20 to 40 in
heifers. Theriogenology 1991;35:351-363.
Kastelic JP, Pierson RA, Ginther OJ. Ultrasonic morphology of CL and central luteal
cavities during the estrous cycle and early pregnancy in heifers. Theriogenology
1990;34:487-498.
Kesler DJ, Elmore RG, Brown EM, Garverick HA. Gonadotropin releasing hormone
treatment of dairy cows with ovarian cysts. I. Gross ovarian morphology and
endocrinology. Theriogenology 1981;16:207-217.
Kesler DJ, Garverick HA. Ovarian cysts in dairy cattle: a review. J Anim Sci
1982;55:1147-1159.


231
with ovarian cysts or anestrus will change, the use of GnRH and progesterone inserts
could be used to synchronize follicular waves and induce CL formation before a
nonpregnancy diagnosis by per rectum examination of the uterus (Day 35-40) and clinical
findings at ultrasonography and per rectum palpation of the reproductive tract can still be
used to select the most effective resynchronization of ovulation and timed insemination
protocol of nonpregnant cows.


201
Pregnancy rate on Days 30, 55 and 90 for cows in anestrus were 25.0% (1/4) in
the CIDR Group, 0.0% (0/3) in the GnRH+CIDR Group, 0.0% (0/2) in the Control
Group, and there were no cows in the GnRH Group. Overall pregnancy rate for anestrus
cows on Days 30, 55 and 90 was 11.1% (1/9).
Discussion
Nonpregnant lactating dairy cows treated with GnRH on Day 23 post
insemination, classified as diestrus at Day 30 (75 % of the cows) obtained similar
pregnancy rate after inducing luteolysis with PGF2a and resynchronizing ovulation for
TAI with either GnRH or ECP. In addition, there was no difference in pregnancy losses
between Days 30-55 and 55-90 between treatments for cows in diestrus.
Initiation of the Ovsynch protocol 7 d before pregnancy diagnosis was used to
resynchronize lactating dairy cows (Moreira et al., 2000; Chebel et al., 2003; Fricke et al.,
2003). Initiation of the Ovsynch on Day 20 after previous insemination for
resynchronization in cows receiving bST tended to reduce pregnancy rate to first
service in bST treated cows but not in control cows, and generated acceptable pregnancy
rate to second service compared with cows reinseminated at detected estrus (Moreira et
al., 2000). Initiation of the Ovsynch protocol on Day 19 after previous insemination did
not affect pregnancy rate to first service but generated a lower pregnancy rate to second
service compared with Ovsynch initiated on Days 26 or 33 (Fricke et al., 2003). In a
study designed to test the potential effect of initiating the Ovsynch at 21 d post
insemination on pregnancy rate and embryo survival, initiation of GnRH at Day 21 after
insemination did not reduce pregnancy rate to first service and resulted in a similar
pregnancy rate to second service when compared to initiating the Ovsynch protocol on


131
on Day 27 that formed accessory CL had a reduced pregnancy loss between Day 27 and
Day 45, while cows that did not form an accessory CL had increased pregnancy loss.
Indeed, cows with an accessory corpus luteum within deslorelin implant group had a
greater P4 concentration in plasma. In contrast, control cows with multiple CL had greater
pregnancy losses and plasma P4 was not elevated.
In the present study, administration of a deslorelin implant on Day 27 increased
the number of corpora ltea (45% of the cows formed accessory CL) and increased
plasma P4 concentration on Day 45 compared to untreated control cows. Those cows of
the deslorelin implant group with an accessory CL had greater concentration of plasma
P4 on Day 45 (10.7 ng/ml versus 6.3 ng/ml). Similarly, administration of hCG between
Day 29 and Day 59 of pregnancy in nonlactating beef cows induce an accessory CL
formation but in 82% of the cases (Bridges et al., 2000).
/
The number of class 2 follicles was reduced in the ovary containing the CL in
pregnant cows after Day 22 of gestation, and the size of the largest follicle was greater in
the ovary contralateral to the CL-bearing ovary (Pierson and Ginther 1987; Driancourt et
al., 1990; Thatcher et al., 1991). In the present study, follicular growth, as evaluated by
the number of class 2 and class 3 ovarian follicles, was reduced in cows treated with a
deslorelin implant compared to untreated control cows. The suppression of follicular
growth is in agreement with a previous study using the same deslorelin implant dose
during the early post partum (Mattos et al., 2001) or different dose of the implant for
induction of ovulation (Ambrose et al., 1998; Bartolom et al., 2004).
In the present study cows that were treated with a 2.1 mg of deslorelin implant on
Day 27 of pregnancy and formed an accessory CL had a reduced pregnancy loss at Day


167
Plasma P4 concentrations on Day 0 for cows in the Ovsynch (2.21 0.33 ng/ml),
Heatsynch (1.64 0.27 ng/ml), or GnRH+Ovsynch (1.78 0.31 ng/ml) groups were not
different. There were no differences in the proportion of cows with plasma P4
concentration lower than 2.0 ng/ml among cows in the Ovsynch (42/67, 62.9%),
Heatsynch (45/59, 76.3%), and GnRH+Ovsynch (47/63, 74.6%) groups. The positive
predictive value for per rectum examination and ultrasonography of the genital tract to
detect cows in metestrus was 70.1% (134/189).
Cows in Proestrus
The distribution of cows by the different variables between groups was not
different (P>0.15), except among inseminators (P<0.01, Table 9.8). Pregnancy rates on
Days 30, 55 and 90 were evaluated by logistic regression adjusting for inseminator.
Pregnancy rates on Days 30, 55 and 90 for cows in the Ovsynch (30.1, 23.9, 21.8%) and
GnRH+Ovsynch (28.0, 23.2, and 20.2 %) groups were not different (Table 9.9).
Pregnancy losses between Days 30-55 and 55-90 for cows in the Ovsynch (19.0 and
5.2%) and GnRH+Ovsynch (15.0 and 5.9%) groups were not different (Table 9.9).
Plasma P4 concentrations on Day 0 for cows in the Ovsynch (0.69+0.13 ng/ml)
and GnRH+Ovsynch (0.47+0.09 ng/ml) groups were not different. There was no
difference in the proportion of cows with plasma P4 concentration lower than 1.0 ng/ml in
the Ovsynch (70/87; 80%) and GnRH+Ovsynch (76/86; 88%) groups. The positive
predictive value for per rectum examination and ultrasonography of the genital tract to
detect cows in proestrus was 84.4% (146/173).


163
Pregnancy Diagnosis to Resynchronized Insemination
Pregnancy diagnosis was conducted on Day 30 (1) after the resynchronized
insemination using a linear ultrasound scanner with a 5 MHz transrectal probe (Aloka
500V, Wallingford, CT 06492). Cows were classified as 1) pregnant: embryo proper with
heartbeats and surrounded by a fluid-filled cavity representing the allantoic cavity as
detected in the uterine lumen (Pierson and Ginther, 1984; Kastelic et al., 1987); 2)
questionable pregnant: fluid in the uterine lumen and CL but no embryo observed, or 3)
nonpregnant: cows that were inseminated at detected estrus before ultrasonography or
nonpregnant at ultrasonography. On Days 55 and 90, pregnancy was reconfirmed by per
rectum examination of the genital tract based on previous described criteria (Zemjanis,
1962). The distribution of interestrus intervals was determined for cows that were
diagnosed not pregnant based on insemination at detected estrus prior to ultrasonography
on Day 30.
Statistical Analysis
Baseline comparisons of distributions for parity (1, 2, 3+), season, DIM
(quartiles) and inseminator (A, B, C) were carried out among groups within each
stage/abnormality of the estrous cycle to establish comparability of the groups using chi-
square test (Proc FREQ, SAS system). Pregnancy rate on Days 30, 55 and 90 and
pregnancy losses between Days 30-55 and 55-90 were compared using either Chi-square
test (Proc FREQ, SAS System) and multiple logistic regression whenever the baseline
data were different between groups and evaluate interactions (Proc GENMOD, SAS
system; Agresti, 1996). Plasma P4 concentrations were evaluated using the least squares
means analysis of variance (Proc GLM, SAS system) and proportions of cows with low


140
No Yes
Accessory CL
1: Control Deslorelin
Figure 7.5. Interaction between Group and presence of
accessory CL at Day 45 on pregnancy losses between Day 27
and Day 45 (P<0.03).


209
Table 10 .1. Distribution of cows by parity, DIM, inseminator and lot for cows in diestrus
subjected to the GnRH and ECP groups.
Variable
Group
P value
GnRH
ECP
%
N
%
N
Parity
1
48.6
107/220
42.6
92/216
0.42
2
21.8
48/220
23.1
50/216
3+
29.5
65/220
34.3
74/216
DIM
95-150
26.4
58/220
27.3
59/216
0.82
151-170
21.8
48/220
25.0
54/216
171-227
26.4
58/220
24.5
53/216
228-899
25.4
56/220
23.1
50/216
Inseminator
A
26.4
58/220
26.4
57/216
0.99
B
39.1
86/220
39.3
85/216
C
34.5
76/220
34.3
74/216
Lot
A
37.3
82/220
35.7
77/216
0.81
B
31.4
69/220
29.6
64/216
C
31.4
69/220
34.7
75/216


114
A treatment with a GnRH analog (buserelin, 8 |ig) on Day 7 after estrus in postpartum
beef cows increased the number of large luteal cells and size of the CL in cyclic cows,
increased the number of large luteal cells of the buserelin-induced CL, and P4 levels in
noncyclic cows (Twagiramungu et al., 1995). Treatment with hCG on Day 5 in dairy
heifers (Schmitt et al., 1996; Diaz et al., 1998) and in lactating dairy cows (Santos et al.,
2001) or on Day 6 in beef cows (Fricke et al., 1993) induced accessory CL formation and
increased P4 concentrations. Induction of accessory CL and increase in P4 levels were
higher in dairy heifers receiving hCG on Day 5 of the estrous cycle compared with a
GnRH analog (buserelin, 8 p.g; Schmitt et al., 1996). In addition, administration of hCG
on Day 5 in lactating dairy cows increased pregnancy rate (Santos et al., 2001).
Administration of a GnRH agonist (fertirelin acetate 100 (ig) between Days 11-14
after insemination increased P4 concentration 4-12 d later and reduced estrogen levels 8-
12 d in association with enhanced luteal function (Retmer et al., 1992a), and increased
pregnancy rate in beef heifers (Retmer et al., 1992b). A meta-analysis indicated an
increase in pregnancy rate for beef and dairy cows receiving GnRH between Days 11 and
14 (Peters et al., 2000). Administration of GnRH (100 (ig gonadorelin diacetate) on Day
11 after TAI in lactating dairy cows subjected to the Ovsynch protocol during a period of
mild heat stress increased plasma P4 concentrations and pregnancy rate (Williard et al.,
2003).
In the present study, treatment with GnRH either on Day 5 or Day 15 in lactating
dairy cows subjected to synchronization of ovulation and TAI did not increase pregnancy
rate on Days 27 and 55, and did not reduce the rate of pregnancy loss between Days 27
and 55. The lack of effect of GnRH either on Day 5 or Day 15 to increase pregnancy rate


149
El-Zarkouny et al., 2001; 2002). These programs require detection of estrus, and fertility
has been reported to be normal (Chenault et al., 2003; El-Zarkouny et al., 2002), or
decreased (El-Zarkouny et al., 2001; Cavestany et al., 2003) when compared to cows
reinseminated at a spontaneous estrus.
Since estrus expression is suppressed in lactating dairy cows of large dairy herds
and estrus detection is low, resynchronization of estrus needs to be combined with
resychronization of ovulation and TAI. Considering that the stage of the estrous cycle
affects the efficacy of timed-insemination protocols (Vasconcelos et al., 1999; Moreira et
al., 2000), different strategies for resynchronization and TAI have been considered.
Pregnancy rates were similar in cows with or without a CL at per rectum examination of
the genital tract and assigned to different protocols that combined estrus detection and
TAI (Bartolom et al., 2002). The use of ultrasonography permitted the initiation of
GnRH for resynchronization at 7 d prior to a nonpregnancy diagnosis at 27-28 d after the
previous insemination (Moreira et al., 2000; Chebel et al., 2003a) or to apply shortened
protocols PGF2a followed either by GnRH (Stevenson et al., 2003) or ECP (Bartolom et
al., 2003; Chebel et al., 2003b) to induce ovulation for TAI.
In the present study, stages and two common abnormalities of the estrous cycle
were evaluated when the resynchronization protocol was initiated. There was a
significant interaction between the Ovsynch and Heatsynch protocols and
stages/abnormalities of the estrous cycle. Cows in metestrus that underwent the
Heatsynch protocol had an increased pregnancy rate compared to the Ovsynch protocol.
It was reported previously that metestrus stage is not ideal to initiate the Ovsynch
protocol (Vasconcelos et al., 1999; Moreira et al., 2000). At this stage a follicular wave


144
Classification in Stages and Two Common Abnormalities of the Estrous Cycle
On Day 0, cows were classified according to different stages and two common
abnormalities of the estrous cycle combining per rectum examination (Zemjanis, 1962)
and ultrasonography of the genital tract (Perison and Ginther 1984b; 1987) to determine
ovarian structures and uterine characteristics (Table 8.1). Detection of a functional CL
included morphology at palpation (i.e., line of demarcation and distortion in the shape of
the ovary) and visualization at ultrasonography. Detection of follicles was based on
ultrasonography, uterine tonicity by per rectum palpation and edema using both
ultrasonography and rectal palpation. Diagnosis of ovarian cysts using per rectum
palpation combined with ultrasonography (Ribadu et al., 1994) was based in three clinical
findings: 1) multiple follicles greater than 18 mm; 2) absence of CL; and 3) lack of
uterine tonicity. Previous studies have used 15 to 20 mm as cut-off value (Zemjanis,
1962; Bierschwal, 1966; Archbald et al., 1991; Hamilton et al., 1995; Bartolom et al.,
2000; Silvia et al., 2002; Gumen et al., 2002; Hatler et al., 2003). Collectively, these
studies indicate that a large follicular size (e.g., 25 mm) is not the sole criteria for cysts
since multiple follicles reach ovulatory size indicating enough gonadotropin to support
follicular development but they fail to ovulate due to lack of GnRH/LH surge. The lack
of uterine tonicity (Zemjanis, 1962; Bierschwal, 1966) is the most important clinical
finding to make a diagnosis in a single exam since lack of tone indicates that regression
of the CL has occurred, the cow has failed to ovulate and the follicles continue to grow in
the absence of high progesterone concentrations.


247
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110
effects of GnRH on Day 5 or Day 15. Cows in Group 1 (n=214) were treated with GnRH
(100 gg, im) on Day 5 post TAI; cows in Group 2 (n=209) were treated with GnRH (100
gg, im) on Day 15 post TAI; cows in Group 3 (n=212) were treated with GnRH (100 gg,
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served as controls.
Since there was an interest in determining any seasonal effects of treatment on
reproduction in these cows, it was decided to divide the year into favorable and
nonfavorable times of fertility. Previous research (Al-Katanani et al., 1999) has shown
that pregnancy rates in Florida are higher during November, December, January and
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February was considered favorable, and March and April were considered unfavorable
for optimum pregnancy rates. In addition, there was also an interest in determining the
/
effect of parity and inseminator on pregnancy rates. Therefore, on Day 0, information
concerning parity, time of year (season), and inseminator were obtained.
Pregnancy Diagnosis
Pregnancy was determined 27 d after insemination using transrectal
ultrasonograhy (Aloka 500V, Wallingford, CT 06492, with a 5 MHz linear probe). Cows
were classified as: 1) pregnant: presence of an embryo exhibiting heartbeats and
surrounded by a fluid-filled cavity (allantoic) in the uterine lumen (Pierson and Ginther,
1984; Kastelic et al., 1988); 2) questionable pregnant: absence of an embryo, but
presence of intrauterine fluid and a corpus luteum on the ovaries; 3) not pregnant: cows
that were inseminated at detected estrus before ultrasonography on Day 27 or diagnosed
not pregnant by ultrasonography. On Day 55, pregnancy was reconfirmed by per rectum


154
Table 8.3. Distribution of cows by inseminator, and stages/abnormalities of the estrous
cycle for both Groups.
Variable
Ovsynch
% (N)
Heatsynch
% (N)
P value
Inseminator
A
7.2 (12/166)
6.0 (10/166)
0.01
B
37.9 (63/166)
27.1 (45/166)
C
9.6 (16/166)
7.2 (12/166)
D
16.9(28/166)
24.1 (40/166)
E
13 .2 (22/166)
27.7(46/166)
F
15.1 (25/166)
7.8 (13/176)
Stages/abnormalities
Diestrus
48.8 (81/166)
43.4(72/166)
0.85
Metestrus
13.2 (22/166)
16.2 (27/166)
Proestrus
21.1 (35/166)
22.9 (38/166)
Ovarian cysts
13.9 (23/166)
15.1 (25/166)
Anestrus
3.0(5/166)
2.4 (4/166)


263
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Vasconcelos JLM, Silcox RW, Lacerda JA, Pursley JR, Wiltbank MC. Pregnancy rate,
pregnancy loss, and response to heat stress after AI at 2 different times from ovulation in
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I want to extend my thanks to my home university for providing the leave during
years away and to the faculty members who were in charge of my classes. My friends in
Argentina, Julio y Silvana, provided unconditional support and help.
Finally, I want to express my love and gratitude to my family (parents, brothers,
sister, brother-in-law, sisters-in-law, nieces and nephews) for their continuous support,
and to my son and daughter, Santiago and Milagros, for their unconditional love.
vi


hco -- Control
Figure 3.2. Plasma P4 concentrations in nonlactating dairy cows after
induction of ovulation with human chorionic gonadotropin (hCG).


75
Figure 3.A.
Figure 3.B.
Figure 4.3. A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (), DESLORELIN 750 () and
DESLORELIN 1000 (A) groups in Experiment 1 (P<0.01). B. Least squares means and
SE for the number of class III follicles after treatment for from Day 0 to Day 16 for
Control (), DESLORELIN 750 () and DESLORELIN 1000 (A) groups in Experiment
1 (P<0.01).


91
of dairy cows on Day 27 (P < 0.001), and pregnant cows had higher plasma progesterone
than cows diagnosed non-pregnant (8.68 vs 6.51 ng/mL; P < 0.001).
During the re-synchronization period in cows diagnosed as non-pregnant to the
first TAI, PR decreased in cows receiving deslorelin compared to controls (12.8 vs
25.2%; P < 0.01). Similarly, cows receiving the higher implant dose had lower re
synchronization PR than those in the control and DESLORELIN 450 combined (9.6 vs
22.3%; P < 0.01).
Body condition score at first postpartum AI tended to affect re-synchronization
PR, with a gradual increase in PR as BCS changed from low to medium to high (11.5 vs
16.1 vs 25.0%; P = 0.12). Plasma progesterone on Day 11 after the TAI also tended to
affect PR during the re-synchronized cycle and cows with plasma concentrations of
progesterone equal to or greater than 1.5 ng/mL had higher PR than those with
/
progesterone below 1.5 ng/mL (19.4 vs 10.7%; P = 0.11).
In cows diagnosed as non-pregnant to the first TAJ and not re-inseminated by Day
27, follicular population was altered by treatment (Table 5.2). Deslorelin reduced the
population of medium and large size follicles and increased the number of follicles less
than 5 mm in diameter. The proportion of cows with a follicle equal to or greater than 10
mm on Day 27 after the initial TAI was higher for control cows than for cows induced to
ovulate with deslorelin (P < 0.001). Similarly, the proportion of non-pregnant and not re
inseminated cows with a CL on Day 27 was lower for deslorelin than controls (P <
0.001). The inhibitory effect of deslorelin on the proportion of non-pregnant cows with a
large follicle or a CL was more pronounced for DESLORELIN 750 than for
DESLORELIN 450. The proportion of cows with ovarian activity (follicle >10 mm or a


244
Greenstein JS, Murray RN, Foley FC. Effect of exogenous hormones on the reproductive
processes of the cycling dairy heifer. J Dairy Sci 1958;41:1834 (abstr.).
Guilbault LA, Dufour JJ, Thatcher WW, Drost M, Haibel GK. Ovarian follicular
development during early pregnancy in cattle. J Reprod Frtil 1986;78:127-135.
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progesterone exposure can induce development of follicular cysts. J Dairy Sci
2002;85:43-50.
Guzeloglu A, Ambrose JD, Kassa T, Diaz T, Thatcher MJ, Thatcher WW. Long-term
follicular dynamics and biochemical characteristics of dominant follicles in dairy cows
subjected to acute heat stress. Anim Rep Sci 2001;66:15-34.
Gwazdauskas FC. Effects of climate on reproduction in cattle. J Dairy Sci
1985;68:15681578.
Hamilton SA, Garverick HA, Keisler DH, Xu ZZ, Loos K, Youngquist RS, Salfen BE.
Characterization of ovarian follicular cysts and associated endocrine profiles in dairy
cows. Biol Reprod 1995;53:890-898.
Hammond J. The physiology of reproduction in the cow. Cambridge University Press,
London, 1927, pp 29-32.
Hansel W. Evaluation of methods for controlling the estrous cycle. In: Proc Conf on
estrous cycle control in domestic animals. USDA Misec. Publ. 1005,1965:1-16
Hansel W. Control of the ovarian cycle in cattle. A review. Aust Vet J. 1967 Oct; 43(10):
441-9.
Hansel W, Dowd JP. New concepts of the control of corpus luteum function. J Reprod
Fert 1986;78:755-768.
Hansel W, Malven PV, Black DL. Estrous cycle regulation in the bovine. J Anim Sci
1961;20:621-625.
Hansel W, Malven PV. Estrous cycle regulation in beef cattle by orally active
progestational agents. J Anim Sci 1960;19:1324 abst.
Hansel W, Seifart KH. Maintenance of luteal function in the cow. J Dairy Sci
1967;50:1948-1958.
Hasashi K, Miyamoto A. Angiotensin II interacts with prostaglandin F2a and endothelin-1
as a local luteolytic factor in the bovine corpus luteum in vitro. Biol Reprod
1999;60:1104-1109.


64
(5-9 mm) and Class III (> 10 mm) follicles (Macmillan and Thatcher, 1991) between Day
0 and Day 16, size of the largest follicle between Day 16 and Day 20 (mm), rate of
growth for largest follicle (mm/day) between Day 16 and Day 28 for DESLORELIN 750
and DESLORELIN 1000 groups, expression of estrus after Day 16 (yes/no), number of
Class II and Class III follicles between Day 16 and Day 28 for DESLORELIN 750 and
DESLORELIN 1000 groups, and response to Ovsynch for cows that did not ovulate after
Day 16.
Experiment 2
Fourteen nonlactating dairy cows were synchronized using GnRH on Day 9 and
two doses of PGF2a 12 h apart on Day 2. On Day 0, cows received either 100 ng of
GnRH (Control, n=8) or a 450 ng deslorelin implant (DESLORELIN 450, n=6). On Day
16, all cows received PGF2ct. Ovaries were evaluated by ultrasonography daily from Day
/
0 to ovulation and every other day to Day 28. Daily blood samples (to determine plasma
P4 concentrations) were collected from Day 0 until PGF2a administration on Day 16. The
Heat Watch system was used to monitor estrus. After Day 16, cows that did not show
estrus and did not ovulate (i.e., based upon ultrasonography) received 100 pg imGnRH at
the time the largest follicle reached 10 mm in diameter, 25 mg im PGF2a 7 d later, and
100 pg im GnRH 2 d after PGF2a (Ovsynch protocol). The outcome variables were
plasma P4 concentrations (ng/mL), first-wave largest follicle (mm), second-wave largest
follicle to Day 16 (mm), number of Class II and Class III follicles between Day 0 and
Day 16, size of the largest follicle (mm) between Day 16 and Day 28, expression of
estrus after Day 16 (yes/no), and response to Ovsynch for cows that did not ovulate after
Day 16.


137
* Control Implant
Figure 7.3. Number of CL on D 27 and D 45 of Pregnancy in
Lactating Dairy Cows after Treatment with 2.1 mg Deslorelin Implant
(Interaction P<0.01)


120
Table 6.2. Pregnancy rates on Days 27 and 55 and pregnancy losses (PL) between Days
27 and 55 for cows in all groups.
Variable
Group 1
%(N)
Group 2
%(N)
Group 3
%(N)
Group 4
%(N)
P
PRD 27
Pregnant
47.7(102/214)
43.5(91/209)
36.8(78/212)
44.4(87/196)
0.12
Open
49.5(106/214)
54.1(113/209)
60.8(129/212)
53.1(104/196)
QP
2.8(6/214)
2.4(5/209)
2.3(5/212)
2.5(5/196)
PL
Yes
19.6(20/102)
13.2(12/91)
23.0(18/78)
19.5(17/87)
0.40
No
80.4(82/102)
86.8(79/91)
77.0(60/78)
80.5(70/87)
PRD 55
Pregnant
39.3(84/214)
38.8(81/209)
28.3(60/212)
36.2(71/196)
0.06
Open
60.7(130/214)
61.2(128/209)
71.7(152/212)
63.8(125/196)
QP=questionable pregnant


142
2002). Another approach for resynchronization after nonpregnancy diagnosis by
ultrasonography was either to initiate the Ovsynch protocol 7 d before ultrasonography
(Moreira et al., 2000; Chebel et al., 2003a; Fricke et al., 2003) or to take advantage of a
natural resynchronization after previous service with application of shortened protocols
that used PGF2a to induce luteolysis and then GnRH (Stevenson et al., 2003) or ECP
(Chebel et al., 2003b; Bartolom et al., 2003) to induce ovulation. An alternative to
consider the stage of the estrous cycle at the time of initiation of the resynchronization
protocol could be to evaluate ovarian structures and uterine characteristics by per rectum
examination (Zemjanis, 1962) and ultrasonography (Pierson and Ginther, 1984b; 1987)
of the genital tract at the time of a nonpregnancy diagnosis.
The objective of this study was to compare pregnancy rates on Days 27, 45 and 90
and pregnancy losses in lactating dairy cows that were diagnosed not pregnant at
/
ultrasonography on Day 27 after a previous insemination and re-inseminated following
either Ovsynch or Heatsynch protocols. In addition, we evaluated the effect of stages
and two common abnormalities (ovarian cysts and anestrus) of the estrous cycle on
pregnancy rates for both treatments.
Materials and Methods
Study Population
The study was conducted from August to December 2001 in a large commercial
dairy with 3,200 milking cows. The herd is in north central Florida and is divided into 14
lots according to levels of production and stage of lactation, and cows from all lots were
included in the study. Cows were housed in free stall barns and dry lots and fed a total
mixed ration (TMR) three times daily. The TMR was formulated to meet or exceed


38
estriases can be considered after any of the PGF2a injections according to management
decisions. Since administration of PGF2oc 12 and 26 days after calving in cows with
abnormal parturition improved conception rate (Risco et al., 1994), the Presynch protocol
inititated early in postpartum may have an additional benefit on cows with uterine
infections.
Another practical alternative of using a presynchronization protocol is to assign
Ovsynch or Heatsynch protocol at the right stage of the estrous cycle based on the
findings at rectal palpation or ultrasonography. Cows with a corpus luteum could be
treated with PGF2a followed by insemination at detected estrus for 14 days and cows not
detected in estrus could be subjected to the Ovsynch protocol. Cows without a corpus
luteum and signs of estrus (follicles and high uterine tonicity) could be subjected to the
Ovsynch or Heatsynch protocol and cows without corpus luteum that are in metestrus,
anestrus or have ovarian cysts could receive GnRH to induce formation of a corpus
luteum and then submitted to the Ovsynch protocols 8 days later. Using this approach,
insemination at detected estrus is combined with TAI and reduces some costs and time
for resynchronization and targets protocols for TAI that are most appropriate for various
stages of the estrous cycle (Bartolom et al., 2002).
The Ovsynch protocol was effective in lactating dairy cows but not dairy heifers
(Pursley et al., 1995; 1997b). However, Schmitt et al., 1996 used a modified Ovsynch
protocol with dairy heifers (Ovsynch protocol with heifers detected in heat at < 39 hours
after PGF2a being inseminated) reported a reduced conception rate, but similar pregnancy
rate compared to a Select Synch protocol involving insemination at detected estrus. The
greater rate of follicular turnover in heifers results in premature expression of estrus.


222
Figure 10.2. The effect of a GnRH treatment on Day 0 on the
proportion of cows with a corpus luteum at PGF2a treatment on
Day 7 (P<0.11)


158
Table 8.7. Pregnancy rate on Day 90, adjusted odd ratios (AOR), 95 % confidence
interval (Cl) and levels of significance for the risk of nonpregnancy for cows in both
groups and at different stages and two common abnormalities of the estrous cycle.
Variable
Pregnancy Rate
N %
AOR
95 % Cl
P
Gtoud
Ovsynch
Stage/Abn.
Diestrus
17.5
14/80
Referent
Referent
Heatsynch
Diestrus
15.3
11/72
1.1
0.5-2.7
0.79
Ovsynch
Metestrus
0.0
0/22
4.9
1.1-36.9
0.03
Heatsynch
Metestrus
32.0
8/25
0.4
0.1-1.1
0.08
Ovsynch
Proestrus
11.4
4/35
1.8
0.5-5.9
0.35
Heatsynch
Proestrus
15.8
6/38
1.2
0.4-3.6
0.71
Ovsynch
Ovarian Cyst
21.7
5/23
0.7
0.2-2.4
0.62
Heatsynch
Ovarian Cyst
4.2
1/24
6.0
0.7-44.7
0.09
DIM
72-160
10.4
8/77
1.5
0.6-4.1
0.38
161-225
13.1
11/84
1.3
0.5-3.1
0.60
226-315
22.2
18/81
0.5
0.2-1.1
0.10
316-724
14.1
12/85
Referent
Referent
-


3
intervals between onset of estrus, luteinizing hormone (LH) surge and time of ovulation after
estrous synchronization protocols which led to development of TAI programs. Timed
artificial insemination increase pregnancy rate despite a slight decrease in conception rate
since 100% of the cows are inseminated (Stevenson, 2001).
After fertilization is achieved, increasing reproductive efficiency depends on embryo
and fetal survival. Several factors (genetics, endocrine and metabolic changes, infectious
diseases, climate) may be responsible for embryo and fetal mortality (Ayalon, 1978). Different
strategies to increase embryo and fetal survival include administration of hCG, GnRH or
GnRH agonists during diestrous and early pregnancy. The aim of these strategies is to
increase plasma P4 concentration, decrease follicular growth, estrogen production, and inhibit
PGF2a release and the luteolytic cascade (Hansel and Seifart, 1967; Schmitt et al., 1996).
The third step to maintain high reproductive efficiency is to detect and rapidly re
inseminate cows that did not conceive or failed to maintain a pregnancy (Stevenson et al.,
2003). Different methods such as nonreturn to estrus, plasma P4 concentrations, detection of
early pregnancy factors, ultrasonograpy and per rectum palpation of the genital tract have
been used to detect nonpregnant cows. At the time of pregnancy diagnosis, when cows are
diagnosed not pregnant, they should be re-inseminated as soon as possible. However, since
estrous detection is not very efficient, protocols for resynchronization and TAI need to be
applied. The clinical findings of the reproductive tract at the time of nonpregnancy diagnosis
allow classifying cows in different stages and abnormalities of the estrus cycle (Zemjanis,
1962; Pierson and Ginther, 1984b; 1987). This information can be used to implement different
resynchronization protocols because the stage of the estrous cycle affects the response to
hormonal treatments.


127
Estrus detection was carried out by visual observation and pedometers (Afikim system,
Kibbutz Afikim, 15148, Israel), and cows pregnant at Day 27 and inseminated at estrus
between Days 27 and 90 were also considered as a pregnancy loss.
Plasma Progesterone Concentration
Blood samples were collected in heparinized vacutainer tubes containing EDTA
(Vacutainer, BD, Franklin Lakes, NJ 07417) from coccygeal vessels by venipuncture
on Day 27 and Day 45, immediately placed on ice, and centrifuged within 24 hours (3000
rpm x 20 minutes). Plasma was separated and stored frozen at -20 C until assayed.
Plasma P4 concentrations were determined using a solid-phase, no-extraction RIA (Coat-
a-Count Progesterone, DPC Diagnostic Products Corporation, Los Angeles, California).
Accuracy of assay procedure was determined by measuring known quantities of
exogenous P4 (0.625, 1.25, 2.5, and 5.0 ng/ml) in cows plasma in seven different assays.
Recovery of added (X) versus measured (Y) P4 concentrations was described by linear
regression (Y=0.57+0.93X;R2=0.89). The regression intercept value (i.e. 0.57 ng/ml)
represented original P4 concentrations measured in a plasma pool prior to addition of
exogenous progesterone. Parallelism of logit plots between the displacement curves for
different volumes of a plasma pool containing 8 ng/ml of P4 (i.e. 25, 50 and 100 ul) and
standard P4 amounts (i.e., 0.1, 0.25, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 ng/ml) was tested by
homogeneity of regression analysis (Wilcox et al., 1990). The linear regression lines for
plasma and P4 standards were parallel (Yp=0.60-1.44X, R2=0.99,Ys=0.21-1.61X,
R2=0.99; where Yp and Ys=ln B/F and X=logio of assay volume or logio standard P4
concentrations, respectively). The slopes were not different.


207
(Archbald and Thatcher, 1992). In the present study, a second treatment of GnRH
(Ovsynch protocol) was used to induce ovulation. The benefit of the CIDR for 7 d
before induction of luteolysis was to elevate P4 concentrations, reduce the frequency of
LH pulses, and improve quality of a newly recruited dominant follicle for a subsequent
synchronization of ovulation. Progesterone treatment reduced plasma LH concentrations
and induced development of a new follicular wave in cows with follicular cysts (Calder et
al., 1999) and cows in anestrus (Nation et al., 2000). Therefore, it was expected that the
CIDR insert would increase ovulation and pregnancy rates after CIDR removal. In the
present study, the CIDR insert did not increase the proportion of CL or plasma P4
concentration on Day 17, but increased pregnancy rates in cows with ovarian cysts.
Body condition score in cows without a CL was considered in this study. Cows
with low BCS (<3.0) were less likely to have a CL on Day 7 and on Day 17 and had
reduced pregnancy rate. Cows that were diagnosed in this study as ovarian cysts based on
the described criteria but with low BCS may have not had follicular waves or a healthy
dominant follicle at the time of resynchronization of ovulation and it may explain the
reduced fertility. Since cows with ovarian cysts develop follicular waves (Hamilton et al.,
1995) perhaps cows with low BCS should be considered in anestrus or at least expect a
reduced fertility to protocols for synchronization of ovulation and TAI.
In summary, a GnRH treatment on Day 23 post insemination results in 75% of the
cows being in diestrus at a nopregnancy diagnosis on Day 30, and similar pregnancy rates
were obtained using either GnRH or ECP for resynchronization of ovulation and TAI. A
different strategy for turning over the follicle may be necessary for cows in metestrus. A
GnRH treatment on Day 0 increased the proportion of CL at Day 7 in cows with ovarian


28
Fertility to PGF2a-synchronized estrus is high compared to cows inseminated after
spontaneous estrus breeding 12 hours after onset of estrus for both cases (Moody and
Lauderdale, 1974). The levels of P4 exposure may have an effect on fertility since P4
values 2 days before the LH surge was higher in cows that conceived compared to cows
that failed to conceive (Erb et al., 1976), cows with higher P4 during the 12 days prior to
estrus had higher conception rate (Fonseca et al., 1983), and cows treated with PGF2otin
late luteal phase had higher fertility (Tanabe et al., 1984; Xu et al., 1997). However, other
reports did not find any relationship between the stage of the estrous cycle at the time of
treatment with PGF2a and fertility (Stevenson et al., 1984).
Combinations of Progesterone or Progestins and PGF2a
Progesterone supplementation was combined with PGF2a in order to reduce the
length of treatment from 7 to 9 days (Beal, 1996), and elevate P4 concentrations before
/
PGF2a administration (Xu et al 1997).
A combination of PGF2a and MGA is commonly used in beef and dairy heifers to
synchronize estrus. It includes administration of MGA for 14 days and heifers express
estrus within 2 to 6 days after MGA removal. Then, heifers are treated with PGF2a 17
days after MGA removal when heifers are in late luteal phase obtaining high fertility after
insemination at detected estrus (Patterson et al., 1989). Several studies using a
combination of progestins and PGF2a showed increased (Xu et al., 1997), acceptable
(Roche,1976; Folman et al., 1990, Ryan et al., 1995; Whittier et al., 1986; Beal and Good
1986) or reduced fertility (Xu et al., 1997).
The use of MGA for 7 days combined with a dose of PGF2a on Day 7 was applied
in order to reduce the length of the treatment (Beal and Good 1986). However, when this


Moreira F, Orlandi C, Risco CA, Lopes F, Mattos R, Thatcher WW. Effects of
presynchronization and bovine somatotropin on pregnancy rates to a timed artificial
insemination protocol in lactating dairy cows. J Dairy Sci 2001;84:1646-1659.
Moreira F, Risco C, Pires MFA, Ambrose JD, Drost M, DeLorenzo M, Thatcher WW.
Effect of body condition on reproductive efficiency of lactating dairy cows receiving a
timed insemination. Theriogenology 53:1305-1319.
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somatotropin in lactating dairy cows receiving timed artificial insemination. J Dairy Sci
2000;83:1237-1247.
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Am J Vet Res 1983;44:888-890.
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controlling the function and life span of the corpus luteum. Physiologycal Reviews
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10
P450 mRNA were only affected when PGF2 was administered in mid-luteal phase (Day
10) but not early in the estrous cycle (Levy et al., 2000).
A clinical sign that may be observed during the first day of metestrus, especially
in heifers and rarely in cows, is a bloody mucous discharge caused by the increased blood
supply as an effect of estrogen during estrus. The increased blood supply results in
uterine edema during metestrus which is one the clinical characteristics at per rectum
examination of the genital tract (Zemjanis, 1962). Another palpable characteristic is the
absence of significant structures in the ovaries and sometimes the presence of an
ovulation depression (Zemjanis, 1962). At ultrasonography, carry-over follicles (did not
ovulate at estrus) may be present, but usually nonsignificant structures are observed. The
uterine horns appear very heteregoneous and a hypoechogenic line can be observed in the
vascular layer of the submucosa reflecting the edema of the uterus (Pierson and Ginther,
1984a; 1987).
Diestrus
The diestrus stage begins on Day 5 (mature CL) and ends with CL regression
between Days 16 and 18. It is characterized by high levels of plasma P4 concentration
secreted by the CL (Schams et al., 1977). Corpus luteum development and differentiation
are supported by tonic secretion of LH from the anterior pituitatry gland (Hansel and
Seifart, 1967). The CL secretes P4 to support embryo development and at the same time
P4 inhibits the amplitude and frequency of LH pulses in a negative feed back mechanism
(Roberson et al., 1989) influencing follicular growth. Follicles grow in a wave pattern
during diestrus (Rajakoski, 1960). Using ultrasonography, two and three follicular waves
have been described (Savio et al., 1988; Sirois ad Fortune., 1988), and oestradiol-17(3


89
which ranged from 2.50 to 4.00, and within each BCS group the frequency of
anovulatory/anestrous was calculated. The number of cows within each BCS group
ranged from 21 to 172 animals. Body condition score was then plotted against the
frequency of anovulatory/anestrous cows using the regression procedure of MINITAB
(MINITAB, 2000) to determine the best-fitted line. Treatment differences with P < 0.05
were considered significant.
Results
Plasma progesterone concentration prior to TAI was not determined in 2 of the
593 cows. Of the remaining 591 cows, 75 were not cycling (12.7%) by 58 DIM, and a
greater proportion of primiparous cows were anovulatory compared to multiparous cows
(18.2 vs 7.4; P < 0.001). Body condition score at AI (P < 0.0001), but not changes in
BCS from Day 30 postpartum to AI (P = 0.16) affected the proportion of cyclic cows in
the first 58 DIM. A linear relationship between BCS at first postpartum AI and the
incidence of anovulatory cows was established; anovulatory cows decreased 3.13% for
every 0.25 unit increase in BCS between 2.50 and 4.00 (Figure 5.2).
Frequency of cycling cows prior to TAI was 88.2, 87.9, and 85.0% for cows
treated with GnRH, DESLORELIN 450 and DESLORELIN 750, respectively (P = 0.90).
Subsequent to TAI, the proportion of cows with plasma progesterone higher than 1.5
ng/mL on Day 11 was 94.0%; and 92.9% of the anovulatory cows resumed cyclicity.
Resumption of cyclicity in anovulatory cows was unaffected by treatment, BCS, or
parity.
Pregnancy rates on Day 27 after TAI did not differ between control cows and
cows treated with DESLORELIN 450 (Table 5.1). However, PR was lower for cows


2
(Fetrow et al., 1990). When estrous detection rates of 35 %, 55 % and 75%, and conception
rates of 42 %, 50 % and 58% were used to model reproductive efficiency, the estrous
detection rate had a major impact (Oltenacu et al., 1981).
Pregnancy rate can be increased by either increasing conception rate or estrous
detection rate. Estrous detection and conception are affected by several factors. Some of them
include level of production, management, nutrition, health problems, climate, and genetic
selection. Even though conception rate is slightly reduced in intensively-managed dairy herds,
estrous detection has been the most affected variable (Nebel et al., 1987). The length and
intensity of estrus have been reduced throughout the years because of genetic selection, level
of production and intensive management (Washburn et al., 2002). Consequently, estrous
detection is a difficult task that usually becomes compromised. Estrous detection rate by
visual observation is very low (-50%) in both intensively-managed dairy herds (King et al.,
1976) and in small herds on pasture-based systems (Williamson et al., 1972). This low estrous
detection rate can have a devastating effect on reproductive performance (Heersche and
Nebel, 1994). In addition, estrous detection could be inaccurate, and results in the
insemination of cows that are not in estrus, which further contributes to reduced conception
rate (Heersche and Nebel, 1994).
Even though estrous synchronization programs were developed to concentrate
expression of estrus in 3 to 4 days to make detection of estrus more efficient, estrous
detection rates continue to be less than 50% in dairy herds, and 5 % to 30% of the cows are
not in estrus at time of insemination (Senger, 1994). Synchronization of ovulation in a group
of cows allows insemination by appointment (timed artificial insemination, TAI) and
eliminates the need for estrous detection. Several studies provided information regarding the


251
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Momcilovic D, Archbald LF, Walters A, Tran T, Kelbert D, Risco CA, Thatcher WW.
Reproductive performance of lactating dairy cows treated with gonadotrophin-releasing
hormone (GnRH) and/or prostaglandin F2a (PGF2a) for synchronization of estrus and
ovulation. Theriogenology 1998;50:1131-1139.
Monniaux D, Monget P, Besnard N, Huet C, Pisselet C. Growth factors and antral
follicular development in domestic ruminants. Theriogenology 1997;47:3-12.
Moody EL, Lauderdale JW. Fertility of cattle following PGF2a controlled ovulation. J
Anim Sci 1977;45 (Suppl. 1): 189 abst.
Moreira F, de la Sota RL, Diaz T, Thatcher WW. Effect of day of the estrous cycle at the
initiation of a timed artificial insemination protocol on reproductive responses in dairy
heifers. J Anim Sci 2000;78:1568-1576.


21
An intravaginal insert consisting of a stainless steel spiral coated with silicone
rubber and impregnated with P4 (Progesterone Releasing Intravaginal Device, PRID,
CEVA, Paris, France) was developed (Mauer et al., 1975) and shown to suppress estrus
and ovulation in heifers and cows (Roche et al., 1976). The CIDR* intravaginal insert is
produced by coating a nylon spine with silicon-based elastomer containing 1.9 g (10%
w/w) of P4 (Macmillan et al., 1991). The retention rates of the CIDR insert are 99% in
heifers when used for 4-15 day periods and 98% in cows when used for a 4-7 day period
(Macmillan et al., 1991). Currently in the USA, the CIDR insert contains 1.38 g of P4
(CIDR-B, Pharmacia, Kalamazoo, MI, 49001) and is approved for use in lactating dairy
cows for resynchronization of estrus administered from Day 14 to Day 21 after
insemination.
GnRH and GnRH Agonists
Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted by the
hypothalamus into hypophyseal portal system and stimulates secretion of gonadotropins
from the anterior pituitary gland (Goodman, 1988). The half-life of GnRH is very short
and is cleared from the circulation in 4 to 7 minutes (Redding et al., 1972). GnRH and
GnRH analogue have been used to stimulate follicular growth, induce follicular turnover,
or ovulation in cattle according to the stage of follicular development at the time of
treatment (Macmillan and Thatcher, 1991, Thatcher et al., 1993). GnRH (native GnRH)
includes gonadorelin diacetate tetrahydrate (Cystorelin; Fertagyl) and gonadorelin
hydrochloride (Factrel). GnRH analogues and agonists include buserelin (D-
Ser(tBu)6,Pro9 Net; Receptal), fertirelin acetate (Ovulyse) and deslorelin ((pGlu-His-
Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-NEHt; Ovuplant). The chemical modification of


53
Therefore, cows develop more large follicles during the summer (Wolfenson et al., 1995)
that can be detrimental for embryo survival.


69
five cows in DESLORELIN 450 Group where subjected to the Ovsynch protocol and
three of them ovulated in response to the second GnRH.
Experiment 3
Overall plasma P4 concentration tended (P<0.07) to be higher for heifers in
DESLORELIN 750 Group (12.9 + 0.7 ng/mL) compared to that of heifers in the Control
Group (11.1 0.7 ng/mL; Figure 4.9). Within the 6-d period after PGF2, estrus
expression was higher (P<0.01) for heifers in Control group (22/22) compared to that for
heifers in DESLORELIN 750 Group (12/22). Among the 12 heifers that did not show
estrus, 11 of them did not have follicles larger than 10 mm in diameter.
Discussion
In the present study, ovulation was synchronized with administration of either
gonadorelin diacetate (GnRH) or a deslorelin implant (GnRH agonist) in nonlactating
/
dairy cows (Experiments 1 and 2) and dairy heifers (Experiment 3). Clearly, deslorelin
implants did not suppress plasma P4 concentrations; rather it increased P4 concentrations
on certain days in late diestrus. However, these increases were sporadic and of minimal
magnitude for all three experiments. The results are in agreement with a previous report
using a 700 jig deslorelin implant to induce ovulation in nonlactating dairy cows
(Ambrose et al., 1998). Size of the CL was not evaluated and there was no effect of
treatment on the number of CL. In a previous study, it was suggested that a deslorelin
implant may increase differentiation and development of the CL, since the number and
size of the CL had no effect on plasma P4 concentration (Ambrose et al., 1998).
Luteinizing hormone (LH) surge and pulsatile secretion from the pituitary gland
are responsible for ovulation, CL development and CL differentiation (Peters et al.,


101
Parturition
1
BCS
BS
BS
BCS
PGF2o PGF2 GnRH PGF2 GnRH or DES TAI BS US + BCS Palpation
Day 0 30 3 44 58 65.5 67.5 68 79 95 109
GnRH + US PGF2dUS GnRH TAI Palpation
1 1 1 1 1
Day 95 102 104 105 143
Figure 5.1. Diagram of activities during the study. BCS=body condition score;
BS=blood samples; DES=deslorelin implant; GnRH=gonadorelin;
TAI= timed-artificial insmeination; US=ultrasound


112
there was no difference in pregnancy rate on Day 27 and pregnancy loss among groups
(Table 6.2).
On Day 27, 2.5% (21/831) of cows were diagnosed as questionable pregnant
(Table 6.2). On Day 55, 76.2% (16/21) of the cows classified as questionable pregnant
were determined to be not pregnant, and 23.8% (5/21) were confirmed pregnant (2 in
Group 1, 2 in Group 2, and one in Group 4).
In the multivariate analysis, the interaction between treatment of cows with GnRH
on Day 5 and Day 15 on pregnancy rate tended to be significant (P<0.10). Administration
of GnRH on Day 15 tended to decrease pregnancy rate on Day 27 only in cows
previously treated with GnRH on Day 5 (Figure 6.1). In addition, there was a significant
(P<0.01) effect of season on pregnancy rate. Therefore, the final model to evaluate
pregnancy rates on Day 27, 55 and pregnancy loss between Days 27 and 55 included
/
group and season.
The number of cows pregnant on Day 27 and Day 55, the risk of nonpregnancy,
adjusted odd ratios (AOR), 95% confidence interval (Cl) and level of significance for all
groups adjusting for season are shown in Tables 6.3 and 6.4, respectively. There was a
reduction in pregnancy rate on Day 27 (AOR=1.4, 95% Cl 0.9-2.1, P<0.03) and on Day
55 (AOR=1.4, 95% Cl 1.0-2.2, P<0.01) for cows in Group 3.
The number of cows which lost a pregnancy between Days 27 and 55, the risk of
pregnancy loss, adjusted odd ratios, 95% confidence interval and level of significance for
all groups adjusting for season are shown in Table 6.5. There was no difference in the
risk of pregnancy loss between Days 27 and 55 among groups.


139
DO
a
a
o
DO
O
Days of pregnancy
Control Implant
Figure 7.4. Plasma P4 Concentration on Days 27 and 45 of
Pregnancy in Lactating Dairy Cows after Treatment with 2.1
mg Deslorelin Implant (Interaction P<0.10; Day 45: P<0.01).


Ill
palpation of the uterus using previously described techniques and criteria (Zemjanis,
1962).
Statistical Analysis
Baseline comparisons for parity (1, 2, 3+), season (November to February and
March/April), and inseminator (A, B, C) were carried out to establish comparability of
groups using a chi-square test. A univariate analysis for the effect of group on pregnancy
rates at Day 27 and Day 55 and pregnancy loss between Days 27 and 55 was conducted
using chi-square test. A multivariate analysis was conducted to evaluate the main effect
of GnRH on Day 5 and Day 15 and the interaction between GnRH on Day 5 and Day 15
on pregnancy rate and pregnancy loss. The model included the main effect of GnRH on
Day 5, the main effect of GnRH on Day 15, the interaction between both GnRH on Day 5
and Day 15, as well as parity, season and inseminator to adjust for residual confounding
/
using the backward elimination procedure (Agresti, 1996) in a logistic regression model
(Proc GENMOD, SAS; SAS, 2001). Variables remained in the model at P<0.15. The
proportion of cows diagnosed not pregnant based on insemination before Day 27 was
analyzed by chi-square. The interval (days) from first service to next insemination was
analyzed by analysis of variance (Wilcox et al., 1990) using Proc GLM of SAS.
Differences between groups with a P value < 0.05 were considered significant.
Results
There was no difference in the distributions of cows by parity, season and
inseminator between all 4 groups (Table 6.1). In the univariate analysis, pregnancy rates
on Day 55 tended (P<0.06) to be lower in cows in Group 3 compared to Group 4, but


155
Table 8.4. Pregnancy rate on Days 27, 45, and 90 and pregnancy losses between Days 27-
45 and 45-90 for cows in the Ovsynch and Heatsynch Groups.
Group
Variable
Ovsynch
% (N)
Heatsynch
% (N)
Pregnancy rate
Day 27
25.2 (32/127)
25.8 (34/132)
Day 45
17.5 (29/166)
19.9 (33/166)
Day 90
13.9 (23/165)
16.1 (26/162)
Pregnancy losses
Day 27-45
25.0 (8/32)
14.7 (5/34)
Day 45-90
17.9(5/28)
10.3 (3/29)


57
In addition, on Day 16, CL tended to be smaller (P<0.08) in cows treated with hCG
(6.96 cm3 0.71) compared to control cows (8.82 cm3 0.76; Figure 3.3).
Discussion
Administration of hCG 10 hours after onset of estrus in nonlactating dairy cows
resulted in a reduction in the size of the CL and plasma P4 concentration. Even though basal
levels of LH can support the development of the CL, LH pulsatility is required for
differentiation and development of the CL during early stages of the luteal phase (Peters et al.,
1994). In the present study, hCG administered during or right after the spontaneous LH surge
may have produced an excess of LH and LH-like activity and negatively affected mitosis and
differentiation of small luteal cells and loss of response to the luteotrophic stimulus of LH. In
fact, administration of hCG in rats induced loss of LH receptors and desensitization of
adenylate cyclase in the ovary (Conti et al., 1976).
s
A previous study reported that administration of hCG (1,000 and 2,000 IU) 12 hours
after estrus increases CL size, plasma P4 concentrations. However, the increment in CL size in
cows treated 12 hours after onset of estrus was less than cows that were treated on Day 3
(Veenhuizen et al., 1972). Using the same treatment, Wagner et al. (1973) reported an
increased in pregnancy rate but a decrease when hCG was administered on Day 3 after first
service. Cows submitted to estrus synchronization using 2 doses of PGF2a 14 days apart and
then treated with GnRH 72 hours after the second dose of PGF2a had reduced P4 levels during
the luteal phase (Lucy and Stevenson, 1986). It was concluded that the administration ofhCG
during or right after the spontaneous LH surge with a high LH and LH-like exposure in the
peri-ovulatory period did not appear to cause a hyper-stimulation of CL function.


208
cysts, and the inclusion of a CIDR insert increased pregnancy rate in cows with ovarian
cysts but reduced pregnancy rate in proestrus cows.


196
Statistical Analysis
For cows in diestrus, baseline comparisons for parity (1,2, 3+), DIM (quartiles),
inseminator (A, B, C), and lot (A=lots with cooling system, B= first calf heifers in
outside lots with shade and ponds, C=multiparous cows in outside lots with shade and
ponds) were carried out to establish comparability between groups using the Chi-square
test (Proc FREQ, SAS System). Pregnancy rates on Days 30, 55 and 90 and pregnancy
losses between Days 30-55 and 55-90 for cows in diestrus were compared using the Chi-
square test. Pregnancy rates for cows in metestrus were not statistically evaluated since
they were assigned to one treatment due to the low number of cows in this category.
For cows without a CL (proestrus, ovarian cysts and anestrus), baseline
comparisons for parity, DIM, inseminator, lot, body condition score (BCS, > or < 3.0)
and stages/abnormalities of the estrous cycle (proestrus, ovarian cysts, anestrus) were
carried out to establish comparability among groups using Chi-square test (Proc FREQ,
SAS System). Since there was an interest in evaluating interactions between treatments
and stages/abnormalities of the estrous cycle, categorical responses (CL on Days 7 and
17, pregnancy rates on Days 30, 55 and 90, and pregnancy losses between Day 30-55 and
55-90) were analyzed by multiple logistic regression using the backward elimination
procedure in SAS (Proc GENMOD, SAS system; Agresti, 1996) including treatment,
stages/abnormalities of the estrous cycle, parity, DIM, inseminator, lot housing, and body
condition score in the model. Continuous responses (rise in plasma P4 concentration from
Day 0 to 7 (ng/ml) and from Day 10 to 17 (ng/ml), were evaluated by the repeated
measures procedure (Mixed models, Proc MIXED, SAS; Littell et al., 1997). Treatment
differences with P<0.05 were considered significant.


81
Figure 4.9. Least squares means and SE for plasma P4 concentration on Days 7, Day 13
and Day 16 for Control () and DESLORELIN 750 () groups (P<0.07) in Experiment 3.


115
or reduce pregnancy loss is in agreement with other studies of cows inseminated at
detected estrus and treated with either hCG or GnRH at specific times. Administration of
hCG on Day 3 (Wagner et al., 1973), or Day 4 (Breuel et al., 1990) in beef heifers, on
Day 4 in lactating dairy cows (Tefera et al., 2001) or on Day 5 in dairy heifers (Schmitt et
al., 1996) failed to increase pregnancy rate. Buserelin acetate administered either on Days
11 to 14 (Stevenson et al., 1993), Day 12 (Tefera et al., 2001) or Day 15 or hCG on Days
10 or 15 (Lewis et al., 1990) failed to increase pregnancy rate in lactating dairy cows.
The increase in pregnancy rates on Day 27 and Day 55 for cows receiving a dose
of GnRH on Day 5 (P<0.11 and P<0.19) and the reduction in pregnancy losses between
Day 27 and Day 55 for cows receiving a dose of GnRH on Day 15 (P<0.11) were not
statistically significant. A previous study showed an increase in pregnancy rate in
lactating dairy cows treated with hCG on Day 5 after insemination at detected estrus (42).
In dairy heifers, hCG administered on Day 5 after detected estrus was more efficient than
GnRH on inducing accessory CL and increasing P4 concentration (30). Perhaps, an
increase in pregnancy rate or reduction in pregnancy losses in cows subjected to
synchronization of ovulation and timed insemination could have been obtained by
administering hCG at either Day 5 or 15.
Treatment with GnRH on both Day 5 and 15 reduced the proportion of cows
diagnosed not pregnant based on expression of estrus and insemination before
ultrasonography on Day 27, and extended the interval between the initial insemination
(TAI) and the next insemination in the present study. This is in agreement with previous
research using buserelin (10 ug) daily from Days 9 to 12 (10 og; Milvae et al., 1984), or
a single dose of buserelin on Day 11 (Seguin et al., 1977), or between Days 11-13 (5 ug,


136
Figure 7.1. Number of Class 2 Follicles on D 27 and D 45 of
Pregnancy in Lactating Dairy Cows after Treatment with 2.1 mg
Deslorelin Implant (Interaction P<0.01)


194
Cows in metestrus (n=35) received 100 pg (i.m.) GnRH on Day 0, 25 mg (i.m.)
PGF2a on Day 7, 1 mg (i.m.) ECP on Day 8, and TAI on Day 9 (Modified Heatsynch
Group).
On Day 0, cows without a CL (n=122) were classified as proestrus, with ovarian
cysts or anestrus according to previously described criteria (12), and assigned randomly
to 4 groups in a factorial arrangement. Cows in the GnRH Group (n=28) received 100 pg
(i.m.) GnRH on Day 0, 25 mg (i.m.) PGF2a on Day 7, 100 pg (i.m.) GnRH on Day 9, and
TAI 16 h later. Cows in the CIDR Group (n=34) received a CIDR device (CIDR inserts,
Pharmacia, Kalamazoo, MI, 49001) on Day 0, 25 mg (i.m.) PGF2(X and removal of the
CIDR on Day 7, 100 pg (i.m.) GnRH on Day 9, and TAI 16 h later. Cows in the
GnRH+CIDR Group (n=32) received 100 pg (i.m.) GnRH and a CIDR device on Day 0,
25 mg (i.m.) PGF2ot and removal of the CIDR on Day 7, 100 pg (i.m.) GnRH on Day 9,
and TAI 16 h later. Cows in the Control Group (n=28) received 25 mg (i.m.) PGF2a on
Day 7, 100 pg (i.m.) GnRH on Day 9, and TAI 16 h later.
On Day 0, parity, time of the year (season), days in milk (DIM), inseminator and
housing lot were recorded for all the cows, and body condition score (BCS) for cows
without CL.
Plasma P4 Concentrations
For cows without a CL, blood samples were collected on Days 0, 7, 10 and 17
from the coccygeal vessels into evacuated tubes containing EDTA (Vacutainer, BD,
Franklin Lakes, NJ 07417) and placed on ice immediately after collection. Samples were
centrifuged at 3000 xg for 30 minutes, and plasma stored at -20C until assayed for P4
using a solid-phase, RIA (Coat-a-Count Progesterone, DPC, Diagnostic Products


202
Day 28 after a nonpregnancy diagnosis by ultrasonography (Chebel et al., 2003). In this
study, GnRH was administered in all cows on Day 23 post insemination and controls
cows were not included since pregnancy rate was reduced after administering a shortened
protocol (PGF2 the follicular wave (Bartolom et al., 2004).
The use of a shortened resynchronization protocol that did not attempt to
synchronize follicle and CL development after a previous insemination, included the use
of PGF2ct on Day 28 to induce luteolysis in nonpregnant cows at ultrasonography, and
synchronization of ovulation with either GnRH (Stevenson et al., 2003) or ECP (Chebel
et al., 2003; Bartolom et al., 2004). Pregnancy rate for cows subjected to a shortened
protocol (PGF2a-GnRH) was similar to cows inseminated after PGF2a combining estrus
detection and TAI at 72-80 h (Stevenson et al., 2003), and similar between cows
subjected to a short protocol (PGF2a-ECP) as compared to cows subjected to a Heatsynch
protocol initiated on Day 28 (Chebel et al., 2003). In contrast, pregnancy rate for cows
subjected to a short protocol (PGF2a-ECP) was reduced compared with cows subjected to
an Ovsynch protocol (Bartolom et al., 2004). Protocols including ECP require an
efficient detection and insemination of cows at estrus before TAI at 48 h later (Pancarci
et al., 2002) and may explain the difference between studies. Perhaps, the success of short
resynchronization protocols that did not consider synchronization of the follicular wave
will depend on estrus detection efficiency.
Early resynchronization and TAI of nonpregnant lactating dairy cows on Days 27-
29 after previous insemination using PGF2a alone or PGF2a and GnRH resulted in a 22-
23 d reduction in the calving to conception interval compared to nontreated cows


187
Table 9.10. Distribution of cows by parity, season, DEM and inseminator for cows with
ovarian cysts from January to December in both groups.
Variable
Ovsynch'
s
GnRH+Ovsynch
P value
%
N
%
N
Parity
1
37.1
36/97
30.3
33/109
0.58
2
25.8
25/97
28.4
31/109
3+
37.1
36/97
41.3
45/109
Season
Mar-Sep
62.9
61/97
57.8
63/109
0.45
Oct-Dec
37.1
36/97
42.2
46/109
DIM
100-172
22.7
22/97
25.7
28/109
0.17
173-219
31.9
31/97
19.3
21/109
220-278
24.7
24/97
25.7
28/109
280-908
20.6
20/97
29.4
32/109
Inseminator
A
35.1
34/97
61.5
67/109
<0.01
B
13.4
13/97
19.3
21/109
C
51.5
50/97
19.3
21/109


LIST OF FIGURES
Figure Page
3-1 Experimental design 59
3-2 Plasma P4 concentrations in nonlactating dairy cows after induction of
ovulation with human chorionic gonadotropin (hCG) 60
3-3 Size of the CL in nonlactating dairy cows treated with human chorionic
gonadotropin (hCG) 8 to 10 hours after onset of estrus 61
4-1 Least squares means and SE for plasma P4 concentration between Day 0
and Day 16 for Control (), DESLORELIN 750 (), DESLORELIN 1000
(A) groups in Experiment 1. DESLORELIN 1000 vs Control on Day 11
(P<0.05), DESLORELIN 750 vs. Control on Day 12 (P<0.05) 73
4-2 Least squares means and SE for the first-wave largest follicles from Day 0
to Day 16 for Control (), DESLORELIN 750 () and DESLORELIN 1000
(A)groups (P<0.02 Control vs. DESLORELIN 1000) and second-wave
largest follicle for Control (), DESLORELIN 750 (O), and DESLORELIN
1000 (A) groups (P<0.01) in Experiment 1 74
4-3 A. Least squares means and SE for the number of Class II follicles after
treatment for from Day 0 to Day 16 for Control (), DESLORELIN 750
() and DESLORELIN 1000 (A) groups in Experiment 1 (P<0.01).
B. Least squares means and SE for the number of class III follicles after
treatment for from Day 0 to Day 16 for Control (), DESLORELIN 750
() and DESLORELIN 1000 (A) groups in Experiment 1 (P<0.01) 75
4-4 Least squares means and SE for the largest follicle after PGF2a on Day 16
for cows in Control (), DESLORELIN 750 () and DESLORELIN 1000
(A) groups in Experiment 1 (P<0.001) 76
4-5 Least squares means and SE for plasma P4 concentration between Day 0
and Day 16 for Control () and DESLORELIN 450 () groups in
Experiment 2. DESLORELIN 450 vs. Control on Day 9 (P<0.10), and
DESLORELIN 450 vs. Control on Day 10 (P<0.05) 77
xiv


245
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133
Table 7.1. Distribution of
insemination in both groups.
cows by parity,
previous
services
and type of artificial
Variable
Control Group
Implant Group
P value
%
N
%
N
Parity
1
55.4
36/65
44.6
29/65
0.65
2
47.8
22/46
52.2
24/46
3+
48.6
34/70
51.4
36/70
Previous services
0
45.7
16/35
54.3
19/35
0.07
1
62.2
23/37
37.8
14/37
2
35.7
15/42
64.3
27/42
3+
56.7
38/67
43.3
29/67
Insemination Type
Standing estrus
70.0
7/10
30.0
3/10
0.10
Pedometers
45.5
55/121
54.5
66/121
TAI
30.0
30/50
20.0
20/50
Month
June
38.9
7/18
61.1
43/91
0.55
July
51.4
37/72
48.6
35/72
August
52.7
48/91
47.3
11/18


I would like to express my gratitude to Dr. Pedro Melendez for his friendship and
for his scientific and field work contributions. Also, to his familyMaria Ester, Elisa,
Diego and Ignacioare now part of my family. I will remember my friends Dr. Antonio
Landaeta and Dr. Martin Giangreco for the time we spent together in graduate school.
Marie Joelle Thatcher provided excellent, very kind, and continuous technical
support in the laboratory and with computer techniques. All of the members of Dr.
Thatcher LaboratoryOscar Hernandez, Dr. Aydin Gulezoglu, Dr. Ricardo Mattos, Dr.
Frederico Moreira, Dr. Metin Pancarci, Todd Bilby, Dr. Ana Meiklehelped me with
discussions and comments about my research. Drs. Alvaro Arteche, Alessandro Sozzi,
and Shun Kamimura gave me invaluable help and joy during my field work at DRU and
North Florida Holstein.
I am very grateful to the faculty, staff, students and residents in Large Animal
Clinical Sciences and to Delores Foreman, for creating a nice environment to study and
for their support, discussions, and friendship. I really appreciated all the help from Dr.
Charles Courtney, Sally OConnell and the Phi Zeta Organization. They were always
ready to help me and solve every problem I had.
There are not enough words to express all the fun, friendship and knowledge I
received during my six years of work at North Florida Holstein. I owe special gratitude
to Mr. Don Bennink, Dr. David Kelbert, and the entire staff of North Florida Holstein and
the invaluable help from Dr. Jennifer MacHale and John Karanja. I am also grateful for
the help and friendship of all the guys at NFH: Gabriel, Gonzalo, Raul, Panfilo, Jorge,
Burt and Concepcion.
v


80
Figure 4.8. Least squares means and SE for the largest follicle after PGF2tt on Day 16 for
cows in Control () and DESLORELIN 450 () groups (P<0.001) in Experiment 2.


256
Reynolds LP, Killilea SD, Grazul-Bilska AT, Redmer DA. Mitogenic factors of corpora
ltea. Progress in Growth Factor Research 1994;5:59-175.
Reynolds LP, Redmer DA. Growth and development of the corpus luteum. J Reprod Fert
1999, Suppl.54:181-191.
Ribadu AY, Dobson FI, Ward WR. Ultrasound and progesterone monitoring of ovarian
follicular cysts in cows treated with GnRH. Br Vet J 1994;150:489-497.
Risco CA, Archbald LF, Elliott J, Tran T, Chavarte P. Effect of hormonal treatment on
fertility in dairy cows with dystocia or retained fetal membranes at parturition. J Dairy
Sci 1994;77:2562-2569.
Risco CA, Donovan GA, Hernandez J. Clinical mastitis associated with abortion in dairy
cows. J Dairy Sci 1999;82:1684-1689.
Risco CA, Drost M, Archbald LF, Moreira F, de la Sota RL, Burke J, Thatcher WW.
Timed artificial insemination in dairy cattle-Part I. Compend Contin Educ Pract Vet
1998;20:280-287.
Rivera RM, Kelley KL, Erdos GW, Hansen PJ. Alterations in ultrastructural morphology
of two-cell bovine embryos produced in vitro and in vivo following a physiologically
relevant heat shock. Biol Reprod 2003;69:2068-2077.
Robbins RK, Sullivan JJ, Pace MM. Timing the insemination of beef cattle.
Theriogenology 1978, 10:247-255.
Roberson MS, Wolfe MW, Stumpf TT, Kittok RJ, Kinder JE. Luteinizing hormone
secretion and corpus luteum function in cows receiving two levels of progesterone. Biol
Reprod 1989;41:997-1003.
Roberts SJ. Veterinary obstetrics and genital diseases. J W Edwards, Ann Arbor,
Michigan, 1956.
Robker RL, Richards JS. Hormone-induced proliferation and differentiation of granulse
cell: a coordinated balance of the cell cycle regulator cyclin D and P27Kipl. Mol Endoc
1998;12:929-940.
Roche JF. Attempts to determine the optimal time of artificial insemination in heifers. J
Reprod Fert 1974a, 41:223-225.
Roche JF. Synchronization of oestrus in heifers with implants of progesterone. J Reprod
Fert 1974b, 41:337-344.
Roche JF. Effect of short-term progesterone treatment of oestrus response and fertility in
heifers. J Reprod Fert 1974c, 40:433-440.


129
insemination type, season and interactions. The effect of treatment, significant variables,
and their higher order interactions with treatment were included in the final model to
examine pregnancy losses. Treatment differences with P < 0.05 were considered
significant.
Results
There was not significant difference in the distribution of cows by parity, previous
services and type of artificial insemination in both groups (Table 1). Pregnancy losses did
not differ between Days 27-45 and 45-90 for cows in the Control Group [15.2% (14/92);
11.0% (8/77)] and cows in the Implant Group [20.2% (18/89); 10.5 % (8/76)]. In
addition, overall pregnancy loss (Day 27 to Day 90) between cows in the Control Group
and the Implant Group (Table 7.2) were similar.
On Day 45, the number of class 2 (0.72 0.19) and class 3 (0.86 0.12) follicles

for cows in the Implant Group were lower (P<0.01) than the number of class 2 (1.90
0.18) and class 3 (1.92 0.12) follicles for cows in the Control Group (Figure 7.1 and
7.2). On Day 45, the number of corpora ltea for cows in the Implant Group was higher
than that for cows in the Control Group (1.80 0.07 > 1.31 0.07; P<0.01; Figure 7.3).
The rise in plasma P4 concentration from Day 27 to Day 45 tended to be higher
for cows in the Implant Group (P<0.10; Figure 7.4). On Day 27, plasma P4
concentrations were similar for cows in the Control Group (6.59 0.30 ng/ml) and the
Implant Group (7.17 0.30 ng/ml). On Day 45, plasma P4 concentration was increased
(PO.01) for cows in the Implant Group (8.03 0.33 ng/ml) compared to cows in the
Control group (6.40 0.31 ng/ml).


Introduction 159
Materials and Methods 160
Results 164
Discussion 169
10 RESYNCHRONIZATION OF OVULATION AND TIMED INSEMINATION
(TAI) IN LACTATING DAIRY COWS, III: ADMINISTRATION OF
GNRH 23 DAYS POST AI AND ULTRASONOGRAPHY FOR
NONPREGNANCY DIAGNOSIS ON DAY 30 191
Introduction 191
Materials and Methods 192
Results 197
Discussion 201
11 SUMMARY AND CONCLUSIONS 226
REFERENCE LIST 232
BIOGRAPHICAL SKETCH 267
IX


48
FGF-2 and platelet-derived growth factor (PDGF) which stimulate pericyte migration and
proliferation. The pericytes produce VEGF which stimulates thecal-derived endothelial
cells (Reynolds and Redmer, 1999). The CL increases in size by hypertrophy of large
luteal cells and increase in number of small luteal cells, fibroblasts and endothelial cells
(Farin et al., 1986).
Cows that become pregnant after insemination had higher plasma P4
concentrations during the luteal phase compared with nonpregnant cows (Lukesewska
and Hansel 1980). In addition, luteal insufficiency has been observed in subfertile cows
compared with heifers (Shelton et al., 1990). The role of P4 on CL maintenance and
embryo survival is carried out by: 1) stimulating embryo development and consequently
stimulating the antiluteolytic signal of interferon-x (Mann and Lamming, 1995); 2)
inhibiting LH pulsatility, follicular growth and estrogen production which contribute to
s
suppression of oxytocin receptors and the luteolytic cascade involving estrogen, oxytocin
and prostaglandin (Vallet and Lamming, 1991); and 3) inhibiting apoptosis of the luteal
cells by a local mechanism (Rueda et al., 2000).
Progesterone production by the CL stimulates secretion of the endometrial glands
necessary for embryo development (Geisert et al., 1992). Insufficient amount of P4 may
result in compromised embryo development, reduction in interferon-x secretion, failure to
inhibit the luteolytic cascade and embryo loss (Mann and Lamming 1995). High levels of
P4 and low levels of estrogen during the luteal phase contribute to attenuation of the
luteolytic cascade (Lamming and Mann, 1995). Progesterone is able to inhibit oxytocin
receptors (Vallet and Lamming, 1991), and cows with low plasma P4 concentrations had
a greater PGF2 release in response to oxytocin administration compared with cows with


22
GnRH agonists makes them more stable and increases their affinity to plasma proteins,
membranes and the GnRH receptor (Conn and Crowley, 1991). Fertirelin acetate is 4 to
10 times and buserelin 50 times more potent than gonadorelin (Chenault et al., 1990).
The recommended or commonly used doses are 10-20 pg for buserelin, 25-50 pg for
fertirelin acetate and 100-200 pg for gonadorelin acetate (Chenault et al., 1990). In a
protocol for synchronization of ovulation and timed insemination (TAI; Ovsynch) the
use of either 100 or 50 pg of gonadorelin acetate was equally effective (Fricke et al.,
1998). Gonadorelin diacetate was more effective in inducing the LH surge in dairy cows
than gonadorelin hydrochloride, but no differences were detected in beef heifers and
difference were observed between commercial preparations of the same analogue
(Martinez et al., 2002). Deslorelin (GnRH analogue, 2,100 ug, Ovuplant, Peptech
Animal Health, North Ryde, Sydney, Australia 2113) is a nonapeptide administered as a
subcutaneous implant and approved for induction of ovulation in horses.
Human Chorionic Gonadotropin (hCG)
Human chorionic gonadotropin is a glycoprotein secreted by the placenta of the
pregnant woman and excreted in the urine. The administration of hCG during diestrus has
been used to stimulate CL function and enhance embryo survival (Wiltbank et al., 1961).
Intravenous injection of hCG (5,000 IU) on Days 5, 10, 15 and 20 of the estrous cycle
had variable results in terms of P4 increase, induced ovulations and prolonged the estrous
cycle (Schomberg et al., 1967). The administration of 1,000 and 2,000 IU of hCG from
Days 1 to 7 of the estrous cycle were also able to stimulate CL function in beef heifers
(Veenhuizen et al., 1972). The administration of 1,000 to 2,000 IU of hCG at estrus or 3
days later has been used to increase pregnancy rate in synchronized beef heifers with


108
Seguin et al., 1977). The ovulation of dominant follicles and formation of accessory CL
increase P4 and reduce estrogen concentrations (Fricke et al., 1993; Schmitt et al., 1996).
There is a paucity of information available on the effect of GnRH administered
coincident with the presence of the dominant follicle of the first (Day 5) and second (Day
15) follicular wave in cows subjected to synchronization of ovulation and TAI.
The hypothesis of this study was that treatment of cows with GnRH at specific
times after insemination will increase pregnancy rate and reduce pregnancy loss in
lactating dairy cows subjected to synchronization of ovulation and TAI. The objectives
of this study were to compare pregnancy rates at Days 27 and 55, and pregnancy loss
between Days 27 and 55 in lactating dairy cows treated with GnRH 5 and 15 d after TAI.
Materials and Methods
Study Population
This study was conducted during the period of November 2001 to
April 2002 in a large commercial dairy herd (approximately 3,000 milking cows) in north
central Florida. All reproductive, health and management records were computerized
using a cow-side computer system. Cows were routinely vaccinated against bovine virus
diarrhea, infectious bovine rhinotracheitis, bovine respiratory syncytial virus,
parainfluenza virus, leptospirosis, campylobacteriosis, clostridiosis, and gram-negative
organisms according to recommendations by attending veterinarians. Cows were housed
in free stall barns and dry lots and fed a total mixed ration (TMR) three times daily. The
TMR was formulated to meet or exceed requirements for lactating cows (NRC, 2001).
Cows were milked three times a day, and had a rolling herd average for milk production
of 10,700 kg. Beginning at 60 d post partum, cows received bST (Posilac, 500 mg


241
Ferguson JD, Galligan DT, Thomsen N. Principal descriptors of body condition score in
Holstein cows. J Dairy Sci 1994;77:2695-2703.
Fernandez-Limia O, Murphy BD, Manns JG. Synchronization of preovulatory LH surges
after PGF2 alpha in beef cattle: the effects of D-ala-GnRH and testosterone benzoate.
Theriogenology 1977;7:73-81.
Fetrow J, McClary D, Harman R, Butcher K, Weaver L, Studer E, Ehrlich J, Etherington
W, Guterbock W, Klingborg D, Reneau J, Williamson N. Calculating selected
reproductive indices: recommendations of the ameriacna association of bovine
practitioners. J Dairy Sci 1990;73:78-90.
Findlay JK, Drummond AE, Fry RC. Intragonadal regulation of follicular development
and ovulation. Anim Rep Sci 1996;42:321-331.
Fitz TA, Hoyer PB, Niswender GD. Interactions of prostaglandins with subpopulations of
ovine luteal cells I. Stimulatory effect of prostaglandins El, E2 and 12. Prostaglandins
1984;28:119-126.
Fitz TA, Mayan MH, Sawyer HR, Niswender GD. Characterization of two steroid cell
types in the ovine corpus luteum. Biol Reprod 1982;27:703-711.
Flint APE, Sheldrick EL. Ovarian secretion of oxytocin is stimulated by prostaglandin.
Nature 1982;297:587-588.
Flint APE, Sheldrick EL. Evidence for a systemic role of ovarian oxytocin in luteal
regression in sheep. J Reprod Fert 1983;67:215-225.
Fogwell RL, Cowley JL, Wortman A, Ames NK, Ireland JJ. Luteal function in cows
following destruction of ovarian follicles at mydcycle. Theriogenology 1985;23:389-398.
Folman Y, Kaim M, Herz Z, Rosenberg M.Comparison of methods for the
synchronization of estrous cycles in dairy cows. 2. Effects of progesterone and parity on
conception. J Dairy Sci 1990;73:2817-2825.
Fonseca FA, Britt JH, McDaniel BT, Wilk JC, Rakes AH. Reproductive traits of
Holsteins and Jerseys. Effects of age, milk yield, and clinical abnormalities on involution
of cervix and uterus, ovulation, estrous cycles, detection of estrus, conception rate, and
days open. J Dairy Sci 1983;66:1128-1147.
Foote RH. Review: dairy cattle reproductive physiology research and management-past
progress and future prospects. J Dairy Sci 1996;79:980-990.
Forar AL, Gay JM, Hancock DD, Gay CC. Fetal loss frequency in ten Holstein dairy
herds. Theriogenology 1996;45:15051513.


32
comparison to administering PGF2a alone, increases estrus synchronization rate, and may
induce estrus in some noncyclic animals and may increase fertility. An extra benefit can
be obtained by including a P4 supplement (CIDR Insert) between the GnRH and the
PGF201 in the Select Synch protocol. This will avoid the occurrence of estrus before
PGF20C treatment (Stevenson et al., 1997) and induce estrus in some noncyclic animals.
Induction of Ovulation and TAI Using GnRH and PGF2 (Ovsynch Protocol)
The administration of GnRH induced an LH surge and ovulation in the cow
(Fernandez Limia et al., 1977; Graves et al., 1974), and therefore it was administered at
estrus or at Al in normal and repeat-breeding cows in order to increase conception rate
(Mee et al., 1990). The results were inconsistent since stage of follicular development at
the time of GnRH treatment was not considered. However, a second dose of GnRH
administered 48 hours after PGF2a injection in the Select Synch protocol will
/
synchronize ovulation and allow for TAI (Pursley et al., 1995; Burke et al., 1996). Once
the follicular wave is controlled, synchronization of ovulation for TAI is possible by
inducing an LH surge since there is a constant interval between LH surge and time of
ovulation (Cumming et al., 1973).
Administration of GnRH at random stages of the estrous cycle induced ovulation
in approximately 70 % of lactating dairy cows (Vasconcelos et al., 1999), and treatment
with PGF20C 7 days later caused luteolysis in 90 to 100% of the cows (Pursley et al., 1995;
Vasconcelos et al., 1999). Injection of GnRH 48 hours later (Ovsynch protocol)
synchronized ovulation within 24 to 32 hours later (Pursley et al., 1995) and TAI is
conducted 16 hours after the second GnRH treatment (Burke et al., 1996). GnRH
administered after PGF2a induces ovulation, but cows usually do not express estrus


160
stages and two common abnormalities of the estrous cycle at the time of nonpregnancy
diagnosis by ultrasonography.
Materials and Methods
Study Population
The study was conducted from January to December 2002 in a large commercial
dairy with 3,200 milking cows. The herd is in north central Florida and is divided into 14
lots according to levels of production and stage of lactation, and cows from all lots were
included in the study. Cows were housed in free stall barns and dry lots and fed a total
mixed ration (TMR) three times daily. The TMR was formulated to meet or exceed
requirements for lactation (NRC, 2001). Cows were milked three times a day and had a
rolling herd average for milk production of 10,700 kg. Beginning at 60 d post partum,
cows received a bST treatment (Posilac, 500 mg sometribove zinc, subcutaneously;
/
Monsanto, St Louis, Missouri) every 14 d during the entire lactation. Reproductive
management consisted of a voluntary waiting period of 75 d that incorporated a
Presynch-Ovsynch program (Moreira et al., 2001) for first service, and estrus detection
using visual observation and a computerized pedometer system (Afimilk , S.A.E.
Afikim, Kibbutz Afikim, 15148, Israel) for subsequent services. The study included 1083
lactating dairy cows determined to be nonpregnant by ultrasonography at Day 30
(experimental Day 0) after artificial insemination. Cows with reproductive abnormalities
(i.e. metritis, pyometra, uterine-ovarian adhesions, granulosa cell tumor) were excluded.
Experimental Design
On Day 0, cows were classified into different stages and two common
abnormalities (ovarian cysts and anestrus) of the estrous cycle as determined by per


85
meet the nutrient requirements for lactating Holstein cows weighing 650 kg and
producing 40 kg of 3.5% fat-corrected milk (NRC, 2001).
The experiment was conducted from March to June of 2001 and from August to
December of 2001. Cows were housed in two free-stall barns, and primiparous and
multiparous cows were grouped separately during the entire study. Cows were milked
twice daily and milk yields were recorded for individual cows once monthly during the
official California dairy herd improvement association test. Body condition of all cows
was scored (Ferguson et al., 1994) at the moment of enrolment in the study (30 DIM), at
insemination (68 DIM), and at the first ultrasound examination (95 DIM). Changes in
BCS from enrollment in the study to AI and to pregnancy diagnosis were calculated.
Also, BCS at AI was categorized as low (BCS < 2.75), medium (BCS = 3.0 to 3.25), and
high (BCS >3.5).
Treatments, Ovarian Ultrasonography, and Pregnancy Diagnosis
Cows were subjected to a pre-synchronization treatment with i.m. injections of
PGF2a (25 mg Dinoprost Tromethamine; Lutalyse, Pharmacia Animal Health,
Kalamazoo, MI) at 30 3 and 44 3 d postpartum (Moreira et al., 2001). Fourteen days
after the second PGF2a, (58 3 DIM), all cows received an i.m. injection of 100 pg of
GnRH (gonadorelin diacetate tetrahydrate; Cystorelin, Merial, Athens, GA). On Day
7.5 after the GnRH injection, cows received a third injection of PGF2a. Forty-eight hours
after the last PGF2a injection, cows received either 100 pg of GnRH (i.m ), or a 450 or
750 pg biodegradable deslorelin implant (Peptech Animal Health, North Ryde, Australia)
subcutaneously in the neck area. Timed AI was performed in all cows 16 to 18 h later; the
same technician artificially inseminated all cows throughout the study. After the initial


197
Results
Cows in Diestrus
For the diestrus stage, 7.6% (18/238) of the cows in the GnRH Group and 5.7%
(13/229) of the cows in the ECP Group were excluded from the study since they either
had not received the complete protocols or they were culled from the herd before
pregnancy diagnosis on Day 30. The distributions of cows by parity, DIM, inseminator
and housing lot for the GnRH (n=220) and the ECP (n=216) groups were not different
(Table 10.1).
At Day 30, in the GnRH Group, 211 cows were diagnosed as either pregnant or
not pregnant, five cows were diagnosed as questionable pregnant, and four cows were not
ultrasounded, in the ECP Group, 210 cows were diagnosed as either pregnant or not
pregnant, one cow was diagnosed as a questionable pregnant, and five cows were not
ultrasounded. One cow in the GnRH Group that was pregnant at Day 30 was culled from
the herd before Day 55.
Pregnancy rates on Days 30, 55 and 90, and pregnancy losses between Days 30-
55 and 55-90 for cows in diestrus subjected to either the GnRH or ECP groups were not
different (P>0.10; Table 10.2).
There was a group of cows (n=40) that were not presented for early
resynchronization on Day 30 after a prior insemination. These cows were either
reinseminated at 12 h after signs of estrus or palpated per rectum at 405 d after AI and
resynchronized using the Ovsynch protocol. The interval between previous insemination
and reinsemination was 51.1 d for cows treated with GnRH on Day 23 (35 cows) and
51.4 d for cows that did not receive the GnRH on Day 23 (five cows). The interval


119
Table 6.1.Baseline comparison for parity, season and inseminator.
Item
Group 1
%(N)
Group 2
%(N)
Group 3
%(N)
Group 4
%(N)
P
Parity
1
31.8(68/214)
31.6(66/209)
25.5(54/212)
32.1(63/196)
0.67
2
34.1(73/214)
34.4(72/209)
35.4(75/212)
30.6(60/196)
3+
34.1(73/214)
34.0(71/209)
39.1(83/212)
37.3(73/196)
Season
N-F
53.3(114/214)
53.6(112/209)
51.9(110/212)
51.5(101/196)
0.96
M/A
46.7(100/214)
46.4(97/209)
48.1(102/212)
48.5(95/196)
Ins.
A
41.6(89/214)
42.1(88/209)
44.3(94/212)
47.9(94/196)
0.80
B
28.5(61/214)
28.7(60/209)
29.7(63/212)
28.1(55/196)
C
29.9(64/214)
29.2(61/209)
25.9(55/212)
20.1(47/196)
N-F=November to February; M/A-=March and April; Ins.=Inseminator


173
did not increase pregnancy rate (Bartolom et al., 2003). It is possible that 7 d was not
long enough for the recruited follicle to acquire ovulatory size and be responsive to the
second GnRH dose. However, an 8 d interval may enhance the ovulatory response such
that a CL is present during the Ovsynch protocol coupled with recruitment of a new
dominant follicle. The advantage of the GnRH+Ovsynch protocol in this study seems to
be in cows with follicular cysts as determined by clinical diagnosis but not based on
levels of P4.
Pregnancy losses between Days 30 and 55 for cows in diestrus during the period
from June to December were higher for cows in the Ovsynch protocol compared to
cows in the Modified Quicksynch protocol. Higher pregnancy losses were reported for
cows time-inseminated using the Ovsynch protocol compared to cows inseminated at
detected estrus after GnRH and PGF2a given 7 d apart during the summer (Cartmill et al.,
2001). In contrast, no differences in pregnancy losses were detected between cows
inseminated at detected estrus after GnRH-PGF2a or time-inseminated using the
Ovsynch protocol (Santos et al., 2003) or inseminated at detected estrus after GnRH-
PGF2a or time- inseminated using the Heatsynch protocol (Cerri et al., 2003).
Plasma P4 concentrations has been used to differentiate luteal from follicular cysts
(Nakao et al., 1983; Dobson et al., 1997) and in combination with ultrasonography gave
an accurate classification of the ovarian cyst (Ribadu et al., 1994; Douthwaite et al.,
2000). In the present study, per rectum examination and ultrasonography of the genital
tract were used to determine the stages/abnormalities of the estrous cycle. The advantage
of using both techniques in detection of ovarian cysts lies in the fact that ultrasonography
allows for determination of luteinized structures (Carroll et at., 1990; Farin et al., 1990)


77
Interval after treatment (d)
Figure 4.5. Least squares means and SE for plasma P4 concentration between Day 0 and
Day 16 for Control () and DESLORELIN 450 () groups in Experiment 2.
DESLORELIN 450 vs. Control on Day 9 (P<0.10), and DESLORELIN 450 vs. Control
on Day 10 (P<0.05).


51
The detrimental effect of low P4 and high estrogen concentrations on early
embryo development may be exacerbated in high-producing dairy cows subjected to
synchronization of ovulation. Comparing lactating dairy cows with nonlacting dairy cows
(de la Sota et al., 1993) or with heifers (Sartori et al., 2002b) it was concluded that
lactating dairy cows have a reduced level of circulating P4. Lactating dairy cows have an
increased feed intake that results in increased liver blood flow and steroid metabolism
(Sangsritavong et al., 2002) that may result in low P4 concentrations. Low levels of P4
during diestrus increase LH pulses (Ireland and Roche, 1982b) which stimulate follicular
growth (Savio et al., 1993; Stock and Fortune, 1993) resulting in elevated concentration
of estradiol. In fact, follicular development is more pronounced in lactating than in
nonlactating dairy cows (Lucy et al., 1992) and it may be detrimental for embryo survival
since elimination of follicles during diestrus is known to extend the lifespan of the CL in
/
cattle (Fogwell et al., 1985), and follicular development is attenuated on the CL-bearing
ovary ipsilateral to the pregnant uterine horn (Guibault et al., 1986). Lactating dairy cows
inseminated at detected estrus with high fertility semen had more nonviable embryos
compared to nonlactating dairy cows (Sartori et al., 2002a).
The pulsatile secretion of LH and follicular development during proestrus are
important for differentiation and development of the CL and P4 production (Quintal-
Franco et al., 1999). In protocols for TAI, the timing between GnRH administration and
follicular development may be altered and could result in ovulation and formation of a
corpus luteum with reduced production of P4 (Vasconcelos et al., 1999; 2001; Peters and
Pursley, 2003), and it may result in increased embryo mortality. Timing of insemination
and protocols for synchronization of ovulation may affect CL formation and indirectly


78
Figure 4.6. Least squares means and SE for the first-wave largest follicles from Day 0 to
Day 16 for Control () and DESLORELIN 450 () groups (P<0.01), and second-wave
largest follicle for control (), DESLORELIN 450 (O) groups (P<0.001) in Experiment
2.


45
the LH surge, there is a hypertrophy of the granulosa cells and nuclear activation, the
basement membrane breaks down and blood vessels from the theca interna invade the
cavity of the rupture follicle (Niswender and Nett, 1994). During CL development, the
vascularized theca cells and the avascular granulosa cells of the ovarian follicle become
the small and large luteal cells, respectively (Donaldson and Hansel, 1965; Alila and
Hansel, 1984), and during advanced stages of luteal phase some of the small luteal cells
are transformed to large luteal cells (Alila and Hansel, 1984).
The amount of large luteal cells in the CL is consistent with the number of
granulosa cells in the preovulatory follicle (Rodgers et al., 1984). Mitosis in the granulosa
cells ends with the LH surge by inhibition of cyclin D and upregulation of p27Kipl and
p21Cipl which are inhibitors of cyclin D complexes (Robker and Richards, 1998). Other
cells that form the CL are fibroblasts, vascular smooth muscle, pericytes and endothelial
/
cells (Niswender and Nett, 1994). Matrix metalloproteinases 2 (MMP2) which are
endopeptidases that cleave extracellular proteins (lattice network of basement membranes
that support epithelial cells and endothelium) are mediators of ovulation and luteinization
facilitating connective tissue projections that provide a framework for cellular migration
and angiogenesis (Gottsch et al., 2002).
The LH surge changes the steroidogenic pathway from the two-cell two-
gonadotropin secretion of estrogen by the follicle to secretion of P4 by small and large
luteal cells (Niswender et al., 2000). Cholesterol from high density lipoproteins (HDL)
and low density lipoproteins (LDL) enters the cell and is transformed to pregnelonone in
the mitochondria (enzyme P450cc) and then to P4 in the endoplasmic reticulum (enzyme
3b-HSD) and P4 diffuses from the cells and the amount stored is very low (Niswender et


68
Experiment 2
Overall plasma P4 concentration was not different among cows in the Control
Group (5.9 0.80 ng/mL) and DESLORELIN 450 Group (7.4 1.0 ng/mL). However,
plasma P4 concentration tended to be higher (P<0.10) on Day 9 for cows in the
DESLORELIN 450 Group (11.4 1.5 ng/mL) compared to cows in Control Group (8.0
1.2 ng/mL), and was higher (P<0.05) on Day 10 for cows in DESLORELIN 450 Group
(13.0 1.5 ng/mL) compared to cows in Control Group (8.2 1.2 ng/mL; Figure 4.5).
The first-wave largest follicle was larger (P<0.01) for cows in Control Group
(11.4 + 1.0 mm) compared to that for cows in DESLORELIN 450 Group (6.0 1.1 mm).
The second-wave largest follicle monitored until Day 16 was larger (P<0.001) for cows
in Control Group (8.5 0.4 mm) compared to cows in DESLORELIN 450 Group (3.3
0.5 mm; Figure 4.6).
The number of Class II follicles from Day 0 to Day 16 was higher (P<0.01) for
cows in Control Group (2.5 0.2) compared to cows in DESLORELIN 450 Group (0.9
0.2, Figure 4.7A). The number of Class III follicles from Day 0 to Day 16 was higher
(PO.01) for cows in Control Group (1.3 0.1) compared to cows in DESLORELIN 450
Group (0.4 0.1; Figure 4.7B).
The average diameter of the largest follicles from Day 16 to Day 28 was larger
(P<0.001) in cows in Control Group (16.5 1.85 mm) compared to cows in
DESLORELIN 450 Group (9.7 1.85 mm; Figure 4.8). The number of cows expressing
estrus and ovulating after PGF2tt on Day 16 until Day 20 was higher (P< 0.001) in
Control Group (8/8) compared to cows in DESLORELIN 450 Group (1/6). One cow in
DESLORELIN 450 Group showed estrus on Day 28, but failed to ovulate. Therefore,


258
present during the period of progestin treatment to synchronize time of estrus in cows and
heifers. Biol Reprod 1993;49:1102-1107.
Sangsritavong S, Combs DK, Sartori R, Armentano LE, Wiltbank MC. High feed intake
increases liver blood flow and metabolism of progesterone and estradiol-17beta in dairy
cattle. J Dairy Sci 2002;85:2831-2842.
Santos JEP, Bartolom JA, Cerri RLA, Juchem SO, Trigg TE, Thatcher, WW. Effect of
deslorelin implant in a timed AI protocol on follicle development, luteal activity and
reproductive performance of dairy cows. J Dairy Sci 2002;85:Suppl l:263(abst. 1052).
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C. Effect of bST and reproductive management on reproductive and lactational
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Sartori R, Fricke PM, Ferreira JCP, Ginther OJ, Wiltbank MC. Follicular deviation and
acquisition of ovulatory capacity in bovine follicles. Biol Reprod 2001;65:1403-1409.
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250
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121
Table 6.3. Pregnancy rates on Day 27, and the adjusted odd ratios (AOR), 95 %
confidence interval (Cl), and level of significance for the risk of nonpregnancy in cows in
all groups.
Variable
Pregnancy
AOR
95 % Cl
P value
%
N
Group
1
47.7
102/214
0.9
0.6-1.3
0.10
2
43.5
91/209
1.1
0.7-1.6
0.92
3
36.8
78/212
1.4
0.9-2.1
0.03
4
Season
44.4
87/196
Referent
Referent
NA
November-February
50.4
215/427
Referent
Referent
NA
March/April
37.3
143/383
1.7
1.3-2.2
0.0002
NA= not applicable


16
concentration of FSH and LH are parallel during most part of the luteal phase, and before
ovulation there is a coincident FSH and LH surge (Rahe et al., 1980). However, there are
two instances during the estrous cycle in which FSH and LH diverge (Turzillo and
Fortune, 1990). Firstly, after luteolysis LH pulsatility increases and FSH decreases (Rahe
et al., 1980), and secondly, early in the luteal phase there is an increase in FSH but no rise
in LH (Dobson, 1978). This post-ovulatory surge of FSH precedes the first follicular
wave (Turzillo and Fortune, 1990) and an increase in FSH precedes the emergence of
each follicular wave by 1 to 2 days (Adams, 1992).
Follicular deviation in cattle occurs during the selection phase when the dominant
follicle is approximately 8.5 mm in diameter (Ginther et al., 2000). The mechanism by
which one or two follicles are selected to be dominant and the rest become subordinate is
not clearly understood and has been the subject of several studies. Intravenous
administration of the proteinaceous fraction of the follicular fluid inhibited follicular
growth (Kastelic et al., 1990a). After deviation (Ginther et al., 2001), the dominant
follicles become dependent on LH pulsatility (Gong et al., 1996), secrete inhibin and
estrogen, and both suppress FSH secretion which may be the mechanism to inhibit
development of subordinate follicles (Badinga et al., 1992; Ginther et al., 2000).
Dominant follicles have a greater aromatase activity and estrogen production than
subordinate follicles with a high ratio of P^estrogen indicating the beginning of atresia
(Badinga et al., 1992). Follicular dominance or terminal follicular development is
characterized by decreased intrafollicular concentrations of IGF- binding proteins
(IGFBP 2, 4 and 5) that result from a decreased synthesis and increased proteolysis
(Monniaux et al., 1997). In contrast, atresia is associated with an increased concentration


265
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ovulation using subcutaneous implants and estrogens in beef cattle. J Anim Sci 1971 ;33:
600-606.
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oestrogen and gonadotrophin. J Reprod Fert 1966;Suppl. 1:1-7.
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activation of protein kinase (PK) A and PKC in ovine small and large luteal cells. Mol
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dairy heifer. J Reprod Fert 1972, 30:333-334.
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activity of cows at oestrus. Veterinary Record 2003;152:239-240.
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follicular cysts and associated changes of plasa LH, FSH, progesterone and estradiol-17b
in cows. Res Vet Sci 1996;61:240-244.


26
Syncro-Mate-B combines a norgestomet ear implant for 9 days and estrogen at
initiation of the treatment and after implant insertion. Therefore, estradiol increases in the
first 24 hours, and remains higher than controls until the end of the treatment, P4
decreases, LH pulses seem to be slightly increased but mean concentrations decrease,
FSH levels are reduced and LH and FSH increase 48 hours after implant removal
(Rathbone et al., 2001). Synchronization of estrus with Syncro-Mate-B resulted in
reduced estrus expression (Roche 1974b, 1974c), when treatment was initiated in cows in
proestrus or metestrus (Roche 1974c). This indicated that many cows did not regress the
CL or the estrogen injection induced ovulation and the formation of CL that did not
regressed after implant removal (Smith and Zimbelman, 1968b). Consequently,
pregnancy rates were variable (Hansel, 1965; Beal, 1996). In addition, fertility after P4
implant (Roche 1974b) or a norgestomet implant (Wiltbank et al., 1971) is variable due to
variation in LH surge and behavioral estrus after cessation of treatment.
Prostaglandin F2a
In 1972, PGF2a was identified as luteolytic in sheep (McCraken et al., 1972) and
used to synchronize estrus in cattle (Lauderdale 1972, Rowson et al., 1972) allowing for
certain control of time of ovulation (Lauderdale et al., 1974). After PGF2a administration,
plasma P4 declines 50% within 7 hours and to less than 0.5 ng/ml within 24 hours,
estradiol increases abruptly 1 to 9 hous after PGF2(X treatment and remains high until the
LH surge occurs and then decreases (Thatcher and Chenault 1975). Animals treated with
PGF2ocusually express estrus in 64 hours with an average of 36 to 96 hours (Dalton et al
2001) and fertility is similar to cows inseminated at spontaneous estrus (Lauderdale 1972,
Rowson et al., 1972).


PHARMACOLOGICAL REGULATION OF OVARIAN FUNCTION IN DAIRY
COWS TO IMPROVE REPRODUCTIVE PERFORMANCE
By
JULIAN ALBERTO BARTOLOME
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
2004


CHAPTER 1
INTRODUCTION
Reproductive efficiency is one of the most important factors affecting dairy herd
productivity (Louca and Legates, 1968; Pellisier, 1976; Oltenacu et al., 1981; Galligan, 1999).
Fertility has been reduced in the last 50 years due to genetic selection for milk production and
intensive management, which are characteristics of modem dairy herds (Butler and Smith,
1989; Stevenson, 2001; Lucy, 2001).
Reproductive efficiency is based on high estrous detection/service rate, high
conception rate, enhanced embryo and fetal survival, and efficient detection and re
insemination of cows that did not conceive or lost their embryos after first and subsequent
services. Therefore, enhancing reproductive efficiency using pharmacological and
management strategies should focus at three different levels. First, by increasing pregnancy
rate (either by increasing estrous detection or conception rate); second, by enhancing embryo
and fetal survival; and third, by detecting and rapidly re-inseminating cows that did not
conceive or failed to maintain pregnancy.
Pregnancy rate for 21 -day periods is the product of estrous detection and conception
rates, and is the most commonly used index to evaluate reproductive performance (Ferguson
and Galligan, 1993). Estrous detection rate is defined as the number of animals detected in
estrus in a 21-day period divided by the number of animals available. Conception rate is
defined as the number of pregnant animals divided by the number of inseminated animals
1


37
phase (Day 2) fails to synchronize follicular recruitment since the dominant follicle of the
first wave is too small to turn over in response to GnRH. The follicle will continue to
grow and be an aged follicle at the time of the second GnRH for induction of ovulation
(Moreira et al., 2000). Extended periods of follicular dominance are associated will low
fertility (Mihn et al., 1994) and higher pregnancy losses (Ahmad et al., 1995). Similarly,
if the Ovsynch protocol is initiated late in the luteal phase (e.g. Day 15) the dominant
follicle of the second wave may be too small and PGF2 will be spontaneously released
approximately 2 days later. This will cause the CL to regress and estrus will be induced
well before the TAI resulting in low fertility (Moreira et al., 2000).
Based on this information, a protocol including two doses of PGF2 given 14 d
apart (Presynch) was developed that allowed initiation of the Ovsynch protocol 12 days
after the second PGF2a injection. Following this protocol, cyclic cows will be between
/
Days 5 and 10 of the estrous cycle when the Ovsynch program is initiated (Moreira et
al., 2001). The Presynch-Ovsynch protocol increased pregnancy rate by 10% in cyclic
cows compared with the Ovsynch protocol with no presynchronization (Moreira et al.,
2001). There was no difference in pregnancy rate between Presynch-Ovsynch and
Presynch-Heatsynch protocols in lactating dairy cows (Pancarci et al., 2002). The
Presynch-Ovsynch protocol is 36 to 38 days long (depending if the Ovsynch is initiated
either 12 or 14 days after the second PGF2ttinjection) and can be initiated any time after
calving according to the designed voluntary waiting period and first insemination.
Initiating the Ovsynch at 14 days after the second PGF injection has been designated as
a modified Presynch-Ovsynch protocol and increases pregnancy rate compared to
Ovsynch with no presynchronization (Fricke et al., 2004). Insemination at detected


Girsh E, Milvae RA, Wang W, Meidan R. Effect of endothelin-1 on bovine luteal cell
function: role in prostaglandin F2a -induced antisteroidogenic action. Endocrinology
1996a;134:1306-1312.
243
Girsh E, Wang W, Mamluk R, Arditi F, Friedman A, Milvae RA, Meidan R. Regulation
of endothelin-1 expression in the bovine corpus luteum: elevation by prostaglandin F2a.
Endocrinology 1996b; 137:5191-5196.
Glencross RG, Munro IB, Senior BE, Pope GS. Concentrations of oestradiol-17p,
oestrone and progesterone in jugular venous plasma of cows during the estrous cycle and
in early pregnancy. Acta Endocrin 1973;73:374-384.
Gong JG. Influence of metabolic hormones and nutrition on ovarian follicle developemtn
in cattle: practica implications. Dom Anim Endoc 2002;23:229-241.
Gong JG, Bramley TA, Gutierrez CG, Peters AR, Webb R. Effects of chronic treatment
with gonadotropin-releasing hormone agonist on peripheral concentrations of FSH and
LH, and ovarian function in heifers. J Reprod Fert 1995;105:263-270.
Gong JG, Campbell BK, Bramley TA, Gutierrez CG, Peters AR, Webb R. Suppression in
the secretion of follicle-stimulating hormone and luteinizing hormone, and ovarian
follicle development in heifers continuously infused with a gonadotropin-releasing
hormone agonist. Biol Reprod 1996;55:68-74.
Goodman AL, Hodgen GD. The ovarian triad of the primate menstrual cycle. Recent
progress in hormone research 1983;39:1-73.
Goodman RL. Neuroendocrine control of the ovine estrous cycle. In: The physiology of
reproduction, edited by Knobil E and Neil J, Raven Press, New York, 1988, Chapter 46,
pp 1929-1969.
Gottsch ML, Vankirk EA, Murdoch WJ. Role of matrix metalloproteinases 2 in the
ovulatory follicle-luteal transition of ewes. Reproduction 2002;124:347-352.
Gouveia Nogueira MF, Mel DS, Carvalho LM, Fuck EJ, Trinca LA, Barros CM. Do
high progesterone concentrations decrease pregnancy rates in embryo recipients
synchronized with PGF2t* and eCG?. Theriogenology 2004 (in press).
Graves CN, Dziuk PJ. Control of ovulation in dairy cattle with human chorionic
gonadotrophin after treatment with 6 alpha-methyl-17 alpha-acetoxy-progesterone. J
Reprod Fert 1968;17:169-172.
Graves NW, Short RE, Randall RD, Bellows RA, Kaltenbach CC, Dunn TG. Estrus and
pregnancy following MPA, PGF2a, and GnRH. J Anim Sci 1974, 39:202 (Abstr.).


20
Progesterone and Progestins
Oral administration of progestins inhibits estrus and ovulation in cattle, and can
be used for estrus synchronization (Hansel et al., 1961). Natural P4 (Lamond, 1964) or
synthetic progestins (Wiltbank, 1968) have been used for estrus synchronization in cattle.
Based on the ability of progestins to synchronize estrus various long-term delivery
intravaginal devices such as PRID (Roche, 1976) and CIDR (Macmillan et al., 1991) or
ear implant such as Synchro Mate B (Burrel et al., 1972) were developed. These devices
together with oral administration of melengestrol acetate (MGA) are the most common
approaches using P4 administration for estrus synchronization.
Melengestrol acetate (MGA; 6a-methyl-6-dehydro-l 6-methylene-17a-acetoxy-
pregn-4,6-diene-3-20-dione) is an oral product with progestin activity and is
recommended at a dose of 0.5 mg/day (Zimbelman and Smith, 1966). Other oral
s
progestins are 16-alpha-17 dihydroxyprogesterone acetophenomide (DHPA) and 6-
chloro-A6-17 cetoxyprogesterone (CAP). They are not commonly used. Oral
administration of 400 mg/head/day of DHPA for 9 days combined with 5 mg of estradiol
benzoate on Day 2 (Wiltbank et al., 1968) or 10 mg/heifer/day of CAP for 14 d (Wagner
et al., 1973) have been used to synchronize estrus in beef cattle.
Norgestomet (SC21009;17a-acetoxy-l 1 (3-methyl-19-nor-preg-4-ene-3, 20-dione)
is a potent synthetic progestin that suppress estrus and ovulation at a daily i.m. dose of
0.14 mg (Wishart, 1972). It was incorporated in a polymer as a ear implant (Burrel et al.,
1972). An implant containing 3 or 6 mg of norgestomet implanted for 9 d and combined
with an i.m. injection of 3 mg of norgestomet and 5 mg of estradiol valerate at the time of
implant insertion (Wishart and Young, 1974) was named Synchro-Mate-B.


145
Pregnancy Diagnosis to Resynchronized Insemination
Pregnancy diagnosis was conducted 27 (1) d after the resynchronized
insemination using a linear ultrasonography scanner with a 5 MHz transrectal probe
(Aloka 500V, Wallingford, CT 06492). Cows were classified as 1) pregnant: embryo
proper with heartbeats and surrounded by a fluid-filled cavity representing the allantoic
cavity as detected in the uterine lumen (Pierson and Ginther, 1984a; Kastelic et al.,
1987); 2) questionable pregnant: fluid in the uterine lumen and CL but no embryo
observed, or 3) not pregnant: cows that were inseminated at detected estrus before
ultrasonography or diagnosed not pregnant at ultrasonography. On Days 45 and 90,
pregnancy was reconfirmed by per rectum examination of the genital tract based on
previously described criteria (Zemjanis, 1962).
Statistical Analysis
/
Baseline comparisons for parity (1,2,3+), season (August and September and
October to December), DIM (quartiles), inseminator (A, B, C, D, E, F) and
stages/abnormalities of the estrous cycle (diestrus, metestrus, proestrus, ovarian cysts,
anestrus) were carried out to establish comparability between groups using a Chi-square
test (Proc FREQ, SAS system). Pregnancy rates on Days 27, 45 and 90 and pregnancy
losses between Days 27 and 45 and 45 and 90 for both groups were compared using Chi-
square. In addition, pregnancy rates on Days 27, 45 and 90 were compared using a
multivariate analysis to evaluate the interaction between groups and stages/abnormalities
of the estrous cycle on pregnancy rate. Parity, season, DIM and inseminator were
included in the model to adjust for residual confounding, and they remained in the model
at P<0.15 using the backward elimination method of the Logistic Regression procedure in


71
release pattern of deslorelin in vitro is over a 4-day period (Mattos et al., 2001), there
appears to be a greater period of deslorelin release in vivo or an extended period of
pituitary desensitization to GnRH that was induced by deslorelin.
Continuous administration of GnRH (Gong et al., 1995; Gong et al., 1996;
Lamming and McLeod, 1988) or a single administration of a deslorelin implant (Mattos
et al., 2001) desensitizes the pituitary gland to the stimulatory effect of GnRH. Chronic
administration of 10 pg of GnRH and 5 pg of buserelin (GnRH agonist) twice a day for
21 d in beef heifers suppressed pulsatile secretion of LH, but not FSH, and inhibited the
growth of follicles greater than 9 mm (Gong et al., 1995). A more prolonged buserelin
administration (using a minipump) suppressed LH and FSH concentrations, and follicles
did not grow more than 5 mm in diameter (Gong et al., 1996). Administration of different
doses of a deslorelin implant during the postpartum period and at various stages of the
estrous cycle suppressed follicular development and delayed first estrus or extended the
interestrus interval (Rajamahendran et al., 1998; DOcchio et al., 1996; Mattos et al.,
2001). Induction of ovulation with a 700 pg deslorelin implant reduced the number of
Class III follicles, did not extend CL lifespan, but some cows failed to ovulate by Day 36
(Ambrose et al., 1998).
The luteolytic effect of free and conjugated estrogens in cattle has been reported
previously (Greenstein et al, 1958; Wiltbank, 1966; Eley et al., 1979). Administration of
estradiol-17(3 on Day 13 (Thatcher et al., 1986) or Day 17 (Knickerbocker et al., 1986) of
the estrous cycle induces PGF2tt secretion that was associated with luteolysis. In addition,
follicular development and estradiol levels have a negative effect on CL lifespan
(Fogwell et al., 1985) and fertility (Maurer and Echternkamp, 1982; Randel et al., 1971).


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216
Table 10.8. LSM and STE for plasma P4 concentration on Days 0, 7, 10 and 17 for cows
in proestrus and with ovarian cysts for all 4 groups.
Group* stage
Ovarian Response
Plasma P4 concentration ng/ml
Day 0
Day 7
Day 10
Day 17
GnRH
Proestrus
0.24 0.54
2.38 0.59
0.13 0.53
3.27
0.58
Ovarian cysts
1.82 0.57
3.02 0.54
0.61 0.50
4.34
0.47
CIDR
Proestrus
0.59 0.57
1.73 0.72a
0.14 0.58
3.54
0.71
Ovarian cysts
1.57 0.60
3.23 0.68
0.12 0.58
1.83
1.00
GnRH+CIDR
Proestrus
0.43 0.60
2.55 0.89
1.00 0.71
2.12
0.71
Ovarian cysts
0.85 0.60
3.85 0.54c
0.32 0.51
2.51
0.45
Control
Proestrus
0.69 0.57
3.02 0.63
0.17 0.58
3.32
0.82
Ovarian cysts
1.41 0.54
2.50 0.56d
1.0 0.50
1.59
0.71
For plasma P4 on Day 7:
c > d P<0.08; c > a P<0.05
For plasma P4 rise from Day 10 to Day 17: GnRH*Stage*Day P<0.06


215
Table 10.7. Number of cows, adjusted odd ratios (AOR), 95% confidence interval (Cl)
and levels of significance for the risk of presence of a CL on Day 17 in cows in proestrus
and with ovarian cysts treated with GnRH on Day 0 adjusting for BCS.
Variable
CL on Day 17
% N
AOR
95% Cl
P value
Treatment*stage
GnRH Ovarian cysts
95.0
19/20
Referent
Referent
-
No GnRH Ovarian cysts
69.6
16/23
0.1
0.01-1.1
0.06
GnRH Proestrus
85.2
23/27
0.2
0.02-2.4
0.22
No GnRH Proestrus
84.4
27/32
0.2
0.02-2.2
0.19
BCS
<3.0
70.8
17/24
Referent
Referent
-
>3.0
85.9
67/78
3.4
1.0-11.9
0.04


103
Table 5.1. Effect of a deslorelin implant in a timed AI protocol on fertility of lactating
dairy cows.
Treatment1
P<
2
Parameter
Control
Deslorelin 450
Deslorelin 750
Cl
C2
PR Day 27 (%)
39.0a
41.3a
27.5b
0.19
0.01
PR Day 41 (%)
33.5a
38.0a
24.8b
0.49
0.01
Pregnancy loss
(%)
12.7C
5.0d
9 5cd
0.18
0.36
PR 2nd AI (%)
25.2ac
16.7d
9.6b
0.01
0.18
P4 Day 11
(ng/mL SEM)
7.08 (0.33)
7.01 ( 0.44)
7.75 (0.38)
0.42
0.16
PR=pregnancy rate
a,b Superscripts within the same row differ (P < 0.05).
c,d Superscripts within the same row tend to differ (P < 0.13).
1 GnRH=100 p.g of gonadorelin; DESLORELIN 450=biodegradable implant containing
450 pg of deslorelin; DESLORELIN 750 = biodegradable implant containing 750 pg of
deslorelin.
2 Cl = control vs deslorelin; C2 = DESLORELIN 450 vs DESLORELIN 750.


228
of pituitary-ovarian tissues (i.e. downregulation or desensitization of GnRH receptors in
the pituitary or LH receptors in the ovarian follicles and CL) that results in detrimental
effects on embryo survival. In contrast, induction of ovulation with a 450 pg deslorelin
implant and induction of accessory CL may result in a reduction of early and late
embryonic mortality to increase pregnancy rates.
Different strategies to induce ovulation and enhance CL function resulted in slight
improvement in plasma P4 concentration and reduction in late embryonic mortality, but
the effect on increasing pregnancy rate or reducing early embryonic mortality were not
highly significant. The reduction in late pregnancy losses in cows forming accessory CL
after deslorelin implant treatment warrants further investigation. A lower dose of
deslorelin implant, short-release deslorelin compounds or other alternatives such a
treatment with hCG at the time of pregnancy diagnosis by ultrasonography (Day 28-30)
may reduce late pregnancy loss without affecting follicular growth.
Resynchronization of ovulation and timed insemination in nonpregnant lactating
dairy cows at Day 27 after insemination using the Ovsynch and Heatsynch protocols
resulted in similar pregnancy rates. However, cows in metestrus had higher pregnancy
rate with the Heatsynch protocol while cows with ovarian cysts had a higher pregnancy
rate with the Ovsynch protocol. Assignment of resynchronization protocols according to
stages of the estrous cycle at the time of nonpregnancy diagnosis resulted in increased
pregnancy rates. Therefore, cows in diestrus and proestrus should be subjected to either
the Ovsynch or Heatsynch protocols, cows in metestrus should be subjected to the
Heatsynch protocol considering efficient estrus detection, and finally alternative
treatment should be considered for cows with ovarian cysts (pre-treatment with GnRH or


249
Lopez-Gatius F, Santolaria P, Yaniz J, Rutllant J, Lopez-Bejar M. Factors affecting
pregnancy loss from gestation Day 38 to 90 in lactating dairy cows from a single herd.
Theriogenology 2002;57:1251 -1261.
Louca A, Legates JE. Production losses in dairy cattle due to days open. J Dairy Sci
1968,51:573-583.
Louis TM, Hafs HD, Morrow DA. Intrauterine administration of prostaglandin F2 alpha
in cows: Progesterone, estrogen, luteinizing hormone, estrus and ovulation. J Anim Sci
1974; 38:347-353.
Lucy MC. Reproductive loss in high-producing dairy cattle: where will it end? J Dairy
Sci 2001, 84:1277-1293.
Lucy MC, Savio JD, Badinga L, de la Sota RL, Thatcher WW. Factors that affect ovarian
follicular dynamics in cattle. J Anim Sci 1992;70:3615-3626.
Lucy MC, Stevenson JS. Gonadotropin-releasing hormone at estrus: luteinizing hormone,
estradiol, and progesterone during periestrual and postinsemination periods in cattle. Biol
Reprod 1986, 35: 300-311.
Lukesewska J, Hansel W. Corpus luteum maintenance during early pregnancy in the cow.
J Reprod Fert 1980;59:485-493.
Lulai C, Kastelic JP, Carruthers TD, Mapletoft RJ. Role of luteal regression in embryo
death in cattle. Theriogenology 1994;41:1081-1089.
Macmillan KL. Prostaglandin responses in dairy herd breeding programmes. NZ Vet J
1983, 31:110-113.
Macmillan KL, Burke CR. Effects of oestrus cycle control on reproductive efficiency.
Anim Reprod Sci 1996, 42:307-320.
Macmillan KL, Curnow RJ, Morris GR. Oestrus synchronization with a prostaglandin
analogue: I. systems in lactating dairy cattle. NZ Vet J 1977, 25:366-372.
Macmillan KL, Day AM, Taufa VK, Gibb M, Pearce MG. Effects of an agonist of
gonadotrophin releasing hormone in cattle I. Hormone concentrations and oestrus cycle
length. Anim Rep Sci 1985;8:203-212.
Macmillan KL, Peterson AJ. A new intravaginal progesterone releasing device for cattle
(CIDR-B) for oestrus synchronization, increasing pregnancy rates, and the treatment of
post-partum anoestrus. Anim Reprod Sci 1993, 33:1-25.


190
Table 9.13. Pregnancy rates at Days 30, 55 and 90 for Ovsynch and GnRH+Ovsynch
groups in cows with ovarian cysts classified according type of cyst and plasma P4
concentration.
Variable
PR at Day 30
PR at Day 55
PR at Day 90
%
N
%
N
%
N
Follicular cyst
Ovsynch
18.6a
13/70
17.2a
15/87
12.9a
11/85
GnRH+Ovsynch
32.1b
26/81
27.0b
27/100
23.0b
23/100
Luteal cyst
Ovsynch
33.3
3/9
30.0
3/10
30.0
3/10
GnRH+Ovsynch
12.5
1/8
22.2
2/9
22.2
2/9
< 1 ng/ml plasma P4
Ovsynch
19.2
10/52
19.1
13/68
13.6
9/66
GnRH+Ovsynch
29.0
18/62
24.4
19/78
20.5
16/78
> 1 ng/ml plasma P4
Ovsynch
20.0
5/25
19.2
5/26
19.2
5/26
GnRH+Ovsynch
33.3
8/24
35.7
10/28
32.1
9/28
PR=pregnancy rate; Interaction Group*Type (follicular or luteal) of cyst P<0.05


172
after administration of ECP (Brtolome et al., 2004). In the present study, only 16% of the
cows were inseminated at detected estrus after ECP, and this may explain the lower
pregnancy rate. This difference between studies also reflects differences in efficacy of
estrus detection as a management variable to consider when using the Heatsynch
protocol. Cows in metestrus have already initiated a follicular wave and GnRH will not
induce follicular turnover. Therefore, these cows may be more fertile if they are either
inseminated at detected estrus following ECP in the Heatsynch protocol or delay the
initiation of the Ovsynch protocol. The benefit of the GnRH+Ovsynch protocol
probably relies on the delay of initiating the Ovsynch protocol, since the first dose of
GnRH will not have any effect, but the Ovsynch is started 8 d later at a more appropriate
stage of the estrous cycle.
For cows in proestrus, there was no difference in pregnancy rates for cows in the
Ovsynch or GnRH+Ovsynch groups. This is in agreement with a previous study where
pregnancy rate was similar for the Ovsynch protocol administered in cows either in
diestrus or proestrus (Bartolom et al., 2004).
In a previous study, the Ovsynch protocol was effective in cows with ovarian
cysts (Bartolom et al., 2000). In the present study, pregnancy rates were higher for cows
with ovarian cysts in the GnRH+Ovsynch protocol compared to cows in the Ovsynch
protocol. Cows with ovarian cysts continue to have follicular waves (Hamilton et al.,
1995; Yoshioka et al., 1996), therefore the dominant follicle may or may not respond to a
GnRH treatment (Kesler et al., 1981). In the GnRH+Ovsynch protocol, cows received
two doses of GnRH and may be more likely to have a CL at the time of PGF2a In a
previous study, however, a pretreatment with GnRH at 7 d before the Ovsynch protocol


122
Table 6.4. Pregnancy rates on Day 55, adjusted odd ratios (AOR), 95 % confidence
interval (Cl), and level of significance for the risk of nonpregnancy in cows in all groups.
Variable
Pregnancy
AOR
95 % Cl
P value
%
N
Group
1
39.3
84/214
0.9
0.6-1.3
0.19
2
38.8
81/209
0.9
0.6-1.4
0.27
3
28.3
60/212
1.4
1.0-2.2
0.01
4
Season
36.2
71/196
Referent
Referent
NA
November-February
42.8
187/437
Referent
Referent
NA
March/April
27.7
109/394
1.9
1.5-2.6
0.0001
NA= not applicable


182
Table 9.5. Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between Days
30-55 and 55-90 for cows in diestrus for the Ovsynch and Modified Quicksynch groups
from June to December.
Group
Variable
Ovsynch
% (N)
Modified Quicksynch
% (N)
Pregnancy Rate
Day 30
34.4 (44/128)
27.1 (32/118)
Day 55
24.0 (37/154)
26.2 (37/141)
Day 90
23.6 (33/153)
21.6(33/140)
Pregnancy Losses
Day 30-55
23.2 (10/43)a
6.4(2/31)
Day 55-90
5.7 (2/35)
8.3 (3/36)
a-b PO.05


18
of bST may contribute to a negative energy balance that may result from increased milk
production which could affect growth of the dominant follicle indirectly, and a direct
effect of bST stimulating subordinate follicles and also caused by the increase in IGF-I
secretion (Lucy et al., 1992). Administration of bST in cattle increases the population of
small ovarian follicles (Gong et al., 2002) and in combination with FSH increases the
superovulatory response of dairy heifers (Kuehner e al., 1993).
Drugs Used for the Pharmacological Control of the Estrous Cycle
Prostaglandin F2a
Prostaglandins (PGs) were first detected in human semen and sheep seminal
plasma. They were named PGs since it was believed that they were produced in the
prostate gland. They were shown to cause smooth muscle contraction and changes in
blood pressure. They are a derivative of the prostanoid acid which is form by five
cyclopentane rings (Zubay et al., 1995). They were reported as luteolytic in pregnant rats
(Pharris and Wingarden, 1969), sheep (McCracken et al., 1972) and consequently either
natural (Lauderdale et al., 1972; Rowson et al., 1972) or synthetic PGF2ct(Tervit et al.,
1973) were used to induce luteolysis in cattle. The half-life of prostaglandins is very short
and that is useful for the organism in order to remove the hormonal action when it is no
longer required (Zubay et al., 1995). The half-life of the primary PGF2a is 1 minute and
the half-life of PGFM is 8 minutes (Kindahl et al., 1976). Natural PGF2a (25 mg i.m.,
dinoprost tromethamine; Lutalyse*, Pharmacia) and synthetic PGF2a (500 pg i.m.,
cloprostenol sodium, Estrumate*, Schering Plough) can be used to induce luteolysis in
cattle.


GnRH
PGFJa
GnRH
TAI
Ovsynch
|
I
16 h i
DO
D7
1
D9
1
DIO
Quicks ynch
PGF2a
I
ECP
1
AIDE
I
TAI
I
1
DO
1
D1
1
D2
1
D3
Modified
Quicks ynch
P(jl*2a
1
-i
1
-4
4 1
DO D1 D2 D3 D4 D14
Heatsynch
GnRH
pgf2i
I
ECP
AIDE TAI
I I
DO
1
D7
D8
1 1
D9 DIO
GnRH+Ovsynch
GnRH
GnRH
pgf2
GnRH
|
TAI
DO
D8
D15
D7
D18
Figure 9.1. Protocols used for resynchronization depending upon stage of the estrous
cycle at the time of a nonpregnancy diagnosis at Day 30 after insemination
AIDE= AI at detected estrus based on visual observation and pedometers


84
(DOcchio et al., 2000). This transient state of insensitivity to GnRH decreases
gonadotropin support for follicle growth and maturation. Collectively, use of a low dose
biodegradable deslorelin implant results in normal or improved luteal function (Ambrose
et al., 1998) associated with a transient suppression in follicle development (Ambrose et
al., 1998; Rajamahendran et al., 1998; DOcchio et al., 2000; Bartolom et al., 2004),
which might benefit fertility.
We hypothesized that the use of an implant of a GnRH agonist deslorelin in a TAI
protocol should synchronize ovulation, delay follicular growth, and improve CL
differentiation and development, as indicated by an increased progesterone concentration
during diestrus. These effects are expected to improve embryo survival in lactating dairy
cows.
The objectives were to evaluate follicular development, luteal activity, PR, and
embryo survival in lactating dairy cows given 450 pg (DESLORELIN 450) or 750 pg
(DESLORELIN 750) deslorelin implant or 100 pg gonadorelin to synchronize ovulation
in the Presynch/Ovsynch protocol (Moreira et al., 2001) during the first postpartum Al.
Materials and Methods
Animals, Housing, and Feeding
Early lactation Holstein cows (293 primiparous and 300 multiparous) on a
commercial dairy farm in the Central Valley of California were assigned to one of two
treatments in a randomized, complete-block design. The average lactation (1.97 0.05),
postpartum interval at the beginning of the study (29.5 0.12 d), and BCS (3.3 0.01)
did not differ among treatments (P > 0.50). The study period lasted for the first two
postpartum Al. During the entire study, all cows received the same total mixed ration to


72
Electrocauterization and x-radiation of ovarian follicles on day 10 of the cycle reduced
estradiol levels and extended CL lifespan (Fogwell et al., 1985). Pregnant heifers had a
lower estradiol/progesterone ratio compared to nonpregnant heifers (Maurer and
Echternkamp, 1982). High levels of estradiol on days 14 to 17 of the cycle may have a
negative effect on fertility (Pritchard et al., 1994). Interestingly, higher conception rate
was reported in cows with three follicular waves than those with two follicular waves
(Ahmad et al., 1997). In addition, cows with follicles smaller than 15 mm in diameter
between days 12 and 14 of the cycle had higher conception rate compared with cows
having follicles larger than 15 mm (Hernandez Cern et al., 2001). The present
experiment indicates that the administration of a deslorelin implant, as part of a timed
insemination protocol, reduced follicular development during this critical period when
the CL is maintained for pregnancy. Indeed a deslorelin implant appears to reduce
s
pregnancy losses from day 27 to day 45 compared to GnRH when used to induce
ovulation in the Ovsynch protocol. However, pregnancy rates at day 27 and day 45 were
not different (Santos et al., 2002).
In conclusion, deslorelin implants induced ovulation, stimulated development of a
normal CL, and delayed follicular growth during the subsequent diestrus period. The
effect of a slight increase in plasma concentrations of P4 and the attenuation of follicular
development on embryo survival warrant further investigation. However, the dose will
have to be adjusted and nonpregnant cows will have to be resynchronized (e.g., at the
time of pregnancy diagnosis) to minimize the abnormal return to estrus and ovulation.


92
CL), in cows treated with deslorelin was dose-dependent. Ovarian activity on Day 27 was
reduced (P<0.05) in treated with DESLORELIN 750 compared to DESLORELIN 450.
A greater proportion of non-pregnant cows were re-inseminated prior to Day 27 in
the control group compared to cows induced to ovulate with deslorelin (50.6 vs 18.5%; P
< 0.001), with a more exacerbated effect of the higher dose of deslorelin (P < 0.01).
Furthermore, re-insemination interval in non-pregnant cows prior to Day 27 after the
initial TAI was longer for cows induced to ovulate with deslorelin than with gonadorelin
(P< 0.001).
On Day 7 of the re-synchronization period (Table 5.3), at the moment of the
PGF2a injection, a lower proportion of deslorelin cows had a follicle with a diameter
equal to or greater than 10 mm compared to control cows (72.3 vs 88.6%; P < 0.01); the
negative effect of deslorelin on the presence of a large follicle tended to be more
/
pronounced in cows initially treated with the higher dose deslorelin (P = 0.07). Similarly,
deslorelin also reduced the proportion of cows with a CL 7 d after the GnRH treatment,
as well as ovarian activity, which was the proportion of cows on Day 7 of the re
synchronization with either a CL or a follicle greater than 10 mm. The diameter of the
largest follicle when PGF2a was given tended to be smaller for cows treated with
deslorelin compared to controls, but no difference was observed for the different
deslorelin doses.
Discussion
This study was designed to determine the effects of replacing gonadorelin with a
GnRH agonist implant deslorelin to induce ovulation in the Ovsynch/TAI protocol on
PR, pregnancy loss, luteal function, and follicular development in lactating dairy cows.


259
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hormonal parameters and time relationships concerning oestrus, ovulation, and electrical
resistance of the vaginal mucus. Acta Endocrin 1977, 86:180-192.
Schmitt EJ-P, Diaz T, Barros CM, de la Sota RL, Drost M, Fredriksson EW, Staples CR,
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Schomberg DW, Coudert SP, Short RV. Effects of bovine lueinizing hormone and human
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Seguin BE, Oxender WD, Britt JH. Effect of human chorionic gonadotropin and
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185
Table 9.8. Distribution of cows by parity, season, DIM and inseminator for cows in
proestrus from January to December in both groups.
Variable
Ovsynch'
B
GnRH+Ovsynch
P value
%
N
%
N
Parity
1
38.2
34/89
41.4
36/87
0.86
2
24.7
22/89
25.3
22/87
3+
37.1
33/89
33.3
29/87
Season
Mar-Sep
60.7
54/89
67.8
59/87
0.32
Oct-Dec
39.3
35/89
32.2
28/87
DIM
90-173
23.6
24/89
23.0
20/87
0.93
174-208
24.7
22/89
25.3
22/87
209-283
24.7
22/89
25.3
22/87
284-552
23.6
21/89
26.4
23/87
Inseminator
A
36.0
32/89
56.3
49/87
<0.01
B
19.1
17/89
19.5
17/87
C
44.9
21/89
24.1
21/87


23
variable results (Wagner et al., 1973). Currently, hCG (i.m., 10,000 IU, Chorulon,
Intervet, Millsboro, DE 19966) is available for the treatment of cystic ovaries.
Estrogens
The naturally occurring estrogens in the cow are estradiol and estrone. Estradiol is
produced by the granulosa cells of mature follicles. The half-life of estradiol is around 5
minutes and metabolites are excreted in feces and urine. Synthetic estrogens are
metabolized more slowly in the liver and half-life is extended (Wright and Malmo, 1992).
Estradiol has been used to induce regression of the CL (Wiltbank et al., 1968), induce
atresia of antral follicles and synchronize ovarian follicular waves (Bo et al., 1995) and
induce ovulation (Smith and Zimbelman, 1968a, Welch et al., 1975). Different salts of
estradiol (estradiol valerate, estradiol cypionate, estradiol benzoate), and estradiol-170
can be used in cattle, but they are not approved for use in domestic farm animals. Doses
s
from 0.5 to 10 mg have been administered at different stages of the estrous cycle either
by intramuscular injections or in intravaginal capsules to synchronize follicular waves.
Estradiol-17(3 (Bo et al., 1994), estradiol benzoate (Macmillan and Burke, 1996),
estradiol valerate (Bo et al., 1993) and estradiol cypionate (Thundatil et al., 1998) have
been used alone or in combination with P4, to synchronize follicular waves in cattle.
Estradiol benzoate (10 mg) in a gelatin capsule placed in the groove of a CIDR device
and administered intravaginally had a variable effect on synchronization of estrus
(Macmillan and Burke, 1996). Estradiol-17(3 and estradiol benzoate have been the most
effective to synchronize the follicular wave in cattle in doses from 2 to 5 mg (Bo et al.,
1995). Estradiol cypionate (Pancarci et al., 2002) and estradiol benzoate 1 mg i.m. (Day,
1998) have been administered 48 h after PGF2a to synchronize ovulation in cattle.


150
has already been recruited and GnRH will fail to induce follicular turnover. Therefore, at
the time of induced ovulation 48 h after PGF2a, the follicle may be aged and fertility
reduced due to an extended period of dominance (Revah and Butler, 1996; Mihn et al.,
1999; Austin et al., 1999) as well as increased embryonic losses (Ahmad et al., 1995). In
the present study, a high proportion of cows in the metestrus stage assigned to Heatsynch
protocol were inseminated earlier at estrus on Day 9 (11/27) which may attenuate this
adverse effect and increase fertility.
In contrast, cows with ovarian cysts had a higher pregnancy rate to the Ovsynch
protocol compared to the Heatsynch protocol. The positive feed-back of estradiol on
inducing the LH surge is compromised in cows with ovarian cysts (Dobson and Alam,
1987; Refsal et al., 1988; De Silva and Reeves, 1988 Kaneko et al., 2002). Abnormal
release of the GnRH surge from the hypothalamus is believed to be the cause of ovarian
cysts in lactating dairy cows (Kesler and Garverick, 1982) and administration of GnRH
induced an LH surge in cows with ovarian cysts (Cantley et al., 1975). Therefore, the
Ovsynch protocol that includes injection of GnRH to induce ovulation may be more
effective than the Heatsynch protocol which attempts to induce hypothalamic release of
GnRH via injection of ECP in cows with an impaired positive feed-back to estradiol. In a
previous study, Ovsynch generated an acceptable pregnancy rate in lactating dairy cows
with ovarian cysts compared to normal cows (Bartolom et al., 2000).
In conclusion, Ovsynch and Heatsynch protocols for resynchronization, initiated
in nonpregnant cows at ultrasonography 27 d post-insemination, achieved similar
pregnancy rates. However, the Heatsynch protocol increased pregnancy rate for cows in
metestrus and the Ovsynch protocol was more effective for cows with ovarian cysts.


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Revah I, Butler WR. Prolonged dominance of follicles and reduced viability of bovine
oocytes. J Reprod Fert 1996;106:39-47.
Reynolds LP. Utero-ovarian interactions during early pregnancy : role of the conceptus-
induced vasodilation. J Anim Sci 1986;62(Suppl.2):47-61.


96
and during the period when the CL is maintained for pregnancy, late pregnancy losses
may be attenuated as observed in the current study. Collectively, these studies suggest
that there might be a period of sensitivity to attenuation of follicular growth on pregnancy
losses.
The ability of deslorelin to suppress follicular growth in cows and heifers has
been demonstrated previously (Ambrose et al., 1998; Rajamahendran et al., 1998;
Bartolom et al., 2004). When given during the early postpartum period, deslorelin
attenuated resumption of cyclicity in lactating dairy cows (Mattos et al., 2001). Similarly,
cows in different stages of the lactation cycle also have suppressed follicular
development after treatment with deslorelin (Bartolom et al., 2004). When non-lactating
cows were treated with a biodegradable deslorelin implant containing either 750 or 1000
pg or with a gonadorelin product as the last injection of GnRH in the Ovsynch protocol,
the first wave dominant follicle was smaller, emergence of the second follicular wave
was not altered, and the largest follicle during the second follicular wave also was
reduced in size during the first 16 d after treatment for the deslorelin cows compared to
cows treated with gonadorelin (Bartolom et al., 2004). The largest follicle during the
second wave of follicular development did not grow to a diameter larger than 7 mm in
cows treated with deslorelin, compared to approximately 15 mm on Day 16 after
treatment in cows that received gonadorelin.
The suppressive effect of deslorelin on follicle development was observed based
on the lower proportion of non-pregnant cows on Day 27 after TAI that had a follicle
with a diameter equal to or greater than 10 mm, and by the reduced follicle population
greater than 5 mm. The effect of deslorelin is dose-dependent (DOcchio et al., 2000); a


105
Table 5.3. Effect of a deslorelin implant in a timed AI protocol on ovarian activity 7 d
after re-synchronization with GnRH on Day 27 after the initial timed insemination.
Treatment1
P <2
Parameter3
GnRH
Deslorelin 450
Deslorelin 750
Cl
C2
Follicle > 10 mm
88.6a
83.3e
66.3bf
0.01
0.07
(%)
Corpus luteum
75 7ac
55.ld
60.9b
0.01
0.86
(%)
Ovarian activity
96.0C
91.8
84.8d
0.03
0.26
(%)
Response to GnRH
76.9
23.5
39.6
0.001
0.34
(%)
Largest follicle
13.5e
12.2
11.7f
0.15
0.73
(mm SEM)
(11)
(1.4)
( 0.9)
a,b Superscripts within the same row differ (P < 0. 01).
c,d Superscripts within the same row differ (P < 0.05).
e,f Superscripts within the same row tend to differ (P < 0.15).
1 GnRH = 100 pg of gonadorelin; DESLORELIN 450 = biodegradable implant
containing 450 pg of deslorelin; DESLORELIN 750 = biodegradable implant containing
750 pg of deslorelin.
2 Cl = control vs deslorelin; C2 = DESLORELIN 450 vs DESLORELIN 750.
3 Ovarian activity = presence of a CL or a follicle greater than 10 mm; Response to
GnRH = cows that had no CL on Day 27 when gonadorelin was given and had a CL on
Day 37.


25
1966). However, P4 supplementation for an extended period of time in beef heifers
reduced pregnancy rate (Hansel, 1967, Roche 1974a). Long term (14-21 days) treatment
with P4 for estrus synchronization results in low levels of P4 which increase LH
pulsatility and extends the growth of the dominant follicle which suppress growth of
other follicle for period of more than 20 days (Savio et al., 1993). Persistent follicles are
associated with follicular and oocyte degeneration (Mihn et al., 1994) and reduced
fertility (Savio et al 1993b). Reduction in the length of the treatment was a strategy to
improve fertility, and an oral progestin (Wiltbank 1968), a silastic subcutaneous implant
of P4 (Roche 1974b, 1974c) or a norgestomet implant (Wiltbank et al., 1971) were
combined with estrogen at initiation of treatment to induce premature regression of the
CL
However, the dose of P4 will influence gonadotropins control (Begfeld et
al.,1996) and treatments with MGA or Syncro-Mate-B generated levels of P4 lower than
mid-luteal phase resulting in greater LH pulsatility, persistent follicles and greater
production of estrogen (Kojima et al., 1992). Low levels (~2 ng/ml) compared with high
levels (~6 ng/ml) of P4 created with PRID inserts generated an acute (6-24 hours)
increase in LH pulsatility and 17P-estradiol concentrations (Bergfeld et al., 1996). The
use of 2 compared with 1 PRID insert during a 9 day period and administration of PGF2a
on Days -9 and -3 to lyse the natural CL, reduce estradiol concentrations and increased
pregnancy rate in the cow (Wehrman et al., 1993). Similarly, norgestomet treated cows
had lower levels of estradiol during treatment and higher conception rate to synchronized
estrus when a CL was present during the period of treatment (Sanchez et al., 1993).


205
synchronize follicular wave emergence is not effective during metestrus (Vasconcelos et
al., 1999; Moreira et al., 2000), therefore these cows probably need to be treated with
PGF2 7 d later and then inseminated at detected estrus to optimize pregnancy rate.
Alternatively, perhaps a CIDR insert (i.e. P4 exposure) and estradiol could induce
follicular turnover to synchronize follicular development for a subsequent TAI.
Cows without a CL (19.5%) were classified on Day 0 as proestrus (10.6%), with
ovarian cysts (7.5%) or anestrus (1.4%) and subjected to protocols that included GnRH
and a CIDR insert 7 d before PGF2 in a 2x2 factorial design. Cows in anestrus had a low
overall pregnancy rate, but the number of cows in this category was not sufficient to
compare differences among groups. Since all cows received a GnRH treatment on Day 23
post insemination, the proportion of cows with ovarian cysts was lower in the present
study (7.5%) compared with previous studies (14.4% and 19.0%; Bartolom et al., 2004;
2004). In addition, the proportion of cows with luteal cysts (56.5%) was higher compared
with a previous study (30.0%) in which GnRH was not given 7 d before diagnosis of
nonpregnancy and diagnosis of the cystic condition (Bartolom et al., 2004).
In cows with ovarian cysts, GnRH on Day 0 did not affect pregnancy rate.
However, GnRH increased the proportion of cows with CL on Day 7 (Figure 10.2), the
rate of ovulation after the second GnRH of the Ovsynch protocol (Figure 10.3), and
plasma P4 concentration on Day 17 (Figure 10.4). The effect of a GnRH treatment on
cows with ovarian cysts has been controversial due to the lack of controls in many
experiments and the proportion of cows recovering spontaneously (Bierschwal et al.,
1975; Cook et al., 1990). Consequently, some studies reported beneficial (Cantley et al.,
1975; Kesler et al., 1981; Nanda et al., 1988) or no effect (lou et al., 1999) of GnRH.


199
pregnancy rate on Day 30, 17.2% (21/122) of the cows were not evaluated since they
were culled from the herd before pregnancy diagnosis (three in the GnRH Groupp, six in
the CIDR Group, nine in the GnRH+CIDR Group, and three in Control Group), and 1.6%
(2/122) of the cows were diagnosed as questionable pregnant (one in the GnRH Group
and one in the GnRH+CIDR Group).
The number and proportion of cows with a CL on Days 7 and 17 for cows in
proestrus and with ovarian cysts for all four groups are described in Table 10.5. There
was a tendency for an interaction between GnRH on Day 0 and stages/abnormalities of
the estrous cycle (P<0.11; Figure 10.2) on the proportion of cows with a CL on Day 7.
GnRH administration on Day 0 increased the proportion of cows with ovarian cysts that
had a CL on Day 7 but cow in proestrus had a high proportion of CL on Day 7 regardless
of whether they had GnRH or not on Day 0 (P<0.01; Table 10.6). Cows with BCS greater
than 3.0 were 3.6 times more likely to have a CL on Day 7 than cows with BCS less than
3.0 (P=0.01; Table 10.6). Cows with high plasma P4 (>1 ng/ml) on Day 0 were 6 times
more likely to have a CL on Day 7 than cows with low plasma P4 on Day 0 (P=0.005,
Table 10.6). There was a tendency for an interaction between GnRH on Day 0 and
stages/abnormalities of the estrous cycle (P<0.12; Figure 10.3) on the proportion of cows
with a CL on Day 17. GnRH administration on Day 0 tended to increase the proportion of
cows with a CL on Day 17 in cows with ovarian cysts but not for cows in proestrus
(P=0.06; Table 10.7). Cows with BCS greater than 3.0 were 3.4 times more likely to have
a CL on Day 17 than cows with a BCS less than 3.0 (P=0.04; Table 10.7). A CIDR insert
from Day 0 to 7 did not increase the proportion of cows with CL on Day 7 and 17.


180
Table 9.3. Pregnancy rates at Days 30, 55 and 90, and pregnancy losses between Days
30-55 and 55-90 for cows in diestrus for the Ovsynch and Quicksynch groups from
January to May.
Group
Variable
Ovsynch
% (N)
Quicksynch
% (N)
Pregnancy Rate
Day 30
35.9 (33/92)a
21.7 (20/92)
Day 55
29.2 (28/96)c
16.7 (16/96)d
Day 90
26.0 (25/96)e
15.6 (15/96)f
Pregnancy Losses
Day 30-55
21.2(7/33)
20.0 (4/20)
Day 55-90
10.7(3/28)
6.2 (1/16)
a-b (PO.05); c-d;e-f (P<0.07)


76
Interval after treatment (d)
Figure 4.4. Least squares means and SE for the largest follicle after PGF2a on Day 16 for
cows in Control (), DESLORELIN 750 () and DESLORELIN 1000 (A) groups in
Experiment 1 (P<0.001).


117
From the results of this study, it was concluded that 1) GnRH treatment on Day 5
or Day 15 did not increase pregnancy rate, or reduce pregnancy loss between Day 27 and
Day 55 after TAJ; 2) Cows treated with GnRH on both Day 5 and Day 15 had a lower
proportion of cows diagnosed not pregnant based on expression of estrus before
ultrasonography on Day 27 (26.5%) compared to control cows (52.9%), and these cows
had an extended interestrus interval (23.4 d vs 21.5 d); and 3) GnRH treatment on both
Day 5 and Day 15 after TAI reduced pregnancy rate on Day 27 and Day 55 (36.8% and
28.3%) compared with control cows (44.4% and 36.2%). Therefore, strategies to
stimulate CL function, induce ovulation of dominant follicles and accessory CL
formation using multiple doses of GnRH during the luteal phase to enhance embryo
survival in lactating dairy cows subjected to induction of ovulation need to consider the
potential negative effects demonstrated in this study.


132
45. This finding is in agreement with a previous study reporting that cows with additional
CL at Day 38 of gestation were less likely to experience fetal loss between pregnancy
diagnosis and Day 90 (Lopez-Gatius et al., 2002). The differential effect of the deslorelin
implant between cows that formed accessory corpora ltea and those that did not may be
related to the amount of luteal tissue and the concentration of LH after treatment. The
presence of ovarian follicles was only recorded on Day 27 and Day 45. Therefore,
dynamics of follicular growth on pregnancy loss could not be fully evaluated. The fact
that cows with follicular suppression on Day 45 experienced a greater pregnancy loss
may reflect the negative effect of the implant in cows that did not form accessory CL.
The potential positive effect of a mild follicular suppression in cows that did form a CL
could not be evaluated since daily follicular dynamic was not recorded between Days 27
and 45. Future research is necessary to test the potential effect of accessory CL and
/
temporary follicular suppression on reducing late embryonic loss.


44
Progesterone administered at the time of follicular deviance inhibits LH secretion
and the largest follicle had reductions in diameter and concentration of estradiol and IGF-
1 in the follicular fluid (Ginther et al., 2001). Progesterone in high doses is able to induce
atresia of the dominant follicle and induce a new follicular wave (Anderson and Day,
1994). However, administration of 200 mg of P4 at the time of insertion of an intravaginal
P4 device to synchronize estrus at random stages of the estrous cycle was not as effective
as estradiol benzoate in inducing follicular turnover (Cavalieri et al., 2003).
Since estradiol can be used to synchronize the emergence of a new follicular wave
(Bo et al., 1995) and at the same time can induce GnRH release, increase GnRH receptors
in the pituitary and induce ovulation when administered during proestrus (Sumpf et al.,
1989; Kinder et al., 1991), protocols involving estradiol on Day 0 and then 24 hours after
P4 withdrawal have been used to synchronize ovulation for TAI. The advantage of
/
estradiol lies in the low cost of the treatment and the increased expression of behavioral
estrus. Estrus and ovulation synchronization with estradiol benzoate (1 mg) 24 hours after
or GnRH (gonadorelin, 0.25 mg) 34 hours after CIDR removal resulted in 97.5% of the
cows that received estradiol and 36.2% of the cows that received GnRH expressing
estrus (Cavalieri and Macmillan, 2002).
Function of the Corpus Luteum
Corpus Luteum Development and Differentiation
The LH surge secreted by the anterior pituitary gland induces ovulation and under
LH stimulation the ovarian follicle becomes the corpus luteum which secretes P4
(Niswender and Nett, 1994). Luteinizing hormone supports CL development,
differentiation and P4 production during the luteal phase (Hansel and Seifart, 1967). After


161
rectum examination and ultrasonography of the genital tract (Bartolom et al., 2004) and
then assigned to various resynchronization protocols (Figure 9.1).
The distribution of cows at different stages/abnormalities of the estrous cycle on
Day 0 was as follows: diestrus 45.2% (490/1083), metestrus 17.9% (194/1083), proestrus
16.2% (176/1083), ovarian cysts 19.0% (206/1083), and anestrus 1.6% (17/1083).
Cows in diestrus from January to May (n=192) were divided randomly into two
groups: Ovsynch Group (N=96) and Quicksynch Group (n=96). Cows in diestrus from
June to December (n=298) were divided randomly into two groups: Ovsynch Group
(n=156) and Modified Quicksynch Group (n=142). Cows in metestrus were divided
randomly into three groups: Ovsynch Group (n-68), Heatsynch Group (n=62), and
GnRH+Ovsynch Group (n=64). Cows in proestrus, or with ovarian cysts, or anestrus
were divided randomly into two groups: Ovsynch Group (proestrus n=89, ovarian cysts
n=97, anestrus n=8) and GnRH+Ovsynch Group (proestrus n=87, ovarian cysts n=109,
anestrus n=9). The hormonal doses utilized were 100 (ig (i.m.) GnRH (Cystorelin, Merial
Limited, Iselin, NJ, 08830), 25 mg (i.m.) PGF2a (Lutalyse, Pharmacia, Kalamazoo, MI,
49001) and 1 mg (i.m.) ECP (Pharmacia, Kalamazoo, MI, 49001). Cows were TAI 16
after GnRH and 48 h after ECP. Cows detected in estrus between PGF2a treatment and
TAI were inseminated and included in the study. On Day 0, parity, time of the year
(season), days in milk (DIM), and inseminator were recorded.
Plasma P4 Concentration
On Day 0 a blood sample was obtained from the coccygeal vessels into evacuated
tubes containing EDTA (Vacutainer, BD, Franklin Lakes, NJ 07417) and placed on ice
immediately after collection. Samples were centrifuged at 3000 Xg for 30 minutes, and


138
Days of Pregnancy
11 Control Implant
Figure 7.3. Number of CL on D 27 and D 45 of Pregnancy in
Lactating Dairy Cows after Treatment with 2.1 mg Deslorelin Implant
(Interaction P<0.01)


213
Table 10.5. Ovarian responses on Days 7 and 17 for cows in different
stages/abnormalities of the estrous cycle in all 4 groups.
Group* Stage
Ovarian Response
CL Day 7
CL Day 17
%
N
%
N
GnRH
Proestrus
75.0
9/12
72.7
8/11
Ovarian cysts
72.7
8/11
100
11/11
CIDR
Proestrus
57.9
11/19
83.3
15/18
Ovarian cysts
45.4
5/11
72.7
8/11
GnRH+CIDR
Proestrus
76.5
13/17
93.7
15/16
Ovarian cysts
81.8
9/11
88.9
8/9
Control
Proestrus
78.6
11/14
85.7
12/14
Ovarian cysts
36.3
4/11
66.7
8/12
For the presence of CL on Day 7: GnRH* Stage P<0.11
For the presence on CL on Day 17: GnRH*Stage P<0.12


Table 10.12. Number of cows, adjusted odd ratios (AOR), 95% confidence interval (Cl)
and levels of significance for the risk of pregnancy on Day 90 in cows in proestrus and
with ovarian cysts treated with CIDR on Day 0 adjusting for BCS.
Variable
PR on Day 90
% N
AOR
95% Cl
P value
Treatment* stage
CIDR Proestrus
12.5
4/32
Referent
Referent
-
No CIDR Proestrus
37.0
10/27
4.6
1.2-19.3
0.02
CIDR Ovarian cysts
31.6
6/19
4.0
0.9-18.0
0.06
No CIDR Ovarian cysts
13.0
3/23
1.0
0.2-5.0
0.98
BCS
<3.0
9.5
2/21
Referent
Referent
-
>3.0
26.3
21/80
4.1
0.8-20.3
0.08
PR=Pregnancy rate


99
presence of a CL on Day 11 after TAI. Interestingly, on Day 27 after TAI, there were 34
cows treated with deslorelin that had luteal concentrations of progesterone (>1.5 ng/mL)
on Day 11 after TAI, but were non-pregnant on Day 27 in the absence of a CL and with
only follicles < 9 mm in diameter. Although we did not ultrasound these cows throughout
the post-insemination period, this response would indicate some cows underwent
luteolysis in the absence of normal follicular development. Pritchard et al. (1994)
observed that high levels of estradiol during late diestrus reduce fertility. Large dominant
follicles are more estrogenic than small subordinate follicles. It is suggested that the
second-wave dominant follicle in lactating cows that reach maximum size at
approximately the same time the CL is maintained for pregnancy and raises circulating
estradiol concentrations is partially responsible for stimulating the luteolytic cascade.
However, luteolysis occurred even in the absence of large follicles during diestrus in
/
cows treated with deslorelin (Ambrose et al., 1998; Rajamahendran et al., 1998).
Therefore, a lower estradiol threshold or an alternate pathway is responsible for
triggering the luteolytic cascade in cows.
In conclusion, deslorelin induced the formation of a CL that resulted in normal
mid-luteal phase plasma P4 concentrations, decreased return to estrus in non-pregnant
cows, reduced follicular population at Day 27 after treatment, and suppressed response to
GnRH during re-synchronization of non-pregnant cows. Treatment with a DESLORELIN
450 resulted in similar pregnancy rate to gonadorelin, but the higher-dose deslorelin
impaired fertility. However, DESLORELIN 450 tended to reduce late embryonic losses
compared to gonadorelin, which warrants further investigation. Nevertheless, additional
studies should determine the ideal dose of deslorelin to minimize the carry over effect of


30
transport takes 6 to 12 hours (Hawk, 1987), sperm viability is 24 to 32 hours, ovum
viability is 8 to 12 hours (Thibault, 1967) and at least 10 accessory sperm per embryo are
necessary to achieve satisfactory embryo quality (Saacke et al., 2000). Based on the
dynamics during periovulatory period, insemination is recommended at 12 hours after the
LH surge (Trimberger and Davis, 1943; Dalton et al., 2001).
Ovulation can be induced by administering GnRH to directly stimulate the
anterior lobe of the pituitary gland to release LH (Reeves et al., 1972; Kaltenbach et al.,
1974), hCG to directly induce the follicle in the ovaries to ovulate (Graves and Dziuk,
1968; Schmitt et al., 1996), or estradiol to stimulate GnRH release from the
hypothalamus and indirectly LH release from the pituitary gland (Hobson and Hansel,
1972; Inskeep, 1973; Roche, 1974). In order to obtain better synchronization of estrus,
two doses of PGF2a could be administered 12 days apart. Although estrus is induced in
s
the majority of the cows, the estrus periods were still spread from Days 2 to 4 after the
last PGF2 After the two doses of PGF2a, TAI without induction of ovulation (Lauderdale et al.,
1974; Stevenson et al., 1987) or after administering GnRH at 48 hours after the last dose
of PGF2a (Lucy and Stevenson, 1986), or GnRH 48 hours after PGF2a (Thatcher and
Chenault, 1975) resulted in reduced fertility, but not when it was combined with estrus
detection. Cows in diestrus treated with PGF2a and inseminated at detected estrus for 72
hours, and cows not detected in estrus treated with GnRH and time-inseminated at 72-80
hours had pregnancy rates similar to cows inseminated after detected spontaneous or
PGF2a-induced estrus (Archbald et al., 1992).


36
Factors Affecting the Efficacy of GnRH to Synchronize Follicular Waves
Several factors such as stage of the estrous cycle, parity, days in milk, heat stress,
bovine somatotropin (bST) treatments, and ovarian abnormalities (anestrus and cystic
ovarian degeneration) may affect pregnancy rate to protocols for TAI using GnRH and
PGF201. The key for success of estrous synchronization and induction of ovulation
protocols using GnRH and PGF201 lies in the effectiveness of the GnRH treatment to
ovulate a follicle present in the ovaries and to recruit a new follicular wave which will
produce a healthy follicle which will ovulate and form a strong CL after PGF2 treatment.
Stage of the estrous cycle affects the response to an injection of a GnRH analogue
or agonist to synchronize follicular wave emergence. Induction of follicular turnover
depends on the presence of a dominant follicle of at least 10 mm in diameter, and the
ability of GnRH to induce ovulation of that follicle (Moreira et al., 2000). The Ovsynch
protocol was more efficient when initiated during mid-cycle (Day 5 to 9) since most of
(K)
the cows ovulated to the first GnRH injection (Moreira et al., 2000). When Ovsynch
was initiated on Days 1-4, 5-9, 10-16 and 17-21, ovulation rate to first GnRH was 23, 96,
54 and 77 %, respectively. The ovulation rate to the second GnRH was 94, 89, 85 and
81% when size of the follicle at second GnRH (i.e. 48 h after PGF2) was 19, 17, 18 and
20 mm, respectively. Overall, 93% of the animals regressed their CL after PGF2a
(Vasconcelos et al., 1999). When cows ovulated to the first GnRH, 92% of them ovulated
to the second GnRH injection, but only 79% ovulated if they failed to ovulate to the first
GnRH injection. When the protocol is initiated in late diestrus, 6% of cows ovulated
before the second GnRH, and 7% failed to ovulate at 48 hours after the second dose of
GnRH (Vasconcelos et al., 1999). The Ovsynch protocol initiated early in the luteal


41
may be in postpartum anestrus and GnRH and PGF2a may not be effective. However,
GnRH based protocols are more effective than PGF2a alone since the GnRH injection
may induce cyclicity in a small proportion of anestrus cows (-15-25%; Day, 1998). An
extra benefit can be obtained by including a P4 supplement (e g. CIDR Insert) between
the GnRH and PGF2a of the Select Synch protocol which collectively avoid estrus
expression before PGF2a, increases estrous synchronization by 10%, and induces estrus in
an additional 20% of anestrus cows (Day, 1998).
However, if noncyclic status of prepuberal heifers and postpartum beef cows are
very pronounced, presynchronization with p P4 may be necessary. Administration of
GnRH induced ovulation in 75% (Thomson et al., 1999) to 83% (Troxel and Kesler,
1984) of the anestrous beef cows between Days 17 to 44 and 21 to 30 postpartum,
respectively. When a progestin was administered for 8 days, GnRH treatment on Day 8
r
induced a greater LH surge, induced ovulation in 100% of the cows, and reduced the
incidence of short luteal phases (Troxel and Kesler, 1984). A progestin between GnRH
and PGF2a given 7 days apart increased the proportion of cows that ovulated and had a
normal luteal phase to a GnRH given 48 hours after the PGF2a (Thompson et al., 1999).
A traditional protocol for estrus synchronization in beef heifers in the USA was to
feed MG A for 14 days and administer PGF2a 19 days after MG A removal (Patterson
et al., 1989). Administration of GnRH 7 days before PGF2a in the MGA protocol
increased synchronization of estrus and is called MGA Select (Patterson et al., 2003).
Reduction in the length of the treatment was achieved by using the 7-11 Synch protocol
(MGA Day 0 to 7, PGF2a Day 7, GnRH Day 11, PGF2a Day 18 and insemination at
detected estrus). The 7-11 Synch program increased estrus synchrony compared to the


61
^Control hCG
Figure 3.3. Size of CL in nonlactating dairy cows treated with human
chorionic gonadotropin (hCG) 8 to 10 h after onset of estrus.


To Milagros and Santiago


210
Table 10.2. Pregnancy rates and pregnancy losses for cows in diestrus and subjected to
the GnRH or ECP groups.
Variable
Group
GnRH
ECP
%
N
%
N
Pregnancy rate
Day 30
27.5
58/211
29.1
61/210
Day 55
26.5
58/219
25.5
55/216
Day 90
24.2
53/219
24.1
52/216
Pregnancy losses
Day 30-55
7.0
4/57
9.8
6/61
Day 55-90
8.6
5/58
5.4
3/55


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1996;1:193-202.
Anderson LH, Day ML. Acute progesterone administration regresses persistent dominant
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232


135
Table 7.3. Number of cows, percentages, adjusted odd ratios and 95% confidence interval
for the risk of pregnancy loss between Day 27 and Day 45 for cows with accessory CL in
the both groups.
Variable
Pregnancy Loss
% N
AOR
95% Cl
P value
Treatment
Control
Accessory CL
No
13.7
11/80
Referent
Referent
NA
Control
Yes
33.3
3/9
3.2
0.7-14.9
0.12
Implant
No
34.0
16/47
2.1
0.8-5.4
0.13
Implant
Yes
5.1
2/39
0.1
0.03-0.9
0.04
Follicular Suppression
No
14.9
23/154
Referent
Referent
NA
Yes
42.9
9/21
6.1
1.7-21.2
0.004


31
The description of follicular waves by macroscopic observation of reproductive
tracts from heifers at different stages of the estrous cycle (Rajakoski, 1960) or by
ultrasonography (Pierson and Ginther, 1984) and a detailed description of follicular
waves during diestrus using ultrasonography (Savio et al., 1988; Sirois and Fortune,
1988) made researchers realize that follicular dynamics during diestrus should be
controlled in order to improve fertility (Thatcher et al., 1989). Through different
mechanisms, GnRH, estradiol, and P4 will induce follicular turnover in cows with ovarian
follicles at different stages. Estradiol suppresses FSH secretion and induces atresia of
follicles that are in the recruitment phase (2-8 mm; Burke et al., 2000). However, P4 will
suppress LH pulsatility and induce atresia in follicles that are larger than 10 mm
(Anderson and Day, 1994), and GnRH will induce LH and FSH release with ovulation of
follicles larger than 10 mm and recruitment of a new follicular wave (Thatcher et al.,
1989). A new follicular wave is recruited approximately 2 days after GnRH
administration (Thatcher et al., 1989), 2 days after hCG (Rajamahendran and
Sianangama, 1992; Niasari-Naslaji et al., 1996) 3-4 days after P4 (Anderson and Day,
1994), and 4-5 days after estrogen administration (Bo et al., 1995).
The Use of GnRH and PGF2ato Synchronize Estrus
Administration of a GnRH agonist (buserelin) induces an immediate release of
FSH and LH from the pituitary, a rise in estrogen, ovulation, luteinization or atresia of
follicles larger than 10 mm, and recruitment of a new follicular wave within 2-3 days
(Thatcher et al., 1989). Administration of PGF2tt 7 days after GnRH (Select Synch;
Thatcher et al., 1989) will induce a more synchronized incresase in estradiol (Wolfenson
et al., 1994) and estrus within 36-72 hours after luteolysis. The Select Synch protocol in


174
and rectal palpation (Zemjanis, 1962) to determine that the structure is in fact a follicle
that never ovulated and not a cystic CL (i.e., CL has a line of demarcation and distortion
in the shape of the ovary). In addition, since CL can still be observed at ultrasonography
at 2 d after regression (Kastelic et al., 1990), per rectum palpation of the ovary will
contribute to detecting a functional CL based on its morphology (Zemjanis, 1962). The
positive predictive value for per rectum examination and ultrasonography of the genital
tract was high for cows in diestrus, proestrus, and anestrus, but not as high for cows in
metestrus. The specificity of per rectum examination and ultrasonography of the genital
tract to detect cows with a luteal cyst was high (98.6%) but the sensitivity was low
(31.5%). Farin et al., (1990) reported high sensitivity (91.5%) and low specificity (70%)
for ultrasonography to detect luteal cysts. The low sensitivity may be due to the cut-off
value for plasma P4 concentration being 0.5 ng/ml, and as a consequence the prevalence
of luteal cysts was 70%. Comparing per rectum examination and ultrasonography of the
genital tract, a high sensitivity and specificity was reported and it was concluded that
ultrasonography is effective for detecting luteal cysts (Farin et al., 1992). Similarly, the
present study indicated that when ultrasonography detects luteal cysts, plasma P4 most
likely will be greater than 1 ng/ml (positive predictive value of 90%).
In summary, the present study suggests that cows in diestrus at the time of
nonpregnancy diagnosis obtain acceptable pregnancy rates to the Ovsynch00 protocol, and
in addition a Modified Quicksynch protocol may be an alternative to combine
insemination at detected estrus with TAI to reduce the time and cost needed for re-
insemination. Cows in proestrus obtained acceptable pregnancy rate to the Ovsynch
protocol. For cows in metestrus, insemination at detected estrus or delaying the Ovsynch


107
The detrimental effect of low P4 and high estrogen concentrations on early
embryo development may be exacerbated in high-producing dairy cows subjected to
synchronization of ovulation. Lactating dairy cows have an increased feed intake that
results in increased liver blood flow and steroid metabolism (Sangsritavong et al., 2002)
that results in low P4 concentrations. Low levels of P4 during diestrus increase luteinizing
hormone (LH) pulses (Ireland and Roche, 1982b) which stimulate follicular growth
(Savio et al., 1993; Stock and Fortune, 1993) resulting in elevated concentration of
estradiol. In fact, follicular development is more pronounced in lactating than in
nonlactating dairy cows (Lucy et al., 1992). In an experiment using timed insemination
experiment in which timing between GnRH administration for induction of ovulation and
follicular development was altered, ovulation resulted in formation of a smaller corpus
luteum with reduced production of progesterone (Vasconcelos et al., 1999; 2001; Peters
and Pursley, 2003).
Early embryonic mortality, indicated by cows returning to estrus before 24 d after
insemination, is approximately 20.5-43.6%, and late embryonic mortality with cows
returning to estrus after 24 d is approximately 8.0-17.5% (Humblot, 2002). Late
embryonic mortality in cows subjected to synchronization of ovulation protocols was
14.6% measured between pregnancy diagnosis on Days 32 and 74 after TAI (Moreira et
al., 2001).
Since LH is luteotrophic in the bovine (Hansel and Seifart, 1967), strategies
involving administration of bovine LH, LH-containing preparations, human chorionic
gonadotropin (hCG), or GnRH have been used to stimulate CL function (Wiltbank et al.,
1961; Simmons and Hansel, 1964; Donaldson and Hansel, 1965; Schomberg et al., 1967;


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
PHARMACOLOGICAL REGULATION OF OVARIAN FUNCTION IN DAIRY
COWS TO IMPROVE REPRODUCTIVE PERFORMANCE
By
Julian A. Bartolom
May 2004
Chairman: Dr. Louis F. Archbald
Cochairman: Dr. William W. Thatcher
Major Department: Veterinary Medical Sciences
Dairy herds experience low pregnancy rates and high embryonic mortality. The
objective was to evaluate pharmacological strategies to improve reproductive
performance. Nonlactating dairy cows (n= 17) received human chorionic gonadotropin
(hCG) at estrus or no treatment. Nonlactating dairy cows (n=20 and n=14) and dairy
heifers (n=44) received a deslorelin implant or gonadotropin releasing homone (GnRH)
for induction of ovulation. Lactating dairy cows (n=593) received a deslorelin implant or
GnRH for induction of ovulation and timed artificial insemination (TAI). Lactating dairy
cows (n=831) received no treatment or GnRH on Day 5, Day 15, or both after TAI.
Lactating dairy cows (n=179) received no treatment or a deslorelin implant at Day 27 of
pregnancy. Lactating dairy cows (n=332) were assigned to the Ovsynch or Heatsynch
protocols after nonpregnancy diagnosis. Lactating dairy cows (n=1083) received different
protocols for resynchronization. Lactating dairy cows (n=624) received GnRH on Day 23
xvi 1


CHAPTER 5
EFFECT OF A DESLORELIN IMPLANT IN A TIMED ARTIFICIAL
INSEMINATION PROTOCOL ON FOLLICLE DEVELOPMENT, LUTEAL
FUNCTION AND REPRODUCTIVE PERFORMANCE OF LACTATING DAIRY
COWS
Introduction
Reproductive efficiency is a major component of economic success in dairy herds.
Pharmacological control of the estrous cycle involves synchronization of follicular
development, control of corpus luteum (CL) regression, and synchronization of ovulation
to improve conception and pregnancy rates (PR). Such pharmacological controls have
allowed development of a successful timed artificial insemination (TAI) protocol with
adequate PR (Pursley et al., 1997a). Systematic breeding programs that involve TAI have
become an integral part of reproductive management in high-producing dairy herds
(Stevenson, 2001). However, TAI protocols have not increased conception rates or
altered pregnancy losses in lactating dairy cows (Santos et al., 2003).
Recently, manipulation of the estrous cycle prior to initiation of the TAI protocol
resulted in increased PR in lactating cyclic cows (Moreira et al., 2001). Optimizing
fertility in TAI protocols should result in higher PR and greater adoption of such
protocols to improve reproductive performance in dairy herds.
Embryo mortality in lactating dairy cows has been reported to be as high as 21%
between 27 and 45 d after TAI (Moreira et al., 2001). Losses of pregnancy in lactating
dairy cows have been similar when insemination is performed at detected estrus or after
the Ovsynch/TAI protocol (Santos et al., 2003). Furthermore, conception rate in cows
82


27
Some of the limitations need to be considered regarding the use of PGF2a to
induce estrus: 1) PGF2a is not effective during the first five days of the estrous cycle
(Lauderdale et al., 1972, Rowson, 1972); 2) the interval from treatment to estrus varies
according to the stage of the estrous cycle at the time of PGF2a treatment (King et al.,
1982; Stevenson et al., 1984; Kastelic et al., 1990); 3) even tough conception rates after
PGF2a-induced estrus are high (Beal, 1996). Reduced or inconsistent conception rates
after PGF2a-induced estrus have been reported in lactating dairy cows on a few occasions
(Macmillan et al., 1977; 1983); and 4) PGF2awill not induce estrus in noncycling cows.
Until 1982 it was believed that all cows will respond to PGF2a after Day 4 of the
estrous cycle (Beal, 1996). However, it was demonstrated that there was an interaction
between dose and stage of the estrous cycle, with high doses being effective in early
stages of the estrus cycle (5-9) and lower doses being also effective in late stages (9-17)
of the estrous cycle (Berardinelli and Adair, 1989). The bovine CL becomes more
sensitive as the cycle progresses, and after Day 4 maximum responsive can be achieved
with a 25 mg dose or multiple administrations with equal doses given 6 to 24 hours apart
(Beal, 1996). Since PGF2a is not effective during the first 4 days of the estrous cycle, a
schedule including 2 doses 11 days apart (heifers) or 14 d apart (cows) will synchronize
the majority of the animals (Beal, 1996).
The stage of the estrous cycle also affects the interval from treatment to
expression of estrus (Beal, 1996). Administration of PGF2a between Days 5 to 8 of the
estrous cycle synchronizes estrus within 48 to 59 h (King et al., 1982) but the average
was 52 to 72 h when administered between Days 12 and 15 of the estrous cycle. The
response depends on the stage of the follicular wave at the time of treatment (Beal, 1996).


73
Interval after treatment (d)
Figure 4.1. Least squares means and SE for plasma P4 concentration between Day 0 and
Day 16 for Control (), DESLORELIN 750 () and DESLORELIN 1000 (A) groups in
Experiment 1. DESLORELIN 1000 vs. Control on Day 11 (P<0.05), DESLORELIN 750
vs. Control on Day 12 (P<0.05).


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and j/s fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
William W. Thatcher
Graduate Research Professor
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Jorge Hernandez
Associate Professor
\
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Carlos A. Risco
Professor
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
%
Lokenga B adinga
Assistant Professor


193
Monsanto, St Louis, Missouri) every 14 d during the entire lactation. Reproductive
management consisted of a voluntary waiting period of 75 d that incorporated a
Presynch-Ovsynch program (Moreira et al., 2001) for first service, and estrus detection
using visual observation and a computerized pedometer system (Afimilk , S.A.E.
Afikim, Kibbutz Afikim, 15148, Israel) for subsequent services. The study included 624
lactating dairy cows that received GnRH 23 1 d after artificial insemination and
detected as nonpregnant at ultrasonography on Day 30 1 (experimental Day 0). Cows
with reproductive abnormalities (i.e., metritis, pyometra, uterine-ovarian adhesions,
granulosa cell tumor) were excluded.
Study Design
On Day 0 cows were classified to different stages and two common abnormalities
of the estrous cycle, as determined by per rectum examination and ultrasonography of the
genital tract (Bartolom et al., 2004), and assigned to different resynchronization
protocols. On Day 0, 74.8% (467/624) of the cows were in diestrus, 5.6% (35/624) in
metestrus, and 19.5% (122/624) without a CL (proestrus, ovarian cysts, anestrus).
Resynchronization protocols for cows in different stages and two common
abnormalities of the estrous cycle are described in Figure 10.1.
Cows in diestrus (n=467) were divided randomly into two experimental groups.
Cows in GnRH Group (n=238) received 25 mg (i.m.) PGF2a on Day 0, 100 pg (i.m.)
GnRH (Cystorelin, Merial Limited, Iselin, NJ, 08830) on Day 2, and TAI 16 h later.
Cows in the ECP Group (n=229) received 25 mg (i.m.) PGF2 (Lutalyse, Pharmacia,
Kalamazoo, MI, 49001) on Day 0, 1 mg (i.m.) ECP (Pharmacia, Kalamazoo, MI, 49001)
on Day 1, and TAI 36 h later.


43
GnRH and PGF2a will induce estrus in noncyclic cows and prepuberal heifers. Protocols
combining GnRH, PGF2a and P4 to synchronize estrus and ovulation in comparison with
those utilizing estrogens and P4 resulted in similar pregnancy rates (Martinez et al.,
2002).
The Use of Estrogen to Synchronize the Follicular Wave
Different salts of estradiols such as estradiol benzoate (Burke et al., 2000),
estradiol valerate (Bo et al., 1991), estradiol cypionate (Thundatil et al., 1998) and
estradiol-17p (Bo et al., 1995) have been used alone or in combination with P4 to
synchronize follicular wave in cattle.
Estradiol valerate administered at different stages of the estrous cycle induced
follicular atresia but follicular wave emergence was variable and inconsistent (Bo et al.,
1993). In contrast, estradiol-17P suppressed the dominant follicle and emergence of a
new follicular wave occurred in 4.3 days (Bo et al., 1995).
Estradiol benzoate induces atresia of the dominant follicle in 36 hours (Burke et
al., 2001) and delays FSH surge in a dose-dependent manner (Burke et al., 2003). After
ablation of the dominant follicle, FSH peaks in 29 hours, but in cows receiving 1, 2 or 4
mg of estradiol benzoate FSH peaks at 53, 80 and 90 hours, respectively, and a new
follicular wave was recruited 15 hours after the FSH peak (Burke et al., 2003).
Administration of 0.5 or 1 mg of ECP with 100 mg of P4 on Day 3 of the estrous
cycle was effective, but 1 mg of ECP with 50 mg of P4 in cows at random stages of the
estrous cycle and treated with a CIDR insert failed to synchronize the follicular wave
(Thundathil et al., 1997).



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CHAPTER 6
STRATEGIC USE OF GONADOTROPHIN-RELEASING HORMONE (GnRH) TO
INCREASE PREGNANCY RATE AND REDUCE PREGNANCY LOSS IN
LACTATING DAIRY COWS SUBJECTED TO SYNCHRONIZATION OF
OVULATION AND TIMED INSEMINATION
Introduction
Pregnancy rate for 21-day periods, which is the product of conception rate and
estrus detection rate, is the most commonly used parameter to evaluate reproductive
performance in dairy herds (Ferguson and Galligan, 1993). Synchronization of ovulation
with TAI (without detection of estrus) is commonly used in lactating dairy cows to
increase pregnancy rate (Pursley et al., 1997a; de la Sota et al., 1998; Momcilovic et al.,
1998). However, pregnancy loss in cows subjected to TAI appear to be as high as that
observed in cows inseminated at detected estrus (Cartmill et al., 2001; Santos et al.,
2002).
After fertilization, corpus luteum (CL) development, follicular growth, and
progesterone (P4) and estrogen concentrations influence embryo survival and CL
maintenance (Thatcher et al., 1997). Corpus luteum development results in secretion of
P4 which influences embryo development, interferon-x production, and inhibition of the
luteolytic cascade (Mann and Lamming, 2001). Destruction of ovarian follicles by
electrocauterization during mid-cycle reduced estrogen levels, extended luteal function
(Fogwell et al., 1985), increased CL weight (Villa-Godoy et al., 19985) and reduced the
efficacy of PGF2a-induced luteolysis (Hughes et al., 1987).
106


168
Cows with Ovarian Cysts
The distribution of cows by the different variables between groups was not
different (P>0.15) except among inseminators (P<0.01, Table 9.10). Pregnancy rates on
Days 30, 55 and 90 were evaluated by logistic regression adjusting for inseminator.
Pregnancy rates on Days 30, 55 and 90 for cows with ovarian cysts in the Ovsynch Group
(20.2, 18.5, and 14.7%) were lower (P<0.05) compared to cows in the GnRH+Ovsynch
Group (30.3, 26.6, and 22.9%; Table 9.11). Pregnancy losses between Days 30-55 and
55-90 for cows in the Ovsynch (12.5 and 12.5 %) and GnRH+Ovsynch (11.1 and 13.8%)
groups were not different (Table 9.11).
Plasma P4 concentrations on Day 0 for cows with ovarian cysts in the Ovsynch
(0.82+0.10 ng/ml) and the GnRH+Ovsynch (0.87+0.13 ng/ml) groups were not different.
The proportions of cystic cows with follicular cysts determined by rectal palpation and
ultrasonography of the genital tract in the Ovsynch (89.7%, 87/97) and GnRH+Ovsynch
(91.7%, 100/109) groups were not different. The proportion of cystic cows with plasma
P4 concentration lower than 1 ng/ml (follicular cysts) in the Ovsynch (72.3%, 68/94) and
the GnRH+Ovsynch (73.5%, 78/106) were not different. Cows that underwent both
diagnostic methods (clinical diagnosis using rectal palpation and ultrasonography and
plasma P4 concentration) were used to calculate sensitivity, specificity and positive
predictive value of the clinical diagnosis to differentiate follicular and luteal cysts
utilizing plasma P4 as the gold standard (Table 9.12). The sensitivity, specificity and
positive predictive value for per rectum examination in combination with
ultrasonography to detect luteal cysts was 31.5, 98.6 and 89.5% and for follicular cysts
98.6, 31.5 and 79.6%, respectively.


126
concentrations during the period of late embryonic and early fetal development in order
to reduce pregnancy loss has not been evaluated in lactating dairy cows. The hypothesis
of this study was that a GnRH agonist (deslorelin implant) inserted on Day 27 of
pregnancy would induce accessory corpora ltea, suppress follicular growth and reduce
pregnancy loss. The objective of the study was to evaluate pregnancy loss between Day
27 and Day 45 and Day 45 to Day 90 in lactating dairy cows receiving a 2.1 mg
Deslorelin implant on Day 27 of pregnancy.
Materials and Methods
Study Population
The study was conducted in a large dairy herd (3,000 milking cows) located in
north central Florida and included cows diagnosed pregnant by ultrasonography 27 days
post-insemination.
Study Design
The study included pregnant cows detected by ultrasonography (5 MHz, Aloka
500) on Day 27 after artificial insemination. A total of 181 pregnant cows was assigned
randomly to receive either a 2.1 mg, s.q. biodegradable deslorelin Implant (Ovuplant ,
Peptech, Animal Health, North Ryde, Australia Implant Group; n=89) on Day 27, or no
treatment (Control Group; n=92).
Examination of the Uterus and Ovaries
A real-time ultrasonographic scanning was conducted on Day 27 and Day 45 to
record the presence of a normal embryo (heartbeats), number of class 2 (6-9 mm) and
class 3 (>10 mm) ovarian follicles, and corpora ltea. On Day 90 cows were re-examined
for pregnancy using per rectum palpation of the reproductive tract (Zemjanis, 1962).


157
Table 8.6. Pregnancy rate on Day 45, adjusted odd ratios (AOR), 95 % confidence
interval (Cl) and levels of significance for the risk of nonpregnancy for cows in both
groups and at different stages and two common abnormalities of the estrous cycle.
Variable
Pregnancy Rate
N %
AOR
95 % Cl
P
Group
Ovsynch
Stage/Abn.
Diestrus
19.7
16/81
Referent
Referent
Heatsynch
Diestrus
16.7
12/72
1.5
0.6-3.5
0.36
Ovsynch
Metestrus
9.1
2/22
2.4
0.5-11.7
0.27
Heatsynch
Metestrus
44.4
12/27
0.3
0.1-0.8
0.01
Ovsynch
Proestrus
14.3
5/35
1.7
0.6-5.3
0.32
Heatsynch
Proestrus
15.8
6/38
1.5
0.5-4.6
0.42
Ovsynch
Ovarian Cyst
26.1
6/23
0.9
0.3-2.7
0.86
Heatsynch
Ovarian Cyst
8.0
2/25
4.2
0.8-20.1
0.07
Season
Aug-Sept
19.9
40/201
0.5
0.2-1.2
0.12
Oct-Dec
16.8
22/131
Referent
Referent
-
Inseminator
A
4.6
1/22
6.8
0.7-69.4
0.10
B
18.5
20/108
1.2
0.3-4.0
0.81
C
14.3
4/28
1.4
0.3-6.8
0.67
D
23.5
16/68
0.7
0.2-2.1
0.51
E
/
22.1
15/68
0.8
0.2-2.4
0.65
F
15.8
6/38
Referent
Referent
-


200
Plasma P4 concentration on Days 0, 7, 10 and 17 for cows in proestrus and with
ovarian cysts in all four groups is described in Table 10.8. Among cows with ovarian
cysts, 43.5% of them had plasma P4 less than 1 ng/ml (follicular cysts) and 56.5% had
plasma P4 greater than 1 ng/ml (luteal cysts). There was no difference in the rise of
plasma P4 concentration from Day 0 to Day 7 for cows receiving GnRH on Day 0.
However, plasma P4 concentration on Day 7 tended to be higher (P<0.08) for cows with
ovarian cysts in the GnRH+CIDR Group (3.85 0.54 ng/ml) compared to cows with
ovarian cysts in the Control Group (2.50 0.56 ng/ml) and higher (P<0.05) than for cows
in proestrus in the CIDR Group (1.73 0.72 ng/ml; Table 10.8). The increase of plasma
P4 concentration from Day 10 to 17 tended to be lower in cows with ovarian cysts that
did not receive GnRH on Day 0 but increased in proestrus cows regardless of GnRH
treatment (GnRH*Stage*Day, P<0.06; Figure 10.4). A CIDR insert from Day 0 to 7 did
not affect the rise in plasma P4 concentrations from Day 0 to 7 or from Day 10 to 17.
Pregnancy rates on Day 30, 55 and 90 for cows in proestrus and with ovarian
cysts for all four groups is described in Table 10.9. GnRH on Day 0 did not affect
pregnancy rates on Days 30, 55 and 90. There was an interaction between CIDR Insert on
Day 0 and stage/abnormality of the estrous cycle on pregnancy rate on Day 30, 55 and 90
(P<0.05; Figure 10.4). CIDR insert on Day 0 increased pregnancy rates on Days 30, 55
and 90 in cows with ovarian cysts but decreased pregnancy rates in cows in proestrus
(Tables 10.10, 10.11, 10.12). Cows with BCS greater than 3.0 tended to be 4.5 times
more likely to get pregnant than cows with BCS less than 3.0 (P<0.10; Tables 10.10,
10.11, 10.12).


253
Pancarci SM, Jordan ER, Risco CA, Schouten MJ, Lopes FL, Moreira F, Thatcher WW.
Use of estradiol cypionate in a pre-synchronized timed artificial insemination program
for lactating dairy cattle. J Dairy Sci 2002;85:122-131.
Pate, J.L. Cellular components involved in luteolysis. J Anim Sc 1994;72:1873-1883.
Patterson DJ, Kirakofe GH, Stevenson JS, Corah LR. Control of the bovine estrous cycle
with melengestrol acetate (MGA): a review. J Anim Sci 1989;67:1895-1906.
Patterson DJ, Kojima F. Estrus synchronization in beef cows and heifers. Select Sires
Think Tank, Columbus, OH, September 21-22, 2003.
Pellisier CL. Dairy cattle breeding problems and their consequences. Theriogenology
1976;6:575-583.
Pescador N, Soumano K, Stocco DM, Price CA, Murphy BD. Steroidogenic acute
regulatory protein in bovine corpora ltea. Biol Reprod 1996;55:485-491.
Pescador N, Stocco DM, Murphy BD. Growth factor modulation of steroidogenic acute
regulatory protein and luteinization in the pig ovary. Biol Reprod 1999;60:1453-1461.
Peters AR, Martinez TA, Cook AJC. A meta-analysis of studies of the effect of GnRH
11-14 days after insemination of pregnancy rates in cattle. Theriogenology
2000;54:1317-1326.
Peters KE, Bergfelt EG, Cupp AS, Kojima FN, Mariscal V, Sanchez T, Wehrman ME,
Grotjan HE, Hamernik DL, Kittok RJ, Kinder JE. Luteinizing hormone has a role in
development of fully functional corpora ltea (CL) but is not required to maintain CL
function in heifers. Biol Reprod 1994;51:1248-1254.
Peters MW, Pursley JR. Timing of final GnRH of the Ovsynch protocol affects ovulatory
follicle size, subsequent lutel function, and fertility in dairy cows. Theriogenology 2003,
60:1197-1204.
Pharriss BB, Wyngarden LJ. The effects of prostaglandin F2a on the progestogen content
of ovaries from pseudopregnant rats. Proc Soc Exp Biol Med 1969, 130:92-94.
Pierson RA and Ginther OJ. Ultrasonography of the bovine ovary. Theriogenology
1984a;21:495-504.
Pierson RA, Ginther OJ. Ultrasonography for detection of pregnancy and study of
embryonic development in heifers. Theriogenology 1984b;22:225-233.
Pierson RA, Ginther OJ. Intraovarian effect of the corpus luteum on ovarian follicles
during early pregnancy in heifers. Anim Reprod Sci 1987;15:53-60.


87
The proportion of cows diagnosed as non-pregnant on Day 27 that were reinseminated
prior to pregnancy diagnosis in each treatment group also was evaluated.
Blood Sample Collection and Analyses
Blood samples (10 mL) were collected from all cows at -24, -10, and 11 d
relative to the day of the first postpartum AI by puncture of the coccygeal vein or artery
into evacuated tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ). Samples
were placed immediately on ice and centrifuged at 1500 x g for 15 min at 10C for
separation of plasma. Plasma was frozen at -25C and later analyzed for progesterone
using a single antibody RIA procedure (Knickerbocker et al., 1986). The assay sensitivity
was 0.10 ng/mL, and the intra- and inter-assay CVs were 9.8 % and 7.9%, respectively.
Plasma progesterone concentrations in plasma on Days -24 and -10 relative to AI
were used to determine whether a cow was cycling or anovulatory/anestrous. Cows with
/
plasma progesterone concentration equal to or greater than 1.5 ng/mL in any of the two
samples were considered as cycling. Cows with progesterone concentrations below 1.5
ng/mL in both plasma samples prior to the TAI protocol were considered as anovulatory.
Plasma progesterone concentrations on Day 11 after AI were used to determine whether
the treatment affected luteal function in lactating dairy cows. Induction of cyclicity after
the TAI was determined by the proportion of cows that were anovulatory prior to the TAI
and subsequently had a plasma progesterone concentration above 1.5 ng/mL on Day 11
after TAI.
Experimental Design and Statistical Analyses
The experimental design was a randomized-complete block design (Kuehl, 1994).
Cows were blocked according to parity, BCS at 30 d postpartum, and previous lactation


97
more pronounced effect on the proportion of non-pregnant cows with either a follicle
greater than 10 mm or a CL on Day 27 was detected for DESLORELIN 750 than
DESLORELIN 450.
The suppressive effect of GnRH agonist on follicular development is related to
the chronic phase of GnRH stimulation. Chronic exposure to GnRH, as observed when
deslorelin implant is given, down-regulates LH and FSH beta subunits gene expression
and reduces mRNA encoding the genes for LH transcription (Anderson, 1996). The
down-regulation of GnRH receptors in the pituitary gonadotrophs creates a period of
GnRH insensitivity (DOcchio et al., 2000). This transient state of insensitivity to GnRH
decreases the gonadotropin support for follicle growth and maturation.
Because of the suppressive effect of deslorelin on follicle development, the need
for re-synchronization of non-pregnant cows with a protocol that resumes follicular
/
growth and induces ovulation at the moment of insemination is very important. We re
synchronized non-pregnant cows that had not been re-inseminated by Day 27 after the
initial TAI with the Ovsynch protocol using only a gonadorelin product, deslorelin
implant reduced the response to GnRH on Day 27, based on detection of a new CL on
Day 34 in cows that did not have a CL at the moment of the gonadorelin injection. This
effect is probably related to the uncoupling of GnRH receptors when the gonadotrophs
are still insensitive to GnRH stimulation.
When lactating cows were treated with a deslorelin implant during the first week
postpartum and challenged with GnRH on Day 14 after calving, the LH response to
injection of GnRH was blocked (Mattos et al., 2001), supporting the concept that
deslorelin desensitizes the gonadotrophs. Furthermore, follicles need to reach a minimum


9
granulosa cells and the release of the oocyte (Espey, 1994). After the oocyte is released,
theca interna and granulosa cells form the small and large cells of the corpus luteum
during a period of rapid mitosis and vascularization under LH stimulation (Hansel and
Dowd, 1986). A second peak of LH and a rise in follicle-stimulating hormone (FSH) at
the beginning of metestrus stimulate the first follicular wave (Adams et al., 1992). The
tonic releases of LH and FSH during the luteal phase are controlled by the arcuate
nucleus, ventromedial nucleus and median eminence in the hypothalamus. Tonic and
pulsatile secretion of LH from the anterior pituitary gland stimulate CL development and
P4 secretion gradually rises (Rahe et al., 1980). Progesterone exerts a negative feedback
on the anterior pituitary gland and inhibits gonadotropin secretion. However, during
metestrus, P4 concentrations are still low and LH pulsatility is greater compared to mid-
luteal phase (Rahe et al., 1980; Peters et al., 1994).
During metestrus, the CL is refractory to a single luteolytic dosage of
PGF201 (Lauderdale et al., 1972; Rowson et al., 1972). The mechanism responsible for this
is not completely understood. One possible explanation involves a mechanism that
contributes to luteolysis by uterine and exogenous PGF2a involving stimulation of
prostaglandin secretion by the large luteal cells (Tsai and Wiltbank, 1997). However, this
mechanism may not be functional in the early luteal phase. Nevertheless, the magnitude
of the luteolytic effect of intraluteal prostaglandin still needs to be demonstrated (Tsai
and Wiltbank, 1998). An alternative mechanism involves the role of endothelin-1 in
luteolysis. Injecting PGF2a in early luteal phase (Day 4) did not induce the expression of
ET-1, its receptor type A (ETA-R), and COX-2 compared with cows injected in midcycle
(Day 10; Levy et al., 2000). In adittion, P4 levels, StaR protein mRNA, and cytochrome


227
concentrations, but late embryonic mortality tended to be reduced in cows induced to
ovulate with a 450 pg deslorelin implant. Cows receiving deslorelin implants had
suppressed follicular development and reduced pregnancy rate to a resynchronized
insemination.
Administration of 100 pg of GnRH either on Day 5 or Day 15 after timed
insemination in lactating dairy cows did not increased pregnancy rate or reduced late
embryonic mortality. Moreover, cows that received GnRH on both Day 5 and Day 15
experienced reduced pregnancy rates at Day 27 and Day 55 post-insemination. Treatment
with two doses of GnRH may have resulted in over-stimulation of the CL with reduction
in LH receptors, lost of luteotrophic support and increased luteolysis. Administration of a
2.1 mg deslorelin implant at 27 days of pregnancy in lactating dairy cows reduced
follicular development, induced accessory corpora ltea, and increased plasma P4
concentrations. In addition, cows that received the deslorelin implant and formed
accessory CL had a reduced pregnancy loss compared to control cows. However, cows
that received the deslorelin implant and did not form an accessory CL had increased
pregnancy loss and return to estrous cyclicity was going to be delay since follicular
growth was suppressed at Day 45.
The slight detrimental effect of administration of hCG at estrus on plasma P4
concentration, the reduced pregnancy rate in cows treated with 100 pg of GnRH on Days
5 and 15 after timed insemination, reduced pregnancy rate in cows induced to ovulate
with a 750 pg deslorelin implant, and finally the increase in late pregnancy losses in cows
that did not form an accessory CL in response to a deslorelin implant on Day 27,
collectively indicate that strategies to stimulate the CL may result in an over-stimulation


6
causes regression of the corpus luteum. Subsequently, FSH and LH increase, and
follicular growth is stimulated (proestrus), and this result in a subsequent estrous period
and ovulation (Schams et al., 1977).
Estrus
The onset of estrus coincides with the LH surge and lasts until the end of the
estrus behavior (receptivity to be mounted; 12-20 h). During estrus, P4 levels are low,
secretion of gonadotropins (FSH and LH) by the anterior pituitary increases, LH pulse
amplitude and frequency are high (Rahe et al., 1980; Stumpf et al., 1989), follicular
growth is accelerated and dominant preovulatory follicles reach 14 to 16 mm in cows
with two or three follicular waves, respectively (Ginther et al., 1989). During estrus,
estradiol concentration reaches a maximum (6-7 pg/ml; Glencross et al., 1973) and under
low P4 levels (Kesner et al., 1981; Schallenberger et al., 1982), induces the GnRH/LH
surge (Ireland and Roche, 1982a). The hypothalamic neurons responsible for the GnRH
surge (preoptic area and anterior hypothalamic nuclei) do not contain estrogen receptors.
However, estrogen binds to its receptor in the central nervous system, and the effect on
GnRH release is mediated by neurotransmitters (Kalra et al., 1993). Estradiol stimulates
GnRH release and increases sensitivity of the pituitary to GnRH (Reeves et al., 1971 in
sheep; Kaltenbach et al., 1974 in cows) by increasing GnRH receptors in the anterior
pituitary gland (Schoenemann et al., 1985). In addition, estrogen increases the expression
of its own receptors in the anterior pituitary gland (Clarke et al., 1981). The onset of
estrus occurs at the time of the LH surge and last for 16.9 hours. Then, estradiol levels
drop after the LH surge and ovulation occurs approximately 24 hours after the LH surge
(Schams et al., 1977).


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Macmillan et al., 1985; 10 ug; Macmillan et al., 1986) which extended the estrous cycle
in lactating dairy cows. However, in the present study, treatment with GnRH on both Day
5 and Day 15 reduced pregnancy rate on both Day 27 and Day 55.
There are several potential mechanisms which may explain the apparent
detrimental effect of GnRH treatment on both Day 5 and Day 15 on pregnancy rate
observed on Day 27 and Day 55. It has been shown that GnRH treatment of heifers on
Day 2 or Day 10 of the estrous cycle reduced P4 levels on Days 12, 14 and 16, and the
number of unoccupied LH receptors in plasma membrane of the CL (Rodger and
Stormshak, 1986). The majority of LH receptors are expressed in the small luteal cells
(Fitz et al., 1982), and small luteal cells are transformed into large luteal cells during CL
development (Alila and Hansel, 1984). Therefore, in the present study, treatment of cows
with GnRH on