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Comparison of Two Short-Term Progestogen-Based Estrous Synchronization Protocols in Yearling Heifers and Suckled Postpar...

Permanent Link: http://ufdc.ufl.edu/UFE0022516/00001

Material Information

Title: Comparison of Two Short-Term Progestogen-Based Estrous Synchronization Protocols in Yearling Heifers and Suckled Postpartum cows of Bos indicus X Bos taurus Breeding
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Mckinniss, Erin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

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: A series of experiments were conducted with suckled cows and yearling heifers of Bos indicus x Bos taurus breeding to compare two synchronization protocols including the Select Synch/CIDR+timed-AI (SSC+TAI) and a modified 7-11 protocol. In Experiment 1, suckled Bos indicus x Bos taurus cows (n = 324) were used in a 2 x 2 factorial design comparing SSC+TAI to a modified 7-11 (7-10) protocol and within each synchronization treatment there were two prostaglandins (PG) treatments including cloprostenol sodium (CPG) and dinoprost tromethamine (DPG). Cows were equally distributed to treatments by body condition score (BCS) and days postpartum (DPP). On day 0, the 7-10 treatment received an autoclaved once-used-CIDR that was removed on day 7 concomitant with DPG followed by GnRH on day 10. On day 17, 7-10 cows received either DPG or CPG. Also on day 10, SSC+TAI treatment received a new CIDR concomitant with GnRH with CIDR removal on day 17 where cows received either DPG or CPG. Estrus was visually detected twice daily (0700 and 1600 h) for 72 h after PGF2? and cows were AI 6 to 12 h after a detected estrus. Non-responders were timed-AI + GnRH 72 to 76 h post PG. In experiment 2, yearling heifers over two breeding seasons (n = 218 and 137) were randomly assigned to the same treatments as Experiment 1, estrous detection and AI were administered in the same manner as Experiment 1. In experiment 1, estrous response (ER), conception (CR), and synchronized pregnancy rate (SPR) were affected (P < 0.05) by synchronization treatment but not (P > 0.05) PG treatment. The SPR were 45.5% and 31.2 for the SSC+TAI and 7-10 treatments, respectively. As BCS increased from ? 4.5 to ? 5.5, ER, CR, SPR, and thirty day pregnancy rates (TDPR) increased (P < 0.05). In experiment 2, ER, CR, TAIPR, SPR, and TDPR were similar (P > 0.05) between the synchronization and PG treatments, with the mean ER, CR, TAIPR, SPR and TDPR being 66.8, 65.0, 16.1, 48.7, and 76.3%, respectively. In Experiment 3, yearling heifers (n = 407) were synchronized with either the SSC +TAI treatment from Experiment 1 or 7-11 treatment that included melengestrol acetate (MGA; 0.5 mg/head/d) for 7 d with GnRH 4 d after the last day of MGA followed by DPG 7 d later. Estrus was detected and heifers were inseminated similar to Experiment 1. Heifers were distributed to treatment by reproductive tract score (RTS; Scale: 1=immature to 5= estrous cycling) and BCS. The 7-11 heifers had greater (P < 0.05) ER (55.2 vs. 41.9%), CR (47.0 vs. 31.3%), and SPR (33.5 vs. 24.8%) compared to SSC+TAI heifers, respectively. Heifers exhibiting estrus at 60 h after PG (61.7%) had a greater (P < 0.05) CR compared to heifers that had exhibited estrus at ? 36 (35.3%), 48 (31.6%), and 72 h (36.2%), which were all similar (P > 0.05) to each other. As RTS increased from ? 2 to a ? 3, ER, CR, SPR, and TDPR all increased (P < 0.05). In summary, there was a differential response to synchronization treatments as cows treated with the SSC+TAI had greater SPR compared to the 7-10 treatment; whereas, heifers synchronized with the 7-11 treatment had greater SPR compared to the SSC+TAI treatment.
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 Erin Mckinniss.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Yelich, Joel V.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022516:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022516/00001

Material Information

Title: Comparison of Two Short-Term Progestogen-Based Estrous Synchronization Protocols in Yearling Heifers and Suckled Postpartum cows of Bos indicus X Bos taurus Breeding
Physical Description: 1 online resource (157 p.)
Language: english
Creator: Mckinniss, Erin
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

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: A series of experiments were conducted with suckled cows and yearling heifers of Bos indicus x Bos taurus breeding to compare two synchronization protocols including the Select Synch/CIDR+timed-AI (SSC+TAI) and a modified 7-11 protocol. In Experiment 1, suckled Bos indicus x Bos taurus cows (n = 324) were used in a 2 x 2 factorial design comparing SSC+TAI to a modified 7-11 (7-10) protocol and within each synchronization treatment there were two prostaglandins (PG) treatments including cloprostenol sodium (CPG) and dinoprost tromethamine (DPG). Cows were equally distributed to treatments by body condition score (BCS) and days postpartum (DPP). On day 0, the 7-10 treatment received an autoclaved once-used-CIDR that was removed on day 7 concomitant with DPG followed by GnRH on day 10. On day 17, 7-10 cows received either DPG or CPG. Also on day 10, SSC+TAI treatment received a new CIDR concomitant with GnRH with CIDR removal on day 17 where cows received either DPG or CPG. Estrus was visually detected twice daily (0700 and 1600 h) for 72 h after PGF2? and cows were AI 6 to 12 h after a detected estrus. Non-responders were timed-AI + GnRH 72 to 76 h post PG. In experiment 2, yearling heifers over two breeding seasons (n = 218 and 137) were randomly assigned to the same treatments as Experiment 1, estrous detection and AI were administered in the same manner as Experiment 1. In experiment 1, estrous response (ER), conception (CR), and synchronized pregnancy rate (SPR) were affected (P < 0.05) by synchronization treatment but not (P > 0.05) PG treatment. The SPR were 45.5% and 31.2 for the SSC+TAI and 7-10 treatments, respectively. As BCS increased from ? 4.5 to ? 5.5, ER, CR, SPR, and thirty day pregnancy rates (TDPR) increased (P < 0.05). In experiment 2, ER, CR, TAIPR, SPR, and TDPR were similar (P > 0.05) between the synchronization and PG treatments, with the mean ER, CR, TAIPR, SPR and TDPR being 66.8, 65.0, 16.1, 48.7, and 76.3%, respectively. In Experiment 3, yearling heifers (n = 407) were synchronized with either the SSC +TAI treatment from Experiment 1 or 7-11 treatment that included melengestrol acetate (MGA; 0.5 mg/head/d) for 7 d with GnRH 4 d after the last day of MGA followed by DPG 7 d later. Estrus was detected and heifers were inseminated similar to Experiment 1. Heifers were distributed to treatment by reproductive tract score (RTS; Scale: 1=immature to 5= estrous cycling) and BCS. The 7-11 heifers had greater (P < 0.05) ER (55.2 vs. 41.9%), CR (47.0 vs. 31.3%), and SPR (33.5 vs. 24.8%) compared to SSC+TAI heifers, respectively. Heifers exhibiting estrus at 60 h after PG (61.7%) had a greater (P < 0.05) CR compared to heifers that had exhibited estrus at ? 36 (35.3%), 48 (31.6%), and 72 h (36.2%), which were all similar (P > 0.05) to each other. As RTS increased from ? 2 to a ? 3, ER, CR, SPR, and TDPR all increased (P < 0.05). In summary, there was a differential response to synchronization treatments as cows treated with the SSC+TAI had greater SPR compared to the 7-10 treatment; whereas, heifers synchronized with the 7-11 treatment had greater SPR compared to the SSC+TAI treatment.
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 Erin Mckinniss.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Yelich, Joel V.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022516:00001


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655e911c2ad01bfb8771b86e5c5bf8cf808f1b64







COMPARISON OF TWO SHORT-TERM PROGESTOGEN BASED ESTROUS
SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS AND SUCKLED
POSTPARTUM COWS OF Bos indicus x Bos taurus BREEDING













By

ERIN N. MCKINNISS


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

2008




























2008 Erin N. McKinniss


































To my family and friends for always giving the support and encouragement I needed.









ACKNOWLEDGMENTS

I would like to offer my genuine appreciation to Dr. Joel V. Yelich, my committee advisor,

for his counsel and guidance, for teaching and sharing his knowledge, and for providing me the

opportunity for an excellent graduate program. Appreciation is also extended to the members of

my supervisory committee (Drs. William W. Thatcher and Owen Rae), for their important

contributions to my work.

Special thanks go to my lab mates (Brad Austin, Regina Esterman, and Steavan Woodall)

for putting in many long hours to create successful research in the field and lab as well as

patiently passing on their skills. I would also like to extend thanks to Bar-L Ranch of Marianna,

FL, Dicks Farms of Lake City, FL, Roger Davis of Davis Farms and Tommy Taylor of Little

Creek Farms, Alachua, FL for providing facilities and cattle used in this research.

I would also like to thank my friends, who all at some point offered guidance and

support, shared laughs and tears, and created memories that will last a lifetime. Without them

this experience would have been unfulfilling. They all have enriched my life and have allowed

me to learn more about who I am.

Last of all I would like to thank my family for giving up a daughter and sister to the

Gators and always welcoming me back home to The "Buckeye State" with open arms regardless

of what colors I wear. The support and pride they provided allowed me to always keep my

spirits high and to keep my goals in sight.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S .................................................................................... .....................4

L IS T O F T A B L E S ................................................................................. 7

L IST O F FIG U R E S ............................................................................... 9

ABSTRACT ................... ............... ......... ... ...... ... .............

CHAPTER

1 INTRODUCTION ............... .......................................................... 13

2 R E V IEW O F L ITER A TU R E .................................................................... ... ....................18

A assisted R production in B eef C attle......................................................................... ... ... 18
T h e E strou s C y cle ........................................................................ .. 19
Hormonal Regulation: The Hypothalamic-Pituitary-Ovarian Axis....................................24
Follicular Development, Selection, and Ovulation...................................... ............... 27
Corpus Luteum Function and Luteolysis...................................................... 32
P ub erty ....................................................................................................37
Post Partum Interval and Return to Estrous....................................... ......................... 41
M manipulation of the Estrous Cycle ................................................. ........................ 42
Prostaglandin F2 .................. ....... ....................................43
P rogestogen s ............................................................... ................. 46
Gonadotropin Releasing Hormone (GnRH) ....................................... ............... 50
Synchronization P rotocols ................................................................ .............. .. .51

3 COMPARISON OF TWO PROGESTOGEN BASED ESTROUS
SYNCHRONIZATION PROTOCOLS AND CLOPROSTENOL SODIUM VS.
DINOPROST TROMETHAMINE IN SUCKLED POST PARTUM COWS AND
YEARLING HEIFERS OF BOS INDICUS x BOS TAURUS BREEDING ..........................58

Introduction ..... ..................................... ..............................................58
M materials and M methods ........................ .. ........................ .. .... ........ ........ 60
E x p e rim e n t 1 ............................................................................................................. 6 0
E x p e rim e n t 2 ............................................................................................................. 6 3
R e su lts ................... ...................6...................5..........
E x p e rim e n t 1 ............................................................................................................. 6 5
E x p e rim e n t 2 ............................................................................................................. 6 7
D iscu ssion .......... ..........................................................68
Im p licatio n s .........................................................................7 9









4 EVALUATION OF TWO PROGESTOGEN BASED ESTROUS
SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS OF BOS INDICUS x
B O S TA U R U S B R E E D IN G .......................................................................................88

Introduction .......... ............................... ...............................................88
M materials and M methods ........................ .. ........................ .. .... ........ ........ 89
R esu lts ................... .............................................................. ................. 92
D iscussion................................................................... 94
Im p lic a tio n s .................................................................................................................... 1 0 3

CONCLUSIONS AND IM PLICATIONS........................................................ ............... 110

APPENDIX

A SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 3................................... 123

B SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 4...............................130

L IST O F R E F E R E N C E S .................................................................................. ..................... 134

B IO G R A PH IC A L SK E T C H ......................................................................... .. ...................... 157









LIST OF TABLES


Table page
3-1 Effect of synchronization treatment and body condition score (BCS) on estrous
response in suckled Bos indicus x Bos taurus cows. .............................. ............... .81

3-2 Effect of synchronization treatment and body condition score (BCS) on conception
rate in suckled Bos indicus x Bos taurus cows. ..................................... ............... 82

3-3 Effect of days postpartum (DPP) on timed-AI pregnancy rate in suckled Bos indicus
x B os taurus cow s. .........................................................................83

3-4 Effect of synchronization treatment, body condition score (BCS), and days
postpartum (DPP) on synchronized pregnancy rate in suckled Bos indicus x Bos
ta urus cow s. ............................................................ ................. 84

3-5 Effect of body condition score (BCS) on thirty day pregnancy rates in suckled Bos
indicus x Bos taurus cow s ..................................................................... ............... 85

3-6 Effectiveness of 7-10 treatment compared to Select Sync/CIDR+timed-AI
(SSC+TAI) treatment on estrous response, conception rate, timed-AI pregnancy rate,
synchronized pregnancy rate, and thirty day pregnancy rate in yearling heifers of Bos
indicus x Bos taurus breeding. ..... ........................... ......................................... 86

4-1 Physical description ofBos indicus x Bos taurus yearling heifers including
reproductive tract score (RTS), body condition score (BCS), and breed composition
for heifers synchronized with two progestogen based estrous synchronization
protocols (L SM means SE) ........ .......... ............................................. ............... 104

4-2 Effectiveness of the 7-11 treatment compared to Select Sync/CIDR+timed-AI
(SSC+TAI) synchronization treatment on estrous response, conception rate, timed-
AI pregnancy rate, synchronized pregnancy rate and thirty day pregnancy rate in
yearling heifers of Bos indicus x Bos taurus breeding............................... ...............06

4-3 Effect of body condition score (BCS) on estrous response in Bos indicus x Bos
taurus yearling heifers. ............................................. .. .. .... ......... ....... 107

4-4 Effect of reproductive tract score (RTS) on estrous response, conception rate, timed-
AI pregnancy rate, synchronized pregnancy rate and thirty day pregnancy rate.............09

A-i Effect of synchronization treatment and prostaglandin treatment on estrous response,
conception rates and pregnancy rates in suckled cows of Bos indicus x Bos taurus
breeding ........ ............................... ................................................ 123

A-2 Estrous, conception and pregnancy rates by prostaglandin treatment of suckled cows
ofBos indicus x Bos taurus breeding synchronized with either a modified 7-11 (7-
10) or Select Synch/CIDR+timed-AI (SSC+TAI) treatment with either Cloprostenol
sodium (Cloprostenol) or Dinoprost tromenthamine (Dinoprost). .................................124









A-3 Estrous response, conception and pregnancy rates by synchronization treatment of
yearling heifers of Bos indicus x Bos taurus breeding synchronized with either a
modified 7-11 (7-10) or Select Synch/CIDR+timed-AI (SSC+TAI) treatment with
either Cloprostenol sodium (Cloprostenol) or Dinoprost tromenthamine (Dinoprost)
for years 1 and 2 Experiment2. ..... ........................... ........................................ 127

A-4 Estrous response, conception and pregnancy rates by prostaglandin treatment of
yearling heifers of Bos indicus x Bos taurus breeding synchronized with either a
modified 7-11 (7-10) or Select Synch/CIDR + timed-AI (SSC+TAI) treatment with
either Cloprostenol sodium (Cloprostenol) or Dinoprost tromenthamine (Dinoprost)
for years 1 and 2 Experiment 2. ..... ........................... .......................................128

A-5 Estrous response, conception and pregnancy rates of yearling heifers of Bos indicus
x Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Select
Synch/CIDR + timed-AI (SSC+TAI) treatment with either Cloprostenol sodium
(Cloprostenol) or Dinoprost tromenthamine (Dinoprost) for years 1 and 2
Experim ent 2 ............... ....... ... ........................................... ....... ......... 129

B-l Description of the reproductive tract score (RTS) used in Chapter 4 ............................130

B-2 Estrous response, conception and pregnancy rates of yearling heifers of Bos indicus
x Bos taurus breeding synchronized with either a 7-11 or Select
Synch/CIDR+timed-AI treatment by location........................ ...... ..... ..........133









LIST OF FIGURES


Figure pe

3-1 Experimental design evaluating the effects of two progestogen and two PGF2a
treatments in Bos indicus x Bos taurus suckled cows and yearling heifers for
Experiments 1 and 2. a) 7-10: on day 0 received a once-used-CIDR that was
removed on d 7 concomitant with dinoprost tromethamine (dinoprost; 25 mg i.m)
followed by GnRH (100 [g i.m.) on day 10. On day 17, females received either
dinoprost or cloprostenol sodium (cloprostenol; 500 pg i.m). b) Select
Synch/CIDR+timed-AI: on day 10 received a new CIDR (1.38 g) concomitant with
GnRH. Day 17 CIDR was removed and females received either females received
either dinoprost or cloprostenol. For all four treatments estrus was detected for 3 d
and females were inseminated 6 to 12 h after detected estrus. Females not exhibiting
estrus by 72 h were timed-AI and received GnRH. ............... ................................. 80

3-2 Effect of interval from PGF2, to onset of estrus on conception rate in Bos indicus x
Bos taurus yearling heifers synchronized with 7-10 or Select Synch/CIDR+timed-AI
(SSC+TAI) treatments. Numbers in parenthesis indicate the number of heifers in
each category. Treatment by interval from PGF2, to onset of estrus (P = 0.02). .............87

4-1 Experimental design evaluating the effects of two progestogen based synchronization
treatments in yearling Bos indicus x Bos taurus heifers. a.) 7-11 heifers were
administered MGA (0.5 mg/hd/d) for 7 d with PGF2a (25 mg, im) on last day of
MGA. GnRH (100 Gg, im) was administered on day 11 and PGF2a was administered
on day 18. The Select Synch/CIDR+timed-AI heifers received the same carrier
supplement without MGA from day 0 to 7. On day 11 a CIDR (Eazi-BreedTM
CIDR) was inserted concomitant with GnRH. The CIDR was removed on day 18
concurrent with PGF2,. For both treatments, estrus was detected for 3 d and heifers
were inseminated 6 to 12 h after a detected estrus. Heifers not exhibiting estrus by
72 h were timed-AI and received GnRH between 72 to 76 h ...........................105

4-2 Conception rates for the different interval from PGF2, to onset of estrus categories in
yearling heifers of Bos indicus x Bos taurus breeding. Means are expressed as a
percentage of total that exhibited estrus and became pregnant. Means without a
common superscript differ (P < 0.05). Interval from PGF2a to onset of estrus (P <
0.05) and synchronization treatment x interval from PGF2, to onset of estrus (P >
0 .0 5). ..................................................................................... 10 8

A-1 Effect of 7-10 or Select Synch/CIDR+timed-AI (SSC) treatment on estrous response
in suckled postpartum Bos indicus xBos taurus cows. Means are expressed as a
percentage of total that exhibited estrus...................................... ........................... 125

A-2 Effect of 7-10 or Select Sych/CIDR+timed-AI (SSC) synchronization treatment and
interval from PGF2a to onset of estrus on conception rate in suckled postpartum Bos
indicus xBos taurus cows. Means are expressed as a percentage of total that exhibited
estrus and became e pregnant ........................................................... ............... 126









B-l Effect of estrous synchronization treatment on estrous response in yearling heifers of
Bos indicus x Bos taurus breeding synchronized with 7-11 or Select
Synch/CIDR+timed-AI (SSC). Means are expresses as a percentage of total that
exhibited estrus. ..........................................................................131

B-2 Effect of estrous synchronization treatment on conception rate in yearling heifers of
Bos indicus x Bos taurus breeding synchronized with 7-11 or Select
Synch/CIDR+timed-AI (SSC). Means are expressed as a percentage of total that
exhibited estrus and became e pregnant. ........................................ ........................ 132









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

COMPARISON OF TWO SHORT-TERM PROGESTOGEN BASED ESTROUS
SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS AND SUCKLED
POSTPARTUM COWS OF Bos indicus x Bos taurus BREEDING

By

Erin N. McKinniss

August 2008

Chair: Joel V. Yelich
Major: Animal Sciences

A series of experiments were conducted with suckled cows and yearling heifers of Bos

indicus x Bos taurus breeding to compare two synchronization protocols including the Select

Synch/CIDR+timed-AI (SSC+TAI) and a modified 7-11 protocol. In Experiment 1, suckled

Bos indicus x Bos taurus cows (n = 324) were used in a 2 x 2 factorial design comparing

SSC+TAI to a modified 7-11 (7-10) protocol and within each synchronization treatment there

were two prostaglandins (PG) treatments including cloprostenol sodium (CPG) and dinoprost

tromethamine (DPG). Cows were equally distributed to treatments by body condition score

(BCS) and days postpartum (DPP). On day 0, the 7-10 treatment received an autoclaved once-

used-CIDR that was removed on day 7 concomitant with DPG followed by GnRH on day 10.

On day 17, 7-10 cows received either DPG or CPG. Also on day 10, SSC+TAI treatment

received a new CIDR concomitant with GnRH with CIDR removal on day 17 where cows

received either DPG or CPG. Estrus was visually detected twice daily (0700 and 1600 h) for 72

h after PGF2a and cows were AI 6 to 12 h after a detected estrus. Non-responders were timed-AI

+ GnRH 72 to 76 h post PG. In experiment 2, yearling heifers over two breeding seasons (n =

218 and 137) were randomly assigned to the same treatments as Experiment 1, estrous detection









and AI were administered in the same manner as Experiment 1. In experiment 1, estrous

response (ER), conception (CR), and synchronized pregnancy rate (SPR) were affected (P <

0.05) by synchronization treatment but not (P > 0.05) PG treatment. The SPR were 45.5% and

31.2 for the SSC+TAI and 7-10 treatments, respectively. As BCS increased from < 4.5 to > 5.5,

ER, CR, SPR, and thirty day pregnancy rates (TDPR) increased (P < 0.05). In experiment 2, ER,

CR, TAIPR, SPR, and TDPR were similar (P > 0.05) between the synchronization and PG

treatments, with the mean ER, CR, TAIPR, SPR and TDPR being 66.8, 65.0, 16.1, 48.7, and

76.3%, respectively.

In Experiment 3, yearling heifers (n = 407) were synchronized with either the SSC +TAI

treatment from Experiment 1 or 7-11 treatment that included melengestrol acetate (MGA; 0.5

mg/head/d) for 7 d with GnRH 4 d after the last day of MGA followed by DPG 7 d later. Estrus

was detected and heifers were inseminated similar to Experiment 1. Heifers were distributed to

treatment by reproductive tract score (RTS; Scale: 1=immature to 5= estrous cycling) and BCS.

The 7-11 heifers had greater (P < 0.05) ER (55.2 vs. 41.9%), CR (47.0 vs. 31.3%), and SPR

(33.5 vs. 24.8%) compared to SSC+TAI heifers, respectively. Heifers exhibiting estrus at 60 h

after PG (61.7%) had a greater (P < 0.05) CR compared to heifers that had exhibited estrus at <

36 (35.3%), 48 (31.6%), and 72 h (36.2%), which were all similar (P > 0.05) to each other. As

RTS increased from < 2 to a > 3, ER, CR, SPR, and TDPR all increased (P < 0.05). In summary,

there was a differential response to synchronization treatments as cows treated with the

SSC+TAI had greater SPR compared to the 7-10 treatment; whereas, heifers synchronized with

the 7-11 treatment had greater SPR compared to the SSC+TAI treatment.









CHAPTER 1
INTRODUCTION

Recent advances in reproductive technologies have made a significant impact on the beef

cattle herd on both a national and global scale. Most notably is the use of artificial insemination

(AI) to expedite genetic gains and to decrease costs associated with housing and purchasing bulls

of superior genetic quality. However, broad dissemination and utilization of AI in the United

States beef cattle herd is limited, primarily due to the time and labor costs associated with

detecting estrous during the 21 day estrous cycle. Incorporation of estrous synchronization into

an AI program decreases the amount of time needed to detect estrus and (or) can eliminate

estrous detection when a fixed breeding time is used. Additional benefits of using estrous

synchronization includes promoting estrous cyclicity in some non-cycling animals, increasing

the percentage of cattle pregnant early in the breeding season, shortening the breeding and

subsequent calving seasons, and increasing uniformity of the calf crop. A more uniform calf

crop is important since calves are worth more at weaning.

Numerous factors can influence the effectiveness of estrous synchronization protocols

including nutritional status, estrous cycling status, stage of the estrous cycle, days postpartum at

the start of the synchronization protocol, and animal genotype. There are essentially three

genotypes used in the beef cattle production systems in the United States including Bos taurus,

Bos indicus, and Bos indicus x Bos taurus. The majority of cattle in the Gulf Coast region of

United States, including other tropical and subtropical climates of the world, are of Bos indicus x

Bos taurus breeding. Cattle with Bos indicus breeding are vital to efficient production in these

environments because the cattle can withstand the hot and humid environment, utilize lower

quality forages common in these environments, and are more resistant to parasites compared to

cattle of Bos taurus breeding. With that said, the majority of the research and development of









estrous synchronization protocols is based on the Bos taurus genotype. Furthermore, limited

research indicates that Bos indicus and Bos indicus x Bos taurus cattle have subtle physiological

and behavioral differences that elicit different responses to estrous synchronization

pharmaceuticals and protocols compared to Bos taurus cattle. Consequently, the primary

objective of the research reported in this thesis will focus on evaluating the effectiveness of two

estrous synchronization protocols in cattle of Bos indicus x Bos taurus breeding.

There are several pharmaceuticals available to beef cattle producers that can be used in

estrous synchronization protocols. Whether used alone or together, the primary goal when using

these products is to manipulate the estrous cycle so that estrus and (or) ovulation is more

predictable and uniform across the group of cattle being synchronized. There are three classes of

pharmaceuticals that are primarily used in estrous synchronization protocols including

prostaglandin F2z (PGF2,), gonadotropin-releasing hormone (GnRH), and progestogens. The

primary function of PGF2, is to induce lysis of the corpus luteum, which results in the expression

of estrus in 3 to 5 d. In contrast, GnRH is used to manage follicle development by stimulating a

pre-ovulatory surge of luteinizing hormone (LH) to initiate ovulation of growing follicles > 10

mm in diameter and stimulation of follicle stimulating hormone (FSH) which will assist

follicular recruitment into the next follicular wave. Treatment with GnRH can be used to either

stimulate follicle turnover to initiate a new follicle wave or to synchronize ovulation in

conjunction with AI. The final pharmaceutical that can be used are progestogens. The primary

functions of progestogens are to prevent expression of estrus and ovulation by preventing the

pre-ovulatory surge of LH. There are two progestogens available for use as estrous

synchronization agents and they are melengestrol acetate (MGA) and the intra-vaginal

progesterone insert known as the CIDR. Progestogens can also have secondary effects by









priming the hypothalamic-pituitary-ovarian-uterine axis, which can serve to initiate estrus in

some anestrous cattle and possibly enhance pregnancy rates in synchronized cattle.

One of the most common estrous synchronization protocols used by beef cattle producers

is the Select Synch protocol. This protocol utilizes GnRH followed by a PGF2, treatment 7 d

later. Estrus is detected for 5 to 7 d post PGF2a with AI occurring 6 to 12 h after an observed

estrus. The down side to the protocol is that some cattle exhibit estrus 1 to 2 d prior to PGF2a,

which increases the amount of estrous detection required. Subsequent research demonstrated

that administration of a progestogen between GnRH and PGF2, eliminated the need for extra

estrous detection before PGF2a without compromising fertility. The progestogen used in the

Select Synch protocol can be either MGA or a CIDR, but the CIDR appears to be the preferred

method for several reasons. First, addition of the CIDR does not increase labor requirements

because the cattle are handled to administer the GnRH and PGF2,. Second, since MGA is

administered in the feed, inadequate MGA consumption can result in cattle that "break through"

and express estrus between the GnRH and PGF2, treatments. And third, the CIDR may provide

more "progesterone like" effects compared to MGA. The "progesterone like" effects include

priming the hypothalamic-pituitary-ovarian-uterine axis, which could induce estrous cyclicity in

some anestrous cattle and quite possibly enhance pregnancy rates. Although, the research is not

definitive on this issue, additional research is needed to validate whether the CIDR is more

effective compared to MGA as a synchronization agent. When a CIDR is used with the Select

Synch system it is called the Select Synch/CIDR synchronization system. Additional research in

cattle of Bos taurus breeding demonstrated that three days of estrous detection and AI combined

with a timed-AI plus GnRH 72 to 80 h after PGF2, for cattle not exhibiting estrus resulted in

similar and more consistent AI pregnancy rates compared to 5 d of estrous detection and AI. The









aforementioned synchronization protocol is called the Select Synch/CIDR+timed-AI protocol,

which has been shown to be an effective protocol in both suckled cows and yearling heifers of

Bos taurus breeding. In contrast, limited research has been conducted on the effectiveness of the

Select Synch/CIDR+timed-AI system in suckled postpartum cows and yearling heifers of Bos

indicus x Bos taurus breeding. Therefore, the first objective of my research was to evaluate the

effectiveness of the Select Synch/CIDR+timed-AI synchronization protocol in suckled

postpartum cows and yearling heifers of Bos indicus x Bos taurus breeding.

One of the problems with utilizing progestogens, particularly long term progestogen

treatments greater than 10 d, is a decrease in fertility of the estrus immediately after progestogen

removal. With short term (< 7 d) progestogen treatments there can be a slight decrease in

fertility, but that decrease can be overcome by administering GnRH at the initiation of the

progestogen treatment in the GnRH + PGF2, protocols. Another way to utilize a progestogen

and avoid the depression in fertility is to use the 7-11 estrous synchronization protocol. The 7-11

protocol consists of 7 d of MGA with PGF2, on the last day of MGA followed by GnRH 4 d

later. This pre-synchronization portion of the 7-11 protocol is designed specifically to

synchronize follicle development in a majority of cattle. Seven days after GnRH, another PGF2,

treatment is administered followed by 5 to 7 d of estrous detection. Research in suckled

postpartum cows and yearling heifers of Bos taurus breeding has demonstrated that the

synchrony of estrus was excellent and a combination of estrous detection and timed-AI could be

incorporated into the 7-11 protocol resulting in acceptable synchronized pregnancy rates.

However, no such research has been conducted with the 7-11 protocol in either suckled

postpartum cows or yearling heifers ofBos indicus x Bos taurus breeding.









The cost of the 7-11 protocol is approximately $10.00/animal to implement compared to

$15.00/animal for the Select Synch/CIDR+timed-AI protocol. The increased cost of the Select

Synch/CIDR+timed-AI is primarily due to the cost of the CIDR, which are approximately $10

each. A cost efficient alternative to using a new CIDR is utilization of a once used CIDR in the

Select Synch+timed-AI protocol. However, limited research has evaluated this alternative and

additional research is needed to determine if the once-used CIDR used in the Select Synch

protocol results in similar responses compared to a new CIDR in both estrous cycling and

anestrous cows. The once used CIDR could also be included in the 7-11 protocol as an

alternative to MGA. This is particularly true in programs where MGA consumption is of

concern or producers do not want to have to provide additional supplements to administer the

MGA. Therefore, a second objective of the research was to evaluate the effectiveness of the 7-

11 estrous synchronization protocol in cattle of Bos indicus x Bos taurus breeding and to

evaluate the effectiveness of incorporating the once-used CIDR into the 7-11 estrous

synchronization protocol.









CHAPTER 2
REVIEW OF LITERATURE

Assisted Reproduction in Beef Cattle

One of the most important goals in beef cattle production is to improve the genetic

quality of the cow herd from year to year in an effective and economic manner. Many recent

improvements in biotechnology and beef cattle management have made this more accessible to

the average producer, namely development of artificial insemination (AI), and estrous

synchronization protocols. Historically, reports of AI date back hundreds of years; however, it

has only been used commercially in the later part of this century (Foote, 2002). Development of

effective semen collection and storage methods, improved methods of delivery of frozen/thawed

semen to females, development of enhanced estrous detection and estrous synchronization

methods as well as a more complete understanding of the reproductive physiology of the male

and female have all been a vital part in developing successful assisted reproduction programs.

Artificial insemination allows producers an unlimited availability to superior sires

without expending costs associated with purchasing and housing a bull. Utilization of AI also

allows for rapid genetic improvement of the cowherd by using specific genetic indicators such as

expected progeny differences (EPD), which serve as indicators of the bull's ability to pass on his

traits to his progeny. However, the costs associated with implementing an AI program including

labor and supplies, frozen/thawed semen, and natural service bull costs have to be weighed with

success of pregnancy and profits (Pace, 1985).

Estrous synchronization used in conjunction with AI is a great way to capitalize on this

technology. Estrous synchronization uses exogenous hormones to manipulate the life span of the

corpus luteum (CL) and (or) to synchronize follicle development. Synchronization of the CL

and (or) follicle development, allows for a synchronization of estrus in most females of the herd









allowing for a shorter window for AI breeding. Treatment with an estrous synchronization

protocol can also increase the number of females pregnant early in the breeding season, which

results in a shorter calving season and a more uniform calf crop at weaning. All of these

variables drive profits and increase an operation's production efficiency. With that said,

additional costs are associated with implementing an estrous synchronization and AI program

including increased labor, synchronization pharmaceuticals, frozen semen, and hiring an AI

technician. Consequently, beef producers want estrous synchronization protocols that require

minimal cattle handling and labor, cost effective, and consistently yield acceptable AI pregnancy

rates. There are numerous factors that influence the effectiveness of an estrous synchronization

protocols including but not limited to onset of puberty, estrous cycling status, parity, lactation

status, nutrition, environment, management techniques, and cattle genotype. These factors can

act either alone or in concert to influence the outcome to an estrous synchronization protocol.

Therefore, the purpose of this literature review is to discuss and summarize many of these factors

as they relate to the implementation of estrous synchronization protocols in cattle of Bos indicus

x Bos taurus breeding

The Estrous Cycle

The bovine estrous cycle is an accretion of hormonal and physiological events which

provides repeated opportunities for mating and quite possibly pregnancy. These reproductive

events will begin at puberty and continue through adult life, only to be interrupted by pregnancy

and the subsequent lactation. The estrous cycle is delineated into two phases, follicular and

luteal, which are recognized by the ovarian structures present. The follicular phase is defined as

the period from regression of the CL to ovulation of a newly developed follicle. The follicular

phase can be further separated into proestrus and estrus. Estradiol secreted from selected

dominant follicles during the follicular phase will drive female receptivity, estrous behavior, and









the surge of luteinizing hormone (LH) which induces ovulation. The longer of the two phases,

the luteal phase, is defined as the period from ovulation until CL regression. The luteal phase

can also be further separated into two stages, metestrus and diestrus. The erupted follicle from

the follicular phase eventually luteinizes and forms a CL, which secretes progesterone and begins

the process of developing the uterus into an environment acceptable for pregnancy. If pregnancy

does not occur, the uterus secretes prostaglandin F2a (PGF2,) and luteolysis occurs and the female

returns to estrus.

The length of the estrous cycle in cattle of Bos taurus breeding is on average 21 d (Hansel

et al., 1973). However, variations in estrous cycle length exist between Bos indicus and Bos

taurus type cattle, with Bos indicus breeds having longer estrous cycles. Mattoni et al. (1988)

reported a mean estrous cycle length of 22.6 d in Zebu cattle and Plasse et al. (1970) reported

27.7 d in Brahman cattle. Gir cows have a mean estrous cycle length of 21.7 d (Moreira-Viana

et al., 2000). Alternatively, Alvarez et al. (2000) reported similar estrous cycle lengths for

Angus (19.5 d), Senepol (20.4 d), and Brahman (19.7 d) cows. Variation in estrous cycle length

has also been observed between heifers and cows of Bos indicus breeding, as Boran cows had a

23 d estrous cycle (Llewelyn et al., 1987), compared 27.7 d in to Brahman heifers (Plasse et al.,

1970). Conversely, Bos taurus cows (Zollers et al., 1989) and heifers (Mihm et al., 2000) exhibit

minimal variation from the 21 d estrous cycle length.

Estrus demarcates the beginning of the estrous cycle. It is the most recognizable stage as it

is diagnosed by behavioral characteristics where the female is sexually receptive to the male.

Estradiol concentrations peak during estrus, causing the behavioral expression of estrus and

initiation of the preovulatory surge ofLH (Wettemann et al., 1972; Short et al., 1973).

Approximately 6 to 8 h after the peak estradiol concentrations, the preovulatory surge of LH









occurs (Walters and Schallenberger, 1984) resulting in ovulation 26 to 48 h after the beginning

of behavioral estrus (Plasse et al., 1970; Looper et al., 1998). Circulating concentrations of

estradiol have been reported to be less in Bos indicus compared to Bos taurus cows during day 7

to 17 of the estrous cycle (Segerson et al., 1984). Visual signs of estrus include but are not

limited to the female standing to be mounted by another animal, vulvular mucous discharge,

Flehmen response, restlessness, sniffing other cow's genitals, and chin resting (Van Eerdenburg

et al., 2002). These signs can occur before, during, or after estrus and should be used as visual

keys in diagnosing the best time for breeding, but should not necessarily be used to determine

exact time of ovulation (Nebel, 2003; Forster et al., 2006).

The duration (length of estrus) and intensity of estrus (number of mounts received during

the duration of estrus) can be influenced by several factors including genotype, estradiol

concentrations, social status, temperature and humidity, and the number of animals in estrus at

one time. Bos indicus breeds tend to have a shorter and less intense estrus (Plasse et al., 1970;

Galina et al., 1982; Mattoni et al., 1988; Pinheiro et al., 1998) compared to Bos taurus breeds

(Stevenson et al., 1996; Richardson et al., 2002). The duration of estrus in Bos taurus breeds

ranges from 3 to 26 h with a mean of 14 h (Schams et al., 1977). Conversely, the duration of

estrus in Bos indicus and Bos indicus x Bos taurus breeds ranges from 1 to 24 h with a mean of 7

h (Mattoni et al., 1988; Rae et al., 1999). Landaeta-Hernandez (2002) also reported that the

duration of a synchronized estrus tended to be shorter in Senepol compared to Angus or

Brahman cows. However, breed had no effect on the duration and total number of mounts

received during either a synchronized or spontaneous estrus; however, duration of estrus and

total number of mounts received during estrus were greater for the synchronized compared to the

spontaneous estrus. The variation in duration and intensity of estrus within and between breed









types may be attributed to circulating estradiol concentrations (Rhodes and Randal, 1978; Lyimo

et al., 2000). Lyimo et al. (2000) reported that increased estradiol concentrations were indicative

of increased behavioral estrus activity. Since circulating estradiol concentrations have been

reported to be greater in Bos taurus compared to Bos indicus cows (Segerson et al., 1984), it

could explain why cattle of Bos indicus breeding have a decreased duration of estrus and estrous

intensity. In contrast, Alvarez (2000) reported similar estradiol concentrations between Angus

and Brahman cows, but the authors did not report on any behavioral estrus data. Social status

within herd can also influence behavioral estrus. Landaeta-Hernandez et al. (2004) reported that

dominant and intermediate cows had a longer duration of estrus compared to subordinate cows

but dominant cows tended to have less total mounts during estrus compared to subordinate and

intermediate cows. The later response suggests that dominant cows probably avoided being

mounted as frequently during estrus compared to subordinate cows.

Increasing the number of cows in estrus at a single point in time increased mounting

activity in both Bos taurus and Bos indicus females (Galina et al., 1994), which can lead to

formation of sexually active groups and make it easier to detect estrus. Humik (1975) reported

that increasing the number of cows in estrus from 1 to > 4 cows increased the number of mounts

received from 11 to 49 in Bos taurus cattle. One of the problems with detecting estrus in cattle

of Bos indicus breeding is the increased incidence of the silent estrus (Plasse et al., 1970;

Dawuda et al., 1989; Lamothe-Zavaleta et al., 1991), high incidence of estrus expression at night

(Pinheiro et al., 1998), increased secondary estrus activities other than mounting such as head

butting (Orihuela et al., 1983), and decreased incidence of mounting activity (Galina et al.,

1982). Therefore, it is imperative for producers that want to AI cattle of Bos indicus breeding

that the cattle be synchronized to increase the opportunity for the cattle to be detected in estrus.









Numerous other factors influence behavioral estrus including environmental conditions

such as temperature and humidity (Landaeta-Hernandez et al., 2002), time of day (Pinheiro et al.,

1998), parity (Flores et al., 2006), and frequency with which cattle are handled through working

facilities (Lemaster et al., 1999). It is unclear whether these variables act alone or in concert to

affect estrous behavior, but it demonstrates the multitude of factors that influence behavioral

estrus.

Ovulation typically occurs between 24 to 36 h after the onset of estrus in Bos taurus

(Looper et al., 1998; Rorie et al., 1999) and Bos indicus cattle (Plasse et al., 1970; Mattoni et al.,

1988; Pinheiro et al., 1998). Estrus and ovulation are followed by metestrus, which lasts from 3

to 5 d and is characterized by formation of a functional CL and synthesis and secretion of

progesterone. Progesterone concentrations remain low until the CL is fully functional. During

metestrus there is a transition from estradiol dominance to progesterone dominance and

increased concentrations of circulating progesterone initiate the next stage, which is diestrus.

Diestrus is the longest stage of the estrous cycle lasting 10 to 14 d and is characterized by

increased progesterone concentrations that are great enough to block the activity of estradiol.

Progesterone also primes the uterus to develop a suitable environment for a potential concepts.

If pregnancy occurs, the CL will be maintained throughout gestation and continue to synthesize

and secrete progesterone. If pregnancy does not occur, the CL will remain functional until day

17 to 18 of the estrous cycle, at which time luteolysis is initiated by PGF2a secreted from the

uterus. At this point, degeneration of the luteal tissue occurs and resumption of a new estrous

cycle begins. The length of diestrus is directly related to life of the CL.

Progesterone concentrations during the luteal phase of the estrous cycle are also different

between Bos taurus and Bos indicus cattle. Adeyemo and Heath (1980) observed higher peak









concentrations of progesterone during the luteal phase in Bos taurus heifers compared to Bos

indicus heifers. Progesterone concentrations were also greater in Bos indicus heifers 1 or 2 d

before estrus. Progesterone concentrations increased from early to mid and late luteal phases,

with the increase being more discernible in the Angus compared to Brahman cows (Segerson et

al., 1984). Mean progesterone concentrations were also greater in Angus compared to Brahman

cows that were either pregnant or not pregnant (Segerson et al., 1984).

Proestrus is the interval from luteolysis until the onset of estrus and is initiated 3 to 4 d

before estrus. Proestrus is characterized by increased follicular growth and is the transition from

progesterone dominance to estrogen dominance (Chenault et al., 1975; Kesner et al., 1982).

During proestrus, a subordinate follicle is selected from a pool of follicles and becomes a

dominant follicle, which will eventually be the ovulatory follicle.

Hormonal Regulation: The Hypothalamic-Pituitary-Ovarian Axis

Estrous cyclicity and overall reproductive function in cattle is highly dependent upon the

complex interactions of hypothalamic and pituitary hormones and the hormone secreting

structures of the reproductive tract. Release of gonadotropin releasing hormone (GnRH) from

the hypothalamus mediates the secretion of the gonadotropins, follicle stimulating hormone

(FSH) and luteinizing hormone (LH), from the anterior pituitary. Gonadotropins act on specific

target tissues and (or) cells of the ovary that synthesize and secrete progesterone and estrogen.

Both positive and negative feedback effects induced by either estradiol or progesterone will

further control synthesis and secretion of gonadotropins from the hypothalamus and pituitary.

Gonadotropin releasing hormone is a neuropeptide (10 amino acids) produced in two sets

of paired nerve cell bodies in the hypothalamus. The GnRH is synthesized and secreted from the

nerve cells located in the tonic and surge centers of the hypothalamus. Nerve axons extend from

the hypothalamic nerve cell bodies to the capillary plexus at the anterior pituitary where GnRH is









released. This unique system is called the hypothalamic-hypophyseal portal system. The portal

system is essential as it allows transfer of GnRH directly to the anterior pituitary instead of the

general circulatory system where it would be quickly degraded. Basal secretion of GnRH is

controlled by the ventromedial and arcuate nuclei or the tonic center of the hypothalamus and is

characterized with low amplitude and high frequency LH pulses during the luteal phase of the

estrous cycle when progesterone concentrations are increased. The tonic center continues to

release basal concentrations of LH until the appropriate positive stimulus are received, which is

increased estrogen preceded by a rapid decline in progesterone. The suprachiasmatic and

preoptic nuclei are part of the surge center which is responsible for the release of GnRH during

the preovulatory phase of the estrous cycle. The surge center mediates high amplitude and low

frequency GnRH pulses, which enhance the LH surge necessary for ovulation. Estradiol

secreted from the dominant follicle precedes this event to enhance the surge of LH (Wettemann

et al., 1972). Each GnRH pulse precedes each LH pulse (Schams et al., 1974).

Luteinizing hormone, a glycoprotein, is the dominant luteotrophic hormone in the bovine.

It is vital to CL function (Simmons and Hansel, 1964; Hoffmann et al., 1974) and is necessary

for progesterone synthesis (Armstrong and Black, 1966). Decreasing LH concentrations

surrounding the LH surge actually inhibits progesterone secretion and the vitality of CL

development (Quintal-Franco et al., 1999). During the early luteal period, LH pulses are

characterized by low amplitude and high frequency, whereas during the midluteal period LH

pulses are characterized by high amplitude and low frequency (Rahe et al., 1980). Changes in

circulating concentrations of estradiol and progesterone can effect LH secretion. Ginther et al.

(1996) reported that decreasing the circulating concentrations of progesterone increased LH

pulse frequency and estradiol concentrations within 6 h. Development of large and persistent









dominant follicles occurs during low progesterone treatment due to an increase in LH pulse

frequency (Roberson et al., 1989; Savio et al., 1990). Although, LH pulses are necessary for

follicle development beyond 9 mm (Gong et al., 1996), decreased LH pulse frequency can result

in atresia of large follicles (Savio et al., 1990).

The physiology of LH secretion is different between Bos indicus and Bos taurus cattle.

Randel (1976) reported a longer interval from an exogenous estradiol treatment to LH peak and

decreased peak LH concentrations in Brahman compared to Hereford cows. Additionally, the

interval from the LH surge to ovulation and interval from the initiation of estrus to ovulation was

decreased in Brahman compared to Angus cows (Randel, 1976). Brahman cows also have a

decreased responsiveness to estradiol and or gonadotropins (Rhodes et al., 1978). Portillo et al.

(2006) reported that as the percentage of Bos indicus breeding increased, the amount of LH

released in response to a GnRH challenge (500 Gg) decreased when administered on day 6 of the

estrous cycle.

The other glycoprotein hormone synthesized and secreted from the anterior pituitary is

FSH. The primary function of FSH is to promote follicular development and production of

estradiol and an FSH surge precedes the initiation of each follicular wave during the estrous

cycle (Adams et al., 1992). Follicle stimulating hormone has receptors located on the granulosa

cells of the growing follicle. Gibbons et al. (1997) demonstrated that the number of follicles that

advanced into a follicular wave was increased by increasing the FSH surge at an exaggerated

amount. As FSH peaks, follicles approach 4 mm in diameter (Ginther et al., 1996) and FSH

must be present in order for a follicle to grow past 9 mm (Gong et al., 1996). The final decline in

FSH is part of the deviation mechanism or selection of a dominant follicle (Ginther et al., 1997).

Growth of the dominant follicle further suppresses FSH secretion (Ginther et al., 1997).









The ovarian steroid hormones, estradiol and progesterone, are also intricately involved in

the regulation of the estrous cycle. The negative effect of progesterone on gonadotropin

secretion decreases as circulating progesterone decreases, which allows for an increase in LH

pulse frequency and a decrease in LH amplitude. This reduction occurs quickly in the three days

approaching estrus (Wettemann et al., 1972) and the change in LH secretion allows for the

dominant follicle to secrete increasing amounts of estradiol (Schallenberger et al., 1984).

Estradiol increases GnRH production as well as GnRH receptors in the anterior pituitary having

a positive effect on LH secretion (Vizcarra et al., 1997; Nett et al., 2002), which allows for

decreased LH pulse frequency, increased LH pulse amplitude, which results in an LH surge and

ovulation of the dominant follicle (Kesner et al., 1981).

Follicular Development, Selection, and Ovulation

Folliculogenesis is the process by which potential ovulatory follicles are formed from a

pool of naive, or primordial follicles (Spicer and Echternkamp, 1986). Folliculogenesis begins

prior to puberty and continues in cycles of development and death throughout the life span of the

animal. Primordial germ cells migrate to the gonadal ridge and are stored in primordial follicles,

and form oogonia during mid gestation (Hirshfield et al., 1991; McGee and Hsueh, 2000).

Lifetime supplies of oogonia within the ovary are established during gestation and the oogonia

replicate by mitosis. The fetal ovary contains growing, preantral, and early antral follicles due to

follicles leaving the resting pool while others are still being formed (Fortune, 2003). At birth,

oogonia are suspended at the first meiotic division and are deemed primary oocytes (Fortune,

1994). In ruminants, the oocytes are arrested at this stage until the LH surge initiates ovulation

and meiosis resumes (Vanderhyden, 2002). Initiation of follicle growth and its subsequent

development are characterized by proliferation of granulosa cells, change in shape of granulosa

cells, enlargement of the oocyte, and formation of the zona pellucida (Braw-Tal, 2002). There









are three stages of follicle development based primarily on the layers of granulosa cells that

comprise the follicle and they include primary, secondary, and tertiary follicles. Primary oocytes

are housed in primordial follicles and are characterized by a single layer of granulosa cells

consisting of 5 to 8 flattened cells, and are often referred to as quiescent follicles (Braw-Tal,

2002). As the follicles leave the resting pool they become primary follicles and the granulosa

cells multiply and become cuboidal and express markers of cell proliferation (Fair et al., 1997;

Fortune, 2003). Development continues to the secondary follicle stage with the addition of a

second layer of granulosa cells, which progresses through the addition of 6 to 7 layers (Fortune,

2003). Additionally, the zona pellucida appears and gap junctions are formed between the

oocyte and granulosa cells (Fair et al., 1997a). Stromal cells are recruited to form the thecal

layer and appear at the mid or late preantral stage (Gougeon, 1996). Folliculogenesis is

independent of gonadotropins (Roche, 2006) until the formation of thecal cells at the secondary

follicle stage (Scaramuzzi et al., 1993; Mihm et al., 2002). The secondary follicle transitions into

a tertiary follicle with the development of an antral cavity. The granulosa cells differentiate into

cumulus and mural cells, the latter of which are associated with steroidogenic functions

(Vanderhyden, 2002). As the follicle continues to develop, the antrum continues to accumulate

follicular fluid increasing the follicle size and is designated as a Graafian follicle.

Follicular development is a cyclic process, and progresses through stages or waves of

recruitment, selection, and dominance (Hodgen, 1982). The aforementioned stages are the basis

for the wave-like phenomenon of follicle development that occurs during the estrous cycle in

cattle. From an evolutionary standpoint, wave-like follicle development provides for an eligible

ovulatory follicle to always be available (Sirois and Fortune, 1988). Cattle have between 1 to 5

follicular waves during one estrous cycle (Viana et al., 2000), although, two to three wave









patterns are more commonly observed (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et

al., 1989).

Recruitment is a process where a cohort of small follicles (2 to 4 mm diameter) begins to

mature with support of sufficient pituitary gonadotropic stimulation (Hodgen, 1982; Adams et

al., 1992a). Stimulation from FSH promotes the growth of the cohort of follicles resulting in

development of 5 to 10 follicles (Lucy et al., 1992) between 4 to 5 mm (Adams et al., 1992a).

Selection is the process whereby a follicle is chosen, avoids atresia, and has the potential to

become an ovulatory follicle (Hodgen, 1982). Atresia is programmed cell death and it is the

most common fate of most follicles as only a few follicles reach dominance and ovulate. Greater

than 99% of ovarian follicles undergo atresia during the lifetime of the cow (Hsueh et al., 1994).

Fortune (1994) reported that atresia most commonly occurred before the final stages of follicular

dominance in cattle, but it can occur at any stage of follicular growth. As FSH concentrations

decline, fewer follicles continue to grow and are committed to atresia (Austin et al., 2001).

Deviation, a part of selection, is when the second largest follicle decreases in size or stops

growing in parallel with the dominant follicle, while the dominant follicle continues to grow

(Ginther et al., 1996; Ginther et al., 1997).

Dominance occurs when the selected follicle dominates through inhibition of recruitment

of a new cohort of follicles (Hodgen, 1982). The dominant follicle has increased estradiol and

inhibin production, the latter of which inhibits the recruitment of follicles of the next follicular

wave (Lucy et al., 1992; Ginther et al., 1999) and there is 3 to 4 d of additional growth of the

dominant follicle. In a majority of estrous cycles the first wave dominant follicle regresses

(Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989a) and a second wave evolves

through the recruitment and selection stages. The regression of the first-wave dominant follicle









is initiated by elevated circulating progesterone concentrations that have a negative effect on LH

secretion, which causes regression of the dominant follicle (Sunderland et al., 1994). In contrast,

if luteolysis occurs, progesterone concentrations decrease and estradiol production from the

dominant follicle initiates an increase in LH secretion and eventually causes an LH surge

resulting in ovulation (Wettemann et al., 1972). Maturation of the second dominant follicle in

two-wave cycles coincides with regression of the CL and this follicle ovulates after luteolysis

(Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991). In three-wave

follicle patterns the second wave dominant follicle becomes atretic and a third wave is recruited,

selected, and becomes the dominant follicle that eventually ovulates.

Follicular dynamics is defined as the continual growth and regression of antral follicles

that lead to the development of the preovulatory follicles (Lucy et al., 1992). Savio et al. (1990)

described follicular dynamics beginning early in the estrous cycle as a cohort of follicles is

recruited out of a pool of smaller antral follicles (2 to 4 mm). After 2 to 4 d of recruitment,

medium-sized follicles (6 to 9 mm) can be detected. After recruitment, selection begins for a

single follicle and this selected follicle continues to grow and the other recruited follicles

decrease in size. The dominant follicle (> 10 mm) of the first wave remains active until day 8 to

11 of the estrous cycle (Ginther et al., 1989a). The first wave dominant follicle prevents other

follicles from reaching a diameter > 5 mm. The first wave dominant follicle will ovulate if

luteolysis is initiated by day 5 to 8 of the estrous cycle (Kastelic et al., 1990); however, in most

cases the first wave dominant follicle regresses (Savio et al., 1988) and a second follicular wave

begins around day 12 to 14 of the estrous cycle (Rajakoski, 1960). Follicular dynamics can be

affected by breed and follicle number and size are most notably affected. The number of small

follicles (2 to 5 mm) is greater in Brahman compared to Angus or Senepol cows (Alvarez et al.,









2000) and Brahman cows have more medium and total number of follicles compared to Angus

cows (Simpson et al., 1994). Dominant follicle size is also different between breeds, as

Figueiredo et al. (1997) reported that Nelore cattle had a smaller dominant follicle compared to

Bos taurus counterparts.

The number of follicular waves during the estrous cycle can be influenced by many factors

such as length of luteal dominance (Ginther et al., 1989b), parity and (or) age (Figueiredo et al.,

1997), plane of nutrition (Rhodes et al., 1995; Mackey et al., 1999), lactation status (Lucy et al.,

1992), and breed (Savio et al., 1988; Ginther et al., 1989b; Rhodes et al., 1995; Viana et al.,

2000; Martinez et al., 2003). In general, Bos indicus cattle have been observed in numerous

studies to have mostly three- and four-wave follicle wave patterns (Rhodes et al., 1995; Zeitoun

et al., 1996; Viana et al., 2000; Martinez et al., 2003) compared to Bos taurus cattle that have

primarily two- and three-wave follicle wave patterns (Savio et al., 1988; Sirois and Fortune,

1988; Ginther et al., 1989b). Follicular dynamics in Gir cattle is characterized by an increased

incidence of estrous cycles with three or four follicle waves, associated with a low persistence of

the dominant follicle (Viana et al., 2000). However, breed has also been shown to have no effect

on the number of follicular waves between Bos indicus and Bos taurus cattle (Figueiredo et al.,

1997; Alvarez et al., 2000).

The length of the estrous cycle can be influenced by the number of follicle waves that

occur during the estrous cycle. The average length of the estrous cycle for cattle with two-wave

follicle patterns is 20 d (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989).

Whereas, cattle with three-wave follicle patterns have estrous cycles that range from 21 to 23 d

(Savio et al., 1988; Ginther et al., 1989; Viana et al., 2000). Taylor and Rajamahendran (1991)

observed that cattle with three follicular waves had a longer estrous cycle due to a longer luteal









phase. The reasons) for the difference in number of follicle wave patterns between breeds is

unclear. One theory suggests it may be due to luteal dominance as Ginther et al. (1989) observed

that luteolysis began later in three-wave estrous cycles compared to two-wave estrous cycles.

Another explanation could be the smaller diameter of the intermediate wave dominant follicles

compared to the diameter of ovulatory follicles, which is associated with a shorter plateau phase

that may contribute to greater number of waves observed in Zebu cattle (Viana et al., 2000).

The number of follicular waves during the estrous cycle could also affect fertility of the

subsequent estrus. Townson et al. (2002) reported that dairy cows had increased fertility after

ovulation of a third wave dominant follicle compared to ovulation of a second wave dominant

follicle. The reason for this could be that two wave patterns have a longer dominance of the

ovulatory follicle, which is associated with decreased fertility and integrity of the follicle. Cattle

that ovulated a dominant follicle from the third wave had follicles that were smaller in diameter,

which is indicative of young growing follicles. Sirois and Fortune, (1990) and Stock and

Fortune, (1993) demonstrated that ovulatory follicles with prolonged dominance, induced by

exogenous progesterone in the absence of luteal progesterone, had significantly lower pregnancy

rates compared to females that ovulated a normal follicle. Austin et al. (1999) reported that

restricting the duration of dominance of pre-ovulatory follicle to 4 d yielded pregnancy rates >

70% in beef heifers.



Corpus Luteum Function and Luteolysis

The preovulatory surge of gonadotropins responsible for ovulation also causes

differentiation of residual follicular cells that form the CL and initiates synthesis and secretion of

progesterone (Niswender et al., 2000). Luteinizing hormone is the major luteotropin responsible

for the development of the CL and production of progesterone (Armstrong and Black, 1966) and









LH pulses are needed for the development of a fully functional CL (McCracken et al., 1999;

Niswender et al., 1994) in cattle. Although, pulsatile LH secretion is not needed for the

maintenance of the CL after day 12 of the estrous cycle (Peters et al., 1994). Initial development

of the CL takes approximately 3 d post ovulation in cattle. Fields and Fields (1996) reported that

the weight of the CL was 640 mg by day 3 of the estrous cycle and increased to 5.1 g by day 14

of the estrous cycle. This growth is primarily due to hypertrophy of the granulosa and thecal

cells. The CL develops two distinct populations of cells consisting of large and small luteal cells

(Hansel et al., 1991), which differ morphologically and physiologically. Large luteal cells are

derived from granulosa cells and small luteal cells from thecal cells (Niswender et al., 2000).

Unlike small luteal cells, large luteal cells do not respond to LH because they do not express LH

receptors (Mamluk et al., 1998). However, large luteal cells are responsible for the majority of

basal progesterone (Fields and Fields, 1996), and are the target of the luteolytic effects of PGF2,

during luteolysis (Pate, 1996). Small luteal cells are primarily responsible for higher magnitude,

LH-stimulated progesterone production (Hansel et al., 1991).

During ovulation, the follicle wall collapses and the basement membrane breaks down

which gives rise to development of an extensive vascular network that invades the antral space of

the follicle. Angiogenesis or formation of new blood vessels plays a key role in formation and

growth of the CL. The mature CL has a dense system of blood vessels and has one of the highest

rates of blood flow per unit of tissue compared to any other organ (Reynolds and Redmer, 1999).

Luteal progesterone has multiple functions including regulating gonadotropin secretion,

preventing ovulation, and maintenance of pregnancy. Progesterone acts on the hypothalamus to

reduce GnRH pulse frequency and preventing the LH surge (Evans et al., 1997). With a

reduction in LH pulse frequency; there is an increase in pulse amplitude which is ideal for luteal









function. Progesterone is also essential to the maintenance of pregnancy by preventing

myometrial contractions to keep the uterus in a "quiescent state". Progesterone also stimulates

the endometrium to produce nutrients and other factors vital for concepts development (Geisert

et al., 1992).

Breed also appears to effect luteal function but the reports are conflicting. Castilho et al.

(2000) and Moreira-Viana et al. (2000) reported that Bos indicus cattle had a decreased luteal

tissue area compared to Bos taurus cattle. Rhodes et al. (1995) and Figueiredo et al. (1997)

observed that the maximum CL diameter in Bos indicus heifers was 17 to 18 mm compared to 25

to 39 mm in Bos taurus heifers (Adams et al., 1993). Similar findings have been reported by

Castilho et al. (2000), Perea et al. (1998), Ruiz-Cortes and Olivera-Anger, (1999). In contrast,

Alvarez et al. (2000) reported that the average and maximal CL diameters of Brahman and

Senepol cows were greater compared to Angus cows. Breed differences have also been reported

for progesterone concentrations. Segerson et al. (1984) observed greater progesterone

concentrations in Angus compared to Brahman cows from d 7 to 17 of the estrous cycle. Randel

et al. (1977) made similar observations, where Brahman and Brahman x Hereford heifers had

circulating progesterone concentrations that were less compared to Hereford heifers. In contrast,

Alvarez et al. (2000) reported similar progesterone concentrations for Brahman, Angus, and

Senepol cows before day 14 of the estrous cycle.

Luteolysis includes the structural and functional demise of the CL, which causes a

decline of progesterone synthesis and secretion (McCracken et al., 1999). Luteolysis allows a

new reproductive cycle to begin and another opportunity for mating and a subsequent pregnancy.

Elevated blood progesterone concentrations during most of the estrous cycle block the uterus

from generating the luteolytic signal, which is PGF2a (Gooding et al., 1972; Inskeep, 1973). The









progesterone block is diminished on approximately day 16 of the estrous cycle and is due to the

loss of progesterone receptors in the luminal and glandular epithelium of the uterus in cattle

(Niswender et al., 2000), which allows the endometrium to produce PGF2,. Progesterone also

promotes the accumulation of phospholipids in the luminal and glandular epithelia of the

endometrium, which serves as a substrate to generate arachidonic acid a precursor for PGF2a

synthesis (Niswender et al., 1994; Spencer, 2004).

The neuroendocrine control of luteolysis has been thoroughly described in the model

proposed by McCracken et al. (1999). Late in the luteal phase there is a down regulation of

progesterone receptors in the hypothalamus and endometrium that causes a decrease in

progesterone action. With the decrease in progesterone dominance, there is a return to the

dominance of estrogen action on these tissues. Estrogen acts to stimulate the hypothalamic

oxytocin pulse generator to secrete oxytocin at high frequency, low amplitude pulses. This

initiates small pulses of PGF2a to be released from the endometrium, which is sufficient to

initiate release of luteal oxytocin. Luteal release of oxytocin assists in amplifying the release of

endometrial PGF2a. With the increased pulses and concentrations of PGF2a, they are high

enough to inhibit progesterone secretion and cause the release secretion of luteal oxytocin,

further reinforcing endometrial PGF2a synthesis. This cascade of events continues until PGF2a

receptor response becomes desensitized and the CL is fully degraded and luteolysis is complete

(Flint et al., 1990).

Prostaglandin F2, travels through a unique countercurrent mechanism which exists in the

utero-ovarian pedicle of the cow (Hixon and Hansel, 1974). In this system, PGF2a travels from

the uterine vein directly to the ovarian artery, thereby avoiding the systemic circulation, which

prevents it from being metabolized systemically. Kawakami et al. (1995) reported the









concentration of PGF2, in ovarian arterial blood increased to greater than 300 times the jugular

venous blood within one minute after the start of PGF2a infusion into the uterine vein.

Prostaglandin F2, has a very short half life (Kindahl, 1980), and will be broken down quickly by

enzymes found in pulmonary tissue.

Many agents have been identified as mediators in structural and functional luteolysis of the

CL. Evidence suggests that tumor necrosis factor-a (TNF-a) induced apoptosis plays a role in

structural degradation in ruminants (Sawyer et al., 1990; Juengel et al., 1993; Zheng et al., 1994).

Ji et al. (1991) and Shaw and Britt, (1995) demonstrated that PGF2a elevates endogenous

concentrations of luteal TNF-a and in vitro. Additionally, the endothelial cells express a TNF-a

receptor and are sensitive to TNF-a induced apoptosis. Although, the exact mechanisms have

not been fully identified for functional luteolysis, it is hypothesized that PGF2, is mediated by

endothelial cell-derived vasoconstrictive peptide, also known as endothelin-1 (ET-1), which

alters normal patterns of progesterone synthesis (Milvae, 2000). Prostaglandin F2a activates the

ET-1 gene in luteal cells thereby stimulating its production. Secretion of ET-1 further stimulates

production of PGF2a from luteal cells, which acts back on the CL to further enhance ET-1

synthesis and secretion (Ohtani et al., 1998; McCracken et al., 1999; Milvae, 2000). Endothelin-

1 binds to specific receptors in both small and large luteal cells and activates a decrease in basal

and LH-induced production of progesterone (Milvae, 2000). This action could be responsible for

the inability of PGF2a to induce luteolysis in the early part of the estrous cycle since there is a

lack of ET-1 and ET-1 only appears in large quantities in the CL just after PGF2a exposure

(Wright et al., 2001). A recent study by Friedman et al. (2000) outlined a positive link of PGF2a

induced ET-1 and TNF-a in functional and structural luteolysis. At luteolysis, ET-1 secretion

from luteal cells and TNF-a production by local macrophages up-regulate one another's









production and they work together to inhibit progesterone production. The low concentrations of

progesterone along with increased expression of the TNF-a receptor facilitate TNF-a apoptosis

of the endothelial cells in the CL. This leads to the functional and structural demise of the CL.

Puberty

Puberty can be defined as the first behavioral estrus accompanied by the development of

a CL that is maintained for a period characteristic of a particular species (Kinder et al., 1987).

The process which culminates in puberty actually starts before birth and continues throughout

prepubertal development. Puberty is a result of the maturation of the endocrine and reproductive

systems of the young animal so it eventually functions similar to an adult. It is proposed that the

primary mechanism regulating pubertal development in heifers is the "gonadostat" theory,

proposed by Ramirez and McCann (1963). The theory suggests that the prepubertal increase in

LH secretion is the result of a decline in the negative feedback of estradiol on hypothalamic

centers that control LH secretion. Day et al. (1987) proposed that the reason for the changes in

LH secretion could be due to a decline in estradiol receptors in the hypothalamus and pituitary,

which results in a decline in the efficacy of estradiol to exert negative feedback effects on LH

secretion. The decline in estradiol receptors permits an increase in pulsatile secretion of LH.

The peripubertal increase in mean LH may result from either a decreased ability of estradiol to

inhibit GnRH secretion and (or) a decrease in negative feedback of estradiol at the pituitary,

thereby increasing pituitary responsiveness to GnRH. Circulating estradiol concentrations will

eventually reach concentrations sufficient to cause the pre-ovulatory surge ofLH. Schams et al.

(1981) reported an increase in LH pulse frequency and a decrease in LH pulse amplitude from 1

month of age until the onset of puberty. Day et al. (1984) also observed an increase in LH pulse

frequency and an increase in mean LH concentration during the 126 d preceding puberty.









Finally, induction of the pre-ovulatory LH surge by estradiol is an essential component for

puberty to occur (Kinder et al., 1987).

Age at puberty is influenced by many variables including genotype, age, nutrition, and

environment. The data are conclusive that Bos indicus breeds reach puberty at older ages and

heavier weights compared to Bos taurus cattle (Wiltbank et al., 1966; Plasse et al., 1968; Laster

et al., 1978; Gregory et al., 1979; Nelson et al., 1982; Dow et al., 1982). Nelsen et al. (1982)

reported Brahman heifers were 428 d old and weighed 287 kg at the onset of puberty compared

to Angus heifers that were 343 d old and weighed 227 kg. Dow et al. (1982) observed that only

17% of Brahman x Hereford heifers reached puberty by 15 mo of age compared to 92% of

Hereford x Red Poll heifers. Heterosis derived from crossbreeding Bos indicus with Bos taurus

cattle decreases the age at puberty compared to the age at puberty in the straight bred Bos indicus

heifers.

Nutrition is a key factor that can dictate the onset of puberty in beef heifers. Early work by

Wiltbank et al. (1969) and Short and Bellows (1971) demonstrated that age-at-puberty was

inversely related to growth rate. In short, as yearling heifer nutrition increased, the age at which

heifers' attained puberty decreased. With this information, the "target weight" principal was

conceptualized by Lamond (1970). This principal dictated that heifers needed to be

approximately 65% of their dam's mature weight to attain puberty. Therefore, a "target weight"

could be estimated and heifers could be placed on a nutritional plan allowing for a specified rate

of gain to attain their "target weight" at the start of the breeding season. Subsequent studies

demonstrated that plane of nutrition was associated with LH secretion. Pre-pubertal heifers

maintained on a low gain diet failed to exhibit an increase in LH pulse frequency compared to

contemporaries fed a growth diet, which resulted in increased LH pulse frequency and decreased









age at puberty (Day et al., 1986). Recent research by Gasser et al. (2006), demonstrated that

precocious puberty can be induced in early weaned heifers fed a high-concentrate diet by

increasing LH pulse frequency. Treatment of heifers with exogenous progestogens has also been

documented to enhance the onset of puberty in some pre-pubertal heifers (Short et al., 1976;

Anderson et al., 1996; Imwalle et al., 1998). The mode of action appears to be associated with

enhancing the pulsatile release of LH after progestogen withdrawal allowing for increased

follicular growth and eventually ovulation and development of a CL (Anderson et al., 1996).

The age at which heifers attain puberty and get pregnant can affect the subsequent

reproductive performance throughout a female's lifetime. Heifers that have their first calf at 2 yr

of age produce more calves during their lifetime compared to heifers that have their first calf at 3

yr of age or greater (Pope, 1967; Donaldson, 1968, Chapman et al., 1978). Furthermore, heifers

that calve early in their first calving season have a greater lifetime calf production compared to

those that calve late (Lemeister et al., 1973). Consequently, cows that calve late in the calving

season tend to calve late or not at all in subsequent years (Burris and Priode, 1958). The

Lemeister et al. (1973) study also demonstrated that calves born late in the calving season

usually weigh less and are worth less economically, which tends to decrease the total

productivity of the dam compared to calves born early in the calving season. In summary, it is

important that heifers attain puberty before the start of the breeding season, get pregnant early in

the breeding season, and calve early the next calving season in order to maintain a yearly calving

interval and optimize a cow's productivity throughout her lifetime in the cowherd. .

Maturation of the endocrine system plays a key role in determining when the onset of

puberty occurs in the yearling heifer. Yet, the remainder of the reproductive axis including the

uterus and ovaries also pay a significant role in the onset of puberty. During the 50 d preceding









puberty, uterine weights increased rapidly and major morphological changes in the ovary

occurred (Day et al., 1987). The reproductive tract score (RTS), which uses rectal palpation to

quantify uterine size and presence of ovarian structures, was developed in yearling heifers of Bos

taurus breeding by Anderson et al. (1991) to predict when and if puberty occurred in yearling

heifers prior to the start of the breeding season. The RTS was developed on a scale of 1 to 5. A

RTS of 1 is characterized with uterine horns less than 20 mm in diameter and no palpable

structures on the ovary and is considered an immature tract. A RTS of 2 is characterized with

uterine horns 20 to 25 mm in diameter with no tone, follicles no greater than 8 mm in diameter,

and is identified as being greater than 30 d from puberty. A RTS of 3 is characterized with

uterine horns of 20 to 25 mm in diameter with slight tone, follicles that are 8 to 10 mm in

diameter, and is identified as being within 30 d of puberty. A RTS of 4 has uterine horns of at

least 30 mm in diameter with good tone, follicles that are greater than 10 mm in diameter with a

possible CL, and is identified as potentially estrous cycling. A RTS of 5 has uterine horns

greater than 30 mm in diameter with good tone, a CL present, and is identified as estrous cycling.

A common management practice used in yearling heifers of Bos taurus breeding is to conduct a

RTS approximately 1 mo prior to the start of the breeding season. The RTS can be used to

predict the response to an estrous synchronization protocol, predict the potential reproductive

performance during the breeding season, and (or) to make culling decisions. In general, as RTS

increases from a 1 to a 5, both the response to a synchronization treatment increases (Patterson et

al., 2000) and breeding season pregnancy rates increase (Randle et al., 2000). For example,

Randle et al. (2000) reported pregnancy rates of 58% for heifers with a RTS of 1 compared to

89% for heifers with a RTS of 5. However, essentially no research has been conducted in

yearling heifers ofBos indicus x Bos taurus breeding utilizing the RTS as a management tool to









either predict the reproductive performance of yearling heifers or as a culling tool. Therefore,

additional research needs to be conducted to determine if the RTS developed in Bos taurus

heifers has the same potential to predict reproductive performance in yearling heifers of Bos

indicus x Bos taurus breeding.

Post Partum Interval and Return to Estrous

There are numerous factors that can influence the return to estrus after calving including

dystocia problems encountered during calving, days since calving, time of weaning, parity,

suckling and lactational status, body condition at calving, and pre and postpartum nutrition. The

interval from calving to first ovulation in suckled cows ranges from 35 to 100 d (Casida, 1971;

Foote, 1974; Wettemann et al., 1986; Hoffman et al., 1996); whereas average intervals to first

estrus ranges from 65 to 104 d (Graves et al., 1968; Wiltbank, 1970; Casida, 1971). Young cows

have a longer interval from calving to first estrus compared to older cows. For example,

Wiltbank (1970) reported that the interval from calving to first estrus was 53.4 d in cows' >5-

years-old, 60.2 d in 4-year-old cows, 66.8 d in 3-year-old cows, and 91.6 d in 2-year-old cows

(Wiltbank, 1970). Body condition score (BCS) and nutritional status at calving tends to be the

most influential factor on the resumption of estrous. Body condition can be used as in indirect

indicator of nutritional status as it estimates the amount of fat that an animal contains and cows

on an increased nutrient intake have greater BCS (Richards et al., 1986). Additionally, body

energy reserves are related to reproductive function of postpartum cows (Dziuk and Bellows,

1983). Most studies suggests a minimum BCS of > 5 at calving is needed to ensure adequate

body stores so peak reproductive performance can be attained during the subsequent breeding

season (Dziuk and Bellows, 1983; Richards et al., 1986). Cows calving at a BCS of 7 to 9 (Scale

1-9) were capable of returning to estrus within 60 d after calving. Dietary restrictions of cows

during the late pre-partum period resulted in weight loss and decreased body fat, which lead to a









decrease in the number of cows returning to estrus early in the breeding season (Whitman, 1975;

Wettemann et al., 1982). Wiltbank et al. (1964) observed that cows fed the recommended level

of TDN averaged 49 d from calving to first estrus as compared with 73 and 72 d for cows

receiving 75% and 150% of the recommended level, respectively.

There is also a relationship between nutrient intake and function of the endocrine system.

Low-energy diets decrease mean LH concentrations in postpartum cows (Terqui et al., 1980)

whereas, cows fed a diet deficient in protein failed to have increased LH pulse frequency as

postpartum intervals increase (Nolan et al., 1989). These studies support the idea that

suppression or lack of LH pulsatility during the postpartum period leads to longer periods of

anestrous. Another factor that affects normal endocrine activity is the suckling action of the calf.

Randel (1981) reported once-daily suckled Bos indicus x Bos taurus heifers had a decreased

postpartum interval by an average of 99 d compared to normal suckled first calf heifers. Mean

interval from calving to estrus for normal suckled heifers was 168.2 13.8 d, compared to 68.9 +

6.2 d for once-daily suckled heifers. Suckling releases opioids that have a negative effect on the

hypothalamus, which function to decrease GnRH release (Malven et al., 1986). This negative

effect on GnRH suppresses LH pulse frequency thereby, preventing an early return to estrus.

Walters et al. (1982) noted that by removing calves from cows 3 wk after calving or later

resulted in a rapid removal of the negative effects of suckling on the tonic release of LH

secretion. Bellows et al. (1974) reported that early weaning calves also decreased the postpartum

interval in cows producing multiple or single calves.

Manipulation of the Estrous Cycle

Traditional AI programs were focused on determining when estrus occurred over a 21 d

estrous cycle to determine when cattle were ready to be inseminated. This process was very time

and labor consuming and limited whether or not producers could implement an AI program.









Development and implementation of estrous synchronization protocols allowed for a large

number of cattle to be in estrus during a short time period compared to the detection of estrus

during the 21 d estrous cycle. For most estrous synchronization protocols behavioral estrus will

be exhibited during a 3 to 7 d period, which allows for more efficient use of AI. Some protocols

have actually eliminated estrous detection and inseminate all cows at a predetermined timed

known as timed-AI. Most synchronization protocols utilize exogenous hormones including

PGF2,, GnRH, and progestogens either alone and (or) in combination to manipulate the estrous

cycle. The primary function of a synchronization protocol is to provide for a tightly

synchronized estrus and or ovulation by manipulating follicle development and luteolysis.

Prostaglandin F2,

It has been well established that prostaglandinF2z is the luteolytic signal in beef cattle

(Gooding et al., 1972; Inskeep, 1973) that originates from the uterus and initiates the demise of

the CL. Rowson et al. (1972) and Lauderdale et al. (1974) demonstrated that an exogenous

administration of PGF2, during the luteal phase of the estrous cycle causes luteolysis of the CL

without compromising fertility of the subsequent estrus (Lauderdale et al., 1974). Consequently,

PGF2, became the first pharmaceutical used for the purpose of estrous synchronization.

Subsequent research by Watts and Fuquay (1985) demonstrated that stage of the estrous

cycle at administration of PGF2, had an effect on estrous response, interval from PGF2, to the

onset of estrus, and conception rate. In this study, heifers in the early stages of the estrous cycle

(day 5 to 7) had an estrous response of 43% and an interval from PGF2, to the onset of estrus 59

h. In contrast, heifers in the middle (day 8 to 11) and later (day 12 to 15) stages of the estrous

cycle had a greater estrous response (84 and 100%; respectively) but longer intervals from PGF2,

to the onset of estrous (71 and 72 h; respectively) compared to heifers in the early stages of the

estrous cycle. First service conception rate also increased as the estrous cycle progressed. The









interval from PGF2a to the onset of estrus was longer for heifers in the mid to late stages of the

estrous cycle, which is due to status of the follicular waves and size of the dominant follicle at

PGF2a (Sirois and Fortune, 1988). During the early stages of the estrous cycle, a dominant

follicle is growing and ready to ovulate if luteolysis is induced compared to mid and late stages

of the estrous cycle where follicles could be in the early stages of development and require

additional time to reach ovulatory capacity and size.

A single dose of PGF2a initiates luteolysis in 85 to 100% of Bos taurus females (King et

al., 1982; Tanabe and Hann, 1984; Kiracofe et al., 1985) resulting in estrous responses between

75 and 95% (Tanabe and Hann, 1984; Watts and Fuquay, 1985). However, there has been

limited research conducted to determine if genotype influences the effectiveness of PGF2a to

initiate luteolysis. Pinheiro et al. (1998) demonstrated that Nelore cattle treated with two

injections of PGF2a 11 d apart had a 33.3% estrous response in heifers and 46.4% estrous

response in cows. Orihuela et al. (1983) reported estrous responses of 56 and 62% in Zebu cows

following PGF2, treatment. It should be noted that in the preceding studies that estrous response

was used as an indirect indicator of luteolysis and no blood samples were collected to evaluate

blood progesterone concentrations to confirm ifluteolysis occurred. Because estrus is difficult to

detect in Bos indicus cattle and there is an increased incidence of silent estrus in Bos indicus type

cattle (Plasse et al., 1970), caution should be taken when using only estrous response when

evaluating effectiveness of PGF2a to initiate luteolysis. It has been hypothesized that the

decreased estrous response in Bos indicus cattle was due to decreased effectiveness of the PGF2a

to initiate complete regression of the CL (Pinheiro et al., 1998; Rekwot et al., 1999) resulting in

rebounding of progesterone production by the CL that block the expression of estrus. Recent

research by Bridges et al. (2005) supported this hypothesis by demonstrating that a PGF2a









induced luteolysis was decreased in yearling heifers ofBos indicus x Bos taurus breeding

compared to yearling heifers of Bos taurus breeding. However, in that same study, they also

demonstrated that there may be an age effect because luteolysis was similar between two-year

old virgin heifers of Bos indicus x Bos taurus breeding compared to two-year old virgin heifers

of Bos taurus breeding.

To combat the inability of PGF2a to initiate luteolysis in a majority of Bos indicus cattle,

researchers added a second treatment of PGF2u 24 h after the first one (Cornwell et al., 1985;

Santos et al., 1988). Administration of the second PGF2, treatment initiated complete luteolysis

and prevented the CL from continuing to secrete progesterone. Bridges et al. (2005)

administered two consecutive PGF2, treatments 24 h apart to yearling heifers in the later stages

of the estrous cycle and observed increased estrous response, timed-AI pregnancy rate, and

synchronized pregnancy rate compared to heifers that received a single dose. Portillo et al.

(2007) administered two half treatments of PGF2a (12.5 mg) 24 h apart to mature suckled Bos

indicus x Bos taurus cows and observed a significantly enhanced luteolysis, although, estrous

response and synchronized pregnancy rates were similar compared to cows that received a single

dose of PGF2,.

There are two common forms of prostaglandins available for commercial use. The trade

names of Lutalyse Sterile Solution, Prostamate, and In-Synch are from the chemical class

dinoprost tromethamine, a naturally occurring prostaglandin, which is administered at a rate of

25 mg (5 cc) per dose. Estrumate and Estroplan are a synthetic analogous of PGF2a made from

cloprostenol sodium. These analogs are slightly more potent and are administered at a rate of

500 tg (2 cc). Both classes of prostaglandins provide for acceptable pregnancy rates when used

in synchronization systems (Johnson, 1978; Seguin et al., 1984; Jackson et al., 1979).









Comparisons of both classes of prostaglandins have been made in Bos taurus animals (Young

and Anderson, 1986; Salverson et al., 2002). Salverson et al. (2002) observed similar results

when cloprostenol sodium was compared to dinoprost tromethamine in Bos taurus heifers

synchronized with the long term MGA + PGF2, protocol. Evaluation of the effectiveness of the

two classes of prostaglandins in cattle of Bos indicus breeding has not been completely

characterized in either suckled cows or yearling heifers. In non-lactating cows ofBos indicus

breeding synchronized with a GnRH + PGF2u based protocol, Heirs et al. (2003) reported a

numerical increase in synchronized pregnancy rates of 5% in cloprostenol sodium treated cows

compared to dinoprost tromethamine treated cows. In a recent study in suckled postpartum Bos

indicus x Bos taurus cows synchronized with GnRH + PGF2, based protocol concomitant with a

CIDR, estrous response, conception rate, and synchronized pregnancy rates were similar

between cows that received either cloprostenol sodium or dinoprost tromethamine (Esterman et

al., 2007). There has been no research evaluating the effectiveness of cloprostenol sodium and

dinoprost tromethamine in GnRH + PGF2, based protocol concomitant with a CIDR in yearling

heifers of to Bos indicus x Bos taurus breeding.

Progestogens

The primary function of a progestogen is to prevent the expression of estrus by

mimicking the actions of the CL. Progestogens act to prevent the ovulatory surge of LH and

thereby prevent ovulation from occurring (Patterson et al., 1989). An additional benefit of using

progestogens is that they can initiate estrous cycles in some anestrous cows (Miksch et al., 1978;

Smith et al., 1987; Fike et al., 1997; Yelich et al., 1997; Yavas and Walton, 2000) and pre-

pubertal heifers (Anderson et al., 1996; Hall et al., 1997). Administration of a progestogen in

anestrous cows and pre-pubertal heifers stimulates LH secretion both during and after

progestogen treatment, which accelerates follicle growth resulting in ovulation after progestogen









withdrawal (Garcia-Winder et al., 1987; Anderson et al., 1996). There are two major classes of

progestogens including natural progesterone and synthetic analogues including norgestomet and

melengestrol acetate (MGA). Progestogens can be administration via injection, implant,

intravaginally, or orally. The two most common progestogens used in estrous synchronization

protocols include the intravaginal progesterone insert or CIDR and MGA, which is orally active

and administered in the feed.

The CIDR is an intravaginal insert made with a progesterone impregnated silicone

coating. When inserted into the vagina, the moisture acts to move the progesterone out of the

silicone coating. In the United States, the CIDR contains 1.38 g of progesterone, and it is

estimated that 0.7 g of progesterone is used in its initial use (Savio et al., 1993), which is

typically 7 d. Most estrous synchronization protocols leave the CIDR in the vagina for 7 d with

administration of PGF2, either one day before (Lucy et al., 2001) or at CIDR removal (Martinez

et al., 2000; Stevenson et al., 2004; Larsen et al., 2006). The primary function of the CIDR is to

improve estrous synchrony compared to a single PGF2, treatment or unsynchronized cattle. An

additional benefit of the CIDR is that it can initiate estrus in anestrous cows and peripubertal

heifers. Lucy et al. (2001) reported that a 7 d CIDR induced estrus in approximately 40% of the

anestrous cows and 40% of the prepubertal heifers. Fike et al. (1997) reported that 70% of

anestrous cows were initiated into estrus or had formation of a CL after a 7 d CIDR treatment.

Since there is approximately 0.7 g (Rathbone et al., 2002) progesterone left in the CIDR

after a single use (Savio et al., 1993), there has been growing interest among producers to use a

once-used CIDR. Beal et al. (2003) reported that utilization of a once-used CIDR for 7 d

effectively suppressed estrus in beef females. Furthermore, autoclaving the once-used CIDR

before re-insertion results in greater serum progesterone concentrations compared to a once-used









CIDR that was only cleaned and not autoclaved (Zulagea and Williams, 2006). Moreover,

Colazo et al. (2004) reported similar pregnancy rates after a fixed timed-AI in heifers that

received either a new or once used CIDR. Therefore, the potential to use a once-used CIDR

exists, but research in this area is limited relative to the effectiveness of a once-used CIDR to

synchronize estrus as well as the ability to induce estrus in anestrous cattle.

Melengestrol acetate is capable of promoting endometrial proliferation, maintenance of

pregnancy, and preventing the expression of estrus (Duncan et al., 1964; Zimbelman and Smith,

1966; Prichard et al., 1969; Schul et al., 1970). One of the advantages of using MGA is that it

can be administered in the feed. The minimal effective dose needed to maintain pregnancy (4

mg/d) is much greater compared to that needed to prevent estrus and ovulation (0.5 mg/d)

(Zimbelman, 1963b, Zimbelman and Smith, 1966). Early research demonstrated that

administering MGA for more than 9 d lead to decreased fertility of the subsequent estrus and

ovulation compared to untreated controls (Hill et al., 1971). Patterson et al. (1989) and Beal et

al. (1988) further demonstrated that administration of MGA for 7 d or 9 d; respectively, also

resulted in decreased fertility in some animals depending on what day of the estrous cycle they

started the MGA treatment. Heifers which began MGA treatment after day 12 of the estrous

cycle had decreased fertility compared to heifers where MGA was initiated prior to day 12 of the

estrous cycle. The reduction in fertility is temporary, as fertility at the subsequent estrus is not

affected (Zimbelman et al., 1970).

The decrease in fertility after a long term MGA treatment is caused by the development of

what is termed a "persistent dominant follicle" (Guthrie et al., 1970). A persistent dominant

follicle is a follicle that has a longer lifespan, increased maximal diameter, and increased

estradiol secretion compared to normal ovulatory follicles (Sirois and Fortune, 1990; Savio et al.,









1993b). Development of the persistent dominant follicle typically occurs with long-term

progestin treatments (9 to 14 d) in the absence of a CL, which results in increased LH pulse

frequency and decreased pulse amplitude (Kojima et al., 1992). The altered LH secretary profile

results in continued follicle growth and development due to the absence of luteal progesterone

that would normally initiate follicle turnover. After MGA withdrawal, estrus occurs within 3 to

7 d. Initial research indicated that the decreased fertility at the estrus after MGA withdrawal was

due to increased estrogen concentrations that altered the uterine environment (Wordinger et al.,

1972; Gibbons et al., 1973) and did not allow for conditions conducive to fertilization and (or)

proper embryo development. However, recent research has demonstrated that fertilization

actually occurs in the majority of cattle and the reduction is fertility is due to decreased embryo

viability (Ahmad et al., 2005) and deceased oocyte competence (Stock and Fortune, 1993; Savio

et al., 1993; Mihm et al., 1994a). Additionally, Mihm et al. (1992) reported that progesterone

concentrations did not vary between ovulation of a normal follicle compared to ovulation of a

persistent dominant follicle indicating that luteal function was not altered and probably does not

contribute to the decrease in fertility.

There can also be a decreases in fertility associated with short term (< 9 d; Beal et al.,

1988); Patterson et al., 1989) progestogen treatments. The decrease in fertility with a short term

progestogen treatment is due to the duration of dominance of a follicle. Austin et al. (1999)

reported that heifers which ovulated follicles between 2 and 8 d of dominance had pregnancy

rates between 71 and 89%. In contrast, when follicles were ovulated that had either 10 or 12 d of

dominance, pregnancy rates were 52 and 12%, respectively. Animals that ovulated follicles with

prolonged dominance had significantly lower pregnancy rates compared to females that ovulated

follicles of normal duration (Sirois and Fortune, 1990; Stock and Fortune, 1993).









Gonadotropin Releasing Hormone (GnRH)

Administration of exogenous GnRH alters follicular development by inducing luteinization

and (or) ovulation of dominant follicles (Macmillan et al., 1985; Thatcher et al., 1989), which

results in synchronization of a new follicular wave (Twagiramungu et al., 1994). Gonadotropin

releasing hormone acts on the pituitary to induce release of the gonadotropes LH and FSH

(Chenault et al., 1990). Gonadotropin releasing hormone is produced commercially as Fertagyl,

Factrel, and Cystorelin and is administered at 2 mL as an either an intravenous or

intramuscular injection.

Concentrations of LH and FSH increase within 2 to 4 h of an endogenous GnRH

treatment (Stevenson et al., 1993) and ovulation occurs 24 to 32 h after treatment (Thatcher et

al., 1989; Pursley et al., 1994b). However, the ability of GnRH to initiate ovulation is stage of

estrous cycle dependent. A dominant growing follicle must be present on the ovary in order for a

GnRH induced ovulation to occur (Moreira et al., 2000). Kohram et al. (1998) demonstrated that

GnRH administered between days 4 to 7 and days 15 to 18 induced ovulation, decreased

estrogen concentrations, and initiated a new wave of follicle development. In another study

using dairy heifers, GnRH induced ovulation less frequently in heifers that were day 2 and dayl0

of the estrous cycle compared to day 5, 15 and 18 of the estrous cycle (Moreira et al., 2000).

Lack of a dominant follicle on day 2 and d 10 is representative of the initiation of follicular

waves in cattle where recruitment and selection is taking place and have not reached at least 10

mm in diameter, which appears to be the minimal size that a follicle needs to be to be ovulated

by GnRH (Sartori et al., 2001). There does not appear to be a breed effect on the ability of

GnRH to ovulate follicles across several stages of the estrous cycle. In a recent study by

Esterman et al. (2008), ovulation rates were similar for Angus and Brangus cows that received

GnRH across several days of the estrous cycle at the initiation of a CIDR treatment.









There are essentially two types of CL that result from a GnRH induced ovulation

including newly developed CL and an accessory CL. An accessory CL is an additional CL that

develops in the presence of an existing CL. The CL that develops from a GnRH induced

ovulation appears to be similar to a naturally formed CL in its capacity to be regressed with

endogenous PGF2, (Twagiramungu et al., 1992). However, mixed results have been reported

about the integrity of progesterone output from an induced ovulation. As would be expected,

induced ovulations that result in development of an accessory CL have increased progesterone

concentrations (Twagiramungu et al., 1994; Wolfenson et al., 1994). However, progesterone

concentrations can also be less following a GnRH treatment compared to concentrations

normally observed during the estrous cycle (Ford and Stormshak, 1978; Rodger and Stormshak,

1986).

The purpose of including GnRH in an estrous synchronization and AI program is

essentially two fold. First, it is used to induce ovulation and to synchronize the subsequent

follicle wave by stimulating FSH to recruit the new follicle wave. In turn, when PGF2a is

administered 7 d after GnRH it results in a very synchronous estrus (Thatcher et al., 1989;

Macmillan and Thatcher, 1991; Pursley et al., 1994). And second, GnRH administered in

conjunction with AI, known as a timed-AI, functions to synchronize ovulation with delivery of

semen into the reproductive tract. The later scenario is particularly effective when used in

conjunction with GnRH followed seven days later with PGF2a and a timed-AI approximately 48

to 60 h after PGF2a (Pursley et al., 1995, 1997; Burke et al., 1996; Schmitt et al., 1996).

Synchronization Protocols

The primary functions of synchronization protocols is to either synchronize estrus so cattle

can be inseminated over a 1 to 5 d period or to synchronize ovulation so cattle can be

inseminated at a predetermined timed know as a timed-AI. Initial synchronization protocols









focused on the use of either a single PGF2 or two PGF2, treatments 10 to 11 d apart. The major

drawback with the PGF2a protocols is that they only work in cattle that are going through estrous

cycles and they require considerable estrous detection. The more preferred methods of

synchronization include combining one of several pharmaceuticals including GnRH, a

progestogen, and PGF2a. Synchronization protocols significantly reduce estrous detection and

(or) eliminate estrous detection completely by incorporating a timed-AI. Furthermore, they can

induce estrus in some anestrous cattle. It appears that some synchronization protocols are more

effective in suckled postpartum cows compared to heifers. The numerous synchronization

protocols available to producers can best be tailored to fit a producers operation. Although, most

producers want synchronization protocols that require minimal animal handling, and result in

acceptable and consistent AI pregnancy rates. The general consensus is that acceptable

pregnancy rates are 50%.

One of the most widely used estrous synchronization programs for yearling heifers is the

14 d melengestrol acetate (MGA) treatment followed by PGF2a 17 d later (MGA-PGF2a; Brown

et al., 1977). The MGA-PGF2, protocol was designed to bypass inseminating heifers at the

infertile estrus immediately after MGA withdrawal and took advantage of administering PGF2a

in the later stages of the estrous cycle when it was more effective in initiating luteolysis.

Subsequent research illustrated that administering PGF2a 19 d after the last day of MGA (Lamb

et al., 2000) improved the synchrony of the PGF2a induced estrus without reducing fertility.

Heifers can be inseminated either after an observed estrus with estrous detection typically lasting

5 to 7 d or estrus can be detected for 3 d with a timed-AI + GnRH on the third day for heifers not

exhibiting estrus with each insemination protocol yielding similar AI pregnancy rates Beef

cattle producers like to use the MGA-PGF2a system because it is easy to implement, cost









effective, requires minimal animal handling, and yields consistent synchronized pregnancy rates

(typically > 50%) in yearling heifers of Bos taurus breeding (Brown et al., 1988; Lamb et al.,

2000; Salverson et al., 2002). In contrast, Bridges et al. (2005) reported synchronized

pregnancy rates of only 34.5% in yearling heifers of Bos indicus x Bos taurus breeding

synchronized with the MGA-PGF2, system where estrus was detected for 3 d with timed-

AI+GnRH on day 3 for heifers not exhibiting estrus. Synchronized pregnancy rate was

improved to 42.5% when the PGF2, treatment (25 mg) was split (12.5 mg; split- PGF2u) and

administered on two consecutive days, which resulted in greater luteolysis for the split-PGF2u

compared to the single PGF2a. Even with the addition of the split-PGF2, treatment to the MGA-

PGF2 system, synchronized pregnancy rates are still less for Bos indicus x Bos taurus heifers

compared to Bos taurus heifers. Recent work in our lab reported that follicle development was

different between Bos taurus (Angus) and Bos indicus x Bos taurus (Brangus) heifers after a 14

d MGA treatment (Woodall et al., 2006). First, ovulation rate after MGA withdrawal tended to

be greater in Angus (100%; n=10/10) compared to Brangus (80%; n=8/10) heifers. Second,

Brangus (n=5/10) had more one, three, and four follicle wave patterns compared to Angus

(n=2/10), which resulted in asynchronous follicle development at PGF2a for Brangus heifers. In

a subsequent experiment, GnRH was given either 3 or 10 d after a 14 d MGA treatment to

synchronize follicle development followed by PGF2a 7 d after GnRH. The day 3 GnRH

treatment resulted in more heifers in estrus during the 3 d after PGF2a compared to GnRH given

10 d after MGA withdrawal (Woodall et al., 2007a). In subsequent field trials, synchronized

pregnancy rates were similar between GnRH given 3 d after a 14 d MGA treatment (34.8%;

n=178) compared to the original MGA-PGF2a system (38.5%; n=174; Woodall et al., 2007b).

Both treatments received a split-PG with estrous detection and AI for 3 d after PGF2a and heifers









not exhibiting estrus by 73 h after PGF2, being timed-AI with GnRH between 73 to 80 h. Since

less than acceptable pregnancy rates are obtained when synchronizing Bos indicus x Bos taurus

heifers with a long-term MGA-PGF2, treatment, other synchronization system must be

evaluated.

Short-term (7 d) CIDR treatments are another alternative to synchronizing estrus. In a

recent set of field trials, Lamb et al., (2006) evaluated the effectiveness of several short term

progestogen treatments in yearling Bos taurus heifers. Lamb et al. (2006) reported that one of

the most consistent synchronization protocols in yearling heifers included administration of

GnRH concurrent with a 7 d Eazi-Breed CIDR with PGF2, at CIDR removal followed by a fixed

timed-AI + GnRH 60 h after PGF2,, which yielded synchronized pregnancy rates of 53%.

Equally effective was the same CIDR synchronization protocol but with 3 d of estrous detection

combined with a timed-AI plus GnRH for heifers not exhibiting estrus by 84 h after PGF2a,

which yielded a synchronized pregnancy rate of 57%. There has been limited research

conducted in yearling heifers of Bos indicus x Bos taurus breeding evaluating short-term CIDR

treatments. Lucy et al. (2001) reported that CIDR+PGF2a treated heifers had a greater estrous

response (84%) during the first 3 d of the breeding period compared to PGF2a treated heifers

(57%). We have recently initiated a multiyear study in our lab comparing the effectiveness of

cloprostenol sodium (Estrumate) compared to dinoprost tromethamine (Lutalyse) in the Select

Synch/CIDR+timed-AI program in Angus, Brahman, and Brahman x Angus 2-year old virgin

heifers. The overall estrous, conception, timed-AI pregnancy, and synchronized pregnancy rates

for the first two years were 48.5 (n=163), 60.9 (n=79), 30.7 (n=84), and 45.3% (n=163),

respectively (Unpublished data). There were no prostaglandin or breed effects on any of the

variables measured. Synchronized pregnancy rates ranged from 40.5 to 54.1% across treatments









and years suggesting that the Select Synch/CIDR+timed-AI synchronization program could work

in yearling heifers of Bos indicus x Bos taurus breeding but additional research is needed to

confirm this.

The 7 d CIDR protocol has also been researched in suckled Bos taurus cows. Larsen et

al. (2006) observed greater pregnancy rates for the Select Synch/CIDR+timed-AI and CO-

Synch+CIDR treatments compared to the CO-Synch without CIDR treatment. Conception rates

were greater for the Select Synch+timed-AI and Select Synch/CIDR+timed-AI cows compared

to cows synchronized with a CIDR alone. The authors concluded that the Co-Synch+CIDR

yielded similar pregnancy rates compared to estrous detection protocols and can be used as an

effective timed-AI protocol in suckled Bos taurus beef cows. Lucy et al. (2006) reported a

tendency for first service conception rate and pregnancy rates to be greater in the CIDR+PGF2a

compared to unsynchronized controls in suckled Bos taurus and Bos indicus x Bos taurus beef

cows from seven different locations.

Utilization of a once-used-CIDR has been shown to be an option to the new CIDR when

used in synchronization protocols. Colazo et al. (2003) conducted three experiments using

heifers and suckled beef cows. Experiment one compared efficacy of a new (1.9 g) or once-used

CIDR inserted for 9 d and estradiol cypionate with or without progesterone at CIDR insertion in

heifers. Pregnancy rate was not affected by number of times a CIDR was used or addition of

progesterone. The second experiment in heifers and cows received either a once- or twice-used

CIDR, or either estradiol benzoate with or without progesterone at CIDR insertion.

Synchronized pregnancy rates were decreased for the twice-used CIDR, but progesterone did not

affect pregnancy rates. In the third experiment, heifers received a new, once-used, twice-used, or

two twice-used CIDR, pregnancy rates were similar for all treatments and there was no affect of









progesterone. These results suggest that incorporation of a once-used CIDR into a

synchronization program has potential. Follow up research conducted by Solorzano et al. (2004,

2008), demonstrated that a once-used CIDR could effectively synchronize estrus in non-lactating

beef cows and yearling heifers that were eventually used for embryo transfer. It should be noted

that the afore mentioned experiments utilized a CIDR containing 1.9 g of progesterone compared

to 1.38 g CIDR which is the only type available for purchase in the United States. Consequently,

it is recommended that CIDR purchased in the United States only be re-used one time. Recent

research also indicates that autoclaving the once-used CIDR actually results in greater circulating

blood progesterone concentrations compared to a once-used CIDR that was not autoclaved

(Eaton et al., 2007).

The 7-11 estrous synchronization protocol consist of a 7 d MGA treatment with PGF2, on

the last day of MGA followed by GnRH 4 d later; seven days after GnRH, PGF2, is administered

to synchronize estrus (Kojima et al., 2000). The concept behind the 7-11 protocol is that the 7 d

MGA treatment prevents cattle from exhibiting estrus and the first PGF2, treatment initiates

luteolysis of any CL present at MGA removal. This pretreatment regime results in a majority of

cattle with an ovulatory sized follicle present when GnRH is administered 4 d after MGA

withdrawal. The GnRH treatment functions to synchronize follicle development so a majority of

cattle will have a healthy growing follicle ready for ovulation at the second PGF2, administered 7

d after GnRH. The 7-11 protocol is an effective protocol for synchronizing estrus for either a

detected estrus in Bos taurus (Kojima et al., 2000) or a timed-AI in Bos taurus cattle (Bader et

al., 2005).

In a recent study conducted in our lab, estrous cycling and anestrous postpartum Brangus

and Angus cows received GnRH 4 d after a 7 d MGA treatment with PGF2a at MGA withdrawal,









GnRH induced ovulation in 94.4% of Brangus (n=17/18) and Angus (n=17/18) cows (Esterman

et al., 2007). The resulting five day estrous response (70.5, 65.8%), conception (67.7, 76.0%),

and synchronized pregnancy rates (47.7, 50.0%) were similar (P > 0.05) for Angus and Brangus,

respectively. Because GnRH effectively ovulated follicles in Brangus cows when administered 4

d after a short term (7 d) progestogen treatment, it appears that the 7-11 synchronization

protocol is an effective synchronization protocol for use in suckled cows of Bos indicus x Bos

taurus breeding. However, additional research will need to be conducted to evaluate the

effectiveness of the 7-11 protocol in yearling heifers of Bos indicus x Bos taurus breeding.

In summary, the following objectives are being proposed and will constitute the research to

be presented in this thesis.

Objective 1: Evaluate the effectiveness of the Select Synch/CIDR+timed-AI
synchronization protocol and a modified 7-11 protocol in yearling heifers and suckled
cows of Bos indicus x Bos taurus breeding.

Objective 2: Evaluate the effectiveness of cloprostenol sodium and dinoprost
tromethamine when used in conjunction with the synchronization treatments outlined in
objective 1.

Objective 3: Evaluate the effectiveness of the 7-11 and Select Synch/CIDR+timed-AI
synchronization protocols in yearling heifers ofBos indicus x Bos indicus breeding.









CHAPTER 3
COMPARISON OF TWO PROGESTOGEN BASED ESTROUS SYNCHRONIZATION
PROTOCOLS AND CLOPROSTENOL SODIUM VS. DINOPROST TROMETHAMINE IN
SUCKLED POST PARTUM COWS AND YEARLING HEIFERS OF Bos indicus x Bos taurus
BREEDING

Introduction

In the Gulf Coast region of the Southeastern US, the majority of cattle contains some Bos

indicus influence because of their superior ability to deal with heat stress, utilization of low

quality forages, and increased parasite tolerance. However, the slight physiological and

behavioral differences that exist in cattle of Bos indicus breeding could cause differential

responses to estrous synchronization protocols that have been developed primarily in cattle of

Bos taurus breeding. Cattle of Bos indicus breeding have an increased sensitivity and respond

differently to exogenous hormones (Randel, 1984; Portillo et al., 2007) compared to cattle of Bos

taurus breeding. Furthermore, estrus is more difficult to detect in cattle of Bos indicus breeding

due to a decreased expression and duration of estrus (Galina et al., 1982) as well as an increased

incidence of silent estruses (Galina et al., 1996).

One of the most consistent synchronization protocols in suckled cows (Larson et al., 2006)

and yearling heifers (Lamb et al., 2006) of Bos taurus breeding includes administration of a 7 d

Eazi-BreedTM CIDR (CIDR) with GnRH at CIDR insertion and PGF2a at CIDR removal,

followed by 3 d of estrous detection and AI with a timed-AI plus GnRH for cattle not exhibiting

estrus by 72 h after PGF2,. This protocol is known as the Select Synch/CIDR+timed-AI

protocol. Lucy and coworkers (2001) conducted a multi-location study in suckled beef cows and

yearling heifers that received a 7 d CIDR with PGF2, one day prior to CIDR removal followed

by 3 d of estrous detection and AI. The AI pregnancy rates were similar for suckled cows and

yearling heifers of Bos indicus x Bos taurus breeding compared to locations with cattle of Bos

taurus breeding. It should be noted that GnRH was not administered at CIDR insertion in the









Lucy et al. (2001) study. Therefore, additional experiments are needed in cattle ofBos indicus x

Bos taurus breeding to evaluate CIDR synchronizations treatments when GnRH is administered

at CIDR insertion and at timed-AI for induction of ovulation in cows not expressing estrus.

The 7-11 synchronization protocol is another short term progestogen synchronization

protocol that is frequently used in cattle of Bos taurus breeding (Kojima et al., 2000). The 7-11

protocol consists of a 7 d melengestrol acetate (MGA) treatment with PGF2, on the last day of

MGA followed by GnRH 4 d later. Seven days after GnRH, PGF2, is administered to

synchronize estrus. The 7-11 synchronization protocol is effective in Bos taurus cattle (Kojima

et al., 2000; Stegner et al., 2004) but no research has been conducted to evaluate the

effectiveness of the 7-11 protocol in yearling heifers of Bos indicus x Bos taurus breeding and

one study has been conducted in suckled cows of Bos indicus x Bos taurus breeding (Esterman et

al., 2007b).

As production costs increase, producers seek to implement new management practices that

either reduce their operating costs and (or) increase productivity. Therefore, there has been

considerable interest in using a once-used CIDR in estrous synchronization protocols.

Utilization of a once-used CIDR for 7 d suppresses estrus in beef females (Beal et al., 2003) and

autoclaving a once-used CIDR increases circulating progesterone concentrations compared to a

non-autoclaved once-used CIDR (Zuluaga and Williams, 2008). Autoclaving also reduces the

risk of disease transmission. Moreover, synchronized pregnancy rates after a fixed timed-AI

were similar in heifers receiving either a new or once-used CIDR (Colazo et al., 2004).

Therefore, there appears to be potential for incorporating a once-used CIDR into some estrous

synchronization protocols. Additionally, a minimal amount of research has been conducted

evaluating the effectiveness of prostaglandin type, cloprostenol sodium compared to dinoprost









tromethamine, when used in GnRH+PGF2, estrous synchronization protocols in cattle of Bos

indicus x Bos taurus breeding. Heirs et al. (2003) reported similar synchronized AI pregnancy

rates for cloprostenol sodium compared to dinoprost tromethamine in non-lactating Bos indicus x

Bos taurus cows synchronized with GnRH+PGF2, protocol combined with MGA and Esterman

et al. (2007a) reported similar results between the two prostaglandins in suckled Bos indicus x

Bos taurus cows synchronized with GnRH/CIDR+PGF2, protocol. However, no direct

comparisons have been made between cloprostenol sodium and dinoprost tromethamine in

yearling Bos indicus x Bos taurus heifers synchronized with GnRH+PGF2, synchronization

protocols.

Therefore, the objectives of these experiments were to evaluate the effectiveness of

cloprostenol sodium compared to dinoprost tromethamine when used in a modified 7-11 protocol

and a Select Synch/CIDR+timed-AI protocol in yearling heifers and suckled cows of Bos indicus

x Bos taurus breeding.

Materials and Methods

Experiment 1

Experiment 1 (Figure 3-1) was conducted from January to March 2007 at the Bar L

Ranch, Marianna, Florida. In Experiment 1, multiparous suckled postpartum Bos indicus x Bos

taurus cows (n = 324) were used. The percentage ofBos indicus breeding of cows utilized

ranged from approximately 7 to 38% with the remainder being Bos taurus breeding. The

experiment was a 2 x 2 factorial design. At the start of the experiment (d 0), cows were equally

distributed by body condition score (BCS; 1 = emaciated, 5 = moderate, 9 = very fat; Richards et

al., 1986) and days post partum (DPP) to one of two progestogen based synchronization

treatments and one of two PGF2, treatments. The synchronization treatments included a









modified 7-11 treatment, which will be termed the 7-10 treatment, and the Select

Synch/CIDR+timed-AI treatment. Within each synchronization treatment, half the cows

received one of two PGF2a treatments including dinoprost tromethamine (dinoprost, 25 mg i.m.;

Prostamate, Agrilabs, St. Joseph, MO) and cloprostenol sodium (cloprostenol, 500 pg i.m.;

Estrumate, Schering-Plough Veterinary Corp., Kenilworth, NJ). The original 7-11

synchronization protocol administers GnRH 4 d after MGA withdrawal (Kojima et al., 2000).

Since an intravaginal CIDR was used in the present study, it was hypothesized that the

disappearance of the negative feedback effect of progesterone on LH secretion should take less

time to clear the circulatory system compared to MGA. Therefore, GnRH was administered on

day 10 instead of day 11 resulting in the name change to the 7-10 synchronization treatment. On

day 0, the 7-10 treatment received an autoclaved once-used CIDR (Eazi-BreedTM CIDR, 1.38 g

progesterone, Pfizer Animal Health, New York, NY) that was removed on day 7 concomitant

with dinoprost followed by GnRH (500 Gg; Cystorelin, Merial Animal Health, Duluth, GA) on

day 10. The once-used autoclaved CIDR was used in place of MGA to test its effectiveness as a

low cost alternative progestogen source in the 7-10 protocol. On day 17, 7-10 cows received

either dinoprost or cloprostenol. Also on day 10, the Select Synch/CIDR+timed-AI treatment

received a new CIDR concomitant with GnRH followed by CIDR removal on day 17 where

cows received either dinoprost or cloprostenol.

To aid in estrous detection, all cows received EstrotectT estrous detection patches

(Rockway, Inc., Spring Valley, WI) on day 18 of the experiment. Estrus was visually detected

twice daily (0700 and 1600 h) for 72 h after PGF2, and cows were inseminated 6 to 12 h after

detection of estrus. Non-responders were timed-AI + GnRH 72 to 76 h post PGF2,. Estrus was

defined as a cow standing to be mounted by another cow, showed signs of visible mucous, and/or









had a half to full red estrous detection patch. Two AI technicians inseminated cows using

frozen-thawed semen from a single sire of known fertility and technicians were equally

distributed across treatments for both cows that either exhibited estrus or were timed-AI. Seven

days after the timed-AI, natural service sires were placed with cows. Using a real-time B-mode

ultrasonography machine (Aloka 500V, Corometrics Medical Systems, Wallingford, CT) with a

5.0 MHz transducer, pregnancy was diagnosed approximately 55 d after AI. Due to the 7 d

period where no cows were exposed to natural service sires, differences in fetal size were used to

determine whether a pregnancy resulted from the synchronized AI breeding (54 and 55 d

pregnant) or natural service sire (< 48 d pregnant). Embryos develop a C shape appearance

approximately day 25 of gestation, which transitions to an L shape embryos by day 32. By day

48, the fetus is approximately 35 mm in length with visible limb buds and by day 55 the fetus is

approximately 50 mm in length and has identifiable ribs (Curran et al., 1986).

Estrous response was defined as the number of cows displaying estrus for 3 d after

prostaglandin and artificially inseminated (AI) divided by the total number of cows treated.

Conception rate was defined as the number of cows that became pregnant to AI divided by the

number of cows that displayed estrus and were AI. Timed-AI pregnancy rate was the number of

cows that failed to display estrus, were timed-AI, and became pregnant divided by the total

number of cows that were timed-AI. Synchronized pregnancy rate was the number of cows

pregnant to AI divided by the total number of cows treated. Thirty-day pregnancy rate was the

number of cows pregnant during the first 30 d of the breeding season divided by the total number

of cows treated.

Binomially distributed data including estrous response, conception rate, timed-AI

pregnancy rate, synchronized pregnancy rate, and 30 d pregnancy rate, were analyzed by logistic









regression using the LOGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). All models

included synchronization treatment (7-10 and Select Synch/CIDR+timed-AI), PGF2a treatment

(cloprostenol and dinoprost), DPP, cow age, BCS, AI technician, and all appropriate interactions.

Days postpartum, cow age, and BCS were analyzed as categorical variables. Days postpartum

was evaluated in three categories < 43 d, 44 to 59 d, and > 60 d. Cow age ranged from 3 to 9 yr

of age resulting in seven categories. Body condition score was grouped into three categories <

4.5, 5, > 5.5. The effect of interval from PGF2a to the onset of estrus (48, 60, and 72 h) on

conception rate also was analyzed with synchronization treatment, PGF2a treatment, interval

from PGF2a to the onset of estrus, DPP, cow age, BCS, and all appropriate interactions included

in the model. Because of a limited number of cows exhibiting estrus between 0 and 48 h after

PGF2a, these times periods were combined to one group of cows' < 48 h. The final logistic

regression model entered variables by a stepwise selection based on the Wald statistics criterion

when P < 0.20 and removed variables based on P < 0.10. Variables were considered significant

at P < 0.05. Variables that were significant were entered into a linear regression model using

GENMOD procedure of SAS to calculate the adjusted odds ratios and 95% confidence intervals.

Odds ratios were reported in tables and were used to calculate relative risk, and the relative risk

was reported in the results.

Experiment 2

Experiment 2 (Figure 3-1) was conducted with yearling Bos indicus x Bos taurus heifers

(13 to 15 mo old) at the Bar L Ranch, Marianna, Florida, during two consecutive years, 2006

(year 1, n = 218) and 2007 (year 2, n = 137). The percentage of Bos indicus breeding ranged

from approximately 7 to 38% with the remainder being Bos taurus breeding. The experiment

was a 2 x 2 factorial design and heifers were equally but randomly assigned to the same









treatments used in Experiment 1. Body condition scores were recorded for heifers at CIDR

insertion.

For all treatments and years, heifers received EstrotectT estrous detection patches on day

18 of the experiment to aid in estrous detection. Estrus detection and insemination protocols

were similar as described in Experiment 1. Heifers were inseminated with frozen-thawed semen

from a single sire in year 1 and two pre-assigned sires in year 2. Heifers were inseminated by the

same AI technician in both years. Seven days after the timed-AI, natural service sires were

placed with heifers in both years. Pregnancy was diagnosed approximately 54 to 56 d after AI

using a real-time B-mode ultrasound (Aloka 500V, Corometrics Medical Systems, Wallingford,

CT) with a 5.0 MHz transducer. Due to the 7 d period in which heifers were not exposed to

natural service sires, differences in fetal size (Curran et al., 1986) as described for Experiment 1

were used to determine whether a pregnancy resulted from the synchronized breeding or the

natural service sire.

Estrous response was defined as the number of heifers displaying estrus for 3 d after

prostaglandin and inseminated divided by the total number of heifers treated. Conception rate

was defined as the number of heifers that became pregnant to AI divided by the number of

heifers that displayed estrus and were AI. Timed-AI pregnancy rate was the number of heifers

that failed to display estrus, were timed-AI, and became pregnant divided by the total number of

heifers that were timed-AI. Synchronized pregnancy rate was the number of heifers pregnant to

AI divided by the total number of heifers treated. Thirty-day pregnancy rate was the number of

heifers pregnant during the first 30 d of the breeding season divided by the total number of

heifers treated.









Binomially distributed data including estrous response, conception rate, timed-AI

pregnancy rate, synchronized pregnancy rate, and thirty-day pregnancy rate were analyzed by

logistic regression using the LOGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). All

models included synchronization treatment (7-10 and Select Synch/CIDR+timed-AI), PGF2,

treatment (cloprostenol and dinoprost), year, BCS, and all appropriate interactions. Body

condition score was analyzed as a categorical variable and was grouped into three categories (<

5, 5.5, > 6). The effect of interval from PGF2, to the onset of estrus (< 48, 60, and 72 h) on

conception rate was also analyzed with synchronization treatment, PGF2, treatment, interval

from PGF2a to the onset of estrus, year, BCS, and all appropriate interactions included in the

model. Because of a limited number of heifers exhibiting estrus between 0 and 48 h after PGF2a,

these time periods were combined into one group of heifers' < 48 h. The final logistic regression

model entered variables by a stepwise selection based on the Wald statistics criterion when P <

0.20 and removed variables based on P < 0.10. Variables were considered significant at P <

0.05. Variables that were significant were entered into a linear regression model using

GENMOD procedure of SAS to calculate the adjusted odds ratios and 95% confidence intervals.

Odds ratios were reported in tables and were used to calculate relative risk, and relative risk was

reported in the results.

Results

Experiment 1

Synchronization treatment affected (P < 0.05) estrous response with the risk of displaying

estrous in the Select Synch/CIDR+timed-AI treated cows being 1.2 times greater compared to 7-

10 cows. Body condition score also affected (P < 0.05) estrous response. Cows in BCS < 4.5

had an estrous response that was 73.7 percent of what cows that were BCS > 5.5 expressed.

Cows with a BCS 5 tended (P = 0.1) to have a decreased estrous response compared to cows in









BCS > 5.5 (Table 3-1). There were no effects (P > 0.05) of PGF2, treatment, age, DPP, and all

appropriate interactions on estrous response.

Synchronization treatment affected (P < 0.05) conception rate as the Select

Synch/CIDR+timed-AI cows had a 1.4 higher risk of becoming pregnant to AI after an observed

estrus compared to 7-10 cows. Body condition also affected (P < 0.05) conception rate. Cows

with BCS > 5.5 had 2 times greater risk of becoming pregnant after an observed estrus compared

to cows that were BCS < 4.5. Cows in BCS 5 tended (P = 0.1) to have decreased conception rate

compared to cows in BCS > 5.5 (Table 3-2). There was also an AI technician effect (P < 0.05)

on conception rate (56.8 vs. 45.2%). There were no effects (P > 0.05) of PGF2a treatment, age,

DPP, interval from PGF2, to estrus, and all appropriate interactions on conception rate.

There were no effects (P > 0.05) of synchronization treatment, PGF2, treatment, BCS,

age, AI technician, and all appropriate interactions on timed-AI pregnancy rate. Timed-AI

pregnancy rates for the 7-10 and Select Synch/CIDR+timed-AI were 17.5 and 20.9%,

respectively. However, DPP tended (P = 0.1) to effect timed-AI pregnancy rate (Table 3-3). In

general, as days from calving increased, timed-AI pregnancy rates also increased.

Synchronization treatment affected (P < 0.05) synchronized pregnancy rate with cows

treated with Select Synch/CIDR+timed-AI having a 1.5 greater risk of becoming pregnant during

the synchronized breeding compared to 7-10 cows (Table 3-4). Body condition score also

affected (P < 0.05) synchronized pregnancy rate (Table 3-4). Cows with a BCS > 5.5 had a 2.1

greater risk of becoming pregnant during the synchronized breeding compared to cows with a

BCS < 4.5. Cows with a BCS > 5.5 also had 1.4 times greater risk of becoming pregnant during

the synchronized breeding compared to cows with BCS 5. An increased (P < 0.05) percentage of

cows with a DPP > 60 got pregnant to the synchronized breeding compared to cows that were <









43 DPP. Cows with a DPP > 60 had a risk of becoming pregnant 1.8 times that of the < 43 DPP

cows. Synchronized pregnancy rates were similar (P > 0.05) for cows 44-59 DPP compared to

cows > 60 DPP (Table 3-4). There were no effects (P > 0.05) of PGF2u treatment, age, AI

technician, and all appropriate interactions on synchronized pregnancy rate.

There were no significant effects of synchronization treatment, PGF2, treatment, age,

DPP, and all appropriate interactions on thirty-day pregnancy rate. However, cows with a BCS 5

and < 4.5 had decreased (P < 0.05) thirty-day pregnancy rates compared to cows with a BCS >

5.5 (Table 3-5).

Experiment 2

Synchronization treatment, PGF2, treatment, year, BCS, and all appropriate interactions

had no effect (P > 0.05) on estrous response, conception rate, timed-AI pregnancy rate,

synchronized pregnancy rate, and thirty-day pregnancy rate (Table 3-6). Across the

synchronization and PGF2, treatments, the mean estrous response, conception rate, timed-AI

pregnancy rate, synchronized pregnancy rate, and thirty-day pregnancy rate were 66.8, 65.0,

16.1, 48.7, and 76.3% respectively. The mean synchronized pregnancy rates for the dinoprost

and cloprostenol treatments were 45.5 and 52.0%, respectively. However, there was a treatment

by interval from PGF2a to the onset of estrus interaction (P < 0.05; Figure 3-2) on conception

rate. For heifers that exhibited estrus 48 h after PGF2,, conception rates were similar (P > 0.05)

between treatments. Whereas, for heifers that exhibited estrus at 60 h, the Select

Synch/CIDR+timed-AI treatment tended (P = 0.10) to have a decreased conception rate

compared to the 7-10 treatment. Conversely, for heifers that exhibited estrus at 72 h, the Select

Synch/CIDR+timed-AI treatment had an increased (P < 0.05) conception rate compared to the 7-

10 treatment.









Discussion

The true measure of the effectiveness of a synchronization treatment is the synchronized

pregnancy rate or the number of cattle pregnant to the synchronized AI breeding. In Experiment

1, 14.3% more suckled Bos indicus x Bos taurus synchronized with the Select

Synch/CIDR+timed-AI treatment became pregnant compared to the 7-10 treatment. The

synchronized pregnancy rate of the 7-10 treatment are considerably less compared to reports in

suckled Bos taurus cows synchronized with 7-11 protocol with either estrous detection and AI

(Kojima et al., 2000) or a timed-AI (Bader et al., 2004). Both studies reported synchronized

pregnancy rates greater than 60% for estrous cycling and anestrous cows. Likewise, the

synchronized pregnancy rate for the Select Synch/CIDR+timed-AI treatment was less (13%)

compared to a report by Larson et al. (2006) in estrous cycling and anestrous suckled Bos taurus

cows synchronized with the same protocol. Larson et al. (2006) observed a range in mean

synchronized pregnancy rates between 51 to 61% for cows across different BCS, estrous cycling

statuses, DPP, and parities. The decreased response of the 7-10 and Select/Synch+timed-AI

treatments in suckled Bos indicus x Bos taurus cows compared to similarly synchronized

suckled Bos taurus cows (Kojima et al., 2000; Bader et al., 2004; Larson et al., 2006) is due to

slight decreases in estrous response, conception rate, and timed-AI pregnancy rates, which

function together to decrease synchronized pregnancy rates in the suckled Bos indicus x Bos

taurus cows. It is difficult to determine the exact reasons) for the reduced estrous response and

conception rates between the suckled Bos indicus x Bos taurus in the present study compared to

what is reported for Bos taurus cows. As with this study and research in the literature, there are

very few instances where cattle of both Bos indicus x Bos taurus and Bos taurus breeding are at

the same location and receiving the same synchronization treatments under similar

environmental conditions. Therefore, making general comparisons about responses to estrous









synchronization protocols between the two genotypes must be made with caution. In general, it

has been well established that estrus is more difficult to detect in cattle ofBos indicus breeding

due to a decreased expression and duration of estrus (Galina et al., 1982) as well as an increased

incidence of silent estrus (Galina et al., 1996), which could play a role in the decreased estrous

response for both synchronization treatments. What role if any, the increased incidence of three

and four-wave follicle growth patterns, which are more frequent in cattle of Bos indicus breeding

(Rhodes et al., 1995; Viana et al., 2000; Martinez et al., 2003), had in the estrous response of a

synchronized estrous is uncertain but should not be discounted.

As mentioned previously, the primary reasons for the greater synchronized pregnancy

rate for the Select Synch/CIDR+timed-AI compared to the 7-10 treatment were significant

increases in estrous response and conception rate for the Select Synch/CIDR+timed-AI

treatment. The three day estrous response for the 7-10 cows was 10.1% less compared to Select

Synch/CIDR+timed-AI cows. Kojima et al. (2000) reported a peak estrous response by 66 h

after PGF2, of 92% in suckled Bos taurus cows synchronized with the 7-11 protocol, which is

43% greater compared to the 72 h estrous for the 7-10 treated Bos indicus x Bos taurus cows.

Esterman et al. (2007) observed similar five day estrous responses between suckled Angus (Bos

taurus) and Brangus (Bos indicus x Bos taurus) cows synchronized with the 7-11 protocol at the

same location. In contrast, the 72 h estrous response (59.9%) for the Select Synch/CIDR+timed-

AI cows was only slightly less compared to the 72 h estrous response (69.3%) reported by

Larson et al. (2006) in suckled Bos taurus cows. One reason for the significantly decreased

estrous response of the 7-10 compared to the Select Synch/CIDR+timed-AI treatment could be

due to pre-synchronization portion of the 7-10 treatment that used a once-used CIDR did not

create the same synchrony of follicular wave development compared to cows treated with the









traditional 7-11 protocol that uses MGA. In Brangus cows synchronized with the 7-11 protocol,

Esterman et al. (2007) reported that GnRH initiated ovulation in 94% of the cows after MGA

treatment similar to reports in Bos taurus cows (Kojima et al., 2000). Since ovulation rate to

GnRH was not determined in the present study, the ability of GnRH to ovulate follicles three

days after CIDR removal is not known. Hence, it is unclear if the timing of GnRH relative to

CIDR removal was appropriate and if a majority of follicles were of ovulatory size for GnRH to

ovulate. It is also possible that the low circulating concentrations of progesterone from the once-

used CIDR altered LH secretary patterns (Kojima et al., 1992) and follicle growth and

development (Bergfeld et al., 1996) that resulted in abnormal follicle development resulting in

development of some persistent dominant follicles that were not ovulated by GnRH (Woodall et

al., 2007a). Any of these scenarios could have resulted in asynchronous follicle development at

the second PGF2, resulting in a low estrous response during the three day estrous detection

period.

The second reason for the decreased synchronized pregnancy rates of the 7-10 treatment

compared to the Select Synch/CIDR+timed-AI treatment was a significant reduction in

conception rate for the 7-10 treatment. Conception rate of the 7-10 treatment is also

considerably less compared to suckled Bos taurus (Kojima et al., 2000) and Bos indicus x Bos

taurus (Esterman et al., 2007) cows synchronized with a 7-11 protocol. In both studies,

conception rates were greater than 65% compared to 45.5% in the current study. In suckled Bos

indicus x Bos taurus cows synchronized with the Select Synch protocol without a CIDR,

Lemaster et al. (2006) reported a conception rate of 57.7%. The conception rates for the Select

Synch/CIDR+timed-AI treatment were similar to a report by Larson et al. (2006) in suckled Bos

taurus cows synchronized with a similar protocol and other reports in Bos taurus synchronized









cows (Kojima et al., 2000; Stegner et al., 2004). It is unclear why the conception rates were

decreased in the 7-10 treatment. Based on the how the 7-10 treatment was designed, the

pretreatment phase with the once-used CIDR followed 3 d later by GnRH should have worked to

synchronize follicle development by ovulating a majority of the follicles so they are

synchronized for the subsequent PGF2a treatment 7 d later, which is essentially the Select Synch

protocol. The newly developed follicles should have an increased fertility compared to

ovulations of "aged" dominant follicles (Mihm et al., 1994) or persistent dominant follicles

(Kinder et al., 1996). Additionally, the hormonal environment that follicles of the Select

Synch/CIDR+timed-AI treatment were exposed to was different compared to the GnRH+PGF2a

portion of the 7-10 treatment. The Select Synch/CIDR+timed-AI would have been exposed to

elevated progesterone concentrations between GnRH and PGF2a; whereas, the 7-10 treatment did

not receive this treatment. Although, addition of a CIDR to the Select Synch+timed-AI protocol

did not improve conception rates to an observed estrus (Larson et al., 2006) in Bos taurus cows.

Whether the progesterone from the CIDR is enhancing conception rates in the Bos indicus x Bos

taurus cows in the present study remains in question and needs to be evaluated further.

The one variable that had a significant effect on conception rate was BCS across both

synchronization treatments. Cows with a BCS 5 had approximately a 25% greater conception

rate compared to cows with a BCS < 4.5, regardless of synchronization treatment. In contrast,

Larson et al. (2006) reported only a 7% increase in conception rate in cows with a BCS > 5

compared to cows with a BCS < 5 in suckled Bos taurus cows synchronized with the Select

Sync/CIDR+timed AI. Body condition can be used as an indirect measure of nutritional status

(Richards et al., 1986), and nutritional status influences estrous cycling status of suckled beef

cows (Smith et al., 1976; Oyedipe et al., 1982) as well as the number of cows in estrus within 60









d after calving (Whitman, 1975; Wettemann et al., 1982). It is possible that a greater percentage

of cows with a BCS > 5 were going through estrous cycles at the start of the synchronization

treatments resulting in greater fertility compared to cows with a BCS < 5. Both Lucy et al.

(2001) and Larsen et al. (2006) observed numerically greater conception rates for estrous cycling

cows compared to anestrous cows when inseminated after a synchronized estrus.

Body condition score at the start of the synchronization treatment also had an effect on

synchronization response, as cows with a body condition score > 5.5 had significantly greater

estrous response, conception rate, synchronized pregnancy rate, and thirty-day pregnancy rate

compared to cows with a BCS < 4.5. This agrees with a report by Lamb et al. (2001) in Bos

taurus cows where for each increase in BCS of one unit, the proportion of cows pregnant to AI

after a synchronized ovulation increased by 23%. In contrasts, Larson et al. (2006) reported only

a 7% increase in conception rate in cows that had BCS > 5 compared to cows with a BCS < 5 in

suckled Bos taurus cows synchronized with the Select Synch/CIDR+timed AI. Although in the

Larson et al. (2006) study, there were a limited number of cows with BCS < 5 that could have

influenced the results. For the Bos indicus x Bos taurus cows in the current experiment,

synchronized pregnancy rates increased by more than 30% for cows with a BCS > 5.5 compared

to a BCS of < 4.5. The importance of BCS on the response to a synchronization treatment has

been reported by others (Yelich et al., 1988; De Jarnette et al., 2004; Larson et al., 2006) and the

common theme among these studies is that cows with BCS > 5, respond well to most

synchronization systems for cattle of either Bos taurus or Bos indicus x Bos taurus breeding.

Therefore, it appears that suckled Bos indicus x Bos taurus cows that have a BCS > 5 at the start

of a synchronization protocol respond favorably to synchronization protocols and have an









excellent opportunity of getting pregnant, similar to what is observed in suckled Bos taurus

cows.

Treatment had no effect on timed-AI pregnancy rates. However, timed-AI pregnancy

rates were numerically less for 7-10 (17%) compared to Select Synch/CIDR+timed-AI (20%).

Larson et al. (2006) reported timed-AI pregnancy rates for the Select Synch+timed-AI without a

CIDR, Select Synch/CIDR+timed-AI, and CIDR+timed-AI of 39, 26, and 38%, respectively in

suckled Bos taurus cows. There certainly appears to be a considerable reduction in timed-AI

pregnancy rates between cattle of Bos indicus x Bos taurus compared to Bos taurus cattle. This

could be due to several reasons ranging from lack of cattle going through estrous cycles and

asynchronous follicle development at CIDR removal for the cattle ofBos indicus x Bos taurus.

Furthermore, the increased incidence of three and four wave follicle wave patterns in Bos indicus

x Bos taurus compared to Bos taurus cattle could result in asynchronous follicle development at

the timed-AI resulting in either aged follicles with reduced fertility (Sirois and Fortune, 1990;

Mihm et al., 1994) ovulating to GnRH or follicles that are not of the adequate size ovulating to

GnRH (Lamb et al., 2001; Perry et al., 2005; Busch et al., 2008), which also have reduced

fertility. A recent study by Esterman et al. (2008) in suckled cows of Bos indicus x Bos taurus

breeding synchronized with the Select Synch/CIDR with estrous detection, reported that

conception rate peaked for cattle that exhibited estrus by 60 h after PGF2, and conceptions rates

decreased in a linear fashion for cows that exhibited estrus between 72 and 96 h post PGF2,.

This suggests that maybe the timed-AI needs to be performed at 60 h for the 7-10 protocol to

avoid development of follicles with longer durations of dominance, which could be more

susceptible to decreased fertility. It should also be noted, the timed-AI groups also include









anestrous cows that did not respond to the synchronization treatments and therefore had no

opportunity to conceive and get pregnant.

Timed-AI and synchronized pregnancy rates were also significantly influenced by DPP at

the start of the synchronization treatment. Timed-AI pregnancy rate increased by 16% and

synchronized pregnancy rate increased by 21% in cows > 60 DPP compared to cows < 43 DPP.

Both, Esterman et al. (2007) in Bos indicus x Bos taurus cows synchronized with a Select

Synch/CIDR+timed-AI protocol and Stevenson et al. (2000) in Bos taurus cows synchronized

with Select Synch concurrent with a norgestomet implant observed an increase in synchronized

pregnancy rates as the interval from calving increased. The present study and others (Stevenson

et al., 2000; Esterman et al., 2007) stress the importance of knowing where cows are in their

postpartum period before starting a synchronization program. However, the interval from

calving to the initiation of the synchronization treatment did not influence thirty day pregnancy

rates, which were similar between the three postpartum interval categories. In contrast, BCS at

the start of the synchronization treatment continued to influence the opportunity for cows to get

pregnant as the breeding season progressed as observed by others (Spitzer et al., 1995; Kunkle et

al., 1998). Many factors affect fertility and response to synchronization protocols and re-

breeding of the postpartum cow, most importantly estrous cycling status. Factors that control the

resumption of estrous cyclicity in postpartum females include endocrine function, (Erb et al.,

1971), nutritional status (Short et al., 1976), BCS (Stevenson et al., 2000), and suckling status

(Randel, 1981).

In Experiment 2, there were no synchronization treatment (7-10 vs. Select

Synch/CIDR+timed-AI) or PGF2a treatment (dinoprost vs. cloprostenol), affects on estrous

response, conception rate, timed-AI pregnancy rate, synchronized pregnancy rate, or thirty day









pregnancy rate for Bos indicus x Bos taurus yearling heifers, which is in stark contrast to the

results obtained for the same synchronization treatments for cows in Experiment 1. In Bos

taurus heifers synchronized with the Select Synch/CIDR+timed-AI protocol, Lamb et al. (2006)

reported synchronized pregnancy rates that were approximately 5% greater compared to the

Select Synch/CIDR+timed-AI treatment and approximately 12% greater compared to the 7-10

treatment of the present study. The 72 h estrous responses were 5.6% less for the Select

Synch+timed-AI and 8.8% less for the 7-10 treatments compared to Bos taurus heifers

synchronized with the Select Synch/CIDR+timed-AI reported by Lamb et al. (2006).

Conception rates were similar between synchronization treatments and were also similar

compared to conception rates reported by Lamb et al. (2006) in similarly synchronized Bos

taurus heifers. Similar to the results observed for cows of Experiment 1, estrous response and

timed-AI pregnancy rates were less in the Bos indicus x Bos taurus synchronized heifers

compared to reports in Bos taurus synchronized heifers (Lamb et al., 2006). In Bos indicus x

Bos taurus heifers synchronized with a 7 d CIDR with PGF2a provided the day before CIDR

removal, Lucy et al. (2001) reported similar conception and synchronized pregnancy rates

compared to estrous cycling Bos taurus heifers. Furthermore, of the Bos indicus x Bos taurus

heifers that were going through estrous cycles at CIDR insertion, 83% exhibited estrus within 3 d

after PGF2a, which was similar to Bos taurus heifers in the same study. There have been no

studies evaluating the 7-11 protocol in yearling heifers ofBos indicus x Bos taurus breeding.

Heifers treated with the 7-10 protocol had slightly decreased conception and synchronized

pregnancy rates compared to long term MGA based protocols in Bos taurus heifers (Wood et al.,

2001; Patterson et al., 1993). Bridges et al. (2005) synchronized yearling heifers of Bos indicus

x Bos taurus breeding with a 14 d MGA treatment followed by two consecutive PGF2a









treatments 19 and 20 d after MGA, and heifers were AI after a detected estrus for 72 h and non-

responders were timed-AI plus GnRH. They reported similar synchronized pregnancy rates

compared to the 7-10 treatment. It should also be mentioned that both estrous response and

subsequent conception rates were considerably less in the Bridges et al. (2005) study compared

to the 7-10 treatment. However, the timed-AI pregnancy rates were nearly doubled in the

Bridges et al. (2005) study, which resulted in similar synchronized pregnancy rates compared to

the current experiment. Hence, it appears that a decreased estrous response is one of the limiting

factors that decrease the overall effectiveness of synchronization systems in heifers of Bos

indicus x Bos taurus breeding compared to yearling Bos taurus heifers. Additional studies need

to be conducted in heifers of Bos indicus x Bos taurus breeding to determine what physiological

and management factors influence the onset of behavioral estrus. Additionally, the relationship

between follicle development during and after a synchronization treatment and what affect it has

on the expression of behavioral estrus after a CIDR treatment needs to be evaluated.

As mentioned previously, conception rates were similar between synchronization and

PGF2a treatments. However, there was a treatment by interval from PGF2a to onset of estrus

effect on conception rate. For the 7-10 treatment, conception rate peaked for heifers that

exhibited estrus at 60 h after PGF2, and decreased at 72 h; whereas, the inverse was observed for

the Select Synch/CIDR+timed-AI heifers. Although the numbers of heifers are limited in each

estrus category, there were no significant year effects on conception rates. Additionally, the

same AI technician inseminated heifers in both years and a single AI sire was used in year 1 and

two AI sires were used in year 2, which had similar conception rates. Therefore, it appears that

conception rates are influenced not only by synchronization treatment but when heifers exhibited

estrus relative to PGF2,. However, it should be noted that since estrous detection ceased at 72 h









post PGF2a it is not known what the conception rates would have been for the heifers exhibiting

estrus after 72 h. Although, it may prove beneficial to move the timed-AI in the 7-10 treatment

from 72 h to approximately 60 to 66 h to take advantage of improved fertility of the 60 h estrus.

The interval to estrus following PGF2a is dependent on the stage of ovarian follicular

growth when PGF2, is administered (Geary et al., 1999; Hittinger et al., 2004). Kastelic et al.

(1990) observed cows with a mature dominant follicle present at PGF2a display estrus earlier

after PGF2C compared to cows with a growing and developing follicle at PGF2a, which takes

longer to exhibit estrus. The 7-10 treated heifers should have a greater synchrony of follicle

development compared to the Select Synch/CIDR+timed-AI group since the GnRH after the

initial PGF2a in the 7-10 treatment should have initiated ovulation in a majority of animals

resulting in a synchronous follicle development compared to GnRH administered at CIDR

insertion in the Select Synch/CIDR treatment. The number of follicles ovulating to GnRH at

CIDR insertion in the Select Synch/CIDR treatment would probably have been less compared to

the 7-10 treatment since there would be follicles at all stages of follicle development at CIDR

insertion and stage of follicle development can influence ovulation rates to GnRH (Moreira et al.,

2000). Therefore, there could have been a mix of follicles at different stages of development at

CIDR removal for the Select Synch/CIDR treatment resulting in ovulation of both newly

developed follicles and aged follicles. Heifers with aged follicles and (or) a greater duration of

dominance should exhibit estrus earlier after PGF2a and fertility of the aged follicles would be

decreased compared to newly developed follicles (Mihm et al., 1994). Bridges et al. (2008)

hypothesized that fertility of the ovulatory follicle is a function of the duration of proestrus and

capacity of the ovulatory follicle to produced increased estradiol concentrations preceding estrus

and AI. They further hypothesized that by reducing the duration of a CIDR treatment from 7 to









5 d could enhance secretion of estradiol by the potential ovulatory follicle even with younger

follicles of reduced diameters. They conducted a series of experiments that synchronized

suckled Bos taurus cows with a 5 d Co-Synch protocol with timed-AI at 72 h compared with a 7

d Co-Synch with timed-AI at 60 h. With the 5 d program, newly developed follicles would be

approximately 3 to 4 d from emergence compared to 5 to 6 d from emergence for the 7 d

program. The 5 d program significantly increased timed-AI pregnancy rates compared to the 7 d

treatment in suckled beef cows. These findings certainly demonstrate the importance of duration

of follicle development on fertility of a synchronization system. Whether these findings will

translate to beef heifers is unknown but it does provide evidence to explain the significant shifts

in fertility observed between synchronization systems based on follicle development.

In Experiments 1 and 2, the response to cloprostenol and dinoprost was similar between

treatments for suckled cows and yearling heifers. This agrees with Hiers et al. (2003) who

reported similar synchronized pregnancy rates in Bos indicus x Bos taurus cows synchronized

with a modified Co-synch protocol with either cloprostenol or dinoprost treatments. Likewise,

Salverson et al. (2002) reported a similar response in Bos taurus heifers treated with either

cloprostenol or dinoprost treatments. Therefore, it appears that cloprostenol or dinoprost are

equally effective when used in either the 7-10 or the Select Synch/CIDR+timed-AI

synchronization protocols in cattle of Bos indicus x Bos taurus breeding.

In summary, the Select Synch/CIDR+timed-AI had increased conception and synchronized

pregnancy rates compared to the 7-10 treatment in suckled cows of Bos indicus x Bos taurus

breeding in Experiment 1. The effectiveness of the treatment was influenced by BCS, as BCS

increased, the estrous response, conception rate, and synchronized pregnancy rates increased.

Days postpartum also influenced effectiveness of the synchronization treatments, as time from









calving to start of synchronization increased, synchronized pregnancy rates increased. There was

no effect of prostaglandin treatment on estrous response, conception rate, timed-AI pregnancy

rate, synchronized pregnancy rate, or thirty day pregnancy rate. In Experiment 2, there were no

synchronization or prostaglandin treatment effects on any of the reproductive responses

measured in the yearling heifers of Bos indicus x Bos taurus breeding.

Implications

The Select Synch/CIDR+timed-AI protocol is a practical synchronization method for Bos

indicus x Bos taurus yearling heifers and postpartum cows that are in good body condition and >

60 d postpartum at the start of the synchronization treatment. The 7-10 synchronization protocol

yields similar results as the Select Synch/CIDR+timed-AI protocol when used in yearling heifers

but decreased results in suckled Bos indicus x Bos taurus cows. Cloprostenol sodium and

dinoprost tromethamine are equally effective and yielded similar synchronized pregnancy rates

in both yearling heifers and suckled Bos indicus x Bos taurus cows.











a) 7-10 treatment with Dinoprost or Cloprostenol PGF2Q on day 17


Dinoprost GnRH


Once-used CIDR


Dinoprost or
Cloprostenol


GnRH +
timed-AI


S
Estrus detect & Al


b) Select Synch/CIDR+timed-AI treatment with Dinoprost or Cloprostenol PGF2, on day 17


Dinoprost or GnRH +


GnRH


Cloprostenol


New CIDR Estrus detect & A
New CIDR Estrus detect & Al


timed-AI


Day of experiment


Experimental design evaluating the effects of two progestogen and two PGF2,
treatments in Bos indicus x Bos taurus suckled cows (Experiment 1) and yearling
heifers (Experiment 2). a) 7-10 treatment: on day 0 received a once-used CIDR that
was removed on d 7 concomitant with dinoprost tromethamine (Dinoprost; 25 mg
i.m.) followed by GnRH (500 [tg i.m.) on day 10. On day 17, females received
either dinoprost or cloprostenol sodium (Cloprostenol; 500 tg i.m.). b) Select
Synch/CIDR+timed-AI treatment: on day 10 received a new CIDR (1.38 g)
concomitant with GnRH. Day 17 CIDR was removed and females received either
Dinoprost or Cloprostenol. For all four treatments estrus was detected for 3 d and
females were inseminated 6 to 12 h after detected estrus. Females not exhibiting
estrus by 72 h were timed-AI and received GnRH.


Figure 3-1.










Table 3-1. Effect of synchronization treatment and body condition score (BCS) on estrous
response in suckled Bos indicus x Bos taurus cows.a
Estrous
Variable response, %0 Odds Ratio 95% CId P-value

Treatment
Select Synch/CIDR+timed-AI 59.9 (100/167) 1.59 1.02-2.48 0.05
7-10 49.0 (77/157) Referent

BCSb
< 4.5 48.8 (59/121) 0.47 0.25-0.89 0.02
5 54.4 (75/138) 0.59 0.32-1.10 0.10
> 5.5 66.2 (43/65) Referent

a Refer to Figure 3-1 for details of treatments.
b Body condition score: 1 = emaciated, 5 = moderate; 9 = very fat.
' Percentage of cows displaying estrus 72 h after PGF2a of the total treated.
d 95% confidence interval.










Table 3-2. Effect of synchronization treatment and body condition score (BCS) on conception
rate in suckled Bos indicus x Bos taurus cows.a

Conception
Variable rate, %0 Odds Ratio 95% CId P-value

Treatment
Select Synch/CIDR+timed-AI 62.0 (62/100) 2.08 1.10-3.95 0.03
7-10 45.5 (35/77) Referent

BCSb
< 4.5 35.6 (21/59) 0.20 0.08-0.47 0.0003
5 60.0 (45/75) 0.51 0.22-0.1.17 0.11
> 5.5 72.1 (31/43) Referent

a Refer to Figure 3-1 for details of treatments.
b BCS: 1 = emaciated, 5 = moderate; 9 = very fat.
' Percentage of cows pregnant to AI of the total that exhibited estrus and were AI.
d 95% confidence interval.










Table 3-3. Effect of days postpartum (DPP) on timed-AI pregnancy rate in suckled Bos
indicus x Bos taurus cows.a

Timed-AI pregnancy
DPP rate, %b Odds Ratio 95% CIc P-value

<43 11.5 (7/61) 0.34 0.12-0.98 0.05

44-59 21.7 (10/46) 0.73 0.27-1.96 0.54

>60 27.5 (11/40) Referent

a Refer to Figure 3-1 for details of treatments.
b Percentage of cows that failed to display estrus, were timed-AI, and became pregnant of the
total number of cows that were timed-AI.
c 95% confidence interval










Table 3-4. Effect of synchronization treatment, body condition score (BCS), and days
postpartum (DPP) on synchronized pregnancy rate in suckled Bos indicus x Bos
taurus cows.a

Synchronized Odds
Variable pregnancy rate, %" Ratio 95% CId P-value

Treatment
Select Synch/CIDR+timed-AI 45.5 (76/167) 2.01 1.25-3.24 0.004
7-10 31.2 (49/157) Referent

BCSb
S4.5 26.5 (32/121) 0.29 0.15-0.55 0.0002
5 40.6 (56/138) 0.52 0.28-0.97 0.04
> 5.5 56.9 (37/65) Referent

DPP
<43 27.8 (32/115) 0.45 0.25-0.82 0.009
44-59 40.5 (45/111) 0.73 0.42-1.30 0.28
> 60 49.0 (48/98) Referent

a Refer to Figure 3-1 for details of treatments.
b BCS: 1 = emaciated, 5 = moderate; 9 = very fat.
' Percentage of cows pregnant during the synchronized breeding of the total treated.
d 95% confidence interval










Table 3-5. Effect of body condition score (BCS) on thirty day pregnancy rates in suckled Bos
indicus x Bos taurus cows.

Variable Thirty day pregnancy rate, %b Odds Ratio 95% CIc P-value

BCSa
< 4.5 72.7 (88/121) 0.32 0.12-0.78 0.01
5 77.5 (107/138) 0.42 0.17-1.00 0.05
> 5.5 89.2 (58/65) Referent

a BCS: 1 = emaciated, 5 = moderate, 9 = very fat.
b Percentage of cows pregnant during the first 30 d of breeding season of the total treated.
c 95% confidence interval.










Table 3-6. Effectiveness of 7-10 treatment compared to Select Sync/CIDR+timed-AI
(SSC+TAI) treatment on estrous response, conception rate, timed-AI pregnancy
rate, synchronized pregnancy rate, and thirty day pregnancy rate in yearling heifers
of Bos indicus x Bos taurus breeding.a

Variable 7-10 SSC+TAI Odds Ratiog 95% CIh P-value

Estrous
rspons b 65.2 (116/178) 68.4 (121/177) 1.26 0.80-1.97 0.32
response, %

Conception
nceptin 62.1 (72/116) 67.8 (82/121) 1.34 0.78-2.30 0.29
rate, %

Timed-AI
pregnancy 14.5 (9/62) 17.9 (10/56) 1.30 0.48-3.42 0.62
rate, %d

Synchronized
pregnancy 45.5 (81/178) 52.0 (92/177) 1.30 0.85-1.97 0.22
rate, %e

Thirty day
pregnancy 74.7 (133/178) 78.0 (138/177) 1.20 0.73-1.96 0.47
rate, %f

a Refer to Figure 3-1 for details of treatments.
b Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
' Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers that failed to display estrus, were timed-AI, and became pregnant of the
total number of heifers that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.
fPercentage of heifers pregnant during the first 30 d of the breeding season of the total number
of heifers treated.
g 7-10 used as referent value
h 95% confidence interval











*P-0.10
*P<*0. 05T



63.8

54.8


77.1


58.8











(34)


* 7-10
0 SSC+TAI


48 60 72
Interval from PGF2a to onset of estrus, hours


Effect of interval from PGF2, to onset of estrus on conception rate in Bos indicus x
Bos taurus yearling heifers synchronized with 7-10 or Select Synch/CIDR+timed-
AI (SSC+TAI) treatments. Numbers in parenthesis indicate the number of heifers
in each category. Treatment by interval from PGF2, to onset of estrus (P = 0.02).


86.2


100

90

80


g 70

S60

so
| 50

40

U 30


20

10


0 --


(58)


Figure 3-2.


-


-


I









CHAPTER 4
EVALUATION OF TWO PROGESTOGEN BASED ESTROUS SYNCHRONIZATION
PROTOCOLS IN YEARLING HEIFERS OF BOS INDICUS x BOS TAURUS BREEDING

Introduction

Estrous synchronization is a reproductive management tool that allows for an increased

number of cattle displaying estrus and ovulating over a 3 to 5 d period, which allows for a

minimal number of days of estrous detection or elimination of estrous detection resulting in a

timed artificial insemination (timed-AI). A common estrous synchronization protocol used in

Bos taurus cattle, the Select Synch protocol, utilizes GnRH with prostaglandin F2a (PGF2,) 7 d

later (Macmillan and Thatcher, 1991; Pursley et al., 1995), followed by 5 d of estrous detection.

However, a common problem with this protocol is premature expression of estrus prior to PGF2a,

resulting in additional estrous detection (DeJarnette et al., 2001). Addition of an exogenous

progestogen between the GnRH and PGF2, eliminates the need for additional estrous detection

(Thompson et al., 1999; DeJarnette et al., 2003). Addition of a progesterone insert like the Eazi-

BreedTM CIDR (CIDR) to the Select Synch protocol, combined with estrous detection for 72 h

after CIDR removal and timed-AI + GnRH for non-responders (Select Synch/CIDR+timed-AI),

is an effective estrous synchronization protocol in yearling Bos taurus heifers (Lamb et al.,

2006). However, the effectiveness of this protocol in yearling Bos indicus x Bos taurus heifers

has not been thoroughly evaluated.

The 7-11 estrous synchronization protocol consists of a 7 d melengestrol acetate (MGA)

treatment with PGF2a on the last day of MGA followed by GnRH 4 d later; 7 d after GnRH,

PGF2a is administered to synchronize estrus (Kojima et al., 2000). The 7-11 protocol is an

effective synchronization protocol in Bos taurus cattle (Kojima et al., 2000; Stegner et al., 2004)

but no research has been conducted evaluating the effectiveness of the 7-11 protocol in yearling

heifers of Bos indicus x Bos taurus breeding. The basis behind the 7-11 treatment is that the









administration of GnRH after MGA results in ovulation in majority of the animals resulting in

very synchronous follicle development pattern. In a recent study conducted by Esterman et al.

(2007), estrous cycling and anestrous suckled postpartum Brangus and Angus cows received the

7-11 protocol, and GnRH induced ovulation in 94.4% of both Brangus and Angus cows. The

resulting 5 d estrous response, conception, and synchronized pregnancy rates were similar

between Angus and Brangus cows. Since GnRH effectively ovulates and synchronizes follicles

when administered 4 d after a short term (7 d) progestogen treatment in Brangus cows, the 7-11

protocol could be an effective synchronization protocol in yearling heifers of Bos indicus x Bos

taurus breeding.

Therefore the objective of this experiment was to evaluate the effectiveness of the 7-11

and Select Synch/CIDR+timed-AI synchronization protocols in yearling heifers ofBos indicus x

Bos taurus breeding.

Materials and Methods

The experiment was conducted at three locations in North Central Florida from January

to March, 2007. Yearling (12 to 15 mo) Bos indicus x Bos taurus heifers (n = 410) were used in

the experiment and the percentage of Bos indicus breeding ranged from approximately 1/4 to 1/2

with the remainder being Bos taurus breeding (Table 4-1). At the start of the experiment (d 0)

heifers were distributed by reproductive tract score (RTS, Scale 1 to 5; 1 = non-cycling, 5 =

estrous cycling; Anderson et al., 1991; See Table B-l for complete RTS score description) and

body condition score (BCS, Scale 1 to 9; 1 = emaciated, 5 = moderate, 9 = very fat; Richards et

al., 1986) to one of two progestogen based synchronization protocols including the 7-11 and

Select Select Synch/CIDR+timed-AI protocols (Figure 4-1). The 7-11 heifers received MGA

(MGA200 premix, Pfizer Animal Health, New York, NY) in a carrier supplement fed at a rate

of 0.9 kg/head/d to deliver 0.5 mg MGA/head/d from day 0 to 7 of the experiment with PGF2,









(25 mg i.m.; Lutalyse Sterile Solution, Pfizer Animal Health, New York, NY) concomitant with

the last day of MGA. On day 11, heifers received GnRH (500 [g i.m.; Cystorelin, Merial,

Duluth, GA) followed by PGF2 on day 18. The Select Synch/CIDR+timed-AI heifers received

the same carrier supplement without MGA from day 0 to 7. On day 11, a CIDR (Eazi-BreedT

CIDR, 1.38 g progesterone; Pfizer Animal Health, New York, NY) was inserted concomitant

with GnRH. The CIDR was removed on day 18 concurrent with PGF2, (25 mg i.m.; Lutalyse).

Heifers from both synchronization treatments received EstrotectT estrous detection patches

(Rockway, Inc., Spring Valley, WI) on day 18 of the experiment to aid in estrous detection and

estrus was visually detected twice daily (0700 and 1600 h) for 72 h after PGF2,. Heifers were

artificially inseminated (AI) 6 to 12 h after detection of estrus. Non-responders were timed-AI

and treated with GnRH 72 to 76 h post PGF2a. Estrus was defined as either a heifer standing to

be mounted by another heifer, showed signs of visible mucous, and (or) had a half to full red

estrous detection patch. One AI technician within each location inseminated heifers using

frozen-thawed semen from multiple sires. Whenever possible, AI sires were equally distributed

across treatments and between heifers that either exhibited estrus and (or) were timed-AI.

Natural service sires were placed with heifers 10 d after timed-AI. Using a real-time B-mode

ultrasonography machine (Aloka 500V, Corometrics Medical Systems, Wallingford, CT) with a

5.0 MHz transducer pregnancy was diagnosed approximately 55 d after AI. Due to the 10 d

period where no cows were exposed to natural service sires, differences in fetal size were used to

determine whether a pregnancy resulted from the synchronized AI breeding (54 and 55 d

pregnant) or natural service sire (< 48 d pregnant). Embryos develop a C shape appearance

approximately day 25 of gestation, which transitions to an L shape embryos by day 32. By day









48, the fetus is approximately 35 mm in length with visible limb buds and by day 55 the fetus is

approximately 50 mm in length and has identifiable ribs (Curran et al., 1986).

Estrous response was defined as the number of heifers displaying estrus for 5 d after PGF2,

and inseminated divided by the total number of heifers treated. Conception rate was defined as

the number of heifers that became pregnant to AI divided by the number of heifers that displayed

estrus and were AI. Timed-AI pregnancy rate was the number of heifers that failed to display

estrus, were timed-AI, and became pregnant divided by the total number of heifers that were

timed-AI. Synchronized pregnancy rate was the number of heifers pregnant to AI divided by the

total number of heifers treated. Thirty-day pregnancy rate was the number of heifers pregnant

during the first 30 d of the breeding season divided by the total number of heifers treated.

Binomially distributed data including estrous response, conception rate, timed-AI

pregnancy rate, synchronized pregnancy rate, and thirty-day pregnancy rate, were analyzed by

logistic regression using the LOGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). All

models included synchronization treatment (7-11 and Select Synch/CIDR+timed-AI), location,

BCS, and all appropriate interactions. Body condition was analyzed as a categorical variable and

was grouped into three categories (< 4.5, 5, > 5.5). The effect of interval from PGF2, to the onset

of estrus (< 36, 48, 60, and 72 h) on conception rate was also analyzed with synchronization

treatment, location, BCS, and all appropriate interactions included in the model. Because of a

limited number of heifers exhibiting estrus between 0 and 36 h after PGF2,, these time periods

were combined into < 36 h. The final logistic regression model entered variables by a stepwise

selection based on the Wald statistics criterion when P < 0.20 and removed variables based on P

< 0.10. Variables were considered significant at P < 0.05. Variables that were significant were

entered into a linear regression model using GENMOD procedure of SAS to calculate the









adjusted odds ratios and 95% confidence intervals. Reproductive tract score (RTS) was not

included in the initial model due to confounding effects with BCS; therefore, a second model

was conducted evaluating the same binomial variables of estrous response, conception rate,

timed-AI pregnancy rate, synchronized pregnancy rate, and thirty-day pregnancy rate and

categorical variables including synchronization treatment, location, RTS, and all appropriate

interactions. Reproductive tract score was evaluated as a categorical variable and was grouped

into three categories (< 2, 3, > 4). The initial analysis indicated that all response variables were

similar between RTS 4 and 5, so the two RTS categories were combined for the final analysis.

The same statistical process as previously described was used to determine the final logistic

regression model as well as the linear regression model. Adjusted odds ratios and 95%

confidence intervals are also reported. Odds ratios were reported in tables and were used to

calculate relative risk, and relative risk was reported in the results.

Results

An increased (P < 0.05) percentage of heifers expressed estrus during the 72 h following

PGF2a for the 7-11 compared to Select Synch/CIDR+timed-AI treatment (Table 4-2). Estrous

response was also affected (P < 0.05) by BCS. Heifers with a BCS < 4.5 and 5 had a decreased

(P< 0.05) estrous response compared to heifers with a BCS > 5.5 (Table 4-3). The relative risk

of heifers with a BCS < 4.5 exhibiting estrus was 60% of heifers with a BCS > 5.5 and heifers

with a BCS 5 exhibiting estrus was 74% of heifers with a BCS > 5.5. Estrous response was not

affected (P > 0.05) by location nor were there any significant interactions.

Conception rates were also affected (P < 0.05) by synchronization treatment. The risk of

heifers being pregnant to an AI after an observed estrus was 1.5 times greater for the 7-11

compared to Select Synch/CIDR+timed-AI heifers (Table 4-2). In addition, conception rate was









also affected (P < 0.05) by the interval from PGF2a to the onset of estrus (Figure 4-2); however,

there was no (P > 0.05) treatment by interval from PGF2a to the onset of estrus effects on

conception rate. Heifers exhibiting estrus at 60 h had an increased (P < 0.05) conception rate

compared to heifers that had exhibited estrus at < 36, 48, and 72 h (Figure 4-2), which were

similar (P > 0.05) to each other. Conception rates were not (P > 0.05) affected by BCS and

location, nor were there any significant interactions.

Timed-AI pregnancy rates were similar (P > 0.05) between the 7-11 and Select

Synch/CIDR+timed-AI treatments (Table 4-2) and there were no effects (P > 0.05) of BCS and

location on timed-AI pregnancy rate nor were there any significant interactions.

Synchronized pregnancy rates were affected (P < 0.05) by synchronization treatment. The

risk of heifers treated with the 7-11 becoming pregnant to the synchronized breeding was 1.35

times greater compared to the Select Synch/CIDR+timed-AI treatment (Table 4-2). Body

condition score had no effect (P > 0.05) on synchronized pregnancy rate. Synchronized

pregnancy rates were not influenced by (P > 0.05) BCS and location, nor were there any

significant interactions. Thirty day pregnancy rates were similar (P > 0.05) between treatments

(Table 4-2) and were not affected (P > 0.05) by BCS, location, nor were there any significant

interactions.

The effect of RTS on response to synchronization treatments by RTS category are

presented in Table (4-4). There were no treatment by RTS effects (P > 0.05) but RTS affected

(P < 0.05) estrous response, conception rate, synchronized pregnancy rate, and thirty-day

pregnancy rate. It should be mentioned that heifers with a RTS > 4, had a greater (P < 0.05)

BCS (5.2 0.02) compared to heifers with a RTS of 3 (5.0 + 0.02) and < 2 (4.7 0.02). Heifers

with a RTS 3 had a greater (P < 0.05) BCS compared to heifers with RTS < 2. Heifers with a









RTS > 4, had a 2.0 times greater risk of exhibiting estrus compared to heifers with a RTS < 2 and

a 1.3 times greater risk to exhibit estrus compared to heifers with a RTS 3 (Table 4-4). For

conception rate, heifers with RTS > 4 had a 6 times greater risk of being pregnant after

exhibiting estrus and being AI compared to heifers with RTS < 2. Conception rates were similar

(P > 0.05) between heifers with a RTS 3 and > 4 (Table 4-4).

Timed-AI pregnancy rates tended (P = 0.10) to be greater for heifers with a RTS > 4

compared to heifers with a RTS < 2 (Table 4-4). Synchronized pregnancy rates were similar (P

< 0.05) for heifers with RTS > 4 and 3. However, heifers with RTS > 4 had a 4 times greater

risk of becoming pregnant to the synchronized breeding compared to heifers with RTS 2 (Table

4-4). Heifers with RTS > 4 had a 2.3 greater risk of being pregnant during the first thirty-days of

the breeding season compared to heifers with RTS < 2 and a 1.2 greater risk compared to heifers

with a RTS 3(Table 4-4).

Discussion

Estrous response was greater for the 7-11 compared to Select Synch/CIDR+timed-AI

heifers. A possible reason for the increased estrous response of the 7-11 treatment could be that

7-11 treatment may do a better job of synchronizing follicle development compared to the Select

Synch/CIDR treatment. The MGA in combination with PGF2a allows for more follicles to be of

ovulatory size at MGA withdrawal resulting in most animals ovulating to GnRH (Kojima et al.,

2001), which provides for a very synchronous wave of follicle development at the subsequent

PGF2a. In contrast, the administration of GnRH at the initiation of the Select

Synch/CIDR+timed-AI protocol probably does not do as an effective job of ovulating and

synchronizing follicle development since animals are at various stages of the follicle

development at CIDR insertion, resulting in GnRH ovulating fewer follicles (Moreira et al.,









2000). Consequently, follicle development at CIDR withdrawal may not be as synchronous for

the Synch/CIDR+timed-AI compared to the 7-11 treatment, resulting in a decreased 72 h estrous

response.

The estrous response for both treatments are considerably less compared to studies in Bos

taurus heifers synchronized with either the Select Synch/CIDR+timed-AI (Lamb et al., 2006),

short term progestogen protocols (Patterson et al., 1989), and CIDR + PGF2a protocol (Lucy et

al., 2001). In all of the aforementioned studies, the 3 d estrous responses were approximately 20

to 30% greater compared to those observed in the present study in yearling Bos indicus x Bos

taurus heifers. However, the 3 d estrous responses for both treatments are similar to that

reported by Bridges et al. (2005) in yearling Bos indicus x Bos taurus heifers synchronized with

a long term (14 d) MGA + PGF2, synchronization protocol. The decreased estrous response is

probably due to heifer genotype and estrous cycling status of heifers at the start of the

synchronization treatment. Patterson et al. (1989) demonstrated that yearling Bos indicus x Bos

taurus heifers going through estrous cycles had a significant reduction in estrous response (22%)

compared to contemporary Bos taurus heifers synchronized with a short term progestogen

treatment. Estrus is more difficult to detect in cattle ofBos indicus breeding due to a decreased

expression and duration of estrus (Galina et al., 1982) as well as an increased incidence of silent

estrus (Galina et al., 1996). However, utilization of the estrous detection patches should have

assisted identifying heifers that did not exhibit a "good" standing estrous during the two times

that estrus was visually detected (0700 and 1600 h). Therefore, the method of estrous detection

is probably not the major factor responsible for the decreased estrous response. Since blood

samples were not taken on the heifers, it is not known how many heifers were going through

estrous cycles at the start of the synchronization treatment. Bos indicus x Bos taurus and Bos









taurus heifers that are going through estrous cycles at the start of a CIDR+PGF2a treatment

(Lucy et al., 2001) had significantly increased estrous responses compared to pre-pubertal

heifers. Additionally, the ability of the synchronization treatments to induce estrous cycles in the

pre-pubertal heifers is unclear. Lucy et al. (2001) reported that a CIDR+PGF2a treatment

induced estrous cycles in prepubertal Bos indicus x Bos taurus and Bos taurus heifers over

several locations ranging from 40 to 100%.

Onset of puberty is related inversely to growth rate (Wiltbank et al., 1969; Short and

Bellows, 1971) and heifers with higher growth rates are typically going to have greater BCS.

Body condition score can be used as an indirect indicator of nutritional status (Herd and Sprott,

1986; Wettemann and Lusby, 1987) and estrous cycling status (Schillo et al., 1992; Hall et al.,

1995) or as an indirect indicator of when heifers attain puberty. Heifers with a BCS > 5.5 had

the greatest estrous response (62.3%), which was significantly greater compared to heifers with a

BCS < 5. The estrous response for heifers with a BCS > 5.5 is comparable to values observed

for Bos taurus heifers synchronized with the Select Synch/CIDR+timed-AI (Lamb et al., 2006)

protocol. Moreover, Lucy et al. (2001) reported a 3 d estrous response of 80% in yearling Bos

indicus x Bos taurus heifers that were going through estrous cycles when synchronized with a

CIDR+PGF2, protocol. Based on reports in the literature and the estrous response of heifers with

a BCS > 5.5 in the present experiment, acceptable estrous responses can be achieved in

synchronized yearling Bos indicus x Bos taurus heifers but they are probably influenced by

pubertal status more than anything else. The onset of puberty is influenced by many factors

including age, genotype, and nutrition. Heifers of Bos indicus breeding are known to reach

puberty at older ages compared to Bos taurus heifers (Dow et al., 1982; Nelsen et al., 1982).

Therefore, the low number of heifers going through estrous cycles at the start of the experiment









was probably the primary reason for the decreased estrous response for the two synchronization

treatments, which was probably due to heifers not being the appropriate weight and age to reach

puberty.

There could also be environmental effects that are not easily quantifiable that could have

influenced estrous response. In estrous cycling Bos indicus x Bos taurus heifers and estrous

cycling and prepubertal Bos taurus heifers synchronized with a long term (14 d) MGA + PGF2,

protocol, Bridges et al. (2005) reported similar 3 d estrous responses (< 50%) for both genotypes

similar to heifers in the current study. Both the current study and Bridges et al. (2005) study

were conducted in sub-tropical environment within a 50 mile radius of each other during a

similar time of the year (January to March). Reproductive function has been observed to

decrease in Bos indicus x Bos taurus cattle in the winter months (Randel, 1984), which could

have negatively affected estrous response. Frequent handling of heifers of Bos indicus x Bos

taurus breeding can also have a negative effect on the intensity of estrus (Lemaster et al., 1999)

and could have altered the behavioral and endocrine physiology associated with estrus (Hardin

and Randel, 1982). The increased frequency of cattle handling could have resulted in increased

cortisol concentrations that may have had a negative effect on LH secretion (Dunlap et al., 1981)

and the preovulatory LH surge (Stoebal and Moberg, 1979). Another indication of effect of

stress on reproductive function has recently been observed by Cooke et al. (2008) in prepubertal

Bos indicus x Bos taurus heifers. They observed that by acclimating heifers to frequent animal

handling for three times a week for a 30 d period approximately 100 d before the start of the

breeding season enhanced the attainment of puberty by the start of the breeding season compared

to non-acclimated contemporaries.









There was a marked increase in conception rates for heifers synchronized with the 7-11

(47.0%) compared to the Select Synch/CIDR+timed-AI treatment (31.3%). Thus the 7-11

treatment may be doing a better job of synchronizing follicle development compared to the

Select Synch/CIDR+timed-AI treatment resulting in follicles that are more fertile. The 7-11

protocol is designed to maximize follicle development at PGF2a treatment (Kojima et al., 2001).

In contrast, the administration of GnRH at the initiation of the Select Synch/CIDR+timed-AI

protocol probably is not as effective at ovulating and synchronizing follicle development since

animals are at various stages of follicle development at CIDR insertion and GnRH will ovulate

fewer follicles (Moreira et al., 2000). Consequently, there is the probability of an increased

population of non-ovulated follicles that have extended durations of dominance at CIDR removal

resulting in ovulation of aged oocytes of decreased fertility (Mihm et al., 1994). Follicles with

prolonged dominance under the influence of exogenous progesterone had significantly lower

pregnancy rates compared with females with a normal ovulatory follicle (Sirois and Fortune,

1990; Stock and Fortune, 1993). Austin et al. (1999) reported that restricting the duration of

dominance of a pre-ovulatory follicle to 4 d consistently yielded pregnancy rates > 70% in

estrous cycling beef heifers. Furthermore, Bridges et al. (2008) reported that reducing the

duration of a CIDR treatment from 7 to 5 d in postpartum Bos taurus beef cows resulted in

significant increases in timed-AI pregnancy rates of a Co-Synch protocol. They attributed the

increased fertility to a decreased duration of dominance of follicles induced to ovulate after

CIDR removal.

Conception rates for the 7-11 treatment were similar to a report by Bridges et al. (2005) in

peripubertal yearling heifers of Bos indicus x Bos taurus breeding synchronized with a long term

14 d MGA + PGF protocol. In contrasts, conception rate for the Select Synch/CIDR+timed-AI









treatment was approximately 20% less compared to Bridges et al. (2005) study. Moreover, both

treatments are considerably less compared to Bos taurus heifers synchronized with variations of

the CIDR + PGF protocols where conception rates ranged from 60 to 80% (Lamb et al., 2006;

Lucy et al., 2001). The conception rates for the 7-11 heifers (47%) were similar to a report by

Patterson et al. (1989) in estrous cycling Bos indicus x Bos taurus heifers synchronized with

either a short term progestogen treatment or unsynchronized controls. Additionally, Patterson et

al. (1989) demonstrated that there was a breed by energy level interaction on conception rates in

estrous cycling Bos indicus x Bos taurus heifers but not similarly treated Bos taurus heifers.

They observed a 30% increase in fertility of the Bos indicus x Bos taurus heifers that were on an

increasing plane of nutrition at breeding compared to Bos indicus x Bos taurus heifers on a low

plane of nutrition. Several studies with synchronized Bos taurus heifers have reported that

fertility to an observed estrus and AI are similar between pre-pubertal and estrous cycling heifers

(Lucy et al., 2001; Lamb et al., 2006). It could be possible that the level of nutrition was not

adequate enough to enhance the fertility of the Bos indicus x Bos taurus heifers in our study.

Body condition had no effect on conception rates across the 7-11 or Select Synch/CIDR+timed-

AI treatments. Although, BCS can be used as an indicator of both pubertal status and nutrition

level, a single record taken at the start of the synchronization treatment is probably not an

accurate indicator of whether any significant changes in the plane of nutrition occurred in the

weeks preceding the onset of treatment. Therefore, it is not clear what if any effects plane of

nutrition and (or) estrous cycling status at the start of treatment had on conception rates.

Timed-AI pregnancy rates were similar between the 7-11 and Select Synch/CIDR+timed-

AI treatments but they were nearly half the values observed for Bos taurus heifers synchronized

with the Select Synch/CIDR+timed-AI protocol (Lamb et al., 2006). It should be noted that









there was an effect of interval from PGF2a to the onset of estrus on conception rate and heifers

that exhibited estrus at 60 h after PGF2, had conception rates that were nearly twice that of

heifers that exhibited estrus < 36, 48 and 72 h. Therefore, the timing of the timed-AI needs to be

re-evaluated and it is possible that it should be conducted at 60 h after PGF2a. However, the

primary reason that the timed-AI pregnancy rates were so low is probably due to the limited

number of heifers that were going through estrous cycles at the start of the experiment.

Furthermore, neither synchronization treatment probably induced estrous cycles in the

prepubertal heifers, which resulted in a substantial number of heifers that were still prepubertal at

the timed-AI.

The 7-11 treatment had a greater synchronized pregnancy rate compared to the Select

Synch/CIDR+timed-AI protocol, which was due to the greater estrous response and conception

rate of the 7-11 compared to the Select Synch/CIDR+timed-AI. As mentioned previously, the 7-

11 treatment probably was more effective at synchronizing follicle waves allowing a new

growing follicle ready to ovulate after PGF2a, which resulted in more heifers exhibiting estrus

compared to the Select Synch/CIDR+timed-AI. The synchronized pregnancy rates of the 7-11

(33.5%) are similar to those reported by Bridges et al. (2005; 34.5%) in yearling Bos indicus x

Bos taurus heifers synchronized with the 14 d MGA+PGF2a protocol with a single PGF2a

treatment. Bridges et al. (2005) significantly improved the synchronized pregnancy rate (42.5%)

by adding an additional PGF2a to the treatment, which enhanced the PGF2a induced luteolysis

resulting in greater estrous response and timed-AI pregnancy rates. It should be noted that

heifers in the Bridges et al. (2005) study were in later stages of the estrous cycle (day 12 to 15)

where PGF2a is supposed to be more effective in initiating luteolysis (Tanabe and Hahn, 1984;

Watts and Fuquay, 1985). A limited amount of research indicates that response to PGF2a is









decreased when administered early in the estrous cycle compared to the middle and late stages of

the estrous cycle in Bos taurus (Tanabe and Hahn, 1984; Watts and Fuquay, 1985) and Bos

indicus cattle (Hardin et al., 1980; Santos et al., 1988). Incomplete luteolysis can lead to

decreased estrous response, which directly affects synchronized pregnancy rates. Whether

incomplete luteolysis occurred in both synchronization treatments is not known since no blood

samples were taken at PGF2a and during the days after PGF2a to confirm if luteolysis occurred.

The synchronized pregnancy rates for both treatments are considerably less compared to 58%

reported by Lamb et al. (2006) in Bos taurus heifers synchronized with Select

Synch/CIDR+timed-AI protocol and 49% reported by Lucy et al. (2001) in Bos taurus and Bos

indicus x Bos taurus heifers synchronized with a CIDR + PGF2, protocol. The primary reason

for the decreased synchronized pregnancy rates was due to the low estrous response, conception

rate, and timed-AI pregnancy rate for the 7-11 and Select Synch/CIDR+timed-AI treatments. All

three of the response variables were probably influenced by estrous cycling status at the start of

the synchronization treatment.

Reproductive tract score (Anderson et al., 1991; Scale 1 to 5) can also be used as an

indirect indicator of pubertal status in yearling heifers. Heifers with a RTS of 1 and 2 are

considered pre-pubertal, while heifers with a RTS of 3 are approximately 1 mo from initiating

estrous cycles, and heifers with a RTS of 4 or 5 are considered to be estrous cycling. Therefore,

conducting a RTS at the initiation of the experiment was used to provide an estimate of the

pubertal status of the heifers before the start of the synchronization treatment. Although, RTS

had an effect on estrous response, conception rate, synchronized pregnancy rate, and thirty-day

pregnancy rate regardless of synchronization treatment, the magnitude in differences between

response variables across the different RTS categories was less than anticipated. Patterson et al.









(2000) reported significant increases in estrous response in Bos taurus heifers as RTS increased

from a 1 to 3 and reaching a plateau for RTS 4 and 5, which is similar to our observations. It is

not surprising that the conception rate for the RTS 1 and 2 was only 6.7%, which was

significantly less compared to heifers with RTS > 3. However, what was unexpected was that

conception rates did not increase as RTS increased from a 3 to either a 4 or 5 as observed in

synchronized yearling Bos taurus heifers (Herd and Sprott, 1986). Additionally, it was

unexpected that synchronized pregnancy rates were similar for heifers with a RTS of 3 compared

to RTS of 4 and 5 as synchronized pregnancy rates typically increase as RTS increase from a 3 to

a RTS of 4 or 5 (Brown et al., 1988). However, thirty day pregnancy rates tended to be greater

for heifers with a RTS of 4 and 5 compared to RTS of 3, which were all greater compared to

RTS 1 and 2. The results from this study suggest that RTS may not be an effective indicator of

pubertal status in yearling heifers of Bos indicus x Bos taurus breeding compared to using it to

estimate pubertal status in yearling Bos taurus heifers. Additional research needs to be

conducted to determine if RTS can be used as an accurate predictor of pubertal status based on

both blood progesterone and transrectal ultrasonography data to confirm the activity and

presence of luteal tissue in yearling heifers of Bos indicus x Bos taurus breeding.

In summary, the 7-11 treatment provided increased estrous response, conception rates, and

synchronized pregnancy rates compared to the Select Synch/CIDR+timed-AI treatment.

Synchronization treatments had similar timed-AI pregnancy rates. Estrous response was

directly affected by BCS at start of synchronization but BCS had no effect on any of the other

response variables. Although, RTS had significant affects on estrous response, conception rate,

synchronized pregnancy rate and thirty day pregnancy rate, the difference was primarily between

heifers with RTS of 1 and 2 compared to heifers with a RTS of 3, 4, and 5.









Implications

The 7-11 synchronization treatment resulted in more yearling heifers of Bos taurus x Bos

indicus breeding pregnant to the synchronized breeding compared to the Select

Synch/CIDR+timed-AI protocol; however, the synchronized pregnancy rates are less compared

to Bos taurus heifers. The role that RTS and BCS play as predictors of pubertal status in Bos

indicus x Bos taurus heifers when synchronizing remains unclear. However, considerations

must be made for timing of initiation of synchronization in heifers especially for Bos taurus x

Bos indicus heifers as they reach puberty at later ages.











Table 4-1. Physical description ofBos indicus x Bos taurus yearling heifers including
reproductive tract score (RTS), body condition score (BCS), and breed composition
for heifers synchronized with two progestogen based estrous synchronization
protocols (LSMeans SE).
Location n RTSa BCSb Breed composition'

1 61 3.8 + 0.14 5.5 + 0.05 Brangus (n=61)

Brangus(n=9)
2 119 3.6 0.10 5.0 +0.04 Brangus(n
Bos indicus crossbred (n= 110)

3 230 3.7 0.07 5.0 + 0.03 Bos indicus crossbred (n=230)

a Reproductive tract score: = non-cycling, 5 = estrous cycling.
b Body condition score: 1 = emaciated, 5 = moderate; 9 = very fat.
Brangus = 3/8 Brahman 5/8 Angus; Bos indicus crossbred approximately 1/4 to 1/2 Bos indicus
breeding with remainder being Bos taurus breeding.











7-11 treatment


PGF2a


GnRH


PGF2,


Select Synch/CIDR +timed-AI treatment

GnRH PGF2a


7 11 18 21
Day of experiment


Experimental design evaluating the effects of two progestogen based
synchronization treatments in yearling Bos indicus x Bos taurus heifers. a) 7-11
treatment was administered MGA (0.5 mg/hd/d) for 7 d with PGF2, (25 mg, i.m.)
on last day of MGA. GnRH (500 ig, i.m.) was administered on day 11 and PGF2a
was administered on day 18. b) Select Synch/CIDR+timed-AI treatment received
the same carrier supplement without MGA from day 0 to 7. On day 11 a CIDR
(Eazi-BreedTM CIDR) was inserted concomitant with GnRH. The CIDR was
removed on day 18 concurrent with PGF2,. For both treatments, estrus was
detected for 3 d and heifers were inseminated 6 to 12 h after a detected estrus.
Heifers not exhibiting estrus by 72 h were timed-AI and received GnRH between 72
to 76 h.


GnRH +
timed-AI


GnRH +
timed-AI


Figure 4-1.










Table 4-2. Effectiveness of the 7-11 treatment compared to Select Sync/CIDR+timed-AI
(SSC+TAI) synchronization treatment on estrous response, conception rate, timed-
AI pregnancy rate, synchronized pregnancy rate, and thirty day pregnancy rate in
yearling heifers of Bos indicus x Bos taurus breeding.a

Variable 7-11 SSC+TAI Odds Ratiog 95% CIh P-value

Estrous
resos %b 55.2 (117/212) 41.9 (83/198) 0.60 0.40-0.89 0.01
response, %

Conception
nceptn 47.0 (55/117) 31.3 (26/83) 0.51 0.29-0.93 0.03
rate, %

Timed-AI
pregnancy 16.8 (16/95) 20.0 (23/115) 1.23 0.61-2.5 0.56
rate, %d

Synchronized
pregnancy 33.5 (71/212) 24.8 (49/198) 0.65 0.43-1.00 0.05
rate, %e

Thirty day
pregnancy 65.7 (138/210) 60.4 (119/197) 0.80 0.53-1.19 0.27
rate, %f

a Refer to Figure 4-1 for details of treatments.
b Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
' Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers that failed to display estrus, were timed-AI, and became pregnant of the
total number of heifers that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.
fPercentage of heifers pregnant during the first 30 d of the breeding season of the total number
of heifers treated.
g 7-11 treatment used as referent value
h 95% confidence interval










Table 4-3. Effect of body condition score (BCS) on estrous response in Bos indicus x Bos
taurus yearling heifers.

BCSa Estrous response, %b Odds Ratio 95% CIc P-value

< 4.5 37.5 (30/80) 0.37 0.20-0.67 0.001

5 46.4 (104/224) 0.54 0.33-0.87 0.01

>5.5 62.3 (66/106) Referent

a Body condition score: 1 = emaciated, 5 = moderate; 9 = very fat.
b Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
c 95% confidence interval











100


90

80

70

60

50 .

40

30

20

10


0


<36


Interval fromPGF2ato onset of estrus, hours


Conception rates for the different interval from PGF2, to onset of estrus categories in
yearling heifers of Bos indicus x Bos taurus breeding. Means are expressed as a
percentage of total that exhibited estrus and became pregnant. Means without a
common superscript differ (P < 0.05). Interval from PGF2a to onset of estrus (P <
0.05) and synchronization treatment x interval from PGF2, to onset of estrus (P >
0.05).


61.7b



36.21
36.4a 31.6a



(22) (57) (47) (69)


* i a a i i U a i -


Figure 4-2.











Table 4-4. Effect of reproductive tract score (RTS) on estrous response, conception rate,
timed-AI pregnancy rate, synchronized pregnancy rate and thirty day pregnancy
rate in yearling heifers of Bos indicus x Bos taurus breeding.a

Variable RTSb Means Odds Ratio 95% CIc P-value


Estrous
response, %


<2 28.3 (15/53)

3 44.1 (56/127)

>4 56.1 (129/230)


0.30

0.60


0.16-0.58

0.39-0.94


0.0004

0.02


Referent


Conception
rate, %e


Timed-AI
pregnancy
rate, %f


Synchronized
pregnancy
rate, %g


Thirty day
pregnancy
rate, %h


S2 6.7 (1/15)


3 44.6 (25/56)

>4 42.6 (55/129)

<2 7.9 (3/38)

3 22.5 (16/71)

>4 19.8 (20/101)

< 2 7.6 (4/53)

3 32.3 (41/127)

>4 32.6 (75/230)

S2 32.1 (17/53)

3 59.1 (75/127)

>4 72.6 (167/230)


a Refer to Figure 4-1 for details of treatments.
b Reproductive tract score (Scale 1-5)
c 95% confidence interval
d Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
e Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
f Percentage of heifers that failed to display estrus, were timed-AI, and became pregnant of the
total number of heifers that were timed-AI.
g Percentage of heifers pregnant during the synchronized breeding of the total treated.
h Percentage of heifers pregnant during the first 30 d of the breeding season of the total number
of heifers treated.


0.07

1.15


0.10-0.60

0.59-2.20


0.01

0.68


Referent


0.34

1.22


0.09-1.24

0.57-2.58


0.10

0.61


Referent


0.17

0.97


0.06-0.48

0.61-1.55


Referent


0.001

0.91


0.22

0.49


0.07-0.68

0.24-1.0


Referent


0.008

0.06









CHAPTER 5
CONCLUSIONS AND IMPLICATIONS

Incorporation of estrous synchronization into an AI program decreases the amount of time

spent detecting estrus and can eliminate estrous detection when a fixed insemination time is

used. Additional benefits of using estrous synchronization includes promoting estrous cyclicity

in some non-cycling cattle, increasing the percentage of cattle pregnant early in the breeding

season, shortening the breeding season, shortening the subsequent calving season, and increasing

the uniformity of the calf crop. A majority of the research and development of estrous

synchronization protocols is based on the Bos taurus genotype. Furthermore, limited research

indicates that Bos indicus and Bos indicus x Bos taurus cattle have subtle physiological and

behavioral differences that can elicit different responses to estrous synchronization products

compared to Bos taurus cattle. Consequently, the primary objective of the research reported in

this thesis focused on evaluating the effectiveness of two estrous synchronization protocols in

cattle of Bos indicus x Bos taurus breeding.

In chapter 3, two experiments were conducted to evaluate the effectiveness of two

prostaglandins, cloprostenol sodium (cloprostenol) compared to dinoprost tromethamine

(dinoprost), when used in either the Select Synch/CIDR+timed-AI or the 7-10 synchronization

programs. In Experiment 1, suckled postpartum Bos indicus x Bos taurus cows were

synchronized with the protocols and yearling heifers of Bos indicus x Bos taurus breeding were

used in Experiment 2.

For both the suckled postpartum cows and the yearling heifers, prostaglandin treatment had

no effect on any of the response variables evaluated. This is similar to reports in cattle of Bos

indicus breeding as well as Bos taurus breeding. It was originally hypothesized that the

cloprostenol, which is a prostaglandin analog, may have been more effective compared to the









natural prostaglandin, dinoprost. However, this does not appear to be the case based on the

results of our experiment. Therefore, based on the limited number of animals tested in these

experiments as well as the reports in the literature, it appears that any subtle difference in

dynamics of CL regression between the two luteolytic drugs is not sufficient to alter either

estrous response or pregnancy rates. Hence, producers can use either type of prostaglandin

without any reduction in reproductive performance.

For the suckled cows in Experiment 1, estrous response, conception rate, and synchronized

pregnancy rate were affected (P < 0.05) by synchronization treatment as the Select

Synch/CIDR+timed-AI treatment had greater (P < 0.05) estrous response, conception rate, and

synchronized pregnancy rate compared to the 7-10 treatment.

Additionally, the synchronized pregnancy rates of both the 7-10 and Select Synch/CIDR

+timed-AI treatments are considerably less compared to reports in suckled Bos taurus cows

synchronized with either the 7-11 or Select Synch/CIDR+timed-AI protocols. The primary

reasons for the decreased response of the 7-10 compared to the Select Synch/CIDR+timed-AI

treatment as well as the decreased response of both treatments compared to similarly

synchronized suckled Bos taurus cows is due to a decrease in the estrous response, conception

rates, and timed-AI pregnancy rates. All of the aforementioned factors acted in concert and

resulted in less than acceptable synchronized pregnancy rates in the suckled Bos indicus x Bos

taurus cows. The first question that needs to be addressed is why the estrous response and

conception rates were significantly reduced in the 7-10 compared to the Select

Synch/CIDR+timed-AI treatment. One reason for the decreased estrous response for the 7-10

cows could be the pre-synchronization portion of the 7-10 treatment is not creating the same

synchrony of follicle development of the follicles being ovulated to GnRH compared to the









traditional 7-11 protocol, where MGA is used instead of a once-used CIDR. Since ovulation rate

to GnRH was not determined in the present study, it is not known how effective GnRH was in

ovulating follicles after removal of the once-used CIDR or if the timing of the GnRH treatment

relative to CIDR removal was appropriate. Type of progestogen treatment and concentration of

circulating progestogen can alter follicle growth and development, which could have also

resulted in asynchronous follicle development at GnRH. Therefore, if ovulation did not occur in

a majority of cows at GnRH in the 7-10 treatment, follicle development would have been

asynchronous at PGF2, resulting in a decreased estrous response compared to the Select Synch

CIDR+timed-AI treatment. Because cattle of Bos indicus influence have an increased incidence

of three and four wave follicle development patterns, additional research needs to be conducted

to determine if follicle wave patterns influence how cattle of Bos indicus breeding respond to

synchronization systems.

The second question that needs to be investigated is why cattle of Bos indicus influence

consistently have decreased estrous response to synchronization treatments compared to Bos

taurus cattle synchronized with similar protocols. One factor that had a significant effect on

estrous response in Experiment 1 was body condition score (BCS) of the cattle at the start of the

synchronization treatments. In summary, as BCS increased from < 4.5 to a > 5, there was a

significant increase in estrous response. Although estrous cycling status was not determined in

the present study, suckled cows with a BCS > 5 are typically cattle that are either close to or are

going through estrous cycles. Therefore, it is imperative for producers that want to use estrous

synchronization and AI that their cattle are in good BCS at the start of the breeding season in

order to get cattle to exhibit estrus and achieve acceptable pregnancy rates. Additionally, it is not

clear what effect environment has on estrous response, since there are a limited number of









studies that actually evaluate the effect of environment on response to synchronization protocols

in both cattle of Bos indicus and Bos taurus breeding under similar environmental conditions.

This would include investigating not only how environmental factors effect an animal's

physiology but also their behavior in response to environmental stressors.

Conception rates were also influenced by synchronization treatment as the 7-10 had

decreased conception rates compared to the Select Synch/CIDR+timed-AI treatment. It is not

clear why conception rates were decreased in the 7-10 treatment. Based on how the 7-10

treatment is designed, pretreatment with the once-used CIDR followed 3 d later by GnRH should

have worked to synchronize follicle development for the subsequent PGF2, treatment. The

newly developed follicles should have had an increased fertility compared to "aged" dominant

follicles or persistent dominant follicles, which could occur in some Select Synch/CIDR+timed-

AI treated cows. Additionally, the hormonal environment that follicles were exposed to during

the GnRH + PGF portion of the 7-10 treatment were certainly different compared to the Select

Synch/CIDR+timed-AI treatment. Whether progesterone from the CIDR affected the follicle

development pattern of the 7-10 treatment enough to decrease the subsequent conception rates is

unclear. Conception rates of the 7-10 treatment were considerably less compared to those

observed in suckled Bos taurus and Bos indicus x Bos taurus cows synchronized with a 7-11

protocol (Esterman et al., 2007b). It is important to note that acceptable conception rates can be

achieved in suckled Bos indicus x Bos taurus that exhibit estrus and inseminated as reflected by

the conception rates of the Select Synch/CIDR+timed-AI treatment, which are comparable to

reports in Bos taurus cows synchronized with the same protocol. As with estrous response, BCS

had a significant effect on conception rates, as cows with a BCS > 5, had approximately a 30%

greater conception rate compared to cows with a BCS < 4.5. Body condition score is an indirect









measure of nutritional status and has a direct influence on reproductive function and estrous

cycling status of suckled beef cows. Cows that receive enough pre-partum nutrition have an

excellent opportunity of returning to estrus within 60 d after calving and get pregnant to a

synchronized AI. Therefore, if producers want to achieve acceptable pregnancy rates to a

synchronized AI, they must make sure that their cattle are in adequate body condition at the start

of the synchronization treatments. However, producers will have to determine if the extra cost

required for the increased nutrition needed to keep cows in good body condition at the start of

the breeding season can be offset by the price obtained for the AI calves that will be produced by

the synchronized breeding.

Treatment had no effect on timed-AI pregnancy rates across the two synchronization

treatments. However, timed-AI pregnancy rates for both treatments are approximately 6 to 20%

less compared to reports for Bos taurus cattle synchronized with similar synchronization

protocols. As previously discussed with estrous response and conception rate, asynchrony of

follicle development at the time of PGF2a could have been part of the reason for the decreased

timed-AI pregnancy rate. It is possible that follicles were not of the correct size to ovulate to

GnRH, were infertile follicles when ovulating to GnRH, or cows were still anestrous and had

follicles of inadequate size to ovulate to GnRH. Timed-AI pregnancy rates across the two

treatments were also influenced by DPP at the start of the synchronization treatment. Timed-AI

pregnancy rate increased by 16% in cows' > 60 DPP compared to cows at or below 43 DPP. In

general, as the interval from calving to start of the breeding season increases, the number of cows

going through estrous cycles also increases. Therefore, some of the decrease in timed-AI

pregnancy rates could be attributed to increased incidence of anestrous compared to estrous

cycling cows. Another possible reason for the decreased timed-AI pregnancy rates could be that









the timing of the timed-AI + GnRH treatment is not synchronized correctly with the follicle

development after CIDR removal. A recent study in our lab with suckled Bos indicus x Bos

taurus cows synchronized with a Select Synch/CIDR protocol demonstrated that conception rate

peaked for cows that exhibited estrus at 60 h post PGF2, and conception rates decreased

significantly for cows that exhibited estrus after 60 h. Therefore, further research is needed to

evaluate if the timed-AI should be conducted at 60 h instead of 72 h after PGF2,.

Synchronization treatment had a significant effect on synchronized pregnancy rate as a

greater number of Select Synch/CIDR+timed-AI cows became pregnant to the synchronized

breeding compared to the 7-10 treatment. Additionally, the synchronized pregnancy rates for

both treatments were less compared to similarly synchronized Bos taurus cows. As already

discussed, a reduction in estrous response and conception rate of the 7-10 treatment were the

primary reasons for the decreased synchronized pregnancy rates for the 7-10 compared to the

Select Synch/CIDR+timed-AI treatment in the present experiment as well as the decreased

response compared to similarly synchronized Bos taurus cows. Nevertheless, acceptable

synchronized pregnancy rates can be achieved in suckled Bos indicus x Bos taurus cows

synchronized with the Select Synch/CIDR+timed-AI protocol if cows are of adequate BCS and

DPP at the start of the breeding season. The reasons) for the less than acceptable synchronized

pregnancy rates for the 7-10 treatment are not clear, and its practicality as a low cost

synchronization protocol is questionable. Future studies need to evaluate what effect the once-

used CIDR has on follicle development and if an asynchrony in follicle development after CIDR

removal effects the response to GnRH administered 3 d after CIDR removal. In conclusion, it

appears that the major factors contributing to the success of the synchronization treatments used

in the present study are BCS and DPP. Therefore, producers need to pay particular attention to









managing body condition prior to the start of the synchronization treatment and wait longer after

calving before cows are started on a synchronization treatment to take advantage of the DPP

effect. Future studies need to fully characterize the effectiveness of the two treatments ability to

induce estrous in anestrous suckled Bos indicus x Bos taurus cows, with an emphasis on the

Select Synch/CIDR+timed-AI protocol. Furthermore, intensive studies need to be conducted in

Bos indicus x Bos taurus and Bos taurus cows in similar environments to completely

characterize follicle wave patterns between the genotypes and to determine if they have any

effect on response to the Select Synch/CIDR+timed-AI protocol.

In Chapter 3, Experiment 2, yearling heifers over two consecutive years were synchronized

with the same estrous synchronization protocols as Experiment 1. The estrous response,

conception rate, timed-AI pregnancy rate, synchronized pregnancy rate, and thirty day pregnancy

rate were similar (P > 0.05) between the 7-10 and Select Synch/CIDR+timed-AI treatment and

between cloprostenol and dinoprost treatment. However, there was a treatment by interval from

PGF2a to the onset of estrus (P < 0.05; Figure 3-2) effect on conception rate. Conception rates

were similar (P > 0.05) between treatments for heifers that exhibited estrus 48 h after PGF2a.

However, the Select Synch/CIDR+timed-AI treatment tended (P = 0.10) to have a decreased

conception rate compared to the 7-10 heifers that exhibited estrus at 60 h after PGF2u. Whereas,

the Select Synch/CIDR+timed-AI treatment had an increased (P < 0.05) conception rate

compared to the 7-10 treatment for heifers that exhibited estrus 72 h after PGF2,.

In Experiment 2, there were no synchronization (7-10 vs. Select Synch/CIDR+timed-AI)

or PGF2a treatments (dinoprost vs. cloprostenol), affects on estrous response, conception rate,

timed-AI pregnancy rate, synchronized pregnancy rate or thirty day pregnancy rate for Bos

indicus x Bos taurus yearling heifers. This is in stark contrast to the results obtained for the









same synchronization treatments used on suckled cows in Experiment 1. Similar to results

observed for cows of Experiment 1, estrous response and timed-AI pregnancy rates were slightly

less in the Bos indicus x Bos taurus heifers compared to reports in Bos taurus and Bos indicus x

Bos taurus heifers synchronized with a 7 d CIDR with PGF2, provided the day before CIDR

removal. However, conception and synchronized pregnancy rates were similar compared to

what has been observed in estrous cycling Bos taurus heifers. Although, estrous cycling status

was not determined in the present study, the final results suggest that most of the heifers were

pubertal and responded favorably to either synchronization treatment and producers have the

option to use either synchronization system. There have been no studies evaluating the 7-11

protocol in yearling heifers of Bos indicus x Bos taurus breeding. However, heifers treated with

the 7-10 protocol had only slightly decreased conception and pregnancy rates compared to long

term MGA based protocols in Bos taurus heifers. As observed with the cows in Experiment 1, it

appears that a decreased estrous response is one of the limiting factors that decrease the overall

effectiveness of synchronization systems in heifers ofBos indicus x Bos taurus breeding

compared to yearling Bos taurus heifers. Additional studies need to be conducted in heifers of

Bos indicus x Bos taurus breeding to determine the relationship between follicle development

both during and after a synchronization treatment on the expression of estrus and subsequent

conception rates. Because there was a treatment by interval from PGF2, to onset of estrus effect

on conception rate, it may be beneficial to determine when the timed-AI should be performed

relative to the PGF2, treatment for both the 7-10 and Select Synch/CIDR+timed-AI treatments.

It will also be of interest to determine if estrous detection can be eliminated, and only a fixed

timed-AI can be used in yearling heifers of Bos indicus x Bos taurus breeding.









In Chapter 4, yearling Bos indicus x Bos taurus heifers were synchronized with either the

Select Synch/CIDR+timed-AI or 7-11 treatment. Heifers were distributed to treatment by

reproductive tract score (RTS) and BCS. The 7-11 heifers had greater (P < 0.05) estrous

response, conception rate, and synchronized pregnancy rate compared to Select

Synch/CIDR+timed-AI heifers. Heifers exhibiting estrus at 60 h had a greater (P < 0.05)

conception rate compared to heifers that had exhibited estrus at < 36, 48, and 72 h, which were

similar (P > 0.05) to each other. As RTS increased from < 2 to a > 3, estrous response,

conception rate, synchronized pregnancy rate, and thirty day pregnancy rate all increased (P <

0.05).

Estrous response was greater for 7-11 heifers compared to Select Synch/CIDR+timed-AI

heifers. The difference observed between treatments could be due to the 7-11 treatment

synchronizing follicular wave development more effectively compared to the Select

Synch/CIDR+timed-AI treatment. The MGA in combination with PGF2, allows most heifers to

have a large dominant follicle present at MGA withdrawal that is ready to be ovulated by GnRH;

thereby, synchronizing follicle development for the subsequent PGF2u, and thereby improving

the estrous response. The Select Synch/CIDR+timed-AI protocol is probably not as effectively

synchronizing follicle development since heifers are at all stages of follicle development at

GnRH and CIDR insertion, which probably results in asynchronous follicle development and a

less synchronous estrus. The estrous response for both treatments are considerably less

compared to studies in yearling Bos taurus and Bos indicus x Bos taurus heifers synchronized

with either the Select Synch/CIDR+timed-AI, short term progestogen protocols, and CIDR +

PGF2, protocol. The decreased estrous response is probably due to a combination of heifer

genotype and the numbers of heifers that are pubertal at the start of the synchronization









treatment, which was probably influenced by level of nutrition. Although, estrus is more

difficult to detect in cattle of Bos indicus breeding due to a decreased expression and duration of

estrus as well as an increased incidence of silent estrus, the use of estrous detection aids probably

made up for any deficiencies that occurred in estrous detection efficiency. Since blood samples

were not taken, it is not known how many heifers were pubertal at the start of the

synchronization treatment. Additionally, it is not known how effective the synchronization

treatments were in inducing estrous cycles in the pre-pubertal heifers. Body condition score, an

indirect indicator of nutritional status and estrous cycling status, had a significant effect on

estrous response as estrous response increased to acceptable levels in heifers that had a BCS >

5.5 at the start of the treatment. Consequently, the low number of estrous cycling heifers was

probably the primary reason for the decreased estrous response for the two synchronization

treatments in the present study.

There was a marked increase in conception rates for heifers synchronized with the 7-11

compared to the Select Synch/CIDR+timed-AI treatment. This suggest that the 7-11 treatment

may be doing a better job of synchronizing follicle development compared to the Select

Synch/CIDR+timed-AI treatment resulting in follicles that are more fertile. The duration of

follicle dominance is negatively associated with follicle fertility as follicles with prolonged

durations of dominance have significantly decreased fertility compared to follicles that are

ovulated with a normal duration of dominance. Therefore, it is possible that a greater number of

7-11 heifers had newly developed follicles present at CIDR removal compared to the Select

Synch/CIDR+timed-AI treatment, which resulted in greater fertility for the 7-11 treatment.

Additionally, conception rates for both treatments are considerably less compared to Bos taurus

heifers synchronized with variations of the CIDR + PGF protocols were the conception rates









ranged from 60 to 80%. For that reason, additional research needs to be conducted to fully

characterize the effects of prolonged follicle dominance on fertility in yearling heifers of Bos

indicus x Bos taurus breeding and to determine why there is such a significant reduction in

conception rates in yearling heifers of Bos indicus x Bos taurus compared to Bos taurus heifers.

Timed-AI pregnancy rates were similar between the 7-11 and Select Synch/CIDR+timed-AI

treatments but they were nearly half the values observed for Bos taurus heifers synchronized

with the Select Synch/CIDR+timed-AI protocol and yearling Bos indicus x Bos taurus heifers

synchronized with the 14 d MGA + PGF2, protocol. The primary reason for decreased timed-AI

pregnancy rates is probably due to the decreased number of heifers going through estrous cycles

at the start of the synchronization treatment resulting in a significant number of pre-pubertal

heifers at the timed-AI. The low timed-AI pregnancy rates also suggest that the synchronization

treatments did not induce estrous cycles in many of the pre-pubertal heifers. It should also be

noted that there was an effect of interval from PGF2u to the onset of estrus on conception rate and

heifers that exhibited estrus at 60 h had conception rates that were nearly twice heifers that

exhibited estrus < 36, 48 and 72 h. Therefore, when the timed-AI is performed needs to be re-

evaluated and it may be beneficial to conduct it at 60 h after PGF2, instead of 72 h.

The 7-11 treatment had greater synchronized pregnancy rates compared to the Select

Synch/CIDR+timed-AI treatment and the primary reasons were that the 7-11 treatment had a

greater estrous response and conception rate compared to the Select Synch/CIDR+timed-AI.

The 7-11 treatment was probably more effective at synchronizing follicle waves allowing a new

follicle to be ready to ovulate after PGF2z compared to the Select Synch/CIDR+timed-AI

treatment. With that said, synchronized pregnancy rates for both treatments were considerably

less compared to what has been observed in Bos taurus heifers synchronized with similar









synchronization protocols. The primary reason for the decreased synchronized pregnancy rates

is probably due to the small percentage of heifers that had reached puberty at the start of the

treatment. Therefore, additional experiments need to be conducted to determine how effective

the 7-11 and Select Synch/CIDR+timed-AI treatments are synchronizing follicle development as

well as how effective the treatments are for inducing estrous cycles in pre-pubertal heifers.

Utilization of the RTS at the start of the experiment was used to try and predict the estrous

cycling status of the heifers without having to work the heifers an additional time to take blood

sample 10 d before the start of the experiment. In general, as RTS increased from a 1 or 2 to > 3,

the estrous response, conception rate, and synchronized pregnancy rate increased. However,

what is interesting is that the reproductive performance did not increase significantly as RTS

increased from a 3 to > 4, with the exception of estrous response, although this increase was only

12%. One would have expected a more significant increase in estrous response and

synchronized pregnancy rate for heifers with an RTS of 4 and 5, which should be pubertal

heifers compared to prepubertal heifers with RTS 3. Utilization of RTS is an often used

management practice in yearling heifers of Bos taurus breeding to predict future reproductive

performance and to cull heifers (i.e., RTS 1 and 2) that will have substandard reproductive

performance. Consequently, it appears that we need to re-evaluate the manner in which RTS is

used to predict potential response to a synchronization treatment in cattle of Bos indicus x Bos

taurus breeding.

In summary, the 7-11 treatment provided increased estrous response, conception rates, and

synchronized pregnancy rates compared to the Select Synch/CIDR+timed-AI treatment. Estrous

response was directly affected by BCS at start of synchronization treatment, but interestingly

enough, BCS did not affect the overall reproductive performance of the heifers. Although RTS









had significant affects on estrous response, conception rate, and synchronized pregnancy rate,

using RTS to predict the potential response to a synchronization treatment was questionable.

Results from this experiment demonstrate the importance of having heifers either going through

estrous cycles or near the onset of puberty before the start of the breeding season in order to

achieve acceptable synchronized pregnancy rates. The synchronization treatments used in the

current study will need to be further tested in a population of heifers of known estrous cycling

status to determine the true response to treatment.

Relative to yearling heifers of Bos indicus x Bos taurus breeding, future research needs to

investigate the reasons for the less than acceptable estrous response and conception rates

observed in heifers synchronized with either the 7-11 or Select Synch/CIDR+timed-AI

treatments. This includes a complete characterization of the effectiveness of synchronization

treatments in peri-pubertal heifers as determined by blood progesterone concentrations. Because

nutrition can have a significant effect on reproductive performance, it will be also be important

to determine what effect nutritional status and different nutritional management strategies have

on conception rates in synchronized heifers. It will also be important to characterize the

utilization of RTS as a reproductive management tool and to determine its use as an indicator of

a heifer's breeding ability based on the heifer's weight and age at the start of the breeding

season. Environmental factors such as temperature, humidity, plane of nutrition, and animal

handling and management can all influence the endocrine, reproductive, and behavioral factors

associated with the expression of estrus as well as the overall treatment response. Future

research must focus on identifying the significance of these variables and determine which

one(s) can be controlled and (or) modified to enhance the onset of puberty and response to a

synchronization treatment.









APPENDIX A
SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 3

Table A-1. Effect of synchronization treatment and prostaglandin treatment on estrous
response, conception rates and pregnancy rates in suckled cows of Bos indicus x
Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Select
Synch/CIDR+timed-AI (SSC+TAI) treatment with either Cloprostenol sodium
(Cloprostenol) or Dinoprost tromethamine (Dinoprost).a
Timed-AI
Estrous Conception pregnancy rate, Synchronized
Variable response, %b rate, %" %d pregnancy rate, %e

7-10 49.0 (77/157) 45.5 (35/77) 17.5 (14/80) 31.2 (49/157)

SSC+TAI 59.9 (100/167) 62.0 (62/100) 20.9 (14/67) 45.5 (76/167)

P-value < 0.05 < 0.05 > 0.05 < 0.05




Cloprostenol 57.0 (94/165) 48.9 (46/94) 23.9 (17/71) 38.2 (63/165)

Dinoprost 52.2 (83/159) 61.5 (51/83) 14.5 (11/76) 39.0 (62/159)

P-value > 0.05 > 0.05 > 0.05 > 0.05

a See Figure 3-1 for details of treatments.
b Percentage of cows displaying estrus 72 h after PGF2a of the total treated.
c Percentage of cows pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of cows pregnant to timed-AI of the total that were timed-AI.
e Percentage of cows pregnant during the synchronized breeding of the total treated










Table A-2. Estrous, conception and pregnancy rates by prostaglandin treatment of suckled
cows ofBos indicus x Bos taurus breeding synchronized with either a modified 7-
11 (7-10) or Select Synch/CIDR+timed-AI (SSC+TAI) treatment with either
Cloprostenol sodium (Cloprostenol) or Dinoprost tromethamine (Dinoprost).a
Timed-AI
Estrous Conception pregnancy Synchronized
Variable n response, %b rate, % rate, %d pregnancy rate, %e
7-10
70 81 58.0 (47/81) 40.4 (19/47) 20.6 (7/34) 32.1 (26/81)
Cloprostenol
7-10
70 76 39.5 (30/76) 53.3 (16/30) 15.2 (7/46) 30.3 (23/76)
Dinoprost
SSC+TAI
Cr + 84 56.0 (47/84) 57.5 (27/47) 27.0 (10/37) 44.1 (37/84)
Cloprostenol
SSC+TAI
I 83 63.9 (53/83) 66.0 (35/53) 13.3 (4/30) 47.0 (39/83)
Dinoprost

P-value < 0.05 > 0.05 > 0.05 0.06

a See Figure 3-1 for details of treatments.
b Percentage of cows displaying estrus 72 h after PGF2a of the total treated.
C Percentage of cows pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of cows pregnant to timed-AI of the total that were timed-AI.
e Percentage of cows pregnant during the synchronized breeding of the total treated.









100


90
90 07-10
80
7 SSC
70 -


32.0


15.6


28.0


16.9


K


67.5



7 43.0


12 24 36 48 60 72

Interval from PGF2, to onset of estrus, hours


Figure A-1. Effect of 7-10 or Select Synch/CIDR+timed-AI (SSC) treatment on estrous response
in suckled postpartum Bos indicus xBos taurus cows. Means are expressed as a
percentage of total that exhibited estrus.










100


70
S70

I50


30


07-10
U SSC


69.2


59.4


16.7


(12)


65.1


641.0


46.2


(13)


(52)


IntervalfromPGF, to onset of estrus, hours


Figure A-2. Effect of 7-10 or Select Synch/CIDR+timed-AI (SSC) synchronization treatment
and interval from PGF2a to onset of estrus on conception rate in suckled postpartum
Bos indicus xBos taurus cows. Means are expressed as a percentage of total that
exhibited estrus and became pregnant.










Table A-3. Estrous response, conception and pregnancy rates by synchronization treatment of
yearling heifers of Bos indicus x Bos taurus breeding synchronized with either a
modified 7-11 (7-10) or Select Synch/CIDR+timed-AI (SSC+TAI) treatment with
either Cloprostenol sodium (Cloprostenol) or Dinoprost tromethamine (Dinoprost)
for years 1 and 2 Experiment2.a

Timed-AI
Estrous response, Conception rate, Synchronized
ob oc pregnancy rate
%b % d pregnancy rate, %
Variable %d

7-10 65.2 (116/178) 62.1 (72/116) 14.5 (9/62) 45.5 (81/178)

Year 1 71.6 65.4 9.68 49.5

Year 2 55.1 55.3 19.4 39.1

SSC +TAI 68.4 (121/177) 67.8 (82/121) 17.9 (10/56) 52.0 (92/177)

Year 1 64.2 71.4 20.5 53.2

Year 2 75.0 62.7 11.8 50.0




Sync p > 0.05 p > 0.05 p > 0.05 p > 0.05

Year p > 0.05 p > 0.05 p > 0.05 p > 0.05

Sync xYear p < 0.05 p > 0.05 p > 0.05 p > 0.05

a See Figure 3-1 for details of treatments.
b Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
' Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI of the total that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.










Table A-4. Estrous response, conception and pregnancy rates by prostaglandin treatment of
yearling heifers of Bos indicus x Bos taurus breeding synchronized with either a
modified 7-11 (7-10) or Select Synch/CIDR + timed-AI (SSC+TAI) treatment with
either Cloprostenol sodium (Cloprostenol) or Dinoprost tromethamine (Dinoprost)
for years 1 and 2 Experiment 2.a

Timed-AI
Estrous Conception rate, pregnancy rate, Synchronized
Variable response, %b %C %d pregnancy rate, %e

Cloprostenol 68.7 (123/179) 67.5 (83/123) 17.9 (10/56) 52.0 (93/179)

Year 1 70.6 71.4 18.8 56.0

Year 2 65.7 60.9 16.7 45.7

Dinoprost 64.8 (114/176) 62.3(71/114) 14.5 (9/62) 45.4 (80/176)

Year 1 65.1 64.8 13.2 46.8

Year 2 64.2 58.1 16.7 43.3




Prostaglandin p > 0.05 p > 0.05 p > 0.05 p > 0.05

Year p > 0.05 p > 0.05 p > 0.05 p > 0.05

Prostaglandin
SYear p > 0.05 p > 0.05 p > 0.05 p > 0.05
x Year

a See Figure 3-1 for details of treatments.
b Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
' Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI of the total that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.











Table A-5. Estrous response, conception and pregnancy rates of yearling heifers of Bos
indicus x Bos taurus breeding synchronized with either a modified 7-11 (7-10) or
Select Synch/CIDR + timed-AI (SSC+TAI) treatment with either Cloprostenol
sodium (Cloprostenol) or Dinoprost tromethamine (Dinoprost) for years 1 and 2
Experiment 2.

Timed-AI Synchronized
Estrous Conception rate, pregnancy rate, pregnancy rate,
Variable n response, %b %c %d %e


7-10
Cloprostenol


Year 1

Year 2


7-10
Dinoprost


Year 1

Year 2


90 71.1(64/90)


55 78.2

35 60.0


88 59.1 (52/88)


54 64.8

34 50.0


62.5 (40/64)


69.8

47.6


61.5 (32/52)


60.0

64.7


15.4 (4/26)


8.33

21.4


13.9 (5/36)


10.5

17.6


48.9 (44/90)


56.4

37.1


42.0 (37/88)


42.6

41.2


SSC+TAI
Cloprostenol


Year 1

Year 2


SSC+TAI
Dinoprost


89 66.3 (59/89)


54 63.0

35 71.4


88 70.5 (62/88)


72.9 (43/59)


73.5

72.0


20.0 (6/30)


25.0

10.0


62.9 (39/62)


15.4(4/26)


55.1 (49/89)


55.6

54.3


48.9 (43/88)


Year 1

Year 2

TRT

Year


TRT x Year


55 65.5

33 78.8


p > 0.05

p > 0.05

p > 0.05


69.4

53.8

p > 0.05

p > 0.05

p > 0.05


15.8

14.3

p > 0.05

p > 0.05

p > 0.05


a Figure 3-1 gives details of treatments.
b Percentage of heifers displaying estrus 72 h after PGF2. of the total treated.
' Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI of the total that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated


50.9

45.5

p > 0.05

p > 0.05

p > 0.05









APPENDIX B
SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 4


Table B-1. Description of the reproductive tract score (RTS) used in Chapter 4

Ovarian Measurement (mm)

Ovarian
RTS Uterine horns Length Height Width structures Description

S Immature <20mm 15 10 No palpable Infantile
1 15 10 8 Infantile
diameter No tone follicles
Prepubertal
20-25 mm diameter 8 mm Prepu
2 Notone18 12 10 follicles (more than 30 d
prepubertal)
Peripubertal
20-25 mm diameter 8-10 mm Peripubertal
3 22 15 10 (within 30 d of
Slight tone follicles t 3
puberty)
0 m d > 10 mm Cycling
30 mm diameter .
4 God tne 30 16 12 follicles CL (Follicular
Good tone
possible Phase)

5 > 30 mm diameter >32 20 15 CL present Cycling (Luteal
phase)
Adapted from Anderson et al., 1991.











100
90
80 7-11 m SSC
70 -
S60
's 50
S 39.0
40 27.4 31.7 23.2 31.9
30 24.8
W 20
10 2.7 3.5 5.3 4.9 4.4
0 0

0 12 24 36 48 60 72

Interval from PGF2a to onset of estrus, hours





Figure B-1. Effect of estrous synchronization treatment on estrous response in yearling heifers
of Bos indicus x Bos taurus breeding synchronized with 7-11 or Select
Synch/CIDR+timed-AI (SSC). Means are expresses as a percentage of total that
exhibited estrus.








100
90
80
a 70
60
' 50
o 40
0.30
u 20
0 10
S0


16.7


41.9




]


643


57.9


44.4
I 28.1


(37)


U(


<36 48 60 72
Internal from PGF,_ onset of estrus, hours



Figure B-2. Effect of estrous synchronization treatment on conception rate in yearling heifers of
Bos indicus x Bos taurus breeding synchronized with 7-11 or Select
Synch/CIDR+timed-AI (SSC). Means are expressed as a percentage of total that
exhibited estrus and became pregnant.










Table B-2 Estrous response, conception and pregnancy rates of yearling heifers of Bos indicus
x Bos taurus breeding synchronized with either a 7-11 or Select
Synch/CIDR+timed-AI treatment by location.a

Timed-AI Synchronized
Estrous Conception rate, pregnancy, rate, pregnancy rate,
Location response, %b %C %d %e

1 57.4 (35/61) 40.0 (14/35) 7.7 (2/26) 26.2 (16/61)

2 42.9 (51/119) 49.2 (25/51) 14.7 (10/68) 29.4 (35/119)

3 49.6 (114/230) 36.8 (42/114) 23.3 (27/116) 30.0 (69/230)

P-value > 0.05 > 0.05 >0.05 > 0.05

a See Figure 4-1 for details of treatments.
b Percentage of heifers displaying estrus 72 h after PGF2a of the total treated.
' Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI.
d Percentage of heifers pregnant to timed-AI of the total that were timed-AI.
e Percentage of heifers pregnant during the synchronized breeding of the total treated.









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BIOGRAPHICAL SKETCH

Erin Nicole McKinniss was born in 1984, in Athens, Ohio. Erin, the oldest daughter of

Michael and Lynn McKinniss, was raised in western Ohio on a small purebred Simmental farm

with her two younger brothers, Matthew and Stephen. She attended Northmont High School and

was active in the varsity softball team, marching and concert bands, the American Junior

Simmental Association, and 4-H. In 2002, she was honored with the title of Ohio Beef

Ambassador and she represented her state at the national contest. That year she traveled to the

Minnesota State Fair as a representative of the program to promote beef to consumers. In 2004

she was elected to the junior board of trustees of the American Junior Simmental Association,

where she represented the eastern region and was later elected to First Vice President of the

board. Erin earned a bachelor's degree in animal sciences from The Ohio State University,

during which time she was an active member of Sigma Alpha, Saddle and Sirloin, and the Ag

council. As part of the animal sciences department, she also was awarded the opportunity to

study abroad in Australia for a quarter learning about agriculture production. Upon graduation

from Ohio State in 2006, she began a Master of Science degree under Dr. Joel Yelich, at the

University of Florida, studying reproductive physiology. Under this appointment Erin held many

offices for the Animal Science Graduate Student Association and also taught an undergraduate

reproduction lab. Erin's future plans are to pursue her Ph.D. at the University of Florida, under

the tutelage of Dr. Joel Yelich and Dr. Matt Hersom.





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COMPARISON OF TWO SHORT-TERM PROGESTOGEN BASED ESTROUS SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS AND SUCKLED POSTPARTUM COWS OF Bos indicus Bos taurus BREEDING By ERIN N. MCKINNISS 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 2008 1

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2008 Erin N. McKinniss 2

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To my family and friends for always gi ving the support and encouragement I needed. 3

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ACKNOWLEDGMENTS I would like to offer my genuine appreciation to Dr. Joel V. Yelich, my committee advisor, for his counsel and guidance, for teaching and sharing his knowledge, an d for providing me the opportunity for an excellent graduate program. Appreciation is also extended to the members of my supervisory committee (Drs. William W. Thatcher and Owen Rae), for their important contributions to my work. Special thanks go to my lab mates (Brad Au stin, Regina Esterman, and Steavan Woodall) for putting in many long hours to create successful research in the field and lab as well as patiently passing on their skills. I would also like to extend tha nks to Bar-L Ranch of Marianna, FL, Dicks Farms of Lake City, FL, Roger Davi s of Davis Farms and Tommy Taylor of Little Creek Farms, Alachua, FL for providing facil ities and cattle used in this research. I would also like to thank my friends, w ho all at some point offered guidance and support, shared laughs and tears, and created memories that will last a lifetime. Without them this experience would have been unfulfilling. Th ey all have enriched my life and have allowed me to learn more about who I am. Last of all I would like to thank my fam ily for giving up a daughter and sister to the Gators and always welcoming me back home to The Buckeye State with open arms regardless of what colors I wear. The support and pride they provided allowed me to always keep my spirits high and to k eep my goals in sight. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................11 CHAPTER 1 INTRODUCTION................................................................................................................. .13 2 REVIEW OF LITERATURE.................................................................................................18 Assisted Reproduction in Beef Cattle .....................................................................................18 The Estrous Cycle ...................................................................................................................19 Hormonal Regulation: The Hypotha lamic-Pituitary-Ovarian Axis ........................................24 Follicular Development, Selection, and Ovulation .................................................................27 Corpus Luteum Function and Luteolysis ................................................................................32 Puberty ....................................................................................................................................37 Post Partum Interval and Return to Estrous ............................................................................41 Manipulation of the Estrous Cycle .........................................................................................42 Prostaglandin F2 .............................................................................................................43 Progestogens ...................................................................................................................46 Gonadotropin Releasing Hormone (GnRH) ...................................................................50 Synchronization Protocols ......................................................................................................51 3 COMPARISON OF TWO PROGESTOGEN BASED ESTROUS SYNCHRONIZATION PROTOCOLS AND CLOPROSTENOL SODIUM VS. DINOPROST TROMETHAMINE IN SUCK LED POST PARTUM COWS AND YEARLING HEIFERS OF BOS INDICUS BOS TAURUS BREEDING..........................58 Introduction .............................................................................................................................58 Materials and Methods ...........................................................................................................60 Experiment 1 ...................................................................................................................60 Experiment 2 ...................................................................................................................63 Results .....................................................................................................................................65 Experiment 1 ...................................................................................................................65 Experiment 2 ...................................................................................................................67 Discussion ...............................................................................................................................68 Implications ............................................................................................................................79 5

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4 EVALUATION OF TWO PROGESTOGEN BASED ESTROUS SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS OF BOS INDICUS BOS TAURUS BREEDING....................................................................................................88 Introduction .............................................................................................................................88 Materials and Methods ...........................................................................................................89 Results .....................................................................................................................................92 Discussion ...............................................................................................................................94 Implications ..........................................................................................................................103 CONCLUSIONS AND IMPLICATIONS ...................................................................................110 APPENDIX A SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 3.....................................123 B SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 4.....................................130 LIST OF REFERENCES .............................................................................................................134 BIOGRAPHICAL SKETCH .......................................................................................................157 6

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LIST OF TABLES Table page 3-1 Effect of synchronization treatment and body condition score (BCS) on estrous response in suckled Bos indicus Bos taurus cows. ........................................................81 3-2 Effect of synchronization treatment and body condition score (BCS) on conception rate in suckled Bos indicus Bos taurus cows.................................................................82 3-3 Effect of days postpartum (DPP) on timed-AI pregnancy rate in suckled Bos indicus Bos taurus cows.............................................................................................................83 3-4 Effect of synchronization treatment, body condition score (BCS), and days postpartum (DPP) on synchronized pregnancy rate in suckled Bos indicus Bos taurus cows........................................................................................................................84 3-5 Effect of body condition score (BCS) on thirty day pregnancy rates in suckled Bos indicus Bos taurus cows. ................................................................................................85 3-6 Effectiveness of 7-10 treatment co mpared to Select Sync/CIDR+timed-AI (SSC+TAI) treatment on estrous response, conception rate, timed-AI pregnancy rate, synchronized pregnancy rate, and thirty day pregnancy rate in yearling heifers of Bos indicus Bos taurus breeding. ..........................................................................................86 4-1 Physical description of Bos indicus Bos taurus yearling heifers including reproductive tract score (RTS), body conditi on score (BCS), and breed composition for heifers synchronized with two pr ogestogen based estr ous synchronization protocols (LSMeans SE). ..............................................................................................104 4-2 Effectiveness of the 711 treatment compared to Select Sync/CIDR+timed-AI (SSC+TAI) synchronization treatment on es trous response, conception rate, timedAI pregnancy rate, synchronized pregnancy rate and thirty day pregnancy rate in yearling heifers of Bos indicus Bos taurus breeding. ...................................................106 4-3 Effect of body condition score (BCS) on estrous response in Bos indicus Bos taurus yearling heifers. ....................................................................................................107 4-4 Effect of reproductive tract score (RTS ) on estrous response, conception rate, timedAI pregnancy rate, synchronized pregnanc y rate and thirty day pregnancy rate. ............109 A-1 Effect of synchronization treatment and prostaglandin treatment on estrous response, conception rates and pregnancy rates in suckled cows of Bos indicus Bos taurus breeding ............................................................................................................................123 A-2 Estrous, conception and pregnancy rates by prostaglandin treatment of suckled cows of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (710) or Select Synch/CIDR+timed-AI (SSC+T AI) treatment with either Cloprostenol sodium (Cloprostenol) or Dinopr ost tromenthamine (Dinoprost). ..................................124 7

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A-3 Estrous response, conception and pregna ncy rates by synchronization treatment of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Se lect Synch/CIDR+timed-AI (SSC+TAI) treatment with either Cloprostenol sodium (Cloprostenol ) or Dinoprost tromenthamine (Dinoprost) for years 1 and 2 Experiment2. ........................................................................................127 A-4 Estrous response, conception and pregna ncy rates by prostaglandin treatment of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Se lect Synch/CIDR + timed-AI (SSC+TAI) treatment with either Cloprostenol sodium (Cloprostenol ) or Dinoprost tromenthamine (Dinoprost) for years 1 and 2 Experiment 2. .......................................................................................128 A-5 Estrous response, conception and pregnancy rates of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Select Synch/CIDR + timed-AI (SSC+TAI) treatm ent with either Cloprostenol sodium (Cloprostenol) or Dinoprost tromenth amine (Dinoprost) for years 1 and 2 Experiment 2. ...................................................................................................................129 B-1 Description of the reproductive tr act score (RTS) used in Chapter 4. .............................130 B-2 Estrous response, conception and pregnancy rates of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a 7-11 or Select Synch/CIDR+timed-AI treatment by location .................................................................133 8

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LIST OF FIGURES Figure page 3-1 Experimental design evaluating the effects of two progestogen and two PGF2 treatments in Bos indicus Bos taurus suckled cows and yearling heifers for Experiments 1 and 2. a) 7-10: on day 0 received a once-used-CIDR that was removed on d 7 concomitant with dinoprost tromethamine (dinoprost; 25 mg i.m) followed by GnRH (100 g i.m.) on day 10. On day 17, females received either dinoprost or cloprostenol sodium (clopr ostenol; 500 g i.m). b) Select Synch/CIDR+timed-AI: on day 10 received a new CIDR (1.38 g) concomitant with GnRH. Day 17 CIDR was removed and fema les received either females received either dinoprost or cloprostenol. For all f our treatments estrus was detected for 3 d and females were inseminated 6 to 12 h after detected estrus. Females not exhibiting estrus by 72 h were timed-AI and received GnRH. ...........................................................80 3-2 Effect of interval from PGF2 to onset of estrus on conception rate in Bos indicus Bos taurus yearling heifers synchronized with 7-10 or Select Synch/CIDR+timed-AI (SSC+TAI) treatments. Numbers in parent hesis indicate the number of heifers in each category. Treatment by interval from PGF2 to onset of estrus ( P = 0.02). .............87 4-1 Experimental design evaluating the eff ects of two progestogen based synchronization treatments in yearling Bos indicus Bos taurus heifers. a.) 7-11 heifers were administered MGA (0.5 mg/hd/d) for 7 d with PGF2 (25 mg, im) on last day of MGA. GnRH (100 g, im) was administered on day 11 and PGF2 was administered on day 18. The Select Synch/CIDR+timed-AI heifers received the same carrier supplement without MGA from day 0 to 7. On day 11 a CIDR (Eazi-Breed CIDR) was inserted concomitant with GnRH. The CIDR was removed on day 18 concurrent with PGF2 For both treatments, estrus was detected for 3 d and heifers were inseminated 6 to 12 h after a detected estrus. Heifers not exhibiting estrus by 72 h were timed-AI and received GnRH between 72 to 76 h. .........................................105 4-2 Conception rates for the di fferent interval from PGF2 to onset of estrus categories in yearling heifers of Bos indicus Bos taurus breeding. Means are expressed as a percentage of total that ex hibited estrus and became pregnant. Means without a common superscript differ ( P < 0.05). Interval from PGF2 to onset of estrus ( P < 0.05) and synchronization treatment interval from PGF2 to onset of estrus ( P > 0.05). ................................................................................................................................108 A-1 Effect of 7-10 or Select Synch/CIDR+timed-AI (SSC) treatment on estrous response in suckled postpartum Bos indicus Bos taurus cows. Means are expressed as a percentage of total th at exhibited estrus. ............................................................................125 A-2 Effect of 7-10 or Select Sych/CIDR+ti med-AI (SSC) synchronization treatment and interval from PGF2 to onset of estrus on concep tion rate in suckled postpartum Bos indicus Bos taurus cows. Means are expressed as a percentage of total that exhibited estrus and became pregnant. ..............................................................................................126 9

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B-1 Effect of estrous synchronization treatment on estrous response in yearling heifers of Bos indicus Bos taurus breeding synchronized with 7-11 or Select Synch/CIDR+timed-AI (SSC). Means are e xpresses as a percentage of total that exhibited estrus. ...............................................................................................................131 B-2 Effect of estrous synchronization treatmen t on conception rate in yearling heifers of Bos indicus Bos taurus breeding synchronized with 7-11 or Select Synch/CIDR+timed-AI (SSC). Means are e xpressed as a percentage of total that exhibited estrus and became pregnant.............................................................................132 10

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Abstract of Thesis Presen ted to the Graduate School of the University of Floridain Partial Fulfillment of the Requirements for the Degree of Master of Science COMPARISON OF TWO SHORT-TERM PROGESTOGEN BASED ESTROUS SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS AND SUCKLED POSTPARTUM COWS OF Bos indicus Bos taurus BREEDING By Erin N. McKinniss August 2008 Chair: Joel V. Yelich Major: Animal Sciences A series of experiments were conducted with suckled cows and yearling heifers of Bos indicus Bos taurus breeding to compare two synchronization protocols including the Select Synch/CIDR+timed-AI (SSC+TAI) and a modified 7-11 protocol. In Experiment 1, suckled Bos indicus Bos taurus cows (n = 324) were used in a 2 2 factorial design comparing SSC+TAI to a modified 7-11 (7-1 0) protocol and within each synchronization treatment there were two prostaglandins (PG) treatments including cloprostenol sodium (CPG) and dinoprost tromethamine (DPG). Cows were equally dist ributed to treatments by body condition score (BCS) and days postpartum (DPP). On day 0, th e 7-10 treatment received an autoclaved onceused-CIDR that was removed on day 7 concomitant with DPG followed by GnRH on day 10. On day 17, 7-10 cows received either DPG or CPG. Also on day 10, SSC+TAI treatment received a new CIDR concomitant with GnRH with CIDR removal on day 17 where cows received either DPG or CPG. Estrus was visually detected twice daily (0700 and 1600 h) for 72 h after PGF 2 and cows were AI 6 to 12 h after a dete cted estrus. Non-responders were timed-AI + GnRH 72 to 76 h post PG. In experiment 2, yearling heifers over two breeding seasons (n = 218 and 137) were randomly assigned to the same tr eatments as Experiment 1, estrous detection 11

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and AI were administered in the same manner as Experiment 1. In experiment 1, estrous response (ER), conception (CR), and synchronized pregnancy rate (SPR) were affected ( P < 0.05) by synchronization treatment but not (P > 0.05) PG treatment. The SPR were 45.5% and 31.2 for the SSC+TAI and 7-10 treatments, respectively. As BCS increased from 4.5 to 5.5, ER, CR, SPR, and thirty day pre gnancy rates (TDPR) increased ( P < 0.05). In experiment 2, ER, CR, TAIPR, SPR, and TDPR were similar ( P > 0.05) between the synchronization and PG treatments, with the mean ER, CR, TA IPR, SPR and TDPR being 66.8, 65.0, 16.1, 48.7, and 76.3%, respectively. In Experiment 3, yearling heifers (n = 407) were synchronized with either the SSC +TAI treatment from Experiment 1 or 7-11 treatm ent that included melengestrol acetate (MGA; 0.5 mg/head/d) for 7 d with GnRH 4 d after the last day of MGA followed by DPG 7 d later. Estrus was detected and heifers were inseminated similar to Experiment 1. Heifers were distributed to treatment by reproductive tract scor e (RTS; Scale: 1=immature to 5= estrous cycling) and BCS. The 7-11 heifers had greater (P < 0.05) ER (55.2 vs. 41.9%), CR (47.0 vs. 31.3%), and SPR (33.5 vs. 24.8%) compared to SSC+TAI heifers, re spectively. Heifers exhibiting estrus at 60 h after PG (61.7%) had a greater ( P < 0.05) CR compared to heifers that had exhibited estrus at 36 (35.3%), 48 (31.6%), and 72 h (36 .2%), which were all similar (P > 0.05 ) to each other. As RTS increased from 2 to a 3, ER, CR, SPR, and TDPR all increased ( P < 0.05). In summary, there was a differential response to synchroniza tion treatments as co ws treated with the SSC+TAI had greater SPR compared to the 7-10 treatment; whereas, heifers synchronized with the 7-11 treatment had greater SPR compared to the SSC+TAI treatment. 12

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CHAPTER 1 INTRODUCTION Recent advances in reproductive technologies ha ve made a significant impact on the beef cattle herd on both a national and global scale. Mo st notably is the use of artificial insemination (AI) to expedite genetic gains a nd to decrease costs associated with housing and purchasing bulls of superior genetic quality. However, broad dissemination and utilization of AI in the United States beef cattle herd is limited, primarily due to the time and labor costs associated with detecting estrous during the 21 day estrous cycle. Incorporation of estr ous synchronization into an AI program decreases the amount of time need ed to detect estrus and (or) can eliminate estrous detection when a fixed breeding time is used. Additional benefits of using estrous synchronization includes promoting estrous cyclic ity in some non-cycling animals, increasing the percentage of cattle pregna nt early in the breeding seaso n, shortening the breeding and subsequent calving seasons, and increasing uniformity of the calf crop. A more uniform calf crop is important since calves are worth more at weaning. Numerous factors can influence the effec tiveness of estrous synchronization protocols including nutritional status, estrous cycling status, stage of the estrous cycle, days postpartum at the start of the synchronization protocol, and animal genotype. There are essentially three genotypes used in the beef cattle production systems in the United States including Bos taurus, Bos indicus, and Bos indicus Bos taurus. The majority of cattle in the Gulf Coast region of United States, including other tropical and subtropical clim ates of the world, are of Bos indicus Bos taurus breeding. Cattle with Bos indicus breeding are vital to efficient production in these environments because the cattle can withstand the hot and humid environment, utilize lower quality forages common in these environments, a nd are more resistant to parasites compared to cattle of Bos taurus breeding. With that said, the majority of the research and development of 13

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estrous synchronization pr otocols is based on the Bos taurus genotype. Furthermore, limited research indicates that Bos indicus and Bos indicus Bos taurus cattle have subtle physiological and behavioral differences th at elicit different responses to estrous synchronization pharmaceuticals and protocols compared to Bos taurus cattle. Consequently, the primary objective of the research reported in this thesis will focus on evaluating the effectiveness of two estrous synchronization pr otocols in cattle of Bos indicus Bos taurus breeding. There are several pharmaceuticals available to beef cattle producers that can be used in estrous synchronization protocols. Whether used alone or togeth er, the primary goal when using these products is to manipulate the estrous cycl e so that estrus and (or) ovulation is more predictable and uniform across th e group of cattle being synchronize d. There are three classes of pharmaceuticals that are primarily used in estrous synchronization protocols including prostaglandin F 2 (PGF 2 ), gonadotropin-releasing hormone (GnRH), and progestogens. The primary function of PGF 2 is to induce lysis of the corpus lu teum, which results in the expression of estrus in 3 to 5 d. In contrast, GnRH is used to manage follicle development by stimulating a pre-ovulatory surge of luteinizing hormone (LH) to initiate ovulation of growing follicles 10 mm in diameter and stimulation of follicle stimulating hormone (FSH ) which will assist follicular recruitment into the next follicular wave Treatment with GnRH can be used to either stimulate follicle turnover to initiate a new follicle wave or to synchronize ovulation in conjunction with AI. The final pharmaceutical that can be used are progestogens. The primary functions of progestogens are to prevent expr ession of estrus and ovulation by preventing the pre-ovulatory surge of LH. There are two progestogens available for use as estrous synchronization agents and they are melenge strol acetate (MGA) and the intra-vaginal progesterone insert known as the CIDR. Proge stogens can also have secondary effects by 14

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priming the hypothalamic-pituitary-ovarian-uterine axis, which can serve to initiate estrus in some anestrous cattle and possibly enhance pregnancy rates in synchronized cattle. One of the most common estrous synchronization protocols used by beef cattle producers is the Select Synch protoc ol. This protocol utilizes GnRH followed by a PGF 2 treatment 7 d later. Estrus is detected for 5 to 7 d post PGF 2 with AI occurring 6 to 12 h after an observed estrus. The down side to the protocol is that some cattle exhibit estrus 1 to 2 d prior to PGF 2 which increases the amount of estrous detection required. Subs equent research demonstrated that administration of a proge stogen between GnRH and PGF 2 eliminated the need for extra estrous detection before PGF 2 without compromising fertility. The progestogen used in the Select Synch protocol can be ei ther MGA or a CIDR, but the CIDR appears to be the preferred method for several reasons. First, addition of the CIDR does not increase labor requirements because the cattle are handled to administer the GnRH and PGF 2 Second, since MGA is administered in the feed, inadequate MGA consum ption can result in cattl e that break through and express estrus between the GnRH and PGF 2 treatments. And third, the CIDR may provide more progesterone like effects compared to MGA. The progesterone like effects include priming the hypothalamic-pituitary-ovarian-uterine axis, which could induce estrous cyclicity in some anestrous cattle and quite possibly enhance pr egnancy rates. Although, the research is not definitive on this issue, additional research is needed to validate whether the CIDR is more effective compared to MGA as a synchronization ag ent. When a CIDR is used with the Select Synch system it is called the Sele ct Synch/CIDR synchronization sy stem. Additional research in cattle of Bos taurus breeding demonstrated that three days of estrous detection and AI combined with a timed-AI plus GnRH 72 to 80 h after PGF 2 for cattle not exhibiting estrus resulted in similar and more consistent AI pr egnancy rates compared to 5 d of estrous detection and AI. The 15

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aforementioned synchronization protocol is called the Select Synch/CIDR+timed-AI protocol, which has been shown to be an effective protoc ol in both suckled cows and yearling heifers of Bos taurus breeding. In contrast limited research has been conduc ted on the effectiveness of the Select Synch/CIDR+timed-AI system in suck led postpartum cows and yearling heifers of Bos indicus Bos taurus breeding. Therefore, the first objective of my research was to evaluate the effectiveness of the Select Synch/CIDR+tim ed-AI synchronization protocol in suckled postpartum cows and yearling heifers of Bos indicus Bos taurus breeding. One of the problems with utilizing progest ogens, particularly long term progestogen treatments greater than 10 d, is a decrease in fe rtility of the estrus im mediately after progestogen removal. With short term ( 7 d) progestogen treatments there can be a slight decrease in fertility, but that decrease can be overcome by administering GnRH at the initiation of the progestogen treatment in the GnRH + PGF 2 protocols. Another way to utilize a progestogen and avoid the depression in fertility is to use the 7-11 estrous synchroniza tion protocol. The 7-11 protocol consists of 7 d of MGA with PGF 2 on the last day of MGA followed by GnRH 4 d later. This pre-synchronizati on portion of the 7-11 protocol is designed specifically to synchronize follicle development in a majority of cattle. Seven days after GnRH, another PGF 2 treatment is administered followed by 5 to 7 d of estrous detection. Research in suckled postpartum cows and yearling heifers of Bos taurus breeding has demonstrated that the synchrony of estrus was excellent and a combina tion of estrous detection and timed-AI could be incorporated into the 7-11 protocol resulting in acceptable synchronized pregnancy rates. However, no such research has been conducted with the 7-11 protocol in either suckled postpartum cows or yearling heifers of Bos indicus Bos taurus breeding. 16

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The cost of the 7-11 protocol is approxima tely $10.00/animal to implement compared to $15.00/animal for the Select Synch/CIDR+timed-AI pr otocol. The increased cost of the Select Synch/CIDR+timed-AI is primarily due to the cost of the CIDR, which are approximately $10 each. A cost efficient alternative to using a new CIDR is utilization of a once used CIDR in the Select Synch+timed-AI protocol. However, limite d research has evaluated this alternative and additional research is needed to determine if the once-used CIDR used in the Select Synch protocol results in similar responses compared to a new CIDR in both estrous cycling and anestrous cows. The once used CIDR could also be included in the 7-11 protocol as an alternative to MGA. This is particularly true in programs where MGA consumption is of concern or producers do not want to have to provide additional supplements to administer the MGA. Therefore, a second objective of the resear ch was to evaluate the effectiveness of the 711 estrous synchronization protocol in cattle of Bos indicus Bos taurus breeding and to evaluate the effectiveness of incorporating the once-used CIDR into the 7-11 estrous synchronization protocol. 17

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CHAPTER 2 REVIEW OF LITERATURE Assisted Reproduction in Beef Cattle One of the most important goals in beef cattle production is to improve the genetic quality of the cow herd from year to year in an effective and economic manner. Many recent improvements in biotechnology and beef cattle mana gement have made this more accessible to the average producer, namely development of artificial insemination (AI), and estrous synchronization protocols. Histor ically, reports of AI date back hundreds of years; however, it has only been used commercially in the later part of this century (Foote, 2002). Development of effective semen collection and storage methods, improved methods of delivery of frozen/thawed semen to females, development of enhanced estrous detection and estrous synchronization methods as well as a more complete understandi ng of the reproductive physiology of the male and female have all been a vital part in deve loping successful assisted reproduction programs. Artificial insemination allows producers an unlimited availability to superior sires without expending costs associated with purchasi ng and housing a bull. Utilization of AI also allows for rapid genetic improvement of the cowh erd by using specific genetic indicators such as expected progeny differences (EPD), which serve as indicators of the bulls ability to pass on his traits to his progeny. However, the costs associated with impl ementing an AI program including labor and supplies, frozen/thawed semen, and natural service bull costs have to be weighed with success of pregnancy and profits (Pace, 1985). Estrous synchronization used in conjunction with AI is a great way to capitalize on this technology. Estrous synchronization uses exogenous hormones to mani pulate the life span of the corpus luteum (CL) and (or) to synchronize fol licle development. S ynchronization of the CL and (or) follicle development, allows for a synchr onization of estrus in most females of the herd 18

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allowing for a shorter window for AI breeding. Treatment with an estrous synchronization protocol can also increase the number of female s pregnant early in the breeding season, which results in a shorter calving season and a more uniform calf crop at weaning. All of these variables drive profits and increase an operati ons production efficiency. With that said, additional costs are associated with implemen ting an estrous synchroni zation and AI program including increased labor, synchronization pharmaceuticals, frozen semen, and hiring an AI technician. Consequently, beef producers want estrous synchroni zation protocols that require minimal cattle handling and labor, co st effective, and consistently yield acceptable AI pregnancy rates. There are numerous factors that influenc e the effectiveness of an estrous synchronization protocols including but not limited to onset of pube rty, estrous cycling stat us, parity, lactation status, nutrition, environment, management tec hniques, and cattle genot ype. These factors can act either alone or in concert to influence the outcome to an estrous synchronization protocol. Therefore, the purpose of this literature review is to discuss and summari ze many of these factors as they relate to the implementation of es trous synchronization protocols in cattle of Bos indicus Bos taurus breeding The Estrous Cycle The bovine estrous cycle is an accretion of hormonal and physiological events which provides repeated opportunities for mating and quite possibly pregnancy. These reproductive events will begin at puberty and continue through adult life, only to be interrupted by pregnancy and the subsequent lactation. The estrous cycl e is delineated into two phases, follicular and luteal, which are recognized by the ovarian structures present. The follicular phase is defined as the period from regression of the CL to ovulation of a newly developed follicle. The follicular phase can be further separated in to proestrus and estrus. Estr adiol secreted from selected dominant follicles during the follicular phase will drive female receptivity, estrous behavior, and 19

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the surge of luteinizing hormone (LH) which i nduces ovulation. The long er of the two phases, the luteal phase, is defined as the period from ovulation until CL regre ssion. The luteal phase can also be further separated into two stages, me testrus and diestrus. The erupted follicle from the follicular phase eventually luteinizes and fo rms a CL, which secretes progesterone and begins the process of developing the uterus into an en vironment acceptable for pregnancy. If pregnancy does not occur, the uterus secretes prostaglandin F 2 (PGF 2 ) and luteolysis occurs and the female returns to estrus. The length of the estrous cycle in cattle of Bos taurus breeding is on average 21 d (Hansel et al., 1973). However, variations in estrous cycle le ngth exist between Bos indicus and Bos taurus type cattle, with Bos indicus breeds having longer estrous cy cles. Mattoni et al. (1988) reported a mean estrous cycle le ngth of 22.6 d in Zebu cattle and Plasse et al. (1970) reported 27.7 d in Brahman cattle. Gir cows have a mean estrous cycle length of 21.7 d (Moreira-Viana et al., 2000). Alternatively, Al varez et al. (2000) reported sim ilar estrous cycle lengths for Angus (19.5 d), Senepol (20.4 d), an d Brahman (19.7 d) cows. Vari ation in estrous cycle length has also been observed between heifers and cows of Bos indicus breeding, as Boran cows had a 23 d estrous cycle (Llewelyn et al., 1987), compared 27.7 d in to Brahman heifers (Plasse et al., 1970). Conversely, Bos taurus cows (Zollers et al., 1989) and he ifers (Mihm et al., 2000) exhibit minimal variation from the 21 d estrous cycle length. Estrus demarcates the beginning of the estrous cycl e. It is the most recognizable stage as it is diagnosed by behavioral characteristics where th e female is sexually receptive to the male. Estradiol concentrations peak during estrus, cau sing the behavioral expression of estrus and initiation of the preovulatory surge of LH (Wettemann et al., 1972; Short et al., 1973). Approximately 6 to 8 h after the peak estradiol concentrations, the preovulatory surge of LH 20

PAGE 21

occurs (Walters and Schallenberger, 1984) resu lting in ovulation 26 to 48 h after the beginning of behavioral estrus (Plasse et al., 1970; Looper et al., 1998). Circulating concentrations of estradiol have been reported to be less in Bos indicus compared to Bos taurus cows during day 7 to 17 of the estrous cycle (Seger son et al., 1984). Visual signs of estrus include but are not limited to the female standing to be mounted by another animal, vulvular mucous discharge, Flehmen response, restlessness, sniffing other co ws genitals, and chin resting (Van Eerdenburg et al., 2002). These signs can o ccur before, during, or after estrus and should be used as visual keys in diagnosing the best time for breeding, bu t should not necessarily be used to determine exact time of ovulation (Nebel 2003; Forster et al., 2006). The duration (length of estrus) and intensity of estrus (number of mounts received during the duration of estrus) can be influenced by several factors includi ng genotype, estradiol concentrations, social status, temperature and hu midity, and the number of animals in estrus at one time. Bos indicus breeds tend to have a shorter and less intense estrus (Plasse et al., 1970; Galina et al., 1982; Mattoni et al., 19 88; Pinheiro et al., 1998) compared to Bos taurus breeds (Stevenson et al., 1996; Richardson et al ., 2002). The duration of estrus in Bos taurus breeds ranges from 3 to 26 h with a mean of 14 h (Sch ams et al., 1977). Conversely, the duration of estrus in Bos indicus and Bos indicus Bos taurus breeds ranges from 1 to 24 h with a mean of 7 h (Mattoni et al., 1988; Rae et al., 1999). Landaeta-Hernandez (2002) also reported that the duration of a synchronized estrus tended to be shorter in Senepol compared to Angus or Brahman cows. However, breed had no effect on the duration and to tal number of mounts received during either a synchronized or spontaneous estrus; however, duration of estrus and total number of mounts received du ring estrus were greater for the synchronized compared to the spontaneous estrus. The variati on in duration and intensity of es trus within and between breed 21

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types may be attributed to circulating estradio l concentrations (Rhodes and Randal, 1978; Lyimo et al., 2000). Lyimo et al. (2000) reported that increased estradio l concentrations were indicative of increased behavioral estrus activity. Since circulating estr adiol concentrations have been reported to be greater in Bos taurus compared to Bos indicus cows (Segerson et al., 1984), it could explain why cattle of Bos indicus breeding have a decreased dur ation of estrus and estrous intensity. In contrast, Alvar ez (2000) reported similar estradio l concentrations between Angus and Brahman cows, but the authors did not report on any behavioral estrus data. Social status within herd can also influence behavioral estrus. Landaeta-Hern andez et al. (2004) reported that dominant and intermediate cows had a longer duration of estrus compared to subordinate cows but dominant cows tended to have less total mounts during estrus compared to subordinate and intermediate cows. The late r response suggests that dominan t cows probably avoided being mounted as frequently during estrus compared to subordinate cows. Increasing the number of cows in estrus at a single point in time increased mounting activity in both Bos taurus and Bos indicus females (Galina et al., 1994), which can lead to formation of sexually active groups and make it easi er to detect estrus. Hurnik (1975) reported that increasing the number of cows in estrus from 1 to 4 cows increased the number of mounts received from 11 to 49 in Bos taurus cattle. One of the problems w ith detecting estrus in cattle of Bos indicus breeding is the increased incidence of the silent es trus (Plasse et al., 1970; Dawuda et al., 1989; Lamothe-Zavaleta et al., 1991), high incidence of estrus expression at night (Pinheiro et al., 1998), increased secondary estrus activities othe r than mounting such as head butting (Orihuela et al., 1983), a nd decreased incidence of moun ting activity (Galina et al., 1982). Therefore, it is imperative for producers that want to AI cattle of Bos indicus breeding that the cattle be synchronized to increase the opp ortunity for the cattle to be detected in estrus. 22

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Numerous other factors influe nce behavioral estrus includ ing environmental conditions such as temperature and humidity (Landaeta-Hern andez et al., 2002), time of day (Pinheiro et al., 1998), parity (Flores et al., 2006) and frequency with which cat tle are handled through working facilities (Lemaster et al., 1999). It is unclear whether these variab les act alone or in concert to affect estrous behavior, but it de monstrates the multitude of fact ors that influence behavioral estrus. Ovulation typically occurs between 24 to 36 h after the onset of estrus in Bos taurus (Looper et al., 1998; Rori e et al., 1999) and Bos indicus cattle (Plasse et al ., 1970; Mattoni et al., 1988; Pinheiro et al., 1998). Estr us and ovulation are followed by me testrus, which lasts from 3 to 5 d and is characterized by formation of a functional CL and synthe sis and secretion of progesterone. Progesterone concentrations remain low until the CL is fully functional. During metestrus there is a transition from estradiol dominance to progesterone dominance and increased concentrations of circ ulating progesterone initiate the next stage, which is diestrus. Diestrus is the longest stage of the estrous cycle lasting 10 to 14 d and is characterized by increased progesterone concentrations that are gr eat enough to block the ac tivity of estradiol. Progesterone also primes the uterus to develop a suitable environment for a potential conceptus. If pregnancy occurs, the CL will be maintained throughout gestation and continue to synthesize and secrete progesterone. If pregnancy does not occur, the CL will remain functional until day 17 to 18 of the estrous cycle, at whic h time luteolysis is initiated by PGF 2 secreted from the uterus. At this point, degenera tion of the luteal tissue occurs and resumption of a new estrous cycle begins. The length of diestrus is directly related to life of the CL. Progesterone concentrations duri ng the luteal phase of the estr ous cycle are also different between Bos taurus and Bos indicus cattle. Adeyemo and Heath (1980) observed higher peak 23

PAGE 24

concentrations of progesterone during the luteal phase in Bos taurus heifers compared to Bos indicus heifers. Progesterone concentr ations were also greater in Bos indicus heifers 1 or 2 d before estrus. Progesterone con centrations increased from early to mid and late luteal phases, with the increase being more disc ernible in the Angus compared to Brahman cows (Segerson et al., 1984). Mean progesterone conc entrations were also greater in Angus compared to Brahman cows that were either pregnant or not pregnant (Segerson et al., 1984). Proestrus is the interval from luteolysis until the onset of estrus and is initiated 3 to 4 d before estrus. Proestrus is characterized by incr eased follicular growth an d is the transition from progesterone dominance to estrogen dominance (C henault et al., 1975; Kesner et al., 1982). During proestrus, a subordinate follicle is selected from a pool of follicles and becomes a dominant follicle, which will eventu ally be the ovulatory follicle. Hormonal Regulation: The Hypoth alamic-Pituitary-Ovarian Axis Estrous cyclicity and overall reproductive f unction in cattle is highly dependent upon the complex interactions of hypothalamic and p ituitary hormones and the hormone secreting structures of the reproductive tract. Release of gonadotropin releasi ng hormone (GnRH) from the hypothalamus mediates the secretion of th e gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), from the ante rior pituitary. Gonadotropins act on specific target tissues and (or) cells of the ovary that synthesize and secrete pr ogesterone and estrogen. Both positive and negative feedback effects induced by either estradiol or progesterone will further control synthesis and secretion of gonadot ropins from the hypothalamus and pituitary. Gonadotropin releasing hormone is a neuropeptide (10 amino acids) produced in two sets of paired nerve cell bodies in the hypothalamus. The GnRH is synthesized and secreted from the nerve cells located in the tonic and surge centers of the hypothalamus. Nerve axons extend from the hypothalamic nerve cell bodies to the capillary plexus at the ante rior pituitary where GnRH is 24

PAGE 25

released. This unique system is called the hypothalamic-hypophyseal portal system. The portal system is essential as it allows transfer of GnRH directly to the anterior pituitary instead of the general circulatory system wher e it would be quickly degraded. Basal secretion of GnRH is controlled by the ventromedial and arcuate nucle i or the tonic center of the hypothalamus and is characterized with low amplitude and high freque ncy LH pulses during the luteal phase of the estrous cycle when progesterone concentrations are increased. The tonic center continues to release basal concentrations of LH until the appr opriate positive stimulus are received, which is increased estrogen preceded by a rapid decline in progesterone. The suprachiasmatic and preoptic nuclei are part of the surge center whic h is responsible for the release of GnRH during the preovulatory phase of the estrous cycle. The surge center mediates high amplitude and low frequency GnRH pulses, which enhance the LH surge necessary for ovulation. Estradiol secreted from the dominant follicle precedes this event to enhance the surge of LH (Wettemann et al., 1972). Each GnRH pulse precedes each LH pulse (Schams et al., 1974). Luteinizing hormone, a glycoprotein, is the dominant luteotrophic hormone in the bovine. It is vital to CL function (Simmons and Hansel, 1964; Hoffmann et al., 1974) and is necessary for progesterone synthesis (Armstrong and Bl ack, 1966). Decreasing LH concentrations surrounding the LH surge actually inhibits progesterone secreti on and the vitality of CL development (Quintal-Franco et al., 1999). During the early luteal period, LH pulses are characterized by low amplitude and high freque ncy, whereas during the midluteal period LH pulses are characterized by high amplitude and lo w frequency (Rahe et al., 1980). Changes in circulating concentrations of estradiol and progest erone can effect LH secr etion. Ginther et al. (1996) reported that decreasing the circulating concentrations of progesterone increased LH pulse frequency and estradiol con centrations within 6 h. Devel opment of large and persistent 25

PAGE 26

dominant follicles occurs during low progesteron e treatment due to an increase in LH pulse frequency (Roberson et al., 1989; Savio et al., 1990). Although, LH pulses are necessary for follicle development beyond 9 mm (Gong et al., 1996) decreased LH pulse frequency can result in atresia of large follicles (Savio et al., 1990). The physiology of LH secretion is different between Bos indicus and Bos taurus cattle. Randel (1976) reported a longer interval from an exogenous estradiol trea tment to LH peak and decreased peak LH concentrations in Brahman compared to Hereford cows. Additionally, the interval from the LH surge to ovulation and interval from the initiation of estrus to ovulation was decreased in Brahman compared to Angus cows (Randel, 1976). Brahman cows also have a decreased responsiveness to estradiol and or gona dotropins (Rhodes et al., 1978). Portillo et al. (2006) reported that as the percentage of Bos indicus breeding increased, the amount of LH released in response to a GnRH challenge (500 g ) decreased when administered on day 6 of the estrous cycle. The other glycoprotein hormone synthesized and secreted from the anterior pituitary is FSH. The primary function of FSH is to promote follicular development and production of estradiol and an FSH surge precedes the initiat ion of each follicular wave during the estrous cycle (Adams et al., 1992). Folli cle stimulating hormone has rece ptors located on the granulosa cells of the growing follicle. Gibbons et al. (19 97) demonstrated that the number of follicles that advanced into a follicular wave was increased by increasing the FSH surge at an exaggerated amount. As FSH peaks, follicles approach 4 mm in diameter (Ginther et al., 1996) and FSH must be present in order for a follicle to grow past 9 mm (Gong et al., 1996). The final decline in FSH is part of the deviation mechanism or select ion of a dominant follicl e (Ginther et al., 1997). Growth of the dominant follicle further suppres ses FSH secretion (Ginther et al., 1997). 26

PAGE 27

The ovarian steroid hormones, estradiol and pr ogesterone, are also intr icately involved in the regulation of the estrous cycle. The ne gative effect of proge sterone on gonadotropin secretion decreases as circulating progesterone decreases, which allows for an increase in LH pulse frequency and a decrease in LH amplitude. This reduction occurs quic kly in the three days approaching estrus (Wettemann et al., 1972) and the change in LH secr etion allows for the dominant follicle to secrete in creasing amounts of estradiol (S challenberger et al., 1984). Estradiol increases GnRH production as well as Gn RH receptors in the anterior pituitary having a positive effect on LH secreti on (Vizcarra et al., 1997; Nett et al., 2002), which allows for decreased LH pulse frequency, increased LH pulse amplitude, which results in an LH surge and ovulation of the dominant follic le (Kesner et al., 1981). Follicular Development, Selection, and Ovulation Folliculogenesis is the process by which poten tial ovulatory follicles are formed from a pool of nave, or primordial follicles (Spicer and Echternkamp, 1986). Folliculogenesis begins prior to puberty and continues in cycles of development and deat h throughout the lif e span of the animal. Primordial germ cells mi grate to the gonadal ridge and are stored in primordial follicles, and form oogonia during mid gestation (Hirshfield et al., 1991; McGee and Hsueh, 2000). Lifetime supplies of oogonia within the ovary are established during gestation and the oogonia replicate by mitosis. The fetal ovary contains gr owing, preantral, and earl y antral follicles due to follicles leaving the resting pool while others are still being formed (Fortune, 2003). At birth, oogonia are suspended at the first meiotic divisi on and are deemed primary oocytes (Fortune, 1994). In ruminants, the oocytes are arrested at this stage until the LH surge initiates ovulation and meiosis resumes (Vanderhyden, 2002). Initia tion of follicle growth and its subsequent development are characterized by pr oliferation of granulosa cells, change in shape of granulosa cells, enlargement of the oocyte, and formation of the zona pellucida (BrawTal, 2002). There 27

PAGE 28

are three stages of follicle development based primarily on the layers of granulosa cells that comprise the follicle and they include primary, s econdary, and tertiary follicles. Primary oocytes are housed in primordial follicles and are characterized by a single layer of granulosa cells consisting of 5 to 8 flattened cells, and are ofte n referred to as quiescen t follicles (BrawTal, 2002). As the follicles leave the resting pool they become primary follicles and the granulosa cells multiply and become cuboidal and express ma rkers of cell proliferat ion (Fair et al., 1997; Fortune, 2003). Development continues to the se condary follicle stage with the addition of a second layer of granulosa cells, which progresses through the addition of 6 to 7 layers (Fortune, 2003). Additionally, the zona pellucida appear s and gap junctions are formed between the oocyte and granulosa cells (Fair et al., 1997a). Stromal cells are recruited to form the thecal layer and appear at the mid or late prean tral stage (Gougeon, 1996). Folliculogenesis is independent of gonadotropins (Roche, 2006) until th e formation of thecal cells at the secondary follicle stage (Scaramuzzi et al., 1993; Mihm et al., 2002). The secondary follicle transitions into a tertiary follicle with the development of an antral cavity. The granulosa cells differentiate into cumulus and mural cells, the latter of which are associated with steroidogenic functions (Vanderhyden, 2002). As the follicle continues to develop, the antrum continues to accumulate follicular fluid increasing the follicle size and is designated as a Graafian follicle. Follicular development is a cyclic process, and progresses through stages or waves of recruitment, selection, and dominance (Hodgen, 1982) The aforementioned stages are the basis for the wave-like phenome non of follicle development that oc curs during the estrous cycle in cattle. From an evolutionary standpoint, wave-l ike follicle development provides for an eligible ovulatory follicle to always be available (Sirois and Fortune, 1988). Cattle have between 1 to 5 follicular waves during one estrous cycle (Via na et al., 2000), although, two to three wave 28

PAGE 29

patterns are more commonly observed (Savio et al., 1988; Sirois and Fo rtune, 1988; Ginther et al., 1989). Recruitment is a process where a cohort of sm all follicles (2 to 4 mm diameter) begins to mature with support of sufficient pituitary gonadotropic stimulation (Hodgen, 1982; Adams et al., 1992a). Stimulation from FSH promotes the gr owth of the cohort of follicles resulting in development of 5 to 10 follicles (Lucy et al., 199 2) between 4 to 5 mm (Adams et al., 1992a). Selection is the process whereby a follicle is chosen, avoids atre sia, and has the potential to become an ovulatory follicle (Hodgen, 1982). Atresia is programm ed cell death and it is the most common fate of most follicles as only a few follicles reach dominance and ovulate. Greater than 99% of ovarian follicles undergo atresia duri ng the lifetime of the cow (Hsueh et al., 1994). Fortune (1994) reported that atresia most commonly occurred before the final stages of follicular dominance in cattle, but it can occur at any stag e of follicular growth. As FSH concentrations decline, fewer follicles continue to grow and are committed to atresia (Austin et al., 2001). Deviation, a part of selection, is when the second largest follic le decreases in size or stops growing in parallel with the dominant follicle, while the dominant follicle continues to grow (Ginther et al., 1996; Ginther et al., 1997). Dominance occurs when the selected follicle dominates through inhibition of recruitment of a new cohort of follicles (Hodgen, 1982). The dominant follicle has increased estradiol and inhibin production, the latter of which inhibits th e recruitment of follicles of the next follicular wave (Lucy et al., 1992; Ginther et al., 1999) and there is 3 to 4 d of additional growth of the dominant follicle. In a major ity of estrous cycles the first wave dominant follicle regresses (Savio et al., 1988; Sirois and Fortune, 1988; Gi nther et al., 1989a) and a second wave evolves through the recruitment and selection stages. The regression of the first-wave dominant follicle 29

PAGE 30

is initiated by elevated circulating progesterone concentrations that have a negative effect on LH secretion, which causes regression of the dominant follicle (Sunderland et al., 1994). In contrast, if luteolysis occurs, progeste rone concentrations decrease and estradiol production from the dominant follicle initiates an increase in LH secretion and eventually causes an LH surge resulting in ovulation (Wettemann et al., 1972). Maturation of th e second dominant follicle in two-wave cycles coincides with regression of the CL and this follicle ovulates after luteolysis (Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991). In three-wave follicle patterns the second wave dominant follicle becomes atretic and a third wave is recruited, selected, and becomes the dominant follicle that eventually ovulates. Follicular dynamics is defined as the continual growth and regression of antral follicles that lead to the development of the preovulatory follicles (Lucy et al., 1992). Savio et al. (1990) described follicular dynamics beginning early in the estrous cycle as a cohort of follicles is recruited out of a pool of smaller antral follicles (2 to 4 mm). After 2 to 4 d of recruitment, medium-sized follicles (6 to 9 mm) can be detected. After recruitment, selection begins for a single follicle and this selected follicle continues to grow a nd the other recruited follicles decrease in size. The dominant follicle (> 10 mm) of the first wave remains active until day 8 to 11 of the estrous cycle (Ginther et al., 1989a). The first wave dominant follicle prevents other follicles from reaching a diameter > 5 mm. The first wave dominant follicle will ovulate if luteolysis is initiated by day 5 to 8 of the estrous cycle (Kastelic et al ., 1990); however, in most cases the first wave dominant follicle regresses (Savio et al., 1988) and a second follicular wave begins around day 12 to 14 of the estrous cycle (Rajakoski, 1960). Follicular dynamics can be affected by breed and follicle number and size are most notably affected. The number of small follicles (2 to 5 mm) is greater in Brahman compared to Angus or Senepol cows (Alvarez et al., 30

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2000) and Brahman cows have more medium and total number of follicles compared to Angus cows (Simpson et al., 1994). Dominant follicl e size is also different between breeds, as Figueiredo et al. (1997) reported th at Nelore cattle had a smaller dominant follicle compared to Bos taurus counterparts. The number of follicular waves during the estr ous cycle can be influenced by many factors such as length of luteal dominance (Ginther et al., 1989b), parity and (or) age (Figueiredo et al., 1997), plane of nutrition (Rhodes et al., 1995; Mackey et al., 1999), la ctation status (Lucy et al., 1992), and breed (Savio et al., 1988; Ginther et al., 1989b; Rhodes et al., 1995; Viana et al., 2000; Martinez et al., 2003). In general, Bos indicus cattle have been observed in numerous studies to have mostly threeand four-wave fo llicle wave patterns (R hodes et al., 1995; Zeitoun et al., 1996; Viana et al., 2000; Mart inez et al., 2003) compared to Bos taurus cattle that have primarily twoand three-wave follicle wave pa tterns (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989b). Follicular dynamics in Gir cattle is charac terized by an increased incidence of estrous cycles with three or four follicle waves, asso ciated with a low persistence of the dominant follicle (Viana et al., 2000). However, breed has also been shown to have no effect on the number of follicular waves between Bos indicus and Bos taurus cattle (Figueiredo et al., 1997; Alvarez et al., 2000). The length of the estrous cycle can be influenced by the number of follicle waves that occur during the estrous cycle. The average length of the estrous cycle for cattle with two-wave follicle patterns is 20 d (Savio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989). Whereas, cattle with three-wave fo llicle patterns have estrous cycles that range from 21 to 23 d (Savio et al., 1988; Ginther et al., 1989; Viana et al., 2000). Taylor and Rajamahendran (1991) observed that cattle with three fo llicular waves had a longer estrous cycle due to a longer luteal 31

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phase. The reason(s) for the difference in numbe r of follicle wave patterns between breeds is unclear. One theory suggests it may be due to luteal dominance as Ginther et al. (1989) observed that luteolysis began later in three-wave estrou s cycles compared to two-wave estrous cycles. Another explanation could be the smaller diamet er of the intermediate wave dominant follicles compared to the diameter of ovulatory follicles, wh ich is associated with a shorter plateau phase that may contribute to greater number of wa ves observed in Zebu cattle (Viana et al., 2000). The number of follicular waves during the estrous cycle could also affect fertility of the subsequent estrus. Townson et al. (2002) reporte d that dairy cows had increased fertility after ovulation of a third wave dominant follicle co mpared to ovulation of a second wave dominant follicle. The reason for this could be that two wave patterns have a longer dominance of the ovulatory follicle, which is associated with decrease d fertility and integrity of the follicle. Cattle that ovulated a dominant follicle from the third wave had follicles that were smaller in diameter, which is indicative of young growing follicles. Sirois and Fortune, (1990) and Stock and Fortune, (1993) demonstrated th at ovulatory follicles with prolonged dominance, induced by exogenous progesterone in the abse nce of luteal progesterone, ha d significantly lower pregnancy rates compared to females that ovulated a norma l follicle. Austin et al. (1999) reported that restricting the duration of dominance of pre-ovulatory follicle to 4 d yielded pregnancy rates > 70% in beef heifers. Corpus Luteum Function and Luteolysis The preovulatory surge of gonadotropins responsible for ovulation also causes differentiation of residual follicula r cells that form the CL and init iates synthesis and secretion of progesterone (Niswender et al., 2000 ). Luteinizing hormone is th e major luteotropin responsible for the development of the CL and production of progesterone (Armst rong and Black, 1966) and 32

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LH pulses are needed for the development of a fully functional CL (M cCracken et al., 1999; Niswender et al., 1994) in cattle. Although, pul satile LH secretion is not needed for the maintenance of the CL after day 12 of the estrous cycle (Peters et al., 1994). Initial development of the CL takes approximately 3 d post ovulation in cattle. Fields and Fiel ds (1996) reported that the weight of the CL was 640 mg by day 3 of the estrous cycle and increased to 5.1 g by day 14 of the estrous cycle. This gr owth is primarily due to hypert rophy of the granulosa and thecal cells. The CL develops two distin ct populations of cells consisting of large and small luteal cells (Hansel et al., 1991), which differ morphologically and physiologically. Large luteal cells are derived from granulosa cells and small luteal cells from thecal cells (Niswender et al., 2000). Unlike small luteal cells, large luteal cells do not respond to LH because they do not express LH receptors (Mamluk et al., 1998). However, large lu teal cells are responsible for the majority of basal progesterone (Fields and Fi elds, 1996), and are the target of the luteolytic effects of PGF 2 during luteolysis (Pate, 1996). Sm all luteal cells are primarily responsible for higher magnitude, LH-stimulated progesterone producti on (Hansel et al., 1991). During ovulation, the follicle wall collapses and the basement membrane breaks down which gives rise to development of an extensive vascular network th at invades the antral space of the follicle. Angiogenesis or formation of new blood vessels plays a key role in formation and growth of the CL. The mature CL has a dense sy stem of blood vessels a nd has one of the highest rates of blood flow per unit of tissue compared to any other organ (Reynolds and Redmer, 1999). Luteal progesterone has multiple functions including regulating gonadotropin secretion, preventing ovulation, and mainte nance of pregnancy. Progester one acts on the hypothalamus to reduce GnRH pulse frequency and preventing th e LH surge (Evans et al., 1997). With a reduction in LH pulse frequency; th ere is an increase in pulse amp litude which is ideal for luteal 33

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function. Progesterone is also essential to the maintenan ce of pregnancy by preventing myometrial contractions to keep the uterus in a quiescent state. Progesterone also stimulates the endometrium to produce nutrients and other f actors vital for conceptus development (Geisert et al., 1992). Breed also appears to effect luteal function but the reports are conflicting. Castilho et al. (2000) and Moreira-Viana et al. (2000) reported that Bos indicus cattle had a decreased luteal tissue area compared to Bos taurus cattle. Rhodes et al. (1995) and Figueiredo et al. (1997) observed that the maxi mum CL diameter in Bos indicus heifers was 17 to 18 mm compared to 25 to 39 mm in Bos taurus heifers (Adams et al., 1993). Sim ilar findings have been reported by Castilho et al. (2000), Perea et al (1998), Ruiz-Cortes and Oliver a-Anger, (1999). In contrast, Alvarez et al. (2000) reported that the average an d maximal CL diameters of Brahman and Senepol cows were greater compared to Angus cows Breed differences have also been reported for progesterone concentrations. Segerson et al. (1984) observed greater progesterone concentrations in Angus compared to Brahman cows from d 7 to 17 of the estrous cycle. Randel et al. (1977) made similar observations, where Brahman and Brahman Hereford heifers had circulating progesterone concentratio ns that were less compared to Hereford heifers. In contrast, Alvarez et al. (2000) reported similar progest erone concentrations for Brahman, Angus, and Senepol cows before day 14 of the estrous cycle. Luteolysis includes the stru ctural and functional demise of the CL, which causes a decline of progesterone synthesis and secretion (McCracken et al ., 1999). Luteolysis allows a new reproductive cycle to begin and another opportunity for mating and a subsequent pregnancy. Elevated blood progesterone concentrations durin g most of the estrous cycle block the uterus from generating the luteolytic signal, which is PGF 2 (Gooding et al., 1972; Inskeep, 1973). The 34

PAGE 35

progesterone block is diminished on approximately day 16 of the estrous cycle and is due to the loss of progesterone receptors in the luminal a nd glandular epithelium of the uterus in cattle (Niswender et al., 2000), which allo ws the endometrium to produce PGF 2 Progesterone also promotes the accumulation of phospholipids in th e luminal and glandular epithelia of the endometrium, which serves as a substrate to generate arachidonic acid a precursor for PGF 2 synthesis (Niswender et al., 1994; Spencer, 2004). The neuroendocrine control of luteolysis has been thoroughly described in the model proposed by McCracken et al. (1999) Late in the luteal phase there is a down regulation of progesterone receptors in the hypothalamus a nd endometrium that causes a decrease in progesterone action. With the decrease in proge sterone dominance, there is a return to the dominance of estrogen action on these tissues. Estrogen acts to stimulate the hypothalamic oxytocin pulse generator to secrete oxytocin at high frequency, low amplitude pulses. This initiates small pulses of PGF 2 to be released from the endometrium, which is sufficient to initiate release of luteal oxytocin. Luteal release of oxytocin assi sts in amplifying the release of endometrial PGF 2 With the increased pulses and concentrations of PGF 2 they are high enough to inhibit progesterone secretion and cause the release secretion of luteal oxytocin, further reinforcing endometrial PGF 2 synthesis. This cascade of events continues until PGF 2 receptor response becomes desensitized and the CL is fully degraded and luteolysis is complete (Flint et al., 1990). Prostaglandin F 2 travels through a unique countercurrent mechanism which exists in the utero-ovarian pedicle of th e cow (Hixon and Hansel, 1974). In this system, PGF 2 travels from the uterine vein directly to the ovarian artery, thereby avoiding the systemic circulation, which prevents it from being metabolized systemically. Kawakami et al (1995) reported the 35

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concentration of PGF 2 in ovarian arterial blood increased to greater than 300 times the jugular venous blood within one minut e after the start of PGF 2 infusion into the uterine vein. Prostaglandin F 2 has a very short half life (Kindahl, 1980), and will be broken down quickly by enzymes found in pulmonary tissue. Many agents have been identified as mediators in structural and functional luteolysis of the CL. Evidence suggests that tumor necrosis factor(TNF) induced apoptosis plays a role in structural degradation in rumina nts (Sawyer et al., 1990; Juengel et al., 1993; Zheng et al., 1994). Ji et al. (1991) and Shaw and Br itt, (1995) demonstrated that PGF 2 elevates endogenous concentrations of luteal TNFand in vitro. Additionally, the endothelial cells express a TNFreceptor and are sensitive to TNFinduced apoptosis. Although, the exact mechanisms have not been fully identified for functional luteolysis, it is hypo thesized that PGF 2 is mediated by endothelial cell-derived vasoc onstrictive peptide, also known as endothelin-1 (ET-1), which alters normal patterns of progesterone s ynthesis (Milvae, 2000). Prostaglandin F 2 activates the ET-1 gene in luteal cells thereby stimulating its production. Secretion of ET-1 further stimulates production of PGF 2 from luteal cells, which acts back on the CL to further enhance ET-1 synthesis and secretion (Ohtani et al., 1998; McCracken et al., 1999; Milvae, 2000). Endothelin1 binds to specific receptors in both small and large luteal cells and activat es a decrease in basal and LH-induced production of progesterone (Milvae, 2000). This action could be responsible for the inability of PGF 2 to induce luteolysis in the early part of the estrous cycle since there is a lack of ET-1 and ET-1 only appears in larg e quantities in the CL just after PGF 2 exposure (Wright et al., 2001). A recent study by Friedman et al. (2000) outlined a positive link of PGF 2 induced ET-1 and TNFin functional and structural luteol ysis. At luteolysis, ET-1 secretion from luteal cells and TNFproduction by local macrophages up-regulate one anothers 36

PAGE 37

production and they work together to inhibit prog esterone production. The low concentrations of progesterone along with increased expression of the TNFreceptor facilitate TNFapoptosis of the endothelial cells in the CL. This leads to the functional and structural demise of the CL. Puberty Puberty can be defined as the first behavi oral estrus accompanied by the development of a CL that is maintained for a period characteristic of a particular specie s (Kinder et al., 1987). The process which culminates in puberty actua lly starts before birt h and continues throughout prepubertal development. Pubert y is a result of the maturation of the endocrine and reproductive systems of the young animal so it eventually functions similar to an adult. It is proposed that the primary mechanism regulating pubertal developmen t in heifers is the gonadostat theory, proposed by Ramirez and McCann (1963). The theory suggests that the pr epubertal increase in LH secretion is the result of a decline in the negative fee dback of estradiol on hypothalamic centers that control LH secreti on. Day et al. (1987) proposed th at the reason for the changes in LH secretion could be due to a decline in estr adiol receptors in the hypothalamus and pituitary, which results in a decline in the efficacy of es tradiol to exert negative feedback effects on LH secretion. The decline in estrad iol receptors permits an increase in pulsatile secr etion of LH. The peripubertal increase in mean LH may result fr om either a decreased ability of estradiol to inhibit GnRH secretion and (or) a decrease in negative feedback of estradiol at the pituitary, thereby increasing pituitary res ponsiveness to GnRH. Circulati ng estradiol concentrations will eventually reach concentrations su fficient to cause the pre-ovulator y surge of LH. Schams et al. (1981) reported an increase in LH pulse frequency and a decrease in LH pulse amplitude from 1 month of age until the onset of puberty. Day et al. (1984) also observed an increase in LH pulse frequency and an increase in mean LH c oncentration during the 126 d preceding puberty. 37

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Finally, induction of the pre-ovul atory LH surge by estradiol is an essential component for puberty to occur (Kinder et al., 1987). Age at puberty is influenced by many vari ables including genotype, age, nutrition, and environment. The data are conclusive that Bos indicus breeds reach puberty at older ages and heavier weights compared to Bos taurus cattle (Wiltbank et al., 1966; Pl asse et al., 1968; Laster et al., 1978; Gregory et al., 1979; Nelson et al., 1982; Dow et al., 1982). Nelsen et al. (1982) reported Brahman heifers were 428 d old and weighed 287 kg at the onset of puberty compared to Angus heifers that were 343 d old and weighe d 227 kg. Dow et al. (1982) observed that only 17% of Brahman Hereford heifers reached puberty by 15 mo of age compared to 92% of Hereford Red Poll heifers. Heterosis derived from crossbreeding Bos indicus with Bos taurus cattle decreases the age at puberty compared to the age at puberty in the straight bred Bos indicus heifers. Nutrition is a key factor that can dictate the onset of puberty in beef heifers. Early work by Wiltbank et al. (1969) and Short and Bellows (1 971) demonstrated that age-at-puberty was inversely related to growth rate. In short, as yearling heifer nu trition increased, the age at which heifers attained puberty decreased. With this information, the target weight principal was conceptualized by Lamond (1970). This princi pal dictated that heifers needed to be approximately 65% of their dams mature weight to attain puberty. Theref ore, a target weight could be estimated and heifers could be placed on a nutritional plan allowing for a specified rate of gain to attain their target weight at the start of the breeding season. Subsequent studies demonstrated that plane of nutrition was associated with LH secretion. Pre-pubertal heifers maintained on a low gain diet failed to exhibit an increase in LH pulse frequency compared to contemporaries fed a growth diet, which resulted in increased LH pulse frequency and decreased 38

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age at puberty (Day et al., 1986). Recent research by Gasser et al. (2006), demonstrated that precocious puberty can be induced in early w eaned heifers fed a high-concentrate diet by increasing LH pulse frequency. Treatment of heif ers with exogenous progestogens has also been documented to enhance the onset of puberty in some pre-pubertal heifer s (Short et al., 1976; Anderson et al., 1996; Imwalle et al., 1998). The mode of action a ppears to be associated with enhancing the pulsatile release of LH after progestogen with drawal allowing for increased follicular growth and eventually ovulation and development of a CL (Anderson et al., 1996). The age at which heifers attain puberty and get pregnant can aff ect the subsequent reproductive performance throughout a females lifetime. Heifers that have their first calf at 2 yr of age produce more calves during their lifetime compar ed to heifers that have their first calf at 3 yr of age or greater (Pope, 1967; Donaldson, 196 8, Chapman et al., 1978). Furthermore, heifers that calve early in their first calving season have a greater life time calf production compared to those that calve late (Lemeister et al., 1973). C onsequently, cows that cal ve late in the calving season tend to calve late or not at all in s ubsequent years (Burris and Priode, 1958). The Lemeister et al. (1973) study also demonstrated that calves born late in the calving season usually weigh less and are worth less economi cally, which tends to decrease the total productivity of the dam compared to calves born early in the calving season. In summary, it is important that heifers attain puberty before the st art of the breeding seaso n, get pregnant early in the breeding season, and calve earl y the next calving season in orde r to maintain a yearly calving interval and optimize a cows productivity th roughout her lifetime in the cowherd. Maturation of the endocrine system plays a key role in determining when the onset of puberty occurs in the yearling heifer. Yet, the remainder of the reproductive axis including the uterus and ovaries also pay a significant role in the onset of puberty. During the 50 d preceding 39

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puberty, uterine weights increased rapidly a nd major morphological changes in the ovary occurred (Day et al., 1987). The reproductive tract score (RTS), which uses rectal palpation to quantify uterine size and presence of ovarian st ructures, was developed in yearling heifers of Bos taurus breeding by Anderson et al. (1991) to predic t when and if puberty occurred in yearling heifers prior to the start of th e breeding season. The RTS was developed on a scale of 1 to 5. A RTS of 1 is characterized with uterine horns less than 20 mm in diameter and no palpable structures on the ovary and is considered an im mature tract. A RTS of 2 is characterized with uterine horns 20 to 25 mm in diameter with no to ne, follicles no greater than 8 mm in diameter, and is identified as being greater than 30 d fr om puberty. A RTS of 3 is characterized with uterine horns of 20 to 25 mm in diameter with slight tone, follicles that are 8 to 10 mm in diameter, and is identified as being within 30 d of puberty. A RTS of 4 has uterine horns of at least 30 mm in diameter with good tone, follicles th at are greater than 10 mm in diameter with a possible CL, and is identified as potentially es trous cycling. A RTS of 5 has uterine horns greater than 30 mm in diameter with good tone, a CL present, and is identified as estrous cycling. A common management practice used in yearling heifers of Bos taurus breeding is to conduct a RTS approximately 1 mo prior to the start of the breeding season. The RTS can be used to predict the response to an estrous synchronizatio n protocol, predict the potential reproductive performance during the breeding season, and (or) to make culling decisions. In general, as RTS increases from a 1 to a 5, both the response to a synchronization treatment increases (Patterson et al., 2000) and breeding season pregnancy rates in crease (Randle et al., 2000). For example, Randle et al. (2000) reported pre gnancy rates of 58% for heifers with a RTS of 1 compared to 89% for heifers with a RTS of 5. However, essentially no research has been conducted in yearling heifers of Bos indicus Bos taurus breeding utilizing the RTS as a management tool to 40

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either predict the reproductive pe rformance of yearling heifers or as a culling tool. Therefore, additional research needs to be conducted to determine if the RTS developed in Bos taurus heifers has the same potential to predict repr oductive performance in yearling heifers of Bos indicus Bos taurus breeding. Post Partum Interval and Return to Estrous There are numerous factors that can influen ce the return to estrus after calving including dystocia problems encountered during calving, days since calving, time of weaning, parity, suckling and lactational status, body condition at calving, and pre and postpartum nutrition. The interval from calving to first ovulation in suck led cows ranges from 35 to 100 d (Casida, 1971; Foote, 1974; Wettemann et al., 1986 ; Hoffman et al., 1996); whereas average intervals to first estrus ranges from 65 to 104 d (Graves et al ., 1968; Wiltbank, 1970; Casida, 1971). Young cows have a longer interval from calving to first es trus compared to older cows. For example, Wiltbank (1970) reported that the interval from calving to first estrus was 53.4 d in cows 5years-old, 60.2 d in 4-year-old cows, 66.8 d in 3-year-old cows, and 91.6 d in 2-year-old cows (Wiltbank, 1970). Body condition score (BCS) and nutri tional status at calving tends to be the most influential factor on the resumption of estr ous. Body condition can be used as in indirect indicator of nutritional st atus as it estimates the amount of fat that an animal contains and cows on an increased nutrient intake have greater BCS (Richards et al., 1986). Additionally, body energy reserves are related to reproductive f unction of postpartum cows (Dziuk and Bellows, 1983). Most studies suggests a minimum BCS of 5 at calving is needed to ensure adequate body stores so peak reproductive performance can be attained during the subsequent breeding season (Dziuk and Bellows, 1983; Richards et al., 1986). Cows calving at a BCS of 7 to 9 (Scale 1-9) were capable of returning to estrus within 60 d after calving. Dietar y restrictions of cows during the late pre-partum period resulted in weight loss and decrea sed body fat, which lead to a 41

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decrease in the number of cows returning to es trus early in the breed ing season (Whitman, 1975; Wettemann et al., 1982). Wiltbank et al. (1964) observed that cows fed the recommended level of TDN averaged 49 d from calving to first es trus as compared with 73 and 72 d for cows receiving 75% and 150% of the r ecommended level, respectively. There is also a relationship between nutrient intake and function of the endocrine system. Low-energy diets decrease mean LH concentrat ions in postpartum cows (Terqui et al., 1980) whereas, cows fed a diet deficien t in protein failed to have in creased LH pulse frequency as postpartum intervals increase (N olan et al., 1989). These studies support the idea that suppression or lack of LH pulsatility during th e postpartum period leads to longer periods of anestrous. Another factor that affects normal endocrine activity is the suckling action of the calf. Randel (1981) reported once-daily suckled Bos indicus Bos taurus heifers had a decreased postpartum interval by an average of 99 d compared to normal suckled first calf heifers. Mean interval from calving to estrus for normal su ckled heifers was 168.2 13.8 d, compared to 68.9 6.2 d for once-daily suckled heifers. Suckling rel eases opioids that have a negative effect on the hypothalamus, which function to decrease GnRH release (Malven et al., 1986). This negative effect on GnRH suppresses LH pulse frequency thereby, preventing an early return to estrus. Walters et al. (1982) noted that by removing cal ves from cows 3 wk after calving or later resulted in a rapid removal of the negative effects of suckling on the tonic release of LH secretion. Bellows et al. (1974) reported that early weaning calv es also decreased the postpartum interval in cows producing mu ltiple or single calves. Manipulation of the Estrous Cycle Traditional AI programs were focused on dete rmining when estrus occurred over a 21 d estrous cycle to determine when cattle were ready to be inseminated. This process was very time and labor consuming and limited whether or not producers could implement an AI program. 42

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Development and implementation of estrous sy nchronization protocols allowed for a large number of cattle to be in estrus during a short time period compar ed to the detection of estrus during the 21 d estrous cycle. For most estrous synchronization protocols behavioral estrus will be exhibited during a 3 to 7 d peri od, which allows for more effici ent use of AI. Some protocols have actually eliminated estrous detection and inseminate all cows at a predetermined timed known as timed-AI. Most synchronization protocols utilize exogenous hormones including PGF 2 GnRH, and progestogens either alone and (o r) in combination to manipulate the estrous cycle. The primary function of a synchronization protocol is to provide for a tightly synchronized estrus and or ovulation by manipulating follicle development and luteolysis. Prostaglandin F 2 It has been well establ ished that prostaglandinF 2 is the luteolytic signal in beef cattle (Gooding et al., 1972; Inskeep, 1973) th at originates from the uterus and initiates the demise of the CL. Rowson et al. (1972) and Lauderdale et al. (1974) demonstrat ed that an exogenous administration of PGF 2 during the luteal phase of the estrous cycle causes luteolysis of the CL without compromising fertility of the subsequent estrus (Lauderdal e et al., 1974). Consequently, PGF 2 became the first pharmaceutical used for the purpose of estrous synchronization. Subsequent research by Watts and Fuquay (1985) demonstrated that stage of the estrous cycle at administration of PGF 2 had an effect on estrous re sponse, interval from PGF 2 to the onset of estrus, and conception rate. In this study, heifers in the early stages of the estrous cycle (day 5 to 7) had an estrous respons e of 43% and an interval from PGF 2 to the onset of estrus 59 h. In contrast, heifers in the middle (day 8 to 11) and later (day 12 to 15) stages of the estrous cycle had a greater estrous res ponse (84 and 100%; resp ectively) but longer intervals from PGF 2 to the onset of estrous (71 and 72 h; respectively) compared to heifers in the early stages of the estrous cycle. First service conc eption rate also increased as th e estrous cycle progressed. The 43

PAGE 44

interval from PGF 2 to the onset of estrus was longer for he ifers in the mid to late stages of the estrous cycle, which is due to status of the fo llicular waves and size of the dominant follicle at PGF 2 (Sirois and Fortune, 1988). During the early stages of the estrous cycle, a dominant follicle is growing and ready to ovulate if luteolys is is induced compared to mid and late stages of the estrous cycle where follicles could be in the early stages of development and require additional time to reach ovulat ory capacity and size. A single dose of PGF 2 initiates luteolysis in 85 to 100% of Bos taurus females (King et al., 1982; Tanabe and Hann, 1984; Kiracofe et al., 1985) resulting in estr ous responses between 75 and 95% (Tanabe and Hann, 1984; Watts and Fuquay, 1985). However, there has been limited research conducted to determine if geno type influences the effectiveness of PGF 2 to initiate luteolysis. Pinheiro et al. (1998) demonstrated that Nelore cattle treated with two injections of PGF 2 11 d apart had a 33.3% estrous res ponse in heifers and 46.4% estrous response in cows. Orihuela et al. (1983) reported estr ous responses of 56 and 62% in Zebu cows following PGF 2 treatment. It should be noted that in the preceding studies that estrous response was used as an indirect indicat or of luteolysis and no blood samp les were collected to evaluate blood progesterone concentrations to confirm if luteolysis occurred. Because estrus is difficult to detect in Bos indicus cattle and there is an increased incidence of silent estrus in Bos indicus type cattle (Plasse et al., 1970), caution should be taken when using only estrous response when evaluating effectiveness of PGF 2 to initiate luteolysis. It has been hypothesized that the decreased estrous response in Bos indicus cattle was due to decrease d effectiveness of the PGF 2 to initiate complete regression of the CL (Pinheiro et al., 1998; Rekwot et al., 1999) resulting in rebounding of progesterone production by the CL that block the expression of estrus. Recent research by Bridges et al. ( 2005) supported this hypothesis by demonstrating that a PGF 2 44

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induced luteolysis was decrea sed in yearling heifers of Bos indicus Bos taurus breeding compared to yearling heifers of Bos taurus breeding. However, in that same study, they also demonstrated that there may be an age effect because luteolysis was similar between two-year old virgin heifers of Bos indicus Bos taurus breeding compared to two-year old virgin heifers of Bos taurus breeding. To combat the inability of PGF 2 to initiate luteolysis in a majority of Bos indicus cattle, researchers added a second treatment of PGF 2 24 h after the first on e (Cornwell et al., 1985; Santos et al., 1988). Admini stration of the second PGF 2 treatment initiated complete luteolysis and prevented the CL from continuing to s ecrete progesterone. Bridges et al. (2005) administered two consecutive PGF 2 treatments 24 h apart to yearling heifers in the later stages of the estrous cycle and obser ved increased estrous response, timed-AI pregnancy rate, and synchronized pregnancy rate compar ed to heifers that received a single dose. Portillo et al. (2007) administered two half treatments of PGF 2 (12.5 mg) 24 h apart to mature suckled Bos indicus Bos taurus cows and observed a significantly e nhanced luteolysis, although, estrous response and synchronized pregnancy rates were similar compared to cows that received a single dose of PGF 2 There are two common forms of prostaglandins available for commercial use. The trade names of Lutalyse Sterile Solution, Prostamate and In-Synch are from the chemical class dinoprost tromethamine, a naturally occurring prosta glandin, which is admini stered at a rate of 25 mg (5 cc) per dose. Estrumate and Estroplan are a synthetic analogous of PGF 2 made from cloprostenol sodium. These analogs are slightly more potent and ar e administered at a rate of 500 g (2 cc). Both classes of prostaglandins provide for acceptable preg nancy rates when used in synchronization systems (Johnson, 1978; Se guin et al., 1984; Jackson et al., 1979). 45

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Comparisons of both classes of prostaglandins have been made in Bos taurus animals (Young and Anderson, 1986; Salverson et al., 2002). Sa lverson et al. (2002) observed similar results when cloprostenol sodium was compar ed to dinoprost tromethamine in Bos taurus heifers synchronized with the long term MGA + PGF 2 protocol. Evaluation of the effectiveness of the two classes of prostagl andins in cattle of Bos indicus breeding has not been completely characterized in either suckled cows or y earling heifers. In non-lactating cows of Bos indicus breeding synchronized with a GnRH + PGF 2 based protocol, Heirs et al. (2003) reported a numerical increase in synchronized pregnancy rates of 5% in cl oprostenol sodium treated cows compared to dinoprost tromethamine treated co ws. In a recent study in suckled postpartum Bos indicus Bos taurus cows synchronized with GnRH + PGF 2 based protocol concomitant with a CIDR, estrous response, conception rate, and synchronized pregnancy rates were similar between cows that received eith er cloprostenol sodium or dinoprost tromethamine (Esterman et al., 2007). There has been no rese arch evaluating the effectiveness of cloprostenol sodium and dinoprost tromethamine in GnRH + PGF 2 based protocol concomitant with a CIDR in yearling heifers of to Bos indicus Bos taurus breeding. Progestogens The primary function of a progestogen is to prevent the expression of estrus by mimicking the actions of the CL. Progestogens act to prevent the ovulatory surge of LH and thereby prevent ovulation from occurring (Patterson et al., 1989). An additional benefit of using progestogens is that they can ini tiate estrous cycles in some anestrous cows (Miksch et al., 1978; Smith et al., 1987; Fike et al., 1997; Yelich et al., 1997; Yavas and Walton, 2000) and prepubertal heifers (Anderson et al., 1996; Hall et al., 1997). Administration of a progestogen in anestrous cows and pre-pubertal heifers stim ulates LH secretion both during and after progestogen treatment, which accelerates follicle growth resulting in ovulation after progestogen 46

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withdrawal (Garcia-Winder et al., 1987; Anderson et al., 1996). There are two major classes of progestogens including natural progesterone an d synthetic analogues in cluding norgestomet and melengestrol acetate (MGA). Progestogens can be administration via injection, implant, intravaginally, or orally. The two most comm on progestogens used in estrous synchronization protocols include the intravaginal progesterone insert or CIDR a nd MGA, which is orally active and administered in the feed. The CIDR is an intravaginal insert made with a progesterone impregnated silicone coating. When inserted into the vagina, the mois ture acts to move the progesterone out of the silicone coating. In the United States, the CI DR contains 1.38 g of progesterone, and it is estimated that 0.7 g of progesterone is used in its initial use (Savio et al., 1993), which is typically 7 d. Most estrous synchronization protocols leave the CIDR in the vagina for 7 d with administration of PGF 2 either one day before (Lucy et al., 2001) or at CIDR removal (Martinez et al., 2000; Stevenson et al., 2004; Larsen et al., 2006). The primary function of the CIDR is to improve estrous synchrony co mpared to a single PGF 2 treatment or unsynchronized cattle. An additional benefit of the CIDR is that it can ini tiate estrus in anestrous cows and peripubertal heifers. Lucy et al. (2001) re ported that a 7 d CIDR induced estr us in approximately 40% of the anestrous cows and 40% of the prepubertal heifers. Fike et al (1997) reported that 70% of anestrous cows were initiated into estrus or had formation of a CL after a 7 d CIDR treatment. Since there is approximately 0.7 g (Rathbone et al., 2002) progesterone left in the CIDR after a single use (Savio et al., 1993), there ha s been growing interest among producers to use a once-used CIDR. Beal et al. (2003) reported that utilization of a once-used CIDR for 7 d effectively suppressed estrus in beef females. Furthermore, autoclaving the once-used CIDR before re-insertion results in gr eater serum progesterone concentrations compared to a once-used 47

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CIDR that was only cleaned and not autocl aved (Zulagea and Williams, 2006). Moreover, Colazo et al. (2004) reported si milar pregnancy rates after a fi xed timed-AI in heifers that received either a new or once used CIDR. Ther efore, the potential to use a once-used CIDR exists, but research in this area is limited relati ve to the effectiveness of a once-used CIDR to synchronize estrus as well as the ability to induce es trus in anestrous cattle. Melengestrol acetate is capa ble of promoting endometrial proliferation, maintenance of pregnancy, and preventing the expression of es trus (Duncan et al., 1964; Zimbelman and Smith, 1966; Prichard et al., 1969; Schul et al., 1970). One of the advantag es of using MGA is that it can be administered in the feed. The minimal effective dose needed to maintain pregnancy (4 mg/d) is much greater compared to that need ed to prevent estrus and ovulation (0.5 mg/d) (Zimbelman, 1963b, Zimbelman and Smith, 1966). Early research demonstrated that administering MGA for more than 9 d lead to de creased fertility of the subsequent estrus and ovulation compared to untreated co ntrols (Hill et al., 1971). Patterson et al. (1989) and Beal et al. (1988) further demonstrated th at administration of MGA for 7 d or 9 d; respectively, also resulted in decreased fertility in some animals depending on what day of the estrous cycle they started the MGA treatment. Heifers which bega n MGA treatment after day 12 of the estrous cycle had decreased fertility compared to heifer s where MGA was initiated prior to day 12 of the estrous cycle. The reduction in fert ility is temporary, as fertility at the subsequent estrus is not affected (Zimbelman et al., 1970). The decrease in fertility afte r a long term MGA treatment is caused by the development of what is termed a persistent dominant follicle (Guthrie et al., 1970). A persistent dominant follicle is a follicle that has a longer lifesp an, increased maximal diameter, and increased estradiol secretion compared to normal ovulator y follicles (Sirois and Fo rtune, 1990; Savio et al., 48

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1993b). Development of the persistent dominan t follicle typically oc curs with long-term progestin treatments (9 to 14 d) in the absen ce of a CL, which results in increased LH pulse frequency and decreased pulse amplitude (Kojima et al., 1992). The altered LH secretory profile results in continued follicle gr owth and development due to the absence of luteal progesterone that would normally initiate follicle turnover. After MGA withdrawal, estrus occurs within 3 to 7 d. Initial research indicated th at the decreased fertility at the estrus after MGA withdrawal was due to increased estrogen concentrations that al tered the uterine environment (Wordinger et al., 1972; Gibbons et al., 1973) and did not allow for conditions conducive to fertilization and (or) proper embryo development. However, recent re search has demonstrated that fertilization actually occurs in the majority of cattle and the reduction is fertility is due to decreased embryo viability (Ahmad et al., 2005) and deceased oocyte competence (Stock and Fortune, 1993; Savio et al., 1993; Mihm et al., 1994a). Additionally, Mi hm et al. (1992) reported that progesterone concentrations did not vary between ovulation of a normal follicle compared to ovulation of a persistent dominant follicle indicating that lute al function was not altered and probably does not contribute to the decrease in fertility. There can also be a decreases in fertility associated with short term ( 9 d; Beal et al., 1988); Patterson et al., 1989) progest ogen treatments. The decrease in fertility with a short term progestogen treatment is due to the duration of dominance of a follicle. Austin et al. (1999) reported that heifers which ovulated follicles be tween 2 and 8 d of dominance had pregnancy rates between 71 and 89%. In contrast, when follicl es were ovulated that ha d either 10 or 12 d of dominance, pregnancy rates were 52 and 12%, respectively. Animal s that ovulated follicles with prolonged dominance had significantly lower pregnancy rates compared to females that ovulated follicles of normal duration (Sirois and Fortune, 1990; Stock and Fortune, 1993). 49

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Gonadotropin Releasing Hormone (GnRH) Administration of exogenous GnRH alters follicular developm ent by inducing luteinization and (or) ovulation of dominant follicles (Macmillan et al., 1985; Thatcher et al., 1989), which results in synchronization of a new follicular wave (Twagiramungu et al., 1994). Gonadotropin releasing hormone acts on the pi tuitary to induce release of the gonadotropes LH and FSH (Chenault et al., 1990). Gonadotr opin releasing hormone is produced commercially as Fertagyl Factrel and Cystorelin and is administered at 2 mL as an either an intravenous or intramuscular injection. Concentrations of LH and FSH increase within 2 to 4 h of an endogenous GnRH treatment (Stevenson et al., 1993) and ovulation occurs 24 to 32 h after treatment (Thatcher et al., 1989; Pursley et al., 1994b). However, the abili ty of GnRH to initiate ovulation is stage of estrous cycle dependent. A domina nt growing follicle must be pres ent on the ovary in order for a GnRH induced ovulation to occur (M oreira et al., 2000). Kohram et al. (1998) demonstrated that GnRH administered between days 4 to 7 a nd days 15 to 18 induced ovulation, decreased estrogen concentrations, and initiated a new wave of follicle development. In another study using dairy heifers, GnRH induced ovulation less frequently in he ifers that were day 2 and day10 of the estrous cycle compared to day 5, 15 and 18 of the estrous cycle (Moreira et al., 2000). Lack of a dominant follicle on day 2 and d 10 is representative of the initiation of follicular waves in cattle where recruitment and selection is taking place and have not reached at least 10 mm in diameter, which appears to be the minimal size that a follicle needs to be to be ovulated by GnRH (Sartori et al., 2001). There does not appear to be a breed effect on the ability of GnRH to ovulate follicles across several stages of the estrous cycle. In a recent study by Esterman et al. (2008), ovulation rates were si milar for Angus and Brangus cows that received GnRH across several days of the estrous cycl e at the initiation of a CIDR treatment. 50

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There are essentially two types of CL th at result from a GnRH induced ovulation including newly developed CL and an accessory CL. An accessory CL is an additional CL that develops in the presence of an existing CL. The CL that develops from a GnRH induced ovulation appears to be similar to a naturally fo rmed CL in its capacity to be regressed with endogenous PGF 2 (Twagiramungu et al., 1992). However, mixed results have been reported about the integrity of progesteron e output from an induced ovulation. As would be expected, induced ovulations that result in development of an accessory CL have increased progesterone concentrations (Twagiramungu et al., 1994; Wolf enson et al., 1994). However, progesterone concentrations can also be less following a GnRH treatment compared to concentrations normally observed during the es trous cycle (Ford and Storms hak, 1978; Rodger and Stormshak, 1986). The purpose of including GnRH in an es trous synchronization and AI program is essentially two fold. First, it is used to i nduce ovulation and to synchronize the subsequent follicle wave by stimulating FSH to recruit the new follicle wave. In turn, when PGF 2 is administered 7 d after GnRH it results in a ve ry synchronous estrus (T hatcher et al., 1989; Macmillan and Thatcher, 1991; Pursley et al., 1994). And second, GnRH administered in conjunction with AI, known as a timed-AI, functi ons to synchronize ovulation with delivery of semen into the reproductive tract. The later scen ario is particularly effective when used in conjunction with GnRH followed seven days later with PGF 2 and a timed-AI approximately 48 to 60 h after PGF 2 (Pursley et al., 1995, 1997; Burke et al., 1996; Schmitt et al., 1996). Synchronization Protocols The primary functions of synchronization protocols is to either synchronize estrus so cattle can be inseminated over a 1 to 5 d period or to synchronize ovulat ion so cattle can be inseminated at a predetermined timed know as a timed-AI. Initial synchronization protocols 51

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focused on the use of either a single PGF 2 or two PGF 2 treatments 10 to 11 d apart. The major drawback with the PGF 2 protocols is that they only work in cattle that are going through estrous cycles and they require considerable estrous detection. The more preferred methods of synchronization include combining one of several pharmaceuticals including GnRH, a progestogen, and PGF 2 Synchronization protocols signific antly reduce estrous detection and (or) eliminate estrous detection completely by inco rporating a timed-AI. Furthermore, they can induce estrus in some anestrous cattle. It appears that some synchronization protocols are more effective in suckled postpartum cows compared to heifers. The numerous synchronization protocols available to producers ca n best be tailored to fit a producers operation. Although, most producers want synchronization protocols that require minimal animal handling, and result in acceptable and consistent AI pregnancy rates. The general consensus is that acceptable pregnancy rates are 50%. One of the most widely used estrous synchr onization programs for yearling heifers is the 14 d melengestrol acetate (MGA) treatment followed by PGF 2 17 d later (MGA-PGF 2 ; Brown et al., 1977). The MGA-PGF 2 protocol was designed to bypass inseminating heifers at the infertile estrus immediately after MGA withdraw al and took advantage of administering PGF 2 in the later stages of the estrous cycle when it was more effec tive in initiating luteolysis. Subsequent research illustra ted that administering PGF 2 19 d after the last day of MGA (Lamb et al., 2000) improved the synchrony of the PGF 2 induced estrus without reducing fertility. Heifers can be inseminated either after an observe d estrus with estrous de tection typically lasting 5 to 7 d or estrus can be detected for 3 d with a timed-AI + GnRH on the third day for heifers not exhibiting estrus with each insemination protocol yielding similar AI pr egnancy rates Beef cattle producers like to use the MGA-PGF 2 system because it is easy to implement, cost 52

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effective, requires minimal animal handling, and yields consistent sync hronized pregnancy rates (typically > 50%) in yearling heifers of Bos taurus breeding (Brown et al., 1988; Lamb et al., 2000; Salverson et al., 2002). In contrast, Bridges et al. (2005) reported synchronized pregnancy rates of only 34.5% in yearling heifers of Bos indicus Bos taurus breeding synchronized with the MGA-PGF 2 system where estrus was detected for 3 d with timedAI+GnRH on day 3 for heifers not exhibiti ng estrus. Synchronized pregnancy rate was improved to 42.5% when the PGF 2 treatment (25 mg) was split (12.5 mg; splitPGF 2 ) and administered on two consecutive days, which resu lted in greater luteolysis for the split-PGF 2 compared to the single PGF 2 Even with the addition of the split-PGF 2 treatment to the MGAPGF 2 system, synchronized pregnancy rates are still less for Bos indicus Bos taurus heifers compared to Bos taurus heifers. Recent work in our lab reported that follicle development was different between Bos taurus (Angus) and Bos indicus Bos taurus (Brangus) heifers after a 14 d MGA treatment (Woodall et al., 2006). First, ovulation rate after M GA withdrawal tended to be greater in Angus (100%; n=10 /10) compared to Brangus (80%; n=8/10) heifers. Second, Brangus (n=5/10) had more one, three, and f our follicle wave patterns compared to Angus (n=2/10), which resulted in asynchronous follicle development at PGF 2 for Brangus heifers. In a subsequent experiment, GnRH was given eith er 3 or 10 d after a 14 d MGA treatment to synchronize follicle development followed by PGF 2 7 d after GnRH. The day 3 GnRH treatment resulted in more heifers in estrus during the 3 d after PGF 2 compared to GnRH given 10 d after MGA withdrawal (Woodall et al., 2007a). In subsequent fiel d trials, synchronized pregnancy rates were similar between GnRH given 3 d after a 14 d MGA treatment (34.8%; n=178) compared to the original MGA-PGF 2 system (38.5%; n=174; Woodall et al., 2007b). Both treatments received a split-PG with estrous detection and AI for 3 d after PGF 2 and heifers 53

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not exhibiting estrus by 73 h after PGF 2 being timed-AI with GnRH between 73 to 80 h. Since less than acceptable pregnancy rate s are obtained when synchronizing Bos indicus Bos taurus heifers with a long-term MGA-PGF 2 treatment, other synchr onization system must be evaluated. Short-term (7 d) CIDR treatments are anothe r alternative to synchr onizing estrus. In a recent set of field trials, Lamb et al., (2006) ev aluated the effectiveness of several short term progestogen treatments in yearling Bos taurus heifers. Lamb et al. (2006) reported that one of the most consistent synchronization protocols in yearling heifers included administration of GnRH concurrent with a 7 d Eazi-Breed CIDR with PGF 2 at CIDR removal followed by a fixed timed-AI + GnRH 60 h after PGF 2 which yielded synchronized pregnancy rates of 53%. Equally effective was the same CIDR synchronization protocol but with 3 d of estrous detection combined with a timed-AI plus GnRH for he ifers not exhibiting es trus by 84 h after PGF 2 which yielded a synchronized pregnancy rate of 57%. There has been limited research conducted in yearling heifers of Bos indicus Bos taurus breeding evaluating short-term CIDR treatments. Lucy et al. (2001) reported that CIDR+PGF 2 treated heifers ha d a greater estrous response (84%) during the first 3 d of the breeding period compared to PGF 2 treated heifers (57%). We have recently initiated a multiyear study in our lab comparing the effectiveness of cloprostenol sodium (Estrumate) compared to din oprost tromethamine (Lutalyse) in the Select Synch/CIDR+timed-AI program in Angus, Brahma n, and Brahman Angus 2-year old virgin heifers. The overall estrous, conception, timed-A I pregnancy, and synchronized pregnancy rates for the first two years were 48.5 (n=163), 60.9 (n=79), 30.7 (n=84), and 45.3% (n=163), respectively (Unpublished data). There were no prostaglandin or breed effects on any of the variables measured. Synchronized pregnancy rates ranged from 40.5 to 54.1% across treatments 54

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and years suggesting that the Se lect Synch/CIDR+timed-AI sync hronization program could work in yearling heifers of Bos indicus Bos taurus breeding but additional research is needed to confirm this. The 7 d CIDR protocol has also been researched in suckled Bos taurus cows. Larsen et al. (2006) observed greater pregnancy rates for the Select Synch/CIDR+timed-AI and COSynch+CIDR treatments compared to the CO-Sync h without CIDR treatme nt. Conception rates were greater for the Select Synch+timed-AI a nd Select Synch/CIDR+timed-AI cows compared to cows synchronized with a CIDR alone. Th e authors concluded that the Co-Synch+CIDR yielded similar pregnancy rates compared to estr ous detection protocols and can be used as an effective timed-AI protocol in suckled Bos taurus beef cows. Lucy et al. (2006) reported a tendency for first service concep tion rate and pregnancy rates to be greater in the CIDR+PGF 2 compared to unsynchronized controls in suckled Bos taurus and Bos indicus Bos taurus beef cows from seven different locations. Utilization of a once-used-CIDR has been show n to be an option to the new CIDR when used in synchronization protocol s. Colazo et al. (2003) cond ucted three experiments using heifers and suckled beef cows. Experiment one compared efficacy of a new (1.9 g) or once-used CIDR inserted for 9 d and estrad iol cypionate with or without pr ogesterone at CIDR insertion in heifers. Pregnancy rate was not affected by num ber of times a CIDR was used or addition of progesterone. The second experime nt in heifers and cows received either a onceor twice-used CIDR, or either estradiol benzoate with or without progesterone at CIDR insertion. Synchronized pregnancy rates were decreased for the twice-used CIDR, but progesterone did not affect pregnancy rates. In the third experiment heifers received a new, once-used, twice-used, or two twice-used CIDR, pregnancy rates were similar for all treatments and there was no affect of 55

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progesterone. These results suggest that incorporation of a once-used CIDR into a synchronization program has potential. Follow up research conducted by Solorzano et al. (2004, 2008), demonstrated that a once-used CIDR could effectively synchronize estrus in non-lactating beef cows and yearling heifers that were eventually used for embryo transfer. It should be noted that the afore mentioned experiments utilized a CIDR containing 1.9 g of progesterone compared to 1.38 g CIDR which is the only type available fo r purchase in the United States. Consequently, it is recommended that CIDR purchased in the Unite d States only be re-used one time. Recent research also indicates that autoclaving the onceused CIDR actually results in greater circulating blood progesterone concentrations compared to a once-used CI DR that was not autoclaved (Eaton et al., 2007). The 7-11 estrous synchronization protocol co nsist of a 7 d MGA treatment with PGF 2 on the last day of MGA followed by GnRH 4 d later; seven days after GnRH, PGF 2 is administered to synchronize estrus (Kojima et al., 2000). The concept behind the 7-11 protocol is that the 7 d MGA treatment prevents cattle from exhibiting estrus and the first PGF 2 treatment initiates luteolysis of any CL present at MGA removal. Th is pretreatment regime re sults in a majority of cattle with an ovulatory sized fo llicle present when GnRH is administered 4 d after MGA withdrawal. The GnRH treatment functions to synchronize follicle development so a majority of cattle will have a healthy growing folli cle ready for ovulatio n at the second PGF 2 administered 7 d after GnRH. The 7-11 protocol is an effectiv e protocol for synchronizi ng estrus for either a detected estrus in Bos taurus (Kojima et al., 2000) or a timed-AI in Bos taurus cattle (Bader et al., 2005). In a recent study conducted in our lab, estrous cycling and anestrous postpartum Brangus and Angus cows received GnRH 4 d af ter a 7 d MGA treatment with PGF 2 at MGA withdrawal, 56

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GnRH induced ovulation in 94.4% of Brangus (n =17/18) and Angus (n=17/18) cows (Esterman et al., 2007). The resulting five day estr ous response (70.5, 65.8%), conception (67.7, 76.0%), and synchronized pregnancy rates (47.7, 50.0%) we re similar (P > 0.05) for Angus and Brangus, respectively. Because GnRH effectively ovulated follicles in Brangus cows when administered 4 d after a short term (7 d) progestogen treatmen t, it appears that the 7-11 synchronization protocol is an effective synchronization protocol for use in suckled cows of Bos indicus Bos taurus breeding. However, additional research wi ll need to be conducte d to evaluate the effectiveness of the 7-11 prot ocol in yearling heifers of Bos indicus Bos taurus breeding. In summary, the following objectives are being proposed and will constitute the research to be presented in this thesis. Objective 1: Evaluate the effectiveness of the Select Synch/CIDR+timed-AI synchronization protocol and a modified 7-11 protocol in yearling heifers and suckled cows of Bos indicus Bos taurus breeding. Objective 2: Evaluate the effectiveness of cloprostenol sodium and dinoprost tromethamine when used in conjunction with the synchronization tr eatments outlined in objective 1. Objective 3: Evaluate the effectiveness of th e 7-11 and Select Synch/CIDR+timed-AI synchronization protocols in yearling heifers of Bos indicus Bos indicus breeding. 57

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CHAPTER 3 COMPARISON OF TWO PROGESTOGEN BASED ESTROUS SYNCHRONIZATION PROTOCOLS AND CLOPROSTENOL SODIUM VS. DINOPROST TROMETHAMINE IN SUCKLED POST PARTUM COWS AND YEARLING HEIFERS OF B os indicus Bos taurus BREEDING Introduction In the Gulf Coast region of the Southeastern US, the majority of cattle contains some Bos indicus influence because of their superior ability to deal with heat stress, utilization of low quality forages, and increased parasite tolerance. However, the slight physiological and behavioral differences th at exist in cattle of Bos indicus breeding could cause differential responses to estrous synchronizatio n protocols that have been deve loped primarily in cattle of Bos taurus breeding. Cattle of Bos indicus breeding have an increased sensitivity and respond differently to exogenous hormones (Randel, 1984; Portillo et al., 2007) co mpared to cattle of Bos taurus breeding. Furthermore, estrus is more difficult to detect in cattle of Bos indicus breeding due to a decreased expression and duration of estr us (Galina et al., 1982) as well as an increased incidence of silent estr uses (Galina et al., 1996). One of the most consistent synchronization pr otocols in suckled cows (Larson et al., 2006) and yearling heifers (Lamb et al., 2006) of Bos taurus breeding includes admi nistration of a 7 d Eazi-Breed CIDR (CIDR) with GnRH at CIDR insertion and PGF 2 at CIDR removal, followed by 3 d of estrous detection and AI with a timed-AI plus GnRH for cattle not exhibiting estrus by 72 h after PGF 2 This protocol is known as the Select Synch/CIDR+timed-AI protocol. Lucy and coworkers (2001) conducted a multi-location study in su ckled beef cows and yearling heifers that received a 7 d CIDR with PGF 2 one day prior to CIDR removal followed by 3 d of estrous detection and AI. The AI preg nancy rates were similar for suckled cows and yearling heifers of Bos indicus Bos taurus breeding compared to locations with cattle of Bos taurus breeding. It should be noted that GnRH wa s not administered at CIDR insertion in the 58

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Lucy et al. (2001) study. Th erefore, additional experiment s are needed in cattle of Bos indicus Bos taurus breeding to evaluate CIDR synchronizations treatments when GnRH is administered at CIDR insertion and at timed-AI for inducti on of ovulation in cows not expressing estrus. The 7-11 synchronization protocol is anot her short term progestogen synchronization protocol that is frequently used in cattle of Bos taurus breeding (Kojima et al., 2000). The 7-11 protocol consists of a 7 d melengestrol acetate (MGA) treatment with PGF 2 on the last day of MGA followed by GnRH 4 d later. Seven days after GnRH, PGF 2 is administered to synchronize estrus. The 7-11 synchr onization protocol is effective in Bos taurus cattle (Kojima et al., 2000; Stegner et al., 2004) but no research has been conducted to evaluate the effectiveness of the 7-11 prot ocol in yearling heifers of Bos indicus Bos taurus breeding and one study has been conducted in suckled cows of Bos indicus Bos taurus breeding (Esterman et al., 2007b). As production costs increase, pr oducers seek to implement new management practices that either reduce their operating co sts and (or) increase productivity. Therefore, there has been considerable interest in using a once-used CIDR in estrous synchr onization protocols. Utilization of a once-used CIDR for 7 d suppresses estrus in beef females (Beal et al., 2003) and autoclaving a once-used CIDR in creases circulating progesterone concentrations compared to a non-autoclaved once-used CIDR (Zuluaga and W illiams, 2008). Autoclaving also reduces the risk of disease transmission. Moreover, synchr onized pregnancy rates after a fixed timed-AI were similar in heifers receivi ng either a new or once-used CIDR (Colazo et al., 2004). Therefore, there appears to be potential for in corporating a once-used CIDR into some estrous synchronization protocols. Additionally, a minimal amount of research has been conducted evaluating the effectiveness of prostaglandin ty pe, cloprostenol sodium compared to dinoprost 59

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tromethamine, when used in GnRH+PGF 2 estrous synchronization protocols in cattle of Bos indicus Bos taurus breeding. Heirs et al. (2003) repor ted similar synchronized AI pregnancy rates for cloprostenol sodium compared to dinoprost tromethamine in non-lactating Bos indicus Bos taurus cows synchronized with GnRH+PGF 2 protocol combined with MGA and Esterman et al. (2007a) reported similar results betw een the two prostaglandins in suckled Bos indicus Bos taurus cows synchronized with GnRH/CIDR+PGF 2 protocol. However, no direct comparisons have been made between clopros tenol sodium and dinoprost tromethamine in yearling Bos indicus Bos taurus heifers synchronized with GnRH+PGF 2 synchronization protocols. Therefore, the objectives of these experiments were to evaluate the effectiveness of cloprostenol sodium compared to dinoprost tromethamine when used in a modified 7-11 protocol and a Select Synch/CIDR+timed-AI protocol in yearling heifers and suckled cows of Bos indicus Bos taurus breeding. Materials and Methods Experiment 1 Experiment 1 (Figure 3-1) was conducte d from January to March 2007 at the Bar L Ranch, Marianna, Florida. In Experi ment 1, multiparous suckled postpartum Bos indicus Bos taurus cows (n = 324) were used. The percentage of Bos indicus breeding of cows utilized ranged from approximately 7 to 38% with the remainder being Bos taurus breeding. The experiment was a 2 2 factorial design. At the st art of the experiment (d 0), cows were equally distributed by body condition score (BCS; 1 = emaciat ed, 5 = moderate, 9 = very fat; Richards et al., 1986) and days post partum (DPP) to one of two progestogen based synchronization treatments and one of two PGF 2 treatments. The synchronization treatments included a 60

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modified 7-11 treatment, which will be te rmed the 7-10 treatment, and the Select Synch/CIDR+timed-AI treatment. Within each synchronization treatment, half the cows received one of two PGF 2 treatments including dinoprost tromet hamine (dinoprost, 25 mg i.m.; Prostamate Agrilabs, St. Joseph, MO) and cloprostenol sodium (cloprostenol, 500 g i.m.; Estrumate Schering-Plough Veterinary Corp., Kenilworth, NJ). The original 7-11 synchronization protocol admini sters GnRH 4 d after MGA wit hdrawal (Kojima et al., 2000). Since an intravaginal CIDR was used in the present study, it was hypothesized that the disappearance of the negative feed back effect of progesterone on LH secretion should take less time to clear the circulatory system compared to MGA. Therefore, Gn RH was administered on day 10 instead of day 11 resulting in the name change to the 7-10 synchronization treatment. On day 0, the 7-10 treatment received an autoclaved once-used CIDR (Eazi-Breed CIDR 1.38 g progesterone, Pfizer Animal Health, New Yor k, NY) that was removed on day 7 concomitant with dinoprost followed by GnRH (500 g; Cystorelin Merial Animal Health, Duluth, GA) on day 10. The once-used autoclaved CIDR was used in place of MGA to test its effectiveness as a low cost alternative progestogen source in the 7-10 protocol. On day 17, 7-10 cows received either dinoprost or cloprostenol Also on day 10, the Select Synch/CIDR+timed-AI treatment received a new CIDR concomitant with Gn RH followed by CIDR removal on day 17 where cows received either din oprost or cloprostenol. To aid in estrous detection, all cows received Estrotect estrous detection patches (Rockway, Inc., Spring Valley, WI) on day 18 of the experiment. Estrus wa s visually detected twice daily (0700 and 1600 h) for 72 h after PGF 2 and cows were inseminated 6 to 12 h after detection of estrus. Non-responders we re timed-AI + GnRH 72 to 76 h post PGF 2 Estrus was defined as a cow standing to be mounted by anothe r cow, showed signs of visible mucous, and/or 61

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had a half to full red estrous detection patch. Two AI technicians inseminated cows using frozen-thawed semen from a single sire of known fertility and tec hnicians were equally distributed across treatments for both cows that ei ther exhibited estrus or were timed-AI. Seven days after the timed-AI, natural service sires were placed with cows. Using a real-time B-mode ultrasonography machine (Aloka 500V, Corometric s Medical Systems, Wallingford, CT) with a 5.0 MHz transducer, pregnancy was diagnosed a pproximately 55 d after AI. Due to the 7 d period where no cows were exposed to natural service sires, differen ces in fetal size were used to determine whether a pregnancy resulted from the synchronized AI breeding (54 and 55 d pregnant) or natura l service sire ( 48 d pregnant). Embryos develop a C shape appearance approximately day 25 of gestation, which transitions to an L shape embryos by day 32. By day 48, the fetus is approximately 35 mm in length with visible limb buds and by day 55 the fetus is approximately 50 mm in length and has id entifiable ribs (Curran et al., 1986). Estrous response was defined as the number of cows displaying estrus for 3 d after prostaglandin and artificially inseminated (AI) divided by the total number of cows treated. Conception rate was defined as the number of co ws that became pregnant to AI divided by the number of cows that displayed estrus and were AI. Timed-AI pregnancy rate was the number of cows that failed to display estrus, were tim ed-AI, and became pregnant divided by the total number of cows that were timed-AI. Synchr onized pregnancy rate wa s the number of cows pregnant to AI divided by the total number of cows treated. Th irty-day pregnancy rate was the number of cows pregnant during the first 30 d of the breeding season divided by the total number of cows treated. Binomially distributed data including estrous response, conception rate, timed-AI pregnancy rate, synchronized preg nancy rate, and 30 d pregnancy ra te, were analyzed by logistic 62

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regression using the LOGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). All models included synchronization treatment (7-10 and Select Synch/CIDR+timed-AI), PGF 2 treatment (cloprostenol and dinoprost), DPP, cow age, BCS, AI technician, and all a ppropriate interactions. Days postpartum, cow age, and BCS were analyzed as categorical variables. Days postpartum was evaluated in three categories 43 d, 44 to 59 d, and 60 d. Cow age ranged from 3 to 9 yr of age resulting in seven categories. Body condition score was grouped into three categories 4.5, 5, 5.5. The effect of interval from PGF 2 to the onset of estr us (48, 60, and 72 h) on conception rate also was analyzed with synchronization treatment, PGF 2 treatment, interval from PGF 2 to the onset of estrus, DPP, cow age, BCS and all appropriate interactions included in the model. Because of a limited number of cows exhibiting estrus between 0 and 48 h after PGF 2 these times periods were combined to one group of cows 48 h. The final logistic regression model entered variables by a stepwise selection based on the Wa ld statistics criterion when P < 0.20 and removed variables based on P < 0.10. Variables were considered significant at P 0.05. Variables that were significant were entered into a linear regression model using GENMOD procedure of SAS to calcu late the adjusted odds ratios a nd 95% confidence intervals. Odds ratios were reported in tables and were used to calculate relative ris k, and the relative risk was reported in the results. Experiment 2 Experiment 2 (Figure 3-1) was conducted with yearling Bos indicus Bos taurus heifers (13 to 15 mo old) at the Bar L Ranch, Marian na, Florida, during tw o consecutive years, 2006 (year 1, n = 218) and 2007 (year 2, n = 137). The percentage of Bos indicus breeding ranged from approximately 7 to 38% with the remainder being Bos taurus breeding. The experiment was a 2 2 factorial design and heifers were equally but randomly assigned to the same 63

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treatments used in Experiment 1. Body condition scores were recorded for heifers at CIDR insertion. For all treatments and years, heifers received Estrotect estrous detection patches on day 18 of the experiment to aid in estrous detecti on. Estrus detection a nd insemination protocols were similar as described in Experiment 1. He ifers were inseminated with frozen-thawed semen from a single sire in year 1 a nd two pre-assigned sires in year 2. Heifers were inseminated by the same AI technician in both years. Seven days after the timed-AI, natural service sires were placed with heifers in both years. Pregnancy was diagnosed approximately 54 to 56 d after AI using a real-time B-mode ultrasound (Aloka 500 V, Corometrics Medical Systems, Wallingford, CT) with a 5.0 MHz transducer. Due to the 7 d period in which heifers were not exposed to natural service sires, differences in fetal size (C urran et al., 1986) as de scribed for Experiment 1 were used to determine whether a pregnancy re sulted from the synchronized breeding or the natural service sire. Estrous response was defined as the number of heifers displaying estrus for 3 d after prostaglandin and inseminated divided by the tota l number of heifers trea ted. Conception rate was defined as the number of heifers that becam e pregnant to AI divi ded by the number of heifers that displayed estrus a nd were AI. Timed-AI pregnancy rate was the number of heifers that failed to display estrus, were timed-AI, a nd became pregnant divided by the total number of heifers that were timed-AI. S ynchronized pregnancy rate was the number of heifers pregnant to AI divided by the total number of heifers treate d. Thirty-day pregnancy rate was the number of heifers pregnant during the fi rst 30 d of the breeding season divided by the total number of heifers treated. 64

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Binomially distributed data including estrous response, conception rate, timed-AI pregnancy rate, synchronized pr egnancy rate, and thirty-day pr egnancy rate were analyzed by logistic regression using the L OGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). All models included synchronization treatment (7 -10 and Select Synch/CIDR+timed-AI), PGF 2 treatment (cloprostenol and dinoprost), year, BCS, and all appropriate interactions. Body condition score was analyzed as a categorical va riable and was grouped in to three categories ( 5, 5.5, 6). The effect of interval from PGF 2 to the onset of estrus ( 48, 60, and 72 h) on conception rate was also analyzed with synchronization treatment, PGF 2 treatment, interval from PGF 2 to the onset of estrus, year, BCS, and all appropriate interacti ons included in the model. Because of a limited number of heifer s exhibiting estrus betw een 0 and 48 h after PGF 2 these time periods were combined into one group of heifers 48 h. The final logistic regression model entered variables by a stepwise selecti on based on the Wald stat istics criterion when P < 0.20 and removed variables based on P < 0.10. Variables were considered significant at P 0.05. Variables that were significant were en tered into a linear regression model using GENMOD procedure of SAS to calcu late the adjusted odds ratios a nd 95% confidence intervals. Odds ratios were reported in tables and were used to calculate relative ris k, and relative risk was reported in the results. Results Experiment 1 Synchronization treatment affected ( P < 0.05) estrous response w ith the risk of displaying estrous in the Select Synch/CIDR+timed-AI treat ed cows being 1.2 times greater compared to 710 cows. Body condition score also affected ( P < 0.05) estrous response. Cows in BCS 4.5 had an estrous response that was 73.7 pe rcent of what cows that were BCS 5.5 expressed. Cows with a BCS 5 tended ( P = 0.1) to have a decreased estrous response compared to cows in 65

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BCS 5.5 (Table 3-1). There were no effects ( P > 0.05) of PGF 2 treatment, age, DPP, and all appropriate interactions on estrous response. Synchronization treatment affected ( P < 0.05) conception rate as the Select Synch/CIDR+timed-AI cows had a 1.4 higher risk of becoming pregnant to AI after an observed estrus compared to 7-10 cows. Body condition also affected ( P < 0.05) conception rate. Cows with BCS 5.5 had 2 times greater risk of becoming pre gnant after an observed estrus compared to cows that were BCS 4.5. Cows in BCS 5 tended ( P = 0.1) to have decr eased conception rate compared to cows in BCS 5.5 (Table 3-2). There was also an AI technician effect ( P < 0.05) on conception rate (56.8 vs. 45.2 %). There were no effects ( P > 0.05) of PGF 2 treatment, age, DPP, interval from PGF 2 to estrus, and all appropriate interactions on conception rate. There were no effects ( P > 0.05) of synchronization treatment, PGF 2 treatment, BCS, age, AI technician, and all appropriate intera ctions on timed-AI pregnancy rate. Timed-AI pregnancy rates for the 7-10 and Select Synch/CIDR+timed-AI were 17.5 and 20.9%, respectively. However, DPP tended ( P = 0.1) to effect timed-AI pre gnancy rate (Table 3-3). In general, as days from calving increased, timed-AI pregnancy rates also increased. Synchronization treatment affected ( P < 0.05) synchronized pregnancy rate with cows treated with Select Synch/CIDR +timed-AI having a 1.5 greater risk of becoming pregnant during the synchronized breeding compared to 7-10 co ws (Table 3-4). Body condition score also affected ( P < 0.05) synchronized pregnancy rate (Table 3-4). Cows with a BCS 5.5 had a 2.1 greater risk of becoming pregna nt during the synchronized breedi ng compared to cows with a BCS 4.5. Cows with a BCS 5.5 also had 1.4 times greater risk of becoming pregnant during the synchronized breeding compared to cows with BCS 5. An increased ( P < 0.05) percentage of cows with a DPP 60 got pregnant to the synchronized breeding compared to cows that were 66

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43 DPP. Cows with a DPP 60 had a risk of becoming pregnant 1.8 times that of the 43 DPP cows. Synchronized pregnancy rates were similar ( P > 0.05) for cows 44-59 DPP compared to cows 60 DPP (Table 3-4). There were no effects ( P > 0.05) of PGF 2 treatment, age, AI technician, and all appropriate intera ctions on synchronized pregnancy rate. There were no significant effect s of synchronization treatment, PGF 2 treatment, age, DPP, and all appropriate interact ions on thirty-day pregnancy rate. However, cows with a BCS 5 and 4.5 had decreased ( P < 0.05) thirty-day pregnancy rates compared to cows with a BCS 5.5 (Table 3-5). Experiment 2 Synchronization treatment, PGF 2 treatment, year, BCS, and all appropriate interactions had no effect ( P > 0.05) on estrous response, concepti on rate, timed-AI pregnancy rate, synchronized pregnancy rate, and thirty-day pregnancy rate (Table 3-6). Across the synchronization and PGF 2 treatments, the mean estrous response, conception rate, timed-AI pregnancy rate, synchronized pregnancy rate, and thirty-day pregnancy rate were 66.8, 65.0, 16.1, 48.7, and 76.3% respectively. The mean sync hronized pregnancy rates for the dinoprost and cloprostenol treatments were 45.5 and 52.0%, re spectively. However, there was a treatment by interval from PGF 2 to the onset of estrus interacti on (P < 0.05; Figure 3-2) on conception rate. For heifers that exhi bited estrus 48 h after PGF 2 conception rates were similar ( P > 0.05) between treatments. Whereas, for heifers that exhibited estrus at 60 h, the Select Synch/CIDR+timed-AI treatment tended ( P = 0.10) to have a decreased conception rate compared to the 7-10 treatment. Conversely, for he ifers that exhibited estr us at 72 h, the Select Synch/CIDR+timed-AI treatment had an increased ( P < 0.05) conception rate compared to the 710 treatment. 67

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Discussion The true measure of the effectiveness of a synchronization treatment is the synchronized pregnancy rate or the number of cattle pregnant to the synchronized AI breeding. In Experiment 1, 14.3% more suckled Bos indicus Bos taurus synchronized with the Select Synch/CIDR+timed-AI treatment became pregnant compared to the 7-10 treatment. The synchronized pregnancy rate of the 7-10 treatment are considerably less compared to reports in suckled Bos taurus cows synchronized with 711 protocol with either estrous detection and AI (Kojima et al., 2000) or a timed-AI (Bader et al ., 2004). Both studies re ported synchronized pregnancy rates greater than 60% for estrous cycling and anestrous cows. Likewise, the synchronized pregnancy rate fo r the Select Synch/CIDR+timed -AI treatment was less (13%) compared to a report by Larson et al. (2006) in estrous cycling and anestrous suckled Bos taurus cows synchronized with the same protocol. Larson et al. (2006) observed a range in mean synchronized pregnancy rates between 51 to 61% for cows across different BCS, estrous cycling statuses, DPP, and parities. The decreased response of the 7-10 a nd Select/Synch+timed-AI treatments in suckled Bos indicus Bos taurus cows compared to similarly synchronized suckled Bos taurus cows (Kojima et al., 2000; Bader et al ., 2004; Larson et al., 2006) is due to slight decreases in es trous response, conception rate, and timed-AI pregnancy rates, which function together to decrease synchroni zed pregnancy rate s in the suckled Bos indicus Bos taurus cows It is difficult to determine the exact r eason(s) for the reduced estrous response and conception rates between the suckled Bos indicus Bos taurus in the present study compared to what is reported for Bos taurus cows. As with this study and res earch in the lite rature, there are very few instances where cattle of both Bos indicus Bos taurus and Bos taurus breeding are at the same location and receiving the same synchronization treatments under similar environmental conditions. Therefore, making ge neral comparisons about responses to estrous 68

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synchronization protocols between the two genotype s must be made with caution. In general, it has been well established that estrus is more difficult to detect in cattle of Bos indicus breeding due to a decreased expression and duration of estr us (Galina et al., 1982) as well as an increased incidence of silent estrus (Ga lina et al., 1996), which could play a role in the decreased estrous response for both synchronization treatments. What role if any, the increa sed incidence of three and four-wave follicle growth patterns, which are more frequent in cattle of Bos indicus breeding (Rhodes et al., 1995; Viana et al., 2000; Martinez et al., 2003), had in th e estrous response of a synchronized estrous is uncertain but should not be discounted. As mentioned previously, th e primary reasons for the gr eater synchronized pregnancy rate for the Select Synch/CIDR +timed-AI compared to the 7-10 treatment were significant increases in estrous response and conception rate for the Select Synch/CIDR+timed-AI treatment. The three day estrous response for the 7-10 cows was 10.1% less compared to Select Synch/CIDR+timed-AI cows. Kojima et al. ( 2000) reported a peak estrous response by 66 h after PGF 2 of 92% in suckled Bos taurus cows synchronized with th e 7-11 protocol, which is 43% greater compared to the 72 h estrous for the 7-10 treated Bos indicus Bos taurus cows. Esterman et al. (2007) observed similar five day estrous responses between suckled Angus ( Bos taurus) and Brangus ( Bos indicus Bos taurus) cows synchronized with the 7-11 protocol at the same location. In contrast, the 72 h estrous response (59.9%) for the Select Synch/CIDR+timedAI cows was only slightly less compared to the 72 h estrous response (69.3%) reported by Larson et al. (2006) in suckled Bos taurus cows. One reason for the significantly decreased estrous response of the 7-10 compared to the Select Synch/CIDR+timed-AI treatment could be due to pre-synchronization portion of the 7-10 tr eatment that used a once-used CIDR did not create the same synchrony of follicular wave de velopment compared to cows treated with the 69

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traditional 7-11 protocol that uses MGA. In Brangus cows synchronized with the 7-11 protocol, Esterman et al. (2007) reported that GnRH in itiated ovulation in 94% of the cows after MGA treatment similar to reports in Bos taurus cows (Kojima et al., 2000). Since ovulation rate to GnRH was not determined in the present study, th e ability of GnRH to ovulate follicles three days after CIDR removal is not known. Hence, it is unclear if the timing of GnRH relative to CIDR removal was appropriate and if a majority of follicles were of ovulatory size for GnRH to ovulate. It is also pos sible that the low circulating concentr ations of progesterone from the onceused CIDR altered LH secretory patterns (K ojima et al., 1992) and follicle growth and development (Bergfeld et al., 1996) that resulted in abnormal follicle development resulting in development of some persistent dominant follic les that were not ovulated by GnRH (Woodall et al., 2007a). Any of these scenarios could have resulted in asynchronous follicle development at the second PGF 2 resulting in a low estrous response during the three day estrous detection period. The second reason for the decreased synchroni zed pregnancy rates of the 7-10 treatment compared to the Select Synch/CIDR+timed-A I treatment was a significant reduction in conception rate for the 7-10 treatment. Conception rate of the 7-10 treatment is also considerably less compared to suckled Bos taurus (Kojima et al., 2000) and Bos indicus Bos taurus (Esterman et al., 2007) cows synchronized w ith a 7-11 protocol. In both studies, conception rates were greater than 65% compared to 45.5% in the current study. In suckled Bos indicus Bos taurus cows synchronized w ith the Select Synch pr otocol without a CIDR, Lemaster et al. (2006) reported a conception rate of 57.7%. The c onception rates for the Select Synch/CIDR+timed-AI treatment were similar to a report by Larson et al. (2006) in suckled Bos taurus cows synchronized with a simila r protocol and other reports in Bos taurus synchronized 70

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cows (Kojima et al., 2000; Stegne r et al., 2004). It is unclear why the conception rates were decreased in the 7-10 treatment. Based on the how the 7-10 treatment was designed, the pretreatment phase with the once-used CIDR foll owed 3 d later by GnRH should have worked to synchronize follicle development by ovulating a majority of the follicles so they are synchronized for the subsequent PGF 2 treatment 7 d later, which is essentially the Select Synch protocol. The newly developed follicles should have an increased fertility compared to ovulations of aged dominant follicles (Mihm et al., 1994) or persistent dominant follicles (Kinder et al., 1996). Additionally, the hormonal environment that follicles of the Select Synch/CIDR+timed-AI treatment were exposed to was different compared to the GnRH+PGF 2 portion of the 7-10 treatment. The Select Sync h/CIDR+timed-AI would have been exposed to elevated progesterone concentrations between GnRH and PGF 2 ; whereas, the 7-10 treatment did not receive this treatment. Although, addition of a CIDR to the Select Synch+timed-AI protocol did not improve conception rates to an obs erved estrus (Larson et al., 2006) in Bos taurus cows. Whether the progesterone from the CIDR is enhancing conception rates in the Bos indicus Bos taurus cows in the present study remains in quest ion and needs to be evaluated further. The one variable that had a significant e ffect on conception rate was BCS across both synchronization treatments. Cows with a BCS 5 had approximately a 25% greater conception rate compared to cows with a BCS 4.5, regardless of synchronizati on treatment. In contrast, Larson et al. (2006) reported only a 7% increase in conception rate in cows with a BCS > 5 compared to cows with a BCS < 5 in suckled Bos taurus cows synchronized with the Select Sync/CIDR+timed AI. Body condition can be used as an indirect measure of nutritional status (Richards et al., 1986), and nutritiona l status influences estrous cy cling status of suckled beef cows (Smith et al., 1976; Oyedipe et al., 1982) as well as the number of cows in estrus within 60 71

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d after calving (Whitman, 1975; Wettemann et al., 1982) It is possible that a greater percentage of cows with a BCS 5 were going through estrous cycles at the start of the synchronization treatments resulting in greater fertility compared to cows with a BCS < 5. Both Lucy et al. (2001) and Larsen et al. (2006) observed numerically greater conception rates for estrous cycling cows compared to anestrous cows when inseminated after a synchronized estrus. Body condition score at the start of the sync hronization treatment also had an effect on synchronization response, as cows with a body condition score 5.5 had significantly greater estrous response, conception rate synchronized pregnancy rate, a nd thirty-day pregnancy rate compared to cows with a BCS 4.5. This agrees with a repo rt by Lamb et al. (2001) in Bos taurus cows where for each increase in BCS of one unit, the proportion of cows pregnant to AI after a synchronized ovulation increa sed by 23%. In contrasts, Lars on et al. (2006) reported only a 7% increase in conception rate in cows that ha d BCS > 5 compared to cows with a BCS < 5 in suckled Bos taurus cows synchronized with the Select Synch/CIDR+timed AI. Although in the Larson et al. (2006) study, there were a limited number of cows with BCS < 5 that could have influenced the results. For the Bos indicus Bos taurus cows in the current experiment, synchronized pregnancy rates increased by more than 30% for cows with a BCS 5.5 compared to a BCS of 4.5. The importance of BCS on the respons e to a synchronization treatment has been reported by others (Yelich et al., 1988; De Jarnette et al., 2004; Larson et al., 2006) and the common theme among these studies is that cows with BCS 5, respond well to most synchronization systems for cattle of either Bos taurus or Bos indicus Bos taurus breeding. Therefore, it appears that suckled Bos indicus Bos taurus cows that have a BCS > 5 at the start of a synchronization protocol respond favorably to synchronization protocols and have an 72

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excellent opportunity of gett ing pregnant, similar to what is observed in suckled Bos taurus cows. Treatment had no effect on timed-AI pregnanc y rates. However, timed-AI pregnancy rates were numerically less for 7-10 (17%) compar ed to Select Synch/CIDR+timed-AI (20%). Larson et al. (2006) reported tim ed-AI pregnancy rates for the Se lect Synch+timed-AI without a CIDR, Select Synch/CIDR+timed-AI, and CIDR +timed-AI of 39, 26, and 38%, respectively in suckled Bos taurus cows. There certainly appears to be a considerable reduction in timed-AI pregnancy rates between cattle of Bos indicus Bos taurus compared to Bos taurus cattle. This could be due to several reasons ranging from lack of cattle going through estrous cycles and asynchronous follicle development at CIDR removal for the cattle of Bos indicus Bos taurus. Furthermore, the increased incidence of thr ee and four wave follicle wave patterns in Bos indicus Bos taurus compared to Bos taurus cattle could result in asynchronous follicle development at the timed-AI resulting in either aged follicles with reduced fertility (Sirois and Fortune, 1990; Mihm et al., 1994) ovulating to GnRH or follicles th at are not of the adequate size ovulating to GnRH (Lamb et al., 2001; Perry et al., 2005; Bu sch et al., 2008), which also have reduced fertility. A recent study by Esterman et al. (2008) in suckled cows of Bos indicus Bos taurus breeding synchronized with the Select Synch/CIDR with estr ous detection, reported that conception rate peaked for cattle that exhibited estrus by 60 h after PGF 2 and conceptions rates decreased in a linear fashion for cows that exhibited estrus betw een 72 and 96 h post PGF 2 This suggests that maybe the timed-AI needs to be performed at 60 h for the 7-10 protocol to avoid development of follicles with longer durations of domi nance, which could be more susceptible to decreased fertil ity. It should also be noted, the timed-AI groups also include 73

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anestrous cows that did not respond to the synchronization tr eatments and therefore had no opportunity to conceive and get pregnant. Timed-AI and synchronized pregnancy rates we re also significantly influenced by DPP at the start of the synchronization treatment. Timed-AI pregnancy rate increased by 16% and synchronized pregnancy rate increased by 21% in cows 60 DPP compared to cows 43 DPP. Both, Esterman et al. (2007) in Bos indicus Bos taurus cows synchronized with a Select Synch/CIDR+timed-AI protocol an d Stevenson et al. (2000) in Bos taurus cows synchronized with Select Synch concurrent with a norgestom et implant observed an increase in synchronized pregnancy rates as the interval from calving increased. The pres ent study and others (Stevenson et al., 2000; Esterman et al., 2007) stress the im portance of knowing where cows are in their postpartum period before starting a synchronization program. However, the interval from calving to the initiation of the synchronization treatment did not influence thirty day pregnancy rates, which were similar between the three postpartum interval categories. In contrast, BCS at the start of the synchronization treatment continued to influence the opportunity for cows to get pregnant as the breeding season progressed as observed by others (Spitzer et al., 1995; Kunkle et al., 1998). Many factors affect fertility and response to synchroniza tion protocols and rebreeding of the postpartum cow, most importantly es trous cycling status. Fa ctors that control the resumption of estrous cyclicity in postpartum females include endocrine function, (Erb et al., 1971), nutritional status (Short et al., 1976), BCS (Stevenson et al ., 2000), and suckling status (Randel, 1981). In Experiment 2, there were no sync hronization treatment (7-10 vs. Select Synch/CIDR+timed-AI) or PGF 2 treatment (dinoprost vs. cloprostenol), affects on estrous response, conception rate, timed-AI pregnancy rate synchronized pregnancy rate, or thirty day 74

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pregnancy rate for Bos indicus Bos taurus yearling heifers, which is in stark contrast to the results obtained for the same synchronization treatments for cows in Experiment 1. In Bos taurus heifers synchronized with th e Select Synch/CIDR+timed-AI protocol, Lamb et al. (2006) reported synchronized pregnancy rates that were approximately 5% greater compared to the Select Synch/CIDR+timed-AI tr eatment and approximately 12% greater compared to the 7-10 treatment of the present study. The 72 h estr ous responses were 5.6% less for the Select Synch+timed-AI and 8.8% less for th e 7-10 treatments compared to Bos taurus heifers synchronized with the Select Synch/CIDR+ timed-AI reported by Lamb et al. (2006). Conception rates were similar between synchron ization treatments and were also similar compared to conception rates reported by La mb et al. (2006) in similarly synchronized Bos taurus heifers. Similar to the results observed fo r cows of Experiment 1, estrous response and timed-AI pregnancy rates were less in the Bos indicus Bos taurus synchronized heifers compared to reports in Bos taurus synchronized heifers (L amb et al., 2006). In Bos indicus Bos taurus heifers synchronized with a 7 d CIDR with PGF 2 provided the day before CIDR removal, Lucy et al. (2001) reported simila r conception and synchr onized pregnancy rates compared to estrous cycling Bos taurus heifers. Furthermore, of the Bos indicus Bos taurus heifers that were going through estr ous cycles at CIDR insertion, 83% exhibited estrus within 3 d after PGF 2 which was similar to Bos taurus heifers in the same study. There have been no studies evaluating the 7-11 prot ocol in yearling heifers of Bos indicus Bos taurus breeding. Heifers treated with the 7-10 pr otocol had slightly decreased conception and synchronized pregnancy rates compared to l ong term MGA based protocols in Bos taurus heifers (Wood et al., 2001; Patterson et al., 1993). Bridges et al. (2005) synchronized yearling heifers of Bos indicus Bos taurus breeding with a 14 d MGA treatme nt followed by two consecutive PGF 2 75

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treatments 19 and 20 d after MGA, and heifers were AI after a detected estrus for 72 h and nonresponders were timed-AI plus GnRH. They reported similar synchronized pregnancy rates compared to the 7-10 treatment. It should also be mentioned that both estrous response and subsequent conception rates were considerably less in the Bridges et al. (2005) study compared to the 7-10 treatment. However, the timed-AI pregnancy rates were nearly doubled in the Bridges et al. (2005) study, which resulted in similar synchronized pregnancy rates compared to the current experiment. Hence, it appears that a decreased estrous response is one of the limiting factors that decrease the overall effectiven ess of synchronization systems in heifers of Bos indicus Bos taurus breeding compared to yearling Bos taurus heifers. Additional studies need to be conducted in heifers of Bos indicus Bos taurus breeding to determine what physiological and management factors influence the onset of be havioral estrus. Additionally, the relationship between follicle development during and after a synchronization treatment and what affect it has on the expression of behavioral estrus after a CIDR treatment needs to be evaluated. As mentioned previously, con ception rates were similar between synchronization and PGF 2 treatments. However, there was a treatment by interval from PGF 2 to onset of estrus effect on conception rate. For the 7-10 treatmen t, conception rate peaked for heifers that exhibited estrus at 60 h after PGF 2 and decreased at 72 h; whereas the inverse was observed for the Select Synch/CIDR+timed-AI heifers. A lthough the numbers of heifers are limited in each estrus category, there were no significant year effects on conception rates. Additionally, the same AI technician inseminated heifers in both y ears and a single AI sire was used in year 1 and two AI sires were used in year 2, which had simila r conception rates. Therefore, it appears that conception rates are influenced not only by sync hronization treatment but when heifers exhibited estrus relative to PGF 2 However, it should be noted that since estrous detection ceased at 72 h 76

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post PGF 2 it is not known what the conception rates would have been for the heifers exhibiting estrus after 72 h. Although, it may pr ove beneficial to move the ti med-AI in the 7-10 treatment from 72 h to approximately 60 to 66 h to take ad vantage of improved fertility of the 60 h estrus. The interval to estrus following PGF 2 is dependent on the stage of ovarian follicular growth when PGF 2 is administered (Geary et al., 1999; Hittinger et al ., 2004). Kastelic et al. (1990) observed cows with a mature dominant follicle present at PGF 2 display estrus earlier after PGF 2 compared to cows with a growing and developing follicle at PGF 2 which takes longer to exhibit estrus. The 7-10 treated heifers should have a greater synchrony of follicle development compared to the Select Synch/ CIDR+timed-AI group since the GnRH after the initial PGF 2 in the 7-10 treatment should have initiat ed ovulation in a majority of animals resulting in a synchronous follicle developmen t compared to GnRH administered at CIDR insertion in the Select Synch/CIDR treatment. The number of follicles ovulating to GnRH at CIDR insertion in the Select S ynch/CIDR treatment would probabl y have been less compared to the 7-10 treatment since there would be follicles at all stages of follicle development at CIDR insertion and stage of follicle development can infl uence ovulation rates to GnRH (Moreira et al., 2000). Therefore, there could have been a mix of follicles at different stages of development at CIDR removal for the Select Synch/CIDR tr eatment resulting in ovulation of both newly developed follicles and aged follicles. Heifers w ith aged follicles and (or) a greater duration of dominance should exhibit estrus earlier after PGF 2 and fertility of the aged follicles would be decreased compared to newly developed follicle s (Mihm et al., 1994). Bridges et al. (2008) hypothesized that fertility of the ovulatory follicle is a function of the duration of proestrus and capacity of the ovulatory follicle to produced increas ed estradiol concentrations preceding estrus and AI. They further hypothesized that by reduci ng the duration of a CIDR treatment from 7 to 77

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5 d could enhance secretion of estradiol by th e potential ovulatory follicle even with younger follicles of reduced diameters. They conducted a series of experiments that synchronized suckled Bos taurus cows with a 5 d Co-Synch protocol with timed-AI at 72 h compared with a 7 d Co-Synch with timed-AI at 60 h. With the 5 d program, newly developed follicles would be approximately 3 to 4 d from emergence compared to 5 to 6 d from emergence for the 7 d program. The 5 d program significantly increased timed-AI pregnancy rates compared to the 7 d treatment in suckled beef cows. These findings certainly demonstrate the importance of duration of follicle development on fertility of a synchronization system. Whether these findings will translate to beef heifers is unknow n but it does provide evidence to explain the sign ificant shifts in fertility observed between synchronization systems based on follicle development. In Experiments 1 and 2, the response to clop rostenol and dinoprost was similar between treatments for suckled cows and yearling heifers. This agrees with Hiers et al. (2003) who reported similar synchronized pregnancy rates in Bos indicus Bos taurus cows synchronized with a modified Co-synch protocol with either cloprostenol or dinoprost treatments. Likewise, Salverson et al. (2002) repor ted a similar response in Bos taurus heifers treated with either cloprostenol or dinoprost treatments. Therefore, it appears that cloprost enol or dinoprost are equally effective when used in either th e 7-10 or the Select Synch/CIDR+timed-AI synchronization protocols in cattle of Bos indicus Bos taurus breeding. In summary, the Select Synch/CIDR+timed-A I had increased conception and synchronized pregnancy rates compared to the 710 treatment in suckled cows of Bos indicus Bos taurus breeding in Experiment 1. The effectiveness of the treatment was influenced by BCS, as BCS increased, the estrous response, conception rate, and synchronized pregnancy rates increased. Days postpartum also influenced effectiveness of the synchronization treatments, as time from 78

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calving to start of synchronizati on increased, synchronized pregnancy rates increased. There was no effect of prostaglandin treatment on estrous response, conception rate, timed-AI pregnancy rate, synchronized pregnancy rate, or thirty day pregnancy rate. In Experiment 2, there were no synchronization or prostaglandi n treatment effects on any of the reproductive responses measured in the yearling heifers of Bos indicus Bos taurus breeding. Implications The Select Synch/CIDR+timed-AI protocol is a practical synchronization method for Bos indicus Bos taurus yearling heifers and postpartum cows that are in good body condition and 60 d postpartum at the start of the synchronizatio n treatment. The 7-10 synchronization protocol yields similar results as the Select Synch/CIDR+t imed-AI protocol when used in yearling heifers but decreased results in suckled Bos indicus Bos taurus cows. Cloprostenol sodium and dinoprost tromethamine are equally effective an d yielded similar synchr onized pregnancy rates in both yearling heifers and suckled Bos indicus Bos taurus cows. 79

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a) 7-10 treatment with Dinopros t or Cloprostenol PGF 2 on day 17 Dinoprost or Cloprostenol GnRH + timed-AI Dinoprost GnRH Once-used CIDR Estrus detect & AI b) Select Synch/CIDR+timed-AI treatment with Dinoprost or Cloprostenol PGF 2 on day 17 Dinoprost or Cloprostenol GnRH + timed-AI GnRH 0 7 10 17 20 Estrus detect & AI New CIDR Day of experiment Figure 3-1. Experimental design evaluating the effects of two progestogen and two PGF 2 treatments in Bos indicus Bos taurus suckled cows (Experiment 1) and yearling heifers (Experiment 2). a) 7-10 treatment: on day 0 received a once-used CIDR that was removed on d 7 concomitant with di noprost tromethamine (Dinoprost; 25 mg i.m.) followed by GnRH (500 g i.m.) on day 10. On day 17, females received either dinoprost or cloprostenol sodium (Cloprostenol; 500 g i.m.). b) Select Synch/CIDR+timed-AI treatment: on day 10 received a new CIDR (1.38 g) concomitant with GnRH. Day 17 CIDR wa s removed and females received either Dinoprost or Cloprostenol. For all four tr eatments estrus was detected for 3 d and females were inseminated 6 to 12 h after detected estrus. Females not exhibiting estrus by 72 h were timed-AI and received GnRH. 80

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Table 3-1. Effect of synchronization trea tment and body condition score (BCS) on estrous response in suckled Bos indicus Bos taurus cows. a Variable Estrous response, % c Odds Ratio 95% CI d P-value Treatment Select Synch/CIDR+timed-AI 7-10 59.9 (100/167) 49.0 (77/157) 1.59 Referent 1.02-2.48 0.05 BCS b 4.5 5 5.5 48.8 (59/121) 54.4 (75/138) 66.2 (43/65) 0.47 0.59 Referent 0.25-0.89 0.32-1.10 0.02 0.10 a Refer to Figure 3-1 for details of treatments. b Body condition score: 1 = emaciate d, 5 = moderate; 9 = very fat. c Percentage of cows displaying estrus 72 h after PGF 2 of the total treated. d 95% confidence interval. 81

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Table 3-2. Effect of synchr onization treatment and body condition score (BCS) on conception rate in suckled Bos indicus Bos taurus cows. a Variable Conception rate, % c Odds Ratio 95% CI d P-value Treatment Select Synch/CIDR+timed-AI 7-10 62.0 (62/100) 45.5 (35/77) 2.08 Referent 1.10-3.95 0.03 BCS b 4.5 5 5.5 35.6 (21/59) 60.0 (45/75) 72.1 (31/43) 0.20 0.51 Referent 0.08-0.47 0.22-0.1.17 0.0003 0.11 a Refer to Figure 3-1 for details of treatments. b BCS: 1 = emaciated, 5 = moderate; 9 = very fat. c Percentage of cows pregnant to AI of th e total that exhibited estrus and were AI. d 95% confidence interval. 82

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Table 3-3. Effect of days postpartum (D PP) on timed-AI pregnancy rate in suckled Bos indicus Bos taurus cows. a DPP Timed-AI pregnancy rate, % b Odds Ratio 95% CI c P-value 43 11.5 (7/61) 0.34 0.12-0.98 0.05 44-59 21.7 (10/46) 0.73 0.27-1.96 0.54 60 27.5 (11/40) Referent a Refer to Figure 3-1 for details of treatments. b Percentage of cows that faile d to display estrus, were timed-A I, and became pregnant of the total number of cows that were timed-AI. c 95% confidence interval 83

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Table 3-4. Effect of sync hronization treatment, body condition score (BCS), and days postpartum (DPP) on synchronized pregnancy rate in suckled Bos indicus Bos taurus cows. a Variable Synchronized pregnancy rate, % c Odds Ratio 95% CI d P-value Treatment Select Synch/CIDR+timed-AI 7-10 45.5 (76/167) 31.2 (49/157) 2.01 Referent 1.25-3.24 0.004 BCS b 4.5 5 5.5 26.5 (32/121) 40.6 (56/138) 56.9 (37/65) 0.29 0.52 Referent 0.15-0.55 0.28-0.97 0.0002 0.04 DPP 43 44-59 60 27.8 (32/115) 40.5 (45/111) 49.0 (48/98) 0.45 0.73 Referent 0.25-0.82 0.42-1.30 0.009 0.28 a Refer to Figure 3-1 for details of treatments. b BCS: 1 = emaciated, 5 = moderate; 9 = very fat. c Percentage of cows pregnant during the synchronized breeding of the total treated. d 95% confidence interval 84

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Table 3-5. Effect of body condi tion score (BCS) on thirty da y pregnancy rates in suckled Bos indicus Bos taurus cows. Variable Thirty day pregnancy rate, % b Odds Ratio 95% CI c P-value BCS a 4.5 5 5.5 72.7 (88/121) 77.5 (107/138) 89.2 (58/65) 0.32 0.42 Referent 0.12-0.78 0.17-1.00 0.01 0.05 a BCS: 1 = emaciated, 5 = moderate, 9 = very fat. b Percentage of cows pregnant during the firs t 30 d of breeding season of the total treated. c 95% confidence interval. 85

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Table 3-6. Effectiveness of 7-10 treatmen t compared to Select Sync/CIDR+timed-AI (SSC+TAI) treatment on estrous response, conception rate, timed-AI pregnancy rate, synchronized pregnancy ra te, and thirty day pregnanc y rate in yearling heifers of Bos indicus Bos taurus breeding. a Variable 7-10 SSC+TAI Odds Ratio g 95% CI h P-value Estrous response, % b 65.2 (116/178) 68.4 (121/177) 1.26 0.80-1.97 0.32 Conception rate, % c 62.1 (72/116) 67.8 (82/121) 1.34 0.78-2.30 0.29 Timed-AI pregnancy rate, % d 14.5 (9/62) 17.9 (10/56) 1.30 0.48-3.42 0.62 Synchronized pregnancy rate, % e 45.5 (81/178) 52.0 (92/177) 1.30 0.85-1.97 0.22 Thirty day pregnancy rate, % f 74.7 (133/178) 78.0 (138/177) 1.20 0.73-1.96 0.47 a Refer to Figure 3-1 for details of treatments. b Percentage of heifers disp laying estrus 72 h after PGF 2 of the total treated. c Percentage of heifers pregna nt to AI of the total that exhibited estrus and were AI. d Percentage of heifers that failed to display estrus, were tim ed-AI, and became pregnant of the total number of heifers 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 bree ding season of the total number of heifers treated. g 7-10 used as referent value h 95% confidence interval 86

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Figure 3-2. Effect of interval from PGF 2 to onset of estrus on conception rate in Bos indicus Bos taurus yearling heifers synchronized with 7-10 or Select Synch/CIDR+timedAI (SSC+TAI) treatments. Numbers in pa renthesis indicate the number of heifers in each category. Treatment by interval from PGF 2 to onset of estrus ( P = 0.02). 87

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CHAPTER 4 EVALUATION OF TWO PROGESTOGEN BASED ESTROUS SYNCHRONIZATION PROTOCOLS IN YEARLING HEIFERS OF BOS INDICUS BOS TAURUS BREEDING Introduction Estrous synchronization is a re productive management tool th at allows for an increased number of cattle displaying estrus and ovulati ng over a 3 to 5 d period, which allows for a minimal number of days of estr ous detection or elimination of estrous detection resulting in a timed artificial insemination (timed-AI). A co mmon estrous synchronizati on protocol used in Bos taurus cattle, the Select Synch protocol, utilizes GnRH with prostaglandin F 2 (PGF 2 ) 7 d later (Macmillan and Thatcher, 1991 ; Pursley et al., 1995), followed by 5 d of estrous detection. However, a common problem with this protocol is premature expression of estrus prior to PGF 2 resulting in additional estrous detection (DeJarnette et al., 2001). Addition of an exogenous progestogen between the GnRH and PGF 2 eliminates the need for additional estrous detection (Thompson et al., 1999; DeJarnette et al., 2003). A ddition of a progesterone insert like the EaziBreed CIDR (CIDR) to the Select Synch protocol, combined with estrous detection for 72 h after CIDR removal and timed-AI + GnRH for non-responders (Select Sy nch/CIDR+timed-AI), is an effective estrous synchr onization protocol in yearling Bos taurus heifers (Lamb et al., 2006). However, the effectiveness of this protocol in yearling Bos indicus Bos taurus heifers has not been thoroughly evaluated. The 7-11 estrous synchronization protocol cons ists of a 7 d melengestrol acetate (MGA) treatment with PGF 2 on the last day of MGA followed by GnRH 4 d later; 7 d after GnRH, PGF 2 is administered to synchronize estrus (Kojima et al., 2000). The 7-11 protocol is an effective synchronization protocol in Bos taurus cattle (Kojima et al., 2000; Stegner et al., 2004) but no research has been conducted evaluating the effectiveness of the 7-11 protocol in yearling heifers of Bos indicus Bos taurus breeding. The basis behind th e 7-11 treatment is that the 88

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administration of GnRH after MGA results in ovul ation in majority of the animals resulting in very synchronous follicle development pattern. In a recent study conducted by Esterman et al. (2007), estrous cycling and anestrous suckled pos tpartum Brangus and Angus cows received the 7-11 protocol, and GnRH induced ovulation in 94.4% of both Brangus and Angus cows. The resulting 5 d estrous response, conception, a nd synchronized pregnanc y rates were similar between Angus and Brangus cows. Since GnRH effectively ovulates and synchronizes follicles when administered 4 d after a short term (7 d) progestogen treatment in Brangus cows, the 7-11 protocol could be an effective synchroni zation protocol in yearling heifers of Bos indicus Bos taurus breeding. Therefore the objective of this experiment wa s to evaluate the effectiveness of the 7-11 and Select Synch/CIDR+timed-AI synchroni zation protocols in yearling heifers of Bos indicus Bos taurus breeding. Materials and Methods The experiment was conducted at three loca tions in North Central Florida from January to March, 2007. Yearling (12 to 15 mo) Bos indicus Bos taurus heifers (n = 410) were used in the experiment and the percentage of Bos indicus breeding ranged from approximately 1/4 to 1/2 with the remainder being Bos taurus breeding (Table 4-1). At the start of the experiment (d 0) heifers were distributed by repr oductive tract score (RTS, Scal e 1 to 5; 1 = non-cycling, 5 = estrous cycling; Anderson et al., 1991; See Tabl e B-1 for complete RTS score description) and body condition score (BCS, Scale 1 to 9; 1 = emaciat ed, 5 = moderate, 9 = very fat; Richards et al., 1986) to one of two proge stogen based synchronization pr otocols including the 7-11 and Select Select Synch/CIDR+timed-AI protocols (Figure 4-1). The 7-11 heifers received MGA (MGA 200 premix, Pfizer Animal Health, New York, NY) in a carrier supplement fed at a rate of 0.9 kg/head/d to deliver 0.5 mg MGA/head/d fr om day 0 to 7 of the experiment with PGF 2 89

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(25 mg i.m.; Lutalyse Sterile Solution, Pfizer Animal Health, New York, NY) concomitant with the last day of MGA. On day 11, heifer s received GnRH (500 g i.m.; Cystorelin Merial, Duluth, GA) followed by PGF 2 on day 18. The Select Synch/CI DR+timed-AI heifers received the same carrier supplement without MGA from day 0 to 7. On day 11, a CIDR (Eazi-Breed CIDR 1.38 g progesterone; Pfizer Animal Health, New York, NY) was inserted concomitant with GnRH. The CIDR was removed on day 18 concurrent with PGF 2 (25 mg i.m.; Lutalyse ). Heifers from both synchronizati on treatments received Estrotect estrous detection patches (Rockway, Inc., Spring Valley, WI) on day 18 of the experiment to aid in estrous detection and estrus was visually detected twice daily (0700 and 1600 h) for 72 h after PGF 2 Heifers were artificially inseminated (AI) 6 to 12 h after dete ction of estrus. Non -responders were timed-AI and treated with GnRH 72 to 76 h post PGF 2 Estrus was defined as either a heifer standing to be mounted by another heifer, showed signs of vi sible mucous, and (or) had a half to full red estrous detection patch. One AI technician within each location inse minated heifers using frozen-thawed semen from multiple sires. Whenev er possible, AI sires were equally distributed across treatments and between heifers that either exhibited estrus and (or) were timed-AI. Natural service sires were placed with heifers 10 d after timed-AI. Using a real-time B-mode ultrasonography machine (Aloka 500V, Corometric s Medical Systems, Wallingford, CT) with a 5.0 MHz transducer pregnancy was diagnosed a pproximately 55 d after AI. Due to the 10 d period where no cows were exposed to natural service sires, differen ces in fetal size were used to determine whether a pregnancy resulted from the synchronized AI breeding (54 and 55 d pregnant) or natura l service sire ( 48 d pregnant). Embryos develop a C shape appearance approximately day 25 of gestation, which transitions to an L shape embryos by day 32. By day 90

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48, the fetus is approximately 35 mm in length with visible limb buds and by day 55 the fetus is approximately 50 mm in length and has id entifiable ribs (Curran et al., 1986). Estrous response was defined as the number of heifers displaying estr us for 5 d after PGF 2 and inseminated divided by the total number of he ifers treated. Concepti on rate was defined as the number of heifers that became pregnant to AI divided by the number of heifers that displayed estrus and were AI. Timed-AI pregnancy rate was the number of heifers that failed to display estrus, were timed-AI, and became pregnant divi ded by the total number of heifers that were timed-AI. Synchronized pregnancy rate was the number of heifers pregnant to AI divided by the total number of heifers treated. Thirty-day preg nancy rate was the number of heifers pregnant during the first 30 d of the br eeding season divided by the tota l number of heifers treated. Binomially distributed data including estrous response, conception rate, timed-AI pregnancy rate, synchronized pr egnancy rate, and thirty-day pr egnancy rate, were analyzed by logistic regression using the L OGISTIC procedure of SAS (SAS Inst. Inc., Cary, NC). All models included synchronization treatment (7-11 and Select Synch/CIDR+timed-AI), location, BCS, and all appropriate interactions. Body condition was analyzed as a categorical variable and was grouped into three categories ( 4.5, 5, 5.5). The effect of interval from PGF 2 to the onset of estrus ( 36, 48, 60, and 72 h) on conception rate wa s also analyzed with synchronization treatment, location, BCS, and all a ppropriate interactions included in the model. Because of a limited number of heifers exhibiting estrus between 0 and 36 h after PGF 2 these time periods were combined into 36 h. The final logistic regression model entered variables by a stepwise selection based on the Wald statistics criterion when P < 0.20 and removed variables based on P < 0.10. Variables were considered significant at P 0.05. Variables that were significant were entered into a linear regression model using GENMOD procedure of SAS to calculate the 91

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adjusted odds ratios and 95% confidence interv als. Reproductive tract score (RTS) was not included in the initial model due to confounding effects with BCS ; therefore, a second model was conducted evaluating the same binomial variab les of estrous response, conception rate, timed-AI pregnancy rate, synchronized pregnanc y rate, and thirty-day pregnancy rate and categorical variables including synchronization treatment, locati on, RTS, and all appropriate interactions. Reproductive tract score was eval uated as a categorical variable and was grouped into three categories ( 2, 3, 4). The initial analysis indicate d that all respons e variables were similar between RTS 4 and 5, so the two RTS categor ies were combined for the final analysis. The same statistical process as previously desc ribed was used to determine the final logistic regression model as well as the linear regression model. Adjusted odds ratios and 95% confidence intervals are also report ed. Odds ratios were reported in tables and were used to calculate relative risk, and relative risk was reported in the results. Results An increased ( P < 0.05) percentage of heifers expre ssed estrus during the 72 h following PGF 2 for the 7-11 compared to Select Synch/CIDR +timed-AI treatment (Table 4-2). Estrous response was also affected ( P < 0.05) by BCS. He ifers with a BCS 4.5 and 5 had a decreased ( P < 0.05) estrous response compared to heifers with a BCS 5.5 (Table 4-3). The relative risk of heifers with a BCS 4.5 exhibiting estrus was 60% of heifers with a BCS 5.5 and heifers with a BCS 5 exhibiting estrus was 74% of heifers with a BCS 5.5. Estrous response was not affected ( P > 0.05) by location nor were there any significant interactions. Conception rates were also affected ( P < 0.05) by synchronization treatment. The risk of heifers being pregnant to an AI after an obs erved estrus was 1.5 times greater for the 7-11 compared to Select Synch/CIDR+ timed-AI heifers (Table 4-2). In addition, conception rate was 92

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also affected (P < 0.05) by the interval from PGF 2 to the onset of estrus (Figure 4-2); however, there was no ( P > 0.05) treatment by interval from PGF 2 to the onset of estrus effects on conception rate. Heifers exhibiting estrus at 60 h had an increased (P < 0.05) conception rate compared to heifers that had exhibited estrus at 36, 48, and 72 h (Figur e 4-2), which were similar ( P > 0.05) to each other. Co nception rates were not ( P > 0.05) affected by BCS and location, nor were there any si gnificant interactions. Timed-AI pregnancy rates were similar ( P > 0.05) between the 7-11 and Select Synch/CIDR+timed-AI treatments (Table 4-2) and there were no effects ( P > 0.05) of BCS and location on timed-AI pregnanc y rate nor were there any significant interactions. Synchronized pregnancy rates were affected ( P < 0.05) by synchronization treatment. The risk of heifers treated with the 7-11 becomi ng pregnant to the sync hronized breeding was 1.35 times greater compared to the Select Synch/ CIDR+timed-AI treatment (Table 4-2). Body condition score had no effect ( P > 0.05) on synchronized pre gnancy rate. Synchronized pregnancy rates were not influenced by ( P > 0.05) BCS and location, nor were there any significant interactions. Thirty day pregnancy rates were simila r (P > 0.05) between treatments (Table 4-2) and were not affected ( P > 0.05) by BCS, location, nor were there any significant interactions. The effect of RTS on response to synchronization treatments by RTS category are presented in Table (4-4). There were no treatment by RTS effects (P > 0.05) but RTS affected ( P < 0.05) estrous response, conception rate, sy nchronized pregnancy rate, and thirty-day pregnancy rate. It should be mentioned that heifers with a RTS 4, had a greater (P < 0.05) BCS (5.2 0.02) compared to heifers with a RTS of 3 (5.0 0.02) and 2 (4.7 0.02). Heifers with a RTS 3 had a greater ( P < 0.05) BCS compared to heifers with RTS 2. Heifers with a 93

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RTS 4, had a 2.0 times greater risk of exhibitin g estrus compared to heifers with a RTS 2 and a 1.3 times greater risk to exhibit estrus compar ed to heifers with a RTS 3 (Table 4-4). For conception rate, heifers with RTS 4 had a 6 times greater risk of being pregnant after exhibiting estrus and being AI compared to heifers with RTS 2. Conception rates were similar ( P > 0.05) between heifers with a RTS 3 and 4 (Table 4-4). Timed-AI pregnancy rates tended (P = 0.10) to be greater for heifers with a RTS 4 compared to heifers with a RTS 2 (Table 4-4). Synchronized pregnancy rates were similar ( P < 0.05) for heifers with RTS 4 and 3. However, heifers with RTS 4 had a 4 times greater risk of becoming pregnant to the synchronized br eeding compared to heifers with RTS 2 (Table 4-4). Heifers with RTS 4 had a 2.3 greater risk of being pregnant during the first thirty-days of the breeding season compared to heifers with RTS 2 and a 1.2 greater risk compared to heifers with a RTS 3(Table 4-4). Discussion Estrous response was greater for the 7-11 co mpared to Select Synch/CIDR+timed-AI heifers. A possible reason for th e increased estrous response of th e 7-11 treatment could be that 7-11 treatment may do a better job of synchronizing follicle development compared to the Select Synch/CIDR treatment. The MGA in combination with PGF 2 allows for more follicles to be of ovulatory size at MGA withdrawal resulting in most animals ovulating to GnRH (Kojima et al., 2001), which provides for a very synchronous wave of follicle development at the subsequent PGF 2 In contrast, the administration of GnRH at the initiation of the Select Synch/CIDR+timed-AI protocol probably does not do as an effective job of ovulating and synchronizing follicle development since animals are at various stages of the follicle development at CIDR insertion, resulting in Gn RH ovulating fewer follicles (Moreira et al., 94

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2000). Consequently, follicle development at CI DR withdrawal may not be as synchronous for the Synch/CIDR+timed-AI compared to the 7-11 tr eatment, resulting in a decreased 72 h estrous response. The estrous response for both treatments are co nsiderably less compared to studies in Bos taurus heifers synchronized with either the Se lect Synch/CIDR+timed-AI (Lamb et al., 2006), short term progestogen protocols (Pa tterson et al., 1989), and CIDR + PGF 2 protocol (Lucy et al., 2001). In all of the aforementioned studies, the 3 d estrous responses were approximately 20 to 30% greater compared to those obser ved in the present study in yearling Bos indicus Bos taurus heifers. However, the 3 d estrous respon ses for both treatments are similar to that reported by Bridges et al. (2005) in yearling Bos indicus Bos taurus heifers synchronized with a long term (14 d) MGA + PGF 2 synchronization protocol. The decreased estrous response is probably due to heifer genotype and estrous cycling status of heifers at the start of the synchronization treatment. Patterson et al. (1989) demonstrated that yearling Bos indicus Bos taurus heifers going through estrous cycles had a si gnificant reduction in estrous response (22%) compared to contemporary Bos taurus heifers synchronized with a short term progestogen treatment. Estrus is more difficult to detect in cattle of Bos indicus breeding due to a decreased expression and duration of estrus (G alina et al., 1982) as well as an increased incidence of silent estrus (Galina et al., 1996). Ho wever, utilization of the estrous detection patches should have assisted identifying heifers that did not exhib it a good standing estrous during the two times that estrus was visually detected (0700 and 1600 h). Therefore, the method of estrous detection is probably not the major factor responsible fo r the decreased estrous response. Since blood samples were not taken on the heifers, it is not known how many heif ers were going through estrous cycles at the start of the synchronization treatment. Bos indicus Bos taurus and Bos 95

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taurus heifers that are going through estrous cycles at the start of a CIDR+PGF 2 treatment (Lucy et al., 2001) had significantly increased estrous responses compared to pre-pubertal heifers. Additionally, the ability of the synchronization treatments to induce estrous cycles in the pre-pubertal heifers is unclear. Lucy et al. (2001) repor ted that a CIDR+PGF 2 treatment induced estrous cycles in prepubertal Bos indicus Bos taurus and Bos taurus heifers over several locations ranging from 40 to 100%. Onset of puberty is related inversely to growth rate (Wiltbank et al., 1969; Short and Bellows, 1971) and heifers with higher growth ra tes are typically going to have greater BCS. Body condition score can be used as an indirect indicat or of nutritional status (Herd and Sprott, 1986; Wettemann and Lusby, 1987) and estrous cycli ng status (Schillo et al., 1992; Hall et al., 1995) or as an indirect indi cator of when heifers attain puberty. Heifers with a BCS 5.5 had the greatest estrous response (62.3% ), which was significantly greater compared to heifers with a BCS 5. The estrous response for heifers with a BCS 5.5 is comparable to values observed for Bos taurus heifers synchronized with the Select Synch/CIDR+timed-AI (Lamb et al., 2006) protocol. Moreover, Lucy et al. (2001) reported a 3 d estrous response of 80% in yearling Bos indicus Bos taurus heifers that were going through estr ous cycles when synchronized with a CIDR+PGF 2 protocol. Based on reports in the literature an d the estrous response of heifers with a BCS 5.5 in the present experiment, acceptable estrous responses can be achieved in synchronized yearling Bos indicus Bos taurus heifers but they are probably influenced by pubertal status more than anything else. The onset of puberty is influenced by many factors including age, genotype, and nutrition. Heifers of Bos indicus breeding are known to reach puberty at older ages compared to Bos taurus heifers (Dow et al., 1982 ; Nelsen et al., 1982). Therefore, the low number of heif ers going through estrous cycles at the start of the experiment 96

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was probably the primary reason for the decreased estrous response for the two synchronization treatments, which was probably due to heifers not being the appropriate we ight and age to reach puberty. There could also be environmental effects that are not easily quantifia ble that could have influenced estrous response. In estrous cycling Bos indicus Bos taurus heifers and estrous cycling and prepubertal Bos taurus heifers synchronized with a long term (14 d) MGA + PGF 2 protocol, Bridges et al. (2005) reported similar 3 d estrous responses (< 50%) for both genotypes similar to heifers in the curre nt study. Both the current st udy and Bridges et al. (2005) study were conducted in sub-tropical environment with in a 50 mile radius of each other during a similar time of the year (January to March) Reproductive function has been observed to decrease in Bos indicus Bos taurus cattle in the winter months (Randel, 1984), which could have negatively affected estrous response. Frequent handling of heifers of Bos indicus Bos taurus breeding can also have a negati ve effect on the intensity of estrus (Lemaster et al., 1999) and could have altered the beha vioral and endocrine physiology a ssociated with estrus (Hardin and Randel, 1982). The increased frequency of cattl e handling could have resulted in increased cortisol concentrations that may have had a nega tive effect on LH secre tion (Dunlap et al., 1981) and the preovulatory LH surge (Stoebal and Mo berg, 1979). Another indi cation of effect of stress on reproductive function has recently been observed by Cooke et al. (2008) in prepubertal Bos indicus Bos taurus heifers. They observed that by acclimating heifers to frequent animal handling for three times a week for a 30 d peri od approximately 100 d before the start of the breeding season enhanced the attainment of puberty by the start of the br eeding season compared to non-acclimated contemporaries. 97

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There was a marked increase in conception ra tes for heifers synchronized with the 7-11 (47.0%) compared to the Select Synch/CIDR+t imed-AI treatment (31.3%). Thus the 7-11 treatment may be doing a better job of synchronizing follicle development compared to the Select Synch/CIDR+timed-AI treatment resulting in follicles that are more fertile. The 7-11 protocol is designed to maximi ze follicle development at PGF 2 treatment (Kojima et al., 2001). In contrast, the administration of GnRH at th e initiation of the Sele ct Synch/CIDR+timed-AI protocol probably is not as e ffective at ovulating and synchronizing follicle development since animals are at various stages of follicle deve lopment at CIDR insert ion and GnRH will ovulate fewer follicles (Moreira et al., 2000). Consequently, there is the probability of an increased population of non-ovulated follicles that have extended durations of dominance at CIDR removal resulting in ovulation of aged oocytes of decreased fertility (Mihm et al., 1994). Follicles with prolonged dominance under the influence of e xogenous progesterone had significantly lower pregnancy rates compared with females with a normal ovulatory follicle (Sirois and Fortune, 1990; Stock and Fortune, 1993). Aus tin et al. (1999) reported that restricting the duration of dominance of a pre-ovulatory follicle to 4 d consistently yielded pregnancy rates > 70% in estrous cycling beef heifers. Furthermore, Br idges et al. (2008) repor ted that reducing the duration of a CIDR treatment fr om 7 to 5 d in postpartum Bos taurus beef cows resulted in significant increases in timed-AI pregnancy rates of a Co-Synch pr otocol. They attributed the increased fertility to a decreased duration of dominance of follicles i nduced to ovulate after CIDR removal. Conception rates for the 7-11 treatment were sim ilar to a report by Brid ges et al. (2005) in peripubertal yearling heifers of Bos indicus Bos taurus breeding synchronized with a long term 14 d MGA + PGF protocol. In contrasts, concep tion rate for the Select Synch/CIDR+timed-AI 98

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treatment was approximately 20% less compared to Bridges et al. (2005) study. Moreover, both treatments are considerably less compared to Bos taurus heifers synchronized with variations of the CIDR + PGF protocols where conception ra tes ranged from 60 to 80% (Lamb et al., 2006; Lucy et al., 2001). The conception rates for the 7-11 heifers (47%) were similar to a report by Patterson et al. (1989) in estrous cycling Bos indicus Bos taurus heifers synchronized with either a short term progestogen treatment or unsynchronized controls. Additionally, Patterson et al. (1989) demonstrated that there was a breed by energy level in teraction on conception rates in estrous cycling Bos indicus Bos taurus heifers but not similarly treated Bos taurus heifers. They observed a 30% increase in fertility of the Bos indicus Bos taurus heifers that were on an increasing plane of nutrition at breeding compared to Bos indicus Bos taurus heifers on a low plane of nutrition. Several studies with synchronized Bos taurus heifers have reported that fertility to an observed estrus and AI are simila r between pre-pubertal and estrous cycling heifers (Lucy et al., 2001; Lamb et al., 2006 ). It could be possible that the level of nutrition was not adequate enough to enhance the fertility of the Bos indicus Bos taurus heifers in our study. Body condition had no effect on conception rates across the 7-11 or Select Synch/CIDR+timedAI treatments. Although, BCS can be used as an indicator of both pubert al status and nutrition level, a single record taken at the start of the synchronizati on treatment is probably not an accurate indicator of whether any significant ch anges in the plane of nutrition occurred in the weeks preceding the onset of treatm ent. Therefore, it is not clear what if any effects plane of nutrition and (or) estrous cycling status at the start of trea tment had on conception rates. Timed-AI pregnancy rates were similar betw een the 7-11 and Select Synch/CIDR+timedAI treatments but they were nearly half the values observed for Bos taurus heifers synchronized with the Select Synch/CIDR+timed-AI protocol (Lamb et al., 2006). It should be noted that 99

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there was an effect of interval from PGF 2 to the onset of estrus on conception rate and heifers that exhibited estrus at 60 h after PGF 2 had conception rates that we re nearly twice that of heifers that exhibited estrus < 36, 48 and 72 h. Therefore, the timing of the timed-AI needs to be re-evaluated and it is possible that it should be conducted at 60 h after PGF 2 However, the primary reason that the timed-AI pregnancy rates were so low is probably due to the limited number of heifers that were going through estrous cycles at th e start of the experiment. Furthermore, neither synchronization treatmen t probably induced estrous cycles in the prepubertal heifers, which resulted in a substantial number of heifer s that were still prepubertal at the timed-AI. The 7-11 treatment had a greater synchronized pregnancy rate compared to the Select Synch/CIDR+timed-AI protocol, which was due to the greater estrous response and conception rate of the 7-11 compared to the Select Synch/CIDR+timed-AI. As mentioned previously, the 711 treatment probably was more effective at synchronizing follicle waves allowing a new growing follicle rea dy to ovulate after PGF 2 which resulted in more heifers exhibiting estrus compared to the Select Synch/CIDR+timed-AI. The synchronized pregnancy rates of the 7-11 (33.5%) are similar to those reported by Bridges et al. (2005; 34.5%) in yearling Bos indicus Bos taurus heifers synchronized with the 14 d MGA+PGF 2 protocol with a single PGF 2 treatment. Bridges et al. (2005) significantl y improved the synchronize d pregnancy rate (42.5%) by adding an additional PGF 2 to the treatment, which enhanced the PGF 2 induced luteolysis resulting in greater estrous res ponse and timed-AI pregnancy rates. It should be noted that heifers in the Bridges et al. (2005) study were in later stages of the estrous cycle (day 12 to 15) where PGF 2 is supposed to be more effective in in itiating luteolysis (T anabe and Hahn, 1984; Watts and Fuquay, 1985). A limited amount of research indicates that response to PGF 2 is 100

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decreased when administered early in the estrous cycle compared to the middle and late stages of the estrous cycle in Bos taurus (Tanabe and Hahn, 1984; Watts and Fuquay, 1985) and Bos indicus cattle (Hardin et al., 1980; Sa ntos et al., 1988). Incomple te luteolysis can lead to decreased estrous response, which directly a ffects synchronized pregnancy rates. Whether incomplete luteolysis occurred in both synchronization treatments is not known since no blood samples were taken at PGF 2 and during the days after PGF 2 to confirm if luteolysis occurred. The synchronized pregnancy rates for both treatm ents are considerably less compared to 58% reported by Lamb et al. (2006) in Bos taurus heifers synchronized with Select Synch/CIDR+timed-AI protocol and 49% reported by Lucy et al. (2001) in Bos taurus and Bos indicus Bos taurus heifers synchronized with a CIDR + PGF 2 protocol. The primary reason for the decreased synchronized pr egnancy rates was due to the lo w estrous response, conception rate, and timed-AI pregnancy rate for the 7-11 an d Select Synch/CIDR+timed-AI treatments. All three of the response variables we re probably influenced by estrous cycling status at the start of the synchronization treatment. Reproductive tract score (Anderson et al., 1991; S cale 1 to 5) can also be used as an indirect indicator of pub ertal status in yearling heifers. Heifers with a RTS of 1 and 2 are considered pre-pubertal, while he ifers with a RTS of 3 are appr oximately 1 mo from initiating estrous cycles, and heifers with a RTS of 4 or 5 ar e considered to be estr ous cycling. Therefore, conducting a RTS at the initiation of the experime nt was used to provide an estimate of the pubertal status of the heifers before the start of the synchronization tr eatment. Although, RTS had an effect on estrous response, conception ra te, synchronized pregnancy rate, and thirty-day pregnancy rate regardless of synchronization treatment, the magnitude in differences between response variables across the different RTS categorie s was less than anticipat ed. Patterson et al. 101

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(2000) reported significant incr eases in estrous response in Bos taurus heifers as RTS increased from a 1 to 3 and reaching a plateau for RTS 4 and 5, which is similar to our observations. It is not surprising that the conception rate fo r the RTS 1 and 2 was only 6.7%, which was significantly less compared to heifers with RTS 3. However, what was unexpected was that conception rates did not increase as RTS increased from a 3 to either a 4 or 5 as observed in synchronized yearling Bos taurus heifers (Herd and Sprott, 1986). Additionally, it was unexpected that synchronized pregnancy rates were similar for heifers with a RTS of 3 compared to RTS of 4 and 5 as synchronized pregnancy rate s typically increase as RTS increase from a 3 to a RTS of 4 or 5 (Brown et al., 1988). However, thirty day pregnancy rates tended to be greater for heifers with a RTS of 4 and 5 compared to RTS of 3, which were all greater compared to RTS 1 and 2. The results from this study suggest that RTS may not be an effective indictor of pubertal status in yearling heifers of Bos indicus Bos taurus breeding compared to using it to estimate pubertal status in yearling Bos taurus heifers. Additional research needs to be conducted to determine if RTS can be used as an accurate predictor of pubertal status based on both blood progesterone and tran srectal ultrasonography data to confirm the activity and presence of luteal tissue in yearling heifers of Bos indicus Bos taurus breeding. In summary, the 7-11 treatment provided increas ed estrous response, conception rates, and synchronized pregnancy rates compared to th e Select Synch/CIDR+timed-AI treatment. Synchronization treatments had similar timedAI pregnancy rates. Estrous response was directly affected by BCS at start of synchronization but BCS had no effect on any of the other response variables. Although, RTS had significant affects on estrous response, conception rate, synchronized pregnancy rate and thirty day pregnancy rate, the difference was primarily between heifers with RTS of 1 and 2 compared to heifers with a RTS of 3, 4, and 5. 102

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Implications The 7-11 synchronization treatment resu lted in more yearling heifers of Bos taurus Bos indicus breeding pregnant to the synchronize d breeding compared to the Select Synch/CIDR+timed-AI protocol; however, the s ynchronized pregnancy ra tes are less compared to Bos taurus heifers. The role that RTS and BCS play as predictors of pubertal status in Bos indicus Bos taurus heifers when synchronizing remains unclear. However, considerations must be made for timing of initiation of synchronization in heifers especially for Bos taurus Bos indicus heifers as they reach pub erty at later ages. 103

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Table 4-1. Physical description of Bos indicus Bos taurus yearling heifers including reproductive tract score (RTS), body conditi on score (BCS), and breed composition for heifers synchronized with two pr ogestogen based estr ous synchronization protocols (LSMeans SE). Location n RTS a BCS b Breed composition c 1 61 3.8 0.14 5.5 0.05 Brangus (n=61) 2 119 3.6 0.10 5.0 0.04 Brangus (n=9) Bos indicus crossbred (n=110) 3 230 3.7 0.07 5.0 0.03 Bos indicus crossbred (n=230) a Reproductive tract score:1 = non -cycling, 5 = es trous cycling. b Body condition score: 1 = emaciate d, 5 = moderate; 9 = very fat. c Brangus = 3/8 Brahman 5/8 Angus; Bos indicus crossbred approximately 1/4 to 1/2 Bos indicus breeding with remainder being Bos taurus breeding. 104

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a) 7-11 treatment b) Select Synch/CIDR +timed-AI treatment PGF2 PGF2 GnRH GnRH + timed-AI Estrus detect & AI MGA 0.5 m g /head/ d Supplement no MGA PGF2 GnRH CID R Estrus detect & AI GnRH + time d -AI 0 7 11 18 21 Day of experiment Figure 4-1. Experimental design evalua ting the effects of two progestogen based synchronization treatments in yearling Bos indicus Bos taurus heifers. a) 7-11 treatment was administered MGA (0.5 mg/hd/d) for 7 d with PGF 2 (25 mg, i.m.) on last day of MGA. GnRH (500 g, i. m.) was administered on day 11 and PGF 2 was administered on day 18. b) Select Synch/CIDR+timed-AI treatment received the same carrier supplement without MGA from day 0 to 7. On day 11 a CIDR (Eazi-Breed CIDR ) was inserted concomitant with GnRH. The CIDR was removed on day 18 concurrent with PGF 2 For both treatments, estrus was detected for 3 d and heifers were insemina ted 6 to 12 h after a detected estrus. Heifers not exhibiting estrus by 72 h were timed-AI and received GnRH between 72 to 76 h. 105

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Table 4-2. Effectiveness of the 7-11 treatment compared to Select Sync/CIDR+timed-AI (SSC+TAI) synchronization treatment on es trous response, conception rate, timedAI pregnancy rate, synchronized pregnancy rate, and thirty day pregnancy rate in yearling heifers of Bos indicus Bos taurus breeding. a Variable 7-11 SSC+TAI Odds Ratio g 95% CI h P-value Estrous response, % b 55.2 (117/212) 41.9 (83/198) 0.60 0.40-0.89 0.01 Conception rate, % c 47.0 (55/117) 31.3 (26/83) 0.51 0.29-0.93 0.03 Timed-AI pregnancy rate, % d 16.8 (16/95) 20.0 (23/115) 1.23 0.61-2.5 0.56 Synchronized pregnancy rate, % e 33.5 (71/212) 24.8 (49/198) 0.65 0.43-1.00 0.05 Thirty day pregnancy rate, % f 65.7 (138/210) 60.4 (119/197) 0.80 0.53-1.19 0.27 a Refer to Figure 4-1 for details of treatments. b Percentage of heifers disp laying estrus 72 h after PGF 2 of the total treated. c Percentage of heifers pregna nt to AI of the total that exhibited estrus and were AI. d Percentage of heifers that failed to display estrus, were tim ed-AI, and became pregnant of the total number of heifers 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 bree ding season of the total number of heifers treated. g 7-11 treatment used as referent value h 95% confidence interval 106

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Table 4-3. Effect of body condition score (BCS) on estrous response in Bos indicus Bos taurus yearling heifers. BCS a Estrous response, % b Odds Ratio 95% CI c P-value 4.5 37.5 (30/80) 0.37 0.20-0.67 0.001 5 46.4 (104/224) 0.54 0.33-0.87 0.01 5.5 62.3 (66/106) Referent a Body condition score: 1 = emaciate d, 5 = moderate; 9 = very fat. b Percentage of heifers disp laying estrus 72 h after PGF 2 of the total treated. c 95% confidence interval 107

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Figure 4-2. Conception rates for th e different interval from PGF 2 to onset of estrus categories in yearling heifers of Bos indicus Bos taurus breeding. Means are expressed as a percentage of total that ex hibited estrus and became pregnant. Means without a common superscript differ ( P < 0.05). Interval from PGF 2 to onset of estrus ( P < 0.05) and synchronization treatment interval from PGF 2 to onset of estrus ( P > 0.05). 108

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Table 4-4. Effect of reproduc tive tract score (RTS) on estr ous response, conception rate, timed-AI pregnancy rate, synchronized pre gnancy rate and thirty day pregnancy rate in yearling heifers of Bos indicus Bos taurus breeding. a Variable RTS b Means Odds Ratio 95% CI c P-value 2 28.3 (15/53) 0.30 0.16-0.58 0.0004 3 44.1 (56/127) 0.60 0.39-0.94 0.02 Estrous response, % d 4 56.1 (129/230) Referent 2 6.7 (1/15) 0.07 0.10-0.60 0.01 3 44.6 (25/56) 1.15 0.59-2.20 0.68 Conception rate, % e 4 42.6 (55/129) Referent 2 7.9 (3/38) 0.34 0.09-1.24 0.10 3 22.5 (16/71) 1.22 0.57-2.58 0.61 Timed-AI pregnancy rate, % f 4 19.8 (20/101) Referent 2 7.6 (4/53) 0.17 0.06-0.48 0.001 3 32.3 (41/127) 0.97 0.61-1.55 0.91 Synchronized pregnancy rate, % g 4 32.6 (75/230) Referent 2 32.1 (17/53) 0.22 0.07-0.68 0.008 3 59.1 (75/127) 0.49 0.24-1.0 0.06 Thirty day pregnancy rate, % h 4 72.6 (167/230) Referent a Refer to Figure 4-1 for details of treatments. b Reproductive tract score (Scale 1-5) c 95% confidence interval d Percentage of heifers disp laying estrus 72 h after PGF 2 of the total treated. e Percentage of heifers pregna nt to AI of the total that exhibited estrus and were AI. f Percentage of heifers that faile d to display estrus, were timed-A I, and became pregnant of the total number of heifers that were timed-AI. g Percentage of heifers pre gnant during the synchronized br eeding of the total treated. h Percentage of heifers pregnant during the first 30 d of the breed ing season of the total number of heifers treated. 109

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CHAPTER 5 CONCLUSIONS AND IMPLICATIONS Incorporation of estrous synchronization into an AI program decrea ses the amount of time spent detecting estrus and can eliminate estrous detection when a fixed insemination time is used. Additional benefits of using estrous synchronization includes prom oting estrous cyclicity in some non-cycling cattle, incr easing the percentage of cattle pregnant early in the breeding season, shortening the breeding season, shortening the subsequent calving season, and increasing the uniformity of the calf crop. A majority of the research and development of estrous synchronization protocols is based on the Bos taurus genotype. Furthermore, limited research indicates that Bos indicus and Bos indicus Bos taurus cattle have subt le physiological and behavioral differences that can elicit different responses to estrous synchronization products compared to Bos taurus cattle. Consequently, the primary objective of the rese arch reported in this thesis focused on evaluating the effectivenes s of two estrous synchronization protocols in cattle of Bos indicus Bos taurus breeding. In chapter 3, two experiments were conduc ted to evaluate the effectiveness of two prostaglandins, cloprostenol sodium (clopros tenol) compared to dinoprost tromethamine (dinoprost), when used in either the Select Synch/CIDR+timed-AI or the 7-10 synchronization programs. In Experiment 1, suckled postpartum Bos indicus Bos taurus cows were synchronized with the protoc ols and yearling heifers of Bos indicus Bos taurus breeding were used in Experiment 2. For both the suckled postpartum cows and the yearling heifers, prostaglandin treatment had no effect on any of the response variables evaluate d. This is similar to reports in cattle of Bos indicus breeding as well as Bos taurus breeding. It was origina lly hypothesized that the cloprostenol, which is a prostaglandin analog, may have been more effective compared to the 110

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natural prostaglandin, dinoprost. However, this does not appear to be the case based on the results of our experiment. Therefore, based on the limited number of animals tested in these experiments as well as the reports in the litera ture, it appears that a ny subtle difference in dynamics of CL regression between the two luteol ytic drugs is not sufficient to alter either estrous response or pregnancy rate s. Hence, producers can use either type of prostaglandin without any reduction in re productive performance. For the suckled cows in Experiment 1, estrous response, conception rate, and synchronized pregnancy rate were affected (P < 0.05) by synchronizati on treatment as the Select Synch/CIDR+timed-AI treatment ha d greater (P < 0.05) estrous re sponse, conception rate, and synchronized pregnancy rate compar ed to the 7-10 treatment. Additionally, the synchronized pregnancy rates of both the 7-10 and Select Synch/CIDR +timed-AI treatments are considerably less compared to reports in suckled Bos taurus cows synchronized with either the 7-11 or Select Synch/CIDR+timed-AI protocols. The primary reasons for the decreased response of the 7-10 compared to the Select Synch/CIDR+timed-AI treatment as well as the decr eased response of both treatmen ts compared to similarly synchronized suckled Bos taurus cows is due to a decrease in the estrous response, conception rates, and timed-AI pregnancy rates. All of the aforementioned factors acted in concert and resulted in less than acceptable sync hronized pregnancy rates in the suckled Bos indicus Bos taurus cows. The first question that needs to be addressed is why the estrous response and conception rates were significantly reduced in the 7-10 compared to the Select Synch/CIDR+timed-AI treatment. One reason fo r the decreased estrous response for the 7-10 cows could be the pre-synchroni zation portion of the 7-10 treatme nt is not creating the same synchrony of follicle development of the follicl es being ovulated to GnRH compared to the 111

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traditional 7-11 protocol, where MGA is used instead of a once-used CIDR. Since ovulation rate to GnRH was not determined in the present st udy, it is not known how effective GnRH was in ovulating follicles after removal of the once-used CIDR or if th e timing of the GnRH treatment relative to CIDR removal was appropriate. Type of progestogen treatment and concentration of circulating progestogen can alter follicle growth and developm ent, which could have also resulted in asynchronous follicle development at Gn RH. Therefore, if ovulation did not occur in a majority of cows at GnRH in the 7-10 treatment, follicle development would have been asynchronous at PGF 2 resulting in a decreased estrous resp onse compared to the Select Synch CIDR+timed-AI treatment. Because cattle of Bos indicus influence have an increased incidence of three and four wave follicle development patterns, additional research needs to be conducted to determine if follicle wave patterns influence how cattle of Bos indicus breeding respond to synchronization systems. The second question that needs to be investigated is why cattle of Bos indicus influence consistently have decreased estrous response to synchronization treatments compared to Bos taurus cattle synchronized with similar protocols. One factor that had a significant effect on estrous response in Experiment 1 was body condition sc ore (BCS) of the cattle at the start of the synchronization treatments. In summary, as BCS increased from 4.5 to a 5, there was a significant increase in estrous res ponse. Although estrous cycling status was not determined in the present study, suckled cows with a BCS > 5 are ty pically cattle that are e ither close to or are going through estrous cycles. Therefore, it is imp erative for producers that want to use estrous synchronization and AI that their cattle are in good BCS at the start of the breeding season in order to get cattle to exhibit estrus and achieve acceptable pregnancy rates. Additionally, it is not clear what effect environment has on estrous response, since there ar e a limited number of 112

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studies that actually evaluate the effect of environment on response to synchronization protocols in both cattle of Bos indicus and Bos taurus breeding under similar environmental conditions. This would include investigating not only how environmental factors effect an animals physiology but also their behavior in response to environmental stressors. Conception rates were also influenced by synchronization treatment as the 7-10 had decreased conception rates compared to the Select Synch/CIDR+timed-AI treatment. It is not clear why conception rates were decreased in the 7-10 treatment. Based on how the 7-10 treatment is designed, pretreatment with the onc e-used CIDR followed 3 d later by GnRH should have worked to synchronize follicle development for the subsequent PGF 2 treatment. The newly developed follicles should have had an increased fertility compared to aged dominant follicles or persistent dominant follicles, which could occur in some Select Synch/CIDR+timedAI treated cows. Additionally, the hormonal envi ronment that follicles were exposed to during the GnRH + PGF portion of the 7-10 treatment were certainly different compared to the Select Synch/CIDR+timed-AI treatment. Whether progesterone from the CIDR affected the follicle development pattern of the 7-10 treatment enough to decrease the subseque nt conception rates is unclear. Conception rates of the 7-10 treatmen t were considerably less compared to those observed in suckled Bos taurus and Bos indicus Bos taurus cows synchronized with a 7-11 protocol (Esterman et al., 2007b). It is importa nt to note that acceptable conception rates can be achieved in suckled Bos indicus Bos taurus that exhibit estrus and inseminated as reflected by the conception rates of the Select Synch/CIDR+t imed-AI treatment, which are comparable to reports in Bos taurus cows synchronized with the same protocol. As w ith estrous response, BCS had a significant effect on conception rates, as cows with a BCS 5, had approximately a 30% greater conception rate comp ared to cows with a BCS 4.5. Body condition scor e is an indirect 113

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measure of nutritional status a nd has a direct influence on reproductive function and estrous cycling status of suckled beef cows. Cows th at receive enough pre-pa rtum nutrition have an excellent opportunity of return ing to estrus within 60 d afte r calving and get pregnant to a synchronized AI. Therefore, if producers want to achieve acceptable pregnancy rates to a synchronized AI, they must make sure that their cattle are in adequate body condition at the start of the synchronization treatments. However, producers will have to determine if the extra cost required for the increased nutrition needed to keep cows in good body condition at the start of the breeding season can be offset by the price obtained for the AI calves that will be produced by the synchronized breeding. Treatment had no effect on timed-AI pre gnancy rates across the two synchronization treatments. However, timed-AI pregnancy rates for both treatments are approximately 6 to 20% less compared to reports for Bos taurus cattle synchronized with similar synchronization protocols. As previously di scussed with estrous response and conception rate, asynchrony of follicle development at the time of PGF 2 could have been part of the reason for the decreased timed-AI pregnancy rate. It is po ssible that follicles were not of the correct size to ovulate to GnRH, were infertile follicles when ovulating to GnRH, or cows were still anestrous and had follicles of inadequate size to ovulate to GnRH Timed-AI pregnanc y rates across the two treatments were also influenced by DPP at the st art of the synchronization treatment. Timed-AI pregnancy rate increased by 16% in cows 60 DPP compared to cows at or below 43 DPP. In general, as the interval from calving to start of the breeding season increa ses, the number of cows going through estrous cycles also increases. Therefore, some of th e decrease in timed-AI pregnancy rates could be attributed to increased incidence of anestrous compared to estrous cycling cows. Another possible re ason for the decreased timed-AI pregnancy rates could be that 114

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the timing of the timed-AI + GnRH treatment is not synchronized correctly with the follicle development after CIDR removal. A recent study in our lab with suckled Bos indicus Bos taurus cows synchronized with a Select Synch/CIDR protocol demonstrated that conception rate peaked for cows that exhibi ted estrus at 60 h post PGF 2 and conception rates decreased significantly for cows that exhibite d estrus after 60 h. Therefore, further research is needed to evaluate if the timed-AI should be conduc ted at 60 h instead of 72 h after PGF 2 Synchronization treatment had a significant e ffect on synchronized pregnancy rate as a greater number of Select Synch/CIDR+timed-AI cows became pregnant to the synchronized breeding compared to the 7-10 treatment. Additionally, the s ynchronized pregnancy rates for both treatments were less compar ed to similarly synchronized Bos taurus cows. As already discussed, a reduction in estrous response and conception rate of the 7-10 treatment were the primary reasons for the decreased synchronized pregnancy rates for the 7-10 compared to the Select Synch/CIDR+timed-AI treat ment in the present experiment as well as the decreased response compared to similarly synchronized Bos taurus cows. Nevertheless, acceptable synchronized pregnancy rates can be achieved in suckled Bos indicus Bos taurus cows synchronized with the Select Synch/CIDR+timed-A I protocol if cows are of adequate BCS and DPP at the start of the breeding season. The r eason(s) for the less than acceptable synchronized pregnancy rates for the 7-10 treatment are no t clear, and its practicality as a low cost synchronization protocol is questionable. Future studies need to evaluate what effect the onceused CIDR has on follicle development and if an asynchrony in follicle development after CIDR removal effects the response to GnRH administer ed 3 d after CIDR removal. In conclusion, it appears that the major factors contributing to th e success of the synchroni zation treatments used in the present study are BCS and DPP. Therefore, producers need to pay particular attention to 115

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managing body condition prior to the start of th e synchronization treatment and wait longer after calving before cows are started on a synchronization treatment to take advantage of the DPP effect. Future studies need to fully characterize the effectiveness of the two treatments ability to induce estrous in anestrous suckled Bos indicus Bos taurus cows, with an emphasis on the Select Synch/CIDR+timed-AI protoc ol. Furthermore, intensive studies need to be conducted in Bos indicus Bos taurus and Bos taurus cows in similar environments to completely characterize follicle wave patterns between the genotypes and to determine if they have any effect on response to the Select Synch/CIDR+timed-AI protocol. In Chapter 3, Experiment 2, yearling heifers over two consecutive year s were synchronized with the same estrous synchronization protocol s as Experiment 1. The estrous response, conception rate, timed-AI pregna ncy rate, synchronized pregnancy rate, and thirty day pregnancy rate were similar (P > 0.05) between the 7-10 and Select Synch/CIDR+timed-AI treatment and between cloprostenol and dinopros t treatment. However, there was a treatment by interval from PGF 2 to the onset of estrus (P < 0.05; Figure 32) effect on conception rate. Conception rates were similar (P > 0.05) between treatments for heifers that exhibited estrus 48 h after PGF 2 However, the Select Synch/CIDR+timed-AI treat ment tended (P = 0.10) to have a decreased conception rate compared to the 7-10 heifers that exhibited estr us at 60 h after PGF 2 Whereas, the Select Synch/CIDR+timed-AI treatment ha d an increased (P < 0.05) conception rate compared to the 7-10 treatment for heifer s that exhibited estrus 72 h after PGF 2 In Experiment 2, there were no synchroniza tion (7-10 vs. Select Synch/CIDR+timed-AI) or PGF 2 treatments (dinoprost vs. cloprostenol), a ffects on estrous respon se, conception rate, timed-AI pregnancy rate, synchronized pregnanc y rate or thirty day pregnancy rate for Bos indicus Bos taurus yearling heifers. This is in stark contrast to the results obtained for the 116

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same synchronization treatments used on suckled cows in Experiment 1. Similar to results observed for cows of Experiment 1, estrous response and timed-AI pregnancy rates were slightly less in the Bos indicus Bos taurus heifers compared to reports in Bos taurus and Bos indicus Bos taurus heifers synchronized with a 7 d CIDR with PGF 2 provided the day before CIDR removal. However, conception and synchronized pregnancy rates were similar compared to what has been observed in estrous cycling Bos taurus heifers. Although, estrous cycling status was not determined in the present study, the final results suggest that most of the heifers were pubertal and responded favorably to either synchronization treat ment and producers have the option to use either synchroniza tion system. There have been no studies evaluating the 7-11 protocol in yearling heifers of Bos indicus Bos taurus breeding. However, heifers treated with the 7-10 protocol had only slig htly decreased concep tion and pregnancy rates compared to long term MGA based protocols in Bos taurus heifers. As observed with the cows in Experiment 1, it appears that a decreased estrous response is one of the limiting factors that decrease the overall effectiveness of synchronizat ion systems in heifers of Bos indicus Bos taurus breeding compared to yearling Bos taurus heifers. Additional studies ne ed to be conducted in heifers of Bos indicus Bos taurus breeding to determine the relationship between follicle development both during and after a synchroni zation treatment on the expression of estrus and subsequent conception rates. Because there was a treatment by interval from PGF 2 to onset of estrus effect on conception rate, it may be beneficial to de termine when the timed-AI should be performed relative to the PGF 2 treatment for both the 7-10 and Select Synch/CIDR+timed-AI treatments. It will also be of interest to determine if es trous detection can be e liminated, and only a fixed timed-AI can be used in yearling heifers of Bos indicus Bos taurus breeding. 117

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In Chapter 4, yearling Bos indicus Bos taurus heifers were synchronized with either the Select Synch/CIDR+timed-AI or 7-11 treatment. Heifers were distributed to treatment by reproductive tract score (RTS) and BCS. The 7-11 heifers had greater (P < 0.05) estrous response, conception rate, and synchronized pregnancy rate compared to Select Synch/CIDR+timed-AI heifers. Heifers exhibi ting estrus at 60 h had a greater (P < 0.05) conception rate compared to heifer s that had exhibited estrus at 36, 48, and 72 h, which were similar (P > 0.05) to each other. As RTS increased from 2 to a 3, estrous response, conception rate, synchronized pregnancy rate, and thirty day pregnancy ra te all increased (P < 0.05). Estrous response was greater for 7-11 heifer s compared to Select Synch/CIDR+timed-AI heifers. The difference observed between tr eatments could be due to the 7-11 treatment synchronizing follicular wave development more effectively compared to the Select Synch/CIDR+timed-AI treatment. The MGA in combination with PGF 2 allows most heifers to have a large dominant follicle present at MGA w ithdrawal that is ready to be ovulated by GnRH; thereby, synchronizing follicle development for the subsequent PGF 2 and thereby improving the estrous response. The Select Synch/CIDR+tim ed-AI protocol is probably not as effectively synchronizing follicle development since heifers are at all stages of follicle development at GnRH and CIDR insertion, which probably resu lts in asynchronous follicle development and a less synchronous estrus. The estrous respons e for both treatments are considerably less compared to studies in yearling Bos taurus and Bos indicus Bos taurus heifers synchronized with either the Select Synch/CIDR+timed-AI, short term proge stogen protocols, and CIDR + PGF 2 protocol. The decreased estrous response is probably due to a combination of heifer genotype and the numbers of heifers that are pubertal at the start of the synchronization 118

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treatment, which was probably influenced by level of nutrition. Although, estrus is more difficult to detect in cattle of Bos indicus breeding due to a decreased expression and duration of estrus as well as an increased incidence of silent estrus, the use of estrous detection aids probably made up for any deficiencies that occurred in estrous detection e fficiency. Since blood samples were not taken, it is not known how many heif ers were pubertal at the start of the synchronization treatment. Additionally, it is not known how effective the synchronization treatments were in inducing estrous cycles in the pre-pubertal heif ers. Body condition score, an indirect indicator of nutritional status and estrous cycling status, had a significant effect on estrous response as estrous response increased to acceptable levels in heifers that had a BCS 5.5 at the start of the treatment. Consequentl y, the low number of estr ous cycling heifers was probably the primary reason for the decreased estrous response for the two synchronization treatments in the present study. There was a marked increase in conception ra tes for heifers synchronized with the 7-11 compared to the Select Synch/CIDR+timed-AI tr eatment. This suggest that the 7-11 treatment may be doing a better job of synchronizing fo llicle development compared to the Select Synch/CIDR+timed-AI treatment resulting in follic les that are more fertile. The duration of follicle dominance is negatively associated with follicle fertility as follicles with prolonged durations of dominance have significantly decr eased fertility compared to follicles that are ovulated with a normal duration of dominance. Ther efore, it is possible that a greater number of 7-11 heifers had newly developed follicles presen t at CIDR removal compared to the Select Synch/CIDR+timed-AI treatment, which resulted in greater fertility for the 7-11 treatment. Additionally, conception rates for both treatments are considerably less compared to Bos taurus heifers synchronized with variations of the CI DR + PGF protocols were the conception rates 119

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ranged from 60 to 80%. For that reason, additional research n eeds to be conducted to fully characterize the effects of prolonged follicle dominance on fertility in yearling heifers of Bos indicus Bos taurus breeding and to determine why there is such a significant reduction in conception rates in yearling heifers of Bos indicus Bos taurus compared to Bos taurus heifers. Timed-AI pregnancy rates were similar betw een the 7-11 and Select Synch/CIDR+timed-AI treatments but they were nearly half the values observed for Bos taurus heifers synchronized with the Select Synch/CIDR+ timed-AI protocol and yearling Bos indicus Bos taurus heifers synchronized with the 14 d MGA + PGF 2 protocol. The primary reason for decreased timed-AI pregnancy rates is probably due to the decreased number of he ifers going through estrous cycles at the start of the synchronization treatment resu lting in a significant number of pre-pubertal heifers at the timed-AI. The low timed-AI pregna ncy rates also suggest that the synchronization treatments did not induce estrous cycles in many of the pre-pubert al heifers. It should also be noted that there was an effect of interval from PGF 2 to the onset of estrus on conception rate and heifers that exhibited estrus at 60 h had conception rates that were nearly twice heifers that exhibited estrus < 36, 48 and 72 h. Therefore, when the timed-AI is performed needs to be reevaluated and it may be beneficial to conduct it at 60 h after PGF 2 instead of 72 h. The 7-11 treatment had greater synchronized pregnancy rates compared to the Select Synch/CIDR+timed-AI treatment and the primary reasons were that th e 7-11 treatment had a greater estrous response and conception rate comp ared to the Select S ynch/CIDR+timed-AI. The 7-11 treatment was probably more effective at synchronizing follicle waves allowing a new follicle to be ready to ovulate after PGF 2 compared to the Sel ect Synch/CIDR+timed-AI treatment. With that said, synchronized pregna ncy rates for both treatments were considerably less compared to what has been observed in Bos taurus heifers synchronized with similar 120

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synchronization protocols. The primary reason for the decreased synchronized pregnancy rates is probably due to the small percentage of heif ers that had reached pubert y at the start of the treatment. Therefore, additional experiments need to be conducted to determine how effective the 7-11 and Select Synch/CIDR+timed-AI treatme nts are synchronizing follicle development as well as how effective the treatments are for induc ing estrous cycles in pre-pubertal heifers. Utilization of the RTS at the start of the experi ment was used to try and predict the estrous cycling status of the heifers w ithout having to work the heifers an additional time to take blood sample 10 d before the start of the experiment. In general, as RTS increased from a 1 or 2 to 3, the estrous response, conception rate, and synchr onized pregnancy rate increased. However, what is interesting is that the reproductive performance did not incr ease significantly as RTS increased from a 3 to 4, with the exception of estrous resp onse, although this increase was only 12%. One would have expected a more si gnificant increase in estrous response and synchronized pregnancy rate for heifers with an RTS of 4 and 5, which should be pubertal heifers compared to prepubertal heifers with RTS 3. Utilization of RTS is an often used management practice in yearling heifers of Bos taurus breeding to predict future reproductive performance and to cull heifers (i.e., RTS 1 and 2) that will have substandard reproductive performance. Consequently, it appears that we need to re-evaluate the manner in which RTS is used to predict potential response to a synchronization treatment in cattle of Bos indicus Bos taurus breeding. In summary, the 7-11 treatment provided increas ed estrous response, conception rates, and synchronized pregnancy rates compared to the Se lect Synch/CIDR+timed-AI treatment. Estrous response was directly affected by BCS at start of synchronization treatme nt, but interestingly enough, BCS did not affect the overall reproductive performance of the heifers. Although RTS 121

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had significant affects on estr ous response, conception rate, a nd synchronized pregnancy rate, using RTS to predict the potential response to a synchronization treatment was questionable. Results from this experiment demonstrate the importance of having heifers either going through estrous cycles or near the onset of puberty before the start of the breeding season in order to achieve acceptable synchronized pregnancy rates. The synchronization treatments used in the current study will need to be further tested in a population of heifers of known estrous cycling status to determine the tr ue response to treatment. Relative to yearling heifers of Bos indicus Bos taurus breeding, future research needs to investigate the reasons for the less than accep table estrous response and conception rates observed in heifers synchronized with either the 7-11 or Select Synch/CIDR+timed-AI treatments. This includes a complete characterization of the effectiveness of synchronization treatments in peri-pubertal heifers as determined by blood progeste rone concentrations. Because nutrition can have a significant e ffect on reproductive performance, it will be also be important to determine what effect nutritional status and different nutritional management strategies have on conception rates in synchronized heifers. It will also be important to characterize the utilization of RTS as a reproductive management t ool and to determine its use as an indicator of a heifers breeding ability based on the heifers weight and age at the start of the breeding season. Environmental factors such as temper ature, humidity, plane of nutrition, and animal handling and management can all influence the endocrine, reproductive, a nd behavioral factors associated with the expression of estrus as well as the overall treatment response. Future research must focus on identifying the signifi cance of these variables and determine which one(s) can be controlled and (or) modified to enhance the onset of puberty and response to a synchronization treatment. 122

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APPENDIX A SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 3 Table A-1. Effect of synchr onization treatment and prostaglandin treatment on estrous response, conception rates and preg nancy rates in suckled cows of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Select Synch/CIDR+timed-AI (SSC+TAI) treatmen t with either Cloprostenol sodium (Cloprostenol) or Dinoprost tromethamine (Dinoprost). a Variable Estrous response, % b Conception rate, % c Timed-AI pregnancy rate, % d Synchronized pregnancy rate, % e 7-10 49.0 (77/157) 45.5 (35/77) 17.5 (14/80) 31.2 (49/157) SSC+TAI 59.9 (100/167) 62.0 (62/ 100) 20.9 (14/67) 45.5 (76/167) P -value < 0.05 < 0.05 > 0.05 < 0.05 Cloprostenol 57.0 (94/165) 48.9 ( 46/94) 23.9 (17/71) 38.2 (63/165) Dinoprost 52.2 (83/159) 61.5 (51/83) 14.5 (11/76) 39.0 (62/159) P -value > 0.05 > 0.05 > 0.05 > 0.05 a See Figure 3-1 for details of treatments. b Percentage of cows displaying estrus 72 h after PGF 2 of the total treated. c Percentage of cows pregnant to AI of th e total that exhibited estrus and were AI. d Percentage of cows pregnant to time d-AI of the total that were timed-AI. e Percentage of cows pregnant during the synchronized breeding of the total treated 123

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Table A-2. Estrous, conception and pregnancy rates by prostagl andin treatment of suckled cows of Bos indicus Bos taurus breeding synchronized with either a modified 711 (7-10) or Select Synch/CIDR+timed -AI (SSC+TAI) treatment with either Cloprostenol sodium (Cloprostenol) or Dinoprost tromethamine (Dinoprost). a Variable n Estrous response, % b Conception rate, % c Timed-AI pregnancy rate, % d Synchronized pregnancy rate, % e 7-10 Cloprostenol 81 58.0 (47/81) 40.4 (19/47) 20.6 (7/34) 32.1 (26/81) 7-10 Dinoprost 76 39.5 (30/76) 53.3 (16/30) 15.2 (7/46) 30.3 (23/76) SSC+TAI Cloprostenol 84 56.0 (47/84) 57.5 (27/47) 27.0 (10/37) 44.1 (37/84) SSC+TAI Dinoprost 83 63.9 (53/83) 66.0 (35/53) 13.3 (4/30) 47.0 (39/83) P -value < 0.05 > 0.05 > 0.05 0.06 a See Figure 3-1 for details of treatments. b Percentage of cows displaying estrus 72 h after PGF 2 of the total treated. c Percentage of cows pregnant to AI of th e total that exhibited estrus and were AI. d Percentage of cows pregnant to time d-AI of the total that were timed-AI. e Percentage of cows pregnant during the synchronized breeding of the total treated. 124

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Figure A-1. Effect of 7-10 or Select Synch/CIDR+timed-AI (SSC) treatment on estrous response in suckled postpartum Bos indicus Bos taurus cows. Means are expressed as a percentage of total th at exhibited estrus. 125

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Figure A-2. Effect of 7-10 or Select Synch/CIDR+timed-AI (SSC) synchronization treatment and interval from PGF 2 to onset of estrus on concepti on rate in suckled postpartum Bos indicus Bos taurus cows. Means are expressed as a percentage of total that exhibited estrus and became pregnant. 126

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Table A-3. Estrous response, conception and pregnanc y rates by synchronization treatment of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Se lect Synch/CIDR+timed-AI (SSC+TAI) treatment with either Cloprostenol sodium (Cloprostenol ) or Dinoprost tromethamine (Dinoprost) for years 1 and 2 Experiment2. a Variable Estrous response, % b Conception rate, % c Timed-AI pregnancy rate % d Synchronized pregnancy rate, % e 7-10 65.2 (116/178) 62.1 (72/116) 14.5 (9/62) 45.5 (81/178) Year 1 71.6 65.4 9.68 49.5 Year 2 55.1 55.3 19.4 39.1 SSC +TAI 68.4 (121/177) 67.8 ( 82/121) 17.9 (10/56) 52.0 (92/177) Year 1 64.2 71.4 20.5 53.2 Year 2 75.0 62.7 11.8 50.0 Sync p > 0.05 p > 0.05 p > 0.05 p > 0.05 Year p > 0.05 p > 0.05 p > 0.05 p > 0.05 Sync Year p < 0.05 p > 0.05 p > 0.05 p > 0.05 a See Figure 3-1 for details of treatments. b Percentage of heifers displaying estrus 72 h after PGF 2 of the total treated. c Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pregnant to timed-AI of the total that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated. 127

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Table A-4. Estrous response, conception and pregnancy rates by prostaglandin treatment of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Se lect Synch/CIDR + timed-AI (SSC+TAI) treatment with either Cloprostenol sodium (Cloprostenol ) or Dinoprost tromethamine (Dinoprost) for years 1 and 2 Experiment 2. a Variable Estrous response, % b Conception rate, % c Timed-AI pregnancy rate, % d Synchronized pregnancy rate, % e Cloprostenol 68.7 (123/179) 67.5 ( 83/123) 17.9 (10/56) 52.0 (93/179) Year 1 70.6 71.4 18.8 56.0 Year 2 65.7 60.9 16.7 45.7 Dinoprost 64.8 (114/176) 62.3(71/ 114) 14.5 (9/62) 45.4 (80/176) Year 1 65.1 64.8 13.2 46.8 Year 2 64.2 58.1 16.7 43.3 Prostaglandin p > 0.05 p > 0.05 p > 0.05 p > 0.05 Year p > 0.05 p > 0.05 p > 0.05 p > 0.05 Prostaglandin Year p > 0.05 p > 0.05 p > 0.05 p > 0.05 a See Figure 3-1 for details of treatments. b Percentage of heifers displaying estrus 72 h after PGF 2 of the total treated. c Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pregnant to timed-AI of the total that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated. 128

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Table A-5. Estrous response, conception a nd pregnancy rates of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a modified 7-11 (7-10) or Select Synch/CIDR + timed-AI (SSC+TA I) treatment with either Cloprostenol sodium (Cloprostenol) or Dinoprost trom ethamine (Dinoprost) for years 1 and 2 Experiment 2. Variable n Estrous response, % b Conception rate, % c Timed-AI pregnancy rate, % d Synchronized pregnancy rate, % e 7-10 Cloprostenol 90 71.1(64/90) 62.5 (40/64) 15.4 (4/26) 48.9 (44/90) Year 1 55 78.2 69.8 8.33 56.4 Year 2 35 60.0 47.6 21.4 37.1 7-10 Dinoprost 88 59.1 (52/88) 61.5 (32/52) 13.9 (5/36) 42.0 (37/88) Year 1 54 64.8 60.0 10.5 42.6 Year 2 34 50.0 64.7 17.6 41.2 SSC+TAI Cloprostenol 89 66.3 (59/89) 72.9 (43/59) 20.0 (6/30) 55.1 (49/89) Year 1 54 63.0 73.5 25.0 55.6 Year 2 35 71.4 72.0 10.0 54.3 SSC+TAI Dinoprost 88 70.5 (62/88) 62.9 (39/62) 15.4 (4/26) 48.9 (43/88) Year 1 55 65.5 69.4 15.8 50.9 Year 2 33 78.8 53.8 14.3 45.5 TRT p > 0.05 p > 0.05 p > 0.05 p > 0.05 Year p > 0.05 p > 0.05 p > 0.05 p > 0.05 TRT Year p > 0.05 p > 0.05 p > 0.05 p > 0.05 a Figure 3-1 gives details of treatments. b Percentage of heifers displaying estrus 72 h after PGF 2 of the total treated. c Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pr egnant to timed-AI of the total that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated 129

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APPENDIX B SUPPLEMENTAL TABLES AND FIGURES FOR CHAPTER 4 Table B-1. Description of the reproductiv e tract score (RTS) used in Chapter 4 Ovarian Measurement (mm) RTS Uterine horns Length Height Width Ovarian structures Description 1 Immature <20mm diameter No tone 15 10 8 No palpable follicles Infantile 2 20-25 mm diameter No tone 18 12 10 8 mm follicles Prepubertal (more than 30 d prepubertal) 3 20-25 mm diameter Slight tone 22 15 10 8-10 mm follicles Peripubertal (within 30 d of puberty) 4 30 mm diameter Good tone 30 16 12 > 10 mm follicles CL possible Cycling (Follicular Phase) 5 > 30 mm diameter >32 20 15 CL present Cycling (Luteal phase) Adapted from Anderson et al., 1991. 130

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Figure B-1. Effect of estrous synchronization treatment on estrous response in yearling heifers of Bos indicus Bos taurus breeding synchronized with 7-11 or Select Synch/CIDR+timed-AI (SSC). Means are e xpresses as a percentage of total that exhibited estrus. 131

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Figure B-2. Effect of estrous synchronization treatment on conception rate in yearling heifers of Bos indicus Bos taurus breeding synchronized with 7-11 or Select Synch/CIDR+timed-AI (SSC). Means are e xpressed as a percentage of total that exhibited estrus and became pregnant. 132

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Table B-2 Estrous response, conception a nd pregnancy rates of yearling heifers of Bos indicus Bos taurus breeding synchronized with either a 7-11 or Select Synch/CIDR+timed-AI treatment by location. a Location Estrous response, % b Conception rate, % c Timed-AI pregnancy, rate, % d Synchronized pregnancy rate, % e 1 57.4 (35/61) 40.0 (14/35) 7.7 (2/26) 26.2 (16/61) 2 42.9 (51/119) 49.2 (25/51) 14.7 (10/68) 29.4 (35/119) 3 49.6 (114/230) 36.8 (42/114 ) 23.3 (27/116) 30.0 (69/230) P -value > 0.05 > 0.05 >0.05 > 0.05 a See Figure 4-1 for details of treatments. b Percentage of heifers displaying estrus 72 h after PGF 2 of the total treated. c Percentage of heifers pregnant to AI of the total that exhibited estrus and were AI. d Percentage of heifers pregnant to timed-AI of the total that were timed-AI. e Percentage of heifers pregnant during the synchronized breeding of the total treated. 133

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BIOGRAPHICAL SKETCH Erin Nicole McKinniss was born in 1984, in Athens, Ohio. Er in, the oldest daughter of Michael and Lynn McKinniss, was raised in west ern Ohio on a small purebred Simmental farm with her two younger brothers, Matthew and Ste phen. She attended Northmont High School and was active in the varsity softball team, marc hing and concert bands, the American Junior Simmental Association, and 4-H. In 2002, sh e was honored with the title of Ohio Beef Ambassador and she represented her state at the nati onal contest. That year she traveled to the Minnesota State Fair as a representative of the program to promote beef to consumers. In 2004 she was elected to the junior board of trustees of the American Junior Simmental Association, where she represented the eastern region and was later elected to First Vice President of the board. Erin earned a bachelors degree in anim al sciences from The Ohio State University, during which time she was an active member of Sigma Alpha, Saddle an d Sirloin, and the Ag council. As part of the animal sciences depa rtment, she also was awarded the opportunity to study abroad in Australia for a quarter learni ng about agriculture production. Upon graduation from Ohio State in 2006, she began a Master of Science degree under Dr Joel Yelich, at the University of Florida, studying reproductive physio logy. Under this appointment Erin held many offices for the Animal Science Graduate Student Association and also taught an undergraduate reproduction lab. Erins future pl ans are to pursue her Ph.D. at the University of Florida, under the tutelage of Dr. Joel Yelich and Dr. Matt Hersom.