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Effect of Long-Term Melengestrol Acetate Treatments on Follicle Dynamics and Response to Gonadotropin-Releasing Hormone ...

Permanent Link: http://ufdc.ufl.edu/UFE0021372/00001

Material Information

Title: Effect of Long-Term Melengestrol Acetate Treatments on Follicle Dynamics and Response to Gonadotropin-Releasing Hormone and Prostaglandin F2alpha Synchronization Treatments in Bos indicus x Bos taurus Heifers
Physical Description: 1 online resource (146 p.)
Language: english
Creator: Woodall, Steaven A, Jr
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In experiment 1, yearling Angus (n = 40) and Brangus (n = 26) heifers received melengestrol acetate (MGA; 0.5 mg/hd/d) for 14 d with prostaglandin F2 alpha (PG) administered either 19 d or 19 and 20 d after MGA withdrawal for Angus and Brangus, respectively. A subgroup of Angus (n=11) and Brangus (n=10) heifers had transrectal ultrasonography conducted daily after MGA withdrawal until 7 d after PG to evaluate follicle development. There tended (P = 0.07) to be more Angus (100%; 11/11) compared to Brangus (80%; 8/10) heifers ovulating within 7 d after MGA withdrawal. Follicle wave patterns between MGA withdrawal and PG consisted of one (0/11; 1/10), two (9/11; 5/10), three (2/11; 3/10) or four (0/11; 1/10) waves for Angus and Brangus, respectively. The number of heifers with follicle > 10 mm on 9 (54.5, 80.0 %), 10 (81.8 %, 70.0 %), and 11 d (90.9, 80.0%) after MGA were similar between Angus and Brangus respectively; but greater (P < 0.05) on 12 (100, 70.0 %) and 13 d (100, 50 %) for Angus compared to Brangus, respectively. Because of the asynchrony of follicle wave patterns from MGA withdrawal to PG for Brangus compared to Angus, the best time to administer GnRH to synchronize follicle development in Brangus heifers may be immediately after MGA withdrawal. In Experiment 2 cycling Bos indicus x Bos taurus (BI x BT) heifers were pre-synchronized to start a 14 d MGA (0.5 mg/hd/d) treatment on d 2 of the estrous cycle. Heifers were randomly assigned to receive GnRH (100 micrograms) either 3 (G3; n = 25) or 10 d (G10; n = 23) after MGA withdrawal with PG (12.5 mg) 7 and 8 d after GnRH. During MGA, heifers within each treatment received no PG or two consecutive PG treatments on d 4 and 5, 8 and 9, or 12 and 13, to simulate different periods of low-level progestogen exposure (SOF). Ovulation to GnRH was 76.0 and 47.8% for the G3 and G10, respectively. For G3 and G10 treatments, heifers in the d 14 SOF group did not respond as effectively as the other SOF groups. Following PG, more (P < 0.05) G3 (76%) heifers exhibited estrus during the first 72 h after PG compared to G10 (43.5%) heifers. In Experiment 3, yearling BIxBT (n=295) heifers at two locations were synchronized with two MGA + PG treatments. Treatment 1 was the same as in Experiment 1 (MGA-PG; n=174) while treatment 2 was the same as the G3 treatment in Experiment 2 (MGA-G-P; n=178). Heifers were AI 8 to 12 h after an observed estrus. Heifers not detected in estrus by 72 h after PG were timed-AI concomitant with GnRH. Estrous response, conception, timed-AI, and synchronized pregnancy rates were similar (P > 0.05) between MGA-PG (48.3, 54.9, 22.4, 38.1%) and MGA-G-PG (56.7, 52.4, 18.8, 37.8%), respectively. In summary follicle dynamics during the 19 d after a long term MGA treatment are different between Angus and Brangus heifers. Although, incorporation of a GnRH treatment 3 d after a 14 d MGA treatment effectively induced ovulation and resulted in a very synchronous estrus when PG was administered 7 d later, it did not improve the AI pregnancy rates compared to the MGA-PG estrous synchronization system.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Steaven A Woodall.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Yelich, Joel V.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021372:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021372/00001

Material Information

Title: Effect of Long-Term Melengestrol Acetate Treatments on Follicle Dynamics and Response to Gonadotropin-Releasing Hormone and Prostaglandin F2alpha Synchronization Treatments in Bos indicus x Bos taurus Heifers
Physical Description: 1 online resource (146 p.)
Language: english
Creator: Woodall, Steaven A, Jr
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: In experiment 1, yearling Angus (n = 40) and Brangus (n = 26) heifers received melengestrol acetate (MGA; 0.5 mg/hd/d) for 14 d with prostaglandin F2 alpha (PG) administered either 19 d or 19 and 20 d after MGA withdrawal for Angus and Brangus, respectively. A subgroup of Angus (n=11) and Brangus (n=10) heifers had transrectal ultrasonography conducted daily after MGA withdrawal until 7 d after PG to evaluate follicle development. There tended (P = 0.07) to be more Angus (100%; 11/11) compared to Brangus (80%; 8/10) heifers ovulating within 7 d after MGA withdrawal. Follicle wave patterns between MGA withdrawal and PG consisted of one (0/11; 1/10), two (9/11; 5/10), three (2/11; 3/10) or four (0/11; 1/10) waves for Angus and Brangus, respectively. The number of heifers with follicle > 10 mm on 9 (54.5, 80.0 %), 10 (81.8 %, 70.0 %), and 11 d (90.9, 80.0%) after MGA were similar between Angus and Brangus respectively; but greater (P < 0.05) on 12 (100, 70.0 %) and 13 d (100, 50 %) for Angus compared to Brangus, respectively. Because of the asynchrony of follicle wave patterns from MGA withdrawal to PG for Brangus compared to Angus, the best time to administer GnRH to synchronize follicle development in Brangus heifers may be immediately after MGA withdrawal. In Experiment 2 cycling Bos indicus x Bos taurus (BI x BT) heifers were pre-synchronized to start a 14 d MGA (0.5 mg/hd/d) treatment on d 2 of the estrous cycle. Heifers were randomly assigned to receive GnRH (100 micrograms) either 3 (G3; n = 25) or 10 d (G10; n = 23) after MGA withdrawal with PG (12.5 mg) 7 and 8 d after GnRH. During MGA, heifers within each treatment received no PG or two consecutive PG treatments on d 4 and 5, 8 and 9, or 12 and 13, to simulate different periods of low-level progestogen exposure (SOF). Ovulation to GnRH was 76.0 and 47.8% for the G3 and G10, respectively. For G3 and G10 treatments, heifers in the d 14 SOF group did not respond as effectively as the other SOF groups. Following PG, more (P < 0.05) G3 (76%) heifers exhibited estrus during the first 72 h after PG compared to G10 (43.5%) heifers. In Experiment 3, yearling BIxBT (n=295) heifers at two locations were synchronized with two MGA + PG treatments. Treatment 1 was the same as in Experiment 1 (MGA-PG; n=174) while treatment 2 was the same as the G3 treatment in Experiment 2 (MGA-G-P; n=178). Heifers were AI 8 to 12 h after an observed estrus. Heifers not detected in estrus by 72 h after PG were timed-AI concomitant with GnRH. Estrous response, conception, timed-AI, and synchronized pregnancy rates were similar (P > 0.05) between MGA-PG (48.3, 54.9, 22.4, 38.1%) and MGA-G-PG (56.7, 52.4, 18.8, 37.8%), respectively. In summary follicle dynamics during the 19 d after a long term MGA treatment are different between Angus and Brangus heifers. Although, incorporation of a GnRH treatment 3 d after a 14 d MGA treatment effectively induced ovulation and resulted in a very synchronous estrus when PG was administered 7 d later, it did not improve the AI pregnancy rates compared to the MGA-PG estrous synchronization system.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Steaven A Woodall.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Yelich, Joel V.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021372:00001


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2cdfeef5a5f29d0f8b36a3e369b19b2a
1c44bd960b4f75f6387446a8b6287b3e9b7ddc8f







EFFECT OF LONG-TERM MELENGESTROL ACETATE TREATMENTS ON FOLLICLE
DYNAMICS AND RESPONSE TO GONADOTROPIN-RELEASING HORMONE AND
PROSTAGLANDIN Fzu SYNCHRONIZATION TREATMENTS IN Bos indicus x Bos taurus
HEIFERS




















By

STEAVEN A.WOODALL, JR.


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2007



































O 2007 Steaven A. Woodall, Jr.




































To my loving parents and sister. For their support, encouragement, and love.









ACKNOWLEDGMENTS

First, I would like to offer my appreciation Dr. Joel Yelich for the opportunity to continue

my education and the support and knowledge he imparted on me. This experience will truly

affect my life. I would also like to acknowledge the members of my supervisory committee, Drs.

William Thatcher and Owen Rae, for their knowledge and contributions in fulfilling my degree.

Sincere appreciation is extended to my lab-mates, Brad Austin and Regina Esterman.

Their willingness to help and put forth long hours to complete research proj ects, but most of their

friendship has been invaluable. I would also like to extend my appreciation to the staff of the

Santa Fe Beef Research Unit and the Beef Research Unit for the willingness to assist and the

care given to the animals.

Additionally, I would like to thank my fellow graduate students, most notably Jeremy

Block, Reinaldo Cooke, and Drew Cotton for their willingness to help when needed. Most of all

I would like to thank them for the laughs and the good times we shared that made my graduate

experience enj oyable.

Finally, I thank my parents for the life lessons and the support they have given me along

the way. They have always encouraged me to pursue my dreams and have been there when I

needed them. I am truly blessed to have them in my life.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............7............ ....


LIST OF FIGURES .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


1 INTRODUCTION ................. ...............12.......... ......


2 REVIEW OF LITERATURE ................. ...............15.......... .....


Endocrine Control of the Estrous Cycle ................ ...............15...............
Puberty ................ ...............18.......... ......
Ovarian Function ...................... ...............22
Follicle Growth and Selection ................... .......... ...............22......

Corpus Luteum (CL) Function and Luteolysis ................. ...............31...............
Bovine Estrous Cycle ....................................... ...............3
Estrous Synchronization through Manipulation of the Estrous Cycle ................. ................41
Progestogens ................. ...............41.................
Prostaglandin Fzu ................ ...............44...
Melengestrol acetate + PGF2u................ ...............48.

3 EVALUATION OF FOLLICULAR DEVELOPMENT BETWEEN A 14 D
MELENGESTROL ACETATE (MGA) TREATMENT WITH PGF2u 19 D AFTER
MGA WITHDRAWAL IN ANGUS AND BRANGUS HEIFERS ................... ...............55


Introducti on ................. ...............55.................
Materials and Methods .............. ...............56....
Re sults ................ ...............62.................
Discussion ................. ...............70.................

Im plications .............. ...............79....

4 REFINEMENT OF THE 14 D MELENGESTROL ACETATE (MGA) TREATMENT
+ PROSTAGLANDIN Fzu (PG) 19 D LATER ESTROUS SYNCHRONIZATION
SYSTEM IN HEIFERS OF Bos indicus x Bos taurus BREEDING............... ................8


Introducti on .................. ...............89._ _._......
Materials and Methods .............. ...............90....
R e sults.................. ...............97....... ......

Experim ent 1. ............. ...............97.....
Experim ent 2. ............. ...............101....













Discussion ................. ...............102................

Implications ................. ...............114......... ......


5 SUMMARY ................. ...............124................


LIST OF REFERENCES ................. ...............127................


BIOGRAPHICAL SKETCH ................. ...............146......... ......









LIST OF TABLES


Table page

2-1 Summary of studies evaluating the melengestrol acetate (MGA) + PGF2a estrous
synchronization system in yearling beef heifers. ....._._.__ ... .... .... .. ...._. ..........54

3-1 Age, body weight (BW), body condition score (BCS), and estrous cycling status
(Cycling) at the initiation of the 14 d melengestrol (MGA) treatment for Angus and
Brangus heifers by ultrasound group (scan vs., non-scan) (LS means & SE).a..................80

3-2 Estrous response, interval to estrus, duration of estrus, and number of mounts
received during a HeatWatch" detected estrus for the 7 d following a 14 d
mel engestrol (MGA) treatment............... ...............8

3-3 Percentage of heifers with a functional CL, progesterone concentration (LSM & SE),
and diameter of the largest follicle (LSM & SE) at the initial PG treatment. .........._.......82

3-4 Effect of breed and cycling status at the initiation of a 14 d melengestrol acetate
treatment on estrous response, conception rate and synchronized pregnancy rates of
Angus and Brangus heifers synchronized with a 14 d melengestrol acetate treatment.....83

4-1 The effect of stage of follicle (SOF) development during a 14 d melengestrol acetate
(MGA) treatment on progesterone concentration (LSM & S.E.) at MGA withdrawal,
diameter of the largest follicle at MGA withdrawal ................ .......... ................1 16

4-2 Effect of treatment (T) and stage of follicle (S) development on largest follicle
diameter at GnRH (LSM & SE), diameter of follicle ovulating to GnRH (LSM & SE),
and ovulation rate for heifers receiving GnRH either 3 d (G3) or 10 d (G10) ................117

4-3 Percentage of heifers with a functional corpus luteum (CL), progesterone
concentration (LSM & S.E.), and diameter of the largest dominant follicle at
prostaglandin Fza (PG: LSM & S.E.) for G3 and G10 heifers ..........___.... .............. ..118

4-4 Three-day estrous response, total estrous response, and interval from prostaglandin
Fza (PG) to onset of estrus following PG treatment for G3 and G10 heifers across
different stages of follicle (SOF) development (Experiment 1).a ............... ................1 19

4-5 Estrous, conception and pregnancy rates ofBos taurus x Bos indicus heifers
synchronized with combinations of melengestrol acetate (MGA), GnRH (G), and
prostaglandin Fza (PG) at two locations (LOC) (Experiment 2). ............. ...................121

4-6 Estrous, conception and pregnancy rates of Angus heifers in Location 1
synchronized with combinations of melengestrol acetate (MGA), GnRH (G), and
prostaglandin Fza (PG) (Experiment 2). ............. ...............122....










4-7 Estrous, conception, timed-AI, pregnancy rates by treatment (TRT) and reproductive
tract score (RTS) for Bos taurus x Bos indicus heifers synchronized with
combinations of melengestrol acetate (MGA), GnRH (G), and prostaglandin Fza .........123










LIST OF FIGURES


Figure page

3-1 Profiles of ovulatory follicles after a 14 d melengestrol acetate (MGA) treatment and
the subsequent first wave dominant follicle growth profiles for A) Angus and B)
Brangus heifers. ............. ...............84.....

3-2 Mean first wave dominant follicle diameter during days 9 to 13 following
withdrawal of a 14 d melengestrol acetate (MGA) treatment for Angus (n = 1 1) and
Brangus (n = 10) heifers in the scan group. ............. ...............85.....

3-3 Mean diameter of the A) first, B) second, and C) third follicle wave following
withdrawal of melengestrol acetate (MGA) for Angus and Brangus heifers. Follicle
wave s were normal zed to the day of wave em ergence ........._.. ....... __ ...............8 6

3-4 Diameter of the eventual ovulatory follicle prior to prostaglandin F200 (PG) treatment
for Angus and Brangus heifers based on the number of follicle waves from the last
day of a 14 d melengestrol acetate treatment to a PG treatment 19 days later. .................87

3-5 Follicle growth patterns for the eventual ovulatory follicle preceding the initial
prostaglandin F2ct (PG) treatment, which occurred on day 19 (indicated by the
arrow) in A) Angus and B) Brangus heifers. ............. ...............88.....

4-1 Estrous response, expressed as a percentage of the total number of heifers in a group,
during the 7 d after the initial PG treatment for G3 (n = 25) and G10 (n = 23)
treatments. NR = no estrous response (Experiment 1) ........... .....................12









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECT OF LONG-TERM MELENGESTROL ACETATE TREATMENTS ON FOLLICLE
DYNAMICS AND RESPONSE TO GONADOTROPIN-RELEASING HORMONE AND
PROSTAGLANDIN Fza SYNCHRONIZATION TREATMENTS IN Bos indicus x Bos taurus
HEIFERS

By

Steaven A. Woodall, Jr.

August 2007

Chair: Joel V. Yelich
Major: Animal Sciences

In experiment 1, yearling Angus (n = 40) and Brangus (n = 26) heifers received

melengestrol acetate (MGA; 0.5 mg/hd/d) for 14 d with prostaglandin Fza (PG) administered

either 19 d or 19 and 20d after MGA withdrawal for Angus and Brangus, respectively. A

subgroup of Angus (n=1 1) and Brangus (n=10) heifers had transrectal ultrasonography

conducted daily after MGA withdrawal until 7 d after PG to evaluate follicle development.

There tended (P = 0.07) to be more Angus (100%; 11/11) compared to Brangus (80%; 8/10)

heifers ovulating within 7 d after MGA withdrawal. Follicle wave patterns between MGA

withdrawal and PG consisted of one (0/11; 1/10), two (9/11; 5/10), three (2/11; 3/10) or four

(0/11; 1/10) waves for Angus and Brangus, respectively. The number of heifers with follicle >

10 mm on 9 (54.5, 80.0 %), 10 (81.8 %, 70.0 %), and 11 d (90.9, 80.0%) after MGA were similar

between Angus and Brangus respectively; but greater (P < 0.05) on 12 (100, 70.0 %) and 13 d

(100, 50 %) for Angus compared to Brangus, respectively. Because of the asynchrony of follicle

wave patterns from MGA withdrawal to PG for Brangus compared to Angus, the best time to

administer GnRH to synchronize follicle development in Brangus heifers may be immediately

after MGA withdrawal. In Experiment 2 cycling Bos indicus x Bos taurus (BI x BT) heifers









were pre-synchronized to start a 14 d MGA (0.5 mg/hd/d) treatment on d 2 of the estrous cycle.

Heifers were randomly assigned to receive GnRH (100 Cpg) either 3 (G3; n = 25) or 10 d (G10; n

= 23) after MGA withdrawal with PG (12.5 mg) 7 and 8 d after GnRH. During MGA, heifers

within each treatment received no PG or two consecutive PG treatments on d 4 and 5, 8 and 9, or

12 and 13, to simulate different periods of low-level progestogen exposure (SOF). Ovulation to

GnRH was 76.0 and 47.8% for the G3 and G10, respectively. For G3 and G10 treatments,

heifers in the d 14 SOF group did not respond as effectively as the other SOF groups. Following

PG, more (P < 0.05) G3 (76%) heifers exhibited estrus during the first 72 h after PG compared to

G10 (43.5%) heifers. In Experiment 3, yearling BIxBT (n=295) heifers at two locations were

synchronized with two MGA + PG treatments. Treatment 1 was the same as in Experiment 1

(MGA-PG; n=174) while treatment 2 was the same as the G3 treatment in Experiment 2 (MGA-

G-P; n=178). Heifers were AI 8 to 12 h after an observed estrus. Heifers not detected in estrus

by 72 h after PG were timed -AI concomitant with GnRH. Estrous response, conception, timed-

AI, and synchronized pregnancy rates were similar (P > 0.05) between MGA-PG (48.3, 54.9,

22.4, 38.1%) and MGA-G-PG (56.7, 52.4, 18.8, 37.8%), respectively. In summary follicle

dynamics during the 19 d after a long term MGA treatment are different between Angus and

Brangus heifers. Although, incorporation of a GnRH treatment 3 d after a 14 d MGA treatment

effectively induced ovulation and resulted in a very synchronous estrus when PG was

administered 7 d later, it did not improve the AI pregnancy rates compared to the MGA-PG

estrous synchronization system.









CHAPTER 1
INTTRODUCTION

Artificial insemination (AI) provides producers with the opportunity to improve their herd

through the use of superior genetics. Additionally, a successful AI program benefits the

producer economically by decreasing the number of bulls needed while potentially increasing the

performance and uniformity of the calf crop. However, the implementation of a successful AI

program requires significant labor, which offset the economic benefits and limits the practicality

of AI. Therefore, a maj or requirement of a successful AI program requires estrous

synchronization systems that result in a large number of cattle that can be AI in a short period of

time.

Numerous estrous synchronization systems have been developed to meet the needs of each

production scenario. Products available for estrous synchronization systems include progestins,

prostaglandin Fza (PGF2a), and gonadotropin-releasing hormone (GnRH). Progestins can be used

to lengthen the estrous cycle by preventing the LH surge, estrus, and ovulation. Prostaglandin

Fza acts to artificially shorten the estrous cycle by initiating luteolysis. Finally, GnRH can be

administered to control follicle wave emergence or to initiate ovulation. Furthermore, these

products can be combined to prevent estrus and ovulation, shorten the estrous cycle, and to

control follicle development. The success of an estrous synchronization system is dependant on

its ability to bring a high percentage (> 75%) of cattle into estrus in a short time period (< 7 d).

Conversely, the effectiveness of these products in synchronizing estrus depend on the genetics of

the herd, body condition, reproductive status (i.e., estrous cycle vs anestrous), stage of the

estrous cycle, environment, and breed-type (Bos taunts vs. Bos indicus). Breed is an important

contributing factor in synchronization systems where most systems in use today have been

designed for cattle ofBos taunts breeding. Therefore, these systems need to be evaluated or new










systems need to be developed to account for the physiological and behavioral differences in

cattle of Bos indicus breeding.

Throughout Florida, the most common form of cattle production is cow/calf operations.

However, the subtropical environment of Florida presents cattle producers with a problem where

elevated temperatures and decreased nutrient availability are not suitable for most breeds of

cattle. Therefore, cattle normally found in Florida contain some degree ofBos indicus breeding.

Cattle of Bos indicus breeding provide the Florida cattlemen many advantages in that they are

adapted to the hot, humid environment, able to survive on low quality forages, and are more

resistant to parasites than cattle of Bos taurus breeding. Conversely, several behavioral and

physiological differences are observed in Bos indicus cattle, resulting in reduced reproductive

performance and decreased effectiveness of commonly used estrous synchronization systems.

In cow/calf operations, the greatest opportunity to implement an estrous synchronization

system is in first service breeding of heifers. Heifers offer many benefits that make them best

suited to for the implementation of an estrous synchronization system. First, heifers are usually

managed in groups supplemented to reach targeted weights and condition scores. Second,

heifers do not have the negative effects of lactation and suckling calf. Third, heifers are usually

cycling prior to the breeding season. Finally, since heifers are managed in groups and do not

have calves, they are easily handled. Estrous synchronization and AI of heifers benefit the

producer by reducing labor required for detecting estrus. Producers can choose to inseminate to

calving-ease sires, therefore, reducing the number of calving-ease bulls needed for natural

service. Moreover, an effective estrous synchronization system allows more heifers the

opportunity to become pregnant early in the first 30 days of the breeding season. More heifers

being exposed early in the breeding season results in more heifers calving early, reducing labor










required during calving season. Also, time of first calving affects lifetime performance of the

cow, where cattle calving as two-year olds will have a greater lifetime production than those

calving at a later date.

One of the most common estrous synchronization systems for heifers utilizes a long term

(14 d) melengestrol acetate (MGA) treatment and PGF2a. administered 19 d after MGA

withdrawal. This estrous synchronization system was developed in Bos taurus heifers and

results in excellent AI pregnancy rates. Conversely, this system is less effective in heifers of Bos

indicus breeding. Recent research has increased the effectiveness of this system in Bos indicus

heifers by altering the delivery of PGF2a, but it does still not result in AI pregnancy rates

observed in Bos taurus heifers. Therefore, this review will focus on the physiological and

behavioral characteristics of reproductive function in cattle of Bos indicus breeding and to

review the estrous synchronization literature in an attempt to identify why there is a reduced

reproductive performance to estrous synchronization systems in cattle ofBos indicus breeding.









CHAPTER 2
REVIEW OF LITERATURE

Endocrine Control of the Estrous Cycle

Regulation of mammalian reproduction is primarily controlled at the level of the

hypothalamus and pituitary. The main hypothalamic hormone involved in regulating the

hyp othal ami c-pituitary -gonadal axi s and reproducti on i s gonadotropi n-rel ea sing- horm one

(GnRH). Gonadotropin-releasing-hormone, a decapeptide consisting of ten amino acids, is

released from the hypothalamus and signals the release of the two gonadotropins, luteinizing

hormone (LH) and follicle-stimulating hormone (FSH), from the anterior pituitary (Schally et al.,

1971). These pituitary derived gonadotropins act on ovarian cells to signal changes in ovarian

function and secretion of hormones. Furthermore, either positive or negative feedback of steroid

hormones on the hypothalamus acts to regulate the release of GnRH and gonadotropins.

Neurons responsible for the secretion of GnRH are loosely dispersed throughout the

hypothalamus and GnRH is secreted from two distinct areas of the hypothalamus in either a

"tonic" fashion or as a "surge". Tonic secretion, as observed during the luteal phase of the

estrous cycle, is characterized by high amplitude, low frequency pulses under the negative

feedback effect of progesterone and it is driven by neurons in the ventromedial and arcuate

nuclei. Whereas, the surge-like secretion of GnRH, as observed during estrus and driven by the

positive feedback of estradiol secretion, is responsible for the LH surge and it is controlled by the

preoptic and suprachiasmatic nuclei (Smith and Jennes, 2001). The GnRH secreted from the

hypothalamus is released from the median eminence where it enters the hypothalamo-

hypophyseal portal system through fenestrations in the capillary walls to be carried to the

anterior pituitary. At the anterior pituitary, GnRH acts through a seven transmembrane, G-

protein coupled receptor, which stimulates the release of gonadotropins (Kakar et al., 1993).









Luteinizing hormone is necessary for the development of many ovarian events such as

corpus luteum (CL) development (Snook et al., 1969), secretion of the gonadal steroid

progesterone (Alila et al., 1988), follicle maturation (Ginther et al., 2001), and ovulation

(Wettemann et al., 1972; Fortune, 1994). Pulses of GnRH stimulate the release of LH in a

pulsatile fashion (Schams et al., 1974) where pulses are characterized by a rapid increase

followed by a gradual decline in LH concentrations (Forrest et al., 1980). Anderson et al. (1981)

reported 3.4 pulses of LH over an 8 h period in prepubertal beef calves. However, LH secretary

patterns are dependent upon the stage of the estrous cycle. During the early luteal phase, LH

secretion is characterized by high frequency, low amplitude pulses; whereas during the mid

luteal phase LH secretion is characterized by high amplitude, low frequency pulses (Rahe et al.,

1980). Walters et al. (1984) observed that pulses of estradiol are observed within 60 min

following pulses of LH, with greater estradiol pulses during the early luteal compared to the mid

luteal phase. Furthermore, estradiol enhances the release of LH from the anterior pituitary (Cupp

et al., 1995) by increasing GnRH receptors in the pituitary (Gregg et al., 1990).

Follicle-stimulating hormone, as its name implies, functions to stimulate the recruitment

and growth of a new follicle wave (Sunderland et al., 1994; Evans et al., 1997). Follicle

development remains dependent on FSH until follicle deviation (Ginther et al., 2000a); and

stimulation of thecal estrogen production requires FSH beyond this point (Mihm et al., 1997).

Following hourly infusion of exogenous GnRH, concentrations of LH increased, however no

increases in the concentration of FSH were observed (Vizcarra et al., 1997) indicating that FSH

secretion is not controlled exclusively by GnRH. Furthermore, Cupp et al. (1995) reported that

concentrations of FSH were greater in ovariectomized and ovariectomized + estradiol treated

cows than in intact controls, demonstrating that the regulation of FSH secretion could be









controlled by the ovaries. Unlike LH, repeated treatment of GnRH did not result in the reduction

of FSH secretion (Schams et al., 1974).

Progesterone, secreted by the corpus luteum (CL), and estradiol, secreted by the dominant

follicle, feedback onto the hypothalamus to regulate the secretion of gonadotropins. Bergfeld et

al. (1995) reported that cows with high progesterone concentrations had fewer LH pulses as well

as lower concentrations of estradiol; whereas, cows with low progesterone concentrations had a

greater frequency of LH pulses. During the mid luteal phase, when progesterone concentrations

are at peak concentrations, LH pulses are high amplitude and low frequency (6-8 pulses/24 h;

Rahe et al., 1980). However, high estradiol concentrations, as observed during the follicular

phase, lead to increased LH pulse frequency (Stumpf et al., 1993). Conversely, progesterone and

estradiol act to regulate FSH secretion differently compared to LH. Ireland and Roche (1982)

and Price and Webb (1988) reported no significant effect of progesterone on FSH secretion.

However, treatment of intact (Ireland and Roche, 1982) and ovariectomized (Price and Webb,

1988) heifers with estradiol significantly decreased FSH secretion. Following follicle ablation,

FSH concentrations were greater in heifers treated with 0 mg estradiol than those treated with 0.5

mg estradiol (Ginther et al., 2000b).

Reproductive function is similar between cattle of Bos taurus and Bos indicus breeding,

however differences have been noted in the secretary patterns of reproductive hormones between

the breeds. Both Bos taurus and Bos indicus cattle exhibit a pulsatile secretion of LH, but a

greater number of LH peaks (3.33 vs. 3.00), magnitude of peaks (overall LH peak height; 10.45

vs. 7.85 ng/mL) and LH pulse heights (the highest LH value minus the lowest LH value; 6.50 vs.

4.28 ng/mL) are observed in Bos taurus compared to Bos indicus cows, respectively (Griffen and

Randel, 1978). Griffen and Randel, (1978) observed that ovariectomized Hereford (Bos taurus)









and Brahman (Bos indicus) cows responded to exogenous GnRH with increased concentrations

of LH, but the increases in LH released were significantly less in Brahman cows. In response to

exogenous estradiol, Brahman heifers have decreased LH secretion compared to Hereford and

Brahman x Hereford heifers (Randel, 1976). In addition, Brahman cows were less responsive

and Brahman x Hereford cows tended to be less responsive compared to Hereford cows treated

with exogenous estradiol as determined by subsequent LH secretion (Rhodes and Randel 1978).

Furthermore, the interval from estradiol treatment to LH response was longer in Brahman

compared to Hereford and Brahman x Hereford heifers. These results are supported by Rhodes

et al. (1978) who reported that Brahman cows secrete less LH in response to exogenous estradiol

and take longer to respond compared to Hereford and Brahman x Hereford cows. Therefore,

decreased secretion of LH in response to estradiol in Bos indicus cattle may be due to a

decreased sensitivity of the hypothalamus to the positive feedback effects of estradiol.

Puberty

Throughout fetal development, the female reproductive tract forms and the ovaries are

populated with gametes. Shortly after birth, ovarian function begins with follicular growth and

development followed by steroid production, but the female does not ovulate. Puberty is defined

as the time when the female first expresses estrus and ovulates. During the peripubertal period,

the secretion of gonadotropins and the feedback effects of steroids on the hypothalamus change

prior to and after the first ovulation. Factors such as body weight gains from weaning to puberty

(Plasse et al., 1968) and age (Nelsen et al., 1985) have been shown to play maj or roles in the

timing of the onset of puberty.

At approximately 3 5 months-of-age, the hypothalamic-pituitary axis of the heifer

becomes functional, as LH secretion can be regulated by the actions of estradiol on the

hypothalamus (Staigmiller et al., 1979). Furthermore, Barnes et al. (1980) reported that heifers










(approximately 3 to 9 months-of-age) were capable of releasing LH in response to exogenous

GnRH, but not in sufficient quantities to cause an increase in follicle development and high

enough estradiol production to stimulate an LH surge. Gonzalez-Padilla et al. (1975) reported

that pituitary and hypothalamic hormones were released in bursts, with LH bursts being of high

amplitude and low frequency in 14.5 mo old prepubertal Angus heifers. As the onset of puberty

approaches, circulating concentrations of LH steadily increase (Swanson et al., 1972; Day et al.,

1984) and the continual increase in LH secretion becomes the primary endocrine factor

regulating the onset of puberty (Kinder et al., 1995). Day et al. (1984) reported that increases in

pulsatile secretion of LH during the peripubertal period was due to a decrease in the negative

feedback effects of estradiol and the decline in sensitivity of the hypothalamus to estradiol was

due to a decrease in the concentration in estradiol receptors in the hypothalamus and anterior

pituitary (Day et al., 1987). Removal of the negative effects of estradiol by ovariectomy results

in an acute increase in LH concentrations (Day et al., 1984; Anderson et al., 1985). Immediately

following ovariectomy, increased circulating LH concentrations were due to an increase in LH

pulse frequency; whereas, later increases in circulating LH concentrations were associated with

an increase in LH pulse amplitude (Anderson et al., 1985). Treatment of prepubertal,

ovariectomized heifers with exogenous estradiol decreased circulating LH concentrations and

termination of the episodic release of LH (Schillo et al., 1982), which was dependent on the

amount of estradiol administered. However, the suppression of LH secretion, by estradiol,

decreases, as the heifer gets older (Schillo et al., 1982). Gonzalez-Padilla et al. (1975) reported a

priming peak of LH approximately -11 to -9 d prior to the pubertal LH peak and the priming

peak was associated with a slight increase in progesterone concentrations. Berardinelli et al.

(1979) subsequently reported that a non-palpable CL accompanied the prepubertal increase in










progesterone concentrations and ovariectomy resulted in decreased progesterone concentrations,

indicating that the increase in progesterone concentrations probably originated from the ovaries.

The authors suggested that the priming peak of LH served as a transition from pre- to

postpubertal LH concentrations and progesterone exposure played a key role in the establishment

of puberty. Consequently, the heifer becomes less responsive to the negative feedback effects of

estradiol as she matures, resulting in increased LH pulse frequency, which ultimately reaches a

threshold to initiate estrus and ovulation followed by secretion of luteal progesterone resulting in

attainment of puberty. Thus a short estrous cycle, accompanied by an increase in progesterone

concentrations, is followed by the first ovulation, and progesterone concentrations increase to

concentrations > 1 ng/mL, resulting from the newly formed CL. At this point the heifer will

continue with regular estrous cycles and ovulations (Schillo et al., 1992).

In addition to maturation of the endocrine system as the female approaches puberty, the

reproductive tissues including the ovaries and uterus also undergo maturational changes.

Following birth, diameters of ovarian follicles increase from 2 to 34 wk of age, with the greatest

increases occurring between 2 to 8 wk of age (Evans et al., 1994). Day et al. (1987) reported that

as puberty approached, there was no change in ovarian weight or in the numbers of small (<3

mm), medium (3 to 6 mm), or large follicles (7 to 12 mm) but there was an increase in the

numbers of follicles >12 mm. However, a follicle >12 mm was only observed in heifers that

were close to reaching puberty. Growth and development of follicles occurs in a wave-like

fashion in prepubertal heifers (Adams et al., 1994) similar to postpubertal heifers (Sirois and

Fortune, 1988). Uterine weight also increases as the heifer nears puberty with the most rapid

increase in the 50 d preceding puberty (Day et al., 1987). The increase in uterine weight is likely









due to increased estradiol secretion from the ovaries, which is associated with the onset of

puberty (Day et al., 1987).

Breed plays a maj or role in the age at which puberty is attained in cattle. Bos indicus and

Bos indicus x Bos taurus cattle reach puberty at older ages and heavier weights than cattle of

Bos taurus breeding (Reynolds et al., 1963; Plasse et al., 1968; Gregory et al., 1979; Baker et al.,

1989; Rodrigues et al., 2002). The range in age at puberty is approximately 14 to 24 mo for Bos

indicus and 15 to 20 mo for Bos indicus x Bos taurus crossbred heifers (Plasse et al., 1968), and

9 to 15 mo for Bos taurus (Wiltbank et al., 1966). Baker et al. (1989) reported that Jersey (255

d) and Holstein (282 d) dairy heifers reached puberty at younger ages compared to Angus (418

d) and Hereford (466 d) heifers, whereas, Brahman heifers were the oldest at puberty (537 d).

Conversely, crossbred Angus x Brahman (442 d) and Hereford x Brahman (472 d) reached

puberty at a younger age than Brahman heifers. In support of the crossbred data, Gregory et al.

(1979) noted that Pinzgaur crossed with Bos taurus heifers attained puberty at 303 d while

Brahman crossed with Bos taurus heifers attained puberty at 398 d. The late attainment of

puberty of Bos indicus heifers is also reflected in the 9% pubertal by 22 months of age in Bos

indicus compared to 62% of Hereford heifers (Hearnshaw et al. 1994). In contrast, 82% of the

Brahman x Hereford heifers reached puberty by 22 mo emphasizing the importance of cross

breeding on decreasing age of puberty in Bos indicus based cattle, where reproductive traits are

enhanced through heterosis. Rodrigues et al. (2002) reported that both Bos indicus and Bos

taurus heifers underwent a cessation of the negative feedback effects of estradiol on LH

secretion but, Bos taurus heifers undergo this cessation at a younger age. However, the extent of

the negative feedback effect of estradiol on LH secretion was not amplified in Bos indicus

heifers.









Ovarian Function


Follicle Growth and Selection

All of the oogonia a female has available during her lifetime are developed during fetal

development where primordial germ cells migrate from the margin of the hindgut to the paired

somatic gonadal primordia where they become oogonium (McGee and Hsueh, 2000). Oogonia

undergo mitosis and the first stages of meiosis before being arrested at prophase of meiosis-1

(Wartenberg et al., 2001; McGee and Hsueh, 2000). After the attainment of puberty, the

preovulatory surge of LH initiates resumption of meiosis and maturation of the oogonia (Hyttel,

et al., 1997).

The first stage of follicle growth involves a change in shape and increased numbers of

granulosa cells; whereas, the second stage of development is associated with an increase in

oocyte diameter and granulosa cell numbers (Braw-Tal, 2002). In the activated primordial

follicle, an assortment of 5 to 14 flattened and cuboidal granulosa cells form a single layer

surrounding the oocyte (Fair et al., 1997a). As development progresses to the primary follicle

stage, a single layer of 8 to 20 cuboidal granulosa cells encompass the oocyte and the first stages

of zona pellucida formation are observed (Fair et al., 1997a; Braw-Tal and Yossefi, 1997). At

this stage, granulosa cells begin to secrete follistatin, which acts to block the effects of the

growth inhibitor activin A (Braw-Tal, 1994). Also, the oocyte secretes factors such as growth

differentiation factor-9 (GDF-9) and bone morphogenic protein-15 (BMP-15), both of which

play roles in granulosa cell proliferation (McGrath et al., 1995; Dube et al., 1998; Braw-Tal,

2002), whereas oocyte growth is promoted by granulosa secretions such as kit ligand (Braw-Tal,

2002). As the follicle progresses to the secondary follicle stage, the oocyte is surrounded by a

partial or complete bilayer of granulosa cells and oocyte transcription is enabled (Fair et al.,

1997b). Transcriptional activity of oocytes remains inactive until stimulated by FSH, at which









time primordial follicles are activated and RNA synthesis is increased (Fair et al., 1997b).

Advancing from the secondary to tertiary stage of development is characterized by the

completion of the zona pellucida as well as the formation of a multi-layered granulosa cell

population and a small antral cavity (Fair et al., 1997b). In addition, granulosa cells differentiate

to form cumulus granulosa cells and mural granulosa cells. Cumulus granulosa cells surround

and are in close contact with the oocyte, while mural granulosa cells line the follicle wall and

come into contact with the basal lamina (Gilchrist et al., 2004). At the tertiary follicle stage,

increasing amounts of follicular fluid collect in the antral cavity and the follicle achieves

ovulatory capacity.

In order for the graafian follicle to reach ovulatory status, it must undergo three distinct

periods of development. The first period is recruitment, where a cohort of follicles is stimulated

to grow under the influence of FSH. The second period is selection, the process of one follicle

continuing to grow while the others become atretic. And the third period is dominance, where

one follicle continues to grow while suppressing the growth of its subordinates (Sirois and

Fortune, 1988; Fortune, 1994; Ginther et al., 2001). Beginning on approximately day 1 to 2 of

the estrous cycle, a pool of 5 to 10 follicles < 4 mm in diameter, are recruited in response to a

surge in FSH (Sirois and Fortune, 1988; Driancourt, 2001; Sunderland et al., 1994; Evans et al.,

1997). The recruited follicles grow beyond a stage that usually results in atresia for other

follicles (Fortune, 1994). Ginther et al. (1997) reported that the future dominant follicle emerges

6 to 7 h earlier than it's subordinates, providing a size advantage for the future dominant follicle

over the other emerging follicles (Kulick et al., 1999). At this point, the future dominant follicle

and subordinate follicles enter a common growth phase until the beginning of deviation (Ginther

et al., 1997; Kulick et al., 1999). Deviation is the continued growth of one follicle with a









cessation of growth and regression (termed atresia) of other ovarian follicles (Kulick et al.,

1999). After the initial surge in FSH, FSH concentrations decline with the simultaneous growth

of follicles from 4 to 8.5 mm in diameter (Ginther et al., 1997; Ginther et al., 1999). Gibbons et

al. (1999) observed that 3 mm follicles did not have any detectable capacity to suppress FSH

secretion, while follicles reaching 5 mm gain the capacity to suppress FSH secretions.

Conversely, growth beyond 5mm in diameter did not result in an increase in FSH suppressing

capacity. The first follicle to reach 8.5 mm becomes the dominant follicle (Ginther et al., 1999;

Kulick et al. 1999), which is coincident with a decrease in circulating FSH concentrations

(Adams et al., 1993; Kulick et al., 1999) and increases in circulating LH concentrations (Kulick

et al., 1999). After follicle deviation, circulating estradiol concentrations increase (Kulick et al.,

1999) while follicles not selected for dominance become atretic.

The ability of one of the recruited follicles to continue growing while others undergo

atresia is still an area of question. A maj or characteristic of the future dominant follicle is its

ability to secrete greater amounts of estrogen (Badinga et al., 1992) around day 5 of the estrous

cycle. This obseravtion supports early work of Ireland and Roche (1983) who reported that

estrogen-active follicles had a lower incidence of atresia than estrogen-inactive follicles.

Compared to subordinate follicles, dominant follicles contain lower amounts of insulin-like

binding protein (IGFBP)-2 (Stewart et al., 1996), IGFBP-4, and follistatin (Austin et al., 2001),

which support the continued growth of the dominant follicle by maintaining the availability of

IGF-1 and activin-A. This is supported by Mihm et al. (2000) who reported that in a pool of

recruited follicles, the future dominant follicle had the highest concentrations of estradiol and the

lowest concentrations of IGFBP-4. Ireland and Roche (1983) observed that granulosa cells of

the selected follicle have a greater ability to bind hCG compared to non-selected follicles on days









5 and 7 of the estrous cycle. The selected follicle could also bind more hCG on day 7 compared

to day 3. Xu et al. (1995) reported that mRNA for LH receptors was present in day 4 follicles

compared to day 2 follicles. These findings suggest that a follicles ability to achieve estrogenic

activity is crucial for follicle selection. Ginther et al. (2001) observed that suppression of LH

secretion did not affect the largest follicle prior to deviation but reduced follicle diameter and

follicular fluid concentrations of IGF-1 and estradiol concentrations following deviation. The

findings of Gong et al. (1995) support this by showing that suppression of LH secretion to basal

concentrations and the abolishment of the pulsatile secretion of LH inhibited follicle growth

beyond 7-9 mm. Furthermore, LH-receptor mRNA was only found in healthy dominant follicles

> 9 mm (Xu et al., 1995). These findings suggest that there is a divergence from dependency

from FSH to LH, but not until after deviation. Therefore, LH plays a maj or role in the growth

and function of the dominant follicle following deviation.

In order for one follicle to establish and maintain dominance over its subordinates, it must

suppress FSH secretion to prevent recruitment of smaller follicles. Administering recombinant

bovine FSH to heifers before selection of the dominant follicle delayed the time for divergence

between dominant and subordinate follicles (Adams et al., 1993). Furthermore, cauterization of

the dominant follicle resulted in a surge of FSH and recruitment of a new pool of growing

follicles soon after ablation (Adams et al., 1992). Treatment of animals with estradiol when the

largest follicle reached 6 mm, around the time that endogenous FSH concentrations are normally

declining, resulted in the suppression of FSH secretion and follicle diameter within 8 h (Ginther

et al., 2000a). Ginther et al. (2000b) also reported that exogenous estradiol given to cattle after

dominant follicle ablation caused a 2 to 3 h delay in the FSH surge. Nett et al. (2002) suggested

that estradiol suppressed FSH secretion by altering the production of activin PB in pituitary cells.









Bleach et al. (2001) reported that as FSH concentrations decline, estradiol and inhibin A

concentrations increase coincident with the growth of a new dominant follicle. Inhibin originates

from granulosa cells and functions to suppress secretion and release of FSH from the anterior

pituitary (Good et al., 1995). Sheep immunized against inhibin showed an increase in FSH

concentration as well as ovulation rate (Wheaton et al., 1992). Treatment of cattle with

antiserum for inhibin and estradiol resulted in increased circulating FSH concentrations for a

longer period of time than giving antiserum for inhibin alone, suggesting a synergistic role of

suppressing FSH by inhibin and estradiol (Kaneko et al., 1995). Suppressing the synthesis and

secretion of FSH with estradiol and inhibin resulted in atresia of subordinate follicles due to their

inability to utilize low concentrations of circulating FSH, which is an environment that the

dominant follicle can survive in (Ginther et al., 2000b; Austin et al., 2001).

Following the establishment of dominance, follicles must achieve ovulatory competence in

order to respond to a pre-ovulatory surge of LH. The dominant follicle becomes more

responsive to LH and gains ovulatory capacity when it reaches approximately 10 mm in diameter

(Sartori et al., 2001), coincident with LH receptor mRNA in granulosa cells of follicles > 9 mm

(Xu et al., 1995). Once the dominant follicle achieves ovulatory competence, it can either

ovulate or become atretic, depending on the stage of the estrous cycle. For the dominant follicle

to ovulate, luteolysis must occur followed by a decline in progesterone secretion followed by

subsequent increases in estradiol secretion, which drives the preovulatory surge of LH resulting

in ovulation (Wettemann et al., 1972; Fortune, 1994). When luteolysis does not occur,

progesterone concentrations remain elevated, which suppress LH pulses resulting in decreased

estradiol secretion (Fortune, 1994; Badinga et al. 1992). In response to decreased estradiol

secretion, the dominant follicle becomes atretic, thereby removing the negative feedback effect









of ovarian progesterone, which allows for an increase in FSH concentrations and recruitment of a

new follicle wave (Fortune, 1994).

Follicle development in cattle occurs in a wave-like pattern, which allows for a steady

supply of ovulatory follicles (Sirois and Fortune, 1988). Each wave is characterized as having

one large dominant follicle with ovulatory capacity and several smaller follicles termed

subordinates (Sirois and Fortune, 1988). During an estrous cycle, the number of follicular waves

varies between animals. Two and three-wave cycles are the most common although one, four,

and five wave cycles have been observed (Sirois and Fortune, 1988; Savio et al., 1988; Viana et

al., 2000). Estrous cycle length is reflected in the number of waves that occur during the estrous

cycle. Estrous cycles with two follicle waves are approximately 20 d in duration; whereas,

estrous cycles with three waves last from 21 to 23 d (Ginther et al., 1989; Viana et al., 2000;

Sirois and Fortune, 1988; Savio et al., 1988).

In cattle with two follicular waves, wave emergence is approximately days 2 and 11 of the

estrous cycle for the first and second wave, respectively; whereas, cattle with three follicular

waves, wave emergence is approximately days 2, 9, and 16 of the estrous cycle for the three

waves, respectively (Sirois and Fortune, 1988). In two wave cycles, the first wave reaches a

maximum diameter about day 6 with regression by day 10 while the second dominant follicle

reaches a maximal diameter by day 19 (Savio et al., 1988). For three wave cycles, the first and

second wave dominant follicles reach a maximum diameter on day 6 and 16, respectively,

followed by regression, while the third wave dominant follicle achieves maximal diameter on

day 21 (Savio et al., 1988).

Differences in the number of waves results in different sizes and ages of dominant follicles

in a wave. In cycling Holstein heifers exhibiting two wave cycles, the first-wave dominant









follicle (17.1 mm) and ovulatory (16.5 mm) dominant follicle reached a similar average maximal

diameter, while the duration between emergence of waves was shorter for the first (9.7 d) than

the ovulatory wave (10.4 d; Ginther et al., 1989). Conversely, Savio et al. (1988) noted that the

maximal diameter of the first wave dominant follicle (14.3 mm) was smaller than the ovulatory

dominant follicle (20.3 mm) in cycling beef heifers over two consecutive estrous cycles. In

cycling Holstein heifers exhibiting three follicle waves, Ginther et al. (1989) observed that

average follicle diameter was smaller for second (12.9 mm) and ovulatory (13.9 mm) wave

dominant follicles compared to the first wave dominant follicle (16.0 mm). The duration

between emergence of waves was similar for the first (9.0 d), second (7.2 d), and ovulatory (6.7

d) waves. Sirois and Fortune (1988) reported that in Holstein heifers displaying normal estrous

cycles, the second wave dominant follicle (10.2 mm) had the smallest maximal diameter of the

three follicles with no differences between the first (12.3 mm) and third (12.8 mm) wave

follicles. Contrary to their findings, Savio et al. (1988) demonstrated that the dominant follicles

of the first two waves were smaller than the ovulatory follicle of the third wave. Townson et al.

(2002) reported that cattle with two follicle waves had larger (17.2 vs. 16.0 mm) and older (6.7

vs. 5.2 d) ovulatory follicles that were less fertile than cattle with three waves, respectively.

Furthermore, differences in the length of the luteal phase between two and three wave estrous

cycles were reported by Ginther et al. (1989) where luteal regression occurred on day 16 and 19,

respectively. Also, the interval from emergence to ovulation was shorter in cows with three

compared to two wave cycles, resulting in a shorter period of dominance for the third wave

ovulatory follicle (Ginther et al., 1989).

The number of follicle waves within an estrous cycle has been shown to vary according to

environmental conditions, nutritional management, and lactation status. Heat stress increased the










proportion of three wave follicular cycles (Wilson et al., 1998), resulted in earlier regression of

the first wave dominant follicle followed by earlier recruitment of the second wave in two wave

estrous cycles (Wolfenson et al., 1995), decreased the first wave dominant follicle diameter

(Badinga et al., 1993), and resulted in earlier emergence of ovulatory follicle and a longer period

of dominance (Wolfenson et al., 1995). Nutritional restriction reduced the growth rate and

diameter of dominant follicles during an estrous cycle in beef heifers (Mackey et al., 1999) as

well as decreased dominant follicle diameter and persistence of the first wave dominant follicle

in Brahman heifers (Rhodes et al., 1995). In contrast, supplemented grazing Bos indicus x Bos

taurus heifers had more large follicles than non-supplemented heifers (Maquivar et al., 2005)

and feeding calcium salts of long chain fatty acids increased the diameter of the dominant follicle

in multiparous Holstein cows (Lucy et al., 1991). Lactational status in dairy cows also effects

follicle development, which appears to be driven by the level of nutrition as well as the resulting

hormone profiles. Lactating dairy cows have decreased concentrations of glucose, IGF-1, and

insulin, which is reflected in fewer class two (6-9 mm) and three (10-15 mm) follicles but more

class four (> 15 mm) follicles that are less estrogenic compared to non-lactating dairy cows (De

La Sota et al., 1993)

Characteristics of follicular growth are also different between Bos taurus and Bos indicus

cattle. Early work by Segerson et al. (1984) before the advent of ultrasonography, reported more

follicles < 5 mm in Brahman cows while Angus cows had more follicles > 5 mm in diameter.

Recent work using ultrasonography during an entire estrous cycle revealed that the numbers of

small (2-5 mm), medium (6-8 mm), and large (> 9 mm) follicles were greater in non-lactating

Brahman (39.0, 5.0, and 1.6) compared to Angus (21, 2.3, and 0.9) cows, respectively (Alvarez

et al., 2000). Alvarez et al. (2000) also observed that Angus cows had a greater FSH surge and









circulating plasma FSH concentrations compared to Brahman cows indicating that Brahman

cows produce more follicles even though they have a smaller FSH surge and lower FSH

concentrations. Alvarez et al. (2000) hypothesized that the greater follicle numbers may be due

to higher concentrations of IGF-1 in Brahman cows. This finding is supported by Simpson et al.

(1994), who reported that Brahman cows had greater circulating IGF-1 concentrations and

IGFBP compared to Angus cows.

Alvarez et al. (2002) also indicated that Brahman cows had dominant follicles with a

greater maximum diameter compared to Angus cows during the first (15.3 vs. 11.4 mm) and

ovulatory (15.6 vs. 12.8 mm) follicle wave, respectively. Growth rate of the first wave dominant

follicle tended to be greater in Brahman (1.6 mm/d) compared to Angus cows (1.2 mm/d),

whereas growth rate was similar for the ovulatory dominant follicle between Brahman (1.4 vs.

1.4 mm/d) and Angus (1.4 mm/d). Aside from these differences, length of the estrous cycle

(19.5 vs. 19.7 d), number of two follicular wave cycles (72.7 vs. 55.6%) and three follicular

wave cycles (27.3 vs. 44.4%) was similar between Angus and Brangus cows, respectively

(Alvarez et al., 2000). Viana et al. (2000) reported maximal diameters for first (11.8 mm) and

ovulatory (12.4 mm) wave follicles in Gir (Bos indicus) cows, which were considerably less than

the Brahman cows in the Alvarez et al. (2002) study. Other studies in Bos indicus cattle reported

three follicular waves during the estrous cycle approximately 66.7% (Rhodes et al., 1995) and

60% (Viana et al., 2000) of the time as well as incidences of four follicle waves approximately 7

to 27% of the estrous cycles (Rhodes et al., 1995; Viana et al., 2000). Of interest, Figueiredo et

al. (1997) reported that Nelore cows commonly have two follicle waves (83.3%), whereas Nelore

heifers had a greater incidence of three follicle waves (64.7%).









Corpus Luteum (CL) Function and Luteolysis

After ovulation, the theca interna and granulosa cells of the ovulatory follicle undergo

morphological and biochemical changes to become the CL. The main function of the CL is to

synthesize and secrete progesterone, which is required for the maintenance of pregnancy and

regulation of the estrous cycle. Corpora lutea are mainly comprised of two cell types, large and

small luteal cells. Alila and Hansel (1984) reported that small luteal cells of the early developing

CL were primarily from thecal origin, whereas large luteal cells were primarily granulosa in

origin. The small luteal cells eventually develop into large luteal cells with age as the original

large luteal cells disappear (Alila and Hansel, 1984). Small luteal cells are highly responsive to

LH and secrete progesterone under the influence of low LH secretion; whereas, large luteal cells

are less responsive to LH and secrete progesterone under high LH secretion and are subj ected to

the luteolytic effects of PGFza (Alila et al., 1988). Furthermore, large luteal cells secrete most of

the progesterone (> 80%) but not under the influence of LH in cattle and sheep (Ursley and

Leymarie, 1979; Fitz et al., 1982; respectively). Hoyer et al. (1984) observed that progesterone

production in large luteal cells is independent of elevated intracellular cAMP levels, suggesting

that large luteal cells are secreting progesterone at a maximal rate lending them unresponsive to

further stimulation. Binding of LH to its receptor on small luteal cells results in the activation of

the second messenger system. Upon activation of the second messenger adenyl cyclase, cyclic

adenosine monophosphate (camp) is synthesized (Hoyer and Niswender, 1986), which activates

protein kinase A and phosphorylate the enzymes necessary for steroidogenesis (Milvae et al.,

1996).

Prostaglandin Fza (PGF2a) is widely known as the primary luteolytic agent in many

species, including cattle (Rowson et al., 1972; Inskeep, 1973; Nancarrow et al., 1973). Early









research demonstrated that hysterectomy of ewes and heifers resulted in maintenance of the CL

(Wiltbank and Casida, 1956), suggesting that the luteolytic signal came from the uterus.

Ligation of the uterine vein ipsilateral to the ovary with the CL resulted in maintenance of the

CL for an extended period (Inskeep and Butcher, 1966). Hixon and Hansel (1974) further

reported that PGF2a acted on the ovaries through a countercurrent exchange between the uterine

vein and ovarian artery. Upon reaching the ovary with the CL, PGF2a initiates the rapid decline

in progesterone secretion resulting in elevated LH concentrations leading to estrus and ovulation

(Stellflug et al., 1977).

Further research reported that pulses of PGF2a WeTO Observed throughout the estrous cycle

without the initiation of luteolysis; however, during luteolysis, pulses of PGF2a Were mOTO

frequent in their release (Zarco et al., 1988). During a spontaneous luteolysis in the cow, pulses

of PGF2a Secretion were observed along with pulses of oxytocin (Vighio and Liptrap, 1986),

suggesting a positive feedback loop between PGF2a and oxytocin (Milvae and Hansel, 1980;

Schallenberger et al., 1984). LaFrance and Goff (1985) demonstrated that an injection of 100 IU

of oxytocin had no significant effect on PGF2a prOduction as measured by its metabolite (PGFM)

on days 3 and 6 of the estrous cycle but when oxytocin was administered on days 17 to 19 of the

estrous cycle there were increased concentrations of PGFM. Treatment with progesterone

followed by estradiol increased the numbers of endometrial oxytocin receptors (Vallet et al.,

1990) and when oxytocin was administered to these animals, concentrations of PGFM increased.

It was further demonstrated that during the late stages of the estrous cycle, progesterone down-

regulates its own receptor in the uterine endometrium, reducing its action and stimulating the

action of estradiol (Robinson et al., 2001). Coincident with the down regulation of uterine

progesterone receptors, uterine oxytocin receptors are increased due to increasing estradiol









concentrations (Vallet et al., 1990). The importance of estradiol had been previously reported

LaFrance and Goff, (1988) who demonstrated that PGFM concentrations following either a 14 or

21 d treatment with progesterone were significantly greater in heifers treated with estradiol

followed by exogenous oxytocin compared to treatments with just oxytocin. Increases in

estradiol concentrations increased the frequency of the pulse generator, driving the release of

sub-luteolytic levels of PGFza fTOm the uterus. Furthermore, PGF2a Secreted from the uterus acts

on the CL to stimulate the release of luteal oxytocin, which amplifies the secretion of uterine

PGF2, to luteolytic levels (McCracken et al., 1999). The luteolytic levels of PGFza activate the

PGF2a receptor, located on both large and small luteal cells, and reduce progesterone

concentrations (McCracken et al., 1999). Oxytocin binding to its receptor in the uterus activates

the inositol 1, 3, 4-triphosphate second messenger system, resulting in the conversion of

diacylglycerol to arachidonic acid (Flint et al., 1986) the precursor to PGF2a Synthesis and

eventual release of PGFza.

Luteolysis is defined as the structural demise of the CL associated with reduced synthesis

and secretion of progesterone, followed by a loss in luteal cells (Niswender et al., 2000). The

process of luteolysis can be attributed to a variety of actions of PGF2a at the cellular level as well

as changes in gene expression. There appears to be downregulation of receptors for luteotropic

hormones, however decreases in LH receptors, determined by hCG binding capacity, are not

observed until after a fall in progesterone (Spicer et al., 1981). Also, there are changes in the

transport of cholesterol into the cell. Following treatment with PGF2a, there is a 50% decrease in

steroidogenic acute regulatory protein (StAR; Pescador et al., 1996), the transporter of

cholesterol across the mitochondrial membrane. Finally, the activity of steroidogenic enzymes,

such as 3 P-hydroxysteroid dehydrogenase (3 P-HSD), required for progesterone synthesis is










decreased within within an hour of PGFza treatment (Hawkins et al., 1993). In response to

PGF2a, changes in gene expression include the inhibition of the LH receptor, StAR, and 3 P-HSD

genes, whereas genes regulating luteal cell gene trancription, such as c-fos and prostaglandin

G/H synthase-2 (PGHS-2), and genes involved in recruiting monocytes, macrophages, and

monocyte chemoattractant protein-1 (MCP-1) are induced (Tsai et al., 2001). Furthermore,

PGF2a Stimulates the luteal secretion of high amounts of the vasoconstrictive agent, endothelial

cell vasoconstrictive peptide endothelin-1 (ET-1), which inhibits luteal progesterone production

(Girsh et al., 1996). All the aforementioned changes precede changes in the cellular makeup of

the CL. Braden et al. (1988) reported that by 36 hours after treatment with PGF2a large luteal

cell numbers remained the same but their diameter decreased; whereas, the number of small

luteal cells decreased by 24 hours after treatment.

The luteolytic actions of exogenous PGF2a On the CL can occur as early as day 5 and as

late as day 16 of the estrous cycle. Henricks et al. (1974) reported that treatment with PGF2a On

days 3 and 4 of the estrous cycle had no effect on plasma progesterone concentrations. However,

the unresponsiveness of the early bovine CL does not appear to be due to the lack of receptors

for PGF2 a S PGF2a receptors appear as early as 2 d after ovulation while receptor numbers and

affinity remained the same through day 10 after ovulation (Wiltbank et al., 1995). Therefore, the

refractory period of the early developing CL to PGF2a is not due to the lack of receptors. Tsai

and Wiltbank (1998) reported that circulating PGF2a reached the early developing CL to the

same extent as the mid-cycle CL, but did not induce intraluteal PGF2a. This could be due to the

observation that the early CL has a greater capacity to catabolize PGF2a into PGFM due to

increased enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH; Silva et al.,

2000). Inhibitors of the second messenger system for PGF2a receptors are also increased during









the early luteal phase of the estrous cycle (Juengel et al., 1998). Conversely, Rao et al. (1979)

reported that specific binding of PGF2u to CL membrane increased from the early luteal phase

(day 3 of estrous cycle) to the greatest levels observed during the late luteal phase (day 20 of

estrous cycle), a time where the CL was actively regressing. Moreover, the authors reported an

increased number of PGF2u receptors during the mid luteal phase (day 13 of estrous cycle) but

the affinity of PGF2u to its receptor was 203 times less than during the late luteal phase.

Additionally, Sakamoto et al. (1995) noted that mRNA for PGF2u receptors increased from the

early luteal phase (days 3-5 of estrous cycle) to the late luteal phase (days 15-18 of estrous cycle)

and was reduced for the regressed CL. Following the initiation of luteolysis, intra-luteal

progesterone secretion began decreasing immediately while intra-luteal PGF2u Slightly increased

and dramatically increased from 24 hr to 300% (Shirasuna et al., 2004).

Characteristics of luteal development and function appear to be different between Bos

taurus and Bos indicus cattle but the data is conflicting. In general Bos indicus cattle have

smaller CL than Bos taurus cattle regardless of whether studies included removal of ovaries

(Irvin et al., 1978; Segerson et al., 1984) or evaluation via ultrasonography (Rhodes et al., 1995).

Although Alvarez et al. (2000) reported larger CL sizes in Bos indicus compared to Bos taurus

cows as determined by ultrasonography. Similarly, there are differences in progesterone

production but these data are also conflicting. Segerson et al. (1984) reported that luteal

progesterone content and serum progesterone concentrations were greater in Bos taurus

compared to Bos indicus cows while Adeyemo and Heath (1980) observed that Bos taurus cows

had greater concentrations of progesterone throughout the estrous cycle compared to Bos indicus

cows. In contrast, Irvin et al. (1978) reported no differences in luteal content or concentrations

of progesterone between Bos taurus and Bos indicus cattle. Likewise, Alvarez et al. (2000)










reported that Bos indicus cows had similar progesterone concentrations compared to Bos taurus

cows even though the Bos indicus cows had greater CL sizes. Alvarez et al. (2000) suggested

that the reason for increased luteal growth in Bos indicus cattle may be a result of increased

concentrations of growth hormone or IGF-1.

Although not well documented, there appears to be differences in the luteolytic response to

PGF2u between Bos taurus and Bos indicus cattle. A single study in Bos indicus (Cornwell et al.,

1985) heifers suggests a decreased response to PGF2u during the early luteal phase compared to

early luteal phase in Bos taurus (Tanabe and Hann, 1984) heifers. In Brahman heifers that did

not undergo luteolysis and exhibit estrus, progesterone concentrations initially declined by 12 hr

after PGF2u but progesterone concentrations began to increase within 48 hr after PGF2u treatment

(Cornwell et al., 1985). Santos et al. (1988) reported an increased estrous response following

two consecutive 12.5 or 25 mg PGF2u treatments administered 24 hr apart in Brahman heifers

and Brangus cows. Furthermore, Bridges et al. (2005) noted that the percentage of heifers

undergoing luteolysis was increased in yearling Bos indicus x Bos taurus heifers following two

consecutive 12.5 mg PGF2u treatments compared to a single 25 mg PGF2u treatments. In the

same report, luteolysis was similar between yearling Bos taurus and 2 yr-old Bos indicus x Bos

taurus heifers that received either two consecutive 12.5 mg PGF2u treatments or a single 25 mg

PGF2u treatments. Therefore, the rate of a PGF2u induced luteolysis appears to be different

between Bos indicus and Bos taurus cattle, and there may well be an effect of age on luteolytic

response in Bos indicus cattle.

Bovine Estrous Cycle

Estrous cycles in cattle start with the expression of estrus followed by ovulation, growth

and development of luteal tissues and follicles, luteolysis, and eventually the onset of estrus

again. Associated with this sequence of events are coordinated exchanges in hormonal and









ovarian events. There are two distinct phases that comprise the estrous cycle: the follicular phase

and the luteal phase. The follicular phase is the period from luteolysis through ovulation and is

further divided into proestrus and estrus. The luteal phase is the period from ovulation to

luteolysis and is comprised of metestrus and diestrus.

The length of the estrous cycle length is approximately 20 to 22 d in Bos taunts (Sirois and

Fortune, 1988; Ginther et al., 1989) and Bos indicus (Rhodes et al., 1995; Figueiredo et al., 1997)

cattle. Likewise, Alvarez et al. (2000) observed similar estrous cycle lengths between Angus

(19.5 d) and Brahman (19.7 d) cows. In stark contrast, Plasse et al. (1970) reported a mean

estrous cycle lengths of 28 d in two-year-old Brahman heifers. Numerous studies have

demonstrated that estrous cycle length is dictated by the number of follicle waves during the

cycle. In cattle with two wave follicle development patterns, estrous cycle length was similar

between Nelore cows and heifers (20.7 d; Figueiredo et al., 1997) compared to Holstein heifers

(20.4 d; Ginther et al., 1989), which were significantly less than Nelore cows and heifers (22.0 d)

and Holstein heifers (22.8 d) with three wave follicle growth patterns. In contrast, Savio et al.

(1988) reported similar estrous cycle lengths between Bos taunts beef heifers exhibiting either

two- (20.5 d) or three- (21.3 d) wave follicle development patterns.

The beginning of the estrous cycle is marked by estrus, where progesterone concentrations

are low (0.33 ng/mL) and estradiol concentrations are increasing, which leads to the LH surge

and ovulation (Wettemann et al., 1972). Interval from peak estradiol concentration to the

preovulatory surge of LH is approximately 6 to 8 h (Walters et al., 1984; Cavalieri et al., 1997).

High estradiol concentrations lead to behavioral changes that are characterized by homosexual

activity of females in estrus. The interval from estrus to ovulation has been shown to be










approximately 28-32 hr in Bos taunts (Walker et al., 1996; Wettemann et al., 1998) and 26 hr in

Bos indicus cattle (Lamothe-Zavaleta et al., 1991; Pinheiro et al., 1998).

Cattle of Bos indicus breeding are more difficult to detect in estrus (Galina et al., 1994) and

exhibit more covert signs of estrus such as head butting and smelling of genitalia (Galina et al.,

1982; Lamothe-Zavaleta et al., 1991). Bos indicus cattle also have an increased incidence of

silent estrus (Plasse et al., 1970; Dawuda et al., 1989), which is one of the reasons why estrus is

difficult to detect. The recent advent of radiotelemetric heat detection aids has also provided an

insight into characteristics of behavioral estrus of cattle. Radiotelemetric heat detection

significantly aids in the efficiency of estrous detection compared to visual observation

(Stevenson et al., 1996), it provides a detailed record of the initiation of estrus (night vs. day),

end of estrus, duration of estrus, and the intensity of estrus based on the number of mounts

received. Several authors have a slightly greater percentage of Bos indicus cattle in estrus during

the night time hours (Pinheiro et al., 1998; Landaeta-Hernandez et al., 2002) compared to Bos

taurus cattle. Therefore, a greater number ofBos indicus cattle exhibiting estrus during the night

time hours may impede the effectiveness of visual estrus detection methods and result in fewer

animals being detected in estrus.

The duration of estrus has also been reported to be effected by breed. Bos indicus cattle

have a shorter duration of estrus (Rhodes and Randel, 1978; Lamothe-Zavaleta et al., 1991; Rae

et al., 1999) than Bos taurus cattle and this appears to be influenced as to whether it is a

synchronized estrus or a spontaneous estrus (Landaeta-Hernandez et al., 2002). For a

synchronized estrus, the duration of estrus has been reported to be 12 hr in Bos taurus heifers

(Richardson et al., 2002) and 6-7 hr in Bos indicus heifers (Rae et al., 1999). Landaeta-

Hernandez et al. (2002) also reported a similar duration of a synchronized estrus between Angus










(19 h) and Brahman (17 h) cows. However, the duration of a subsequent spontaneous estrus was

greater for Angus (11 h) compared to Brahman cows (6 h).

The duration of estrus also appears to be correlated with the number of mounts received

during estrus as animals with low mounting activity have shorter durations of estrus (Rae et al.,

1999). Reports on mounts received during estrus are conflicting between Bos indicus and Bos

taurus cattle. Galina et al. (1982) reported that Bos indicus crossbred cows (1.6 mounts/hr)

received fewer mounts compared to Bos taurus cows (2.8 mounts/hr). Rae et al. (1999) reported

that Brahman (25 mounts) heifers received more total mounts compared to Angus (19 mounts)

while Brahman x Angus (37 mounts) heifers received more mounts compared to the Angus and

Brahman heifers. It should be noted that in the Rae et al. (1999) study the heifers were managed

in a single synchronized group and the heifers were not separated by breed. Landaeta-Hernandez

et al. (2002) reported a similar number of mounts for Angus (30 mounts) and Brahman (33

mounts) cows during a synchronized estrus but a greater number of mounts for Angus (1 1

mounts) than Brahman (7 mounts) cows during a spontaneous estrus when the cows were

managed in the same pasture. Therefore, the increased number of mounts observed during a

synchronized estrus is probably due to an increased number of animals in estrus at a given time,

resulting in more mounts and a longer duration. In summary, both the duration of estrus and the

number of mounts received during estrus are greater during a synchronized estrus compared to a

spontaneous estrus, which supports the conclusion AI programs in Bos indicus influenced cattle

should be focused around a synchronized estrus.

Environmental effects have also been reported to play a role in the duration and intensity

of estrus. Landaeta-Hernandez et al. (2002) reported that the duration of estrus and number of

mounts were reduced when the temperature-humidity index was increased. Also, Lamothe-









Zavaleta et al. (1991) reported that the duration of estrus was shorter when temperatures were

above 270C. Furthermore, Plasse et al. (1970) reported an increased incidence of ovulation

without estrus in 2-year-old Brahman heifers during the winter months. In addition to

environmental effects, social hierarchy can influence the duration and intensity of estrus between

Bos indicus and Bos taunts cattle. Dominant Brahman cows took longer to exhibit estrus

compared to dominant Angus cows (Landaeta-Hernandez et al., 2002). However, subordinate

Angus cows had a longer interval from PGF2a treatment to the onset of estrus than subordinate

Brahman cows (Landaeta-Hernandez et al., 2002). The Landaeta-Hernandez et al. (2002) report

suggests that social dominance could play a maj or role in the expression of behavioral estrus.

Following estrus is the period known as metestrus, which is the period from the end of

estrus (day 0.5) to the formation of a functional CL (day 3 to 5). During metestrus, progesterone

secretion is low and increases slowly until complete formation of the CL, which marks the end of

metestrus and the beginning of diestrus. Diestrus lasts 10 to 14 d and is characterized by high

circulating concentrations of progesterone, which suppress the actions of estradiol by preventing

any preovulatory surge of LH. Harms et al. (1969) reported that from day 2 to 9 of the estrous

cycle, progesterone concentrations increased from 2.8 to 14.1ng/mL in Bos taunts heifers.

Additionally, Alvarez et al. (2000) noted similar maximal progesterone concentrations for Angus

(4.3 ng/mL) and Brahman (4.4 ng/mL) cows. Henricks et al. (1971) observed that peak

progesterone concentrations ranged from 5 to 12 ng/mL in Bos taunts cattle, while Ruiz-Cortez

and Olivera-Angel (1999) reported that peak progesterone concentrations ranged from 1 to 8

ng/mL in Bos indicus cattle.

The initiation of luteolysis (day 16 to 19) marks the beginning of proestrus. During

proestrus, circulating progesterone concentrations decline while estradiol increases coincident










with dominant follicle development (Henricks et al., 1971). Increasing estradiol concentrations

lead to the onset of estrus.

Estrous Synchronization through Manipulation of the Estrous Cycle

Synchronization of the estrous cycle is a management tool that allows for beef and dairy

producers to increase the opportunity for success of an artificial insemination (AI) program.

Benefits of estrous synchronization include an increased percentage of cattle pregnant early in

the breeding season, shortened AI breeding season, shortened calving season, and increased calf

crop uniformity. Labor expenses can also be reduced through estrous synchronization by

decreasing the time and labor required for estrous detection and breeding as well during the

subsequent calving periods. Estrous synchronization can be achieved through the use of several

exogenous hormones including prostaglandin Fza (PGF2a) to shorten the luteal phase; progestins

to prevent estrus and ovulation; and gonadotropin releasing hormone (GnRH) to synchronize

follicle wave development or to ovulate the dominant follicle in conjunction with AI. These

hormones can be combined for control of follicle dynamics, initiate luteal regression,

synchronize estrus, and (or) implementation of a timed AI program, eliminating the need for

estrus detection.

Progestogens

As mentioned previously, progesterone secreted form the CL acts to prevent the

preovulatory surge of LH and expression of estrus during the estrous cycle. Consequently,

exogenously administered progestogens such as melengestrol acetate (MGA), norgestomet

(Syncro-mate B; SMB), controlled intravaginal progesterone releasing device (CIDR), and

inj ectable progesterone have been used to mimick the actions of progesterone throughout the

duration of their administration.









Melengestrol acetate is an orally active progestogen that is used in feedlot heifers to

prevent the expression of estrus and as a synchronization agent in yearling beef heifers. Early

research utilizing MGA demonstrated that fertility at the subsequent estrus after MGA

withdrawal was reduced with both short (< 9 d; Beal et al., 1988) and long term (> 9 d; Hill et

al., 1971) MGA treatments. Although, the reduction in fertility was temporary as fertility

returned to normal at the subsequent estrus. Hill et al. (1971) suggested that the reduction in

fertility was due to several factors including altered cervical mucus, ovulation of abnormal ova,

and (or) fertilization failure. In addition, few normal follicles, some hyperplastic follicles, atretic

follicles, and atretic follicles with thickened theca interna were observed on the ovaries of MGA

treated heifers lacking a functional CL during MGA treatment (Lamond et al., 1971). More

recent research demonstrated that fertility decreased in cows and heifers without the presence of

a CL during progestin treatment compared to cows with a CL present (Sanchez et al., 1993).

Subsequent research demonstrated that low level progestogen exposure, supplied by a

CIDR, in the absence of a functional CL produced an endocrine environment that permitted the

dominant follicle to persist on the ovary while suppressing the development of new dominant

follicles (Sirois and Fortune, 1990). Sirois and Fortune (1990) concluded that in the absence of

endogenous progesterone from the CL, low levels of progesterone supplied by the CIDR resulted

in increased LH pulse frequency, which resulted in enhanced follicle development. However,

when two CIDR' s were administered to mimic normal luteal phase progesterone concentrations

resulting in decreased LH pulse frequency and dominant follicle turnover. The type of

progestogen administered as well as the presence of a function CL can also dictate whether there

is follicle turnover. Custer et al. (1994) treated cows with a progesterone-releasing intravaginal

device (PRID) resulting in regression of the dominant follicle present at the initiation of









treatment and recruitment of a new follicle wave. In contrast, dominant follicle turnover was not

observed in cows treated with MGA in the absence of luteal function, which was a result of

increased LH pulse frequency and development of large single non-ovulatory follicle. Kojima et

al. (1992) treated cows with either a CIDR, MGA, or norgestomet in the absence of a functional

CL resulting in high frequency low amplitude pulses of LH, which sustained dominant follicle

growth and increased circulating concentrations of estradiol. The large follicles that develop

under low endogenous progesterone exposure are larger than normal ovulatory follicles and

produce greater concentrations of estradiol (Savio et al., 1993) and are commonly referred to as

persistent dominant follicles.

The decreased fertility observed with development of persistent dominant follicles is

mediated primarily at the level of the follicle. Mihm et al. (1994) demonstrated that as

dominance of follicles is greater than 4 d, fertility is increasingly reduced. Ahmad et al. (1995)

observed that ovulation of persistent dominant follicles resulted in fewer embryos reaching the

16-cell stage and fewer total embryos collected. Moreover, Revah and Butler (1996) determined

that oocytes ovulated from persistent dominant follicles underwent premature maturation in vivo.

In addition to altered follicle development, the increased estrogen production of the persistent

dominant follicle results in an altered hormonal milieu. The altered hormonal environment alter

the synthesis and secretion of oviductal proteins, which create a less than optimal ovarian

microenvironment leading to decreased pregnancy rates by altering oviductal function,

fertilization, and early embryonic development (Binelli et al., 1999). Therefore, disparities in

embryo development and ovarian microenvironment collectively add to the reduction in fertility

during long-term low-level progestogen exposure. Conversely, Wehrman et al. (1997) reported

similar pregnancy rates following embryo transfer in cattle ovulating either persistent dominant









follicles or normal follicles. Furthermore, progesterone concentrations 7 and 12 d after ovulation

were similar between cattle ovulating a persistent dominant follicle and those ovulating a normal

follicle (Mihm et al., 1994). Therefore, oocytes ovulated from a persistent dominant follicle lead

to decreased fertility while no detrimental effects on CL function are observed following

ovulation of a persistent dominant follicle compared to a normal ovulation.

Persistent dominant follicles developed during long term MGA treatments can be regressed

either through treatment with exogenous estrogen (Yelich et al., 1997) or progesterone

(Anderson and Day, 1994; Garcia et al., 2004) during the MGA treatment. The subsequent

follicle ovulated after MGA withdrawal has normal fertility.

Prostaglandin F~a

As previously mentioned, PGF2a is the luteolytic hormone in beef cattle. The luteolytic

actions of exogenous PGF2a On the CL are effective from day 5 to 16 of the estrous cycle

(Rowson et al., 1972; Inskeep, 1973; Kiracofe et al., 1985). As a result administration of

exogenous PGF2a Such as alfaprostal, chloprostenol, dinaprost tromethamine, and luprostial have

been used to synchronize estrus in beef and dairy cattle. In a review of early studies by Inskeep

(1973), both CL size and progesterone concentrations were reduced within 24 hr following

treatment with exogenous PGF2a. Subsequent research reported that progesterone concentrations

were reduced to < 0.5 ng/mL within 24 hr after a 30 mg inj section of PGF2a TOSulting in increased

estradiol concentrations (Chenault et al., 1976) and the subsequent expression of estrus. The

estrous response in cattle ofBos taurus breeding undergoing normal estrous cycles following a

single administration of PGFza rangeS from 65 to 91% (Lauderdale et al., 1974; Tanabe and

Hann, 1984) with estrus being observed within 2 to 7 d. In contrasts, the estrous response in

cycling cattle of Bos indicus breeding ranges from 56 to 62% in Zebu cattle (Orihuela et al.,

1983) and 46.3 to 54.8% in Nelore cattle (Landivar et al., 1985). The variability in estrous









response and interval to the onset of estrus is due to several factors including estrous cycling

status, stage of the estrous cycle at PGF2u administration, and breed.

The actions of PGF2u are Only seen in cattle that are undergoing normal estrous cycles with

a CL present on the ovary. In estrous cycling cattle, estrous response and interval from PGF2u to

the onset of estrus are affected by the stage of estrous cycle at PGF2u administration (Tanabe and

Hann, 1984; Watts and Fuquay, 1985). Tanabe and Hann (1984) treated Holstein heifers with

PGF2u during the early (d 7), mid (d 11), and late (d 15) luteal phase of the estrous cycle and

reported a higher percentage of heifers exhibiting estrus within an 80 hr period for each

advancing stage of the estrous cycle (86.0, 90.0, and 98.0%; respectively). Of heifers that

exhibited estrus, 100.0% of early, 95.9% of late luteal phase, and only 48.9% of mid luteal phase

heifers exhibited estrus within 72 hr following PGF2u treatment. These findings are in agreement

with Watts and Fuquay (1985) who treated heifers with PGF2u during the early (days 5-7), mid

(days 8-11), and late (days 12-15) luteal phase and observed 72 hr estrous response and interval

from PGF2u to the onset of estrus of 43.0% and 59 hr, 83.6% and 70 hr, and 78.3% and 72 hr,

respectively. Macmillan and Henderson (1984) also reported a similar trend with over 70% of

cows treated with PGF2u On day 7 (early diestrous) and 16 (late diestrous) in estrus within 48-72

hr following PGF2u treatment but only 30% of cows treated on days 11 or 12 (mid diestrous) in

estrus within 48-72 hr following treatment.

Sirois and Fortune (1988) reported a negative correlation between size of the preovulatory

follicle at luteolysis and the interval to ovulation. Furthermore, Kastelic et al. (1990) observed

that the interval from PGF2u to the onset of estrus was shorter for heifers administered PGF2u On

day 5 compared to day 12 of the estrous cycle. Consequently, follicle wave development at the

time of a PGF2u treatment affects the synchrony of the subsequent estrus. With this in mind, it









points out the importance of synchronizing follicle development in conjunction with either luteal

regression or removal of a progestogen.

A PGF2u induced luteolysis does not appear to have any negative effects on fertility of the

subsequent estrus. Macmillan and Day (1982) reported pregnancy rates of 69% in PGF2u treated

cows compared to 60% for non-treated cows with over 2,000 animals in each group. Lauderdale

et al. (1974) reported similar pregnancy rates between cows receiving PGF2u COmpared to those

not receiving PGF2u. Moreover, Tanabe and Hann (1984) reported similar pregnancy rates in

PGF2u treated (77.4%) and non-treated heifers (76.0%). Additionally, Hardin et al. (1980) noted

that pregnancy rates were similar between PGF2u treated (30 and 37%) and non- PGF2u treated

(37 and 38%) Bos indicus cattle. However, stage of the estrous cycle when cattle receive PGF2u

may affect fertility. Pregnancy rates for heifers were lowest (56.8%) for heifers receiving PGF2u

early (d 5-7) in the estrous cycle compared to heifers receiving PGF2u in the middle (d 8-11;

62. 1%) or late (d 12-15; 78.3%) stages of the estrous cycle (Watts and Fuquay, 1985).

There is a limited amount of research that suggests that a PGF2u induced luteolysis maybe

compromised in cattle of Bos indicus breeding. Hansen et al. (1987) reported that Brahman

heifers required a greater dose of PGF2u to achieve luteolysis compared to Brahman cows.

Furthermore, the crossbred Brahman heifers required a greater dose of PGF2u to achieve

luteolysis from days 8 tol0 of the estrous cycle compared to days 11 to 13 of the estrous cycle.

Pinheiro et al. (1998) reported that 48 % (12/25) of Nelore cows with a functional CL, failed to

respond to a luteolytic dose of PGFzu between day 6 to 8 of the estrous cycle. Furthermore, the

authors hypothesized that administration of PGF2u fTOm days 5 to 9 of the estrous cycle may

cause partial luteolysis followed by recovery of luteal activity. Cornwell et al. (1985) reported

that Brahman heifers treated with PGF2u On day 7 and 10 of the estrous cycle had estrous









responses of 50 and 67%, respectively; whereas, 100% of the heifers exhibited estrus when

treated with PGF2a On days 14 and 18 of the estrous cycle. For heifers not responding to PGF2a

on days 7 and 10, progesterone concentrations decreased by 12 hr after PGF2a followed by

increased concentrations of progesterone within 48 hr of PGF2a treatment. Therefore, the early

developing CL (day 5-10 of the estrous cycle) appears to be somewhat refractory to the actions

of a single treatment of PGF2a in cattle on Bos indicus breeding.

Several researchers have addressed the issue of incomplete luteolysis in females of Bos

indicus breeding by changing the administration of PGF2a fTOm a single to two consecutive doses

of PGFza. COrnwell et al. (1985) administered PGF2a to Brahman heifers on day 7 and 8 of the

estrous cycle and reported a significant increase in estrous response (97%) compared to a single

inj section (69%). When a single PGF2a treatment was administered during the early luteal phase

(day 7 and 8) of the estrous cycle, Santos et al. (1988) reported a similar estrous response and

interval from PGF2a to the onset of estrus when either 12.5 mg (84%; 94 hr) or 25 mg (83.1%;

100 hr), respectively. Santos et al. (1988) conducted an additional study to test the effectiveness

of either two consecutive 12.5 mg or 25 mg PGF2a inj sections compared to a single 25 mg

inj section of PGF2a in Brangus (Bos taurus x Bos indicus) cows, which had previously received a

single 25 mg injection of PGFza 11 d earlier. Estrous response and conception rates were greater

for cows receiving two 12.5 mg (82; 73%) and two 25 mg (73; 59%) injections of PGFza

compared to a single 25 mg injection (55; 32%), respectively. The interval from the initial PGF

treatment to the onset of estrus was significantly shorter for groups receiving the two consecutive

12.5 mg (79.0 hr) and 25 mg (73.2 hr) inj sections compared to a single 25 mg (93.9 hr) inj section.

A recent study by Bridges et al. (2005) reported increased luteolysis when two consecutive 12.5

mg PGF2a treatments (92.5%) were administered 24 hr apart compared to a single 25 mg PGF2a









treatment (79. 1%) in Bos indicus x Bos taunts heifers during the mid and late stages of the

estrous cycle. Conversely, no increase in luteolysis was observed in yearling Angus (Bos taunts)

or 2-year-old Bos indicus x Bos taunts heifers receiving the same PGF2a treatments. In

summary, modifying the administration of PGF2a fTOm a single 25 mg treatment to two

consecutive 12.5 mg treatments 24 hr apart appears to increase estrous responses by increasing

the rate of luteolysis in yearling heifers of Bos indicus breeding.

Melengestrol acetate + PGF2,

As early as the 1960's, oral progestogens were used for controlling estrous cycles in cattle

(Hansel et al., 1961; Zimbelman, 1963; Wiltbank et al., 1967). An early study by Zimbelman

and Smith (1966) reported that the optimal dosage of MGA needed to prevent estrus and

ovulation was 0.5 mg/hd/d. However, the negative side of using MGA was the development of a

persistent dominant follicle (Sirois and Fortune, 1990; Savio et al., 1993) that resulted in reduced

fertility (Hill et al., 1971; Ahmad et al., 1995) at the estrus after MGA withdrawal due to

improper embryo development (Ahmad et al., 1995). Furthermore, administration of MGA at

levels (1.0 and 1.5 ng/mL) above the optimal level to inhibit the expression of estrus (0.5

mg/hd/d) did not provide a progesterone environment similar to mid-luteal progesterone

concentrations that would regulate the pulsatile release of LH and stimulate follicle turnover

(Kojima et al., 1995). Administering more MGA did result in a greater interval from MGA to

the onset of estrus (Zimbelman and Smith, 1966; Hill et al., 1971). The mean interval to estrus

was decreased in heifers treated with 0.2 mg/hd/d (2.7 d) compared to heifers treated with 2.0

mg/hd/d (6.3 d) (Zimbelman and Smith, 1966).

Brown et al. (1988) developed a system utilizing MGA and PGF2a that was designed to

circumvent the reduction in fertility following long term (14 d) MGA treatment. The MGA was

administered for 14 d (0.5 mg/hd/d) and heifers were allowed to exhibit estrus but not









inseminated at the estrus after MGA withdrawal. Seventeen days after the termination of MGA,

which placed heifers in the late luteal phase of their estrous cycle, heifers received 25 mg of

PGF2u (MGA + PGF2u). The MGA + PGF2u System was compared to Syncro-mate B (SMB),

which consisted of a 9 d norgestomet implant with an estradiol valerate inj section concurrent with

implant insertion. Estrous response was similar between the MGA + PGF2u (83.4%) and SMB

(90.2%) treatments but conception (68.7 vs. 40.6%) and synchronized pregnancy rates (57.3 vs.

36.6%) were greater for the MGA + PGF2u COmpared to SMB heifers, respectively.

Additionally, estrous response and synchronized pregnancy rate were significantly greater for

cycling (91.6 and 68.4%) than non-cycling (71.0 and 40.3%) MGA + PGF2u treated heifers, but

were similar in cycling (92.4 and 44.6%) and non-cycling (85.2 and 26.2%) SMB treated heifers.

Patterson and Corah (1992) observed a greater 6 d estrous response (79.0 vs. 32.0%), similar

conception rates (64.0 vs. 67.0%), and increased synchronized pregnancy rates (50.0 vs. 21.0%)

for MGA + PGF2u treatment compared to untreated controls, respectively. Similarly, Jaeger et

al. (1992) reported an increased 6 d estrous response (77.0 vs. 25.0%), similar conception rates

(64.0 vs. 50.0%), and increased synchronized pregnancy rates (48.7 vs. 14.0%) for the MGA +

PGF2u treatment compared to the untreated controls, respectively. Numerous follow-up studies

testing the efficacy of the MGA + PGF2u System compared to untreated control heifers have also

been conducted and are summarized in Table 2-1.

Other investigators have varied the original MGA + PGF2u System by increasing the

interval from MGA withdrawal to PGF2u administration from 17 to 19 d (Nix et al., 1998;

Deutscher et al., 2000; Lamb et al., 2000). In a large field study conducted by Lamb et al.

(2000), estrous response was similar for heifers treated with PGF2u either 17 (68.3%) or 19

(68.1%) d following MGA withdrawal. However, the synchrony of estrus was improved with









the 19 d treatment as 99% of the 19 d heifers exhibited estrus within 72 hr of PGF2u COmpared to

74% of 17 d heifers. Moreover, the interval to estrus was reduced for heifers treated 19 d (56.2

hr) compared to 17 d (73.1 hr) after MGA withdrawal. Conception and pregnancy rates were

similar for heifers treated 17 (75.9; 51.8%) or 19 d (81.4; 55.4%) following MGA withdrawal,

respectively. Lamb et al. (2000) hypothesized that by increasing the interval from MGA

withdrawal to PGF2u fTOm 17 to 19 d resulted in a more mature preovulatory follicle at PGF2u,

which resulted in an earlier expression of estrus.

The MGA + PGF2u System has also been used in a timed AI system with or without GnRH.

In two experiments, Larson et al. (1996) subj ected heifers to the MGA + PGF2u (17 d) system

and compared two different AI protocols including estrous detection and AI for 72 hr following

PGF2u to a single Eixed-time AI 72 hr following PGF2u for all heifers. In the first experiment,

pregnancy rates were similar for heifers bred to an observed estrus (31.0%) compared to fixed

time AI 72 hr after PGF2u treatment (36.4%). A second experiment was performed to determine

the effects of estrous detection and AI for 72 hr combined with time AI at 72 hr following PGF2u

for heifers not exhibiting estrus. Results from the second experiment suggest that combining

estrus detection and timed AI yield increased pregnancy rate (48.4%) where heifers not

expressing estrus by the third day after PGF2u reduced estrous detection without a reduction in

fertility. Therefore, combining estrous detection with Eixed time AI subj ects all heifers to AI and

the opportunity to become pregnant during the synchronized period.

A recent study by Salverson et al. (2002) evaluated the effectiveness of type of PGF2u

(cloprostenol vs. dinoprost tromethamine) in the MGA + PGF2u (19 d) system and observed

similar estrous (89 vs. 86%), conception (67 vs. 67%) and pregnancy (61 vs. 57%) rates between

the two types of PGF2u. Another refinement to the MGA + PGF2u System included









administration of GnRH 12 d following MGA withdrawal followed 7 d later with PGF2a (Wood

et al., 2001). The purpose of the GnRH was to ovulate or luteinize most large follicles so as to

synchronize follicle development when PGF2a was administered with the theory that the

subsequent estrus could be more tightly synchronized and allow for a timed-AI. Estrous

response from 48 to 60 hr following PGF2a was significantly increased in GnRH treated (71.0%)

compared to non-GnRH treated (35.0%) heifers, while 7 d estrous response (100.0 vs. 94.0 %)

and the interval from PGF2a to estrus (67.0 vs. 71.0 hr) was similar between treatments,

respectively. However, fertility was not evaluated in this study. DeJarnette et al. (2004)

conducted a study where heifers were treated with either the protocol described by Wood et al.

(2001; MGA + G + PGF2a) Or a short term MGA (STMGA) protocol where heifers were

administered MGA for six days, with GnRH the day before MGA and PGF2a the day after the

last day of MGA. In both treatments, estrus detection and AI were performed for 72 hr, at which

time all heifers not detected in estrus were timed-AI and received GnRH. DeJarnette et al.

(2004) reported increased synchronized pregnancy rates for MGA + G + PGF2a (65%; 55/85)

compared to STMGA (46%; 40/87) treated heifers. Therefore, it appears that fertility is not

compromised following the MGA + G + PGF2a System.

Melengestrol acetate has also been implicated in the induction of cyclicity in prepubertal

heifers. Patterson et al. (1990) noted that 71% of prepubertal Bos taunts and 41% of prepubertal

Bos taunts x Bos indicus crossbred heifers had progesterone concentrations > 1 ng/mL following

a 7 d MGA treatment. Jaeger et al. (1992) reported that a significantly greater percentage

(72.0%) of prepubertal heifers treated with a 14 d MGA treatment reached puberty prior to

PGF2a 19 d after MGA withdrawal compared to un-treated heifers in the same period (45.0%).

Following a 14 d MGA feeding, Deutscher et al. (2000) observed a 15 to 20% increase in the










percentage of heifers cycling. Imwalle et al. (1998) suggests that in prepubertal heifers treated

with MGA that an increased mean LH concentration and LH pulse frequency result in an

increased diameter of the largest follicle during MGA treatment, which results in a follicle large

enough to ovulate after MGA withdrawal resulting in the formation of a CL and initiation of

estrous cycles.

Minimal research has been conducted on the effectiveness of the MGA + PGF2u System for

synchronization of estrus in yearling heifers ofBos indicus x Bos taurus breeding. Stevenson et

al. (1996) synchronized Bos indicus x Bos taurus heifers with MGA (14 d) + PGF2u (17 d)

system where estrus was detected for 72 hr and all heifers that were not in estrus were timed-AI

at 72 hr. The synchronized pregnancy rate was 42.9% (21/49).

Bridges et al. (2005) conducted a study in yearling Bos indicus x Bos taurus heifers using

the MGA + PGF2u System comparing the effectiveness of a single (25 mg) PGF2u treatment 19 d

after MGA withdrawal compared to two (12.5 mg) consecutive PGF2u treatments 24 hr apart on

daysl9 and 20 d following MGA withdrawal. Estrus was detected for 72 hr followed by timed-

AI in conjunction with GnRH for heifers not exhibiting estrus by 72 hr. Conception rates (51.5

vs. 48.8%) were similar while estrus response (50.1 vs. 43.2%), TAI pregnancy rate (33.5 vs.

23.9%), and total pregnancy rate (42.5 vs. 34.5%) were significantly improved by modifying the

delivery of PGF2u to two consecutive split treatments compared to a single treatment,

respectively. The increase total pregnancy rate was due to an increased rate of luteolysis in the

two consecutive PGF2u treatments compared to the single PGF2u treatment. Therefore, the MGA

+ PGF2u System in yearling Bos indicus x Bos taurus crossbred heifers can be improved by

modifying the delivery of PGF2u fTOm a single to split PGF2u treatment; however, the resulting










synchronized pregnancy rates of the MGA + PGF2u System in Bos indicus x Bos taurus heifers

are still considerably less than those observed in Bos taurus heifers.

There could be several reasons for the differences in response to the MGA + PGF2u in

yearling Bos indicus x Bos taurus heifers compared to Bos taurus heifers. Cattle ofBos indicus

breeding have a shorter duration and less intense estrus (Rhodes and Randel, 1978; Rae et al.,

1999; Landaeta-Hernandez et al., 2002), have an increased incidence of three and four follicle

waves (Rhodes et al., 1995; Viana et al., 2000), and reach puberty at older ages (Plasse et al.,

1968; Patterson et al., 1991). One area where research has been limited in cattle of Bos indicus

breeding is evaluating what effect of follicle wave development has on the effectiveness of

estrous synchronization treatments. An asynchrony in follicle development at PGF2u Can TOSult

in large variations in the interval to estrus, undermining the overall effectiveness of a

synchronization system and prevent the use of a fixed timed-AI. One way that that the

asynchrony of follicle development can be altered is by the administration of GnRH to

synchronize follicle development. Therefore, incorporating GnRH during the period between

MGA withdrawal and PGF2u in the MGA- PGF2u System in Bos indicus based heifers could be

used to improve the synchrony of follicle development at PGF2u. By increasing the synchrony of

follicle development at PGF2u, a greater number of heifers should have dominant follicles ready

to ovulate, improve the synchrony of estrus, and allow for a fixed timed-AI. Utilizing a fixed-

timed-AI would allow for the elimination of estrous detection and allow all animals an

opportunity to be inseminated, particularly cattle that have a silent estrus or exhibit estrus more

covertly. Therefore, synchronization systems need to be re-evaluated and tailored to better

synchronize follicle development in Bos indicus based cattle.
































5

5

5

5

5

5

5

5

3 +
72 hr timed-AI

3 +
72 hr timed-AI/GnRH

3 +
72 hr timed-AI/GnRH


19 (12.5 mg) & 20 (12.5 mg)


50.1 42.5


Table 2-1. Summary of studies evaluating the melengestrol acetate (MGA) + PGF2a estrous synchronization system in yearling
beef heifers.


I


I


Estrous
response
(%)
83.4

71.4

79.0

64.2

75.1

86.7

77.6

92.4

87.6

68.3

68.1

77.0

89.0

86.0

68.2


43.2


Synchronized
Pregnancy rate
(%)
57.3

54.3

50.0

55.4

51.8

49.2

53.8

57.1

61.4

51.8

55.4

47.0

61.0

57.0

42.9


34.5


Day PF'CF was administered after
MGA (14 d) withdrawal
17


Study

Brown et al., 1988

Jaeger et al., 1992

Patterson and Corah 1992

Nix et al., 1998



Deutscher et al., 2000







Lamb et al., 2000



Funston et al., 2002

Salverson et al., 2002



Stevenson et al., 1996


Bridges et al., 2005


Breed-type
Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos taunts

Bos indictis x
Bos taunts

Bos indictis x
Bos taunts

Bos indictis x
Bos taunts


Days of estrus detection
5


17

19

19

17

19

17

19 (Estrumate)

19 (Lutalyse)

17


19 (25 mg)









CHAPTER 3
EVALUATION OF FOLLICULAR DEVELOPMENT BETWEEN A 14 D MELENGESTROL
ACETATE (MGA) TREATMENT WITH PGF2u 19 D AFTER MGA WITHDRAWAL IN
ANGUS AND BRANGUS HEIFERS

Introduction

Artificial insemination (AI) provides producers with the opportunity to use genetically

superior AI sires and AI is routinely used in conjunction with estrous synchronization. One of

the major limitations of an AI and estrous synchronization program is the amount of time and

labor required to implement and carry out the program. Therefore, estrous synchronization

programs need to be developed that require minimal animal handling and result in a high

percentage of cattle in estrus during a 2 to 3 d period. One way to minimize animal handling is

to use the orally active progestogen melengestrol acetate in a synchronization program.

Melengestrol acetate (MGA) administered for 14 d, coupled with prostaglandin Fzu (PG)

17 d after the last day of MGA is an effective estrous synchronization system (MGA-PG) that

was developed in yearling Bos taurus heifers (Brown et al., 1988). The interval from MGA

withdrawal to PG was increased from 17 to 19 d resulting in a shorter interval to peak estrus and

more synchronous estrus (Lamb et al., 2000). The effectiveness of the MGA-PG system as

measured by synchronized pregnancy rate is less in yearling Bos indicus x Bos taurus (Bridges et

al., 2005) compared to Bos taurus heifers (Brown et al., 1988; (Lamb et al., 2000). Even though

synchronized pregnancy rates in Bos indicus x Bos taurus heifers can be increased slightly by

modifying the delivery of PG from a single PG treatment to two consecutive PG treatments 24

hours apart (Bridges et al., 2005), the synchronized pregnancy rates are still considerably less in

Bos indicus x Bos taurus compared to Bos taurus heifers.

The reasons) for the decreased effectiveness of the MGA-PG synchronization program in

heifers of Bos indicus x Bos taurus breeding may be attributed to physiological differences









between Bos indicus x Bos taurus compared to Bos taurus heifers. Bos indicus cattle have a

greater incidence of 3 and 4 follicle waves (Rhodes et al., 1995; Viana et al., 2000) during the

estrous cycle and this could result in asynchronous follicle development when PG is

administered in the MGA-PG program. Consequently, asynchronous follicle development could

be one of the reasons why the estrous response and subsequent synchronized pregnancy rates

after PG in the MGA-PG synchronization program are low in Bos indicus x Bos taurus heifers.

Therefore, the obj ectives of this study were to evaluate follicular development from withdrawal

of a 14 d MGA treatment until PG administration 19 d later in order to evaluate the possibility of

administering GnRH to synchronize follicle development, and to evaluate follicle development

and the subsequent estrous response after PG in yearling Angus (Bos taurus) and Brangus (Bos

indicus x Bos taurus) heifers.

Materials and Methods

Yearling Angus (Bos taurus; n = 40) and Brangus (Bos indicus x Bos taurus; n = 26)

heifers, at the University of Florida Santa Fe Beef Research Unit were used in the experiment,

which was conducted from February to April of 2004. Average heifer age, BW, body condition

score (BCS; Richards et al., 1986), and percentage of heifers having estrous cycles at the

initiation of the experiment are summarized in Table 3-1. Blood samples were collected -16, -7,

and 0 d before initiation of a 14 d melengestrol acetate (MGA) treatment to determine estrous

cycling status. The start of the experiment was designated as day 0. Heifers were deemed to be

having estrous cycles (cycling) if blood plasma progesterone concentrations were > 1.5 ng/mL at

two of the three blood samples while heifers were classified as not having estrous cycles (non-

cycling) if progesterone concentrations were < 1.5 ng/mL at all three samples. A progesterone

concentration of > 1.5 ng/mL was chosen after a retrospective analysis revealed that several

heifers had progesterone concentrations between 1.0 1.5 ng/mL in the absence of luteal tissue









as determined by ultrasonography on day 0 of the experiment. On day 0 of the experiment,

Angus and Brangus heifers were distributed by breed, cycling status, and BW into two groups.

One group included cycling Angus and Brangus heifers that were to have daily ultrasonography

(scan) conducted from MGA withdrawal until 5 d after PG and the other group included the

remaining Angus and Brangus heifers (cycling and non-cycling) that would have no daily

ultrasonography conducted (non-scan; Table 3-1). Also on day 0, the scan and non-scan heifers

were started on a 14 d MGA (0.5 mg~hd- *d- ; MGA" 200 Premix, Pfizer, Inc., New York, NY)

treatment, which was administered in a high protein pellet fed at a rate of 2 lbs~hd- *d- The

cycling scan and non-scan heifers were at random stages of the estrous cycle at the start of the

MGA treatment. All heifers received prostaglandin Fzu (PG; Lutalyse" Sterile Solution, Pfizer,

Inc. New York, NY) starting on d 19 as described by Bridges et al. (2005). Angus heifers

received a single 25 mg i.m. PG treatment 19 d after MGA while Brangus heifers received 12.5

mg i.m. PG on both 19 and 20 d following MGA withdrawal. Bridges et al. (2005) reported that

a split PG treatment enhanced luteolysis compared to a single PG treatment in yearling Bos

indicus x Bos taurus heifers and the split PG treatment in yearling Bos indicus x Bos taurus

heifers resulted in a similar rate of luteolysis compared to a single PG treatment in Bos taurus

heifers. Therefore, the breed specific PG treatment was used in the present experiment to ensure

that the PG induced luteolysis would be similar between Angus and Brangus heifers.

The scan group consisted of cycling Angus (n = 1 1) and Brangus (n = 10) heifers that were

used to evaluate daily follicular development using transrectal ultrasonography (equipped with a

7.5 MHz linear array transducer) from MGA withdrawal to 5 d after PG. Scan heifers were

handled through the working facilities three times a week during MGA treatment to acclimate

them to frequent handling to reduce any stress related affects associated with frequent handling.









At the start of the ultrasonography exams, two Brangus heifers had to be replaced because of

physical injuries that prevented daily ultrasonography from being conducted. The two heifers

were replaced with two non-cycling Brangus heifers of similar age and BW. Non-cycling

Brangus heifers were chosen because there were no cycling Brangus heifers available that had a

large enough rectal size to allow for daily ultrasonography exams to be performed The two

Brangus heifers that were removed from the scan group were placed in the non-scan group where

they recovered from their injuries. At each ultrasonography evaluation, height and width of all

luteal structures, luteal cavities, and follicles > 3 mm in diameter were measured using the

internal calipers of the ultrasonography machine, and their locations on the ovaries were

recorded. All ultrasonography exams were conducted by a single technician. Volume of the

corpus luteum (CL) was calculated using the formula for volume of a sphere (nd3/6). When a

luteal cavity was present, its volume was subtracted from the volume of the outer sphere

resulting in net luteal volume (CL volume) represented by luteal tissue. Ovarian maps were

evaluated retrospectively to determine follicle growth patterns. Emergence of a follicle wave

was defined as the time when the eventual dominant follicle could first be identified by

ultrasonography. Maximal diameter of a dominant follicle was defined as the maximum

diameter that the dominant follicle reached during the follicle wave. The day of maximal

diameter was defined as the day following emergence that the dominant follicle reached a

maximal diameter. Growth rate of the dominant follicle was determined by subtracting the

diameter of the dominant follicle at emergence from the maximal diameter of the dominant

follicle divided by the number of days from emergence to maximal diameter. Additionally,

blood samples were collected via jugular veinipuncture to determine plasma progesterone

concentrations at each ultrasonography exam. During the ultrasonography exams after MGA









withdrawal, ovulation was defined as disappearance of the largest follicle followed by its

absence at two consecutive ultrasonography exams. Ovulation rate after MGA for the scan

heifers was defined as the number of heifers ovulating a follicle within 7 d after MGA

withdrawal divided by the total number of scan heifers.

The non-scan heifers underwent transrectal ultrasonography (equipped with a 7.5 MHz

linear array transducer) at MGA withdrawal and at the initial PG. At each ultrasonography

evaluation, height and width of all luteal structures, luteal cavities, and follicles 5 mm in

diameter were measured using the internal calipers of the ultrasonography machine, and their

locations on the ovaries were recorded. Additionally, blood samples were collected via jugular

veinipuncture to determine plasma progesterone at MGA withdrawal, at the initial PG, and 3 d

following the initial PG treatment to determine if luteolysis occurred. Both scan and non-scan

heifers were deemed to have a functional CL at PG if plasma progesterone concentrations were >

1.5 ng/mL with the presence of luteal tissue as determined by ultrasonography. The PG induced

luteolysis for scan and non-scan heifers was defined as a heifer having a functional CL at PG

followed by progesterone concentrations < 1.5 ng/mL 3 d after PG. After the onset of the PG

induced estrus for the scan heifers, ovulation was defined as disappearance of the largest follicle

at the subsequent ultrasonography exam. Ovulation was confirmed by the presence of a

functional CL 8 d later as determined by ultrasonography and a blood plasma sample was also

collected to determine plasma progesterone concentrations.

Blood plasma samples were collected into evacuated tubes containing an anticoagulant

(EDTA; Becton, Dickinson and Company, Franklin Lakes, NJ). After collection, blood samples

were immediately placed on ice until they were centrifuged (3000 x g for 15 min). Plasma was

separated and stored at -200C until further analysis. Progesterone concentrations were









determined by RIA (Seals et al., 1998) using DPC kits (Diagnostic Products Corp., Los Angeles,

CA) in multiple assays with intra- and interassay CV of 3.5 and 4.9%, respectively. Sensitivity

of the assay was 0.01 ng/mL of plasma assayed.

Estrus was detected throughout the experiment using radiotelemeric estrous detection

devices (HeatWatch", Cow Chips, Denver, CO; Dransfield et al., 1998), which were fitted to all

heifers at the initiation of MGA treatment. Estrus was detected from the initiation of MGA until

5 d after PG. Heifers were artificially inseminated (AI) by one of two AI technicians with

frozen-thawed semen 8 to 12 h after the onset of the PG induced estrus. Angus heifers were

inseminated to two AI sires that were pre-assigned to heifers prior to insemination and the

Brangus heifers were inseminated to a single AI sire. Pregnancy was determined approximately

30 d after AI by transrectal ultrasonography, using a real-time, B-mode ultrasound (Aloka 500v,

Corometrics Medical Systems, Wallingford, CT) equipped with a 5.0 MHz transducer.

Interval to estrus, duration of estrus, and number of mounts received during estrus were

recorded for the estrus following MGA withdrawal and for the estrus after PG administration.

For both the estrus after MGA withdrawal and PG, onset of estrus was defined as the first of 3

mounts in a 3 h period and the end of estrus was defined as the time of the last mount recorded

during estrus prior to a period of extended inactivity of at least 8 h (Landaeta et al., 1999). The

duration of estrus was calculated by subtracting the date and time of the initial mount of estrus

from the last mount of estrus. Interval to estrus following MGA withdrawal was calculated by

subtracting the approximate date and time of MGA withdrawal from the date and time of the

initial mount of estrus. Estrous response after MGA withdrawal was defined as the total number

of heifers that exhibited behavioral estrus in 7 d divided by the total number of heifers treated.

Interval from PG to the onset of estrus was calculated by subtracting the date and time of the









initial PG from the date and time of the initial mount of estrus. Estrous response after PG was

defined as the total number of heifers that exhibited behavioral estrus in 5 d after the initial PG

divided by the total number of heifers treated. Conception rate was defined as the total number

of heifers that exhibited estrus after PG that were inseminated and became pregnant, divided by

the total number of heifers that exhibited estrus and were inseminated. Synchronized pregnancy

rate was defined as the total number of heifers that became pregnant to the AI divided by the

total number of heifers treated.

Dependent variables tested using the GENMOD procedure of SAS (SAS Inst. Inc., Cary,

NC) included cycling status, estrous response after MGA, estrous response after PG, conception

rate, synchronized pregnancy rate, occurrence of a functional CL at PG, PG induced luteolysis,

and occurrence of heifers with follicles > 10 mm in diameter at PG. Independent variables tested

were breed, ultrasound group (scan vs. non-scan), and breed x ultrasound group. Cycling status

was also evaluated as an independent variable using the model of breed, cycling status (cycling

vs. non-cycling), and breed x cycling status for the aforementioned dependent variables.

Additionally, ovulation rate following MGA withdrawal in scan heifers was evaluated with the

independent variable tested being breed. Diameter of the largest follicle at MGA withdrawal,

diameter of the largest follicle at PG, progesterone concentration at PG, and behavioral estrus

data including interval to estrus, duration of estrus, and number of mounts received during estrus

after MGA withdrawal and after PG were tested using the GLM procedure in SAS. Independent

variables tested were breed, ultrasound group and breed x ultrasound group; additionally, breed,

cycling status, and breed x cycling status were evaluated. Diameter of the first, second, and third

wave dominant follicles normalized for day of emergence, and diameter of the dominant follicle

on days 9-13 after MGA withdrawal (d 0) for scan heifers were evaluated with analysis of









variance for repeated measures using the MIXED procedure in SAS. Independent variables

tested were breed, day, and breed x day. Additionally, diameter of the eventual ovulatory

follicle normalized to PG for scan heifers exhibiting two or three follicle waves was evaluated

with analysis of variance for repeated measures using the MIXED procedure in SAS.

Independent variables tested were breed, day, number of waves, and all possible interactions.

Day of emergence, maximal diameter, and day of maximal diameter for the first, second, and

third wave dominant follicles in scan heifers were evaluated using the GLM procedure in SAS.

The independent variable tested was breed. In scan heifers exhibiting either two or three follicle

waves, estrous response following PG, conception rate, and synchronized pregnancy rate were

evaluated using the GENMOD procedure of SAS, while interval from PG to the onset of estrus

was tested in the GLM procedure of SAS. Independent variables tested were breed, number of

waves and breed x number of waves. One non-scan Brangus heifer exhibited estrus during the

MGA treatment so the data was excluded from the estrous response analysis after MGA

withdrawal, but was included in all analyses at and after PG. Two non-scan Angus heifers did

not have ultrasonography data collected at MGA withdrawal, so they were removed from the

ultrasonography analysis. Four non-scan Angus heifers exhibited estrus before PG and they

were excluded from the post PG data analysis. Three non-scan Angus heifers did not have

ultrasonography data collected at PG so they were removed from the ultrasonography data

analysis at PG.

Results

The physical description of the heifers is presented in Table 3-1. The Angus heifers were

older (P = 0.03), had a greater (P = 0.02) BCS, and had a greater (P = 0.02) percentage cycling at









the initiation of the experiment compared to Brangus heifers. There were no breed x ultrasound

group effects (P > 0.05) on age, BW, BCS, and percentage cycling at the start of the experiment.

With all scan and non-scan heifers included in the analysis, diameter of the largest follicle

present at MGA withdrawal was similar (P = 0.72) for Angus (n= 38; 13.7 & 0.5 mm) and

Brangus (n= 25; 13.5 & 0.5 mm) heifers. There were no (P > 0.05) effects of cycling status or

breed x cycling status on diameter of the largest follicle at MGA withdrawal. The percentage of

Angus and Brangus heifers in estrus during the 7 d after MGA withdrawal was similar (P = .88;

Table 3-2). The interval from MGA withdrawal to the onset of estrus, duration of estrus, and

number of mounts received during estrus were also similar (P > 0.05) between Angus and

Brangus heifers (Table 3-2). The scan heifers had a greater (P = 0.001) estrous response

compared to non-scan heifers, and scan heifers tended (P = 0.09) to have a longer interval from

MGA withdrawal to the onset of estrus compared to non-scan heifers (Table 3-2). Whether

heifers were scan or non-scan did not influence (P > 0.05) the duration of estrus or the number of

mounts received during the estrus after MGA withdrawal (Table 3-2). There were no (P > 0.05)

breed x ultrasound group effects on estrous response, interval from MGA withdrawal to the

onset of estrus, duration of estrus, and number of mounts received during estrus (Table 3-2).

More (P = 0.001) cycling heifers (n = 22/3 5; 62.9%) exhibited estrus after MGA withdrawal

compared to non-cycling heifers (n = 7/30; 23.3%), but there was no (P = 0.55) breed x cycling

status effect. Interval from MGA withdrawal to the onset of estrus (87:03 & 09: 19; 64:36 &

17:01 h:m), duration of estrus (10:24 & 01:25; 1 1:52 & 02:35 h:m), and number of mounts

received (53 & 9; 70 & 17) were similar (P > 0.05) between cycling and non-cycling heifers,

respectively. There were no (P > 0.05) breed x cycling status effects for interval from MGA

withdrawal to the onset of estrus and duration of estrus, but there was (P < 0.05) a breed x









cycling status effect on number of mounts received during estrus. The number of mounts

received for the cycling Angus, non-cycling Angus, cycling Brangus, and non-cycling Brangus

were 68 & 10; 47 & 28; 38 & 15; 94 & 18, respectively.

For scan heifers that exhibited estrus within 7 d after MGA withdrawal, ovulation rate

tended (P = 0.07) to be greater in Angus (11/11 = 100.0%) compared to Brangus (8/10 = 80.0%)

heifers (Figure 3-1). Diameter of the ovulatory follicle was similar (P = 0.93) between Angus

(17.0 + 0.9 mm) and Brangus (17.1 + 1.0) heifers. Two Brangus heifers did not exhibit estrus

within 7 d after MGA withdrawal. One heifer had progesterone < 1.5 ng/mL at MGA

withdrawal and had initiated regression of a persistent dominant follicle prior to MGA

withdrawal. This heifer developed a new follicle wave, which ovulated 11 d after MGA

withdrawal (Figure 3-1). The other Brangus heifer had progesterone > 1.5 ng/mL at MGA

withdrawal and had initiated a new follicle wave after MGA withdrawal. The newly developed

follicle ovulated 12 d after MGA withdrawal (Figure 3-1). Between MGA withdrawal and

administration of PG, 81.8% of Angus (n = 9/1 1) and 50.0% of Brangus (n = 5/10) heifers

displayed two waves of follicle growth, while 18.2% of Angus (n = 2/1 1) and 30.0% of Brangus

(n = 3/10) heifers displayed three waves of follicle growth. Of the two remaining Brangus

heifers, one heifer had a single follicle wave and the other heifer had four follicle waves.

One of the obj ectives of the experiment was to characterize follicle dynamics after MGA

withdrawal to determine the optimum timing to administer GnRH to initiate ovulation and

synchronize follicle wave development in Brangus heifers. Follicle development patterns for the

first follicle wave after MGA withdrawal for Angus and Brangus scan heifers are presented in

Figure 3-1. Emergence of the first wave dominant follicle after MGA withdrawal was similar (P









= 0.27) for Angus (5.7 & 0.5 d; range 4 to 8 d) compared to Brangus (4.9 & 0.6 d; range 3 to 9 d)

heifers.

Diameters of the first wave dominant follicle for the Angus and Brangus scan heifers are

presented in Figure 3-2. There was an effect (P = 0.001) of day on the diameter of the dominant

follicle 9 to 13 d after MGA withdrawal but there were no (P > 0.05) breed or breed x day

effects. The percentage of heifers with follicles > 10 mm was also evaluated for d 9 to 13 after

MGA withdrawal. A diameter of 10 mm was chosen since follicles can be ovulated by

exogenous GnRH at approximately > 10 mm diameters (Moreira et al., 2000). The percentage of

heifers with follicles > 10 mm was similar (P > 0.05) for Angus (n=1 1) and Brangus (n=10)

heifers on d 9 (54.5 vs. 80.0 %), 10 (81.8 vs. 70.0 %), and 11 (90.9 vs. 80.0%), respectively.

However, there were more (P = 0.02) Angus (n= 100) with follicles > 10 mm on d 12 compared

to Brangus (70.0%) and there were more (P = 0.002) Angus (100%) with follicles > 10 mm on d

13 compared to Brangus (50.0%). One Brangus heifer ovulated on d 11 and one on d 12 after

MGA withdrawal.

When normalized to the day of emergence for the first follicle wave, there was an effect of

day (P = 0.001) on diameter of the dominant follicle (Figure 3-3) but there were no (P > 0.05)

breed or breed x day effects. Dominant follicles reached a maximal diameter on a similar (P =

0.15) day following emergence and at a similar (P = 0.61) diameter for Angus (7.5 & 0.7 d; 14.5

& 0.7 mm) and Brangus (5.9 & 0.8 d; 14.0 + 0.8 mm), respectively. From day of emergence to

maximal diameter, follicle growth rates were similar (P = 0.42) for Angus (1.6 & 0.2 mm/d) and

Brangus (1.8 & 0.2 mm/d) heifers.

When the second follicle wave was normalized to the day of emergence (Figure 3-3), there

was an effect of day (P = 0.001) on diameter of the dominant follicle but there were no (P >









0.05) breed or breed x day effects. Breed tended (P = 0. 10) to affect the day of emergence after

MGA withdrawal of the second wave dominant follicle where emergence occurred on d 1 1.7 &

0.8 (range 8 to 16 d) for Brangus compared to d 13.5 & 0.7 (range 10 to 16 d) for Angus.

Dominant follicles reached a maximal diameter on a similar (P = 0.68) day following emergence

for Angus (6.5 & 0.6) compared to Brangus (6. 1 & 0.7 d), but maximal diameter of the dominant

follicle tended (P = 0.06) to be greater in Angus (15.5 & 0.7 mm) compared to Brangus (13.3 &

0.8 mm). There was no (P = 0.3 5) effect of breed on follicle growth rate from day of emergence

to maximal diameter of the second wave dominant follicle. Follicle growth rate for the Angus

was 1.9 & 0.2 mm/d and 1.7 & 0.2 mm/d for the Brangus heifers.

For the third follicle wave, there was an effect of day (P = 0.001) on diameter of the

dominant follicle when normalized to the day of emergence (Figure 3-3). There were no (P >

0.05) breed or breed x day effects on mean diameter of the third wave dominant follicle. Breed

had no (P = 0.24) effect on day of emergence of the third wave dominant follicle after MGA

withdrawal where emergence occurred 17.5 A 1.6 d (range 17 to 18 d) for Angus and 14.8 & 1.2

d (range 12 to 17 d) for Brangus. Third wave dominant follicles had a similar (P = 0.40)

maximal diameter on a similar (P = 0.76) day after emergence for Angus (13.0 + 0.9 mm; 5.5 &

0.6 d) and Brangus (14.0 + 0.6 mm; 5.3 & 0.5 d), respectively. Additionally, follicle growth rate

from day of emergence to maximal diameter of the third wave dominant follicle was similar (P =

0.50) for Angus (1.8 mm/d) and Brangus (2.1 mm/d) heifers.

On the day of PG administration, diameter of the largest follicle tended (P = 0.09) to be

greater for Angus compared to Brangus heifers (Table 3-3). Cycling status and breed x cycling

status had no (P > 0.05) effect (Table 3-2) on diameter of the largest follicle at PG. Likewise,

there were no (P > 0.05) ultrasound group and breed x ultrasound group effects. A similar (P =









0. 13) percentage of Angus (30/33; 90.9%) and Brangus (22/26; 84.6%) heifers had follicles 10

mm in diameter at PG. There were no (P > 0.05) ultrasound group, breed x ultrasound group,

cycling status, and breed x cycling status effects on percentage of heifers with follicles > 10 mm

at PG.

The effect of follicle wave pattern (two-wave vs. three-wave) on follicle development

during the 6 d prior to PG was also evaluated with follicle development being normalized

retrospectively from day of PG (Figure 3-4). Breed tended (P = 0.07) to effect follicle

development and day (P = 0.001) did effect follicle development. The number of follicle waves

between MGA withdrawal and PG also affected (P = 0.01) follicle development (Figure 3-4).

There were no (P > 0.05) breed x day, breed x wave, day x wave, or breed x day x wave effects.

Additionally, day of emergence after MGA withdrawal of the eventual ovulatory follicle was

similar (P = 0.97) for the Angus (14.7 & 0.7 d) and Brangus (14.7 & 0.8 d) heifers (Figure 3-5).

A greater (P = 0.001) percentage of heifers that were cycling at the start of the MGA

treatment had a functional CL at PG compared to non-cycling heifers (Table 3-3). However,

there was neither (P < 0.05) a breed nor breed x cycling group effect on whether heifers had a

functional CL at PG (Table 3-3). A greater (P = 0.001) percentage of scan (20/21 = 95.2%)

compared to non-scan (14/38 = 55.3%) heifers had a functional CL at PG, but there was no (P =

0.37) breed x ultrasound group effect. Progesterone concentrations at PG were similar (P =

0.50) for Angus and Brangus heifers (Table 3-3). Whereas, progesterone concentrations at PG

tended (P = 0.07) to be greater in cycling compared to non-cycling heifers, but there was no (P =

0.31) breed x cycling group effect. Furthermore, progesterone concentrations at PG tended (P =

0.08) to be greater in scan (6.29 & 0.9 ng/mL) compared to non-scan (4.44 & 0.6 ng/mL) heifers

but there was no (P = 0.95) breed x ultrasound group effect. For both the scan and non-scan









heifers with functional CL at PG, luteolysis rates were similar (P > 0.05) for Angus (25/25) and

Brangus (17/17) heifers.

The effect of breed and cycling status at the initiation of MGA treatment on estrous,

conception and synchronized pregnancy rates are presented in Table 3-4. Estrous response,

conception rate, and synchronized pregnancy rate were similar (P > 0.05) for Angus and Brangus

heifers (Table 3-4). Cycling status affected (P = 0.001) estrous response as more cycling heifers

exhibited estrus compared to non-cycling heifers (Table 3-4). There was no (P = 0.93) breed x

cycling status effect on estrous response. When analyzed by ultrasound group, more (P = 0.001)

scan heifers exhibited estrus (95.2%; n=20/21) compared to non-scan (56. 1%; n=23/41) heifers.

There tended (P = 0. 10) to be a breed x ultrasound group effect on estrous response. Estrous

responses for Angus scan, Angus non-scan, Brangus scan and Brangus non-scan were 90.9%

(n=11), 68.0 (n=25), 100.0 (n=10), and 37.5% (n=16), respectively. Breed, cycling status, and

breed cycling status had no effect on conception rate (P > 0.05), which was also the case for

ultrasound group, and breed x ultrasound group. The effect of AI sire (P = 0.69) and AI

technician (P = 0.38) did not affect conception rate. Synchronized pregnancy rates were similar

(P = 0.52) for Angus and Brangus heifers but more (P = 0.05) cycling heifers became pregnant

during the synchronized breeding compared to non-cycling heifers. There were no (P > 0.05)

ultrasound group and breed x cycling status effects on synchronized pregnancy rates. However,

there was (P < 0.05) a breed x ultrasound group effect on synchronized pregnancy rate.

Synchronized pregnancy rates for Angus scan, Angus non-scan, Brangus scan and Brangus non-

scan were 60.0% (n=1 1), 36.0 (n=25), 60.0 (n=10), and 18.8% (n=16), respectively.

Interval from PG to the onset of estrus (61:49 & 5:30; 63:14 & 6: 19 h:m), duration of estrus

(1 1:44 & 1:05; 10:08 & 1:15 h:m), and the number of mounts received during estrus (57 & 9; 42 &










10) were similar (P > 0.05) for Angus and Brangus heifers, respectively. There were no (P >

0.05) cycling status or breed x cycling status effects on interval from PG to the onset of estrus,

duration of estrus, or number of mounts received during estrus. Likewise, there were no (P >

0.05) ultrasound group and breed x ultrasound group effects for interval from PG to the onset of

estrus, duration of estrus, or number of mounts received during estrus. Diameter of the largest

follicle at PG was negatively correlated (r = -0.60; P = 0.01) with the interval from PG to estrus

in Angus heifers; whereas, diameter of the largest follicle at PG only tended to be negatively

correlated (r = -0.33; P = 0.09) with the interval from PG to the onset of estrus in Brangus

heifers.

For the scan heifers, number of follicle waves between MGA withdrawal and PG had no

(P = 0.68) effect on estrous response after PG. Estrous response was 88.9% (8/9) and 100.0%

(2/2) for two- and three-wave Angus heifers, respectively, and 100.0% (5/5) and 100.0% (3/3)

for two- and three-wave Brangus heifers, respectively. Neither the number of waves nor breed x

number of waves effected (P > 0.05) estrous response after PG. The number of waves tended (P

= 0.09) to affect the interval from PG to the onset of estrus and interval from PG to the onset of

estrus was affected by breed (P = 0.02) and breed x number of waves (P = 0.01). Interval from

PG to the onset of estrus was 63:21 & 5:12 h:m and 101:56 & 10:24 h:m for two- and three-wave

Angus heifers, respectively, and 67:43 & 6:35 h:m and 57:33 & 8:30 h:m for two- and three-wave

Brangus heifers, respectively. There tended (P = 0.07) to be an effect of breed on conception

rates but number of waves and breed x number of waves did not affect (P > 0.05) conception

rate. Conception rates were 25.0% (2/8) and 0% (0/2) for two- and three-wave Angus heifers,

respectively, and 40.0% (2/5) and 66.7% (2/3) for two- and three-wave Brangus, respectively.

Synchronized pregnancy rate was not affected (P > 0.05) by breed, number of waves, or breed x









number of waves. Synchronized pregnancy rates were 22.2% (2/9) and 50.0% (1/2) for two- and

three-wave Angus heifers, respectively and 40.0% (2/5) and 66.7% (2/3) for two- and three-wave

Brangus heifers, respectively.

In heifers undergoing daily ultrasound evaluations, 100% of the Angus (n=1 1) and

Brangus (n=10) heifers ovulated after PG. Diameter of the ovulatory follicle before ovulation

was similar (P = 0.86) for Angus (13.1 & 0.4 mm) compared to Brangus (13.2 & 0.5 mm) heifers.

Volume of the CL 8 d following ovulation was similar (P = 0.20) for Angus (4095.6 & 410 mm3)

compared to Brangus (4876.3 & 430 mm3) heifers; however, progesterone concentrations were (P

= 0.05) greater for Brangus (7. 10 + 0.53 ng/mL) compared to Angus (5.56 & 0.51 ng/mL) heifers.

Discussion

In order for the MGA-PG system (Brown et al., 1988) to be effective, heifers need to

exhibit estrus and (or) ovulate so they are in the late luteal phase of the estrous cycle at PG,

which means a maj ority of heifers must exhibit estrus with 3 to 7 d after MGA withdrawal as

reported by Hill et al. (1971). In the present study, one-hundred percent of the scan Angus

heifers, but only 80% of the scan Brangus heifers exhibited estrus and (or) ovulated within 7 d of

MGA withdrawal. Wood et al. (2001) reported that 81.5% ofBos taurus heifers ovulated within

12 d of MGA withdrawal. The two Brangus heifers not exhibiting estrus within 7 d after MGA

withdrawal, eventually exhibited estrus and ovulated 11 and 12 d after MGA withdrawal. One of

the two Brangus heifers had a functional CL (> 1.5 ng/mL) at MGA withdrawal, which regressed

shortly after MGA withdrawal. The heifer was not detected in estrus during MGA; although,

cattle ofBos indicus breeding are noted for having a "silent estrus" (Galina et al., 1982; Orihuela

et al., 1983). The heifer was probably at the beginning of the estrous cycle at the initiation of

MGA, which would have placed the heifer in the late luteal phase of the estrous cycle at MGA

withdrawal. The other Brangus heifer had apparently started regression the largest follicle at









MGA withdrawal resulting in initiation of a new follicle wave, which ovulated 12 d after MGA

withdrawal. The reasons for this pattern of follicle development after a long term MGA

treatment are unclear. Kojima et al. (1992) reported that some cows receiving a MGA treatment

failed to have a preovulatory LH surge within 100 h of MGA withdrawal, which may be due to

luteinization (Guthrie et al., 1970) of persistent follicles capable of secreting enough

progesterone to prevent the LH surge. This does not appear to be likely since the Brangus heifer

had progesterone concentrations < 1 ng/mL for several days prior to and after MGA withdrawal.

After MGA withdrawal, diameters of the largest follicles present were similar between

Angus and Brangus heifers and similar to those reported by Wood et al. (2001) in cycling Bos

taunts heifers. The estrous response after MGA withdrawal was similar between Angus and

Brangus heifers but it was considerably less than another study using a long term MGA

treatment (Yelich et al., 1997) in yearling Bos taunts heifers. The decreased estrous response

was primarily due to the decreased percentage of heifers that were cycling (39.6%) at the start of

MGA. More cycling heifers (62.9%) exhibited estrus after MGA withdrawal compared to non-

cycling heifers (23.3%). These results and others (Brown et al., 1988; Patterson and Corah,

1992; Wood-Follis et al., 2004) point out the importance of having heifers going through estrous

cycles at the start of the MGA treatment. With that said, long-term MGA treatments induce

estrous cycles in some non-cycling heifers (Patterson et al., 1989) and the MGA treatment

induced estrous cycles in 5 1.9% of the non-cycling heifers in the present experiment.

Additionally, the percentage of non-cycling heifers that were cycling at PG was similar between

Angus and Brangus heifers suggesting that the MGA treatment was equally effective at inducing

estrus in non-cycling heifers across the two breed types.









The incidence of a "silent estrus" (Galina et al., 1982; Orihuela et al., 1983) after MGA

withdrawal also contributed to the decreased estrous response. Of the heifers that were cycling

at the start of MGA, 84% had a functional CL at PG; however, only 63.9% of the cycling heifers

exhibited estrus after MGA withdrawal. Therefore, several Angus and Brangus heifers did not

exhibit estrus after MGA withdrawal. Because heifers were fitted with radiotelemeric estrous

detection devices, it is unlikely that the method of estrous detection was the reason for decreased

estrous response. The incidence of "silent estrus" is well documented in Bos indicus cattle

(Galina et al., 1982; Orihuela et al., 1983), but it is unclear why so many heifers did not exhibit

estrus after MGA withdrawal since persistent follicles have increased estrogen concentrations

(Henricks et al., 1973; Kojima et al., 1992). Increased ambient temperatures have been reported

to increase the incidence of ovulation without estrus in dairy heifers (Gwazdauskas et al, 1981).

However, it is unlikely that elevated temperatures caused the increased incidence of "silent

estrus" since the experiment was conducted from February to April when ambient temperatures

were probably to low to initiate heat stress.

One of the primary obj ectives of the experiment was to determine the follicular wave

pattern from MGA withdrawal to PG in Brangus compared to Angus heifers. There was

considerable variation between Brangus compared to Angus heifers in the number of follicle

waves between MGA withdrawal and PG. Eight-two percent of Angus heifers had two follicle

wave patterns with the remaining 18% having three follicle wave patterns. In comparison, only

50% of the Brangus heifers had two follicle waves and the remaining heifers had either one,

three, or four follicle waves. Therefore, based on the number of follicle waves, there is

considerable variation in follicle development patterns for Brangus compared to Angus heifers.

The increased incidence of three and four follicle wave patterns in Brangus heifers is similar to










reports in cattle of Bos indicus breeding having more three and four follicular wave patterns

(Rhodes et al., 1995; Viana et al., 2000) during a normal estrous cycle compared to two-wave

patterns.

Growth and development of the first wave dominant follicle after MGA withdrawal was

similar between Angus and Brangus heifers with respect to day of emergence, growth rate, day

of maximal diameter, and maximal diameter of the dominant follicle. With respect to the first

wave follicle development patterns in the current study, they are in agreement with other

observations reported in Bos taurus cattle (Sirois and Fortune, 1988; Ginther et al., 1989).

Conversely, Viana et al. (2000) reported that emergence of the first follicle wave occurred

earlier, had a reduced growth rate and maximal diameter in non-lactating Bos indicus cows

compared to the Brangus heifers in the present study. Emergence of the second follicle wave

tended to occur later in Angus compared to Brangus heifers. Additionally, Angus heifers had a

greater maximal diameter of the second wave dominant follicle compared to Brangus heifers.

The increased incidence of three and four follicle wave patterns is likely influenced by day of

emergence and maximal follicle diameter of the second wave dominant follicle. Second wave

dominant follicles emerged later (Sirois and Fortune, 1988) and reached a greater maximal

diameter (Ginther et al., 1989) in cattle displaying two compared to three wave follicle growth

profiles. No differences were observed between Angus and Brangus heifers with regard to the

developmental characteristics of the third wave dominant follicle. Characteristics of follicle

development, particularly the second and third waves, should be interpreted with discretion since

PG was administered before some of the heifers were allowed to complete a normal estrous

cycle.









Another research obj ective was to determine the best time to incorporate GnRH into the

MGA-PG system. Wood et al. (2001) conducted an experiment where Bos taurus heifers

received GnRH 12 d after the withdrawal of a 14 d MGA treatment with PG 7 d after GnRH

compared to the MGA-PG system with PG administered 19 d after MGA withdrawal. They

hypothesized that GnRH would induce a new follicle wave, which would increase the synchrony

of follicle development at PG resulting in a more synchronous estrus. As presented in Figure 1-

2, there was considerable variation in the growth and developmental profies of the first wave

dominant follicle in Brangus compared to Angus heifers. For the eleven scan Angus heifers,

100% of the heifers had first wave dominant follicles that were in the growing phase by d 12

after MGA withdrawal. In contrasts, only 60% of the Brangus heifers had a first wave dominant

follicle in the growing phase by d 12 after MGA withdrawal. Furthermore, two Brangus heifers

had follicles that began to go through atresia by d 10 and another heifer had follicle emergence

on d 9 after MGA withdrawal. The asynchrony of follicle development after MGA withdrawal

for Brangus heifers was further reflected in the percentage of heifers with follicles > 10 mm

between d 9 to 13 after MGA withdrawal, which is important since GnRH is typically effective

in growing follicles > 10 mm in diameter (Moreira et al., 2000). By d 11 after MGA withdrawal,

91% of Angus heifers had follicles 10 mm and 100% by d 12 and 13. In contrasts, by d 1 1

after MGA withdrawal, 80% of Brangus heifers had follicles 10 mm and 70.0% by d 12 and

50% by d 13. The reduction in follicles > 10 mm by d 12 and 13 was a result of heifers

exhibiting estrus and ovulating, and several heifers having first wave dominant follicles that

began to enter atresia and regress by d 12. Therefore, the optimal time to administer GnRH to

Brangus heifers should occur approximately 10 d following MGA withdrawal for a couple of

reasons. First, a greater percentage of Brangus heifers would have follicles > 10 mm diameter









and in the growing phase of follicle development. Second, for heifers failing to exhibit estrus

and ovulate within 7 d after MGA withdrawal the heifers would develop a new follicle wave that

should be in the growing phase by d 10 and responsive to GnRH.

Because of the asynchrony of follicle development by d 12 after MGA withdrawal in

Brangus heifers, administering GnRH within 2 to 4 d after MGA withdrawal is an option that

should also be investigated. By 4 d after MGA withdrawal, the variation in follicle development

appears minimal and most heifers have follicles > 10 mm in diameter. However, the

effectiveness of GnRH to ovulate a maj ority of the large persistent dominant follicles needs to be

addressed in further experiments.

At PG, diameter of the eventual ovulatory follicle was similar between Angus and Brangus

heifers, which agree with a report by Wood et al. (2001) in Bos taurus heifers synchronized with

MGA-PG. Although the range in day of emergence of the eventual ovulatory follicle was

considerable for Angus and Brangus heifers, diameter and growth rate of the eventual ovulatory

follicle during the 6 d prior to PG was similar for Angus and Brangus heifers. It is also

interesting to note that even with the asynchronous follicle development that Brangus heifers

experienced between MGA withdrawal and PG, follicle development at PG was similar between

Angus and Brangus heifers. Additionally, the interval from PG to the onset of estrus was similar

between breeds, which can be attributed to the similar diameter of the eventual ovulatory follicle

at PG for the Angus and Brangus heifers. With that said, there was a significant interaction

between breed and the number of follicle waves at PG on the interval from PG to the onset of

estrus. Angus and Brangus heifers that had two follicle waves had a similar interval from PG to

the onset of estrus. Growth and development of the eventual ovulatory follicle was similar

between two wave Angus and Brangus heifers, resulting in similar diameters of the eventual









ovulatory follicle at PG. However, Angus heifers with three follicle waves had a longer interval

from PG to the onset of estrus compared to Brangus heifers with three follicle waves. The

greater interval from PG to the onset of estrus for the three wave Angus heifers was due to the

fact that the follicles were in the early stages of follicle development and diameter of the

eventual ovulatory follicle was considerably less than compared to three wave Brangus heifers,

which had a more mature follicle and a shorter interval from PG to the onset of estrus. Similarly,

Sirois and Fortune (1988) reported that the size of the eventual ovulatory follicle at luteolysis

was negatively correlated with the interval to estrus.

Angus and Brangus heifers were synchronized with the MGA-PG protocol described by

Lamb et al. (2000) where PG was administered 19 d after the last day of MGA; although, there

was a slight modification as the Brangus heifers received a split-PG 19 (12.5 mg) and 20 (12.5

mg) d following MGA withdrawal. For the Angus and Brangus heifers that had a functional CL

at PG, PG initiated luteolysis in 100% of the heifers similar to a report by Bridges et al. (2005).

Estrous response, conception, and synchronized pregnancy rates were similar between Angus

and Brangus heifers. In contrast, the estrous response and synchronized pregnancy rates for

Angus and Brangus heifers are considerably less than those observed by other authors (Nix et al.,

1998; Lamb et al, 2000; Salverson et al., 2002) in Bos taurus heifers synchronized with the

MGA-PG system.

The decreased estrous response and subsequently decreased synchronized pregnancy rates

can be attributed to the decreased percentage of heifers that were cycling at the initiation of the

MGA treatment. Both Angus and Brangus heifers that were cycling prior to the beginning of

MGA had a significantly greater estrous response and synchronized pregnancy rate compared to

non-cycling heifers. Furthermore, the cycling Angus and Brangus heifers had estrous responses









and synchronized pregnancy rates that are similar to those reported for experiments in Bos taurus

synchronized with a similar MGA-PG system (Nix et al., 1998; Lamb et al, 2000; Salverson et

al., 2002). The importance of cycling status on the overall effectiveness of the MGA-PG system

is supported by this and other studies (Brown et al., 1988; Patterson et al., 1989). Attainment of

puberty prior to the beginning of the breeding season is also important since fertility increases as

the number of estrous cycles a heifer has prior to the initiation of the breeding season increases

(Byerley et al., 1987; Galina et al., 1996). Results from the current study suggest that having

heifers of Bos indicus x Bos taurus breeding cycling prior to the start of the breeding season

maybe one of the most important, if not the most important factors, in getting Bos indicus x Bos

taurus heifers pregnant to a synchronized AI breeding. Although, having a high percentage of

Bos indicus x Bos taurus heifers cycling at the start of the breeding season can be difficult since

cattle of Bos indicus breeding reach puberty at a later age than Bos taurus heifers (Plasse et al.,

1968; Baker et al., 1989; Rodrigues et al., 2002).

Cycling status had no effect on conception rate, although, conception rates for the Angus

and Brangus heifers was still substantially less than other studies (Brown et al., 1988; Jaeger et

al., 1992; Lamb et al., 2000) in Bos taurus heifers synchronized with the MGA-PG system.

Conversely, conception rates of Angus and Brangus heifers are similar to conception rates

reported by Bridges et al. (2005) in Bos indicus x Bos taurus heifers synchronized with the

MGA-PG system. However, within the Bridges et al. (2005) study, there was a significant breed

effect on conception rates with cycling Angus heifers having a 28.6% greater conception rate

compared to cycling Bos indicus x Bos taurus heifers. The reason (s) for the considerable

variation in conception rates within and between studies is difficult to evaluate and could be due

to several factors including fertility of the AI sire (DeJarnette et al., 2004), AI technician










(Garcia-Ispierto et al., 2007), stage of follicle development at PG (Austin et al., 1999; Townson

et al., 2002), estrous cycling status at the start of a breeding season (Byerley et al., 1987), and

environmental conditions in which the studies are conducted (Wolfenson et al., 1995). What, if

any, effects that frequent working of cattle had on fertility are unclear. Conception rate was 7%

numerically greater in non-scan compared to scan heifers. Within scan and non-scan heifers,

each breed responded differently. Conception rates were nearly 33% greater in non-scan

compared to scan Angus heifers but only 10% greater in scan compared to non-scan Brangus

heifers. To our knowledge, there are no studies evaluating what effect frequent ultrasonography

examinations have on fertility. However, frequent ultrasound examinations can have a negative

effect on luteal function after ovulation in Bos indicus x Bos taunts cattle (Lemaster et al., 1999).

Luteal function in the scan heifers does not appear to be compromised as all scan heifers

developed a functional CL that secreted progesterone 8 d after the PG induced estrus. However,

luteal function was not evaluated in the non-scan heifers so no comparison can be made between

heifers that were or were not frequently handled.

In the scan heifers, Brangus heifers had greater progesterone concentrations compared to

Angus heifers but CL volume was only numerically greater in Brangus compared to Angus

heifers. These results are in agreement with Alvarez et al (2000) but do not agree with a report

that Bos indicus cattle have a smaller CL (Irving et al., 1978) and decreased progesterone

concentrations (Segerson et al., 1984).

In summary, follicle development from MGA withdrawal to PG is different between

Angus and Brangus heifers. A decreased percentage of Brangus heifers exhibited estrus and

ovulated within 7 d after MGA withdrawal. The increased incidence of three and four follicle

wave patterns in Brangus heifers contributed to the altered follicle wave dynamics compared to










Angus heifers. Although, diameter of the first wave follicle wave from d 9 to 13 after MGA

withdrawal was similar between Angus and Brangus heifers, the number of follicles > 10 mm

and in the growing phase were decreasing in Brangus heifers compared to Angus heifers by d 11

and 12 after MGA withdrawal. These results suggest that addition of GnRH to the MGA-PG

system for Brangus heifers may need to occur prior to d 12 after MGA withdrawal. Furthermore,

administering, GnRH immediately after MGA withdrawal (i.e., day 3 to 4) may actually work

better to synchronize follicle development. Even though follicle development was slightly

different between Angus and Brangus heifers, the estrous response, conception rate, and

synchronized pregnancy rate were similar between breeds. Angus and Brangus heifers that were

cycling prior to the start of the MGA treatment had the greatest synchronized pregnancy rates.

Implications

Follicle development during the period between MGA withdrawal and PG was different

for Brangus compared to Angus heifers. The most opportune time to administer GnRH after a

14 d MGA treatment to synchronize follicle development in Bos indicus x Bos taurus heifers

may be within 3 to 4 d after MGA withdrawal. Additional research will need to be conducted in

Bos indicus x Bos taurus heifers to evaluate the effectiveness of adding GnRH to the MGA-PG

system. Regardless of breed, heifers that were cycling prior to the start of the MGA treatment

had greater synchronized pregnancy rates compared to non-cycling heifers. Therefore, producers

need to make sure that a maj ority of yearling Bos indicus x Bos taurus heifers are going through

estrous cycles at the start of a synchronization treatment to achieve acceptable pregnancy rates to

a synchronized breeding.











Table 3-1. Age, body weight (BW), body condition score (BCS), and estrous cycling status
(Cycling) at the initiation of the 14 d melengestrol (MGA) treatment for Angus and
Brangus heifers by ultrasound group (scan vs., non-scan) (LS means & SE).a

Variable n Age, d BW, kg BCSb Cycling, %"


aMeasurements taken on initial day of MGA feeding, experimental day 0.
bBCS: 1 emaciated, 5 moderate; 9 obese.
"Cycling status determined by blood samples collected d -16, -7. and 0 of experiment. Heifers
were classified as cycling if blood plasma progesterone concentrations were > 1.5 ng/mL at two
of three blood samples and classified as non-cycling if blood plasma progesterone concentrations
were < 1.5 ng/mL at all three blood samples.


Angus


40 384 f 2.5 348 & 5.4 6.3 & 0.1 25/40 = 62.5

26 376 f 2.9 356 & 6.1 6.1 & 0.1 11/26 = 42.3


45 377 f 2.2 347 & 4.7 6.1 & 0.1 17/45 = 37.8

21 384 f 3.1 357 & 6.6 6.2 & 0.1 19/21 = 90.5


29 383 f 2.6 342 & 5.6 6.2 & 0.1 14/29 = 48.3

11 386 f 4.3 353 & 9.2 6.3 & 0.1 11/11 = 100.0


Brangus


Non-scan


Scan


Angus non-scan


Angus scan


Brangus non-scan

Brangus scan


16 370 f 3.6 352 & 7.6 6.1 & 0.1

10 382 f 4.5 360 + 9.6 6.1 & 0.1


3/16 = 18.8

8/10 = 80.0


P values


Breed

Group


0.03

0.07

0.26


0.32

0.24

0.91


0.02

0.47

0.38


0.02

0.001


Breed x Group


0.31











Table 3-2. Estrous response, interval to estrus, duration of estrus, and number of mounts
received during a HeatWatch" detected estrus for the 7 d following a 14 d
melengestrol (MGA) treatment by breed, ultrasound group (scan vs., no-scan), and
breed x ultrasound group.a

Estrous Interval from Number
response, MGA withdrawal Duration of of
Variable n %b to estrus, h:m estrus, h:m mounts


aUltrasound group included scan heifers, which had daily ultrasonography starting the day of
MGA withdrawal and continued for 19 d and non-scan heifers did not receive any daily
ultrasonography
bNumber of heifers exhibiting estrus within 7 d of MGA withdrawal divided by the total number
of heifers treated.


Angus


40 42.5

25 48.0


21 76.2

44 29.6


11 72.7

29 31.0

10 80.0

15 26.7


91:11 & 9:37

71:25 A 12:07


94:52 & 9:54

67:45 A 11:53


103:37 & 13:59

78:46 & 13:11

86:07 & 13:59

56:43 A 19:47


12:55 A 1:33

10:15 A1:57


11:53 A1:35

11:17 &1:55


14:21 & 2:15

11:29 & 2:07

9:24 & 2:15

11:05 & 3:11


66 & 11

63 113


65 A 11

64 & 13


72 & 15

60 & 15

58 & 15

69 & 22


Brangus


Scan


Non-scan


Angus scan

Angus non-scan

Brangus scan

Brangus non-scan


P-values


Breed


0.88

0.001


0.62


0.21

0.09


0.88


0.29

0.81


0.37


0.89

0.98


0.53


Ultrasound group

Breed x Ultrasound
group










Table 3-3. Percentage of heifers with a functional CL, progesterone concentration (LSM &
SE), and diameter of the largest follicle (LSM & SE) at the initial PG treatment.a

Progesterone Diameter of
Functional concentration, largest follicle,
Variable n CL, %b ng/mL mm

Angus 33 75.8 5.5 & 0.7 11.8 & 0.3

Brangus 26 61.5 4.8 & 0.8 11.1 & 0.3


Cycling 32 84.4 6.1 & 0.7 11.5 & 0.3

Non-cycling 27 51.9 4.2 & 0.8 11.4 & 0.3


Angus cycling 21 81.0 5.9 & 0.8 11.9 & 0.4

Angus non-cycling 12 66.7 5.1 + 1.1 11.8 & 0.5

Brangus cycling 11 90.9 6.2 & 1.2 11.2 & 0.5

Brangus non-cycling 15 40.0 3.3 A 1.0 11.0 + 0.4


P-values

Breed 0.87 0.50 0.09

Cycling group 0.01 0.07 0.84
Breed x Cycling group 0.15 0.31 0.88

alnitial PG treatment was administered 19 d following the withdrawal of a 14 d MGA treatment.
Angus heifers received a single (25 mg) PG treatment while Brangus heifers received split (12.5
mg) PG treatments on d 19 and 20.
bA heifer was deemed to have a functional CL if progesterone concentrations were > 1.5 ng/mL
in the presence of luteal tissue as determined by ultrasonography.
" Cycling status determined by blood samples collected d -16, -7. and 0 of experiment with MGA
treatment starting on d 0. Heifers were classified as cycling if blood plasma progesterone
concentrations were > 1.5 ng/mL at two of three blood samples and classified as non-cycling if
blood plasma progesterone concentrations were < 1.5 ng/mL at all three blood samples.












Table 3-4. Effect of breed and cycling status at the initiation of a 14 d melengestrol acetate
treatment on estrous response, conception rate and synchronized pregnancy rates of
Angus and Brangus heifers synchronized with a 14 d melengestrol acetate
treatment followed by either a single (Angus) or split (Brangus) prostaglandin Fzu
treatment 19 d later.a

Estrous Conception Synchronized
Variable response, %b rae c pregnancy rate %d


a Cycling status determined by blood samples collected d -16, -7. and 0 of experiment with MGA
treatment starting on d 0. Heifers were classified as cycling if blood plasma progesterone
concentrations were > 1.5 ng/mL at two of three blood samples and classified as non-cycling if
blood plasma progesterone concentrations were < 1.5 ng/mL at all three blood samples.
bPercentage of heifers exhibiting estrus during the 5 d following the initial prostaglandin Fzu
treatment out of the total number treated.
"Percentage of heifers that were pregnant to AI of the total number of heifers that exhibited
estrous and were AI.
dPercentage of the heifers that were pregnant to AI out of the total number treated.


Angus


27/36 = 75.0

16/26 = 61.5


29/34 = 85.3

14/28 = 50.0


20/23 = 87.0

7/13 = 53.8

9/11 = 81.8

7/15 = 46.7


12/27 = 44.4

9/16 = 56.3


15/29 = 51.7

6/14 = 42.9


10/20 = 50.0

2/7 = 28.6

5/9 = 55.6

4/7 = 57.1


12/36 = 33.3

9/26 = 34.6


15/34 = 44.1

6/28 = 21.4


10/23 = 43.5

2/13 = 15.4

5/11 = 45.5

4/15 = 26.7


Brangus


Cycling


Non-cycling


Angus cycling


Angus non-cycling

Brangus cycling

Brangus non-cycling


P-values


Breed


0.59

0.001

0.93


0.30

0.54

0.48


0.52

0.05

0.61


Cycling group

Breed x Cycling group




















S15


10







O 4 8 12 16 20 24
Days after MGA


B)









10






2 0.



O 4 8 12 16 20 24
Days after MGA


Figure 3-1. Profiles of ovulatory follicles after a 14 d melengestrol acetate (MGA) treatment and
the subsequent first wave dominant follicle growth profiles for A) Angus and B)
Brangus heifers. Stars indicate day of ovulation and the two dashed lines indicate the
potential range when GnRH could be administered to ovulate the first wave follicle
after MGA withdrawal to synchronize the next follicle wave.












14

12






6, Angus

4~ Brangus





9 10 11 12 13

Days after MGA


Figure 3-2. Mean first wave dominant follicle diameter during days 9 to 13 following
withdrawal of a 14 d melengestrol acetate (MGA) treatment for Angus (n = 1 1) and
Brangus (n = 10) heifers in the scan group. One Brangus heifer ovulated on day 11
and another on day 12. Breed (P > 0.05), Day (P < 0.05), Breed x Day (P > 0.05)



















E 10







oo F4 5





AAngus BBrangus


01234567 012345 012345

Day after Wave Emergence

Figure 3-3. Mean diameter of the A) first, B) second, and C) third follicle wave following withdrawal of melengestrol acetate (MGA)
for Angus and Brangus heifers. Follicle waves were normalized to the day of wave emergence. For all three follicle wave
patterns: Breed (P > 0.05), Day (P < 0.05), Breed x Day (P > 0.05).











20

+ Angus two wave Angus three wave

15 Brangus two wave Brangus three wave


10







0 I I I~ I





-6 -5 -4 -3 -2 -1 0

Days Before PG

Figure 3-4. Diameter of the eventual ovulatory follicle prior to prostaglandin F200 (PG)
treatment for Angus and Brangus heifers based on the number of follicle waves from
the last day of a 14 d melengestrol acetate treatment to a PG treatment 19 days later.
Breed (P = 0.07), Day (P = 0.001), Number of Waves (P = 0.01), Breed x Day (P =
0.85), Breed x Number of Waves (P = 0. 19), Number of Waves x Day (P = 11i),
Breed x Day x Number of Waves (P = 0.38).





























10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Days After MGA


10 11 12 13 14 15


16 17 18 19 20 21 22 23 24


Days After MGA


Figure 3-5. Follicle growth patterns for the eventual ovulatory follicle preceding the initial
prostaglandin F2a (PG) treatment, which occurred on day 19 (indicated by the arrow)
in A) Angus and B) Brangus heifers. Solid lines indicate two-wave follicle growth
patterns, long dashed lines indicate three-wave follicle growth patterns and short
dashed lines indicate either a single- or four-wave follicle growth pattern.









CHAPTER 4
REFINEMENT OF THE 14 D MELENGESTROL ACETATE (MGA) TREATMENT +
PROSTAGLANDIN Fzu (PG) 19 D LATER ESTROUS SYNCHRONIZATION SYSTEM IN
HEIFERS OF Bos indicus x Bos taurus BREEDING

Introduction

Melengestrol acetate (MGA) administered for 14 d with prostaglandin Fzu (PG)

administered 17 d after MGA withdrawal is one of the most widely used estrous synchronization

systems (MGA-PG) in yearling beef heifers (Brown et al., 1988; Patterson and Corah, 1992).

The MGA-PG synchronization system is an effective and predictable system in Bos taurus

heifers (Brown et al., 1988; Patterson and Corah, 1992; Lamb et al., 2000) but is less effective in

Bos indicus x Bos taurus heifers (Bridges et al., 2005). Modifying the delivery of PG from a

single to two consecutive split PG treatments improved the estrous response and synchronized

AI pregnancy rates in Bos indicus x Bos taurus heifers (Bridges et al., 2005), but the

synchronized AI pregnancy rates are still less than values observed in Bos taurus heifers.

Wood et al. (2000) incorporated gonadotropin-releasing hormone (GnRH) into the MGA-

PG system by administering GnRH 12 d after the end of MGA treatment followed by PG 7 d

later and reported an increased synchrony of estrus compared to the traditional MGA-PG system

(Lamb et al., 2000). Recent research in our lab (See Chapter 3) indicated that follicle

development between the MGA withdrawal and PG treatment was not as synchronous in Bos

indicus x Bos taurus (Brangus) heifers compared to Bos taurus (Angus) heifers, which resulted

from more three wave follicle development patterns in Brangus compared to Angus heifers.

Results from Chapter 3 raised the possibility that the most effective time to introduce GnRH into

the MGA-PG system may be either a couple of days after MGA withdrawal or 10 d after MGA.

Therefore, two experiments were conducted to evaluate: 1) the effect of GnRH administered

either 3 or 10 d after the last day of a 14 d MGA treatment with PG 7d after to synchronize









estrus, and 2) evaluate the most effective GnRH treatment from Experiment 1 in a field trial

utilizing yearling Bos indicus x Bos taunts and Bos taunts heifers.

Materials and Methods

Experiment 1 was conducted at the University of Florida, Beef Research Unit from

October to December, 2004 using 2-year-old Bos indicus x Bos taunts (n = 58) heifers. Breed

composition of the heifers consisted of 3 to 78% Brahman (Bos indicus) breeding with the

remainder being Angus (Bos taurus) breeding. Heifers were equally distributed by percentage

Brahman breeding to one of two treatments prior to the start of the experiment. Mean (LSM &

S.E.) age, BW, and body condition score (BCS: Richards et al., 1986) of the heifers were 640 &

3.7 d, 395 & 6.3 kg, and 4.8 & 0.1 for one group designated as the G3 treatment, and 641 & 3.8 d,

406 & 6.3 kg, and 4.9 & 0.1 for the other group designated as the G10 treatment, respectively.

Treatment groups were maintained in adj acent pastures throughout the experiment. Prior to the

start of the experiment, both treatments were pre-synchronized according to the protocol

described by Lemaster et al. (1999), so that heifers would start a 14 d melengestrol acetate

(MGA) treatment at d 2 of the estrous cycle. Briefly, heifers received a progesterone insert

(EAZI-BREED CIDR@; Pfizer Animal Health, New York, NY) concurrent with 2 mg (i.m.)

estradiol benzoate. Seven days later, CIDR were removed and heifers received 25 mg (i.m.)

prostaglandin Fza (PG; Lutalyse Sterile Solution, Pfizer Animal Health, New York, NY)

followed 24 h later with 0.5 mg (i.m.) estradiol benzoate to synchronize estrus and initiate

ovulation. Two days after the expression of estrus, both treatments received MGA (0.5 mg~hd-

*~d- ; MGA" Premix, Pfizer Animal Health, New York, NY) in a total mixed ration and the

MGA was administered for 14 d. Three days after the last day of MGA, heifers in the G3 (n =

30) treatment received GnRH (100 Cpg i.m. Cystorelin, Merial, Inc., Duluth, GA); whereas, ten









days after the last day of MGA, heifers in the G10 (n=28) treatment received GnRH. Seven and

eight days after GnRH treatment, heifers in each respective treatment received 12.5 mg PG (i.m.;

Lutalyse" Sterile Solution) on each day. The experiment was designed so that PG was

administered on the same days for both the G3 and G10 treatment.

Heifers were observed for behavioral estrus for approximately 1 h at 0700 and 1700 h daily

from MGA withdrawal until GnRH was administered for the G3 and G10 treatments. The same

estrous detection protocol was also implemented during the 7 d after the initial PG treatment.

All heifers received Kamar detectors (Kamar" Marketing Group, Steamboat Springs, CO) at

MGA withdrawal and a new detector at the initial PG treatment to assist in estrous detection.

Behavioral estrus was defined as a heifer standing to be mounted by another heifer and (or) signs

of vaginal mucous. If a heifer was not detected in estrus by visual observation but had a Kamar

that was one-quarter to completely activated, the heifer was considered to have been in

behavioral estrus. A three-day estrous response after MGA withdrawal was determined for the

G3 and G10 heifers combined and was defined as the total number of heifers exhibiting estrus

within 3 d after MGA withdrawal divided by the total number of heifers in the G3 and G10

treatments. A five-day estrous response after MGA withdrawal was also determined for the G10

treatment and was defined as the total number of heifers exhibiting estrus within 5 d after MGA

withdrawal divided by the total number of heifers in the G10 treatments. Estrous response after

PG was determined as both a three-day estrous response and total estrous response within the G3

and G10 treatments heifers. Three-day estrous response after PG was defined as the total

number of heifers exhibiting estrus within 3 d after the initial PG divided by the total treated.

Total estrous response was defined as the total number of heifers displaying estrus within 7 d

after the initial PG divided by the total number treated.









To evaluate what effect stage of follicle development (SOF) may have had on the

effectiveness of GnRH to initiate ovulation, heifers within the G3 and G10 treatments were

assigned to receive either no PG (G3, n = 6; G10, n = 6) or PG (12.5 mg i.m.; Lutalyses Sterile

Solution) on d 4 and 5 (G3, n = 5; G10, n = 5), 8 and 9 (G3, n = 8; G10, n = 7), or 12 and 13 (G3,

n = 6, G10, n = 5) of MGA treatment to initiate luteolysis. These days were chosen to simulate

variable periods of low level progesterone exposure during MGA comparable to heifers being at

d 2 (no PG), 6 (PG d 12/13), 10 (PG d 8/9), or 14 (PG d 4/5) of the estrous cycle at the initiation

of MGA treatment. The variable lengths of low level progestogen exposure were used to vary

the duration of persistence of the dominant preovulatory follicle prior to MGA withdrawal.

Blood plasma samples were collected via jugular veinipuncture from all heifers before they

received PG during MGA to confirm the presence of a corpus luteum (CL) and at MGA

withdrawal to confirm that luteolysis had occurred. Heifers in the d 6, 10, and 12 SOF groups

were determined to be at the assigned SOF development if progesterone concentrations were > 1

ng/mL at PG during MGA treatment followed by progesterone concentrations < 1 ng/mL at

MGA withdrawal with the absence of a corpus luteum (CL). Heifers in the no PG group would

have had progesterone concentrations that were > 1 ng/mL at MGA withdrawal. Presence or

absence of a CL was determined by transrectal ultrasonography (Aloka 500v, Corometrics

Medical Systems, Wallingford, CT) equipped with a 7.5 MHz linear array transducer.

Ovaries of all heifers were evaluated using transrectal ultrasonography (equipped with a

7.5 MHz linear array transducer) at MGA withdrawal, GnRH, 48 h after GnRH, initial PG after

GnRH, and 8 d after the expression of estrus for heifers that were observed in estrus after PG. At

each ultrasonography evaluation, height and width of all luteal structures, luteal cavities, and

follicles > 3 mm in diameter were measured using the internal calipers of the ultrasonography









machine and their locations on the ovaries were recorded. Volume of the CL was calculated

using the formula for volume of a sphere (nd3/6). When a luteal cavity was present, its volume

was subtracted from the volume of the outer sphere resulting in net luteal volume (CL volume).

Additionally, blood plasma samples were collected via jugular veinipuncture at each

ultrasonography examination and 48 h after the initial PG to determine plasma progesterone

concentrations. Blood plasma samples were collected into evacuated tubes containing an

anticoagulant (EDTA; Becton, Dickinson and Company, Franklin Lakes, NJ). After collection,

blood plasma samples were immediately placed on ice until they were centrifuged (3 000 x g for

15 min) within 3 h after collection. Plasma was separated and stored at -200C until analysis for

progesterone concentrations, in multiple assays, as previously described. Intra- and interassay

CV of the assays were 5.4 and 5.6%, respectively, and sensitivity of the assay was 0.1 ng/mL.

Ovulation to GnRH was defined as the largest follicle present at GnRH followed by its

disappearance at the ultrasonography exam 48 h after GnRH. Seven days after GnRH, the

location of the ovulated follicle was verified by presence of a CL as determined by

ultrasonography. The CL was deemed functional if progesterone concentrations were > 1

ng/mL. Eight-days after the PG induced estrus, ovulation was confirmed by the presence of a

functional CL as previously described. Heifers not exhibiting estrus within 7 d after PG

underwent ultrasonography and blood sampling 10 d after the initial PG to assess ovarian

function.

Because the G3 and G10 treatments were not started until either 3 or 10 d after MGA

withdrawal, a three-day estrous response after MGA withdrawal for the G3 and G10 treatments

combined was tested using the GENMOD procedure with SAS (SAS Inst. Inc., Cary, NC) with

SOF development being the independent variable. The GENMOD procedure of SAS was also









used to test SOF effects within each treatment. Independent variables tested were treatment,

SOF, and treatment x SOF effects. Dependent variables tested were ovulation rate to GnRH,

ovulation rate after PG, estrous response after MGA withdrawal, percentage of heifers with

follicles > 10mm at GnRH, percentage of heifers with follicles > 10mm at PG, and percentage of

heifers with progesterone concentrations > 1 ng/mL at PG. Additionally, three-day estrous

response and total estrous response after PG was tested using the GENMOD procedure.

Progesterone concentration and diameter of the largest follicle at MGA withdrawal for the G3

and G10 treatments combined were tested using the GLM procedure of SAS, with the

independent viable being SOF development. Diameter of the largest follicle present at GnRH,

diameter of the largest follicle present at PG, progesterone concentrations at PG, and

progesterone concentrations 8 d following behavioral estrus were also tested using the GLM

procedure of SAS. Independent variables tested were treatment, SOF, and treatment x SOF

effects. Five heifers from the G3 and G10 treatments did not conform to the assigned SOF as

mentioned previously and they were removed from all analyses.

Experiment 2 was conducted at two locations from December, 2004 to April, 2005.

Yearling Bos taurus (Angus; n = 57) and Bos taurus x Bos indicus crossbred heifers (n = 178)

from the Dicks' Brothers Farm, Lake City, FL (Location 1) and yearling Bos indicus x Bos

taurus heifers (n = 117) from the Davis Ranch, Alachua, FL (Location 2) were used in

Experiment 2. Prior to the start of the experiment, heifers at Location 1 were randomly

distributed to one of two treatments and a BCS was recorded; whereas, heifers at Location 2

were equally distributed to one of two treatments by BCS and reproductive tract score (RTS;

Anderson et al., 1991). Mean BCS (LSM & S.E.) were 5.0 + 0.05 for the Bos taurus and 5.0 +

0.03 for Bos indicus x Bos taurus heifers at Location 1 and 5.4 & 0.04 for Bos indicus x Bos









taurus heifers at Location 2. Mean RTS (LSM & S.E.) was 3.8 & 0.8 for heifers at Location 2.

Heifers received MGA (0.5 mg~hd- *d l) for 14 d in a total mixed ration at Location 1 and in a

high protein pellet fed at a rate of 2 lbs~hd- *d-l at Location 2. Heifers in treatment 1 (MGA-PG)

received 12.5 mg prostaglandin Fza i.m. (PG; Prostamate" Sterile Solution; Agri Laboratories,

Ltd. St. Joseph, MO.) 19 and 20 d following MGA withdrawal. Heifers in treatment 2 (MGA-G-

PG) received 100 Cpg GnRH i.m. (Cystorelin, Merial, Inc., Duluth, GA) 3 d following MGA

withdrawal with 12.5 mg PG i.m. (Prostamate") 7 and 8 d after GnRH (MGA-G-PG). The MGA

and GnRH treatments were staggered so the PG was delivered on the same days for both

treatments. To aid in estrus detection, heifers received an Estrus Alerto heat detection patch

(Estrus Alert", Western Point, Inc, Merrifield, MN) at the second PG.

Estrus was visually detected two times daily at 0700 and 1700 for 3 d after the initial PG.

Estrus was defined as a heifer standing to be mounted by another heifer and/or a 1/4 to full red

Estrus Alert" patch. Eight to twelve hours after being detected in estrus, heifers were AI with

frozen-thawed semen by a single AI technician at both locations. Multiple AI sires were used

within each location and equally distributed between treatments. Heifers not detected in estrus

by 72 h after the initial PG were timed inseminated (timed-AI) and received 100 Cpg GnRH i.m.

(Cystorelin). Bulls were placed with heifers 10 d after timed-AI for both locations. Pregnancy

to AI was determined approximately 55 d following timed-AI for both locations, using a real-

time B mode ultrasound (Aloka 500v) equipped with a 5.0 MHz rectal transducer. Due to the 10

d interval where no heifers were bred by AI or the clean-up bull, differences in fetal size (Curran

et al., 1986) were used to distinguish between a pregnancy resulting from AI or clean-up bull.

Three-day estrous response was defined as the number of heifers that exhibited estrus

during the 3 d between PG and timed-AI, divided by the total number of heifers treated.










Conception rate was defined as the number of heifers observed in estrus, inseminated, and

became pregnant, divided by the total number of heifers inseminated. The timed-AI pregnancy

rate was defined as the number of heifers that became pregnant to timed-AI, divided by the total

number of heifers timed-AI. Synchronized pregnancy rate was defined as the total number of

heifers that became pregnant to either AI following estrus detection or timed-AI, divided by the

total number of heifers treated. Thirty-day pregnancy rate was defined as the total number of

heifers pregnant in the first 30 d of the breeding season divided by the total number of heifers

treated.

The GENMOD procedure of SAS was used for the statistical analysis in Experiment 2.

Within Location 1 data were initially analyzed with breed in the model to evaluate breed effects.

The independent variables included treatment, breed, and treatment x breed, while the dependent

variables were estrous response, conception rate, timed-AI pregnancy rate, synchronized

pregnancy rate, and 30 d pregnancy rate. If the breed and treatment x breed effects were

significant (P < 0.05), data for the Angus heifers were analyzed separately from the Bos indicus

x Bos taurus heifers for Location 1. Data from the Bos indicus x Bos taurus heifers from

Locations 1 and 2 were combined and the independent variables included treatment, location,

and treatment x location while the dependent variables included estrous response, conception

rate, timed-AI pregnancy rate, synchronized pregnancy rate, and 30 d pregnancy rate. Six heifers

from Location 1 were removed from analysis. Three were determined to be freemartins at AI,

one developed a vaginal infection prior to AI, and two were not present for pregnancy detection.

Within Location 2 the effect of RTS was also evaluated. Independent variables included

treatment, RTS, and treatment x RTS while the dependent variables included estrous response,

conception rate, timed-AI pregnancy rate, synchronized pregnancy rate, or 30 d pregnancy rates.









Heifers with missing RTS data (n = 5) and those with a RTS of 2 (n = 2) were removed from the

RTS analysis for Location 2.

Results

Experiment 1.

Stage of follicle development affected (P < 0.05) progesterone concentrations and

diameter of the largest follicle at MGA withdrawal across the G3 and G10 treatments (Table 4-

1). Heifers that did not receive PG during MGA were the only SOF group that had progesterone

concentrations > 1 ng/mL at MGA withdrawal. Progesterone concentrations were greater (P <

0.001) for d 2 compared to the d 6, 10, and 14 SOF groups, which were similar (P > 0.05) to

each other. Diameter of the largest follicle at MGA withdrawal increased (P = 0.001) as the

length of time that a follicle was exposed to a low-level progestin environment increased (Table

4-1). At MGA withdrawal, the d 14 SOF group had a greater (P < 0.05) follicle diameter

compared to d 2, 6, and 10 SOF groups, and the d 10 SOF group had a greater (P < 0.05) largest

follicle diameter compared to d 2 and 6 SOF groups, which were similar (P > 0.05) to each other.

Stage of follicle development also affected (P = 0.02) the three-day estrous response after

MGA withdrawal across the G3 and G10 treatments. The estrous response was similar (P >

0.05) for the d 6 and 10 SOF groups, which were both greater (P < 0.05) compared to the d 2 and

14 SOF groups. The d 2 and 14 SOF groups had similar (P > 0.05) estrous responses. When

only the five-day estrous response was evaluated for the G10 treatment, SOF development did

not effect (P = 0.27) the five-day estrous response. The five-day estrous response was 65.2%

(15/23) and the estrous responses for the d 2, 6, 10, and 14 SOF groups were 50.0% (3/6), 80.0%

(4/5), 85.7% (6/7), and 40.0% (2/5), respectively.

Diameter of the largest follicle at GnRH was affected (P < 0.05) by treatment and SOF

development but there was no treatment x SOF effect (P = 0.21; Table 4-2). Within the G3









treatment, the d 14 SOF group had a larger (P < 0.05) follicle diameter at GnRH compared to the

d 2, 6, and 10 SOF groups, which were all similar (P > 0.05) to each other. Conversely within

the G10 treatment, the d 2 SOF group had a smaller (P < 0.05) follicle diameter at GnRH

compared to the d 6, 10, and 14 SOF groups, which were all similar (P > 0.05) to each other.

When diameter of the largest follicle at GnRH was evaluated across the G3 and G10 treatments,

diameter of the largest follicle at GnRH was similar (P > 0.05) for the d 2, 6, and 10 SOF groups.

Whereas, heifers in the G3 treatment from the d 14 SOF group had a greater (P < 0.05) follicle

diameter compared to the G10 treatment. Diameter of follicle ovulating to GnRH was not

affected (P > 0.05) by treatment, SOF development, or treatment x SOF effect.

Total ovulation rate was affected (P = 0.02; Table 4-2) by treatment but not (P > 0.05) by

SOF or treatment x SOF. More (P = 0.02) G3 (76.0%) heifers ovulated compared to G10

(47.8%) heifers. When treatments were compared across SOF groups, G3 heifers in the d 6 and

10 SOF groups had greater (P < 0.05) ovulation rates compared to G10 heifers in the d 6 and 10

SOF groups. Whereas, ovulation rate for the d 2 and 14 SOF groups were similar (P > 0.05) for

the G3 and G10 treatments. Seventy-six percent (19/25) of G3 heifers had follicles 10 mm at

GnRH but there was no SOF effect (P = 0.27) on follicles > 10 mm for heifers in the d 2 (5/6;

83.3%), 6 (4/6; 66.7%), 10 (5/8; 62.5%), and 14 (5/5; 100.0%) SOF groups. For the G10 heifers,

73.9% had follicles > 10 mm in diameter at GnRH and there was no SOF (P = 0.13) effect. The

percentage of G10 heifers with follicles > 10 mm at GnRH was 50.0 (3/6), 60.0 (3/5), 85.7 (6/7),

and 100.0% (5/5) for d 2, 6, 10, and 14 SOF groups, respectively.

The percentage of heifers with a functional CL at PG was similar (P = 0.59; Table 4-3)

between the G3 (84.0%) and G10 (78.3%) treatments. There was a SOF effect (P = 0.01), but no

(P = 0.89) treatment x SOF effect on the percentage of heifers with a functional CL at PG.









Across SOF groups, more (P < 0.01) d 2 (100%), 6 (91%), and 10 (86.7%) heifers had a

functional CL at PG compared to d 14 (40%) heifers. Additionally, more (P = .01) G10 (43.5%)

compared to G3 (12.0%) heifers had two CL on their ovaries at PG. There were no (P > 0.05)

SOF or treatment x SOF effects on the incidence of heifers with two CL on their ovaries at PG.

Progesterone concentrations at PG were greater (P = 0.01) for G10 compared to G3 heifers

(Table 4-3). Stage of follicle development also affected (P = 0.01) progesterone concentrations

at PG but there was no (P = 0.93) treatment x SOF effect on mean progesterone concentration at

PG. Across SOF groups, d 14 (2.4 & 1.2 ng/mL) had decreased (P = 0.01) progesterone

concentrations compared to d 2 (6.8 & 1.1 ng/mL), 6 (7.0 & 1.2 ng/mL), and 10 (7.7 & 1.0 ng/mL)

of which the later three were similar (P > 0.05) to each other. Of the heifers with a functional

CL at PG, the PG induced luteolysis occurred in a similar (P > 0.05) percentage of G3 (21/21)

and G10 (18/18) heifers.

Diameter of the largest follicle at PG was similar (P = 0.67; Table 4-3) for G3 and G10

treatments but SOF affected (P = 0.01) the diameter of the largest follicle at PG. Diameter of the

largest follicle was greater (P < 0.05) for the d 14 (15.4 & 1.0 mm) SOF group compared to d 2

(13.1 & 0.9 mm), 6 (12.2 & 0.9 mm), and 10 (11.2 & 0.8 mm) SOF groups, whereas, diameter of

the largest follicle was similar (P > 0.05) for the d 2, 6, and 10 SOF groups. There was no (P =

0.54) treatment x SOF effect on diameter of the largest follicle at PG.

More heifers (P = 0.01) in the G3 (76.0%) treatment exhibited estrus within 3 d after the

initial PG compared to the G10 (43.5%) treatment (Table 4-4; Figure 4-1). In contrast, total

estrous response was similar (P = 0. 12) between the G3 and G10 treatments. There were no (P >

0.05) SOF or treatment x SOF effects on the 3 d and total estrous responses. Interval from PG to

the onset of estrus was not affected (P > 0.05) by treatment or treatment x SOF (Table 4-4).









Conversely, there was (P = 0.04) a SOF effect on interval from PG to onset of estrus. The d 14

(60.0 & 7.4 h) SOF group had a shorter (P < 0.05) interval from PG to the onset of estrus

compared to d 2 (91.2 & 7.0 h) and d 10 (79.7 & 6.1 h) SOF groups, but the d 14 interval was (P >

0.05) similar compared to the d 6 interval (76.5 & 7.1 h).

Of heifers that exhibited estrus after PG, a similar (P = 0. 19) percentage of G3 (91.3%;

21/23) and G10 (100.0%; 19/19) heifers formed a functional CL by d 8 after the onset of estrus.

There were two G3 heifers that exhibited estrus but failed to form a functional CL. Neither of

these heifers had a functional CL at PG and both were in the d 14 SOF group. There were two

G3 heifers that did not exhibit estrus after PG. There was a single heifer in the d 2 group that

had a functional CL at PG and had regressed the CL by d 10 after PG. The other G3 heifer was

in the d 10 SOF group, and this heifer did not have a functional CL at PG or 10 d following PG.

There were four G10 heifers that were not observed in estrus. One heifer from each from the d 2

and 6 SOF groups had a functional CL at PG while the heifers from the d 2 SOF group did not

have a functional CL 10 d after PG the heifers from the d 6 SOF group had a functional CL. The

remaining two G10 heifers were from the d 10, and 14 SOF groups both of which had a

functional CL at PG but did not have a functional CL 10 d after PG.

Progesterone concentrations 8 d after the PG induced estrus were similar (P = 0.50)

between the G3 (4.8 & 0.5 ng/mL) and G10 (5.2 & 0.5 ng/mL) treatments and there were no (P >

0.05) SOF or treatment x SOF effects. Progesterone concentrations 8 d following the

synchronized estrus for the d 2, 6, 10, and 14 groups were 5.3 A 1.0, 5.3 A 1.0, 4.8 & 0.8, and 3.6

& 1.0 ng/mL for G3 and 4.9 & 1.0, 5.7 & 1.0, 4.7 & 0.9, and 5.6 & 1.0 ng/mL for G10,

respectively.










Experiment 2.

Within Location 1, there was (P = 0.02) a breed effect on estrous response and there were

treatment x breed effects (P < 0.05) on timed-AI and synchronized pregnancy rates. Therefore,

the Angus data was analyzed separately for Location1 and data for the Bos indicus x Bos taurus

heifers in Locations 1 and 2 were analyzed together.

The three-day estrous response was not affected (P > 0.05) by treatment or treatment x

location for the Bos indicus x Bos taurus heifers (Table 4-5). However, estrous response was

greater (P = 0.04) for Location 2 (70/117; 59.8%) compared to Location 1 (85/178; 47.8%). For

Angus heifers at Location 1, treatment had no effect (P = 0.16) on estrous response (Table 4-6).

There were no (P > 0.05) treatment or location effects on conception rate in Bos indicus x

Bos taurus heifers; however, there was a (P = 0.01) treatment x location effect (Table 4-5) on

conception rates. Conception rates were greater for the MGA-PG treatment at Location 2 and

the MGA-G-PG treatment at Location 1 compared to the MGA-PG treatment at Location 1 and

MGA-G-PG treatment at Location 2. Treatment had no effect (P = 0.42) on conception rates in

Angus heifers at Location 2 (Table 4-6). For the Bos indicus x Bos taurus heifers, there were no

(P > 0.05) treatment, location, and treatment x location effects on timed-AI pregnancy rates. For

the Angus heifers, timed-AI pregnancy rate was greater (P = 0.01) for the MGA-PG compared to

MGA-G-PG treated heifers (Table 4-6).

There were no (P > 0.05) treatment, location, or treatment x location effects on

synchronized and 30 d pregnancy rates for Bos indicus x Bos taurus heifers. For the Angus

heifers, synchronized pregnancy rates tended to be greater (P = 0.08) and 30 d pregnancy rates

were similar (P = 0.76) for the MGA-PG compared to the MGA-G-PG treated heifers (Table 4-

6).









Within Location 2, reproductive tract scores (RTS) were taken at the initiation of the

experiment to evaluate the effect of RTS on response to the synchronization treatments. There

were no (P > 0.05) treatment, RTS, and treatment x RTS effects on estrous response, timed-AI

pregnancy rate, synchronized pregnancy rate, and 30 d pregnancy rate (Table 4-7). In general, as

RTS increased from a 3 to either a 4 or 5, estrous response and synchronized pregnancy rate

increased numerically. However, conception rate was greater (P = 0.02) for the MGA-PG

(68.8%) treatment compared to the MGA-G-PG (41.9%) treatment. There were no (P > 0.05)

RTS or treatment x RTS effects on conception rate.

Discussion

Recent research by Wood and co-workers (2001) indicated that yearling Bos taurus

heifers treated with MGA for 14 d with GnRH 12 d after the last day of MGA followed by PG 7

d later had an improved synchrony of estrus compared to heifers synchronized with the

traditional MGA-PG system. The reason for the increased synchrony of estrus was attributed to

the ability of GnRH to synchronize follicle development. Recent research from our lab (See

Chapter 3) indicated that follicle wave development during the 19 d after a 14 d MGA treatment

was more variable in Brangus compared to Angus heifers. It was concluded that administering

GnRH 12 d after MGA withdrawal in Brangus heifers may be too late because some heifers had

already initiated a second follicle wave resulting in fewer heifers with follicle in the growing

phase capable of ovulating to GnRH (Moreira et al., 2000). Therefore, it was hypothesized that

administering GnRH 10 d after MGA withdrawal instead of 12 d may be more effective in

Brangus heifers. It was also theorized that administration of GnRH 3 d after MGA withdrawal

may actually be more effective since there was less variation in follicle development

immediately after MGA withdrawal and a maj ority of follicles should be large enough to ovulate

to GnRH. For that reason, Experiment 1 was designed to determine the effectiveness of









administering GnRH either 3 or 10 d after a 14 d MGA treatment in Bos indicus x Bos taurus

heifers. Because follicle development can be significantly influenced by the stage of luteal

development that animals are under the influence of during a long-term MGA treatment (Sirois

and Fortune, 1990; Kojima et al., 1992), it was also of interest to evaluate what effect stage of

follicle development at the end of the MGA treatment would have on response to the GnRH.

At MGA withdrawal, only the d 2 SOF group had progesterone concentrations indicative

of luteal activity compared to d 6, 10, or 14 SOF groups, which all received PG during MGA.

Diameters of the largest follicles at MGA withdrawal were smallest in the d 2 and 6 SOF groups

compared to the d 10 and 14 SOF groups. The d 2 SOF group had the smallest follicle diameter,

as they would have been near the end of the estrous cycle and probably in the middle of a follicle

wave (Ginther et al., 1989) due to high luteal progesterone that initiated follicle turnover (Sirois

and Fortune, 1990) just before MGA withdrawal. The d 6 SOF group had their CL regressed

two days before MGA withdrawal resulting in the presence of newly developing dominant

follicle under the influence of a low progesterone environment provided by MGA for

approximately 2 d. In contrasts, the d 10 and 14 SOF groups had the largest follicle diameters at

MGA withdrawal, which was a result of the dominant follicles being under low progesterone

environments for approximately 5 d in the d 10 SOF group and 9 d in the d 14 SOF group.

Consequently, the d 10 and 14 SOF groups had dominant follicles that continued to grow,

develop, and persist on the ovaries during MGA due to the increased frequency of LH pulses

observed during a low progesterone environment (Sirois and Fortune, 1990; Kojima et al., 1992).

Therefore, the experimental model was effective in altering follicle development at the end of a

14 d MGA treatment.









The three-day estrous response following MGA withdrawal was approximately 53%

across the d 6 and 10 SOF groups. Hill et al. (1971) reported that estrus occurred primarily

between 3 to 7 d after treatment with a 14 d MGA withdrawal. It is likely that three days was an

inadequate amount of time to allow for a LH surge and the onset of estrus in all animals, which is

supported by the observation that approximately 83% of the d 6 and 10 heifers in the G10

treatment exhibited estrus within 5 d after MGA withdrawal. Therefore, heifers with follicles

that have been under a low progesterone environment for approximately 2 to 5 d have an

excellent opportunity to express estrus and ovulate within 5 d after the end of a 14 d MGA

treatment. Consequently, exposure of dominant follicles to a low progesterone environment for

approximately 2 to 5 d does not appear to compromise the ability of the follicles to secrete

estrogen and ovulate after MGA withdrawal. In contrast, the three-day estrous response for the d

2 SOF group was only 8.3% but it increased to 83.3% by 5 d after MGA withdrawal in the G10

treatment. Heifers in the d 2 SOF group that did not exhibit estrus by d 3 were probably at the

start of a new follicle wave at MGA withdrawal due to high luteal progesterone that initiated

follicle turnover (Sirois and Fortune, 1990), which would have resulted in a delayed interval to

estrus as the new follicle wave developed (Ginther et al., 1989). The d 14 SOF group also had a

decreased 3 d estrous response of 20% and the estrous response increased to only 40% within by

d 5 d after PG in the G10 treatment. In contrast to the d 2 SOF group, the d 14 SOF group had

the largest follicle diameter at MGA withdrawal due to the development of long-term persistent

dominant follicles (Sirois and Fortune, 1990; Kojima et al., 1992), which should have been

secreting enough estrogen (Kojima et al., 1995) to induce an LH surge and the onset of estrus.

However, this was not the case and the reason (s) for the decreased estrous response of the long-









term persistent dominant follicles in the d 14 SOF group could be several fold and will be

discussed in subsequent paragraphs.

Across the four SOF groups in the G3 treatment, ovulation rate was a respectable 76%;

although, whether ovulation occurred or did not occur tended to be influenced by SOF

development at MGA withdrawal. For the G3 treatment, 93% (13/14) of the heifers in the d 6

and 10 SOF groups ovulated to GnRH compared to only 66.7% in the d 2 SOF group. The two

heifers in the d 2 SOF group that did not ovulate to GnRH were probably heifers that were in the

middle of a follicle wave. This is supported by the observation that they had elevated

progesterone concentrations at MGA withdrawal, which probably initiated follicle turnover

(Ginther et al., 1989). As a result, they did not have a growing follicle that was capable of

ovulating to GnRH (Moriera et al., 2000). In contrasts, the d 14 group had the lowest percentage

of heifers ovulating at only 40%, indicating that GnRH was not very effective in ovulating

persistent dominant follicles that had been under low progesterone exposure for approximately 9

d. Reasons for the failure of GnRH to initiate ovulation in a majority of the d 14 SOF group is

unclear. The increased LH pulse frequency and resulting increased estradiol concentrations

observed when no CL is present during an MGA treatment (Kojima et al., 1995) may have

depleted the stores of LH in the anterior pituitary. In cows that had previously been treated with

MGA for 9 d, Kojima et al. (1992) did not detect a LH surge in 80% of cows after MGA

withdrawal. Furthermore, the LH stores may be more easily depleted in cattle of Bos indicus

breeding since they have less LH released in response to an exogenous GnRH treatment

compared to cattle of Bos taurus breeding (Griffen and Randel, 1978; Portillo et al., 2007). One

could also speculate that the increased LH pulse frequency observed during a low progesterone

environment may have lead to down regulation of LH receptors in the granulosa cells of the










persistent dominant follicles leading to ovulation failure. Another explanation may be that long-

term persistent follicles may become cystic (Sirois and Fortune, 1990; Mihm et al., 1994), and

cannot undergo ovulation (Cook et al., 1990). Therefore, these results suggest that persistent

dominant follicles that develop during a long-term MGA treatment have altered ovulatory

capacities and the capacity to ovulate is probably influenced by the period of time that the

follicles are under the influence of a low progesterone environment. Consequently, if the goal is

to initiate ovulation in a maj ority of follicles to synchronize follicle development after an MGA

treatment, it may prove advantageous to use short-term (7-9 d) MGA treatments to decrease the

incidence of large persistent dominant follicles present during long term (14 d) MGA treatments.

Additional research will need to be conducted to evaluate this.

Estrous response during the five days after MGA withdrawal for the G10 heifers was

only 65.2%, which is similar to the seven day estrous response observed in Angus and Brangus

heifers (See Chapter 3), but slightly less than Bos taurus heifers treated with a 14 d MGA

treatment (Yelich et al., 1997). One explanation for the decreased estrous response could be the

incidence of a "silent estrus", which is a frequent occurrence in cattle of Bos indicus breeding

(Galina et al., 1982; Orihuela et al., 1983) and was observed in similar study conducted in our

lab (See Chapter 3). This also appears to be the case in Experiment 1, since two of five G10

heifers that were not observed in estrus had a CL at GnRH. The ovulation rate to GnRH was

only 47.8% in G10 heifers compared to 76.0% for the G3 heifers. Even though all SOF groups

except the d 2 group had a follicle diameter > 10 mm for the G10 treatment, it did not equate into

a large percentage of follicles ovulating to GnRH for the G10 treatment. The decreased

ovulation to GnRH suggest that there was significant asynchrony of follicle development by d 10

after MGA withdrawal resulting in a limited number of follicles in the growing phase that were









capable of ovulating to GnRH (Moreira et al., 2000). Part of the reason for the follicle

asynchrony was dictated by the low estrous response observed during the 5 d after MGA

withdrawal. This is supported by the observation that more heifers that exhibited estrus after

MGA withdrawal ovulated to GnRH compared to heifers not exhibiting estrus within 5 d of

MGA withdrawal. Hence, the effectiveness of administering GnRH 10 d after MGA withdrawal

is largely predicated on heifers exhibiting estrus within 5 d of MGA withdrawal. Certainly, d 10

is not the appropriate time to administer GnRH and it is unclear if waiting until d 12 would have

provided a better response.

There was a similar percentage of G3 and G10 heifers with a functional CL at PG, but

SOF development influenced the percentage of heifers with a functional CL. It should be noted

that two G3 heifers in the d 2 group that did not ovulate to GnRH, had functional CL's at PG.

The two heifers must have ovulated sometime after GnRH and been in the early luteal phase at

PG. The d 14 heifers had significantly lower percentage of functional CL at PG than all other

SOF groups for both the G3 and G10 treatments. Not only did GnRH not initiate ovulation in

the d 14 SOF group in the G3 treatment, the presence of a persistent dominant follicle altered

follicle development enough so that there was not a new follicle wave available to ovulate by d

10 after MGA in the G10 treatment. Clearly dealing with the persistent dominant follicle in

cattle of Bos indicus breeding is difficult. It is unknown if similar responses are observed in Bos

taurus cattle treated with a 14 d MGA treatment.

Heifers in the G10 treatment had increased progesterone concentrations at PG compared

to G3 treated heifers, which was likely due to the increased incidence of heifers in the G10

treatment having two CL on their ovaries (Diaz et al., 1998) compared to G3 treated heifers.

Therefore, some of the G10 heifers would have a CL from the estrus after MGA withdrawal and









a CL from the GnRH treatment. Two G10 heifers in the d 14 SOF group, one of which ovulated

prior to GnRH and the other to GnRH, did not have a functional CL at PG. Lemaster et al.,

(1999) reported the presence of luteal tissue without any progesterone production in frequently

worked cattle of Bos indicus breeding. However, since both heifers exhibited estrus shortly after

PG, both heifers may have undergone a short estrous cycle accompanied by a short-lived luteal

structure.

Treatment did not influence the diameter of the largest follicle at PG. However, diameter

of the largest follicle of the Bos indicus x Bos taurus heifers in the present experiment are

considerably less than the diameters observed by Wood et al. (2001) in MGA-G-PG treated Bos

taurus heifers. Wood et al. (2001) reported a high percentage of heifers ovulating to GnRH

administered 12 d after a 14 d MGA treatment resulting in a new follicle wave that was reaching

maximal diameter when PG was administered 7 d after GnRH. In the current study, a high

percentage of heifers that received GnRH 10 d after MGA did not ovulate to GnRH, which

probably resulted in asynchronous follicle wave development when PG was administered 7 d

after GnRH resulting follicles of various sizes. Diameters of the largest follicles at PG were

greatest in d 14 SOF heifers for both the G3 and G10 treatments. Because daily ultrasound

examinations were not conducted between MGA withdrawal and PG, it is difficult to determine

if the largest follicle present at PG was either from a new follicle wave initiated by the GnRH

treatment or the presence of persistent dominant follicles that were present at MGA withdrawal

and still on the ovaries at PG. It is interesting to note that progesterone concentrations were < 1

ng/mL for G3 heifers in the d 14 SOF group, which may have resulted in increased pulsatile

secretion of LH secretion resulting in increased follicle development (Sirois and Fortune, 1990;

Kojima et al., 1992).









The G3 treatment proved to be more effective in synchronizing estrus as 76% of the

heifers exhibited estrus during the three-days after PG, which was similar to the estrous response

observed by Wood et al. (2001) in Bos taunts heifers receiving the MGA-G-PG treatment where

GnRH was administeredl2 d after MGA withdrawal. It is interesting to note that all G3 heifers

in the d 14 SOF group exhibited estrus by 3 d after PG. This suggests that the d 14 SOF group

that did not respond to GnRH must have initiated a new follicular wave that was capable of

initiating estrus and ovulating by approximately 12 d after MGA withdrawal. A similar type of

follicle growth pattern was also observed for heifers that developed persistent dominant follicles

during a 14 d MGA treatment and failed to ovulate the follicles within 5 d after MGA

withdrawal (See Chapter 3). In comparison to other studies, the G3 treatment produced a

substantially greater 3 d estrous response than the MGA-PG system in Bos taunts (Wood et al.,

2001; Bridges et al., 2005) and Bos indicus x Bos taunts (Stevenson et al., 1996; Bridges et al.,

2005) heifers. The percentage of heifers exhibiting estrus within the 7 d after PG is similar to

other studies synchronizing Bos taunts heifers with either the MGA-PG (Brown et al., 1988,

Lamb et al., 2000) or the MGA-PG system with GnRH (Wood et al., 2001). It should be noted

that all of the heifers used in the present study were going through normal estrous cycles at the

start of the experiment. Therefore, inducing an effective estrous response in Bos indicus x Bos

taurus heifers with the MGA-PG estrous synchronization maybe predicated more by the estrous

cycling status of heifers (Brown et al., 1988; Patterson et al., 1989) than by manipulation of the

follicle wave dynamics of the heifers. Additional research will need to be conducted to

completely characterize what effects cycling status (cycling vs., non-cycling) has on modifying

follicle dynamics in the MGA-PG synchronization protocol.









Interval from PG to the onset of estrus in the current study was comparable to MGA-G-

PG (GnRH administered 12 d after MGA withdrawal) treated Bos taunts heifers (Wood et al.,

2001), but increased compared to MGA-PG treated Angus and Brangus heifers in chapter 3.

Additionally, heifers that would have begun MGA at d 14 of their estrous cycle had the shortest

interval from PG to estrus regardless of GnRH treatment. One reason for the shorter interval was

the fact that the diameter of the dominant follicle at PG was greatest in d 14 SOF group

regardless of GnRH treatment, which is supported by the observation made by Sirois and

Fortune (1988) where size of the eventual ovulatory follicle at PG was negatively correlated with

the interval to estrus.

The percentage of heifers with a functional CL 8 d after an observed estrus was similar

between G3 and G10 treatments. It should be noted that three G10 treated heifers not detected in

estrus had a CL present 10 d following PG. Furthermore, there were some abnormalities in

ovulation and luteal development that occurred in the G3 heifers. Two G3 heifers that exhibited

estrus failed to ovulate, which was confirmed by lack of a CL and progesterone concentrations <

Ing/mL 8 d after the onset of estrus. Certainly, some of the G10 heifers had a "silent estrus"

(Galina et al., 1996) but it is not clear why the G3 heifers failed to ovulate and form a CL. The

frequent working of the heifers could have resulted in abnormal luteal development where a CL

is formed but it does not secrete any progesterone (Lemaster et al., 1999).

Results from Experiment 1 suggested that administering GnRH 3 d after a 14 d MGA

treatment resulted in the most synchronous estrus. Therefore, the objective of Experiment 2 was

to evaluate the effectiveness of the G3 (MGA-G-PG) treatment compared to a MGA-PG

treatment in a field trial utilizing yearling Bos indicus x Bos taunts and Bos taunts heifers.









The three-day estrous response of the Bos indicus x Bos taurus heifers in Experiment 2

was similar between the MGA-PG and the MGA-G-PG treatments and comparable to a report by

Bridges et al. (2005) but substantially less compared to a report by Stevenson et al. (1996) in Bos

indicus x Bos taurus heifers synchronized with the MGA-PG system. One factor that influences

estrous response is the number of heifers going through estrous cycles at the start of the

synchronization treatment (See Chapter 3; Brown et al., Patterson and Corah, 1992). The

importance of having heifers going through estrous cycles is also supported by the results of

Experiment 1 where 72% of the heifers exhibited estrus in the first 3 d after PG. Although,

estrous cycling status was not determined in Experiment 2, the RTS data from Location 2

suggest that cycling status had a maj or influence on estrous response. Heifers with a RTS of 4

and 5, indicative of heifers that are probably going through normal estrous cycles, had an estrous

response of 70% in the MGA-G-PG heifers compared to 42.9% in MGA-G-PG heifers with a

RTS of 3.

Conception rates were similar between the MGA-PG and the MGA-G-PG treatments.

Although no studies utilizing a similar MGA-G-PG treatment are available for comparison,

conception rates for the MGA-G-PG were similar to Bos indicus x Bos taurus heifers

synchronized with the MGA-PG treatment (Bridges et al., 2005). However, there was a

significant treatment by location interaction indicating that the treatment responses were

different between locations. Exact reasons for the differential response to treatments between

locations are unclear. It does not appear that AI technician or AI sire had an effect on conception

rates, since a single AI technician inseminated all heifers at both locations and AI sires were

equally stratified across treatments. However, when conception rates were evaluated within

Location 2 for the Bos indicus x Bos taurus heifers, the MGA-PG heifers had a 28% greater









conception rate compared to the MGA-G-PG. The trend for decreased conception rates for the

MGA-G-PG was consistent across the three RTS. Additionally, the MGA-G-PG Angus heifers

had a lower conception rate compared to MGA-PG Angus heifers. The similar trend of

decreased conception rates in both the Angus and Bos indicus x Bos taurus heifers is of concern.

It is possible that some of the heifers that exhibited estrus after PG could be heifers that did not

ovulate to GnRH but the heifers initiated a new follicle wave that initiated the expression of

estrus and ovulation around the time of PG. The questions that need to be answered are if these

follicles are fertile or not and if the lack of progesterone exposure before estrus and ovulation

resulted in short estrous cycles after the PG (Berardinelli et al., 1979; Evans et al., 1994).

One of the reasons that producers like to use the MGA-PG system is that it consistently

results in excellent conception rates during the synchronized estrus (Brown et al., 1988; Lamb et

al., 2000) in heifers of Bos taurus breeding. In contrasts, conception rates of Bos indicus x Bos

taurus heifers synchronized with MGA-PG protocols (See Chapter 3; Bridges et al., 2005) are

highly variable and are still considerably less than studies in Bos taurus heifers synchronized

with MGA-PG systems (Brown et al., 1988; Lamb et al., 2000). Age at puberty may have

something to do with this since cattle of Bos indicus breeding (Plasse et al., 1968; Baker et al.,

1989) attain puberty at older ages than Bos taurus breeds (Wiltbank et al., 1966; Baker et al.,

1989). As a result, reaching puberty at older ages may have decreased the number of estrous

cycles heifers had before synchronization and AI resulting in decreased conception rates

(Byerley et al., 1987; Galina et al., 1996).

A similar percentage of MGA-PG and MGA-G-PG treated Bos indicus x Bos taurus

heifers became pregnant to the timed-AI, but the timed-AI pregnancy rate was considerably less

compared to an experiment conducted by Bridges and coworkers (2005) in yearling Bos indicus









x Bos taurus heifers synchronized with an MGA-PG protocol receiving consecutive split

treatments of PG. Additionally, timed-AI pregnancy rates were substantially reduced in MGA-

G-PG compared to MGA-PG treated Angus heifers. The reduced timed-AI pregnancy rate in the

MGA-G-PG compared to MGA-PG treatment may be a result of when the timed-AI was

conducted relative to the ability of GnRH to induce ovulation (Moreira et al., 2000).

Furthermore, the timed-AI groups also contain heifers that are not cycling, did not respond to

PG, or did not have any follicles large enough to ovulate to GnRH. Any of these three scenarios

would result in decreased pregnancy rates to the timed-AI

Synchronized pregnancy rates were similar between the MGA-PG and MGA-G-PG

treated Bos indicus x Bos taurus heifers in Experiment 2, which are similar to synchronized

pregnancy rates of MGA-PG treated Bos indicus x Bos taurus heifers (Bridges et al., 2005). As

observed with estrous response, cycling status probably has more influence on synchronized

pregnancy rates than any other single variable (Brown et al., 1988; Patterson et al., 1989). This

can be partially explained by the reproductive tract score data from Location 2. Heifers with a

RTS of 4 or 5, indicative of heifers that, by definition, are going through normal estrous cycles,

had a synchronized pregnancy rate of 44.4% across the MGA-PG and MGA-G-PG treatments

compared to 34.0% in the heifers with a RTS of 3. For Angus heifers, synchronized pregnancy

rates tended to be greater in MGA-PG compared to MGA-G-PG. The maj or reasons for the

decreased synchronized pregnancy rate for the Angus heifers were due to the numerically lower

conception rate and the significantly lower timed-AI pregnancy rate for the MGA-G-PG heifers

compared to the MGA-PG heifers.

In summary, the G3 treatment improved the synchrony of estrus compared to G10

treatment in Bos indicus x Bos taurus heifers in Experiment 1. Stage of follicle development at









the end of a 14 d MGA treatment had significant effects on largest follicle size at MGA

withdrawal and the subsequent estrous response after MGA withdrawal. Heifers that developed

a long-term (> 9 d) persistent dominant follicle under the influence of a low progesterone

environment had a detrimental effect on the subsequent estrous response after MGA withdrawal

and ability to ovulate to GnRH, suggesting that long-term persistent follicles have a reduced

capacity to ovulation capacity. Additional experiments will need to be evaluated to either

regress or ovulate long-term persistent follicles developed under a 14 MGA environment, or

utilization of short-term (< 9 d) progestin treatments may need to be used to prevent the

occurrence of long-term persistent follicles. In Experiment 2, the MGA-G-PG (G3) protocol

failed to increase estrous response over the MGA-PG system in Bos indicus x Bos taurus heifers.

Synchronized pregnancy rates of the MGA-G-PG and MGA-PG systems were similar but the

synchronized pregnancy rates from Experiment 2 are still considerably less compared to other

reports in Bos taurus and Bos indicus x Bos taurus heifers synchronized with the MGA-PG

sy stem.

Implications

The addition of GnRH 3 d after withdrawal of a 14 d MGA treatment resulted in a greater

synchrony of estrus compared to GnRH 10 d after MGA. However, heifers that would have

started MGA late in the luteal phase of the estrous cycle and developed persistent dominant

follicles did not respond well to the GnRH treatments. Therefore, future research needs to

address dealing with persistent dominant follicles developed in low progesterone environments

in relation to their prevention and (or) removal from the ovary in heifers of Bos indicus x Bos

taurus breeding. The estrous response, conception rate, and synchronized pregnancy rate were

similar for MGA-G-PG compared to the traditional MGA-PG system. Therefore, administering









GnRH 3 d after a long term MGA treatment does not appear to improve the effectiveness of the

MGA-PG estrous synchronization system in heifers ofBos indicus x Bos taurus breeding.









Table 4-1. The effect of stage of follicle (SOF) development during a 14 d melengestrol
acetate (MGA) treatment on progesterone concentration (LSM & S.E.) at MGA
withdrawal, diameter of the largest follicle at MGA withdrawal (LSM & S.E.), and
three day estrous response following MGA withdrawal. Data are combined for G3
and G10 heifers (Experiment 1). a

Progesterone Diameter of largest Three day estrous
SOF n concentration, ng/mL follicle, mm response, % b

d 2 12 5.5 & 0.4 c 12.6 & 1.0 0 8.3 c

d 6 11 0.3 & 0.5 d 13.8 & 0.9" 54.5 d

d 10 15 0.2 & 0.4 d 16.4 & 0.8 d 53.3 d

d 14 10 0.3 & 0.5 d 20.4 & 1.0 e 20.0 0

P-value 0.001 0.001 0.02

a See material and methods for SOF descriptions.
b Estrous response is the total number of heifers exhibiting estrus out of the total number treated
within 3 d of MGA withdrawal.
c,d,e Means within a column without a common superscript differ (P < 0.05)










Table 4-2. Effect of treatment (T) and stage of follicle (S) development on largest follicle diameter at GnRH (LSM & SE), diameter
of follicle ovulating to GnRH (LSM & SE), and ovulation rate for heifers receiving GnRH either 3 d (G3) or 10 d (G10)
after withdrawal of a 14 d melengestrol acetate (MGA) treatment (Experiment 1).

Stage of follicle development group a P-values
Variable 2 6 10 14 T S Tx S


a See material and methods for description of stage of follicle development at MGA withdrawal.
b Four G3 heifers exhibited estrus before GnRH so their data were not include in the size of follicle ovulating to GnRH analysis but
the heifers were included in the ovulation rate data.
" Ovulation rate for G3 defined as number of heifers ovulating after an observed estrus within 3 d after MGA withdrawal and (or)
GnRH 3 d after MGA withdrawal divided by the total in the group. Ovulation rate for G10 treatment defined as the number of heifers
ovulating to GnRH 10 d after MGA withdrawal divided by the total in the group
d~e~fileans within a variable and row without a common superscript differ (P < 0.05).
x~y~zMeans within a variable and column without a common superscript differ (P < 0.05)


Largest follicle GnRH, mm


0.01 0.01 0.21


12.5 A 1.3 (6)d"x

9.7 & 1.3 (6)d"x


12.8 & 1.0 (4)


13.2 & 1.3 (5)d"x


13.3 A 1.2 (8)d"x


G3 (25)

G10 (23)


20.2 & 1.4 (5)e~x

14.2 & 1.4 (5)"



11.5 A 1.4 (2)


12.6 & 1.4 (5)efx 12.0 & 1.2 (7)e"."x


Follicle ovulating to GnRH, mm

G3 (15) b


0.32 0.20 0.11


15.0 & 1.0 (4)

13.7 & 1.2 (3


100.0 (6)e"x

60.0 (5)d"y


15.6 & 0.9 (5)

12.0 & 1.2 (3)


G10 (11)
Ovulation rate, % e

G3 (25)


0.02 0.12 0.34


66.7 (6)d"x
50.0 (6)dx


87.5 (8)d"x

42.9 (7)d"y


40.0 (5)d.x

40.0 (5)d.x


G10 (23)











Table 4-3. Percentage of heifers with a functional corpus luteum (CL), progesterone
concentration (LSM & S.E.), and diameter of the largest dominant follicle at
prostaglandin Fza (PG: LSM & S.E.) for G3 and G10 heifers across different stages
of follicle (SOF) development (Experiment 1).a

Progesterone Diameter of the
Treatment
Functional concentration largest follicle
(SOF)b
n CL at PG, %b at PG, ng/mL at PG, mm

G3 mean 25 84.0 4.3 & 0.8 12.8 & 0.6

d 2 6 100.0 5.61 1.6 12.5 A 1.2
d 6 6 100.0 5.0 & 1.6 12.2 & 1.2
d 10 8 87.5 5.7 & 1.4 10.3 A 1.1
d 14 5 40.0 1.0 & 1.7 16.2 & 1.4

G10 mean 23 78.3 7.7 & 0.8 13.2 & 0.6


d 2 6 100.0 8.0 & 1.6 13.7 & 1.2
d 6 5 80.0 9.1 + 1.7 12.2 & 1.4
d 10 7 85.7 9.8 & 1.5 12.1 + 1.1
d 14 5 40.0 4.0 & 1.7 14.6 & 1.4

P-values
Treatment 0.59 0.01 0.67
SOF 0.01 0.01 0.01
Treatment x SOF 0.89 0.93 0.54
a Heifers administered MGA for 14 d followed by GnRH either 3 (G3) or 10 d (G10) after MGA
withdrawal. Seven days after GnRH, heifers received PG.
bFunctional CL at PG defined as a heifer having progesterone concentrations > 1 ng/mL
combined with the presence of a CL as determined by ultrasonography











Table 4-4. Three-day estrous response, total estrous response, and interval from prostaglandin
Fza (PG) to onset of estrus following PG treatment for G3 and G10 heifers across
different stages of follicle (SOF) development (Experiment 1).a

Treatment Three-day estrous Total estrous Interval from
(SOF) n response, %b response (%)" PG to estrus, h

G3 mean 25 76.0 92.0 71.7 & 4.6

d 2 6 50.0 83.3 79.2 & 9.9
d 6 6 83.3 100.0 72.0 + 9.0
d 10 8 75.0 87.5 75.4 & 8.3
d 14 5 100.0 100.0 60.0 + 9.9


G10 mean 23 43.5 82.6 82.1 & 5.1

d 2 6 33.3 83.3 103.2 & 9.9
d 6 5 40.0 80.0 81.0 &11.0
d 10 7 42.9 85.7 84.0 + 9.0
d 14 5 60.0 80.0 60.0 & 11.0


P-values
Treatment 0.01 0.12 0.14
SOF 0.17 0.67 0.04
Treatment x SOF 0.63 0.52 0.69

aHeifers administered melengestrol acetate (MGA) for 14 d followed by GnRH either 3
(G3) or 10 d (G10) after MGA withdrawal. Heifers received 12.5 mg of PG on both d 7
and 8 after GnRH.
bThree-day estrous response is the number of heifers observed in estrus within 3 d of the
initial PG treatment divided by the total number of heifers treated.
cTotal estrous response is the number of heifers observed in estrus within 7 d of PG treatment
divided by the total number of heifers treated.
















40




S30*



20




10





12 24 36 48 60 72 84 96 108 120 132 144 156 168 ?OR

Interval from PG to the onset of estrus, h

Figure 4-1. Estrous response, expressed as a percentage of the total number of heifers in a group, during the 7 d after the initial PG
treatment for G3 (n = 25) and G10 (n = 23) treatments. NR = no estrous response (Experiment 1) .









Table 4-5. Estrous, conception and pregnancy rates ofBos taurus x Bos indicus heifers synchronized with combinations of
melengestrol acetate (MGA), GnRH (G), and prostaglandin Fza (PG) at two locations (LOC) (Experiment 2).

Estrous Conception Timed-AI Synchronized 30 d
Response rate pregnancy rate Pregnancy pregnancy rate
Treatment (TRT)a n LOC (%)b o%) c od o te o> (%f


a Both treatments received MGA for 14 d. MGA-PG heifers received PG 19 (12.5 mg) and 20(12.5 mg) d after MGA withdrawal.
The MGA-G-PG heifers received GnRH (100 Gig) 3 d after MGA withdrawal with PG 7 (12.5 mg) and 8 (12.5 mg) d after GnRH.
Estrus was detected for 3 d, and heifers exhibiting estrus were AI 8-12 h later. Heifers not displaying estrus were timed-AI 72-80 h
and received GnRH.
b Percentage of heifers displaying estrus 3 d after PG of the total treated.
" Percentage of heifers that were pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.
f Percentage of heifers pregnant during the first 30 d of the synchronized breeding of the total treated.


89 1 38/89 (42.7)
58 2 33/58 (57.0)
147 71/147 (48.3)

89 1 47/89 (52.8)
59 2 37/59 (62.7)
148 84/148 (56.7)


16/38 (42.1)
23/33 (69.7)
39/71 (54.9)

28/47 (59.6)
16/3 7 (43 .2)
44/84 (52.4)


0.55
0.46


13/51 (25.5)
4/25 (16.0)
17/76 (22.4)

7/42 (16.7)
5/22 (22.7)
12/64 (18.8)


0.91
0.83


29/89 (32.6)
27/58 (46.6)
56/147 (38.1)

35/89 (39.3)
21/59 (35.6)
56/148 (37.8)


0.74
0.38


62/89 (69.7)
41/58 (70.7)
103/147 (70.1)

62/89 (69.7)
43/59 (72.9)
105/148 (71.0)


0.84
0.69


MGA-PG
MGA-PG
Overall means

MGA-G-PG
MGA-G-PG
Overall means
P-values


TRT
LOC


0.18
0.04
0.73


TRT x LOC


0.01


0.28


0.13


0.84










Table 4-6. Estrous, conception and pregnancy rates of Angus heifers in Location 1 synchronized with combinations of melengestrol
acetate (MGA), GnRH (G), and prostaglandin Fza (PG) (Experiment 2).

Estrous Conception Timed-AI Synchronized 30 d
response rate pregnancy rate pregnancy rate pregnancy rate
Treatment a oA()b o%) c od o )e o)f

MGA-PG 27 15/27 (55.6) 6/15 (40.0) 5/12 (41.7) 11/27 (40.7) 19/27 (70.4)


MGA-G-PG 30 22/30O (73 .3) 6/22 (27.3) 0/8 (0.0) 6/30 (20.0) 20/30 (66.7)
P-values
Treatment 0.16 0.42 0.01 0.09 0.76

a5 Both treatments received MGA for 14 d. MGA-PG heifers received PG 19 (12.5 mg) and 20 (12.5 mg) d after MGA withdrawal.
The MGA-G-PG heifers received GnRH (100 Gig) 3 d after MGA withdrawal with PG 7 (12.5 mg) and 8 (12.5 mg) d after GnRH.
Estrus was detected for 3 d, and heifers exhibiting estrus were AI 8-12 h later. Heifers not displaying estrus were timed-AI 72-80 h
and received GnRH.
b Percentage of heifers displaying estrus 3 d after PG of the total treated.
Percentage of heifers that were pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.
f Percentage of heifers pregnant during the first 30 d of the synchronized breeding of the total treated.










Table 4-7. Estrous, conception, timed-AI, pregnancy rates by treatment (TRT) and reproductive tract score (RTS) for Bos taunts x
Bos indicus heifers synchronized with combinations of melengestrol acetate (MGA), GnRH (G), and prostaglandin Fza
(PG) at Location 2 (Experiment 2).a

Estrous Conception Timed-AI Synchronized 30 d
response rate pregnancy rate pregnancy rate pregnancy rate
RTS TRT (%)b oc) od oe of
3 MGA-PG 15/26 = 57.7 10/15 = 66.7 0/11 = 0.0 10/26 = 38.5 17/26 = 65.4
MGA-G-PG 9/21 = 42.9 3/9 = 33.3 3/12 = 25.0 6/21 = 28.6 13/21 = 61.9

4 MGA-PG 12/19 = 63.2 8/12 = 66.7 1/7 = 14.3 9/19 = 47.4 14/19 = 73.7
MGA-G-PG 14/21 = 66.7 6/14 = 42.9 1/7 = 14.3 7/21 = 33.3 19/21 = 90.5

5 MGA-PG 5/11 = 45.5 4/5 = 80.0 3/6 = 50.0 7/11 = 63.6 9/11 = 81.8
MGA-G-PG 9/12 = 75.0 4/9 = 44.4 1/3 = 33.3 5/12 = 41.7 7/12 = 58.3

Total MGA-PG 32/56 = 57.1 22/32 = 68.8 4/24 = 16.7 26/56 = 46.4 40/56 = 71.4
MGA-G-PG 32/54 = 59.3 13/32 = 40.6 5/22 = 22.7 18/54 = 33.3 39/54 = 72.2


P-valites TRT 0.51 0.02 0.27 0.12 0.95
RTS 0.37 0.75 0.11 0.31 0.11
TRT x RTS 0.20 0.90 0.15 0.91 0.17

a Both treatments received MGA for 14 d. MGA-PG heifers received PG 19 (12.5 mg) and 20(12.5 mg) d after MGA withdrawal.
The MGA-G-PG heifers received GnRH (100 Gig) 3 d after MGA withdrawal with PG 7 (12.5 mg) and 8 (12.5 mg) d after GnRH.
Estrus was detected for 3 d, and heifers exhibiting estrus were AI 8-12 h later. Heifers not displaying estrus were timed-AI 72-80 h
and received GnRH.
b Percentage of heifers displaying estrus 3 d after PG of the total treated.
" Percentage of heifers that were pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.
f Percentage of heifers pregnant during the first 30 d of the synchronized breeding of the total treated.









CHAPTER 5
SUMMARY

The primary obj ective of Experiment 1 (Chapter 3) was to evaluate follicular development

in yearling Angus and Brangus heifers during the 19 d period between MGA withdrawal and PG,

and to evaluate the follicle development and estrous response following PG. A primary

obj ectives of Experiments 2 and 3 (Chapter 4) were to determine the optimal timing to

implement GnRH during the period from MGA withdrawal to PG and to evaluate the subsequent

estrous response and fertility in Bos indicus x Bos taurus heifers.

In Experiment 1, yearling Angus and Brangus heifers were synchronized with the MGA-

PG system with PG was administered 19 after MGA withdrawal. During the period between

MGA withdrawal and PG, follicle development patterns were characterized by daily

ultrasonography and the follicle development patterns were different between Angus and

Brangus heifers. Factors contributing to the differences in follicle development were the

decreased number of Brangus heifers that exhibited estrus during the 7 d after MGA withdrawal

compared to Angus heifers and the increased incidence of three and four follicle wave patterns in

Brangus compared to Angus heifers. From days 9 to 13 after MGA withdrawal, the percentage

of follicle > 10 mm steadily increased to 100% by d 13 in Angus heifers but increased in

Brangus heifers up to d 10 but started to decrease to a low of 50% by d 13 in Brangus heifers.

Although follicle development between MGA withdrawal and PG was different between Angus

and Brangus heifers, diameter of the largest follicle at PG, estrous response, and interval from

PG to estrus were similar between Angus and Brangus heifers. Conversely, when the number of

follicle waves was evaluated, there was an interaction between breed and number of follicle

waves on the interval from PG to estrus. Angus heifers displaying three follicle waves had a

longer interval from PG to estrus compared to two wave Angus and Brangus and three wave









Brangus heifers. Therefore, the variation in follicle wave development between MGA

withdrawal and the PG administered 19 d later needs to be altered in order to improve the

synchrony of estrus following PG.

In a recent report by Wood and coworkers (2001), GnRH was included in the MGA-PG

estrous synchronization system 12 d after MGA withdrawal in Bos taunts heifers to synchronize

follicle development at the PG treatment. However, it does not appear that administering GnRH

12 d after MGA withdrawal would be as effective in Brangus heifers due to a decreased

percentage of Brangus heifers with large growing follicles capable of ovulating to GnRH 12 d

after MGA withdrawal. It appears that introducing a GnRH treatment would need to occur

approximately 10 d after MGA withdrawal, when a high percentage of Brangus have large

growing follicles capable of ovulating to GnRH. Furthermore, it may actually be better to

administer GnRH soon after MGA (3 to 4 d) withdrawal where most heifers have large follicles

and variation in follicle development is minimal.

In Experiment 2, cycling Bos indicus x Bos taunts heifers that were on day two of the

estrous cycle were administered GnRH either 3 (G3) or 10 (G10) d after the last day of a 14 d

MGA treatment followed by PG 7 d after GnRH. In addition, three groups of heifers received

two consecutive PG treatments at predetermined days during MGA to imitate heifers starting

MGA at different stages of the estrous cycle (SOC). The four stages included d 2, 6, 10, and 14.

Administering GnRH three days after the last day of MGA withdrawal was more effective in

initiating ovulation compared to d 10 after MGA withdrawal. The decreased ovulation rate to

GnRH in G10 likely resulted in asynchronous follicle development at PG, which was partially

due to the poor estrous during the five days after MGA withdrawal. Furthermore, the GnRH

treatment was not very effective at inducing ovulation in long-term persistent follicles (d 14










SOC) regardless of treatment, which demonstrated the negative effects persistent follicles have

on an estrous synchronization system. The synchrony of estrus following PG was substantially

improved for G3 compared to G10 as evidence by the greater 72 h estrus response of the G3

treated heifers.

Because administering GnRH 3 d following MGA withdrawal provided the greatest

synchrony of estrus when PG was administered 7 d after GnRH, it was decided to evaluate the

fertility of the G3 treatment in a field trial in Experiment 3. Experiment 3 was conducted to

evaluate the MGA-G-PG (G3) system compared to the traditional MGA-PG system in Angus

and Bos indicus x Bos taurus heifers. The only exception was heifers received estrus detection

and AI for 72 h, at which time all non-responders were timed-AI and received GnRH. Unlike

Experiment 2, the MGA-G-PG treatment failed to increase the percentage of Bos indicus x Bos

taurus heifers in estrus within 72 h after PG. Conception, timed-AI pregnancy, and

synchronized pregnancy rates were similar for MGA-G-PG and MGA-PG treatments.










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BIOGRAPHICAL SKETCH

Steaven A. Woodall, Jr. was born in Tampa, Florida on December 02, 1978 to Steve and

Paula Woodall of Plant City, Florida. Steaven has one sibling, Priscilla, and he is the oldest of

the two children. Steaven attended several schools during his childhood and graduated from

Durant Senior High School where he was an active member of the Durant FFA. Throughout his

high school years, Steaven was a member of the Florida Junior Limousin Association and was

active in showing cattle throughout the Southeast. After high school, Steaven was employed by

Sun State International Trucks while attending Hillsborough Community College on a Florida

Bright Futures Scholarship, where he received his A.A. degree. In August 2002, Steaven

enrolled at the University of Florida to pursue his B.S. At the University of Florida, Steaven

joined the Alpha Gamma Rho fraternity, where he held the office of Vice Noble Ruler of

Management and Operations, and served as kitchen manager. Steaven graduated in August 2004

and accepted a graduate assistant position, starting the next semester, in the Department of

Animal Sciences at the University of Florida under the direction of Dr. Joel Yelich. In addition

to his own research duties, Steaven had the opportunity to conduct a laboratory section of the

Reproductive Physiology course as well as assist research in many aspects of reproductive

physiology. Steaven's future plans are to pursue a career in the beef cattle industry focusing on

bovine reproduction.





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1 EFFECT OF LONG-TERM MELENGESTRO L ACETATE TREATMENTS ON FOLLICLE DYNAMICS AND RESPONSE TO GONAD OTROPIN-RELEASING HORMONE AND PROSTAGLANDIN F2 SYNCHRONIZATION TREATMENTS IN Bos indicus Bos taurus HEIFERS By STEAVEN A.WOODALL, JR. A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2007

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2 2007 Steaven A. Woodall, Jr.

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3 To my loving parents and sister. For their support, encouragement, and love.

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4 ACKNOWLEDGMENTS First, I would like to offer my appreciation Dr. Joel Yelich for the opportunity to continue my education and the support and knowledge he imparted on me. This experience will truly affect my life. I would also like to acknowledge the members of my supervisory committee, Drs. William Thatcher and Owen Rae, for their knowledge and contributions in fulfilling my degree. Sincere appreciation is extende d to my lab-mates, Brad Austin and Regina Esterman. Their willingness to help and put forth long hours to complete research projects, but most of their friendship has been invaluable. I would also lik e to extend my appreciation to the staff of the Santa Fe Beef Research Unit and the Beef Res earch Unit for the willingness to assist and the care given to the animals. Additionally, I would like to thank my fellow graduate students, most notably Jeremy Block, Reinaldo Cooke, and Drew Cott on for their willingness to help when needed. Most of all I would like to thank them for the laughs and the good times we shared that made my graduate experience enjoyable. Finally, I thank my parents for the life less ons and the support they have given me along the way. They have always encouraged me to pursue my dreams and have been there when I needed them. I am truly blessed to have them in my life.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 2 REVIEW OF LITERATURE.................................................................................................15 Endocrine Control of the Estrous Cycle.................................................................................15 Puberty........................................................................................................................ ............18 Ovarian Function............................................................................................................... .....22 Follicle Growth and Selection.........................................................................................22 Corpus Luteum (CL) Function and Luteolysis................................................................31 Bovine Estrous Cycle........................................................................................................... ..36 Estrous Synchronization through Mani pulation of the Estrous Cycle....................................41 Progestogens................................................................................................................... .41 Prostaglandin F2 .............................................................................................................44 Melengestrol acetate + PGF2 ..........................................................................................48 3 EVALUATION OF FOLLICULAR DEVELOPMENT BETWEEN A 14 D MELENGESTROL ACETATE (MGA) TREATMENT WITH PGF2 19 D AFTER MGA WITHDRAWAL IN ANG US AND BRANGUS HEIFERS.......................................55 Introduction................................................................................................................... ..........55 Materials and Methods.......................................................................................................... .56 Results........................................................................................................................ .............62 Discussion..................................................................................................................... ..........70 Implications................................................................................................................... .........79 4 REFINEMENT OF THE 14 D MELENGESTROL ACETATE (MGA) TREATMENT + PROSTAGLANDIN F2 (PG) 19 D LATER ESTROUS SYNCHRONIZATION SYSTEM IN HEIFERS OF Bos indicus Bos taurus BREEDING......................................89 Introduction................................................................................................................... ..........89 Materials and Methods.......................................................................................................... .90 Results........................................................................................................................ .............97 Experiment 1...................................................................................................................97 Experiment 2.................................................................................................................101

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6 Discussion..................................................................................................................... ........102 Implications................................................................................................................... .......114 5 SUMMARY........................................................................................................................ ..124 LIST OF REFERENCES.............................................................................................................127 BIOGRAPHICAL SKETCH.......................................................................................................146

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7 LIST OF TABLES Table page 2-1 Summary of studies evaluating the melengestrol acetate (MGA) + PGF2 estrous synchronization system in yearling beef heifers................................................................54 3-1 Age, body weight (BW), body condition scor e (BCS), and estrous cycling status (Cycling) at the initiation of the 14 d me lengestrol (MGA) treatment for Angus and Brangus heifers by ultrasound group (s can vs., non-scan) (LS means SE).a..................80 3-2 Estrous response, interval to estrus, duration of estrus, and number of mounts received during a HeatWatch detected estrus for the 7 d following a 14 d melengestrol (MGA) treatment..........................................................................................81 3-3 Percentage of heifers with a functional CL, progesterone concen tration (LSM SE), and diameter of the largest follicle (L SM SE) at the initial PG treatment.....................82 3-4 Effect of breed and cycling status at the initiation of a 14 d melengestrol acetate treatment on estrous response, conception ra te and synchronized pregnancy rates of Angus and Brangus heifers synchronized w ith a 14 d melengestrol acetate treatment.....83 4-1 The effect of stage of follicle (SOF) de velopment during a 14 d melengestrol acetate (MGA) treatment on progesterone concentra tion (LSM S.E.) at MGA withdrawal, diameter of the largest fo llicle at MGA withdrawal........................................................116 4-2 Effect of treatment (T) and stage of follicle (S) development on largest follicle diameter at GnRH (LSM SE), diameter of follicle ovulating to GnRH (LSM SE), and ovulation rate for heif ers receiving GnRH either 3 d (G3) or 10 d (G10)................117 4-3 Percentage of heifers with a func tional corpus luteum (CL), progesterone concentration (LSM S.E.), and diamet er of the largest dominant follicle at prostaglandin F2 (PG: LSM S.E.) for G3 and G10 heifers.........................................118 4-4 Three-day estrous response, total estrous response, and interval from prostaglandin F2 (PG) to onset of estrus following PG treatment for G3 and G10 heifers across different stages of follicle (SOF) development (Experiment 1).a....................................119 4-5 Estrous, conception and pregnancy rates of Bos taurus x Bos indicus heifers synchronized with combinations of mele ngestrol acetate (MGA) GnRH (G), and prostaglandin F2 (PG) at two locations (LOC) (Experiment 2).....................................121 4-6 Estrous, conception and pregnancy ra tes of Angus heifers in Location 1 synchronized with combinations of mele ngestrol acetate (MGA) GnRH (G), and prostaglandin F2 (PG) (Experiment 2)...........................................................................122

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8 4-7 Estrous, conception, timed-AI, pregnancy rates by treatment (TRT) and reproductive tract score (RTS) for Bos taurus Bos indicus heifers synchronized with combinations of melengestrol acetate (MGA), GnRH (G), and prostaglandin F2 .........123

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9 LIST OF FIGURES Figure page 3-1 Profiles of ovulatory follic les after a 14 d melengestrol acetate (MGA) treatment and the subsequent first wave dominant follicle growth profiles for A) Angus and B) Brangus heifers................................................................................................................ ..84 3-2 Mean first wave dominant follicle diameter during days 9 to 13 following withdrawal of a 14 d melenge strol acetate (MGA) treatment for Angus (n = 11) and Brangus (n = 10) heifers in the scan group........................................................................85 3-3 Mean diameter of the A) first, B) second, and C) third follicle wave following withdrawal of melengestrol acetate (MGA) fo r Angus and Brangus heifers. Follicle waves were normalized to the day of wave emergence.....................................................86 3-4 Diameter of the eventual ovulator y follicle prior to prostaglandin F2 (PG) treatment for Angus and Brangus heifers based on the number of follicle waves from the last day of a 14 d melengestrol acetate treatme nt to a PG treatment 19 days later..................87 3-5 Follicle growth patterns for the eventual ovulatory follicle preceding the initial prostaglandin F2 (PG) treatment, which occurred on day 19 (indicated by the arrow) in A) Angus and B) Brangus heifers......................................................................88 4-1 Estrous response, expressed as a percenta ge of the total number of heifers in a group, during the 7 d after the initial PG treatm ent for G3 (n = 25) and G10 (n = 23) treatments. NR = no estrous response (Experiment 1) ..................................................120

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10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECT OF LONG-TERM MELENGESTRO L ACETATE TREATMENTS ON FOLLICLE DYNAMICS AND RESPONSE TO GONAD OTROPIN-RELEASING HORMONE AND PROSTAGLANDIN F2 SYNCHRONIZATION TREATMENTS IN Bos indicus Bos taurus HEIFERS By Steaven A. Woodall, Jr. August 2007 Chair: Joel V. Yelich Major: Animal Sciences In experiment 1, yearling Angus (n = 40) and Brangus (n = 26) heifers received melengestrol acetate (MGA; 0.5 mg/hd/ d) for 14 d with prostaglandin F2 (PG) administered either 19 d or 19 and 20d after MGA withdraw al for Angus and Brangus, respectively. A subgroup of Angus (n=11) and Brangus (n=10) heifers had transr ectal ultrasonography conducted daily after MGA withdrawal until 7 d af ter PG to evaluate follicle development. There tended (P = 0.07) to be more Angus ( 100%; 11/11) compared to Brangus (80%; 8/10) heifers ovulating within 7 d af ter MGA withdrawal. Follicle wave patterns between MGA withdrawal and PG consisted of one (0/11; 1/10), two (9/11; 5/10), three (2 /11; 3/10) or four (0/11; 1/10) waves for Angus and Brangus, respec tively. The number of heifers with follicle 10 mm on 9 (54.5, 80.0 %), 10 (81.8 %, 70.0 %), a nd 11 d (90.9, 80.0%) after MGA were similar between Angus and Brangus re spectively; but greater ( P < 0.05) on 12 (100, 70.0 %) and 13 d (100, 50 %) for Angus compared to Brangus, respec tively. Because of the asynchrony of follicle wave patterns from MGA withdrawal to PG for Brangus compared to Angus, the best time to administer GnRH to synchronize follicle development in Brangus heifers may be immediately after MGA withdrawal. In Experiment 2 cyclin g Bos indicus x Bos taurus (BI BT) heifers

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11 were pre-synchronized to start a 14 d MGA (0.5 mg/ hd/d) treatment on d 2 of the estrous cycle. Heifers were randomly assigned to receive GnRH (100 g) either 3 (G3; n = 25) or 10 d (G10; n = 23) after MGA withdrawal with PG (12.5 mg) 7 and 8 d after GnRH. During MGA, heifers within each treatment received no PG or two consecutive PG treatments on d 4 and 5, 8 and 9, or 12 and 13, to simulate different periods of low-le vel progestogen exposure (SOF). Ovulation to GnRH was 76.0 and 47.8% for the G3 and G10, respectively. For G3 and G10 treatments, heifers in the d 14 SOF group did not respond as effectively as the other SOF groups. Following PG, more (P < 0.05) G3 (76%) heifers exhibited es trus during the first 72 h after PG compared to G10 (43.5%) heifers. In E xperiment 3, yearling BIBT (n=295) heifers at two locations were synchronized with two MGA + PG treatments. Tr eatment 1 was the same as in Experiment 1 (MGA-PG; n=174) while treatment 2 was the same as the G3 treatment in Experiment 2 (MGAG-P; n=178). Heifers were AI 8 to 12 h after an observed estrus. Heifers not detected in estrus by 72 h after PG were timed -AI concomitant wi th GnRH. Estrous response, conception, timedAI, and synchronized pregnancy rates were similar (P > 0.05) between MGA-PG (48.3, 54.9, 22.4, 38.1%) and MGA-G-PG (56.7, 52.4, 18.8, 37.8%), respectively. In summary follicle dynamics during the 19 d after a long term MGA treatment are different between Angus and Brangus heifers. Although, incorporation of a GnRH treatment 3 d after a 14 d MGA treatment effectively induced ovulation and resulted in a very synchronous estrus when PG was administered 7 d later, it did not improve the AI pregnancy rates compared to the MGA-PG estrous synchronization system.

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12 CHAPTER 1 INTRODUCTION Artificial insemination (AI) provi des producers with the opportuni ty to improve their herd through the use of superior genetics. Additi onally, a successful AI program benefits the producer economically by decreasing the number of bulls needed while potentially increasing the performance and uniformity of the calf crop. Ho wever, the implementation of a successful AI program requires significant labor, which offset th e economic benefits and limits the practicality of AI. Therefore, a major requirement of a successful AI program requires estrous synchronization systems that result in a large numbe r of cattle that can be AI in a short period of time. Numerous estrous synchronization systems have been developed to meet the needs of each production scenario. Products available for estr ous synchronization systems include progestins, prostaglandin F2 (PGF2 ), and gonadotropin-releasing hormone (GnRH). Progestins can be used to lengthen the estrous cycle by preventing the LH surge, estr us, and ovulation. Prostaglandin F2 acts to artificially shorten the estrous cycle by initiating luteolysis. Finally, GnRH can be administered to control follicle wave emergence or to initiate ovulation. Furthermore, these products can be combined to prevent estrus a nd ovulation, shorten the estrous cycle, and to control follicle development. The success of an estrous synchronization system is dependant on its ability to bring a high percentage (> 75%) of cattle into estr us in a short time period (< 7 d). Conversely, the effectiveness of these products in synchronizing estrus de pend on the genetics of the herd, body condition, reproductive status (i.e., estrous cycle vs anestrous), stage of the estrous cycle, environm ent, and breed-type ( Bos taurus vs. Bos indicus ). Breed is an important contributing factor in synchronization systems where most systems in use today have been designed for cattle of Bos taurus breeding. Therefore, these system s need to be evaluated or new

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13 systems need to be developed to account for the physiological and behavioral differences in cattle of Bos indicus breeding. Throughout Florida, the most co mmon form of cattle production is cow/calf operations. However, the subtropical environment of Florida presents cattle producers with a problem where elevated temperatures and decreased nutrient av ailability are not suitable for most breeds of cattle. Therefore, cattle normally fo und in Florida contain some degree of Bos indicus breeding. Cattle of Bos indicus breeding provide the Florida cattlemen many advantages in that they are adapted to the hot, humid environment, able to survive on low quality forages, and are more resistant to parasites than cattle of Bos taurus breeding. Conversely, se veral behavioral and physiological differences are observed in Bos indicus cattle, resulting in reduced reproductive performance and decreased effectiveness of co mmonly used estrous synchronization systems. In cow/calf operations, the greatest opportuni ty to implement an estrous synchronization system is in first service breedi ng of heifers. Heifers offer many benefits that make them best suited to for the implementation of an estrous sy nchronization system. Firs t, heifers are usually managed in groups supplemented to reach targ eted weights and condition scores. Second, heifers do not have the negative e ffects of lactation and suckling ca lf. Third, heifers are usually cycling prior to the breeding s eason. Finally, since heifers are managed in groups and do not have calves, they are easily ha ndled. Estrous synchronization a nd AI of heifers benefit the producer by reducing labor required for detecting es trus. Producers can choose to inseminate to calving-ease sires, therefore, reducing the num ber of calving-ease bulls needed for natural service. Moreover, an effective estrous s ynchronization system allows more heifers the opportunity to become pregnant early in the firs t 30 days of the breedi ng season. More heifers being exposed early in the breed ing season results in more heif ers calving earl y, reducing labor

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14 required during calving season. Al so, time of first calving affect s lifetime performance of the cow, where cattle calving as two-year olds wi ll have a greater lifetime production than those calving at a later date. One of the most common estrous synchronizati on systems for heifers utilizes a long term (14 d) melengestrol acetate (MGA) treatment and PGF2 administered 19 d after MGA withdrawal. This estrous synchr onization system was developed in Bos taurus heifers and results in excellent AI pregnancy rates. Conversely, this system is less effective in heifers of Bos indicus breeding. Recent research has increased the effectiveness of this system in Bos indicus heifers by altering the delivery of PGF2 but it does still not re sult in AI pregnancy rates observed in Bos taurus heifers. Therefore, this review will focus on the physiological and behavioral characteristics of re productive function in cattle of Bos indicus breeding and to review the estrous synchronization literature in an attempt to identify why there is a reduced reproductive performance to estrous s ynchronization systems in cattle of Bos indicus breeding.

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15 CHAPTER 2 REVIEW OF LITERATURE Endocrine Control of the Estrous Cycle Regulation of mammalian reprodu ction is primarily controlled at the level of the hypothalamus and pituitary. The main hypotha lamic hormone involved in regulating the hypothalamic-pituitary-gonadal axis and reproduction is gona dotropin-releasinghormone (GnRH). Gonadotropin-releasing-hormone, a decap eptide consisting of ten amino acids, is released from the hypothalamus and signals the release of the two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the anterior pituitary (Schally et al., 1971). These pituitary derived gon adotropins act on ovarian cells to signal changes in ovarian function and secretion of hormones. Furthermore, either positive or negati ve feedback of steroid hormones on the hypothalamus acts to regulate the release of GnRH and gonadotropins. Neurons responsible for the secretion of GnRH are loosely dispersed throughout the hypothalamus and GnRH is secreted from two di stinct areas of the hypothalamus in either a tonic fashion or as a surge. Tonic secre tion, as observed during th e luteal phase of the estrous cycle, is characterized by high amp litude, low frequency pulses under the negative feedback effect of progesterone and it is driven by neurons in the ventromedial and arcuate nuclei. Whereas, the surge-like secretion of Gn RH, as observed during estrus and driven by the positive feedback of estradiol secretion, is responsi ble for the LH surge and it is controlled by the preoptic and suprachiasmatic nuclei (Smith and Jennes, 2001). The GnRH secreted from the hypothalamus is released from the median eminence where it enters the hypothalamohypophyseal portal system through fenestrations in the capillary walls to be carried to the anterior pituitary. At the anterior pituitar y, GnRH acts through a seven transmembrane, Gprotein coupled receptor, which stimulates the release of gonadot ropins (Kakar et al., 1993).

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16 Luteinizing hormone is necessary for the de velopment of many ovarian events such as corpus luteum (CL) development (Snook et al., 1969), secretion of the gonadal steroid progesterone (Alila et al., 1988) follicle maturation (Ginther et al., 2001), and ovulation (Wettemann et al., 1972; Fortune, 1994). Pulses of GnRH stimulate the release of LH in a pulsatile fashion (Schams et al., 1974) where pulses are characterized by a rapid increase followed by a gradual decline in LH concentrations (Forrest et al ., 1980). Anderson et al. (1981) reported 3.4 pulses of LH over an 8 h period in pre pubertal beef calves. However, LH secretory patterns are dependent upon the stag e of the estrous cycle. During the early luteal phase, LH secretion is characterized by high frequency, low amplitude pulses; whereas during the mid luteal phase LH secretion is characterized by hi gh amplitude, low frequency pulses (Rahe et al., 1980). Walters et al. (1984) observed that pu lses of estradiol are observed within 60 min following pulses of LH, with greate r estradiol pulses during the early luteal compared to the mid luteal phase. Furthermore, estradiol enhances th e release of LH from th e anterior pituitary (Cupp et al., 1995) by increasing GnRH receptors in the pituitary (Gregg et al., 1990). Follicle-stimulating hormone, as its name implie s, functions to stimulate the recruitment and growth of a new follicle wave (Sunderland et al., 1994; Evans et al., 1997). Follicle development remains dependent on FSH until folli cle deviation (Ginther et al., 2000a); and stimulation of thecal estrogen production requi res FSH beyond this point (Mihm et al., 1997). Following hourly infusion of exogenous GnRH, c oncentrations of LH increased, however no increases in the concentration of FSH were observed (Vizcarra et al., 1997) indicating that FSH secretion is not controlled excl usively by GnRH. Furthermore, C upp et al. (1995) reported that concentrations of FSH were greater in ovariec tomized and ovariectomized + estradiol treated cows than in intact controls, demonstrating that the regulation of FSH secretion could be

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17 controlled by the ovaries. Unlike LH, repeated tr eatment of GnRH did no t result in the reduction of FSH secretion (Schams et al., 1974). Progesterone, secreted by the corpus luteum (CL), and estradiol, secreted by the dominant follicle, feedback onto the hypothalamus to regulate the secretion of gonadotropins. Bergfeld et al. (1995) reported that cows with high progesterone concentrations had fewer LH pulses as well as lower concentrations of estradiol; whereas, co ws with low progesterone concentrations had a greater frequency of LH pulses. During the mid luteal phase, when proge sterone concentrations are at peak concentrations, LH pulses are high amplitude and low frequency (6-8 pulses/24 h; Rahe et al., 1980). However, high estradiol c oncentrations, as observed during the follicular phase, lead to increased LH pulse frequency (S tumpf et al., 1993). Conversely, progesterone and estradiol act to regulate FSH secretion differently compared to LH. Ireland and Roche (1982) and Price and Webb (1988) reporte d no significant effect of pr ogesterone on FSH secretion. However, treatment of intact (Ireland and Roche, 1982) and ovariectomized (Price and Webb, 1988) heifers with estradiol significantly decreas ed FSH secretion. Following follicle ablation, FSH concentrations were greater in heifers treated with 0 mg estrad iol than those treated with 0.5 mg estradiol (Ginther et al., 2000b). Reproductive function is si milar between cattle of Bos taurus and Bos indicus breeding, however differences have been noted in the secr etory patterns of repr oductive hormones between the breeds. Both Bos taurus and Bos indicus cattle exhibit a pulsatile secretion of LH, but a greater number of LH peaks (3.33 vs. 3.00), magnit ude of peaks (overall LH peak height; 10.45 vs. 7.85 ng/mL) and LH pulse heights (the highest LH value minus the lowest LH value; 6.50 vs. 4.28 ng/mL) are observed in Bos taurus compared to Bos indicus cows, respectively (Griffen and Randel, 1978). Griffen and Randel, (1978) observed that ovariectomized Hereford ( Bos taurus )

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18 and Brahman ( Bos indicus ) cows responded to exogenous GnRH with increased concentrations of LH, but the increases in LH released were si gnificantly less in Brahman cows. In response to exogenous estradiol, Brahman heif ers have decreased LH secretion compared to Hereford and Brahman Hereford heifers (Randel, 1976). In addition, Brahman cows were less responsive and Brahman Hereford cows tended to be less responsive compared to Hereford cows treated with exogenous estradiol as determined by subs equent LH secretion (Rhodes and Randel 1978). Furthermore, the interval from estradiol treatment to LH response was longer in Brahman compared to Hereford and Brahman Hereford heifers. These results are supported by Rhodes et al. (1978) who reported that Br ahman cows secrete less LH in response to exogenous estradiol and take longer to respond compared to Herefo rd and Brahman Hereford cows. Therefore, decreased secretion of LH in response to estradiol in Bos indicus cattle may be due to a decreased sensitivity of the hypothalamus to th e positive feedback effects of estradiol. Puberty Throughout fetal development, the female re productive tract forms and the ovaries are populated with gametes. Shortly after birth, ovari an function begins with follicular growth and development followed by steroid production, but th e female does not ovulate. Puberty is defined as the time when the female first expresses es trus and ovulates. During the peripubertal period, the secretion of gonadotropins and the feedback effects of steroids on the hypothalamus change prior to and after the first ovula tion. Factors such as body weight gains from weaning to puberty (Plasse et al., 1968) and age (Nel sen et al., 1985) have been show n to play major roles in the timing of the onset of puberty. At approximately 3 5 months-of-age, the hypothalamic-pituitary axis of the heifer becomes functional, as LH secretion can be regulated by the actions of estradiol on the hypothalamus (Staigmiller et al., 1979). Furthermor e, Barnes et al. (1980) reported that heifers

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19 (approximately 3 to 9 months-of-age) were capab le of releasing LH in response to exogenous GnRH, but not in sufficient quantities to cause an increase in follicle development and high enough estradiol production to stimulate an LH su rge. Gonzalez-Padilla et al. (1975) reported that pituitary and hypothalamic hormones were rele ased in bursts, with LH bursts being of high amplitude and low frequency in 14.5 mo old prepube rtal Angus heifers. As the onset of puberty approaches, circulating concentrations of LH st eadily increase (Swanson et al., 1972; Day et al., 1984) and the continual increase in LH secret ion becomes the primary endocrine factor regulating the onset of puberty (Kin der et al., 1995). Day et al. ( 1984) reported that increases in pulsatile secretion of LH duri ng the peripubertal period was due to a decrease in the negative feedback effects of estradiol a nd the decline in sensitivity of the hypothalamus to estradiol was due to a decrease in the concentration in estr adiol receptors in the hypothalamus and anterior pituitary (Day et al., 1987). Removal of the ne gative effects of estradiol by ovariectomy results in an acute increase in LH concentrations (Day et al., 1984; Anderson et al., 1985). Immediately following ovariectomy, increased ci rculating LH concentrations we re due to an increase in LH pulse frequency; whereas, later increases in circ ulating LH concentrations were associated with an increase in LH pulse amplitude (Anders on et al., 1985). Treatme nt of prepubertal, ovariectomized heifers with exoge nous estradiol decreased circul ating LH concentrations and termination of the episodic release of LH (S chillo et al., 1982), which was dependent on the amount of estradiol administered. However, the suppression of LH secretion, by estradiol, decreases, as the heifer gets ol der (Schillo et al., 1982). Gonzalez -Padilla et al. (1975) reported a priming peak of LH approximately -11 to -9 d prior to the pubertal LH peak and the priming peak was associated with a slight increase in progesterone concentrations. Berardinelli et al. (1979) subsequently reported that a non-palpab le CL accompanied the prepubertal increase in

PAGE 20

20 progesterone concentrations and ovariectomy resu lted in decreased proges terone concentrations, indicating that the increase in pr ogesterone concentrations probably originated from the ovaries. The authors suggested that the priming peak of LH served as a transition from preto postpubertal LH concentrations a nd progesterone exposure played a key role in the establishment of puberty. Consequently, the heifer becomes less responsive to the negative feedback effects of estradiol as she matures, resulting in increase d LH pulse frequency, which ultimately reaches a threshold to initiate estrus and ovulation followed by secretion of luteal pr ogesterone resulting in attainment of puberty. Thus a short estrous cycl e, accompanied by an increase in progesterone concentrations, is followed by the first ovulation, and progesterone concen trations increase to concentrations 1 ng/mL, resulting from the newly formed CL. At this point the heifer will continue with regular estrous cycles and ovulations (Schillo et al., 1992). In addition to maturation of the endocrine system as the female approaches puberty, the reproductive tissues including the ovaries and ut erus also undergo maturational changes. Following birth, diameters of ovarian follicles increase from 2 to 34 wk of age, with the greatest increases occurring between 2 to 8 wk of age (Evans et al., 1994). Day et al. (1987) reported that as puberty approached, there was no change in ov arian weight or in th e numbers of small (<3 mm), medium (3 to 6 mm), or large follicles (7 to 12 mm) but there was an increase in the numbers of follicles >12 mm. However, a foll icle >12 mm was only observed in heifers that were close to reaching puberty. Growth and de velopment of follicles occurs in a wave-like fashion in prepubertal heifers (Adams et al., 1994) similar to postpubert al heifers (Sirois and Fortune, 1988). Uterine weight al so increases as the heifer ne ars puberty with the most rapid increase in the 50 d preceding puberty (Day et al., 1987). The increase in ut erine weight is likely

PAGE 21

21 due to increased estradiol secretion from the ova ries, which is associat ed with the onset of puberty (Day et al., 1987). Breed plays a major role in the age at which puberty is a ttained in cattle. Bos indicus and Bos indicus Bos taurus cattle reach puberty at older ages and heavier weight s than cattle of Bos taurus breeding (Reynolds et al., 1963; Plasse et al., 1968; Gregor y et al., 1979; Baker et al., 1989; Rodrigues et al., 2002). The range in ag e at puberty is approximately 14 to 24 mo for Bos indicus and 15 to 20 mo for Bos indicus Bos taurus crossbred heifers (Plasse et al., 1968), and 9 to 15 mo for Bos taurus (Wiltbank et al., 1966). Baker et al. (1989) reported that Jersey (255 d) and Holstein (282 d) dairy heifers reached puberty at younger ages compared to Angus (418 d) and Hereford (466 d) heifers, whereas, Brahma n heifers were the oldest at puberty (537 d). Conversely, crossbred Angus Brahman (442 d) and Hereford Brahman (472 d) reached puberty at a younger age than Brahma n heifers. In support of the crossbred data, Gregory et al. (1979) noted that Pinz gaur crossed with Bos taurus heifers attained puberty at 303 d while Brahman crossed with Bos taurus heifers attained puberty at 398 d. The late attainment of puberty of Bos indicus heifers is also reflected in the 9% pubertal by 22 months of age in Bos indicus compared to 62% of Hereford heifers (Hearnshaw et al. 1994) In contrast, 82% of the Brahman x Hereford heifers reached puberty by 22 mo emphasizing the importance of cross breeding on decreasing age of puberty in Bos indicus based cattle, where reproductive traits are enhanced through heterosis. Rodrigue s et al. (2002) reported that both Bos indicus and Bos taurus heifers underwent a cessation of the negati ve feedback effects of estradiol on LH secretion but, Bos taurus heifer s undergo this cessation at a younger age. However, the extent of the negative feedback effect of estradiol on LH secret ion was not amplified in Bos indicus heifers.

PAGE 22

22 Ovarian Function Follicle Growth and Selection All of the oogonia a female has available dur ing her lifetime are developed during fetal development where primordial germ cells migrate from the margin of the hindgut to the paired somatic gonadal primordia where they beco me oogonium (McGee and Hsueh, 2000). Oogonia undergo mitosis and the first stages of meiosis be fore being arrested at prophase of meiosis-1 (Wartenberg et al., 2001; McGee and Hsueh, 2000) After the attainment of puberty, the preovulatory surge of LH initiates resumption of meiosis and maturation of the oogonia (Hyttel, et al., 1997). The first stage of follicle growth involves a change in shape and increased numbers of granulosa cells; whereas, the sec ond stage of development is asso ciated with an increase in oocyte diameter and granulosa cell numbers (Braw-Tal, 2002). In the activated primordial follicle, an assortment of 5 to 14 flattened and cuboidal granulosa cells form a single layer surrounding the oocyte (Fair et al., 1997a). As development progresses to the primary follicle stage, a single layer of 8 to 20 cuboidal granulos a cells encompass the oocyt e and the first stages of zona pellucida formation are observed (Fai r et al., 1997a; Braw-Tal and Yossefi, 1997). At this stage, granulosa cells begin to secrete follistatin, which acts to block the effects of the growth inhibitor activin A (BrawTal, 1994). Also, the oocyte secr etes factors such as growth differentiation factor-9 (GDF-9) and bone morp hogenic protein-15 (BMP-15), both of which play roles in granulosa cell pr oliferation (McGrath et al., 199 5; Dube et al., 1998; Braw-Tal, 2002), whereas oocyte growth is promoted by granul osa secretions such as kit ligand (Braw-Tal, 2002). As the follicle progresses to the secondary follicle stage, the oocyte is surrounded by a partial or complete bilayer of granulosa cells and oocyte transcri ption is enabled (Fair et al., 1997b). Transcriptional activity of oocytes rema ins inactive until stimulated by FSH, at which

PAGE 23

23 time primordial follicles are activated and RNA synthesis is increased (Fair et al., 1997b). Advancing from the secondary to tertiary st age of development is characterized by the completion of the zona pellucida as well as the formation of a multi-layered granulosa cell population and a small antral cavity (Fair et al., 1 997b). In addition, granulosa cells differentiate to form cumulus granulosa cells and mural gr anulosa cells. Cumulus granulosa cells surround and are in close contact with th e oocyte, while mural granulosa cells line the follicle wall and come into contact with the basal lamina (Gilchrist et al., 2004). At the tertiary follicle stage, increasing amounts of follicular fluid collect in the antral cavity and the follicle achieves ovulatory capacity. In order for the graafian follicle to reach ovulatory status, it must undergo three distinct periods of development. The first period is recr uitment, where a cohort of follicles is stimulated to grow under the influence of FSH. The second period is selection, the process of one follicle continuing to grow while the others become at retic. And the third period is dominance, where one follicle continues to grow while suppressing the growth of its subordinates (Sirois and Fortune, 1988; Fortune, 1994; Gint her et al., 2001). Beginning on approximately day 1 to 2 of the estrous cycle, a pool of 5 to 10 follicles < 4 mm in diameter, are recruited in response to a surge in FSH (Sirois and Fortune, 1988; Driancour t, 2001; Sunderland et al ., 1994; Evans et al., 1997). The recruited follicles grow beyond a stag e that usually results in atresia for other follicles (Fortune, 1994). Ginther et al. (1997) reported that the fu ture dominant follicle emerges 6 to 7 h earlier than its subordinates, providi ng a size advantage for the future dominant follicle over the other emerging follicles (Kulick et al., 1999). At this point, the future dominant follicle and subordinate follicles enter a common growth phase until the beginning of deviation (Ginther et al., 1997; Kulick et al., 1999). Deviation is the continued gr owth of one follicle with a

PAGE 24

24 cessation of growth and regression (termed atresi a) of other ovarian fo llicles (Kulick et al., 1999). After the initial surge in FSH, FSH concentrations declin e with the simultaneous growth of follicles from 4 to 8.5 mm in diameter (Gin ther et al., 1997; Ginther et al., 1999). Gibbons et al. (1999) observed that 3 mm follicles did not have any detectable capacity to suppress FSH secretion, while follicles reaching 5 mm gain the capacity to suppress FSH secretions. Conversely, growth beyond 5mm in diameter did not result in an increase in FSH suppressing capacity. The first follicle to reach 8.5 mm beco mes the dominant follicle (Ginther et al., 1999; Kulick et al. 1999), which is coincident with a decrease in circulating FSH concentrations (Adams et al., 1993; Kulick et al., 1999) and incr eases in circulating LH concentrations (Kulick et al., 1999). After follicle deviation, circulating estradiol concentrations increase (Kulick et al., 1999) while follicles not selected for dominance become atretic. The ability of one of the r ecruited follicles to continue growing while others undergo atresia is still an area of questi on. A major characteristic of the future dominant follicle is its ability to secrete greater amount s of estrogen (Badinga et al., 1992) around day 5 of the estrous cycle. This obseravtion supports early work of Ireland and Roche (1983) who reported that estrogen-active follicles had a lower incidence of atresia than estrogen-inactive follicles. Compared to subordinate follicles, dominant fo llicles contain lower amounts of insulin-like binding protein (IGFBP)-2 (Stewart et al., 1996), IGFBP-4, and follistatin (Austin et al., 2001), which support the continued growth of the dominant follicle by ma intaining the av ailability of IGF-1 and activin-A. This is supported by Mihm et al. (2000) who report ed that in a pool of recruited follicles, the future dominant follicle ha d the highest concentrations of estradiol and the lowest concentrations of IGFBP-4. Ireland and Roche (1983) observed that granulosa cells of the selected follicle have a grea ter ability to bind hCG compared to non-selected follicles on days

PAGE 25

25 5 and 7 of the estrous cycle. The selected foll icle could also bind more hCG on day 7 compared to day 3. Xu et al. (1995) re ported that mRNA for LH receptors was present in day 4 follicles compared to day 2 follicles. These findings suggest that a follicles ability to achieve estrogenic activity is crucial for follicle selection. Ginther et al. (2001) observed that suppression of LH secretion did not affect the largest follicle prio r to deviation but reduced follicle diameter and follicular fluid concentrations of IGF-1 and es tradiol concentrations following deviation. The findings of Gong et al. (1995) suppo rt this by showing that suppre ssion of LH secretion to basal concentrations and the abolishment of the pulsat ile secretion of LH i nhibited follicle growth beyond 7-9 mm. Furthermore, LH-receptor mRNA was only found in healthy dominant follicles > 9 mm (Xu et al., 1995). These findings suggest that there is a divergence from dependency from FSH to LH, but not until after deviation. Th erefore, LH plays a majo r role in the growth and function of the dominant follicle following deviation. In order for one follicle to establish and main tain dominance over its subordinates, it must suppress FSH secretion to prevent recruitment of smaller follicles. Administering recombinant bovine FSH to heifers before se lection of the dominant follicle delayed the time for divergence between dominant and subordinate follicles (Adams et al., 1993). Furthermore, cauterization of the dominant follicle resulted in a surge of FSH and recruitment of a new pool of growing follicles soon after ablation (Adams et al., 1992). Treatment of animals with estradiol when the largest follicle reached 6 mm, around the time that endogenous FSH concentrations are normally declining, resulted in the suppressi on of FSH secretion and follicle diameter within 8 h (Ginther et al., 2000a). Ginther et al. (2000b) also reported that exogenous estradiol given to cattle after dominant follicle ablation caused a 2 to 3 h delay in the FSH surge. Nett et al. (2002) suggested that estradiol suppressed FSH secreti on by altering the production of activin B in pituitary cells.

PAGE 26

26 Bleach et al. (2001) reported th at as FSH concentrations decl ine, estradiol and inhibin A concentrations increase coincident with the growth of a new dominant follicle. Inhibin originates from granulosa cells and functions to suppress se cretion and release of FSH from the anterior pituitary (Good et al., 1995). Sheep immunized against inhibi n showed an increase in FSH concentration as well as ovulation rate (Wheat on et al., 1992). Treatment of cattle with antiserum for inhibin and estradiol resulted in increased circulating FSH concentrations for a longer period of time than giving antiserum for inhibin alone, suggesting a synergistic role of suppressing FSH by inhibin and estradiol (Kaneko et al., 1995). Suppressing the synthesis and secretion of FSH with estr adiol and inhibin resulted in atresia of subordinate follicles due to their inability to utilize low concentr ations of circulating FSH, which is an environment that the dominant follicle can survive in (Ginth er et al., 2000b; Au stin et al., 2001). Following the establishment of dominance, fol licles must achieve ovulatory competence in order to respond to a pre-ovulatory surge of LH. The dominant fo llicle becomes more responsive to LH and gains ovulatory capacity when it reaches approximately 10 mm in diameter (Sartori et al., 2001), coincident with LH recep tor mRNA in granulosa cells of follicles > 9 mm (Xu et al., 1995). Once the dominant follicle ac hieves ovulatory competence, it can either ovulate or become atretic, depending on the stage of the estrous cycle. For the dominant follicle to ovulate, luteolysis must occur followed by a decline in progesterone secretion followed by subsequent increases in estradio l secretion, which drives the pre ovulatory surge of LH resulting in ovulation (Wettemann et al., 1972; Fortune, 19 94). When luteolysis does not occur, progesterone concentrations remain elevated, wh ich suppress LH pulses resulting in decreased estradiol secretion (For tune, 1994; Badinga et al. 1992). In response to decreased estradiol secretion, the dominant follicle be comes atretic, thereby removing the negative feedback effect

PAGE 27

27 of ovarian progesterone, which allows for an incr ease in FSH concentrations and recruitment of a new follicle wave (Fortune, 1994). Follicle development in cattle occurs in a wave-like pattern, which allows for a steady supply of ovulatory follicles (Sir ois and Fortune, 1988). Each wave is characterized as having one large dominant follicle with ovulatory capacity and several smaller follicles termed subordinates (Sirois and Fortune, 1988). During an estrous cycle, the number of follicular waves varies between animals. Two and three-wave cycles are the most common although one, four, and five wave cycles have been observed (Siroi s and Fortune, 1988; Savio et al., 1988; Viana et al., 2000). Estrous cycle length is reflected in the number of wave s that occur during the estrous cycle. Estrous cycles with two follicle waves are approximately 20 d in duration; whereas, estrous cycles with three waves last from 21 to 23 d (Ginther et al., 1989; Viana et al., 2000; Sirois and Fortune, 1988; Savio et al., 1988). In cattle with two follicular waves, wave emergence is approximately days 2 and 11 of the estrous cycle for the first and second wave, resp ectively; whereas, cattle with three follicular waves, wave emergence is approximately days 2, 9, and 16 of the estrous cycle for the three waves, respectively (Sirois and Fortune, 1988). In two wave cycles, the first wave reaches a maximum diameter about day 6 with regression by day 10 while the second dominant follicle reaches a maximal diameter by day 19 (Savio et al., 1988). For three wave cycles, the first and second wave dominant follicles reach a maxi mum diameter on day 6 and 16, respectively, followed by regression, while the third wave dom inant follicle achieves maximal diameter on day 21 (Savio et al., 1988). Differences in the number of waves results in di fferent sizes and ages of dominant follicles in a wave. In cycling Holstein heifers exhibi ting two wave cycles, th e first-wave dominant

PAGE 28

28 follicle (17.1 mm) and ovulatory (1 6.5 mm) dominant follicle reached a similar average maximal diameter, while the duration betwee n emergence of waves was shorter for the first (9.7 d) than the ovulatory wave (10.4 d; Ginther et al., 1989). Conversely, Savi o et al. (1988) noted that the maximal diameter of the first wave dominant follicle (14.3 mm) was sm aller than the ovulatory dominant follicle (20.3 mm) in cy cling beef heifers over two cons ecutive estrous cycles. In cycling Holstein heifers exhibiting three follicle waves, Ginther et al (1989) observed that average follicle diameter was smaller fo r second (12.9 mm) and ovul atory (13.9 mm) wave dominant follicles compared to the first wa ve dominant follicle (16.0 mm). The duration between emergence of waves was similar for the first (9.0 d), second (7.2 d), and ovulatory (6.7 d) waves. Sirois and Fortune (1988) reported that in Holstein heifers displaying normal estrous cycles, the second wave dominant follicle (10.2 mm) had the smallest maximal diameter of the three follicles with no differences between the first (12.3 mm) and third (12.8 mm) wave follicles. Contrary to their findings, Savio et al. (1988) demonstrated that the dominant follicles of the first two waves were smaller than the ovulat ory follicle of the third wave. Townson et al. (2002) reported that cattle with two follicle waves had larger (17.2 vs. 16.0 mm) and older (6.7 vs. 5.2 d) ovulatory follicles that were less fertile than cattle w ith three waves, respectively. Furthermore, differences in the length of the lu teal phase between two and three wave estrous cycles were reported by Ginther et al. (1989) where luteal regr ession occurred on day 16 and 19, respectively. Also, the interval from emergen ce to ovulation was shorter in cows with three compared to two wave cycles, resulting in a shorter period of dominance for the third wave ovulatory follicle (Gin ther et al., 1989). The number of follicle waves within an estrous cycle has been shown to vary according to environmental conditions, nutritional management, a nd lactation status. H eat stress increased the

PAGE 29

29 proportion of three wave follicular cycles (Wilson et al., 1998), resulted in earlier regression of the first wave dominant follicle followed by earlier recruitment of the second wave in two wave estrous cycles (Wolfenson et al ., 1995), decreased the first wave dominant follicle diameter (Badinga et al., 1993), and resulted in earlier emergence of ovulat ory follicle and a longer period of dominance (Wolfenson et al., 1995). Nutri tional restriction reduced the growth rate and diameter of dominant follicles during an estrou s cycle in beef heifers (Mackey et al., 1999) as well as decreased dominant follicle diameter and pe rsistence of the first wave dominant follicle in Brahman heifers (Rhodes et al., 1995) In contrast, supplemented grazing Bos indicus Bos taurus heifers had more large follicles than nonsupplemented heifers (Maquivar et al., 2005) and feeding calcium salts of long chain fatty acids increased the diameter of the dominant follicle in multiparous Holstein cows (Lucy et al., 1991). Lactational status in da iry cows also effects follicle development, which appears to be driven by the level of nutrition as well as the resulting hormone profiles. Lactating dairy cows have d ecreased concentrations of glucose, IGF-1, and insulin, which is reflected in fewer class two (6-9 mm) and three (10-15 mm) follicles but more class four (> 15 mm) follicles that are less estr ogenic compared to non-lactating dairy cows (De La Sota et al., 1993) Characteristics of follicular growth are also different between Bos taurus and Bos indicus cattle. Early work by Segerson et al. (1984) before th e advent of ultrasonography, reported more follicles < 5 mm in Brahman cows while Angus cows had more follicles > 5 mm in diameter. Recent work using ultrasonography during an entire estrous cycle revealed that the numbers of small (2-5 mm), medium (6-8 mm), and large ( 9 mm) follicles were gr eater in non-lactating Brahman (39.0, 5.0, and 1.6) compared to Angus (21, 2.3, and 0.9) cows, respectively (Alvarez et al., 2000). Alvarez et al. ( 2000) also observed that Angus co ws had a greater FSH surge and

PAGE 30

30 circulating plasma FSH concentrations compar ed to Brahman cows indicating that Brahman cows produce more follicles even though they have a smaller FSH surge and lower FSH concentrations. Alvarez et al (2000) hypothesized that the great er follicle numbers may be due to higher concentrations of IGF1 in Brahman cows. This finding is supported by Simpson et al. (1994), who reported that Brahman cows had gr eater circulating IGF-1 concentrations and IGFBP compared to Angus cows. Alvarez et al. (2002) also indicated that Brahman cows had dominant follicles with a greater maximum diameter compared to Angus cows during the first (15.3 vs. 11.4 mm) and ovulatory (15.6 vs. 12.8 mm) follicle wave, respectively. Growth rate of the first wave dominant follicle tended to be greater in Brahman ( 1.6 mm/d) compared to Angus cows (1.2 mm/d), whereas growth rate was similar for the ovulat ory dominant follicle between Brahman (1.4 vs. 1.4 mm/d) and Angus (1.4 mm/d). Aside from th ese differences, length of the estrous cycle (19.5 vs. 19.7 d), number of two follicular wave cycles (72.7 vs. 55.6%) and three follicular wave cycles (27.3 vs. 44.4%) was similar be tween Angus and Brangus cows, respectively (Alvarez et al., 2000). Viana et al. (2000) reported maximal di ameters for first (11.8 mm) and ovulatory (12.4 mm) wave follicles in Gir ( Bos indicus ) cows, which were considerably less than the Brahman cows in the Alvarez et al. (2002) study. Other studies in Bos indicus cattle reported three follicular waves during the estrous cycl e approximately 66.7% (R hodes et al., 1995) and 60% (Viana et al., 2000) of the time as well as in cidences of four follicle waves approximately 7 to 27% of the estrous cycles (R hodes et al., 1995; Viana et al., 2000) Of interest, Figueiredo et al. (1997) reported that Nelore cows commonly have two follicle waves (83.3%), whereas Nelore heifers had a greater incidence of three follicle waves (64.7%).

PAGE 31

31 Corpus Luteum (CL) Function and Luteolysis After ovulation, the theca inte rna and granulosa cells of the ovulatory follicle undergo morphological and biochemical changes to become the CL. The main function of the CL is to synthesize and secrete progesterone, which is re quired for the maintenance of pregnancy and regulation of the estrous cycle. Corpora lutea are mainly compri sed of two cell types, large and small luteal cells. Alila and Hans el (1984) reported that small lute al cells of the early developing CL were primarily from thecal origin, whereas la rge luteal cells were primarily granulosa in origin. The small luteal cells eventually develop into large luteal cells with age as the original large luteal cells disappear (Al ila and Hansel, 1984). Small lut eal cells are highly responsive to LH and secrete progesterone under the influence of low LH secretion; wher eas, large luteal cells are less responsive to LH and secrete progesteron e under high LH secretion and are subjected to the luteolytic effects of PGF2 (Alila et al., 1988). Furthermore, large luteal cells secrete most of the progesterone (> 80%) but not under the infl uence of LH in cattle and sheep (Ursley and Leymarie, 1979; Fitz et al., 1982; respectively). Hoyer et al. ( 1984) observed that progesterone production in large luteal cells is independent of elevated intrac ellular cAMP levels, suggesting that large luteal cells are secr eting progesterone at a maximal rate lending them unresponsive to further stimulation. Binding of LH to its receptor on small luteal cells resu lts in the activation of the second messenger system. Upon activation of the second messenger adenyl cyclase, cyclic adenosine monophosphate (camp) is synthesized (Hoyer and Niswender, 1986), which activates protein kinase A and phosphorylate the enzymes n ecessary for steroidogenesis (Milvae et al., 1996). Prostaglandin F2 (PGF2 ) is widely known as the prim ary luteolytic agent in many species, including cattle (Rowson et al., 1972; Inskeep, 1973; Nan carrow et al., 1973). Early

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32 research demonstrated that hyste rectomy of ewes and heifers resu lted in maintenance of the CL (Wiltbank and Casida, 1956), suggesting that the luteolytic signal came from the uterus. Ligation of the uterine vein ipsilateral to the ova ry with the CL resulted in maintenance of the CL for an extended period (Inskeep and Butc her, 1966). Hixon and Hansel (1974) further reported that PGF2 acted on the ovaries through a countercu rrent exchange between the uterine vein and ovarian artery. Upon r eaching the ovary with the CL, PGF2 initiates the rapid decline in progesterone secretion resulting in elevated LH concentrations leading to estrus and ovulation (Stellflug et al., 1977). Further research reporte d that pulses of PGF2 were observed throughout the estrous cycle without the initiation of luteolysis; ho wever, during luteolysis, pulses of PGF2 were more frequent in their release (Zarco et al., 1988). During a spontaneous luteolysis in the cow, pulses of PGF2 secretion were observed along with pulse s of oxytocin (Vighio and Liptrap, 1986), suggesting a positive feedback loop between PGF2 and oxytocin (Milvae and Hansel, 1980; Schallenberger et al., 1984). La France and Goff (1985) demonstrated that an injection of 100 IU of oxytocin had no signi ficant effect on PGF2 production as measured by its metabolite (PGFM) on days 3 and 6 of the estrous cycle but when oxyt ocin was administered on days 17 to 19 of the estrous cycle there were increa sed concentrations of PGFM. Treatment with progesterone followed by estradiol increased the numbers of e ndometrial oxytocin rece ptors (Vallet et al., 1990) and when oxytocin was administered to these animals, concentrations of PGFM increased. It was further demonstrated that during the late stages of the estrous cycle, progesterone downregulates its own receptor in the uterine endom etrium, reducing its action and stimulating the action of estradiol (Robinson et al., 2001). Coincident with the down regulation of uterine progesterone receptors, uterine oxytocin receptors are increased due to increasing estradiol

PAGE 33

33 concentrations (Vallet et al., 1990). The importance of estradio l had been previously reported LaFrance and Goff, (1988) who demonstrated that PGFM concentrations following either a 14 or 21 d treatment with progesterone were significant ly greater in heifers treated with estradiol followed by exogenous oxytocin compared to tr eatments with just oxytocin. Increases in estradiol concentrations increased the frequency of the pulse generator, driving the release of sub-luteolytic levels of PGF2 from the uterus. Furthermore, PGF2 secreted from the uterus acts on the CL to stimulate the release of luteal oxy tocin, which amplifies the secretion of uterine PGF2 to luteolytic levels (McCracken et al ., 1999). The luteolytic levels of PGF2 activate the PGF2 receptor, located on both large and sma ll luteal cells, and reduce progesterone concentrations (McCracken et al., 1999). Oxytocin binding to its receptor in the uterus activates the inositol 1, 3, 4-triphospha te second messenger system, re sulting in the conversion of diacylglycerol to arachidonic acid (Flint et al., 1986) the precursor to PGF2 synthesis and eventual release of PGF2 Luteolysis is defined as the structural demise of the CL associated with reduced synthesis and secretion of progesterone, followed by a loss in luteal cells (Niswe nder et al., 2000). The process of luteolysis can be attribut ed to a variety of actions of PGF2 at the cellular level as well as changes in gene expression. There appears to be downregulation of re ceptors for luteotropic hormones, however decreases in LH receptors determined by hCG binding capacity, are not observed until after a fall in progesterone (Spice r et al., 1981). Also, there are changes in the transport of cholesterol into the cell. Following treatment with PGF2 there is a 50% decrease in steroidogenic acute regulatory pr otein (StAR; Pescador et al ., 1996), the transporter of cholesterol across the mitochondria l membrane. Finally, the activ ity of steroidogenic enzymes, such as 3 -hydroxysteroid dehydrogenase (3 -HSD), required for progesterone synthesis is

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34 decreased within within an hour of PGF2 treatment (Hawkins et al., 1993). In response to PGF2 changes in gene expression include the i nhibition of the LH receptor, StAR, and 3 -HSD genes, whereas genes regulating luteal cell gene trancription, such as cfos and prostaglandin G/H synthase-2 (PGHS-2), and genes involve d in recruiting monocyt es, macrophages, and monocyte chemoattractant protein-1 (MCP-1) are induced (Tsai et al., 2001). Furthermore, PGF2 stimulates the luteal secreti on of high amounts of the vasoc onstrictive agent, endothelial cell vasoconstrictive peptide endothelin-1 (ET-1) which inhibits luteal progesterone production (Girsh et al., 1996). All the aforementioned changes precede changes in th e cellular makeup of the CL. Braden et al. (1988) reported that by 36 hours after treatment with PGF2 large luteal cell numbers remained the same but their diameter decreased; whereas, the number of small luteal cells decreased by 24 hours after treatment. The luteolytic actions of exogenous PGF2 on the CL can occur as early as day 5 and as late as day 16 of the estrous cycle. Henricks et al. (1974) reported th at treatment with PGF2 on days 3 and 4 of the estrous cycle had no effect on plasma progesterone concentrations. However, the unresponsiveness of the early bovine CL does not appear to be due to the lack of receptors for PGF2 as PGF2 receptors appear as early as 2 d af ter ovulation while receptor numbers and affinity remaned the same through day 10 after ovulation (Wiltbank et al., 1995). Therefore, the refractory period of the ear ly developing CL to PGF2 is not due to the lack of receptors. Tsai and Wiltbank (1998) reported that circulating PGF2 reached the early developing CL to the same extent as the mid-cycle CL, but did not induce intraluteal PGF2 This could be due to the observation that the early CL has a greater capacity to catabolize PGF2 into PGFM due to increased enzymatic activity of 15-hydroxypros taglandin dehydrogenase (PGDH; Silva et al., 2000). Inhibitors of the second messenger system for PGF2 receptors are also increased during

PAGE 35

35 the early luteal phase of the estrous cycle (Jue ngel et al., 1998). Conversely, Rao et al. (1979) reported that specific binding of PGF2 to CL membrane increased from the early luteal phase (day 3 of estrous cycle) to the greatest levels observed during the late luteal phase (day 20 of estrous cycle), a time where th e CL was actively regressing. Mo reover, the authors reported an increased number of PGF2 receptors during the mid luteal phase (day 13 of estrous cycle) but the affinity of PGF2 to its receptor was 203 times less than during the late luteal phase. Additionally, Sakamoto et al. (1995) noted that mRNA for PGF2 receptors increased from the early luteal phase (days 3-5 of es trous cycle) to the late luteal phase (days 15-18 of estrous cycle) and was reduced for the regressed CL. Following the initiation of luteolysis, intra-luteal progesterone secretion began decreasing immediately while intra-luteal PGF2 slightly increased and dramatically increased from 24 hr to 300% (Shirasuna et al., 2004). Characteristics of luteal development and function appear to be different between Bos taurus and Bos indicus cattle but the data is conflicting. In general Bos indicus cattle have smaller CL than Bos taurus cattle regardless of whether st udies included removal of ovaries (Irvin et al., 1978; Segerson et al., 1984) or ev aluation via ultrasonography (Rhodes et al., 1995). Although Alvarez et al. (2000) re ported larger CL sizes in Bos indicus compared to Bos taurus cows as determined by ultrasonography. Sim ilarly, there are differences in progesterone production but these data are also conflicting. Segerson et al. (1984) re ported that luteal progesterone content and serum progester one concentrations were greater in Bos taurus compared to Bos indicus cows while Adeyemo and Heath (1980) observed that Bos taurus cows had greater concentrations of progesterone throughout the estrous cycle compared to Bos indicus cows. In contrast, Irvin et al. (1978) reported no differences in lu teal content or concentrations of progesterone between Bos taurus and Bos indicus cattle. Likewise, Alvarez et al. (2000)

PAGE 36

36 reported that Bos indicus cows had similar progesterone concentrations compared to Bos taurus cows even though the Bos indicus cows had greater CL sizes. Alvarez et al. (2000) suggested that the reason for increa sed luteal growth in Bos indicus cattle may be a result of increased concentrations of growth hormone or IGF-1. Although not well documented, there appears to be differences in the luteolytic response to PGF2 between Bos taurus and Bos indicus cattle. A single study in Bos indicus (Cornwell et al., 1985) heifers suggests a d ecreased response to PGF2 during the early luteal phase compared to early luteal phase in Bos taurus (Tanabe and Hann, 1984) heifers. In Brahman heifers that did not undergo luteolysis and exhibit estrus, progesterone concentrati ons initially declined by 12 hr after PGF2 but progesterone concentrations bega n to increase within 48 hr after PGF2 treatment (Cornwell et al., 1985). Santos et al. (1988) reported an incr eased estrous response following two consecutive 12.5 or 25 mg PGF2 treatments administered 24 hr apart in Brahman heifers and Brangus cows. Furthermore, Bridges et al (2005) noted that the percentage of heifers undergoing luteolysis was increased in yearling Bos indicus Bos taurus heifers following two consecutive 12.5 mg PGF2 treatments compared to a single 25 mg PGF2 treatments. In the same report, luteolysis was similar between yearling Bos taurus and 2 yr-old Bos indicus Bos taurus heifers that received either two consecutive 12.5 mg PGF2 treatments or a single 25 mg PGF2 treatments. Therefore, the rate of a PGF2 induced luteolysis a ppears to be different between Bos indicus and Bos taurus cattle, and there may well be an effect of age on luteolytic response in Bos indicus cattle. Bovine Estrous Cycle Estrous cycles in cattle star t with the expression of estr us followed by ovulation, growth and development of luteal tissues and follicles, luteolysis, and eventually the onset of estrus again. Associated with this sequence of even ts are coordinated exchanges in hormonal and

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37 ovarian events. There are two dis tinct phases that comprise the estr ous cycle: the follicular phase and the luteal phase. The follicular phase is th e period from luteolysis through ovulation and is further divided into proestrus and estrus. Th e luteal phase is the period from ovulation to luteolysis and is comprised of metestrus and diestrus. The length of the estrous cycle lengt h is approximately 20 to 22 d in Bos taurus (Sirois and Fortune, 1988; Ginther et al., 1989) and Bos indicus (Rhodes et al., 1995; Fi gueiredo et al., 1997) cattle. Likewise, Alvarez et al. (2000) observed similar estr ous cycle lengths between Angus (19.5 d) and Brahman (19.7 d) cows. In stark co ntrast, Plasse et al. (1970) reported a mean estrous cycle lengths of 28 d in two-year-old Brahman heifer s. Numerous studies have demonstrated that estrous cycle length is dict ated by the number of follicle waves during the cycle. In cattle with two wave follicle devel opment patterns, estrous cycle length was similar between Nelore cows and heifers (20.7 d; Figueir edo et al., 1997) compared to Holstein heifers (20.4 d; Ginther et al., 1989), whic h were significantly less than Ne lore cows and heifers (22.0 d) and Holstein heifers (22.8 d) with three wave follicle growth patterns. In contrast, Savio et al. (1988) reported similar estrous cycle lengths between Bos taurus beef heifers exhibiting either two(20.5 d) or three(21.3 d) wave follicle development patterns. The beginning of the estrous cycle is marked by estrus, where progesterone concentrations are low (0.33 ng/mL) and estradio l concentrations are increasing, which leads to the LH surge and ovulation (Wettemann et al., 1972). Interval from peak estradiol concentration to the preovulatory surge of LH is appr oximately 6 to 8 h (Walters et al., 1984; Cavalieri et al., 1997). High estradiol concentrations l ead to behavioral changes that are characterized by homosexual activity of females in estrus. The interval fr om estrus to ovulation has been shown to be

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38 approximately 28-32 hr in Bos taurus (Walker et al., 1996; Wettema nn et al., 1998) and 26 hr in Bos indicus cattle (Lamothe-Zavaleta et al., 1991; Pinheiro et al., 1998). Cattle of Bos indicus breeding are more difficult to detect in estrus (Galina et al., 1994) and exhibit more covert signs of estrus such as h ead butting and smelling of genitalia (Galina et al., 1982; Lamothe-Zavaleta et al., 1991). Bos indicus cattle also have an increased incidence of silent estrus (Plasse et al., 1970; Dawuda et al., 1989), which is one of the reasons why estrus is difficult to detect. The recent advent of radiotel emetric heat detection aids has also provided an insight into characteristics of behavioral estr us of cattle. Radiotelemetric heat detection significantly aids in the efficiency of estrous detection compared to visual observation (Stevenson et al., 1996), it provides a detailed record of the initiation of es trus (night vs. day), end of estrus, duration of estrus, and the inte nsity of estrus based on the number of mounts received. Several authors have a slightly greater percentage of Bos indicus cattle in estrus during the night time hours (Pinheiro et al., 1998; Landa eta-Hernandez et al., 2002) compared to Bos taurus cattle. Therefore, a greater number of Bos indicus cattle exhibiting estr us during the night time hours may impede the effectiveness of visual estrus detection methods and result in fewer animals being detected in estrus. The duration of estrus has also been reported to be effected by breed. Bos indicus cattle have a shorter duration of estrus (Rhodes and Randel, 1978; Lamothe-Zava leta et al., 1991; Rae et al., 1999) than Bos taurus cattle and this appears to be in fluenced as to whether it is a synchronized estrus or a spontaneous estrus (Landaeta-Hernandez et al., 2002). For a synchronized estrus, the duration of estr us has been reported to be 12 hr in Bos taurus heifers (Richardson et al., 2002) and 6-7 hr in Bos indicus heifers (Rae et al., 1999). LandaetaHernandez et al. (2002) also repo rted a similar duration of a sy nchronized estrus between Angus

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39 (19 h) and Brahman (17 h) cows. However, the duration of a subsequent spontaneous estrus was greater for Angus (11 h) compared to Brahman cows (6 h). The duration of estrus also appears to be co rrelated with the numb er of mounts received during estrus as animals with low mounting activit y have shorter durations of estrus (Rae et al., 1999). Reports on mounts received dur ing estrus are c onflicting between Bos indicus and Bos taurus cattle. Galina et al (1982) reported that Bos indicus crossbred cows (1.6 mounts/hr) received fewer mounts compared to Bos taurus cows (2.8 mounts/hr). R ae et al. (1999) reported that Brahman (25 mounts) heifers received more total mounts compared to Angus (19 mounts) while Brahman x Angus (37 mount s) heifers received more mount s compared to the Angus and Brahman heifers. It should be noted that in the Rae et al. (1999) study the heifers were managed in a single synchronized group and the heifers we re not separated by breed. Landaeta-Hernandez et al. (2002) reported a simila r number of mounts for Angus (30 mounts) and Brahman (33 mounts) cows during a synchroni zed estrus but a greater numb er of mounts for Angus (11 mounts) than Brahman (7 mounts) cows during a spontaneous estrus when the cows were managed in the same pasture. Therefore, the increased number of mounts observed during a synchronized estrus is probably due to an increased number of animals in estrus at a given time, resulting in more mounts and a l onger duration. In summary, both the duration of estrus and the number of mounts received during estrus are greater during a sync hronized estrus compared to a spontaneous estrus, which supports the conclusion AI programs in Bos indicus influenced cattle should be focused around a synchronized estrus. Environmental effects have also been reported to play a role in the duration and intensity of estrus. Landaeta-Hernandez et al. (2002) reported that the dur ation of estrus and number of mounts were reduced when the temperature-hu midity index was increased. Also, Lamothe-

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40 Zavaleta et al. (1991) re ported that the duration of estrus was shorter when temperatures were above 27 C. Furthermore, Plasse et al. (1970) re ported an increased incidence of ovulation without estrus in 2-year-old Brahman heifer s during the winter months. In addition to environmental effects, social hierarchy can influe nce the duration and intens ity of estrus between Bos indicus and Bos taurus cattle. Dominant Brahman cows took longer to exhibit estrus compared to dominant Angus cows (LandaetaHernandez et al., 2002). However, subordinate Angus cows had a longer interval from PGF2 treatment to the onset of estrus than subordinate Brahman cows (Landaeta-Hernandez et al., 2002). The Landaeta-Hernandez et al. (2002) report suggests that social dominance could play a major role in the expression of behavioral estrus. Following estrus is the period known as metest rus, which is the period from the end of estrus (day 0.5) to the formati on of a functional CL (day 3 to 5) During metestrus, progesterone secretion is low and increases slowly until complete formation of the CL, which marks the end of metestrus and the beginning of diestrus. Diestr us lasts 10 to 14 d and is characterized by high circulating concentrations of progesterone, whic h suppress the actions of estradiol by preventing any preovulatory surge of LH. Harms et al. (1969) reported that from day 2 to 9 of the estrous cycle, progesterone concentrations increased from 2.8 to 14.1ng/mL in Bos taurus heifers. Additionally, Alvarez et al. (2000) noted similar maximal progester one concentrations for Angus (4.3 ng/mL) and Brahman (4.4 ng/mL) cows. He nricks et al. (1971) observed that peak progesterone concentrations ranged from 5 to 12 ng/mL in Bos taurus cattle, while Ruiz-Cortez and Olivera-Angel (1999) reported that peak progesterone concen trations ranged from 1 to 8 ng/mL in Bos indicus cattle. The initiation of luteolysis (day 16 to 19) marks the begi nning of proestrus. During proestrus, circulating progesteron e concentrations decline while estradiol increases coincident

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41 with dominant follicle development (Henricks et al., 1971). Increasing estradiol concentrations lead to the onset of estrus. Estrous Synchronization through Manipulation of the Estrous Cycle Synchronization of the estrous cycle is a manage ment tool that allows for beef and dairy producers to increase the opportunity for success of an artificial insemination (AI) program. Benefits of estrous synchronizati on include an increased percentage of cattle pregnant early in the breeding season, shortened AI breeding seas on, shortened calving seas on, and increased calf crop uniformity. Labor expenses can also be reduced through estrous synchronization by decreasing the time and labor required for estr ous detection and breed ing as well during the subsequent calving periods. Estr ous synchronization can be achie ved through the use of several exogenous hormones including prostaglandin F2 (PGF2 ) to shorten the lut eal phase; progestins to prevent estrus and ovulati on; and gonadotropin releasing hormone (GnRH) to synchronize follicle wave development or to ovulate the dominant follicle in conjunction with AI. These hormones can be combined for control of follicle dynamics, initiat e luteal regression, synchronize estrus, and (or) implementation of a timed AI program, eliminating the need for estrus detection. Progestogens As mentioned previously, progesterone secr eted form the CL acts to prevent the preovulatory surge of LH and expression of estr us during the estrous cy cle. Consequently, exogenously administered progestogens such as melengestrol acetate (MGA), norgestomet (Syncro-mate B; SMB), controlle d intravaginal progesterone releasing device (CIDR), and injectable progesterone have been used to mi mick the actions of progesterone throughout the duration of their administration.

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42 Melengestrol acetate is an orally active progest ogen that is used in feedlot heifers to prevent the expression of estrus a nd as a synchronization agent in yearling beef heifers. Early research utilizing MGA demonstrated that fe rtility at the subseque nt estrus after MGA withdrawal was reduced with both short (< 9 d; B eal et al., 1988) and long term (> 9 d; Hill et al., 1971) MGA treatments. Although, the reducti on in fertility was te mporary as fertility returned to normal at the subsequent estrus. Hill et al. (1971) suggested that the reduction in fertility was due to several f actors including altered cervical mu cus, ovulation of abnormal ova, and (or) fertilization failure. In addition, few nor mal follicles, some hyperplastic follicles, atretic follicles, and atretic follicles with thickened theca interna were observed on the ovaries of MGA treated heifers lacki ng a functional CL during MGA treatme nt (Lamond et al., 1971). More recent research demonstrated that fertility decreas ed in cows and heifers without the presence of a CL during progestin treatment co mpared to cows with a CL present (Sanchez et al., 1993). Subsequent research demonstrated that low level progestogen exposure, supplied by a CIDR, in the absence of a functional CL produced an endocrine environment that permitted the dominant follicle to persist on the ovary while su ppressing the development of new dominant follicles (Sirois and Fortune, 1990). Sirois and Fort une (1990) concluded that in the absence of endogenous progesterone from the CL, low levels of progesterone supplied by the CIDR resulted in increased LH pulse frequency, which resulted in enhanced follicle development. However, when two CIDRs were administered to mimic no rmal luteal phase proge sterone concentrations resulting in decreased LH pulse frequency and dominant follicle tur nover. The type of progestogen administered as well as the presence of a function CL can also dictate whether there is follicle turnover. Custer et al. (1994) treated cows with a pr ogesterone-releasing intravaginal device (PRID) resulting in regression of the dominant follicle presen t at the initiation of

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43 treatment and recruitment of a new follicle wave. In contrast, dominant fo llicle turnover was not observed in cows treated with MGA in the abse nce of luteal function, which was a result of increased LH pulse frequency and development of large single non-ovulatory follicle. Kojima et al. (1992) treated cows with eith er a CIDR, MGA, or norgestomet in the absence of a functional CL resulting in high frequency low amplitude pulses of LH, which sustained dominant follicle growth and increased circulating concentrations of estradiol. The large follicles that develop under low endogenous progesterone exposure are larger than nor mal ovulatory follicles and produce greater concentrations of estradiol (Savio et al., 1993) and are commonly referred to as persistent dominant follicles. The decreased fertility observed with development of persistent dominant follicles is mediated primarily at the level of the follicle. Mihm et al. (1994) demonstrated that as dominance of follicles is greater than 4 d, fertility is increasingl y reduced. Ahmad et al. (1995) observed that ovulation of persistent dominant fo llicles resulted in fewer embryos reaching the 16-cell stage and fewer total embryos collected. Moreover, Revah and Butler (1996) determined that oocytes ovulated from persis tent dominant follicles underwent premature maturation in vivo. In addition to altered follicle development, th e increased estrogen production of the persistent dominant follicle results in an altered hormonal milieu. The alte red hormonal environment alter the synthesis and secretion of oviductal protei ns, which create a less than optimal ovarian microenvironment leading to decreased pre gnancy rates by altering oviductal function, fertilization, and early embryonic development (Binelli et al., 1999) Therefore, disparities in embryo development and ovarian microenvironment collectively add to the reduction in fertility during long-term low-level progestogen exposure. Conversely, Wehrman et al. (1997) reported similar pregnancy rates following embryo transfer in cattle ovulating either persistent dominant

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44 follicles or normal follicles. Furthermore, proge sterone concentrations 7 and 12 d after ovulation were similar between cattle ovulating a persiste nt dominant follicle a nd those ovulating a normal follicle (Mihm et al., 1994). Therefore, oocytes ovulated from a persistent dominant follicle lead to decreased fertility while no detrimental effects on CL function are observed following ovulation of a persistent dominant follicle compared to a normal ovulation. Persistent dominant follicles developed duri ng long term MGA treatments can be regressed either through treatment with exogenous estrogen (Yelich et al., 1997) or progesterone (Anderson and Day, 1994; Garcia et al., 2004) du ring the MGA treatment. The subsequent follicle ovulated after MGA w ithdrawal has normal fertility. Prostaglandin F2 As previously mentioned, PGF2 is the luteolytic hormone in beef cattle. The luteolytic actions of exogenous PGF2 on the CL are effective from day 5 to 16 of the estrous cycle (Rowson et al., 1972; Inskeep, 1973; Kiracofe et al., 1985). As a resu lt administration of exogenous PGF2 such as alfaprostal, chloprostenol, di naprost tromethamine, and luprostial have been used to synchronize estrus in beef and dairy cattle. In a review of early studies by Inskeep (1973), both CL size and progesterone concentrat ions were reduced within 24 hr following treatment with exogenous PGF2 Subsequent research reported th at progesterone concentrations were reduced to < 0.5 ng/mL within 24 hr after a 30 mg injection of PGF2 resulting in increased estradiol concentrations (Chenault et al., 1976) and the subs equent expression of estrus. The estrous response in cattle of Bos taurus breeding undergoing normal es trous cycles following a single administration of PGF2 ranges from 65 to 91% (Laude rdale et al., 1974; Tanabe and Hann, 1984) with estrus being observed within 2 to 7 d. In contrasts, the estrous response in cycling cattle of Bos indicus breeding ranges from 56 to 62% in Zebu cattle (Orihuela et al., 1983) and 46.3 to 54.8% in Nelore cattle (Landiva r et al., 1985). The va riability in estrous

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45 response and interval to the onset of estrus is due to several factors including estrous cycling status, stage of the estrous cycle at PGF2 administration, and breed. The actions of PGF2 are only seen in cattle that are unde rgoing normal estrous cycles with a CL present on the ovary. In estrous cycling cattle, estrous response a nd interval from PGF2 to the onset of estrus are affected by the stage of estrous cycle at PGF2 administration (Tanabe and Hann, 1984; Watts and Fuquay, 1985). Tanabe and Hann (1984) treated Holstein heifers with PGF2 during the early (d 7), mid (d 11), and late (d 15) luteal phase of the estrous cycle and reported a higher percentage of heifers exhibi ting estrus within an 80 hr period for each advancing stage of the estrous cycle (86.0, 90.0, and 98.0%; respectively). Of heifers that exhibited estrus, 100.0% of early, 95. 9% of late luteal phase, and only 48.9% of mid luteal phase heifers exhibited estrus within 72 hr following PGF2 treatment. These findings are in agreement with Watts and Fuquay (1985) w ho treated heifers with PGF2 during the early (days 5-7), mid (days 8-11), and late (days 12-15) luteal phase and observed 72 hr estrous response and interval from PGF2 to the onset of estrus of 43.0% and 59 hr, 83.6% and 70 hr, and 78.3% and 72 hr, respectively. Macmillan and Henderson (1984) also reported a similar trend with over 70% of cows treated with PGF2 on day 7 (early diestrous) and 16 (lat e diestrous) in estrus within 48-72 hr following PGF2 treatment but only 30% of cows treated on days 11 or 12 (mid diestrous) in estrus within 48-72 hr following treatment. Sirois and Fortune (1988) reported a negative correlation between size of the preovulatory follicle at luteolysis and the interval to ovulati on. Furthermore, Kastelic et al. (1990) observed that the interval from PGF2 to the onset of estrus was shor ter for heifers administered PGF2 on day 5 compared to day 12 of the estrous cycle. Consequently, follicle wave development at the time of a PGF2 treatment affects the synchrony of the s ubsequent estrus. With this in mind, it

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46 points out the importance of synchr onizing follicle development in conjunction with either luteal regression or removal of a progestogen. A PGF2 induced luteolysis does not appear to have any negative effects on fertility of the subsequent estrus. Macmillan and Day (1982) reported pregnancy rates of 69% in PGF2 treated cows compared to 60% for non-treated cows wi th over 2,000 animals in each group. Lauderdale et al. (1974) reported similar pregna ncy rates between cows receiving PGF2 compared to those not receiving PGF2 Moreover, Tanabe and Hann (1984) reported similar pregnancy rates in PGF2 treated (77.4%) and non-treated heifers (76.0%). Additionally, Hardin et al. (1980) noted that pregnancy rates were similar between PGF2 treated (30 and 37%) and nonPGF2 treated (37 and 38%) Bos indicus cattle. However, stage of the estrous cycle when cattle receive PGF2 may affect fertility. Pregnancy rates for heifer s were lowest (56.8%) fo r heifers receiving PGF2 early (d 5-7) in the estrous cycl e compared to heifers receiving PGF2 in the middle (d 8-11; 62.1%) or late (d 12-15; 78.3%) stages of the estrous cy cle (Watts and Fuquay, 1985). There is a limited amount of rese arch that suggests that a PGF2 induced luteolysis maybe compromised in cattle of Bos indicus breeding. Hansen et al. ( 1987) reported that Brahman heifers required a greater dose of PGF2 to achieve luteolysis compared to Brahman cows. Furthermore, the crossbred Brahman he ifers required a greater dose of PGF2 to achieve luteolysis from days 8 to10 of the estrous cycle comp ared to days 11 to 13 of the estrous cycle. Pinheiro et al. (1998) repo rted that 48 % (12/25) of Nelore cows with a functional CL, failed to respond to a luteolytic dose of PGF2 between day 6 to 8 of the estrous cycle. Furthermore, the authors hypothesized that administration of PGF2 from days 5 to 9 of the estrous cycle may cause partial luteolysis followed by recovery of luteal activity. Cornwell et al. (1985) reported that Brahman heifers treated with PGF2 on day 7 and 10 of the estrous cycle had estrous

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47 responses of 50 and 67%, respectively; whereas, 100% of the heifers exhibited estrus when treated with PGF2 on days 14 and 18 of the estrous cycl e. For heifers no t responding to PGF2 on days 7 and 10, progesterone concentrations decreased by 12 hr after PGF2 followed by increased concentrations of proge sterone within 48 hr of PGF2 treatment. Therefore, the early developing CL (day 5-10 of the estrous cycle) a ppears to be somewhat refractory to the actions of a single treatment of PGF2 in cattle on Bos indicus breeding. Several researchers have addressed the issue of incomplete luteolysis in females of Bos indicus breeding by changing the administration of PGF2 from a single to two consecutive doses of PGF2 Cornwell et al. (1985) administered PGF2 to Brahman heifers on day 7 and 8 of the estrous cycle and reported a sign ificant increase in estrous respons e (97%) compared to a single injection (69%). When a single PGF2 treatment was administered during the early luteal phase (day 7 and 8) of the estrous cycle, Santos et al. (1988) reported a similar estrous response and interval from PGF2 to the onset of estrus when either 12.5 mg (84%; 94 hr) or 25 mg (83.1%; 100 hr), respectively. Santos et al. (1988) conducted an additional study to test the effectiveness of either two consecutive 12.5 mg or 25 mg PGF2 injections compared to a single 25 mg injection of PGF2 in Brangus ( Bos taurus Bos indicus ) cows, which had previously received a single 25 mg injection of PGF2 11 d earlier. Estrous response and conception rates were greater for cows receiving two 12.5 mg (82; 73%) a nd two 25 mg (73; 59%) injections of PGF2 compared to a single 25 mg injection (55; 32%), respectively. The interval from the initial PGF treatment to the onset of estrus was significantl y shorter for groups receiv ing the two consecutive 12.5 mg (79.0 hr) and 25 mg (73.2 hr) injections compared to a si ngle 25 mg (93.9 hr) injection. A recent study by Bridges et al. (2005) reported increased luteolysis when two consecutive 12.5 mg PGF2 treatments (92.5%) were administered 24 hr apart compared to a single 25 mg PGF2

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48 treatment (79.1%) in Bos indicus Bos taurus heifers during the mid and late stages of the estrous cycle. Conversely, no increase in luteolysis was observed in yearling Angus ( Bos taurus ) or 2-year-old Bos indicus Bos taurus heifers receiving the same PGF2 treatments. In summary, modifying the administration of PGF2 from a single 25 mg treatment to two consecutive 12.5 mg treatments 24 hr apart appears to increase estrous responses by increasing the rate of luteolysis in yearling heifers of Bos indicus breeding. Melengestrol acetate + PGF2 As early as the 1960s, oral progestogens were used for controlling estrous cycles in cattle (Hansel et al., 1961; Zimbelman, 1963; Wiltbank et al., 1967). An early study by Zimbelman and Smith (1966) reported that the optimal dos age of MGA needed to prevent estrus and ovulation was 0.5 mg/hd/d. However, the negative side of using MGA was the development of a persistent dominant follicle (Sirois and Fortune, 1990; Savio et al., 1993) that resulted in reduced fertility (Hill et al., 1971; Ahmad et al., 1995) at the estrus after MGA withdrawal due to improper embryo development (Ahmad et al., 1995) Furthermore, administration of MGA at levels (1.0 and 1.5 ng/mL) above the optimal leve l to inhibit the expr ession of estrus (0.5 mg/hd/d) did not provide a progesterone envi ronment similar to mid-luteal progesterone concentrations that would regulate the pulsatile release of LH and stim ulate follicle turnover (Kojima et al., 1995). Administering more MGA di d result in a gr eater interval from MGA to the onset of estrus (Zimbelman and Smith, 1966; Hill et al., 1971). The mean interval to estrus was decreased in heifers treate d with 0.2 mg/hd/d (2.7 d) compar ed to heifers treated with 2.0 mg/hd/d (6.3 d) (Zimbelman and Smith, 1966). Brown et al. (1988) developed a system utilizing MGA and PGF2 that was designed to circumvent the reduction in fertility following lo ng term (14 d) MGA treatment. The MGA was administered for 14 d (0.5 mg/hd/d) and heifer s were allowed to e xhibit estrus but not

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49 inseminated at the estrus after MGA withdrawal. Seventeen days after the termination of MGA, which placed heifers in the late luteal phase of their estrous cy cle, heifers received 25 mg of PGF2 (MGA + PGF2 ). The MGA + PGF2 system was compared to Syncro-mate B (SMB), which consisted of a 9 d norgestomet implant with an estradiol valerate injection concurrent with implant insertion. Estrous response was similar between the MGA + PGF2 (83.4%) and SMB (90.2%) treatments but conception (68.7 vs. 40.6%) and synchronized pregnancy rates (57.3 vs. 36.6%) were greater for the MGA + PGF2 compared to SMB heifers, respectively. Additionally, estrous response and synchronized pregnanc y rate were signif icantly greater for cycling (91.6 and 68.4%) than noncycling (71.0 and 40.3%) MGA + PGF2 treated heifers, but were similar in cycling (92.4 and 44.6%) and non-c ycling (85.2 and 26.2%) SMB treated heifers. Patterson and Corah (1992) obser ved a greater 6 d estrous res ponse (79.0 vs. 32.0%), similar conception rates (64.0 vs. 67.0%), and increase d synchronized pregnanc y rates (50.0 vs. 21.0%) for MGA + PGF2 treatment compared to untreated contro ls, respectively. Similarly, Jaeger et al. (1992) reported an increased 6 d estrous response (77.0 vs. 25.0%), similar conception rates (64.0 vs. 50.0%), and increased synchronized pr egnancy rates (48.7 vs. 14.0%) for the MGA + PGF2 treatment compared to the untreated contro ls, respectively. Numerous follow-up studies testing the efficacy of the MGA + PGF2 system compared to untreated control heifers have also been conducted and are summarized in Table 2-1. Other investigators have vari ed the original MGA + PGF2 system by increasing the interval from MGA withdrawal to PGF2 administration from 17 to 19 d (Nix et al., 1998; Deutscher et al., 2000; Lamb et al., 2000). In a large field study conducted by Lamb et al. (2000), estrous response was simila r for heifers treated with PGF2 either 17 (68.3%) or 19 (68.1%) d following MGA withdrawal. However, the synchrony of estrus was improved with

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50 the 19 d treatment as 99% of the 19 d heifer s exhibited estrus within 72 hr of PGF2 compared to 74% of 17 d heifers. Moreover, the interval to estrus was reduced for heifers treated 19 d (56.2 hr) compared to 17 d (73.1 hr) after MGA withdrawal Conception and pregnancy rates were similar for heifers treated 17 (75.9; 51.8%) or 19 d (81.4; 55.4%) following MGA withdrawal, respectively. Lamb et al. (2000) hypothesized that by increasing the interval from MGA withdrawal to PGF2 from 17 to 19 d resulted in a more mature preovulatory follicle at PGF2 which resulted in an earlier expression of estrus. The MGA + PGF2 system has also been used in a timed AI system with or without GnRH. In two experiments, Larson et al. (19 96) subjected heifers to the MGA + PGF2 (17 d) system and compared two different AI protocols includ ing estrous detection and AI for 72 hr following PGF2 to a single fixed-time AI 72 hr following PGF2 for all heifers. In the first experiment, pregnancy rates were similar for heifers bred to an observed estrus ( 31.0%) compared to fixed time AI 72 hr after PGF2 treatment (36.4%). A second experiment was performed to determine the effects of estrous detection and AI for 72 hr combined with time AI at 72 hr following PGF2 for heifers not exhibiting estrus. Results from the second experiment suggest that combining estrus detection and timed AI yield increas ed pregnancy rate ( 48.4%) where heifers not expressing estrus by the third day after PGF2 reduced estrous detecti on without a reduction in fertility. Therefore, combining es trous detection with fixed time AI subjects all heifers to AI and the opportunity to become pregna nt during the synchronized period. A recent study by Salverson et al. (2002) evaluated the effectiveness of type of PGF2 (cloprostenol vs. dinoprost tromethamine) in the MGA + PGF2 (19 d) system and observed similar estrous (89 vs. 86%), conception (67 vs 67%) and pregnancy (61 vs. 57%) rates between the two types of PGF2 Another refinement to the MGA + PGF2 system included

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51 administration of GnRH 12 d following MGA w ithdrawal followed 7 d later with PGF2 (Wood et al., 2001). The purpose of the GnRH was to ovulate or luteinize most large follicles so as to synchronize follicle development when PGF2 was administered with the theory that the subsequent estrus could be more tightly sync hronized and allow for a timed-AI. Estrous response from 48 to 60 hr following PGF2 was significantly increase d in GnRH treated (71.0%) compared to non-GnRH treated (35.0%) heifers, while 7 d es trous response (100.0 vs. 94.0 %) and the interval from PGF2 to estrus (67.0 vs. 71.0 hr) wa s similar between treatments, respectively. However, fertility was not evalua ted in this study. DeJa rnette et al. (2004) conducted a study where heifers were treated with either the protocol described by Wood et al. (2001; MGA + G + PGF2 ) or a short term MGA (STMGA) protocol where heifers were administered MGA for six days, with GnRH the day before MGA and PGF2 the day after the last day of MGA. In both treatments, estrus de tection and AI were perf ormed for 72 hr, at which time all heifers not detected in estrus were tim ed-AI and received GnRH. DeJarnette et al. (2004) reported increased synchronized pregnancy rates for MGA + G + PGF2 (65%; 55/85) compared to STMGA (46%; 40/87) treated heifers. Therefore, it appears that fertility is not compromised following the MGA + G + PGF2 system. Melengestrol acetate has also been implicated in the induction of cyc licity in prepubertal heifers. Patterson et al. (1990) noted that 71% of prepubertal Bos taurus and 41% of prepubertal Bos taurus Bos indicus crossbred heifers had pr ogesterone concentrations 1 ng/mL following a 7 d MGA treatment. Jaeger et al. (1992) repor ted that a significantl y greater percentage (72.0%) of prepubertal heifers treated with a 14 d MGA treatment reached puberty prior to PGF2 19 d after MGA withdrawal compared to un-tr eated heifers in the same period (45.0%). Following a 14 d MGA feeding, Deutscher et al. (2000) observed a 15 to 20% increase in the

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52 percentage of heifers cycling. Im walle et al. (1998) suggests that in prepubertal heifers treated with MGA that an increased mean LH concentr ation and LH pulse frequency result in an increased diameter of the largest follicle duri ng MGA treatment, which results in a follicle large enough to ovulate after MGA withdr awal resulting in the formati on of a CL and initiation of estrous cycles. Minimal research has been conducted on the effectiveness of the MGA + PGF2 system for synchronization of estrus in yearling heifers of Bos indicus Bos taurus breeding. Stevenson et al. (1996) synchronized Bos indicus Bos taurus heifers with MGA (14 d) + PGF2 (17 d) system where estrus was detected for 72 hr and all heifers that were not in estrus were timed-AI at 72 hr. The synchronized pre gnancy rate was 42.9% (21/49). Bridges et al. (2005) con ducted a study in yearling Bos indicus Bos taurus heifers using the MGA + PGF2 system comparing the effectiv eness of a single (25 mg) PGF2 treatment 19 d after MGA withdrawal compared to two (12.5 mg) consecutive PGF2 treatments 24 hr apart on days19 and 20 d following MGA withdrawal. Estr us was detected for 72 hr followed by timedAI in conjunction with GnRH for heifers not exhibiting estrus by 72 hr Conception rates (51.5 vs. 48.8%) were similar while estrus response (50.1 vs. 43.2%), TAI pregnancy rate (33.5 vs. 23.9%), and total pregnancy rate (42.5 vs. 34.5% ) were significantly improved by modifying the delivery of PGF2 to two consecutive split treatment s compared to a single treatment, respectively. The increase total pr egnancy rate was due to an increa sed rate of luteolysis in the two consecutive PGF2 treatments compared to the single PGF2 treatment. Therefore, the MGA + PGF2 system in yearling Bos indicus Bos taurus crossbred heifers can be improved by modifying the delivery of PGF2 from a single to split PGF2 treatment; however, the resulting

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53 synchronized pregnancy ra tes of the MGA + PGF2 system in Bos indicus Bos taurus heifers are still considerably less than those observed in Bos taurus heifers. There could be several reasons for the di fferences in response to the MGA + PGF2 in yearling Bos indicus Bos taurus heifers compared to Bos taurus heifers. Cattle of Bos indicus breeding have a shorter duration and less intens e estrus (Rhodes and Ra ndel, 1978; Rae et al., 1999; Landaeta-Hernandez et al., 200 2), have an increased inciden ce of three and four follicle waves (Rhodes et al., 1995; Viana et al., 2000), and reach puberty at older ages (Plasse et al., 1968; Patterson et al., 1991). One area wher e research has been limited in cattle of Bos indicus breeding is evaluating what effect of follicle wave development has on the effectiveness of estrous synchronization treatments. An as ynchrony in follicle development at PGF2 can result in large variations in the interval to estr us, undermining the overall effectiveness of a synchronization system and prevent the use of a fixed timed-AI. On e way that that the asynchrony of follicle development can be alte red is by the administration of GnRH to synchronize follicle development. Therefore, incorporating GnRH during the period between MGA withdrawal and PGF2 in the MGAPGF2 system in Bos indicus based heifers could be used to improve the synchrony of follicle development at PGF2 By increasing the synchrony of follicle development at PGF2 a greater number of heifers should have dominant follicles ready to ovulate, improve the synchrony of estrus, and allow for a fixed timed-AI. Utilizing a fixedtimed-AI would allow for the elimination of estrous detection and allow all animals an opportunity to be inseminated, partic ularly cattle that have a silent estrus or exhibit estrus more covertly. Therefore, synchronization systems need to be re-evaluated and tailored to better synchronize follicle development in Bos indicus based cattle.

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54Table 2-1. Summary of st udies evaluating the melengestrol acetate (MGA) + PGF2 estrous synchronization system in yearling beef heifers. Study Breed-type Day PGF2 was administered after MGA (14 d) withdrawal Days of estrus detection Estrous response (%) Synchronized Pregnancy rate (%) Brown et al., 1988 Bos taurus 17 5 83.4 57.3 Jaeger et al., 1992 Bos taurus 17 6 71.4 54.3 Patterson and Corah 1992 Bos taurus 17 6 79.0 50.0 Nix et al., 1998 Bos taurus 17 5 64.2 55.4 Bos taurus 19 5 75.1 51.8 Deutscher et al., 2000 Bos taurus 17 5 86.7 49.2 Bos taurus 17 5 77.6 53.8 Bos taurus 19 5 92.4 57.1 Bos taurus 19 5 87.6 61.4 Lamb et al., 2000 Bos taurus 17 5 68.3 51.8 Bos taurus 19 5 68.1 55.4 Funston et al., 2002 Bos taurus 17 5 77.0 47.0 Salverson et al., 2002 Bos taurus 19 (Estrumate) 5 89.0 61.0 Bos taurus 19 (Lutalyse) 5 86.0 57.0 Stevenson et al., 1996 Bos indicus Bos taurus 17 3 + 72 hr timed-AI 68.2 42.9 Bridges et al., 2005 Bos indicus Bos taurus 19 (25 mg) 3 + 72 hr timed-AI/GnRH 43.2 34.5 Bos indicus Bos taurus 19 (12.5 mg) & 20 (12.5 mg) 3 + 72 hr timed-AI/GnRH 50.1 42.5

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55 CHAPTER 3 EVALUATION OF FOLLICULAR DEVELOPM ENT BETWEEN A 14 D MELENGESTROL ACETATE (MGA) TREATMENT WITH PGF2 19 D AFTER MGA WITHDRAWAL IN ANGUS AND BRANGUS HEIFERS Introduction Artificial insemination (AI) provides producers with the opportunity to use genetically superior AI sires and AI is routinely used in conjunction wi th estrous synchronization. One of the major limitations of an AI and estrous s ynchronization program is the amount of time and labor required to implement and carry out the program. Therefore, estrous synchronization programs need to be developed that require minimal animal handling and result in a high percentage of cattle in estrus during a 2 to 3 d period. One way to minimize animal handling is to use the orally active progestogen melengest rol acetate in a sync hronization program. Melengestrol acetate (MGA) administered for 14 d, coupled with prostaglandin F2 (PG) 17 d after the last day of MGA is an effectiv e estrous synchronization system (MGA-PG) that was developed in yearling Bos taurus heifers (Brown et al., 1988). The interval from MGA withdrawal to PG was increased from 17 to 19 d resu lting in a shorter interv al to peak estrus and more synchronous estrus (Lamb et al., 2000). The effectiveness of the MGA-PG system as measured by synchronized pregna ncy rate is less in yearling Bos indicus Bos taurus (Bridges et al., 2005) compared to Bos taurus heifers (Brown et al., 1988; (Lamb et al., 2000). Even though synchronized pregnancy rates in Bos indicus Bos taurus heifers can be increased slightly by modifying the delivery of PG fr om a single PG treatment to two consecutive PG treatments 24 hours apart (Bridges et al., 2005), the synchronized pr egnancy rates are still considerably less in Bos indicus Bos taurus compared to Bos taurus heifers. The reason(s) for the decreased effectivene ss of the MGA-PG synchronization program in heifers of Bos indicus Bos taurus breeding may be attributed to physiological differences

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56 between Bos indicus Bos taurus compared to Bos taurus heifers. Bos indicus cattle have a greater incidence of 3 and 4 follicle waves (R hodes et al., 1995; Viana et al., 2000) during the estrous cycle and this could result in as ynchronous follicle development when PG is administered in the MGA-PG program. Conseque ntly, asynchronous follic le development could be one of the reasons why the estrous response and subsequent synchronized pregnancy rates after PG in the MGA-PG synchronization program are low in Bos indicus Bos taurus heifers. Therefore, the objectives of this study were to evaluate follicular development from withdrawal of a 14 d MGA treatment until PG administration 19 d later in order to evaluate the possibility of administering GnRH to synchronize follicle deve lopment, and to evaluate follicle development and the subsequent estrous respons e after PG in yearling Angus ( Bos taurus ) and Brangus ( Bos indicus Bos taurus ) heifers. Materials and Methods Yearling Angus ( Bos taurus ; n = 40) and Brangus ( Bos indicus Bos taurus ; n = 26) heifers, at the University of Fl orida Santa Fe Beef Research Unit were used in the experiment, which was conducted from February to April of 2004. Average heifer age, BW, body condition score (BCS; Richards et al., 1986) and percentage of heifers having estrous cycles at the initiation of the experiment are summarized in Table 3-1. Blood samples were collected -16, -7, and 0 d before initiation of a 14 d melengestrol acetate (MGA) treatment to determine estrous cycling status. The start of the experiment was designated as day 0. Heifers were deemed to be having estrous cycles ( cycling ) if blood plasma progester one concentrations were 1.5 ng/mL at two of the three blood samples wh ile heifers were classified as not having estrous cycles ( noncycling ) if progesterone concentrations were < 1.5 ng/mL at all three samples. A progesterone concentration of 1.5 ng/mL was chosen after a retrospec tive analysis reveal ed that several heifers had progesterone concentr ations between 1.0 1.5 ng/mL in the absence of luteal tissue

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57 as determined by ultrasonography on day 0 of the experiment. On day 0 of the experiment, Angus and Brangus heifers were distributed by breed, cycling stat us, and BW into two groups. One group included cycling Angus and Brangus heif ers that were to ha ve daily ultrasonography ( scan ) conducted from MGA withdrawal until 5 d after PG and the othe r group included the remaining Angus and Brangus heifers (cycling and non-cycling) that would have no daily ultrasonography conducted ( non-scan; Table 3-1). Also on day 0, the scan and non-scan heifers were started on a 14 d MGA (0.5 mghd-1d-1; MGA 200 Premix, Pfizer, Inc., New York, NY) treatment, which was administered in a high protein pellet fed at a rate of 2 lbshd-1d-1. The cycling scan and non-scan heifers were at random stages of the es trous cycle at the start of the MGA treatment. All heifers received prostaglandin F2 (PG; Lutalyse Sterile Solution, Pfizer, Inc. New York, NY) starting on d 19 as descri bed by Bridges et al. (2005). Angus heifers received a single 25 mg i.m. PG treatment 19 d after MGA while Brangus heifers received 12.5 mg i.m. PG on both 19 and 20 d following MGA wit hdrawal. Bridges et al (2005) reported that a split PG treatment enhanced luteolysis co mpared to a single PG treatment in yearling Bos indicus Bos taurus heifers and the split PG treatment in yearling Bos indicus Bos taurus heifers resulted in a similar rate of luteol ysis compared to a single PG treatment in Bos taurus heifers. Therefore, the breed specific PG treatme nt was used in the present experiment to ensure that the PG induced luteolysis would be similar between Angus and Brangus heifers. The scan group consisted of cycling Angus (n = 11) and Brangus (n = 10 ) heifers that were used to evaluate daily follicular development us ing transrectal ultrasono graphy (equipped with a 7.5 MHz linear array transducer) fr om MGA withdrawal to 5 d af ter PG. Scan heifers were handled through the working facil ities three times a week duri ng MGA treatment to acclimate them to frequent handling to re duce any stress related a ffects associated with frequent handling.

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58 At the start of the ultrasonogra phy exams, two Brangus heifers had to be replaced because of physical injuries that prevented daily ultras onography from being conduct ed. The two heifers were replaced with two non-cycling Brangus he ifers of similar age and BW. Non-cycling Brangus heifers were chosen because there were no cycling Brangus heifers available that had a large enough rectal size to allow for daily u ltrasonography exams to be performed The two Brangus heifers that were removed from the s can group were placed in the non-scan group where they recovered from their injuries. At each ultrasonography evaluation, height and width of all luteal structures, luteal cavities, and follicles 3 mm in diameter were measured using the internal calipers of the ultr asonography machine, and their lo cations on the ovaries were recorded. All ultrasonography exam s were conducted by a single technician. Volume of the corpus luteum (CL) was calculated usi ng the formula for volume of a sphere ( d3/6). When a luteal cavity was present, its volume was subt racted from the volume of the outer sphere resulting in net luteal volume (CL volume) repr esented by luteal tissue. Ovarian maps were evaluated retrospectively to determine follicle growth patterns. Emergence of a follicle wave was defined as the time when the eventual dom inant follicle could first be identified by ultrasonography. Maximal diameter of a domi nant follicle was defined as the maximum diameter that the dominant follicle reached during the follicle wave. The day of maximal diameter was defined as the day following emer gence that the dominant follicle reached a maximal diameter. Growth rate of the dominant follicle was determined by subtracting the diameter of the dominant follicle at emergence from the maximal diameter of the dominant follicle divided by the number of days from emergence to maximal diameter. Additionally, blood samples were collected via jugular vein ipuncture to determine plasma progesterone concentrations at each ultr asonography exam. Du ring the ultrasonography exams after MGA

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59 withdrawal, ovulation was define d as disappearance of the largest follicle followed by its absence at two consecutive ultrasonography exam s. Ovulation rate after MGA for the scan heifers was defined as the num ber of heifers ovulating a fol licle within 7 d after MGA withdrawal divided by the tota l number of scan heifers. The non-scan heifers underwent transrect al ultrasonography (equipped with a 7.5 MHz linear array transducer) at MGA withdrawal a nd at the initial PG. At each ultrasonography evaluation, height and width of all luteal structures, lu teal cavities, and follicles 5 mm in diameter were measured using the internal cal ipers of the ultrasonography machine, and their locations on the ovaries were recorded. Additio nally, blood samples were collected via jugular veinipuncture to determine plasma progesterone at MGA withdrawal, at the initial PG, and 3 d following the initial PG treatment to determine if luteolysis occurred. Both scan and non-scan heifers were deemed to have a functional CL at PG if plasma progesteron e concentrations were > 1.5 ng/mL with the presence of luteal tissue as determined by ultrasonography. The PG induced luteolysis for scan and non-scan heifers was de fined as a heifer having a functional CL at PG followed by progesterone concentrations < 1.5 ng/mL 3 d after PG. After the onset of the PG induced estrus for the scan heifer s, ovulation was defined as disappe arance of the largest follicle at the subsequent ultrasonography exam. O vulation was confirmed by the presence of a functional CL 8 d later as determined by ultr asonography and a blood plasma sample was also collected to determine plasma progesterone concentrations. Blood plasma samples were collected into ev acuated tubes containing an anticoagulant (EDTA; Becton, Dickinson and Company, Franklin Lakes, NJ). After collection, blood samples were immediately placed on ice until they were centrifuged (3000 g for 15 min). Plasma was separated and stored at 20C until further analysis. Progesterone concentrations were

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60 determined by RIA (Seals et al., 1998) using DPC kits (Diagnostic Products Corp., Los Angeles, CA) in multiple assays with in traand interassay CV of 3.5 a nd 4.9%, respectively. Sensitivity of the assay was 0.01 ng/mL of plasma assayed. Estrus was detected throughout the experiment using radiotelemeric estrous detection devices (HeatWatch, Cow Chips, Denver, CO; Dransfield et al., 1998), which were fitted to all heifers at the initiation of MGA treatment. Estrus was detected from the initiation of MGA until 5 d after PG. Heifers were artificially insemi nated (AI) by one of two AI technicians with frozen-thawed semen 8 to 12 h after the onset of the PG induced estrus. Angus heifers were inseminated to two AI sires that were pre-as signed to heifers prior to insemination and the Brangus heifers were inseminated to a single AI sire. Pregnancy was determined approximately 30 d after AI by transrectal u ltrasonography, using a real-time, B-mode ultrasound (Aloka 500v, Corometrics Medical Systems, Wallingford, CT) equipped with a 5.0 MHz transducer. Interval to estrus, duration of estrus, and number of mounts received during estrus were recorded for the estrus following MGA withdrawal and for the estrus after PG administration. For both the estrus after MGA wit hdrawal and PG, onset of estrus was defined as the first of 3 mounts in a 3 h period and the end of estrus was defined as the time of the last mount recorded during estrus prior to a period of extended inactivity of at least 8 h (Landaeta et al., 1999). The duration of estrus was calculated by subtracting the date and time of the ini tial mount of estrus from the last mount of estrus. Interval to estrus following MG A withdrawal was calculated by subtracting the approximate date and time of MG A withdrawal from the date and time of the initial mount of estrus. Estrous response after MGA withdrawal was defined as the total number of heifers that exhibite d behavioral estrus in 7 d divided by the total num ber of heifers treated. Interval from PG to the onset of estrus was calculated by subtracting th e date and time of the

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61 initial PG from the date and time of the initial mount of estrus. Estrous response after PG was defined as the total number of heif ers that exhibited behavioral estr us in 5 d after the initial PG divided by the total number of he ifers treated. Conception rate was defined as the total number of heifers that exhibited estrus after PG that were inseminate d and became pregnant, divided by the total number of heifers that exhibited estr us and were inseminated. Synchronized pregnancy rate was defined as the total number of heifers that became pregnant to the AI divided by the total number of heifers treated. Dependent variables tested us ing the GENMOD procedure of SAS (SAS Inst. Inc., Cary, NC) included cycling status, estrous response af ter MGA, estrous respons e after PG, conception rate, synchronized pregnancy rate occurrence of a functional CL at PG, PG induced luteolysis, and occurrence of heifers with follicles 10 mm in diameter at PG. Independent variables tested were breed, ultrasound group (scan vs. non-scan), and breed ultrasound group. Cycling status was also evaluated as an indepe ndent variable using the model of breed, cycling status (cycling vs. non-cycling), and breed cycling status fo r the aforementioned dependent variables. Additionally, ovulation rate following MGA withdraw al in scan heifers was evaluated with the independent variable tested being breed. Diamet er of the largest follicle at MGA withdrawal, diameter of the largest follicle at PG, progester one concentration at PG, and behavioral estrus data including interval to estrus duration of estrus, and number of mounts received during estrus after MGA withdrawal and after PG were tested using the GLM procedure in SAS. Independent variables tested were breed, ultrasound group and breed ultr asound group; additionally, breed, cycling status, and breed cycling status were evaluated. Diameter of the first, second, and third wave dominant follicles normalized for day of em ergence, and diameter of the dominant follicle on days 9-13 after MGA withdrawal (d 0) for s can heifers were evaluated with analysis of

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62 variance for repeated measures using the MIXE D procedure in SAS. Independent variables tested were breed, day, and breed day. Additionally, diameter of the eventual ovulatory follicle normalized to PG for scan heifers exhibi ting two or three follicle waves was evaluated with analysis of variance fo r repeated measures using th e MIXED procedure in SAS. Independent variables tested were breed, day, number of waves, and all possible interactions. Day of emergence, maximal diameter, and day of maximal diameter for the first, second, and third wave dominant follicles in scan heifers we re evaluated using the GLM procedure in SAS. The independent variable tested was breed. In scan heifers exhibiting either two or three follicle waves, estrous response following PG, conception rate, and synchronized pregnancy rate were evaluated using the GENMOD procedure of SAS, while interval from PG to the onset of estrus was tested in the GLM procedure of SAS. Inde pendent variables tested were breed, number of waves and breed number of waves. One non-scan Brangus heifer exhibi ted estrus during the MGA treatment so the data was excluded from the estrous response analysis after MGA withdrawal, but was included in all analyses at and after PG. Two non-scan Angus heifers did not have ultrasonography data collected at MGA withdrawal, so they were removed from the ultrasonography analysis. Four nonscan Angus heifers exhibited estrus before PG and they were excluded from the post PG data analysis Three non-scan Angus heifers did not have ultrasonography data collected at PG so they were removed from the ultrasonography data analysis at PG. Results The physical description of the heifers is pres ented in Table 3-1. The Angus heifers were older ( P = 0.03), had a greater ( P = 0.02) BCS, and had a greater ( P = 0.02) percentage cycling at

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63 the initiation of the experiment compared to Brangus heifers. There were no breed ultrasound group effects ( P > 0.05) on age, BW, BCS, and percentage cy cling at the start of the experiment. With all scan and non-scan heifer s included in the anal ysis, diameter of the largest follicle present at MGA withdrawal was similar ( P = 0.72) for Angus (n= 38; 13.7 0.5 mm) and Brangus (n= 25; 13.5 0.5 mm) heifers. There were no ( P > 0.05) effects of cycling status or breed cycling status on diameter of the largest follicle at MGA withdrawal. The percentage of Angus and Brangus heifers in estrus during the 7 d after MGA withdrawal was similar ( P = .88; Table 3-2). The interval from MGA withdrawal to the onset of estrus, duration of estrus, and number of mounts received duri ng estrus were also similar ( P > 0.05) between Angus and Brangus heifers (Table 3-2). Th e scan heifers had a greater ( P = 0.001) estrous response compared to non-scan heifers, and scan heifers tended ( P = 0.09) to have a longer interval from MGA withdrawal to the onset of estrus compar ed to non-scan heifers (Table 3-2). Whether heifers were scan or non -scan did not influence ( P > 0.05) the duration of estrus or the number of mounts received during the estrus after MGA withdrawal (Table 3-2). There were no ( P > 0.05) breed ultrasound group effects on estrous respon se, interval from MGA withdrawal to the onset of estrus, duration of estr us, and number of mounts received during estrus (Table 3-2). More ( P = 0.001) cycling heifers (n = 22/35; 62.9%) exhibited es trus after MGA withdrawal compared to non-cycling heifers (n = 7/30; 23.3%), but there was no ( P = 0.55) breed cycling status effect. Interval from MGA withdrawal to the onset of estrus (87:03 09:19; 64:36 17:01 h:m), duration of estrus (10:24 01:25; 11:52 02:35 h:m), and number of mounts received (53 9; 70 17) were similar ( P > 0.05) between cycling a nd non-cycling heifers, respectively. There were no ( P > 0.05) breed cycling status effects for interval from MGA withdrawal to the onset of estrus a nd duration of estrus but there was ( P < 0.05) a breed

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64 cycling status effect on number of mounts received during es trus. The number of mounts received for the cycling Angus, non-cycling An gus, cycling Brangus, and non-cycling Brangus were 68 10; 47 28; 38 15; 94 18, respectively. For scan heifers that exhibited estrus with in 7 d after MGA withdrawal, ovulation rate tended ( P = 0.07) to be greater in Angus (11/11 = 100.0%) compared to Brangus (8/10 = 80.0%) heifers (Figure 3-1). Diameter of the ovulatory follicle was similar ( P = 0.93) between Angus (17.0 0.9 mm) and Brangus (17.1 1.0) heifers. Two Brangus he ifers did not exhibit estrus within 7 d after MGA withdrawal. One heifer had progesterone < 1.5 ng/mL at MGA withdrawal and had initiated regression of a persistent dominant follicle prior to MGA withdrawal. This heifer de veloped a new follicle wave, wh ich ovulated 11 d after MGA withdrawal (Figure 3-1). Th e other Brangus heifer had pr ogesterone > 1.5 ng/mL at MGA withdrawal and had initiated a new follicle wave after MGA withdrawal. The newly developed follicle ovulated 12 d after MGA withdrawal (F igure 3-1). Between MGA withdrawal and administration of PG, 81.8% of Angus (n = 9/ 11) and 50.0% of Brangus (n = 5/10) heifers displayed two waves of follicle growth, while 18. 2% of Angus (n = 2/11) and 30.0% of Brangus (n = 3/10) heifers displayed three waves of fo llicle growth. Of the two remaining Brangus heifers, one heifer had a single follicle wave and the other heifer had four follicle waves. One of the objectives of the experiment wa s to characterize follicle dynamics after MGA withdrawal to determine the optimum timing to administer GnRH to initiate ovulation and synchronize follicle wave development in Brangus heifers. Follicle development patterns for the first follicle wave after MGA w ithdrawal for Angus and Brangus scan heifers are presented in Figure 3-1. Emergence of the fi rst wave dominant follicle afte r MGA withdrawal was similar ( P

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65 = 0.27) for Angus (5.7 0.5 d; range 4 to 8 d) comp ared to Brangus (4.9 0.6 d; range 3 to 9 d) heifers. Diameters of the first wave dominant follicle for the Angus and Brangus scan heifers are presented in Figure 3-2. There was an effect ( P = 0.001) of day on the diameter of the dominant follicle 9 to 13 d after MGA withdrawal but there were no ( P > 0.05) breed or breed day effects. The percentage of heifers with follicles 10 mm was also evaluated for d 9 to 13 after MGA withdrawal. A diameter of 10 mm was chosen since follicles can be ovulated by exogenous GnRH at approximately 10 mm diameters (Moreira et al ., 2000). The percentage of heifers with follicles 10 mm was similar ( P > 0.05) for Angus (n=11) and Brangus (n=10) heifers on d 9 (54.5 vs. 80.0 %), 10 (81.8 vs. 70.0 %), and 11 (90.9 vs. 80.0%), respectively. However, there were more ( P = 0.02) Angus (n=100) with follicles 10 mm on d 12 compared to Brangus (70.0%) and there were more ( P = 0.002) Angus (100%) with follicles 10 mm on d 13 compared to Brangus (50.0%). One Brangus heifer ovulated on d 11 and one on d 12 after MGA withdrawal. When normalized to the day of emergence for the first follicle wave, there was an effect of day ( P = 0.001) on diameter of the dominant fo llicle (Figure 3-3) but there were no ( P > 0.05) breed or breed day effects. Dominant folli cles reached a maximal diameter on a similar ( P = 0.15) day following emergence and at a similar ( P = 0.61) diameter for Angus (7.5 0.7 d; 14.5 0.7 mm) and Brangus (5.9 0.8 d; 14.0 0.8 mm), respectively. From day of emergence to maximal diameter, follicle growth rates were similar ( P = 0.42) for Angus (1.6 0.2 mm/d) and Brangus (1.8 0.2 mm/d) heifers. When the second follicle wave was normalized to the day of emergence (Figure 3-3), there was an effect of day ( P = 0.001) on diameter of the domin ant follicle but there were no ( P >

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66 0.05) breed or breed day effects. Breed tended (P = 0.10) to affect the day of emergence after MGA withdrawal of the second wave dominant follicle where emergence occurred on d 11.7 0.8 (range 8 to 16 d) for Brangus compared to d 13.5 0.7 (range 10 to 16 d) for Angus. Dominant follicles reached a maximal diameter on a similar ( P = 0.68) day following emergence for Angus (6.5 0.6) compared to Brangus (6.1 0.7 d), but maximal diameter of the dominant follicle tended ( P = 0.06) to be greater in Angus (15.5 0.7 mm) compared to Brangus (13.3 0.8 mm). There was no ( P = 0.35) effect of breed on follicle growth rate from day of emergence to maximal diameter of the second wave dominan t follicle. Follicle grow th rate for the Angus was 1.9 0.2 mm/d and 1.7 0.2 mm/d for the Brangus heifers. For the third follicle wave, there was an effect of day ( P = 0.001) on diameter of the dominant follicle when normalized to the day of emergence (Figure 3-3). There were no ( P > 0.05) breed or breed day effects on mean diamet er of the third wave dominant follicle. Breed had no ( P = 0.24) effect on day of emergence of th e third wave dominant follicle after MGA withdrawal where emergence occurred 17.5 1. 6 d (range 17 to 18 d) for Angus and 14.8 1.2 d (range 12 to 17 d) for Brangus. Third wa ve dominant follicles had a similar ( P = 0.40) maximal diameter on a similar ( P = 0.76) day after emergence for Angus (13.0 0.9 mm; 5.5 0.6 d) and Brangus (14.0 0.6 mm; 5.3 0.5 d), resp ectively. Additionally, follicle growth rate from day of emergence to maximal diameter of the third wave dominant follicle was similar ( P = 0.50) for Angus (1.8 mm/d) and Brangus (2.1 mm/d) heifers. On the day of PG administration, diamet er of the largest follicle tended ( P = 0.09) to be greater for Angus compared to Br angus heifers (Table 3-3). Cyc ling status and breed cycling status had no ( P > 0.05) effect (Table 3-2) on diameter of the largest follicle at PG. Likewise, there were no ( P > 0.05) ultrasound group and breed ultr asound group effects. A similar ( P =

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67 0.13) percentage of Angus (30/33; 90.9%) and Brangus (22/26; 84.6%) heifers had follicles 10 mm in diameter at PG There were no ( P > 0.05) ultrasound group, breed ultrasound group, cycling status, and breed cycling status e ffects on percentage of heifers with follicles 10 mm at PG. The effect of follicle wave pattern (two-wave vs. three-wave) on follicle development during the 6 d prior to PG was also evaluate d with follicle development being normalized retrospectively from day of PG (Figure 3-4). Breed tended ( P = 0.07) to effect follicle development and day ( P = 0.001) did effect follicle developmen t. The number of follicle waves between MGA withdrawal and PG also affected ( P = 0.01) follicle development (Figure 3-4). There were no ( P > 0.05) breed day, breed wave, day wave, or breed day wave effects. Additionally, day of emergence af ter MGA withdrawal of the ev entual ovulatory follicle was similar ( P = 0.97) for the Angus (14.7 0.7 d) and Br angus (14.7 0.8 d) heifers (Figure 3-5). A greater ( P = 0.001) percentage of heifers that we re cycling at the start of the MGA treatment had a functional CL at PG compared to non-cycling heifers (Table 3-3). However, there was neither ( P < 0.05) a breed nor breed x cycling group effect on whether heifers had a functional CL at PG (Table 3-3). A greater ( P = 0.001) percentage of scan (20/21 = 95.2%) compared to non-scan (14/38 = 55.3%) heifers ha d a functional CL at PG, but there was no ( P = 0.37) breed ultrasound group effect. Progester one concentrations at PG were similar ( P = 0.50) for Angus and Brangus heifers (Table 3-3). Whereas, progesterone c oncentrations at PG tended ( P = 0.07) to be greater in cycling compared to non-cycling heifers, but there was no ( P = 0.31) breed cycling group effect. Furthermore, progesterone concentrations at PG tended ( P = 0.08) to be greater in scan (6.29 0.9 ng/mL) co mpared to non-scan (4.44 0.6 ng/mL) heifers but there was no ( P = 0.95) breed ultrasound group effect For both the scan and non-scan

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68 heifers with functional CL at PG, luteolysis rates were similar ( P > 0.05) for Angus (25/25) and Brangus (17/17) heifers. The effect of breed and cycling status at the initiation of MGA treatment on estrous, conception and synchronized pregnancy rates ar e presented in Table 34. Estrous response, conception rate, and synchronized pregnancy rate were similar ( P > 0.05) for Angus and Brangus heifers (Table 3-4). Cy cling status affected ( P = 0.001) estrous response as more cycling heifers exhibited estrus compared to non-cycling heifers (Table 3-4). There was no ( P = 0.93) breed cycling status effect on es trous response. When analyzed by ultrasound group, more ( P = 0.001) scan heifers exhibited estrus ( 95.2%; n=20/21) compared to non-scan (56.1%; n=23/41) heifers. There tended ( P = 0.10) to be a breed ultrasound group effect on estrous response. Estrous responses for Angus scan, Angus non-scan, Br angus scan and Brangus non-scan were 90.9% (n=11), 68.0 (n=25), 100.0 (n=10), and 37.5% (n= 16), respectively. Bree d, cycling status, and breed cycling status had no e ffect on conception rate ( P > 0.05), which was also the case for ultrasound group, and breed ultrasound group. The effect of AI sire ( P = 0.69) and AI technician ( P = 0.38) did not affect conception rate. Synchronized pregnancy rates were similar ( P = 0.52) for Angus and Brangus heifers but more ( P = 0.05) cycling heifers became pregnant during the synchronized breedi ng compared to non-cycling heifers. There were no ( P > 0.05) ultrasound group and breed cycling status effects on synchronized pregnancy rates. However, there was ( P < 0.05) a breed ultrasound group effect on synchronized pregnancy rate. Synchronized pregnancy rates for Angus scan, Angus non-scan, Brangus scan and Brangus nonscan were 60.0% (n=11), 36.0 (n=25), 60.0 (n=10), and 18.8% (n =16), respectively. Interval from PG to the onset of estrus ( 61:49 5:30; 63:14 6:19 h: m), duration of estrus (11:44 1:05; 10:08 1:15 h:m), and the number of mounts received during estrus (57 9; 42

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69 10) were similar ( P > 0.05) for Angus and Brangus heifer s, respectively. There were no ( P > 0.05) cycling status or breed cycl ing status effects on interval fr om PG to the onset of estrus, duration of estrus, or number of mounts receiv ed during estrus. Likewise, there were no ( P > 0.05) ultrasound group and breed ultrasound group eff ects for interval from PG to the onset of estrus, duration of estrus, or number of mounts r eceived during estrus. Diameter of the largest follicle at PG was negatively correlated (r = -0.60; P = 0.01) with the interval from PG to estrus in Angus heifers; whereas, diameter of the la rgest follicle at PG only tended to be negatively correlated (r = -0.33; P = 0.09) with the interval from PG to the onset of estrus in Brangus heifers. For the scan heifers, number of follicle waves between MGA withdrawal and PG had no ( P = 0.68) effect on estrous response after PG. Estrous response was 88.9% (8/9) and 100.0% (2/2) for twoand three-wave Angus heifers, respectively, and 100.0% (5/5) and 100.0% (3/3) for twoand three-wave Brangus heifers, respec tively. Neither the number of waves nor breed number of waves effected ( P > 0.05) estrous response after PG. The number of waves tended ( P = 0.09) to affect the interval from PG to the onset of estrus and interval from PG to the onset of estrus was affected by breed ( P = 0.02) and breed number of waves ( P = 0.01). Interval from PG to the onset of estrus was 63:21 5:12 h:m and 101:56 10:24 h:m for twoand three-wave Angus heifers, respectively, a nd 67:43 6:35 h:m and 57:33 8:30 h:m for twoand three-wave Brangus heifers, respectively. There tended ( P = 0.07) to be an effect of breed on conception rates but number of waves and breed number of waves did not affect ( P > 0.05) conception rate. Conception rates were 25.0% (2/8) and 0% (0/2) for twoand three-wave Angus heifers, respectively, and 40.0% (2/5) and 66.7% (2/3) fo r twoand three-wave Brangus, respectively. Synchronized pregnancy rate was not affected ( P > 0.05) by breed, number of waves, or breed

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70 number of waves. Synchronized pregnancy rate s were 22.2% (2/9) and 50 .0% (1/2) for twoand three-wave Angus heifers, resp ectively and 40.0% (2/5) and 66.7% (2/3) for twoand three-wave Brangus heifers, respectively. In heifers undergoing daily ultrasound ev aluations, 100% of the Angus (n=11) and Brangus (n=10) heifers ovulated after PG. Diameter of the ovul atory follicle before ovulation was similar ( P = 0.86) for Angus (13.1 0.4 mm) compared to Brangus (13.2 0.5 mm) heifers. Volume of the CL 8 d following ovulation was similar ( P = 0.20) for Angus (4095.6 410 mm3) compared to Brangus (4876.3 430 mm3) heifers; however, progester one concentrations were ( P = 0.05) greater for Brangus (7.10 0.53 ng/mL) co mpared to Angus (5.56 0.51 ng/mL) heifers. Discussion In order for the MGA-PG system (Brown et al., 1988) to be effectiv e, heifers need to exhibit estrus and (or) ovulate so they are in the late luteal ph ase of the estrous cycle at PG, which means a majority of heifers must exhibit estrus with 3 to 7 d after MGA withdrawal as reported by Hill et al. (1971). In the present study, one-hundred percent of the scan Angus heifers, but only 80% of the scan Brangus heifers exhibited estrus and (or) ovulated within 7 d of MGA withdrawal. Wood et al. (2001) reported that 81.5% of Bos taurus heifers ovulated within 12 d of MGA withdrawal. The two Brangus heifer s not exhibiting estrus within 7 d after MGA withdrawal, eventually exhibited estrus and ovulat ed 11 and 12 d after MGA withdrawal. One of the two Brangus heifers had a functional CL ( 1.5 ng/mL) at MGA withdr awal, which regressed shortly after MGA withdrawal. The heifer was not detected in estrus during MGA; although, cattle of Bos indicus breeding are noted for having a silent estrus (Galina et al., 1982; Orihuela et al., 1983). The heifer was pr obably at the beginning of the estr ous cycle at the initiation of MGA, which would have placed the heifer in the late luteal phase of the estrous cycle at MGA withdrawal. The other Brangus heifer had appare ntly started regression th e largest follicle at

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71 MGA withdrawal resulting in initiation of a new follicle wave, which ovulated 12 d after MGA withdrawal. The reasons for this pattern of follicle development after a long term MGA treatment are unclear. Kojima et al. (1992) repor ted that some cows receiving a MGA treatment failed to have a preovulatory LH surge within 100 h of MGA withdrawal, which may be due to luteinization (Guthrie et al ., 1970) of persistent follicles capable of secreting enough progesterone to prevent the LH surg e. This does not appear to be likely since the Brangus heifer had progesterone concentrations < 1 ng/mL for se veral days prior to a nd after MGA withdrawal. After MGA withdrawal, diameter s of the largest follicles present were similar between Angus and Brangus heifers and similar to th ose reported by Wood et al. (2001) in cycling Bos taurus heifers. The estrous response after MGA withdrawal was similar between Angus and Brangus heifers but it was considerably le ss than another study using a long term MGA treatment (Yelich et al., 1997) in yearling Bos taurus heifers. The decreased estrous response was primarily due to the decreased percentage of heifers that were cycling (39.6%) at the start of MGA. More cycling heifers (62.9%) exhibited estrus after MGA withdr awal compared to noncycling heifers (23.3%). These results and ot hers (Brown et al., 1988; Patterson and Corah, 1992; Wood-Follis et al., 2004) poi nt out the importance of ha ving heifers going through estrous cycles at the start of the M GA treatment. With that said, long-term MGA treatments induce estrous cycles in some non-cycling heifers (P atterson et al., 1989) and the MGA treatment induced estrous cycles in 51.9% of the non-cy cling heifers in the present experiment. Additionally, the percentage of noncycling heifers that were cyc ling at PG was similar between Angus and Brangus heifers sugges ting that the MGA treatment was equally effective at inducing estrus in non-cycling heifers across the two breed types.

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72 The incidence of a silent estrus (Galina et al., 1982; Orihuela et al., 1983) after MGA withdrawal also contributed to the decreased estr ous response. Of the heifers that were cycling at the start of MGA, 84% had a functional CL at PG; however, only 63.9% of the cycling heifers exhibited estrus after MGA withdrawal. Theref ore, several Angus and Brangus heifers did not exhibit estrus after MGA withdrawal. Because he ifers were fitted with radiotelemeric estrous detection devices, it is unlikely that the method of estrous detection was the reason for decreased estrous response. The incidence of silent estrus is well documented in Bos indicus cattle (Galina et al., 1982; Orihuela et al., 1983), but it is unclear why so many heifers did not exhibit estrus after MGA withdrawal since persistent fo llicles have increased estrogen concentrations (Henricks et al., 1973; Kojima et al., 1992). Increas ed ambient temperatures have been reported to increase the incidence of ovulation without estr us in dairy heifers (Gwa zdauskas et al, 1981). However, it is unlikely that elevated temperat ures caused the increased incidence of silent estrus since the experiment was conducted from February to April when ambient temperatures were probably to low to initiate heat stress. One of the primary objectives of the experiment was to determine the follicular wave pattern from MGA withdrawal to PG in Bra ngus compared to Angus heifers. There was considerable variation between Brangus compared to Angus heifers in the number of follicle waves between MGA withdrawal and PG. Eight-t wo percent of Angus heifers had two follicle wave patterns with the remaining 18% having three follicle wave patterns. In comparison, only 50% of the Brangus heifers had two follicle wa ves and the remaining heifers had either one, three, or four follicle waves. Therefore, based on the number of follicle waves, there is considerable variation in follicle development patterns for Brangus compared to Angus heifers. The increased incidence of three and four follicle wave patterns in Brangus heifers is similar to

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73 reports in cattle of Bos indicus breeding having more three and four follicular wave patterns (Rhodes et al., 1995; Viana et al ., 2000) during a normal estrous cycle compared to two-wave patterns. Growth and development of the first wave dominant follicle afte r MGA withdrawal was similar between Angus and Brangus heifers with re spect to day of emergence, growth rate, day of maximal diameter, and maximal diameter of the dominant follicle. With respect to the first wave follicle development patterns in the cu rrent study, they are in agreement with other observations reported in Bos taurus cattle (Sirois and Fortune, 1988; Ginther et al., 1989). Conversely, Viana et al. (2000) re ported that emergence of the first follicle wave occurred earlier, had a reduced growth rate an d maximal diameter in non-lactating Bos indicus cows compared to the Brangus heifers in the presen t study. Emergence of the second follicle wave tended to occur later in Angus co mpared to Brangus heifers. Additionally, Angus heifers had a greater maximal diameter of the second wave dominant follicle compared to Brangus heifers. The increased incidence of three and four follicle wave patterns is likely influenced by day of emergence and maximal follicle diameter of the second wave dominant follicle. Second wave dominant follicles emerged later (Sirois a nd Fortune, 1988) and reached a greater maximal diameter (Ginther et al., 1989) in cattle displayi ng two compared to three wave follicle growth profiles. No differences were observed between Angus and Brangus heifers with regard to the developmental characteristics of the third wave dominant follicle. Characteristics of follicle development, particularly the second and third wa ves, should be interpreted with discretion since PG was administered before some of the heif ers were allowed to complete a normal estrous cycle.

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74 Another research objective was to determine th e best time to incorporate GnRH into the MGA-PG system. Wood et al. ( 2001) conducted an experiment where Bos taurus heifers received GnRH 12 d after the withdrawal of a 14 d MGA treatment with PG 7 d after GnRH compared to the MGA-PG system with PG ad ministered 19 d after MGA withdrawal. They hypothesized that GnRH would induce a new follic le wave, which would increase the synchrony of follicle development at PG resulting in a mo re synchronous estrus. As presented in Figure 12, there was considerable variati on in the growth and developmen tal profiles of the first wave dominant follicle in Brangus comp ared to Angus heifers. For the eleven scan Angus heifers, 100% of the heifers had first wa ve dominant follicles that we re in the growing phase by d 12 after MGA withdrawal. In contra sts, only 60% of the Brangus heif ers had a first wave dominant follicle in the growing phase by d 12 after MGA withdrawal. Furthermore, two Brangus heifers had follicles that began to go through atresia by d 10 and another heifer had follicle emergence on d 9 after MGA withdrawal. The asynchrony of follicle development after MGA withdrawal for Brangus heifers was further reflected in the percentage of heifers with follicles 10 mm between d 9 to 13 after MGA wit hdrawal, which is important sinc e GnRH is typically effective in growing follicles 10 mm in diameter (Moreira et al ., 2000). By d 11 after MGA withdrawal, 91% of Angus heifers had follicles 10 mm and 100% by d 12 and 13. In contrasts, by d 11 after MGA withdrawal, 80% of Brangus heifers had follicles 10 mm and 70.0% by d 12 and 50% by d 13. The reduction in follicles 10 mm by d 12 and 13 was a result of heifers exhibiting estrus and ovulating, and several heifers having first wave dominant follicles that began to enter atresia and regres s by d 12. Therefore, the optimal time to administer GnRH to Brangus heifers should occur approximately 10 d following MGA withdrawal for a couple of reasons. First, a greater percentage of Brangus heifers would have follicles 10 mm diameter

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75 and in the growing phase of follicle development. Second, for heifers failing to exhibit estrus and ovulate within 7 d after MGA withdrawal the heifers would develop a new follicle wave that should be in the growing phase by d 10 and responsive to GnRH. Because of the asynchrony of follicle deve lopment by d 12 after MGA withdrawal in Brangus heifers, administering GnRH within 2 to 4 d after MGA withdraw al is an option that should also be investigated. By 4 d after MGA withdrawal, the variation in follicle development appears minimal and most heifers have follicles 10 mm in diameter. However, the effectiveness of GnRH to ovulate a majority of the large persistent dominant follicles needs to be addressed in further experiments. At PG, diameter of the eventual ovulatory fo llicle was similar between Angus and Brangus heifers, which agree with a report by Wood et al. (2001) in Bos taurus heifers synchronized with MGA-PG. Although the range in day of emergen ce of the eventual ovulatory follicle was considerable for Angus and Brangus heifers, diam eter and growth rate of the eventual ovulatory follicle during the 6 d prior to PG was similar for Angus and Brangus heifers. It is also interesting to note that even with the asynchronous follicle development that Brangus heifers experienced between MGA withdrawal and PG, fo llicle development at PG was similar between Angus and Brangus heifers. Additionally, the interv al from PG to the onset of estrus was similar between breeds, which can be attributed to the si milar diameter of the eventual ovulatory follicle at PG for the Angus and Brangus heifers. With that said, there was a significant interaction between breed and the number of follicle waves at PG on the interval from PG to the onset of estrus. Angus and Brangus heifers that had two follicle waves had a similar interval from PG to the onset of estrus. Growth and development of the eventual ovulator y follicle was similar between two wave Angus and Brangus heifers, re sulting in similar diameters of the eventual

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76 ovulatory follicle at PG. However, Angus heifers with three follicle waves had a longer interval from PG to the onset of estrus compared to Brangus heifers with three follicle waves. The greater interval from PG to the onset of estrus for the three wa ve Angus heifers was due to the fact that the follicles were in the early stages of follicle development and diameter of the eventual ovulatory follicle was c onsiderably less than compared to three wave Brangus heifers, which had a more mature follicle and a shorter interv al from PG to the onset of estrus. Similarly, Sirois and Fortune (1988) reported that the size of the eventual ovulatory follicle at luteolysis was negatively correlated with the interval to estrus. Angus and Brangus heifers were synchronize d with the MGA-PG protocol described by Lamb et al. (2000) where PG was administered 19 d after the last day of MGA; although, there was a slight modification as the Brangus he ifers received a split-PG 19 (12.5 mg) and 20 (12.5 mg) d following MGA withdrawal. For the Angus and Brangus heifers that had a functional CL at PG, PG initiated luteolysis in 100% of the heifers similar to a report by Bridges et al. (2005). Estrous response, conception, and synchronized pregnancy rates were similar between Angus and Brangus heifers. In cont rast, the estrous response and sy nchronized pregnancy rates for Angus and Brangus heifers are cons iderably less than those observ ed by other authors (Nix et al., 1998; Lamb et al, 2000; Sa lverson et al., 2002) in Bos taurus heifers synchronized with the MGA-PG system. The decreased estrous response and subsequent ly decreased synchronized pregnancy rates can be attributed to the decrease d percentage of heifers that were cycling at the initiation of the MGA treatment. Both Angus and Brangus heifers that were cycling prior to the beginning of MGA had a significantly greater es trous response and synchronized pregnancy rate compared to non-cycling heifers. Furthermor e, the cycling Angus and Brangus heifers had estrous responses

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77 and synchronized pregnancy rates that are si milar to those reported for experiments in Bos taurus synchronized with a similar MGA-PG system (Nix et al., 1998; Lamb et al, 2000; Salverson et al., 2002). The importance of cycling status on th e overall effectiveness of the MGA-PG system is supported by this and other studies (Brown et al., 1988; Patterson et al ., 1989). Attainment of puberty prior to the beginning of th e breeding season is also important since fertility increases as the number of estrous cycles a he ifer has prior to the initiation of the breeding season increases (Byerley et al., 1987; Galina et al., 1996). Results from the curre nt study suggest that having heifers of Bos indicus Bos taurus breeding cycling prior to th e start of the breeding season maybe one of the most important, if not the most important factors, in getting Bos indicus Bos taurus heifers pregnant to a synchronized AI br eeding. Although, having a high percentage of Bos indicus Bos taurus heifers cycling at the start of the breeding season can be difficult since cattle of Bos indicus breeding reac h puberty at a later age than Bos taurus heifers (Plasse et al., 1968; Baker et al., 1989; R odrigues et al., 2002). Cycling status had no effect on conception rate, although, conception rates for the Angus and Brangus heifers was still subs tantially less than other studies (Brown et al., 1988; Jaeger et al., 1992; Lamb et al., 2000) in Bos taurus heifers synchronized with the MGA-PG system. Conversely, conception rates of Angus and Bran gus heifers are similar to conception rates reported by Bridges et al. (2005) in Bos indicus Bos taurus heifers synchronized with the MGA-PG system. However, within the Bridges et al. (2005) study, there was a significant breed effect on conception rates with cycling Angus heifers having a 28.6% greater conception rate compared to cycling Bos indicus Bos taurus heifers. The reason (s) for the considerable variation in conception rates with in and between studies is difficu lt to evaluate and could be due to several factors including fer tility of the AI sire (DeJarnette et al., 2004), AI technician

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78 (Garcia-Ispierto et al., 2007), stage of follicle development at PG (Austin et al., 1999; Townson et al., 2002), estrous cycling stat us at the start of a breeding se ason (Byerley et al., 1987), and environmental conditions in which the studies ar e conducted (Wolfenson et al., 1995). What, if any, effects that frequent working of cattle had on fertility are unclear. Conception rate was 7% numerically greater in non-scan compared to scan heifers. W ithin scan and non-scan heifers, each breed responded differently. Conception ra tes were nearly 33% greater in non-scan compared to scan Angus heifers but only 10% gr eater in scan compared to non-scan Brangus heifers. To our knowledge, there are no studies evaluating what effect frequent ultrasonography examinations have on fertility. However, freque nt ultrasound examinations can have a negative effect on luteal func tion after ovulation in Bos indicus Bos taurus cattle (Lemaster et al., 1999). Luteal function in the scan heifers does not a ppear to be compromised as all scan heifers developed a functional CL that secreted progester one 8 d after the PG induced estrus. However, luteal function was not evaluated in the non-scan heifers so no comparison can be made between heifers that were or were not frequently handled. In the scan heifers, Brangus heifers had grea ter progesterone concentrations compared to Angus heifers but CL volume was only numerical ly greater in Brangus compared to Angus heifers. These results are in agreement with Al varez et al (2000) but do not agree with a report that Bos indicus cattle have a smaller CL (Irving et al., 1978) and decreased progesterone concentrations (Segerson et al., 1984). In summary, follicle development from MGA withdrawal to PG is different between Angus and Brangus heifers. A decreased percen tage of Brangus heifer s exhibited estrus and ovulated within 7 d after MGA withdrawal. The increased incidence of three and four follicle wave patterns in Brangus heifers contributed to the altered follicle wave dynamics compared to

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79 Angus heifers. Although, diameter of the firs t wave follicle wave from d 9 to 13 after MGA withdrawal was similar between Angus and Brangus heifers, the number of follicles 10 mm and in the growing phase were decreasing in Br angus heifers compared to Angus heifers by d 11 and 12 after MGA withdrawal. These results su ggest that addition of GnRH to the MGA-PG system for Brangus heifers may need to occur pr ior to d 12 after MGA withdrawal. Furthermore, administering, GnRH immediately after MGA wit hdrawal (i.e., day 3 to 4) may actually work better to synchronize follicle development. Even though follicle development was slightly different between Angus and Brangus heifers, the estrous response, conception rate, and synchronized pregnancy rate were similar between breeds. Angus and Brangus heifers that were cycling prior to the start of the MGA treatment had the greatest synchronized pregnancy rates. Implications Follicle development during the period between MGA withdrawal and PG was different for Brangus compared to Angus heifers. The mo st opportune time to administer GnRH after a 14 d MGA treatment to synchronize follicle development in Bos indicus Bos taurus heifers may be within 3 to 4 d after MGA withdrawal. Additional research will need to be conducted in Bos indicus Bos taurus heifers to evaluate the effectiven ess of adding GnRH to the MGA-PG system. Regardless of breed, heifers that were cycling prior to the start of the MGA treatment had greater synchronized pregnanc y rates compared to non-cycling heifers. Therefore, producers need to make sure that a majority of yearling Bos indicus Bos taurus heifers are going through estrous cycles at the st art of a synchronization treatment to achieve acceptable pregnancy rates to a synchronized breeding.

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80 Table 3-1. Age, body weight (BW), body condition score (BCS), and estrous cycling status (Cycling) at the initiation of the 14 d me lengestrol (MGA) treatment for Angus and Brangus heifers by ultrasound group (s can vs., non-scan) (LS means SE).a Variable n Age, d BW, kg BCSb Cycling, %c Angus 40 384 2.5 348 5.4 6.3 0.1 25/40 = 62.5 Brangus 26 376 2.9 356 6.1 6.1 0.1 11/26 = 42.3 Non-scan 45 377 2.2 347 4.7 6.1 0.1 17/45 = 37.8 Scan 21 384 3.1 357 6.6 6.2 0.1 19/21 = 90.5 Angus non-scan 29 383 2.6 342 5.6 6.2 0.1 14/29 = 48.3 Angus scan 11 386 4.3 353 9.2 6.3 0.1 11/11 = 100.0 Brangus non-scan 16 370 3.6 352 7.6 6.1 0.1 3/16 = 18.8 Brangus scan 10 382 4.5 360 9.6 6.1 0.1 8/10 = 80.0 P values Breed 0.03 0.32 0.02 0.02 Group 0.07 0.24 0.47 0.001 Breed Group 0.26 0.91 0.38 0.31 aMeasurements taken on initial day of MGA feeding, experimental day 0. bBCS: 1 = emaciated, 5 = moderate; 9 = obese. cCycling status determined by blood samples collect ed d -16, -7. and 0 of experiment. Heifers were classified as cycling if blood pl asma progesterone concentrations were 1.5 ng/mL at two of three blood samples and classified as non-cycl ing if blood plasma proge sterone concentrations were < 1.5 ng/mL at all three blood samples.

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81 Table 3-2. Estrous response, interval to estr us, duration of estrus, and number of mounts received during a HeatWatch detected estrus for the 7 d following a 14 d melengestrol (MGA) treatment by breed, ultrasound group (scan vs., no-scan), and breed ultrasound group.a Variable n Estrous response, %b Interval from MGA withdrawal to estrus, h:m Duration of estrus, h:m Number of mounts Angus 40 42.5 91:11 9:37 12:55 1:33 66 11 Brangus 25 48.0 71:25 12:07 10:15 1:57 63 Scan 21 76.2 94:52 9:54 11:53 1:35 65 11 Non-scan 44 29.6 67:45 11:53 11:17 1:55 64 13 Angus scan 11 72.7 103:37 13:59 14:21 2:15 72 15 Angus non-scan 29 31.0 78:46 13:11 11:29 2:07 60 15 Brangus scan 10 80.0 86:07 13:59 9:24 2:15 58 15 Brangus non-scan 15 26.7 56:43 19:47 11:05 3:11 69 22 P-values Breed 0.88 0.21 0.29 0.89 Ultrasound group 0.001 0.09 0.81 0.98 Breed Ultrasound group 0.62 0.88 0.37 0.53 aUltrasound group included scan heifers, which had daily ultrasonography starting the day of MGA withdrawal and continued for 19 d and non-scan heifers did not receive any daily ultrasonography bNumber of heifers exhibiting es trus within 7 d of MGA withdr awal divided by the total number of heifers treated.

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82 Table 3-3. Percentage of heif ers with a functional CL, proge sterone concentration (LSM SE), and diameter of the largest follicle (LSM SE) at the initial PG treatment.a Variable n Functional CL, %b Progesterone concentration, ng/mL Diameter of largest follicle, mm Angus 33 75.8 5.5 0.7 11.8 0.3 Brangus 26 61.5 4.8 0.8 11.1 0.3 Cyclingc 32 84.4 6.1 0.7 11.5 0.3 Non-cycling 27 51.9 4.2 0.8 11.4 0.3 Angus cycling 21 81.0 5.9 0.8 11.9 0.4 Angus non-cycling 12 66.7 5.1 1.1 11.8 0.5 Brangus cycling 11 90.9 6.2 1.2 11.2 0.5 Brangus non-cycling 15 40.0 3.3 1.0 11.0 0.4 P-values Breed 0.87 0.50 0.09 Cycling group 0.01 0.07 0.84 Breed Cycling group 0.15 0.31 0.88 aInitial PG treatment was administered 19 d follo wing the withdrawal of a 14 d MGA treatment. Angus heifers received a single (25 mg) PG treatment while Bra ngus heifers received split (12.5 mg) PG treatments on d 19 and 20. bA heifer was deemed to have a functional CL if progesterone concentrations were 1.5 ng/mL in the presence of luteal tissue as determined by ultrasonography. c Cycling status determined by blood samples coll ected d -16, -7. and 0 of experiment with MGA treatment starting on d 0. Heifers were classi fied as cycling if blood plasma progesterone concentrations were 1.5 ng/mL at two of three blood samp les and classified as non-cycling if blood plasma progesterone concentrations we re < 1.5 ng/mL at all three blood samples.

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83 Table 3-4. Effect of breed and cycling status at the initiati on of a 14 d melengestrol acetate treatment on estrous response, conception ra te and synchronized pregnancy rates of Angus and Brangus heifers synchroni zed with a 14 d melengestrol acetate treatment followed by either a single (A ngus) or split (Brangus) prostaglandin F2 treatment 19 d later.a Variable Estrous response, %b Conception rate, %c Synchronized pregnancy rate %d Angus 27/36 = 75.0 12/27 = 44.4 12/36 = 33.3 Brangus 16/26 = 61.5 9/16 = 56.3 9/26 = 34.6 Cycling 29/34 = 85.3 15/29 = 51.7 15/34 = 44.1 Non-cycling 14/28 = 50.0 6/14 = 42.9 6/28 = 21.4 Angus cycling 20/23 = 87.0 10/20 = 50.0 10/23 = 43.5 Angus non-cycling 7/13 = 53.8 2/7 = 28.6 2/13 = 15.4 Brangus cycling 9/11 = 81.8 5/9 = 55.6 5/11 = 45.5 Brangus non-cycling 7/15 = 46.7 4/7 = 57.1 4/15 = 26.7 P-values Breed 0.59 0.30 0.52 Cycling group 0.001 0.54 0.05 Breed Cycling group 0.93 0.48 0.61 a Cycling status determined by blood samples coll ected d -16, -7. and 0 of experiment with MGA treatment starting on d 0. Heifers were classi fied as cycling if blood plasma progesterone concentrations were 1.5 ng/mL at two of three blood samp les and classified as non-cycling if blood plasma progesterone concentrations we re < 1.5 ng/mL at all three blood samples. bPercentage of heifers exhibiti ng estrus during the 5 d followi ng the initial prostaglandin F2 treatment out of the total number treated. cPercentage of heifers that were pregnant to AI of the total nu mber of heifers that exhibited estrous and were AI. dPercentage of the heifers that were pregna nt to AI out of the total number treated.

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84 A) 0 5 10 15 20 25 04812162024 Days after MGAFollicle size, mm B) 0 5 10 15 20 25 04812162024 Days after MGAFollicle size, mm Figure 3-1. Profiles of ovulatory follicles after a 14 d melengestro l acetate (MGA) treatment and the subsequent first wave dominant follicle growth profiles for A) Angus and B) Brangus heifers. Stars indi cate day of ovulation and the tw o dashed lines indicate the potential range when GnRH could be admini stered to ovulate the first wave follicle after MGA withdrawal to sync hronize the next follicle wave.

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85 0 2 4 6 8 10 12 14 16 910111213 Days after MGAFollicle diameter, mm Angus Brangus Figure 3-2. Mean first wave dominant follicle diameter during days 9 to 13 following withdrawal of a 14 d melenge strol acetate (MGA) treatment for Angus (n = 11) and Brangus (n = 10) heifers in the scan gr oup. One Brangus heifer ovulated on day 11 and another on day 12. Breed (P > 0.05), Day (P < 0.05), Breed Day (P > 0.05)

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86 0 5 10 15 01234567012345012345 Day after Wave EmergenceFollicle Diameter, mm Angus Brangus Figure 3-3. Mean diameter of the A) firs t, B) second, and C) third follicle wave following withdrawal of melengestrol acetate (MGA) for Angus and Brangus heifers. Follicle wa ves were normalized to the day of wave emergence. For all three follicle wave patterns: Breed (P > 0.05), Day (P < 0.05), Breed Day (P > 0.05). A) B) C)

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87 0 5 10 15 20 -6-5-4-3-2-10 Days Before PGFollicle size, mm Angus two wave Angus three wave Brangus two wave Brangus three wave Figure 3-4. Diameter of th e eventual ovulatory follicle prior to prostaglandin F2 (PG) treatment for Angus and Brangus heifers ba sed on the number of follicle waves from the last day of a 14 d melengest rol acetate treatment to a PG treatment 19 days later. Breed (P = 0.07), Day (P = 0.001), Number of Waves (P = 0.01), Breed Day (P = 0.85), Breed Number of Waves (P = 0.19) Number of Waves Day (P = 11), Breed Day Number of Waves (P = 0.38).

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88 A) 0 5 10 15 20 101112131415161718192021222324 Days After MGAFollicle Size, m m B) 0 5 10 15 20 101112131415161718192021222324 Days After MGAFollicle Size, m m Figure 3-5. Follicle growth patterns for the eventual ovulatory follicle preceding the initial prostaglandin F2 (PG) treatment, which occurred on day 19 (indicated by the arrow) in A) Angus and B) Brangus heifers. Solid lines indicate two-wave follicle growth patterns, long dashed lines indicate thr ee-wave follicle growth patterns and short dashed lines indicate either a singleor four-wave follicle growth pattern.

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89 CHAPTER 4 REFINEMENT OF THE 14 D MELENGESTROL ACETATE (MGA) TREATMENT + PROSTAGLANDIN F2 (PG) 19 D LATER ESTROUS SYNCHRONIZATION SYSTEM IN HEIFERS OF Bos indicus Bos taurus BREEDING Introduction Melengestrol acetate (MGA) administer ed for 14 d with prostaglandin F2 (PG) administered 17 d after MGA withdr awal is one of the most wide ly used estrous synchronization systems (MGA-PG) in yearling beef heifers (Bro wn et al., 1988; Patterson and Corah, 1992). The MGA-PG synchronization system is an effective and predictable system in Bos taurus heifers (Brown et al., 1988; Patt erson and Corah, 1992; Lamb et al., 2000) but is less effective in Bos indicus Bos taurus heifers (Bridges et al., 2005). Modi fying the delivery of PG from a single to two consecutive split PG treatments improved the estrous resp onse and synchronized AI pregnancy rates in Bos indicus Bos taurus heifers (Bridges et al., 2005), but the synchronized AI pregnancy rates are still less than values observed in Bos taurus heifers. Wood et al. (2000) incorporat ed gonadotropin-releasing hor mone (GnRH) into the MGAPG system by administering GnRH 12 d afte r the end of MGA treatment followed by PG 7 d later and reported an increased synchrony of estr us compared to the traditional MGA-PG system (Lamb et al., 2000). Recent research in our lab (See Chapter 3) indicated that follicle development between the MGA withdrawal a nd PG treatment was not as synchronous in Bos indicus Bos taurus (Brangus) heifers compared to Bos taurus (Angus) heifers, which resulted from more three wave follicle development patter ns in Brangus compared to Angus heifers. Results from Chapter 3 raised the possibility that the most effective time to introduce GnRH into the MGA-PG system may be either a couple of da ys after MGA withdrawal or 10 d after MGA. Therefore, two experiments were conducted to evaluate: 1) the effect of GnRH administered either 3 or 10 d after the last day of a 14 d MGA treatment with PG 7d after to synchronize

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90 estrus, and 2) evaluate the most effective GnRH treatment from Experiment 1 in a field trial utilizing yearling Bos indicus Bos taurus and Bos taurus heifers. Materials and Methods Experiment 1 was conducted at the University of Florida, Beef Research Unit from October to December, 2004 using 2-year-old Bos indicus Bos taurus (n = 58) heifers. Breed composition of the heifers consisted of 3 to 78% Brahman ( Bos indicus ) breeding with the remainder being Angus ( Bos taurus ) breeding. Heifers were equa lly distributed by percentage Brahman breeding to one of two treatments prior to the start of the experiment. Mean (LSM S.E.) age, BW, and body condition score (BCS: Richards et al., 1986) of the heifers were 640 3.7 d, 395 6.3 kg, and 4.8 0.1 for one group desi gnated as the G3 treatment, and 641 3.8 d, 406 6.3 kg, and 4.9 0.1 for the other group desi gnated as the G10 treatment, respectively. Treatment groups were maintained in adjacent pa stures throughout the experiment. Prior to the start of the experiment, both treatments were pre-synchronized according to the protocol described by Lemaster et al. (1999), so that heifers would st art a 14 d melengestrol acetate (MGA) treatment at d 2 of the estrous cycle. Briefly, heifers received a progesterone insert (EAZI-BREED CIDR ; Pfizer Animal Health, New York, NY) concurrent with 2 mg (i.m.) estradiol benzoate. Seven days later, CIDR we re removed and heifers received 25 mg (i.m.) prostaglandin F2 (PG; Lutalyse Sterile Solution, Pf izer Animal Health, New York, NY) followed 24 h later with 0.5 mg (i.m.) estradiol benzoate to synchronize estrus and initiate ovulation. Two days after the expression of estrus, both trea tments received MGA (0.5 mghd1d-1; MGA Premix, Pfizer Animal Health, New York, NY) in a total mixed ration and the MGA was administered for 14 d. Th ree days after the la st day of MGA, heifers in the G3 (n = 30) treatment received GnRH (100 g i.m. Cystorelin, Merial, Inc., Duluth, GA); whereas, ten

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91 days after the last day of MGA, heifers in th e G10 (n=28) treatment received GnRH. Seven and eight days after GnRH treatment, heifers in each respective treatment rece ived 12.5 mg PG (i.m.; Lutalyse Sterile Solution) on each day. The experiment was designed so that PG was administered on the same days for both the G3 and G10 treatment. Heifers were observed for behavioral estrus for approximately 1 h at 0700 and 1700 h daily from MGA withdrawal until GnRH was administered for the G3 and G10 treatments. The same estrous detection protocol was al so implemented during the 7 d after the initial PG treatment. All heifers received Ka mar detectors (Kamar Marketing Group, Steamboat Springs, CO) at MGA withdrawal and a new detector at the initial PG treatment to assist in estrous detection. Behavioral estrus was defined as a heifer standing to be mounted by another heifer and (or) signs of vaginal mucous. If a heifer was not detected in estrus by visual observation but had a Kamar that was one-quarter to completely activated, the heifer was consider ed to have been in behavioral estrus. A three-day estrous respons e after MGA withdrawal was determined for the G3 and G10 heifers combined and was defined as the total number of heifers exhibiting estrus within 3 d after MGA withdrawal divided by th e total number of heifers in the G3 and G10 treatments. A five-day estrous response after MGA withdrawal was also determined for the G10 treatment and was defined as the total number of heifers exhibiting estrus within 5 d after MGA withdrawal divided by the total num ber of heifers in the G10 trea tments. Estrous response after PG was determined as both a three-day estrous re sponse and total estrous response within the G3 and G10 treatments heifers. Three-day estrou s response after PG was defined as the total number of heifers exhibiting estr us within 3 d after the initial PG divided by the total treated. Total estrous response was defined as the total nu mber of heifers displaying estrus within 7 d after the initial PG divided by the total number treated.

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92 To evaluate what effect stage of follic le development (SOF) may have had on the effectiveness of GnRH to initia te ovulation, heifers within th e G3 and G10 treatments were assigned to receive either no PG (G3, n = 6; G10, n = 6) or PG (12.5 mg i.m.; Lutalyse Sterile Solution) on d 4 and 5 (G3, n = 5; G10, n = 5), 8 and 9 (G3, n = 8; G10, n = 7), or 12 and 13 (G3, n = 6, G10, n = 5) of MGA treatment to initiate lu teolysis. These days were chosen to simulate variable periods of low level pr ogesterone exposure during MGA co mparable to heifers being at d 2 (no PG), 6 (PG d 12/13), 10 (PG d 8/9), or 14 (P G d 4/5) of the estrous cycle at the initiation of MGA treatment. The variable lengths of low level progestogen exposure were used to vary the duration of persistence of the dominant pre ovulatory follicle prior to MGA withdrawal. Blood plasma samples were collected via jugular veinipuncture from all heifers before they received PG during MGA to confirm the pres ence of a corpus luteum (CL) and at MGA withdrawal to confirm that lute olysis had occurred. Heifers in the d 6, 10, and 12 SOF groups were determined to be at the assigned SOF de velopment if progesterone concentrations were 1 ng/mL at PG during MGA treatment followed by progesterone concentrations < 1 ng/mL at MGA withdrawal with the absence of a corpus luteum (CL). Heifers in the no PG group would have had progesterone concentrations that were 1 ng/mL at MGA withdrawal. Presence or absence of a CL was determined by transrec tal ultrasonography (Al oka 500v, Corometrics Medical Systems, Wallingford, CT) equipped with a 7.5 MHz linear array transducer. Ovaries of all heifers were evaluated using transrectal ul trasonography (equipped with a 7.5 MHz linear array transducer) at MGA withdrawal, GnRH, 48 h after GnRH, initial PG after GnRH, and 8 d after the expression of estrus for heif ers that were observed in estrus after PG. At each ultrasonography evaluation, height and width of all luteal structures, luteal cavities, and follicles 3 mm in diameter were meas ured using the internal cal ipers of the ultrasonography

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93 machine and their locations on the ovaries were recorded. Volume of the CL was calculated using the formula for volume of a sphere ( d3/6). When a luteal cavity was present, its volume was subtracted from the volume of the outer sphe re resulting in net lut eal volume (CL volume). Additionally, blood plasma samples were coll ected via jugular veinipuncture at each ultrasonography examination and 48 h after the initial PG to determine plasma progesterone concentrations. Blood plasma samples were co llected into evacuated tubes containing an anticoagulant (EDTA; Becton, Dickinson and Compa ny, Franklin Lakes, NJ). After collection, blood plasma samples were immediately placed on ice until they were centrifuged (3000 g for 15 min) within 3 h after collection. Plasma was separated and stored at -20C until analysis for progesterone concentrations, in multiple assays, as previously described. Intraand interassay CV of the assays were 5.4 and 5.6%, respectivel y, and sensitivity of the assay was 0.1 ng/mL. Ovulation to GnRH was defined as the largest follicle present at GnRH followed by its disappearance at the ultrasonogra phy exam 48 h after GnRH. Seven days after GnRH, the location of the ovulated follicle was verified by presence of a CL as determined by ultrasonography. The CL was deemed functiona l if progesterone concentrations were 1 ng/mL. Eight-days after the PG induced estrus ovulation was confirme d by the presence of a functional CL as previously described. Heifers not exhibiting estrus within 7 d after PG underwent ultrasonography and bloo d sampling 10 d after the ini tial PG to assess ovarian function. Because the G3 and G10 treatments were not started until either 3 or 10 d after MGA withdrawal, a three-day estrous response after MGA withdrawal fo r the G3 and G10 treatments combined was tested using the GENMOD procedur e with SAS (SAS Inst. Inc., Cary, NC) with SOF development being the independent variable. The GENMOD procedure of SAS was also

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94 used to test SOF effects within each treatment. Independent variables tested were treatment, SOF, and treatment SOF effects. Dependent variables tested were ovulation rate to GnRH, ovulation rate after PG, estrous response after MGA withdrawal, percenta ge of heifers with follicles 10mm at GnRH, percentage of heifers with follicles 10mm at PG, and percentage of heifers with progesterone concentrations 1 ng/mL at PG. Additionally, three-day estrous response and total estrous response after PG was tested using the GENMOD procedure. Progesterone concentration and diameter of the la rgest follicle at MGA withdrawal for the G3 and G10 treatments combined were tested using the GLM procedure of SAS, with the independent viable being SOF development. Diam eter of the largest follicle present at GnRH, diameter of the largest follicle present at PG, progesterone concentrations at PG, and progesterone concentrations 8 d following behavi oral estrus were also tested using the GLM procedure of SAS. Independent variables test ed were treatment, SOF, and treatment SOF effects. Five heifers from the G3 and G10 tr eatments did not conform to the assigned SOF as mentioned previously and they we re removed from all analyses. Experiment 2 was conducted at two locations from December, 2004 to April, 2005. Yearling Bos taurus (Angus; n = 57) and Bos taurus Bos indicus crossbred heifers (n = 178) from the Dicks Brothers Farm, Lake City, FL (Location 1) and yearling Bos indicus Bos taurus heifers (n = 117) from the Davis Ranch, Al achua, FL (Location 2) were used in Experiment 2. Prior to the start of the e xperiment, heifers at Lo cation 1 were randomly distributed to one of two treatments and a BCS was recorded; whereas, heifers at Location 2 were equally distributed to one of two trea tments by BCS and reproduc tive tract score (RTS; Anderson et al., 1991). Mean BCS (L SM S.E.) were 5.0 0.05 for the Bos taurus and 5.0 0.03 for Bos indicus Bos taurus heifers at Location 1 and 5.4 0.04 for Bos indicus Bos

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95 taurus heifers at Location 2. Mean RTS (LSM S.E.) was 3.8 0.8 for heifers at Location 2. Heifers received MGA (0.5 mghd-1d-1) for 14 d in a total mixed ration at Location 1 and in a high protein pellet fed at a rate of 2 lbshd-1d-1 at Location 2. Heifers in treatment 1 (MGA-PG) received 12.5 mg prostaglandin F2 i.m. (PG; Prostamate Sterile Solution; Agri Laboratories, Ltd. St. Joseph, MO.) 19 and 20 d following MGA w ithdrawal. Heifers in treatment 2 (MGA-GPG) received 100 g GnRH i.m. (Cystorelin, Merial, Inc., Duluth, GA) 3 d following MGA withdrawal with 12.5 mg PG i.m. (Prostamate) 7 and 8 d after GnRH (MGA-G-PG). The MGA and GnRH treatments were staggered so the PG was delivered on the same days for both treatments. To aid in estrus detect ion, heifers received an Estrus Alert heat detection patch (Estrus Alert, Western Point, Inc, Merrif ield, MN) at the second PG. Estrus was visually detected two times daily at 0700 and 1700 for 3 d after the initial PG. Estrus was defined as a heifer standing to be mounted by another heifer and/or a 1/4 to full red Estrus Alert patch. Eight to twelve hours after being de tected in estrus, heifers were AI with frozen-thawed semen by a single AI technician at both locations. Multiple AI sires were used within each location and equally distributed between treatments. Heifers not detected in estrus by 72 h after the initial PG were timed inseminated (timed-AI) and received 100 g GnRH i.m. (Cystorelin). Bulls were placed with heifers 10 d after timed-AI for both locations. Pregnancy to AI was determined approximately 55 d follo wing timed-AI for both locations, using a realtime B mode ultrasound (Aloka 500 v) equipped with a 5.0 MHz rectal transducer. Due to the 10 d interval where no heifers were bred by AI or th e clean-up bull, differences in fetal size (Curran et al., 1986) were used to distinguish between a pregnancy resulting from AI or clean-up bull. Three-day estrous response was defined as the number of he ifers that exhibited estrus during the 3 d between PG and timed-AI, divide d by the total number of heifers treated.

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96 Conception rate was defined as the number of heifers observed in estrus, inseminated, and became pregnant, divided by the total number of heifers inseminated. The timed-AI pregnancy rate was defined as the number of heifers that became pregnant to time d-AI, divided by the total number of heifers timed-AI. Synchronized pregna ncy rate was defined as the total number of heifers that became pregnant to either AI follo wing estrus detection or timed-AI, divided by the total number of heifers treated. Thirty-day preg nancy rate was defined as the total number of heifers pregnant in the first 30 d of the breeding season divide d by the total num ber of heifers treated. The GENMOD procedure of SAS was used for th e statistical analysis in Experiment 2. Within Location 1 data were initia lly analyzed with breed in the m odel to evaluate breed effects. The independent variables included treatment, br eed, and treatment breed, while the dependent variables were estrous response, conception rate, timed-AI pregna ncy rate, synchronized pregnancy rate, and 30 d pregnanc y rate. If the breed and treatment x breed effects were significant (P < 0.05), data for the Angus heif ers were analyzed separately from the Bos indicus Bos taurus heifers for Location 1. Data from the Bos indicus Bos taurus heifers from Locations 1 and 2 were combined and the inde pendent variables include d treatment, location, and treatment location while the dependent va riables included estrous response, conception rate, timed-AI pregnancy rate, synchronized pregna ncy rate, and 30 d pregna ncy rate. Six heifers from Location 1 were removed from analysis. Th ree were determined to be freemartins at AI, one developed a vaginal infection prior to AI, an d two were not present fo r pregnancy detection. Within Location 2 the effect of RTS was also evaluated. Independent variables included treatment, RTS, and treatment RTS while the dependent variables included estrous response, conception rate, timed-AI pregnanc y rate, synchronized pregnancy ra te, or 30 d pregnancy rates.

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97 Heifers with missing RTS data (n = 5) and those w ith a RTS of 2 (n = 2) were removed from the RTS analysis for Location 2. Results Experiment 1. Stage of follicle development affected ( P < 0.05) progesterone concentrations and diameter of the largest follicle at MGA withdraw al across the G3 and G10 treatments (Table 41). Heifers that did not receive PG during M GA were the only SOF group that had progesterone concentrations 1 ng/mL at MGA withdrawal. Progest erone concentrations were greater ( P < 0.001) for d 2 compared to the d 6, 10, and 14 SOF groups, which were similar ( P > 0.05) to each other. Diameter of the largest follicle at M GA withdrawal increased ( P = 0.001) as the length of time that a follicle was exposed to a low-level progestin environment increased (Table 4-1). At MGA withdrawal, the d 14 SOF group had a greater ( P < 0.05) follicle diameter compared to d 2, 6, and 10 SOF groups, and the d 10 SOF group had a greater ( P < 0.05) largest follicle diameter compared to d 2 and 6 SOF groups, which were similar ( P > 0.05) to each other. Stage of follicle development also affected ( P = 0.02) the three-day estrous response after MGA withdrawal across the G3 and G10 treatme nts. The estrous response was similar ( P > 0.05) for the d 6 and 10 SOF groups, which were both greater ( P < 0.05) compared to the d 2 and 14 SOF groups. The d 2 and 14 SOF groups had similar ( P > 0.05) estrous responses. When only the five-day estrous response was evaluate d for the G10 treatment, SOF development did not effect ( P = 0.27) the five-day estrous response. The five-day estrous response was 65.2% (15/23) and the estrous responses for the d 2, 6, 10, and 14 SOF groups were 50.0% (3/6), 80.0% (4/5), 85.7% (6/7), and 4 0.0% (2/5), respectively. Diameter of the largest folli cle at GnRH was affected ( P < 0.05) by treatment and SOF development but there was no treatment SOF effect ( P = 0.21; Table 4-2). Within the G3

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98 treatment, the d 14 SOF group had a larger ( P < 0.05) follicle diameter at GnRH compared to the d 2, 6, and 10 SOF groups, which were all similar ( P > 0.05) to each other. Conversely within the G10 treatment, the d 2 SOF group had a smaller ( P < 0.05) follicle diameter at GnRH compared to the d 6, 10, and 14 SOF groups, which were all similar ( P > 0.05) to each other. When diameter of the largest follicle at GnRH was evaluated across the G3 and G10 treatments, diameter of the largest follicle at GnRH was similar ( P > 0.05) for the d 2, 6, and 10 SOF groups. Whereas, heifers in the G3 treatment from the d 14 SOF group had a greater ( P < 0.05) follicle diameter compared to the G10 treatment. Di ameter of follicle ovulating to GnRH was not affected ( P > 0.05) by treatment, SOF development, or treatment SOF effect. Total ovulation rate was affected ( P = 0.02; Table 4-2) by treatment but not ( P > 0.05) by SOF or treatment SOF. More ( P = 0.02) G3 (76.0%) heifers ovulated compared to G10 (47.8%) heifers. When treatments were compared across SOF groups, G3 heifers in the d 6 and 10 SOF groups had greater ( P < 0.05) ovulation rates compared to G10 heifers in the d 6 and 10 SOF groups. Whereas, ovulati on rate for the d 2 and 14 SOF groups were similar ( P > 0.05) for the G3 and G10 treatments. Seventy-six percent (19/25) of G3 heifers had follicles 10 mm at GnRH but there was no SOF effect ( P = 0.27) on follicles 10 mm for heifers in the d 2 (5/6; 83.3%), 6 (4/6; 66.7%), 10 (5/8; 62. 5%), and 14 (5/5; 100.0%) SOF groups. For the G10 heifers, 73.9% had follicles 10 mm in diameter at GnRH and there was no SOF ( P = 0.13) effect. The percentage of G10 he ifers with follicles 10 mm at GnRH was 50.0 (3/6), 60.0 (3/5), 85.7 (6/7), and 100.0% (5/5) for d 2, 6, 10, and 14 SOF groups, respectively. The percentage of heifers with a functional CL at PG was similar (P = 0.59; Table 4-3) between the G3 (84.0%) and G10 (78.3%) tr eatments. There was a SOF effect ( P = 0.01), but no ( P = 0.89) treatment SOF effect on the percentage of heifers with a functional CL at PG.

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99 Across SOF groups, more ( P < 0.01) d 2 (100%), 6 (91%), and 10 (86.7%) heifers had a functional CL at PG compared to d 14 (40%) heifers. Additionally, more ( P = .01) G10 (43.5%) compared to G3 (12.0%) heifers had two CL on their ovaries at PG. There were no ( P > 0.05) SOF or treatment SOF effects on the incidence of heifers with two CL on their ovaries at PG. Progesterone concentrations at PG were greater ( P = 0.01) for G10 compared to G3 heifers (Table 4-3). Stage of follicle development also affected ( P = 0.01) progesterone concentrations at PG but there was no ( P = 0.93) treatment SOF effect on m ean progesterone concentration at PG. Across SOF groups, d 14 (2.4 1.2 ng/mL) had decreased ( P = 0.01) progesterone concentrations compared to d 2 (6.8 1.1 ng/mL ), 6 (7.0 1.2 ng/mL), and 10 (7.7 1.0 ng/mL) of which the later three were similar ( P > 0.05) to each other. Of the heifers with a functional CL at PG, the PG induced luteol ysis occurred in a similar ( P > 0.05) percentage of G3 (21/21) and G10 (18/18) heifers. Diameter of the largest fo llicle at PG was similar ( P = 0.67; Table 4-3) for G3 and G10 treatments but SOF affected ( P = 0.01) the diameter of the largest follicle at PG. Diameter of the largest follicle was greater ( P < 0.05) for the d 14 (15.4 1.0 mm) SOF group compared to d 2 (13.1 0.9 mm), 6 (12.2 0.9 mm), and 10 (11.2 0.8 mm) SOF groups, whereas, diameter of the largest follicle was similar ( P > 0.05) for the d 2, 6, and 10 SOF groups. There was no ( P = 0.54) treatment SOF effect on diameter of the largest follicle at PG. More heifers (P = 0.01) in the G3 (76.0%) trea tment exhibited estrus within 3 d after the initial PG compared to the G10 (43.5%) treatment (Table 4-4; Fi gure 4-1). In contrast, total estrous response was similar (P = 0.12) between the G3 and G10 tr eatments. There were no (P > 0.05) SOF or treatment SOF effects on the 3 d and to tal estrous responses. Interval from PG to the onset of estrus was not affected ( P > 0.05) by treatment or treatment SOF (Table 4-4).

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100 Conversely, there was ( P = 0.04) a SOF effect on interval from PG to onset of estrus. The d 14 (60.0 7.4 h) SOF group had a shorter (P < 0.05) in terval from PG to the onset of estrus compared to d 2 (91.2 7.0 h) and d 10 (79.7 6.1 h) SOF groups, but the d 14 interval was (P > 0.05) similar compared to th e d 6 interval (76.5 7.1 h). Of heifers that exhibited estrus after PG, a similar ( P = 0.19) percentage of G3 (91.3%; 21/23) and G10 (100.0%; 19/19) heifers formed a func tional CL by d 8 after the onset of estrus. There were two G3 heifers that exhibited estrus but failed to fo rm a functional CL. Neither of these heifers had a functional CL at PG and bot h were in the d 14 SOF group. There were two G3 heifers that did not exhibit es trus after PG. There was a singl e heifer in the d 2 group that had a functional CL at PG and had regressed the CL by d 10 after PG. The other G3 heifer was in the d 10 SOF group, and this heifer did not have a functional CL at PG or 10 d following PG. There were four G10 heifers that were not observe d in estrus. One heifer from each from the d 2 and 6 SOF groups had a functional CL at PG wh ile the heifers from the d 2 SOF group did not have a functional CL 10 d after PG the heifers from the d 6 SOF group had a functional CL. The remaining two G10 heifers were from the d 10, and 14 SOF groups both of which had a functional CL at PG but did not have a functional CL 10 d after PG. Progesterone concentrations 8 d after the PG induced estrus were similar (P = 0.50) between the G3 (4.8 0.5 ng/mL) and G10 (5.2 0.5 ng/mL) treatments and there were no ( P > 0.05) SOF or treatment SOF effects. Pr ogesterone concentrations 8 d following the synchronized estrus for the d 2, 6, 10, and 14 groups were 5.3 1.0, 5.3 1.0, 4.8 0.8, and 3.6 1.0 ng/mL for G3 and 4.9 1.0, 5.7 1.0, 4.7 0.9, and 5.6 1.0 ng/mL for G10, respectively.

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101 Experiment 2. Within Location 1, there was ( P = 0.02) a breed effect on estrous response and there were treatment breed effects (P < 0.05) on timed-AI and synchronized pregnancy rates. Therefore, the Angus data was analyzed separately for Location1 and data for the Bos indicus Bos taurus heifers in Locations 1 and 2 were analyzed together. The three-day estrous response was not affected ( P > 0.05) by treatment or treatment location for the Bos indicus Bos taurus heifers (Table 4-5). Howe ver, estrous response was greater ( P = 0.04) for Location 2 (70/117; 59.8%) compar ed to Location 1 (85/178; 47.8%). For Angus heifers at Location 1, treatment had no effect ( P = 0.16) on estrous response (Table 4-6). There were no ( P > 0.05) treatment or location effects on conception rate in Bos indicus Bos taurus heifers; however, there was a ( P = 0.01) treatment location effect (Table 4-5) on conception rates. Conception rates were greate r for the MGA-PG treatment at Location 2 and the MGA-G-PG treatment at Location 1 compared to the MGA-PG treatment at Location 1 and MGA-G-PG treatment at Location 2. Treatment had no effect ( P = 0.42) on conception rates in Angus heifers at Location 2 (Table 4-6). For the Bos indicus Bos taurus heifers, there were no ( P > 0.05) treatment, location, and treatment loca tion effects on timed-AI pr egnancy rates. For the Angus heifers, timed-AI pregnancy rate was greater ( P = 0.01) for the MGA-PG compared to MGA-G-PG treated heifers (Table 4-6). There were no ( P > 0.05) treatment, location, or treatment location effects on synchronized and 30 d pregnancy rates for Bos indicus Bos taurus heifers. For the Angus heifers, synchronized pregnancy rates tended to be greater ( P = 0.08) and 30 d pregnancy rates were similar ( P = 0.76) for the MGA-PG compared to th e MGA-G-PG treated heifers (Table 46).

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102 Within Location 2, reproductive tract scores (RTS) were taken at the initiation of the experiment to evaluate the effect of RTS on re sponse to the synchroniza tion treatments. There were no (P > 0.05) treatment, RTS, and treatmen t RTS effects on estrous response, timed-AI pregnancy rate, synchronized pregna ncy rate, and 30 d pregnancy rate (Table 4-7). In general, as RTS increased from a 3 to either a 4 or 5, es trous response and synchronized pregnancy rate increased numerically. However, conception rate was greater (P = 0.02) for the MGA-PG (68.8%) treatment compared to the MGA-G-PG (41.9%) treatment. There were no (P > 0.05) RTS or treatment RTS effects on conception rate. Discussion Recent research by Wood and co-worke rs (2001) indicated that yearling Bos taurus heifers treated with MGA for 14 d with GnRH 12 d after the last day of MGA followed by PG 7 d later had an improved synchrony of estrus co mpared to heifers synchronized with the traditional MGA-PG system. The reason for the in creased synchrony of estrus was attributed to the ability of GnRH to synchronize follicle deve lopment. Recent research from our lab (See Chapter 3) indicated that follicle wave development during the 19 d after a 14 d MGA treatment was more variable in Brangus co mpared to Angus heifers. It was concluded that administering GnRH 12 d after MGA withdrawal in Brangus heif ers may be too late because some heifers had already initiated a second follicle wave resulting in fewer heifers with follicle in the growing phase capable of ovulating to GnRH (Moreira et al., 2000). Therefore, it was hypothesized that administering GnRH 10 d after MGA withdrawal instead of 12 d may be more effective in Brangus heifers. It was also theorized that administration of GnRH 3 d after MGA withdrawal may actually be more effective since there was less variation in follicle development immediately after MGA withdrawal and a majority of follicles should be large enough to ovulate to GnRH. For that reason, Experiment 1 wa s designed to determine the effectiveness of

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103 administering GnRH either 3 or 10 d after a 14 d MGA treatment in Bos indicus Bos taurus heifers. Because follicle development can be si gnificantly influenced by the stage of luteal development that animals are under the influenc e of during a long-term MGA treatment (Sirois and Fortune, 1990; Kojima et al., 1992), it was also of interest to evaluate what effect stage of follicle development at the end of the MGA tr eatment would have on response to the GnRH. At MGA withdrawal, only the d 2 SOF group ha d progesterone concentrations indicative of luteal activity compared to d 6, 10, or 14 SOF groups, which all received PG during MGA. Diameters of the largest follicles at MGA withdraw al were smallest in the d 2 and 6 SOF groups compared to the d 10 and 14 SOF groups. The d 2 SOF group had the smallest follicle diameter, as they would have been near the end of the es trous cycle and probably in the middle of a follicle wave (Ginther et al., 1989) due to high luteal pr ogesterone that initiated follicle turnover (Sirois and Fortune, 1990) just before MGA withdrawal The d 6 SOF group had their CL regressed two days before MGA withdrawal resulting in the presence of newly developing dominant follicle under the influence of a low proge sterone environment provided by MGA for approximately 2 d. In contrasts, the d 10 and 14 SOF groups had the largest follicle diameters at MGA withdrawal, which was a result of the dominant follicles being under low progesterone environments for approximately 5 d in the d 10 SOF group and 9 d in the d 14 SOF group. Consequently, the d 10 and 14 SOF groups had do minant follicles that continued to grow, develop, and persist on the ovaries during MGA due to the increased fr equency of LH pulses observed during a low progesterone environment (Sirois and Fort une, 1990; Kojima et al., 1992). Therefore, the experimental model was effective in altering follicle development at the end of a 14 d MGA treatment.

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104 The three-day estrous response following MGA withdrawal was approximately 53% across the d 6 and 10 SOF groups. Hill et al. ( 1971) reported that estrus occurred primarily between 3 to 7 d after treatment with a 14 d MGA w ithdrawal. It is likely that three days was an inadequate amount of time to allow for a LH surge a nd the onset of estrus in all animals, which is supported by the observation that approximately 83% of the d 6 and 10 heifers in the G10 treatment exhibited estrus within 5 d after MGA withdrawal. Therefore, heifers with follicles that have been under a low progesterone enviro nment for approximately 2 to 5 d have an excellent opportunity to express estrus and ovulate within 5 d after the end of a 14 d MGA treatment. Consequently, exposure of dominant follicles to a low progesterone environment for approximately 2 to 5 d does not appear to comp romise the ability of the follicles to secrete estrogen and ovulate after MGA withdr awal. In contrast, the threeday estrous response for the d 2 SOF group was only 8.3% but it increased to 8 3.3% by 5 d after MGA withdrawal in the G10 treatment. Heifers in the d 2 SOF group that di d not exhibit estrus by d 3 were probably at the start of a new follicle wave at MGA withdrawal due to high luteal progesterone that initiated follicle turnover (Sirois and Fortune, 1990), which w ould have resulted in a delayed interval to estrus as the new follicle wave developed (Ginth er et al., 1989). The d 14 SOF group also had a decreased 3 d estrous response of 20% and the estrous response increased to only 40% within by d 5 d after PG in the G10 treatment. In c ontrast to the d 2 SOF group, the d 14 SOF group had the largest follicle diameter at MGA withdrawal due to the devel opment of long-term persistent dominant follicles (Sirois a nd Fortune, 1990; Kojima et al ., 1992), which should have been secreting enough estrogen (Kojima et al., 1995) to induce an LH surge and the onset of estrus. However, this was not the case and the reason (s ) for the decreased estrous response of the long-

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105 term persistent dominant follicles in the d 14 SOF group could be several fold and will be discussed in subsequent paragraphs. Across the four SOF groups in the G3 treatm ent, ovulation rate wa s a respectable 76%; although, whether ovulation occurred or did not occur tended to be influenced by SOF development at MGA withdrawal. For the G3 trea tment, 93% (13/14) of the heifers in the d 6 and 10 SOF groups ovulated to GnRH compared to only 66.7% in the d 2 SOF group. The two heifers in the d 2 SOF group that did not ovulate to GnRH were proba bly heifers that were in the middle of a follicle wave. This is supporte d by the observation that they had elevated progesterone concentrations at MGA withdraw al, which probably initiated follicle turnover (Ginther et al., 1989). As a result, they did not have a growing follicle that was capable of ovulating to GnRH (Moriera et al ., 2000). In contrasts, the d 14 group had the lowest percentage of heifers ovulating at only 40% indicating that GnRH was not very effective in ovulating persistent dominant follicles that had been unde r low progesterone exposure for approximately 9 d. Reasons for the failure of GnRH to initiate ovulation in a majority of the d 14 SOF group is unclear. The increased LH pulse frequency and resulting increased estradiol concentrations observed when no CL is present during an M GA treatment (Kojima et al., 1995) may have depleted the stores of LH in the anterior pituitary. In cows that had previously been treated with MGA for 9 d, Kojima et al. (1992) did not det ect a LH surge in 80% of cows after MGA withdrawal. Furthermore, the LH stores ma y be more easily depleted in cattle of Bos indicus breeding since they have less LH released in response to an exogenous GnRH treatment compared to cattle of Bos taurus breeding (Griffen and Randel, 1978; Portillo et al., 2007). One could also speculate that the increased LH pulse frequency observed during a low progesterone environment may have lead to down regulation of LH receptors in the granulosa cells of the

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106 persistent dominant follicles leading to ovulation failure. Another explanation may be that longterm persistent follicles may become cystic (S irois and Fortune, 1990; Mihm et al., 1994), and cannot undergo ovulation (Cook et al., 1990). Therefor e, these results sugge st that persistent dominant follicles that develop during a l ong-term MGA treatment have altered ovulatory capacities and the capacity to ovulate is probabl y influenced by the period of time that the follicles are under the influence of a low progesterone environment. Consequently, if the goal is to initiate ovulation in a majority of follicles to synchronize follicle development after an MGA treatment, it may prove advantageous to use shor t-term (7-9 d) MGA treatments to decrease the incidence of large persistent dominant follicles present during long term (14 d) MGA treatments. Additional research will need to be conducted to evaluate this. Estrous response during the five days afte r MGA withdrawal for the G10 heifers was only 65.2%, which is similar to the seven day es trous response observed in Angus and Brangus heifers (See Chapter 3), but slightly less than Bos taurus heifers treated with a 14 d MGA treatment (Yelich et al., 1997). One explanation for the decreased estrous response could be the incidence of a silent estrus, which is a frequent occurrence in cattle of Bos indicus breeding (Galina et al., 1982; Orihuela et al., 1983) and was observed in similar study conducted in our lab (See Chapter 3). This also appears to be th e case in Experiment 1, since two of five G10 heifers that were not observed in estrus had a CL at GnRH. The ovulation rate to GnRH was only 47.8% in G10 heifers compared to 76.0% for the G3 heifers. Even though all SOF groups except the d 2 group had a follicle diameter > 10 mm for the G10 treatment, it did not equate into a large percentage of follicles ovulating to GnRH for the G10 treatment. The decreased ovulation to GnRH suggest that there was significant asynchrony of follicle development by d 10 after MGA withdrawal resulting in a limited numbe r of follicles in the growing phase that were

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107 capable of ovulating to GnRH (Moreira et al ., 2000). Part of the reason for the follicle asynchrony was dictated by the low estrous response observed duri ng the 5 d after MGA withdrawal. This is supported by the observation that more heifer s that exhibited estrus after MGA withdrawal ovulated to GnRH compared to heifers not exhibiting es trus within 5 d of MGA withdrawal. Hence, the effectiveness of administering GnRH 10 d after MGA withdrawal is largely predicated on heifers exhibiting estrus within 5 d of MGA withdrawal. Certainly, d 10 is not the appropriate time to administer GnRH and it is unclear if waiting until d 12 would have provided a better response. There was a similar percentage of G3 and G 10 heifers with a func tional CL at PG, but SOF development influenced the percentage of he ifers with a functional CL It should be noted that two G3 heifers in the d 2 group that did no t ovulate to GnRH, had fu nctional CLs at PG. The two heifers must have ovulated sometime after GnRH and been in the early luteal phase at PG. The d 14 heifers had significantly lower per centage of functional CL at PG than all other SOF groups for both the G3 and G10 treatments. Not only did GnRH not initiate ovulation in the d 14 SOF group in the G3 treatment, the pres ence of a persistent dominant follicle altered follicle development enough so that there was not a new follicle wave available to ovulate by d 10 after MGA in the G10 treatment. Clearly dealing with the persistent dominant follicle in cattle of Bos indicus breeding is difficult. It is unknown if similar responses are observed in Bos taurus cattle treated with a 14 d MGA treatment. Heifers in the G10 treatment had increased pr ogesterone concentrations at PG compared to G3 treated heifers, which was likely due to the increased incidence of heifers in the G10 treatment having two CL on their ovaries (Diaz et al., 1998) compar ed to G3 treated heifers. Therefore, some of the G10 heifers would have a CL from the estrus after MGA withdrawal and

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108 a CL from the GnRH treatment. Two G10 heifer s in the d 14 SOF group, one of which ovulated prior to GnRH and the other to GnRH, did not ha ve a functional CL at PG. Lemaster et al., (1999) reported the presence of luteal tissue wi thout any progesterone production in frequently worked cattle of Bos indicus breeding. However, since both he ifers exhibited estrus shortly after PG, both heifers may have undergone a short estr ous cycle accompanied by a short-lived luteal structure. Treatment did not influence the diameter of the largest follicle at PG. However, diameter of the largest follicle of the Bos indicus Bos taurus heifers in the present experiment are considerably less than the di ameters observed by Wood et al. (2001) in MGA-G-PG treated Bos taurus heifers. Wood et al. (2001) reported a hi gh percentage of heif ers ovulating to GnRH administered 12 d after a 14 d MGA treatment resu lting in a new follicle wave that was reaching maximal diameter when PG was administered 7 d after GnRH. In the current study, a high percentage of heifers that received GnRH 10 d after MGA did not ovulate to GnRH, which probably resulted in asynchronous follicle wave development when PG was administered 7 d after GnRH resulting follicles of various sizes. Diameters of the largest follicles at PG were greatest in d 14 SOF heifers for both the G3 and G10 treatments. Because daily ultrasound examinations were not conducted between MGA w ithdrawal and PG, it is difficult to determine if the largest follicle present at PG was either from a new follicle wave initiated by the GnRH treatment or the presence of persistent dominant follicles that were pres ent at MGA withdrawal and still on the ovaries at PG. It is interesting to note that progesterone concentrations were < 1 ng/mL for G3 heifers in the d 14 SOF group, whic h may have resulted in increased pulsatile secretion of LH secret ion resulting in increased follicle development (Sirois and Fortune, 1990; Kojima et al., 1992).

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109 The G3 treatment proved to be more effec tive in synchronizing es trus as 76% of the heifers exhibited estrus during th e three-days after PG, which was similar to the estrous response observed by Wood et al. (2001) in Bos taurus heifers receiving the MGA-G-PG treatment where GnRH was administered12 d after M GA withdrawal. It is interesti ng to note that all G3 heifers in the d 14 SOF group exhibited estrus by 3 d af ter PG. This suggests that the d 14 SOF group that did not respond to GnRH mu st have initiated a new follicu lar wave that was capable of initiating estrus and ovulating by approximately 12 d after MGA withdrawal A similar type of follicle growth pattern was also observed for heifers that developed persistent dominant follicles during a 14 d MGA treatment and failed to ovu late the follicles within 5 d after MGA withdrawal (See Chapter 3). In comparison to other studies, the G3 treatment produced a substantially greater 3 d estrous response than the MGA-PG system in Bos taurus (Wood et al., 2001; Bridges et al., 2005) and Bos indicus Bos taurus (Stevenson et al., 1996; Bridges et al., 2005) heifers. The percentage of heifers exhibiting estrus within the 7 d after PG is similar to other studies synchronizing Bos taurus heifers with either the MGA-PG (Brown et al., 1988, Lamb et al., 2000) or the MGA-PG system with Gn RH (Wood et al., 2001). It should be noted that all of the heifers used in the present study were going through normal estrous cycles at the start of the experiment. Therefore, i nducing an effective estrous response in Bos indicus Bos taurus heifers with the MGA-PG estrous synchroni zation maybe predicated more by the estrous cycling status of heifers (Brown et al., 1988; Patterson et al., 1 989) than by mani pulation of the follicle wave dynamics of the heifers. Add itional research will need to be conducted to completely characterize what effects cycling st atus (cycling vs., non-cycling) has on modifying follicle dynamics in the MGA-PG synchronization protocol.

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110 Interval from PG to the onset of estrus in the current study was comparable to MGA-GPG (GnRH administered 12 d af ter MGA withdrawal) treated Bos taurus heifers (Wood et al., 2001), but increased compared to MGA-PG treated Angus and Brangus heifers in chapter 3. Additionally, heifers that would have begun MGA at d 14 of their estrous cycle had the shortest interval from PG to estrus regardless of GnRH treatment. One reason for the shorter interval was the fact that the diameter of the dominant follicle at PG was greatest in d 14 SOF group regardless of GnRH treatment, which is s upported by the observation made by Sirois and Fortune (1988) where size of the eventual ovulatory follicle at PG was negatively correlated with the interval to estrus. The percentage of heifers with a functional CL 8 d after an observed estrus was similar between G3 and G10 treatments. It should be noted that three G10 treated heifers not detected in estrus had a CL present 10 d following PG. Fu rthermore, there were some abnormalities in ovulation and luteal development that occurred in the G3 heifers. Two G3 heifers that exhibited estrus failed to ovulate, which was confirmed by lack of a CL and progesterone concentrations < 1ng/mL 8 d after the onset of estrus. Certainly, some of the G10 heifers had a silent estrus (Galina et al., 1996) but it is not clear why the G3 heifers failed to ovulate and form a CL. The frequent working of the heifers could have resu lted in abnormal luteal development where a CL is formed but it does not secrete a ny progesterone (Lemas ter et al., 1999). Results from Experiment 1 suggested that administering GnRH 3 d after a 14 d MGA treatment resulted in the most synchronous estrus. Therefore, the objectiv e of Experiment 2 was to evaluate the effectiveness of the G3 (MGA-G-PG) treatment compared to a MGA-PG treatment in a field trial utilizing yearling Bos indicus Bos taurus and Bos taurus heifers.

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111 The three-day estrous response of the Bos indicus Bos taurus heifers in Experiment 2 was similar between the MGA-PG and the MGA-GPG treatments and comparable to a report by Bridges et al. (2005) but substantially less comp ared to a report by Stevenson et al. (1996) in Bos indicus Bos taurus heifers synchronized with the MGA-PG system. One factor that influences estrous response is the number of heifers going through estrous cycles at the start of the synchronization treatment (See Chapter 3; Br own et al., Patterson and Corah, 1992). The importance of having heifers going through estrous cycles is also supported by the results of Experiment 1 where 72% of the heifers exhibite d estrus in the first 3 d after PG. Although, estrous cycling status was not determined in Experiment 2, the RTS data from Location 2 suggest that cycling status had a major influence on estrous response. Heifers with a RTS of 4 and 5, indicative of heifers that are probably going through normal estrous cycles, had an estrous response of 70% in the MGA-G-PG heifers compared to 42.9% in MGA-G-PG heifers with a RTS of 3. Conception rates were similar between the MGA-PG and the MGA-G-PG treatments. Although no studies utilizing a similar MGA-GPG treatment are available for comparison, conception rates for the MGA-G-PG were similar to Bos indicus Bos taurus heifers synchronized with the MGA-PG treatment (Bri dges et al., 2005). However, there was a significant treatment by location interaction indicating that th e treatment responses were different between locations. Exact reasons for the differential response to treatments between locations are unclear. It does not appear that AI technician or AI sire had an effect on conception rates, since a single AI technici an inseminated all heifers at both locations and AI sires were equally stratified across treatments. However, when conception rates were evaluated within Location 2 for the Bos indicus Bos taurus heifers, the MGA-PG heifers had a 28% greater

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112 conception rate compared to the MGA-G-PG. Th e trend for decreased conception rates for the MGA-G-PG was consistent across the three RTS. Additionally, the MGA-G-PG Angus heifers had a lower conception rate compared to MGAPG Angus heifers. The similar trend of decreased conception rates in both the Angus and Bos indicus Bos taurus heifers is of concern. It is possible that some of the heifers that exhibi ted estrus after PG could be heifers that did not ovulate to GnRH but the heifers initiated a new follicle wave that init iated the expression of estrus and ovulation around the time of PG. The questions that need to be answered are if these follicles are fertile or not and if the lack of progesterone exposur e before estrus and ovulation resulted in short estrous cycles after the PG (Berardinelli et al., 1979; Evans et al., 1994). One of the reasons that producers like to use the MGA-PG system is that it consistently results in excellent conception ra tes during the synchronized estrus (Brown et al., 1988; Lamb et al., 2000) in heifers of Bos taurus breeding. In contrast s, conception rates of Bos indicus Bos taurus heifers synchronized with MGA-PG protocol s (See Chapter 3; Bridges et al., 2005) are highly variable and are still cons iderably less than studies in Bos taurus heifers synchronized with MGA-PG systems (Brown et al., 1988; Lamb et al., 2000). Age at puberty may have something to do with this since cattle of Bos indicus breeding (Plasse et al ., 1968; Baker et al., 1989) attain puberty at older ages than Bos taurus breeds (Wiltbank et al., 1966; Baker et al., 1989). As a result, reaching puberty at older ag es may have decreased the number of estrous cycles heifers had before synchronization and AI resulting in decreased conception rates (Byerley et al., 1987; Galina et al., 1996). A similar percentage of MGA-PG and MGA-G-PG treated Bos indicus Bos taurus heifers became pregnant to the timed-AI, but th e timed-AI pregnancy rate was considerably less compared to an experiment conducted by Bridges and coworkers (2005) in yearling Bos indicus

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113 Bos taurus heifers synchronized with an MGA-PG protocol receiving consecutive split treatments of PG. Additionall y, timed-AI pregnancy rates were substantially reduced in MGAG-PG compared to MGA-PG treated Angus heifers. The reduced timed-AI pregnancy rate in the MGA-G-PG compared to MGA-PG treatment ma y be a result of when the timed-AI was conducted relative to the abil ity of GnRH to induce ovulati on (Moreira et al., 2000). Furthermore, the timed-AI groups also contain heifers that are not cy cling, did not respond to PG, or did not have any follicles large enough to ovulate to GnRH. Any of these three scenarios would result in decreased pre gnancy rates to the timed-AI Synchronized pregnancy rates were sim ilar between the MGA-PG and MGA-G-PG treated Bos indicus Bos taurus heifers in Experiment 2, which are similar to synchronized pregnancy rates of MGA-PG treated Bos indicus Bos taurus heifers (Bridges et al., 2005). As observed with estrous response, cycling status probably has more influence on synchronized pregnancy rates than any other si ngle variable (Brown et al., 1988; Patterson et al., 1989). This can be partially explained by the reproductive trac t score data from Location 2. Heifers with a RTS of 4 or 5, indicative of he ifers that, by definition, are goin g through normal estrous cycles, had a synchronized pregnancy rate of 44.4% acr oss the MGA-PG and MGA-G-PG treatments compared to 34.0% in the heifers with a RTS of 3. For Angus heifers, synchronized pregnancy rates tended to be greater in MGA-PG compared to MGA-G-PG. The major reasons for the decreased synchronized pregnancy rate for the Angus heifers were due to the numerically lower conception rate and the significantly lower timedAI pregnancy rate for the MGA-G-PG heifers compared to the MGA-PG heifers. In summary, the G3 treatment improved the synchrony of estrus compared to G10 treatment in Bos indicus Bos taurus heifers in Experiment 1. Stage of follicle development at

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114 the end of a 14 d MGA treatment had significan t effects on largest follicle size at MGA withdrawal and the subsequent estrous response after MGA withdrawal. Heifers that developed a long-term (> 9 d) persistent dominant folli cle under the influence of a low progesterone environment had a detrimental effect on the subs equent estrous response after MGA withdrawal and ability to ovulate to GnRH, suggesting that long-term persis tent follicles have a reduced capacity to ovulation capacity. Additional experi ments will need to be evaluated to either regress or ovulate long-term persistent follic les developed under a 14 MGA environment, or utilization of short-term (< 9 d) progestin treatments may need to be used to prevent the occurrence of long-term persistent follicles. In Experiment 2, the M GA-G-PG (G3) protocol failed to increase estrous respons e over the MGA-PG system in Bos indicus Bos taurus heifers. Synchronized pregnancy rates of the MGA-G-PG and MGA-PG sy stems were similar but the synchronized pregnancy rates from Experiment 2 are still considerably less compared to other reports in Bos taurus and Bos indicus Bos taurus heifers synchronized with the MGA-PG system. Implications The addition of GnRH 3 d afte r withdrawal of a 14 d MGA treatment resulted in a greater synchrony of estrus compared to GnRH 10 d afte r MGA. However, heifers that would have started MGA late in the luteal phase of the es trous cycle and develope d persistent dominant follicles did not respond well to the GnRH treatment s. Therefore, future research needs to address dealing with persistent dominant follicl es developed in low pr ogesterone environments in relation to their preven tion and (or) removal from the ovary in heifers of Bos indicus Bos taurus breeding. The estrous response, conception rate, and synchronized pregnancy rate were similar for MGA-G-PG compared to the traditio nal MGA-PG system. Therefore, administering

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115 GnRH 3 d after a long term MGA treatment does not appear to improve the effectiveness of the MGA-PG estrous synchronization system in heifers of Bos indicus Bos taurus breeding.

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116 Table 4-1. The effect of stage of follicle (SOF) development during a 14 d melengestrol acetate (MGA) treatment on progesterone concentration (LSM S.E.) at MGA withdrawal, diameter of the largest follicl e at MGA withdrawal (LSM S.E.), and three day estrous response following MGA w ithdrawal. Data are combined for G3 and G10 heifers (Experiment 1). a SOF n Progesterone concentration, ng/mL Diameter of largest follicle, mm Three day estrous response, % b d 2 12 5.5 0.4 c 12.6 1.0 c 8.3 c d 6 11 0.3 0.5 d 13.8 0.9 c 54.5 d d 10 15 0.2 0.4 d 16.4 0.8 d 53.3 d d 14 10 0.3 0.5 d 20.4 1.0 e 20.0 c P -value 0.001 0.001 0.02 a See material and methods for SOF descriptions. b Estrous response is the total numbe r of heifers exhibiting estrus out of the tota l number treated within 3 d of MGA withdrawal. c,d,e Means within a column without a common superscript differ ( P < 0.05)

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117Table 4-2. Effect of treatment (T) and st age of follicle (S) development on largest fo llicle diameter at GnRH (LSM SE), diam eter of follicle ovulating to GnRH (LSM SE), and ovulation rate for heifers receiving GnRH either 3 d (G3) or 10 d (G10) after withdrawal of a 14 d melengestrol acetate (MGA) treatment (Experiment 1). Stage of follicle development group a P-values Variable 2 6 10 14 T S T S Largest follicle GnRH, mm 0.01 0.01 0.21 G3 (25) 12.5 1.3 (6)d,x 13.2 1.3 (5)d,x 13.3 1.2 (8)d,x 20.2 1.4 (5)e,x G10 (23) 9.7 1.3 (6)d,x 12.6 1.4 (5)e,f,x 12.0 1.2 (7)e,f,x 14.2 1.4 (5)f,y Follicle ovulating to GnRH, mm 0.32 0.20 0.11 G3 (15) b 12.8 1.0 (4) 15.0 1.0 (4) 15.6 0.9 (5) 11.5 1.4 (2) G10 (11) 11.3 1.2 (3) 13.7 1.2 (3 12.0 1.2 (3) 14.5 1.4 (2) Ovulation rate, % c 0.02 0.12 0.34 G3 (25) 66.7 (6)d,x 100.0 (6)e,x 87.5 (8)d,x 40.0 (5)d,x G10 (23) 50.0 (6)d,x 60.0 (5)d,y 42.9 (7)d,y 40.0 (5)d,x a See material and methods for description of st age of follicle development at MGA withdrawal. b Four G3 heifers exhibited estrus before GnRH so their data were not include in the size of follicle ovulating to GnRH analysis but the heifers were included in the ovulation rate data. c Ovulation rate for G3 defined as number of heifers ovulating after an ob served estrus within 3 d af ter MGA withdrawal and (or) GnRH 3 d after MGA withdrawal divided by th e total in the group. Ovulati on rate for G10 treatment de fined as the number of hei fers ovulating to GnRH 10 d after MGA withdr awal divided by the total in the group d,e,fMeans within a variable and row w ithout a common superscript differ ( P < 0.05). x,y,zMeans within a variable and column without a common superscript differ ( P < 0.05)

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118 Table 4-3. Percentage of heifers with a functional corpus luteum (CL), progesterone concentration (LSM S.E.), and diamet er of the largest dominant follicle at prostaglandin F2 (PG: LSM S.E.) for G3 and G10 heifers across different stages of follicle (SOF) development (Experiment 1).a Treatment (SOF) n Functional CL at PG, %b Progesterone concentration at PG, ng/mL Diameter of the largest follicle at PG, mm G3 mean 25 84.0 4.3 0.8 12.8 0.6 d 2 6 100.0 5.6 1.6 12.5 1.2 d 6 6 100.0 5.0 1.6 12.2 1.2 d 10 8 87.5 5.7 1.4 10.3 1.1 d 14 5 40.0 1.0 1.7 16.2 1.4 G10 mean 23 78.3 7.7 0.8 13.2 0.6 d 2 6 100.0 8.0 1.6 13.7 1.2 d 6 5 80.0 9.1 1.7 12.2 1.4 d 10 7 85.7 9.8 1.5 12.1 1.1 d 14 5 40.0 4.0 1.7 14.6 1.4 P -values Treatment 0.59 0.01 0.67 SOF 0.01 0.01 0.01 Treatment SOF 0.89 0.93 0.54 a Heifers administered MGA for 14 d followed by GnRH either 3 (G3) or 10 d (G10) after MGA withdrawal. Seven days after GnRH, heifers received PG. bFunctional CL at PG defined as a heif er having progesterone concentrations 1 ng/mL combined with the presence of a CL as determined by ultrasonography

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119 Table 4-4. Three-day estrous resp onse, total estrous response, a nd interval from prostaglandin F2 (PG) to onset of estrus following PG treatment for G3 and G10 heifers across different stages of follicle (SOF) development (Experiment 1).a Treatment (SOF) n Three-day estrous response, %b Total estrous response (%)c Interval from PG to estrus, h G3 mean 25 76.0 92.0 71.7 4.6 d 2 6 50.0 83.3 79.2 9.9 d 6 6 83.3 100.0 72.0 9.0 d 10 8 75.0 87.5 75.4 8.3 d 14 5 100.0 100.0 60.0 9.9 G10 mean 23 43.5 82.6 82.1 5.1 d 2 6 33.3 83.3 103.2 9.9 d 6 5 40.0 80.0 81.0 11.0 d 10 7 42.9 85.7 84.0 9.0 d 14 5 60.0 80.0 60.0 11.0 P -values Treatment 0.01 0.12 0.14 SOF 0.17 0.67 0.04 Treatment SOF 0.63 0.52 0.69 aHeifers administered melengestrol acetate (MGA) for 14 d followed by GnRH either 3 (G3) or 10 d (G10) after MGA withdrawal. Heifers received 12.5 mg of PG on both d 7 and 8 after GnRH. bThree-day estrous response is th e number of heifers observed in estrus within 3 d of the initial PG treatment divided by th e total number of heifers treated. cTotal estrous response is the numbe r of heifers observed in estrus within 7 d of PG treatment divided by the total numbe r of heifers treated.

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120 0 10 20 30 40 50 1224364860728496108120132144156168NR Interval from PG to the onset of estrus, hPercentage G3 G10 Figure 4-1. Estrous response, expressed as a percentage of the tota l number of heifers in a gr oup, during the 7 d after the in itial PG treatment for G3 (n = 25) and G 10 (n = 23) treatments. NR = no es trous response (Experiment 1) .

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121Table 4-5. Estrous, concepti on and pregnancy rates of Bos taurus x Bos indicus heifers synchronized w ith combinations of melengestrol acetate (MGA), GnRH (G), and prostaglandin F2 (PG) at two locations (LOC) (Experiment 2). Treatment (TRT)a n LOC Estrous Response (%)b Conception rate (%)c Timed-AI pregnancy rate (%)d Synchronized Pregnancy rate (%)e 30 d pregnancy rate (%)f MGA-PG 89 1 38/89 (42.7) 16/38 (42.1) 13/51 (25.5) 29/89 (32.6) 62/89 (69.7) MGA-PG 58 2 33/58 (57.0) 23/33 (69.7) 4/25 (16.0) 27/58 (46.6) 41/58 (70.7) Overall means 147 71/147 (48.3) 39/71 (54.9) 17/76 (22.4) 56/147 (38.1) 103/147 (70.1) MGA-G-PG 89 1 47/89 (52.8) 28/47 (59.6) 7/42 (16.7) 35/89 (39.3) 62/89 (69.7) MGA-G-PG 59 2 37/59 (62.7) 16/37 (43.2) 5/22 (22.7) 21/59 (35.6) 43/59 (72.9) Overall means 148 84/148 (56.7) 44/84 (52.4) 12/64 (18.8) 56/148 (37.8) 105/148 (71.0) P -values TRT 0.18 0.55 0.91 0.74 0.84 LOC 0.04 0.46 0.83 0.38 0.69 TRT LOC 0.73 0.01 0.28 0.13 0.84 a Both treatments received MGA for 14 d. MG A-PG heifers received PG 19 (12.5 mg) a nd 20(12.5 mg) d after MGA withdrawal. The MGA-G-PG heifers received GnRH (100 g) 3 d after MGA withdrawal with PG 7 (12.5 mg) and 8 (12.5 mg) d after GnRH. Estrus was detected for 3 d, and heifers exhi biting estrus were AI 8-12 h later. Heif ers not displaying estrus were timed-AI 7 2-80 h and received GnRH. b Percentage of heifers displaying estrus 3 d after PG of the total treated. c Percentage of heifers that were pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pregnant to timed-AI that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated. f Percentage of heifers pregnant du ring the first 30 d of the synchroni zed breeding of the total treated.

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122 Table 4-6. Estrous, conception a nd pregnancy rates of Angus heif ers in Location 1 synchronized w ith combinations of melengestr ol acetate (MGA), GnRH (G), and prostaglandin F2 (PG) (Experiment 2). Treatment a n Estrous response (%)b Conception rate (%)c Timed-AI pregnancy rate (%)d Synchronized pregnancy rate (%)e 30 d pregnancy rate (%)f MGA-PG 27 15/27 (55.6) 6/15 (40.0) 5/12 (41.7) 11/27 (40.7) 19/27 (70.4) MGA-G-PG 30 22/30 (73.3) 6/22 (27.3) 0/8 (0.0) 6/30 (20.0) 20/30 (66.7) P -values Treatment 0.16 0.42 0.01 0.09 0.76 a Both treatments received MGA for 14 d. MG A-PG heifers received PG 19 (12.5 mg) a nd 20 (12.5 mg) d after MGA withdrawal. The MGA-G-PG heifers received GnRH (100 g) 3 d after MGA withdrawal with PG 7 (12.5 mg) and 8 (12.5 mg) d after GnRH. Estrus was detected for 3 d, and heifers exhi biting estrus were AI 8-12 h later. Heif ers not displaying estrus were timed-AI 7 2-80 h and received GnRH. b Percentage of heifers displaying estrus 3 d after PG of the total treated. c Percentage of heifers that were pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pregnant to timed-AI that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated. f Percentage of heifers pregnant du ring the first 30 d of the synchroni zed breeding of the total treated.

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123Table 4-7. Estrous, conception, timed-AI, pregnancy rates by treatment (TRT) and reproductive tract score (RTS) for Bos taurus Bos indicus heifers synchronized with combinations of melengest rol acetate (MGA), GnRH (G ), and prostaglandin F2 (PG) at Location 2 (Experiment 2).a RTS TRT Estrous response (%)b Conception rate (%)c Timed-AI pregnancy rate (%)d Synchronized pregnancy rate (%)e 30 d pregnancy rate (%)f 3 MGA-PG 15/26 = 57.7 10/15 = 66.7 0/11 = 0.0 10/26 = 38.5 17/26 = 65.4 MGA-G-PG 9/21 = 42.9 3/9 = 33.3 3/12 = 25.0 6/21 = 28.6 13/21 = 61.9 4 MGA-PG 12/19 = 63.2 8/12 = 66.7 1/7 = 14.3 9/19 = 47.4 14/19 = 73.7 MGA-G-PG 14/21 = 66.7 6/14 = 42.9 1/7 = 14.3 7/21 = 33.3 19/21 = 90.5 5 MGA-PG 5/11 = 45.5 4/5 = 80.0 3/6 = 50.0 7/11 = 63.6 9/11 = 81.8 MGA-G-PG 9/12 = 75.0 4/9 = 44.4 1/3 = 33.3 5/12 = 41.7 7/12 = 58.3 Total MGA-PG 32/56 = 57.1 22/32 = 68.8 4/24 = 16.7 26/56 = 46.4 40/56 = 71.4 MGA-G-PG 32/54 = 59.3 13/32 = 40.6 5/22 = 22.7 18/54 = 33.3 39/54 = 72.2 P-values TRT 0.51 0.02 0.27 0.12 0.95 RTS 0.37 0.75 0.11 0.31 0.11 TRT RTS 0.20 0.90 0.15 0.91 0.17 a Both treatments received MGA for 14 d. MG A-PG heifers received PG 19 (12.5 mg) a nd 20(12.5 mg) d after MGA withdrawal. The MGA-G-PG heifers received GnRH (100 g) 3 d after MGA withdrawal with PG 7 (12.5 mg) and 8 (12.5 mg) d after GnRH. Estrus was detected for 3 d, and heifers exhi biting estrus were AI 8-12 h later. Heif ers not displaying estrus were timed-AI 7 2-80 h and received GnRH. b Percentage of heifers displaying estrus 3 d after PG of the total treated. c Percentage of heifers that were pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pregnant to timed-AI that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated. f Percentage of heifers pregnant du ring the first 30 d of the synchroni zed breeding of the total treated.

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124 CHAPTER 5 SUMMARY The primary objective of Experiment 1 (Chapter 3) was to evaluate follicular development in yearling Angus and Brangus heifers during th e 19 d period between MGA withdrawal and PG, and to evaluate the follicle development a nd estrous response following PG. A primary objectives of Experiments 2 and 3 (Chapter 4) were to determine the optimal timing to implement GnRH during the period from MGA withdr awal to PG and to evaluate the subsequent estrous response and fertility in Bos indicus Bos taurus heifers. In Experiment 1, yearling Angus and Brangus heifers were synchronized with the MGAPG system with PG was administered 19 af ter MGA withdrawal. During the period between MGA withdrawal and PG, follicle development patterns were characterized by daily ultrasonography and the follicle development pa tterns were different between Angus and Brangus heifers. Factors contributing to the differences in follicle development were the decreased number of Brangus heif ers that exhibited estrus duri ng the 7 d after MGA withdrawal compared to Angus heifers and th e increased incidence of three a nd four follicle wave patterns in Brangus compared to Angus heifers. From days 9 to 13 after MGA wit hdrawal, the percentage of follicle 10 mm steadily increased to 100% by d 13 in Angus heifers but increased in Brangus heifers up to d 10 but started to decrease to a low of 50% by d 13 in Brangus heifers. Although follicle development between MGA withdr awal and PG was different between Angus and Brangus heifers, diameter of the largest foll icle at PG, estrous response, and interval from PG to estrus were similar between Angus and Br angus heifers. Conversely, when the number of follicle waves was evaluated, there was an interaction between breed and number of follicle waves on the interval from PG to estrus. Angus heifers displaying thr ee follicle waves had a longer interval from PG to estrus compared to two wave Angus and Brangus and three wave

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125 Brangus heifers. Therefore, the variati on in follicle wave development between MGA withdrawal and the PG administered 19 d later needs to be altered in order to improve the synchrony of estrus following PG. In a recent report by Wood and coworkers ( 2001), GnRH was included in the MGA-PG estrous synchronization system 12 d after MGA withdrawal in Bos taurus heifers to synchronize follicle development at the PG treatment. Howeve r, it does not appear that administering GnRH 12 d after MGA withdrawal would be as effec tive in Brangus heifer s due to a decreased percentage of Brangus heifers with large grow ing follicles capable of ovulating to GnRH 12 d after MGA withdrawal. It appe ars that introducing a GnRH tr eatment would need to occur approximately 10 d after MGA withdrawal, when a high percentage of Brangus have large growing follicles capable of ovul ating to GnRH. Furthermore, it may actually be better to administer GnRH soon after MGA (3 to 4 d) wit hdrawal where most heifers have large follicles and variation in follicle development is minimal. In Experiment 2, cycling Bos indicus Bos taurus heifers that were on day two of the estrous cycle were administered GnRH either 3 (G3) or 10 (G10) d after the last day of a 14 d MGA treatment followed by PG 7 d after GnRH. In addition, three groups of heifers received two consecutive PG treatments at predetermined days during MGA to imitate heifers starting MGA at different stages of the estrous cycle (SOC). The four stages included d 2, 6, 10, and 14. Administering GnRH three days after the last day of MGA wit hdrawal was more effective in initiating ovulation compared to d 10 after M GA withdrawal. The decreased ovulation rate to GnRH in G10 likely resulted in asynchronous foll icle development at PG, which was partially due to the poor estrous during the five days after MGA withdrawal. Furthermore, the GnRH treatment was not very effectiv e at inducing ovulation in long-t erm persistent follicles (d 14

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126 SOC) regardless of treatment, which demonstrated the negative effects persistent follicles have on an estrous synchronization system. The sync hrony of estrus following PG was substantially improved for G3 compared to G10 as evidence by the greater 72 h estrus response of the G3 treated heifers. Because administering GnRH 3 d following MGA withdrawal provided the greatest synchrony of estrus when PG was administered 7 d after GnRH, it was de cided to evaluate the fertility of the G3 treatment in a field trial in Experiment 3. Experiment 3 was conducted to evaluate the MGA-G-PG (G3) sy stem compared to the traditi onal MGA-PG system in Angus and Bos indicus Bos taurus heifers. The only exception was he ifers received estrus detection and AI for 72 h, at which time all non-responde rs were timed-AI and received GnRH. Unlike Experiment 2, the MGA-G-PG treatment fa iled to increase the percentage of Bos indicus Bos taurus heifers in estrus within 72 h after PG. Conception, timed-AI pregnancy, and synchronized pregnancy rates were similar for MGA-G-PG and MGA-PG treatments.

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146 BIOGRAPHICAL SKETCH Steaven A. Woodall, Jr. was born in Tampa, Florida on December 02, 1978 to Steve and Paula Woodall of Plant City, Florida. Steaven has one sibling, Priscilla, a nd he is the oldest of the two children. Steaven attended several sc hools during his childhood and graduated from Durant Senior High School where he was an act ive member of the Durant FFA. Throughout his high school years, Steaven was a member of the Florida Junior Limousin Association and was active in showing cattle throughout the Southeas t. After high school, Steaven was employed by Sun State International Trucks while attendi ng Hillsborough Community College on a Florida Bright Futures Scholarship, wh ere he received his A.A. degree. In August 2002, Steaven enrolled at the University of Florida to pursue hi s B.S. At the University of Florida, Steaven joined the Alpha Gamma Rho fraternity, where he held the office of Vice Noble Ruler of Management and Operations, and served as kitc hen manager. Steaven graduated in August 2004 and accepted a graduate assistant position, starti ng the next semester, in the Department of Animal Sciences at the University of Florida unde r the direction of Dr. Joel Yelich. In addition to his own research duties, Steaven had the oppor tunity to conduct a laboratory section of the Reproductive Physiology course as well as assi st research in many as pects of reproductive physiology. Steavens future plans are to pursue a career in the beef cattle industry focusing on bovine reproduction.