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REPRODUCTIVE STRATEGIES IN THE POSTPARTUM DAIRY COW WITH
REFERENCE TO ANOVULATION AND POSTPARTUM UTERINE HEALTH
KATHERINE ELIZABETH MAY HENDRICKS
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
Katherine Elizabeth May Hendricks
Dedicated to my mother, Elsa, and my husband, Gregory
I would like to express my appreciation and thanks to Dr. Louis F. Archbald for the
opportunity to join his graduate program. Dr. Archbald's passion for this subject area,
knowledge and dedication as an advisor have been a great motivating force in aiding the
completion of this thesis. His professionalism and discipline have been an example that I
hope to emulate personally and professionally. I would like to express my thanks to Dr
Charles Courtney for funding my program.
I would like to take this opportunity to thank the members of my committee, Dr.
Peter Hansen, Dr. J. Hernandez, Dr. Pedro Melendez and Dr. Carlos Risco, for their
insights and valuable suggestions throughout my study.
Special thanks go to my parents, Elsa May Wilmot-Binns and Lloyd Ivanhoe
Binns, for believing in my abilities to do anything I set my mind to and for advising me
to stick with my life-long dreams of becoming a veterinarian. Thanks go to my
Godparents, Dermoth and Merle Bingham, for their sacrifices and invaluable assistance
in helping me create the foundation for entry into the University of Florida. I thank my
friends who stood by me during my DVM, Merlene Mercano, Dr. Denice Lewis, Dr.
Nicole Tull, Dr. Susan McClennon and Dr. Rosemary Murray
I would like to acknowledge Dr. Julian Bartolome for his help and support in
execution of the project.
I would like to express my thanks and appreciation to Ingo Kreig for use of the
dairy herd, Roger Rowe and the entire staff at Mecklenberg Dairy. I would like to thank
Don Bennink for the use of the dairy herd and the entire staff at North Florida Holstein
I thank all who contributed to this study but were not mentioned.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF TABLES ....................... .. .. .. ................................ ix
LIST OF FIGURES ........................................ .............. xi
ABSTRACT ................................................... ................. xii
1 IN T R O D U C T IO N ................................................. .............................................. .
2 L IT E R A TU R E R E V IE W .................................................................. ..................... 5
Gonadotropin-releasing Hormone (GnRH) ............................................................5...
Chem ical N ature of the H orm one..................................................... ...............5...
Neurotransmitters and GnRH Secretion..........................................................6...
C control of Secretion .............. .................. ................................................ 7
Function of G nR H .............. .................. ............................................... 10
M echanism of A action .................. ............................................................ 12
P ro stag lan d in s ............................................................................................................. 13
B io sy n th e sis ......................................................................................................... 14
M etab o lism .......................................................................................................... 16
Mechanism of Action ........................................... ......... ............... 16
Function in the Reproductive Tract of the Cow ............................................. 17
Prostaglandin F2u. and its Uses in the Dairy Cow ........................................19
E strou s C y cle of th e C ow ...........................................................................................2 1
F ollicular P hase .......................................................................................... 2 1
L u te al P h a se ......................................................................................................... 2 2
Hormonal Control of the Estrous Cycle .........................................................23
Effect of Season.................................................. ..................... ........ .... ........ ..... 25
Folliculogenesis and Ovarian Dynamics in the Dairy Cow...................................29
F ollicular D ev elopm ent .......................................................................................2 9
Follicular Waves ............ .. .......... ..... ................................ 31
Regulation of Follicular Growth by FSH and LH.........................................33
P rodu action of E strog en ........................................................................................34
C orpus L uteum in the C ow ......................................... ........................ ................ 34
C orpus Luteum D evelopm ent......................................................... ................ 35
Function of the C orpus Luteum ...................................................... ................ 37
The Corpus Luteum and Pregnancy ............................................... ................ 38
L uteolysis ...................................................................................................... 40
Involution of the Bovine Uterus ......................................................... 44
M acroscopic C hanges.......................................... ........................ ................ 44
M icroscopic C changes ..................................................................... ................ 47
Factors A affecting U terine Involution ............................................. ................ 50
M ethods to A ssess U terine Involution ........................................... ................ 56
Endocrinology of the Immediate Postpartum Period in the Cow...............................58
Resumption of Ovarian Cyclicity Post Partum............... .....................................60
Cystic Ovarian D disease in the D airy Cow ............................................. ................ 63
P redisposing F actors......................................... .. ....................... .. ........... .. 64
P ath o g en e sis ........................................................................................................ 6 5
C y st D y n am ics..................................................................................................... 6 7
D ia g n o sis ............................................................................................................. 6 8
Treatm ent................... .... .... ......... ..... .. ......... .................. 69
Timed Artificial Insemination Programs in the Dairy Cow ..................................71
P ro staglan din F 2 a ............... ... ............................ ............ ..... .... .............. 73
Combination of Gonadotropin-releasing Hormone (GnRH) and PGF2a ...........73
Presynch-Ovsynch ................................................................... 75
Postpartum Endom etritis ................... .............................................................. 75
P ath o lo g y ............................................................................................................. 7 6
P ath o g en e sis ........................................................................................................ 7 8
D ia g n o sis ............................................................................................................. 7 9
3 EXPERIMENT 1: AN EVALUATION OF PRETREATMENT WITH GNRH ON
OVARIAN RESPONSE AND PREGNANCY RATE OF LACTATING DAIRY
COWS WITH OVARIAN CYSTS SUBJECTED TO THE OVSYNCH
P R O T O C O L ............................................................................................................... 8 4
In tro d u ctio n ............................................................................................................... .. 8 4
M materials and M ethods .. ..................................................................... ................ 85
Statistical A n aly sis...................................................................................................... 87
R e su lts....................................................................................................... ....... .. 8 8
D isc u ssio n ............................................................................................................... ... 9 2
4 EXPERIMENT 2:EFFECT OF SEQUENTIAL ADMINISTRATION OF PGF2ca
ON THE PREVALENCE OF MUCOPURULENT DISCHARGE, SIZE OF THE
CERVIX, SIZE OF THE PREVIOUSLY PREGNANT UTERINE HORN AND
FIRST SERVICE PREGNANCY RATE IN LACTATING DAIRY COWS ............98
In tro d u ctio n ................................................................................................................ 9 8
Experim ent 2 Part A .................... .............................................................. 102
M materials and M ethods ................. ......................................................... 102
T re atm en t........................................................................................................... 10 4
Exclusion Criteria .................................................. ........ ................. 105
Blood Sampling and Horm one Assay ....... .......... ...................................... 105
V aginoscopy .............. ..... ... .... ... ............... ....................... 107
Transrectal Palpation of the Reproductive Tract.................... .................. 107
Statistical A analysis .............. .... ............. .............................................. 108
E xperim ent 2 P art B .. ..................................................................... ............... 109
T re atm en t........................................................................................................... 1 10
E exclusion C riteria ......................................................... .... .. .............. 111
Pregnancy D iagnosis .. ..................................................... ... ... ............ .. 111
Statistical A analysis .............. .... ............. .............................................. 112
R esults...................................................................................................... . 112
E xperim ent 2 Part A ..................................... ....................... ............... 112
D ay 8 post partum ...................................................... .... ....... ................ 112
D ay 16 post partum ........................................................ 112
D ay 22 post partum ........................................................ 113
D ay 36 post partum ........................................................ 116
D ay 58 post partum ........................................................ 117
M o d e l 1 ...................................................................................................... 1 1 9
M o d e l 2 ...................................................................................................... 12 0
M odel 3 ........................................................... .. ............... 123
First service pregnancy rate................................................... ............... 126
Experiment 2 Part B : Conception Rate to First Service............................. 127
D isc u ssio n ............................................................................................................... .. 12 9
5 SUM M ARY AN D CON CLU SION S ............................................................................134
APPENDIX VAGINOSCOPY AND TRANSRECTAL PALPATION FORM .......... 138
L IST O F R E F E R E N C E S ................................................................................................. 140
BIOGRAPHICAL SKETCH ................................................................................ 163
LIST OF TABLES
3-1 Baseline data for heifers and cows that were diagnosed with ovarian cysts and
successfully completed Experiment 1 for parity (1, 2, 3+), time of year (March-
September/October-February), days in milk (DIM) and progesterone
concentration (< 0.5 ng/ml / > 0.5 ng/ml ) on Day 0........................... ................ 89
3-2 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the
risk of finding a CL on Day 7 in cows with ovarian cysts treated with GnRH on
D ay 0 ................................................................................................................. . 9 0
3-3 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the
risk of finding progesterone concentration > 1.0 ng/ml on Day 7 in cows with
ovarian cysts treated with GnRH on Day 0......................................... ................ 90
3-4 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the
risk of pregnancy in lactating dairy cows with ovarian cysts treated with GnRH
o n D a y 0 ............................................................................................................. .. 9 1
4-1 Distribution of cows enrolled in the study between June and August 2003 within
Groups 1 and 2 based on days in milk (DIM), parity (primiparous/multiparous),
dystocia (yes/no) and retained fetal membranes (yes/no) ................................106
4-2 Baseline data for heifers and cows that successfully completed the study for
parity (primiparous/multiparous), days in milk, dystocia (no/yes), retained fetal
membranes (no/yes) and progesterone concentration (< 1.0 ng/ml / >1.0 ng/ml)
at 8 days post partum ......................................................................... 113
4-3 Model 1-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy on Day 22 post partum...............................121
4-4 Model 1-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy on Day 58 post partum...............................121
4-5 Model 2-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy and a cervix > 50 mm in diameter by
transrectal palpation on Day 22 post partum ....... ... ...................................... 122
4-6 Model 2-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy and a cervix > 30 mm in diameter by
transrectal palpation on Day 58 post partum ....... ........................................ 123
4-7 Model 3-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy, cervix > 50 mm in diameter and previously
pregnant horn (PPH) > 30 mm by transrectal palpation on Day 22 post partum... 125
4-8 Model 3-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy, cervix > 30 mm in diameter and previously
pregnant horn (PPH) > 30 mm by transrectal palpation on Day 58 post partum... 126
4-9 Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence
interval (CI) and P-value for the risk of pregnancy in dairy cows treated
sequentially with PGF2ca in the early postpartum period to first service
post partum .............. ............................................................................ 127
4-10 Baseline data for cows within Group 1 and Group 2 based on parity (2, 3+),
dystocia (yes/no), retained fetal membranes (yes/no) and abnormal calving
(RFM and/or dystocia) that successfully completed the study.............................128
4-11 Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence
interval (CI) and P-value for the risk of pregnancy in dairy cows treated
sequentially with PGF2ca in the early postpartum period and subjected to the
Presynch-Ovsynch protocol. ........................................................ 129
LIST OF FIGURES
3-1 Experim ent 1 Experim ental D esign ................................................. ................ 87
4-1 Experimental 2 Part A: Experimental Design.......................... ...................105
4-2 Experiment 2- Part B: Experimental Design........................................................ 111
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
REPRODUCTIVE STRATEGIES IN THE POSTPARTUM DAIRY COW WITH
REFERENCE TO ANOVULATION AND POSTPARTUM UTERINE HEALTH
Katherine E. M. Hendricks
Chair: L. F. Archbald.
Major Department: Veterinary Medical Sciences
In Experiment 1, a total of 155 cows with ovarian cysts was sequentially allocated
to 3 groups on the day of diagnosis (Day 0). Cows in Group 1 (n = 55) were treated with
GnRH (100 [g, im) on Days 0 and 7, PGF2ca (25 mg, im) on Day 14, GnRH (100 [g, im)
on Day 16, and timed inseminated (TAI) 16-20 h later. Cows in Group 2 (n = 49) were
treated with GnRH on Day 0, PGF2ca on Day 7, GnRH on Day 9, and TAI 16-20 h later.
Cows in Group 3 (n = 51) were treated with GnRH on Day 7, PGF2ca on Day 14, GnRH
on Day 16, and TAI 16-20 h later. Pregnancy was determined by rectal palpation 45-50 d
after TAI. On both Days 0 and 7, cows in all groups were subjected to ultrasonography
(U/S) and blood samples were obtained for determination of progesterone concentration
(P4). Cows in Groups 1 and 2 were more likely to have a CL and high P4 on Day 7
compared to cows in Group 3. There was no significant difference in pregnancy rate (PR)
between cows in all groups, but cows with a CL on Day 7 were more likely to become
pregnant. It was concluded that administering GnRH to cows with ovarian cysts 7 days
prior to the initiation of the Ovsynch protocol did not increase PR, but cows with a CL on
Day 7 were more likely to become pregnant.
In Experiment 2 (Part A), a total of 228 postpartum (pp) dairy cows was
sequentially allocated to 2 groups between Days 7 and 9 pp. Cows in Group 1 (n= 114)
were treated twice with PGF2ca (25 mg, im) 8 h apart on Days 8 and 15 pp, and once on
Days 22 and 36 pp. Cows in Group 2 (n= 114) served as untreated controls. Vaginoscopy
and rectal palpation were done on Days 22 and 58 pp. Cows in both groups were
inseminated at estrus, or TAI approximately 130 to 134 days pp. Pregnancy was
determined by rectal palpation between 45-50 d after insemination. It was concluded that
sequential administration of PGF2ca in the immediate postpartum period reduced the
prevalence of a mucopurulent discharge, and both the size of the cervix and previously
pregnant uterine horn only on Day 58 pp. There was no difference in PR between cows in
In Experiment 2 (Part B), a total of 418 postpartum dairy cows was sequentially
allocated to 2 groups on Day 7 pp. Cows in Group 1 (n=209) were treated twice with
PGF2ca (25 mg, im) 8 h apart on Days 7 and 14 pp, and once on Days 21 and 35 pp.
Cows in Group 2 (n=209) served as untreated controls. All cows were subjected to the
Presynch and Ovsynch protocols on Days 49 and 75 pp, respectively. Pregnancy was
determined by U/S between Days 29 and 32 after TAI. There was no significant
difference in the conception rate to first service between the groups.
The profitability of a commercial dairy farm is based in part on the calving interval
of the cows. In order to maximize the economic profitability of the farm, cows must
return to ovarian cyclicity, express estrus and be bred within 85 days postpartum. The
optimal calving interval is 365 days.
There are two physiologic factors which influence reproductive success in the
postpartum dairy cow. The first is ovarian cyclicity, and the second is uterine health.
Parturition is a very traumatic event, and the ability to control ovarian and uterine events
in the postpartum cow could play an important role in achieving subsequent fertility. In
this study, two experiments were performed. The first experiment investigated the use of
gonadotropin releasing hormone (GnRH) to improve ovarian cyclicity in lactating dairy
cows diagnosed with ovarian follicular cysts. The second experiment investigated the use
of prostaglandin F2uc in the early postpartum period to improve uterine health and
improve fertility in lactating dairy cow.
Ovarian follicular cysts, also known as cystic ovarian degeneration, cystic ovaries
or anovular follicles, is the most common abnormality of follicular function in cattle
(Farin and Estill, 1993). It is most often seen in the postpartum dairy cow within the first
60 days post partum (Farin and Estill, 1993). Approximately 6-19% of dairy cows
develop ovarian follicular cysts (McLeod and Williams, 1991; Garverick 1997; Silvia et
al., 2002). Cystic ovarian degeneration increases days open by 22 to 64 days and
increases the number of cows culled from the herd. Each occurrence of ovarian follicular
cysts has been estimated to cost $137 in reduced milk production and veterinary expenses
(Silvia et al., 2002). The development of programs to prevent and/or treat cystic ovarian
degeneration will benefit the dairy farmer.
The resumption of ovarian cyclicity is dependent on a number of factors including
clearance of bacterial contamination from the uterus (Sheldon et al., 2002). Bacterial
contamination of the uterus occurs within the first week post partum (Elliott et al., 1968)
with spontaneous contamination, clearance and recontamination occurring up to seven
weeks post partum (Griffin et al., 1974). Some cows have the ability to clear these
infections but others do not and the reasons for this variability between cows are
unknown. Bacterial contamination of the uterus has a direct effect on the ability of the
cow to conceive and maintain a concepts. Conception and the maintenance of pregnancy
are, therefore, dependent on a healthy uterine environment.
The incidence of endometritis is greatest during the first 14 days post partum based
on cultures of uterine fluids and uterine biopsies (Griffin et al., 1974). Failure to clear
bacterial contamination by first ovulation post partum and corpus luteum formation could
place the contaminated uterus under the influence of progesterone. Progesterone makes
the uterus more prone to uterine infection (Hawk et al., 1964) with the incidence of
severe endometritis increasing around Day 15 to Day 21 postpartum. This increase in the
severity of endometritis coincides with the time of first post partum ovulation (i.e., 15 to
28 days post partum).
The hypothesis of Experiment 1 is based on research done in cows without ovarian
cysts. This research demonstrated that cows in the early luteal phase of the estrous cycle
at the time of initiation of the Ovsynch protocol had a higher pregnancy rate compared to
cows at other stages of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000a).
The hypothesis of Experiment 1 was that GnRH administered to cows at the time of
diagnosis of ovarian cysts would induce an early luteal phase and thus increase
pregnancy rates to a protocol for synchronization of ovulation and timed insemination
(Ovsynch protocol). The Ovsynch protocol consists of a single intramuscular injection of
100 pg of GnRH, followed seven days later by PGF2a (25 mg, intramuscularly). The
second dose of GnRH (100 rg, im) is administered 48 hours after PGF2a with artificial
insemination occurring 16 to 24 hours later. The objectives of Experiment 1 were two-
fold. The first objective was to evaluate the ovarian response 7 days following diagnosis
and treatment of cows with ovarian cysts with GnRH. The second objective was to
determine the pregnancy rate of cows with ovarian cysts subjected to treatment with
GnRH 7 days prior to the initiation of the Ovsynch protocol.
The hypothesis of Experiment 2 was that sequential administration of PGF2a
during the early postpartum period would reduce the incidence of mucopurulent
discharge, size of the cervix, size of the previously pregnant uterine horn and increase
first service pregnancy rates. The objective of Experiment 2, Part A was to determine the
effect of sequential administration of PGF2a in the immediate postpartum period on the
incidence of mucopurulent discharge, size of the cervix and size of the previously
pregnant uterine horn and first service pregnancy rates. The objective of Experiment 2,
Part B was to evaluate the effect of sequential administration of PGF2a in the immediate
postpartum period on first service conception rate in postpartum dairy cows subjected to
a timed insemination protocol consisting of Presynch-Ovsynch protocol. The Presynch
portion of the timed insemination protocol consisted of two intramuscular injection of 25
mg PGF2a given 14 days apart. The Ovsynch protocol began 12 days after the second
dose of PGF2a.
Gonadotropin-releasing Hormone (GnRH)
Chemical Nature of the Hormone
Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone
releasing hormone (LHRH), is a decapeptide produced in the arcuate, suprachiasmatic
and preoptic nuclei of the hypothalamus in response to hormonal and integrated neuronal
signals. The amino acid sequence of GnRH was first elucidated as early as 1971. The
primary structure of GnRH has been established in the pig (Matsuo et al., 1971), sheep
(Amoss et al., 1971; Burgus et al. 1972) and human placenta (Tan and Rousseau, 1982).
GnRH is synthesized from a precursor molecule, which begins with a 23 amino acid
signal sequence, followed by the GnRH decapeptide and ending with a 56 amino acid
GnRH-agonist associated peptide.
There are between 800 and 2500 GnRH neurons located throughout the brain of
vertebrates. These neurons originate in the olfactory placode outside the CNS and
migrate inwards during fetal development (Silverman, 1984). Immunohistochemical
techniques have been used to elucidate the location of the GnRH containing cell in the
bovine hypothalamus and infundibulum. GnRH-positive cell bodies have been located
singly or in small clusters in the infundibular nucleus (INFN), and in large discrete
clusters in the ventromedial nucleus (VMN) with their axons projecting into the rostral
hypothalamus (the grey matter of the lamina terminalis and medial preoptic area). The
majority of axons were found within the middle region of the hypothalamus within the
dorsomedial nucleus, VMN, INFN, and lateral hypothalamic area, the periventricular
nucleus (Dess and McArthur, 1981).
Embryonic GnRH neurons in primary cultures of olfactory placodes from ovine
embryos begin pulsatile secretion of GnRH by 17 to 24 days, indicating that these
neurons must mature prior to the onset of pulsatility (Duittoz and Batailler, 2000). GnRH
is synthesized in neuroendocrine cell bodies and transported down axons that terminate in
the median eminence where it is released in a pulsatile manner (Silverman, 1984;
Rodriquez and Wise, 1989; Naor et al., 1998). In the median eminence, GnRH is released
into the surrounding capillaries that drain into the hypothalamo-hypophyseal portal
vessels and transported to the pituitary where is stimulates the secretion of luteinizing
hormone (LH) and follicle stimulating hormone (FSH) from gonadotrophs (Garverick
and Smith, 1993).
Luteinizing hormone and FSH are released into the systemic circulation,
transported to the ovary where they stimulate follicular growth, ovulation, formation of
the corpus luteum and steroidogenesis. The ovary synthesizes estrogen, progesterone,
inhibin and activin. The gonadotropins and ovarian products regulate the release of
GnRH by both negative and positive feedback mechanisms.
Neurotransmitters and GnRH Secretion
Neurotransmitters, growth-factors, neuromodulatory peptides and ovarian steroids
positively and negatively affect GnRH and/or LH release (Kordon et al., 1994;Gore and
Roberts, 1997; Gazal et al., 1998; Moenter et al., 2003). Most studies have been carried
out in rats and immortalized GnRH neuron (GT1 cell lines). Since some of these
substances do not have receptors on the GnRH neuron, it is thought that they influence
GnRH gene expression through interneuronal pathways, or by directly binding to a
specific membrane receptor. Receptor binding leads to the activation of second-
messenger pathways to cause GnRH and/or LH release (Gore and Roberts, 1997).
Glutamate and norepinephrine (NE) have been shown to stimulate GnRH/LH
release and gene expression in vivo, and GnRH release in GT1 cells. A single injection of
the neurotransmitter glutamate analog, N-Methyl-D-L-aspartic acid (NMDA) causes a
30% increase in GnRH levels within 15 to 60 minutes (Gore and Roberts, 1997). A
similar increase in GnRH mRNA and plasma LH is seen in GnRH neurons (Petersen et
al., 1991;Liaw and Barraclough, 1993; Gore and Roberts, 1997). The catecholamine,
norepinephrine, stimulates GnRH release from intact rats and rabbits and plays a
physiological role in the onset of puberty (Gore and Roberts, 1997). Dopamine stimulates
and inhibits GnRH release in vivo while dopamine receptor-1 (D ) stimulates GnRH
release in GT1 cells (Gore and Roberts, 1997).
There are direct connections between gamma-amino-butyric acid-ergic
(GABAergic) and GnRH neurons. It is thought that through this direct connection,
GABA plays an inhibitory role in GnRH/LH release and causes a blockage of the LH
surge (Gore and Roberts, 1997). Opiates have also been shown to inhibit the GnRH/LH
release and gene expression in rats and rabbits (Gore and Roberts, 1997).
Cannulation of the third ventricle of adult cattle has allowed for sampling, detection
and quantification of GnRH secretion and has provided a means to monitor the central
regulation of reproduction (Gazal et al., 1998). Gazal et al. (1998) demonstrated that
GnRH is secreted into the cerebrospinal fluid in a pulsatile manner.
Control of Secretion
The GnRH neurosecretory system is diffuse and contains individual neurons
located in different areas of the brain (Silverman, 1984). The activity of GnRH neurons is
regulated by integrated signals from the brain and these include photoperiod, steroid
hormones, nutrition and stress (Moenter et al., 2003). However, in the absence of other
cells types, in pure GnRH neuronal cell cultures, and GT1 cell, GnRH is secreted
spontaneously in a rhythmic pattern (Martinez de la Escalera et al., 1992). Martinez de la
Escalera et al. (1992) suggested that a model of cell-to-cell communication via
intracellular contacts could explain synchronization of pulses in experiments where a
single cell-coated coverslip per superfusion chamber was used. It was also suggested that
there is a diffusible mediator, which would explain how two separated coverslips
synchronized GnRH pulses, and that GnRH itself may be the diffusible mediator
(Martinez de la Escalera et al., 1992).
The release of GnRH is episodic in both sexes. In females, however, there is an
interruption in the episodic release of GnRH by a GnRH surge. This GnRH surge
coincides with the pre-ovulatory LH surge, and lasts longer than the LH surge. Yoshioka
et al. (2001) demonstrated that there is a preovulatory increase in GnRH secretion into
the cerebrospinal fluid (CSF) in the third ventricle following the administration of
PGF2uc in heifers. The GnRH surge was shown to coincide with the LH surge and also
with the onset of standing estrus (Yoshioka et al., 2001).
While it is known that GnRH is secreted in a pulsatile manner, little is known about
the mechanisms generating this rhythmic secretion. It has been shown that GnRH
neurons produce a high-frequency rhythm characterized by oscillations in intracellular
calcium levels, burst firing of action potentials, and a low-frequency rhythm with a
period corresponding to neurosecretion of GnRH (Moenter et al., 2003). Low frequency
rhythms result in secretion of GnRH and can be modified by the effect of steroids
(Moenter et al., 2003).
The frequency of GnRH secretion varies during the estrous cycle and the
reproductive state of the animal from once every 30 minutes to once every few hours
(Moenter et al., 2003). Secretion of GnRH can be controlled at many levels. It may be
regulated at the transcriptional, post-transcriptional and post-translational levels (Gore
and Roberts, 1997).
The biosynthesis and release of GnRH are strictly controlled by afferent neurons to
the GnRH neurons in the hypothalamus (Peter and Burbach, 2002). Cell specific
expression and biosynthetic regulation rely on transcription from the gene promoter for
which the 5'-flanking region of the peptidergic gene contains essential elements (Peter
and Burbach, 2002). The amount of GnRH secreted from GnRH-containing neurons is
controlled by GnRH gene expression. GnRH gene expression is limited by the rate of
transcription of GnRH gene, polyadenylation and 5' capping of the RNA, processing of
its primary transcript to the mature mRNA, and transport of the mRNA from the nucleus
into the cytoplasm. Once in the cytoplasm, mRNA levels are determined by the turnover
of the molecule. Further regulation takes place at the level of mRNA translation into
proGnRH peptide, maturation of GnRH and the rate of degradation and release of GnRH
into the portal circulation, which affect the level of GnRH reaching the gonadotrophs
(Gore and Roberts, 1997).
Estrogen controls LH and FSH synthesis through a negative feedback loop to the
anterior pituitary and hypothalamus. Estrogen has been shown to downregulate mRNA
levels and reporter gene activity in immortalized hypothalamic GnRH neurons (Peter and
Burbach, 2002). Progesterone also causes repression of GnRH promoter activity (Peter
and Burbach, 2002). Neuropeptide Y (NPY) is a potent orexigenic substance, produced in
the hypothalamus, anterior pituitary and adipose tissue. Neuropeptide Y forms a
neuromodulatory link between nutritional status and the central reproductive axis
(McShane et al., 1992). Infusion of high concentrations of NPY into the third ventricle
inhibits GnRH pulses (Gazal et al., 1998).
Function of GnRH
The number of GnRH receptors in the anterior pituitary changes throughout the
estrous cycle. Maximum numbers of the GnRH receptor occur just prior to the
preovulatory surge in the cow, and their numbers decline thereafter (Nett et al., 1987;
Turzillo and Nett, 1999). Luteolysis and decreasing progesterone concentrations may be
the trigger for increased expression of the GnRH receptor gene (Turzillo and Nett, 1999).
The GnRH pulse frequency increases when progesterone concentrations decline, and the
negative feedback signal due to high concentration of P4 is removed. This increase in
GnRH pulse frequency increases the expression of GnRH receptors in the anterior
The preovulatory follicle produces large amounts of estradiol, which enhance
GnRH receptor gene expression. The increasing levels of estradiol and decreasing levels
of progesterone increase the numbers of GnRH receptors on the gonadotrophs, and
increases pituitary sensitivity to GnRH (Turzillo and Nett, 1999). In the rat, the number
of GnRH receptors is less during pregnancy and lactation compared to the estrous cycle.
It has been reported that when up to 50% of GnRH receptors are blocked with a GnRH
antagonist, ewes still respond fully to GnRH with LH release (Wise et al., 1984).
Ovarian hormones influence the numbers of GnRH receptor. In ewes, estradiol-17P3
has been shown to increase the number of GnRH receptors (Clarke et al., 1988). In vitro
studies using ovine pituitary cell cultures showed an increase in GnRH receptor
expression in response to estrogen and decreased expression in response to progesterone
(Laws et al, 1990a, 1990b). Inhibin caused an increase in the expression of GnRH
receptors and, when combined with estradiol-17p3, led to greater expression of GnRH
receptors that either hormone singly (Laws et al, 1990b).
In the immediate postpartum period, the pituitary response to GnRH is reduced.
Within the second week post partum, however, the pituitary responds to endogenous and
exogenous administration of GnRH with release of LH in dairy cows (Cummins et al
1975). Garverick et al. (1980) showed that administration of GnRH within the second
week post partum to dairy cows with large follicles was followed by ovulation and
subsequent formation of a CL. Similarly, a single injection of 100 micrograms of GnRH
12 to 14 days post partum initiated cyclic ovarian activity as evidenced by a palpable
corpus luteum and plasma progesterone > 1.0 ng/ml by Day 9 post-treatment (Zaied et al
1980). Zaied et al. (1980) also suggest that GnRH treatment 12 to 14 days post partum
may be useful in reducing abnormal ovarian activity. They found that 30% of non-treated
postpartum cows developed ovarian cysts prior to conception compared to 12.5% GnRH-
treated cows. Kittock et al. (1973) demonstrated that administration of GnRH to cows
with ovarian follicular cysts led to an increase in LH secretion, return to normal cyclicity,
and all cows expressed estrus 20 to 24 days after treatment.
Mechanism of Action
GnRH binds to receptors in the cell membrane of the gonadotrophs in the anterior
pituitary. The GnRH receptor is a member of the seven transmembrane domain receptor
coupled to a G-protein (Gq). Receptor binding leads to sequential activation of different
phospholipases to provide calcium (Ca2+) and lipid-derived messenger molecules.
Initially, phospholipase C is activated followed by activation of phospholipase A2 (PLA2)
and phospholipase D (PLD). Following receptor binding, GnRH stimulates a GTP-
protein (Gq). Activation of the G-protein results in Ca2+ -independent stimulation of
phospholipase C (PLC). Activation of PLC leads to the generation of second messenger
inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,
5-bisphosphate (PIP2). Phosphatidylinositol 4, 5-bisphosphate is a minor phospholipid
constituting approximately 0.5% of the membrane phospholipids. Phosphatidylinositol 4,
5-bisphosphate and DAG are required for Ca2+ mobilization and PKC activation. Calcium
is mobilized from intracellular pools stored in the endoplasmic recticulum (ER) and by
means of L-type voltage-sensitive Ca2+ channels in the cell membrane (Naor et al., 1998;
Ruf et al., 2003).
During exocytosis, Ca2+ and phosphokinase C (PKC) act in parallel and exert an
additive response on LH and FSH secretion. Calcium and PKC are also required during
stimulated mitogen-activated protein kinase (MAPK) activity. Mitogen-activated protein
kinase can activate transcription factors such as c-fos to mediate gene transcription with
subsequent protein synthesis. In addition, MAPK can lead to stimulation of
phosphokinase A2 (PKA2)with subsequent LH and FSH synthesis and release (Naor et
al., 1998; Ruf et al., 2003).
The signal cascade leading to the LH and FSH gene regulation is still not fully
understood. Activation of the second messenger system leads to protein phosphorylation,
gene transcription and biosynthesis of FSH and LH. Pituitary release of LH and FSH is
down-regulated in the presence of continuous GnRH secretion (Belchetz et al, 1978).
High frequency GnRH pulses favor LH release while low frequency pulses favor FSH
release (Wildt et al., 1981). The changes in GnRH pulse frequency are essential for
normal reproductive function (Moenter et al., 2003).
Prostaglandins were independently isolated from human seminal plasma in the
1930s by Goldblatt and von Euler. Prostaglandins are formed by most mammalian tissues
and by tissues of lower vertebrates and certain invertebrates (Samuelsson et al., 1978).
All mammalian cells types have the capacity for converting the membrane bound fatty
acids into prostaglandins (Granstrom, 1981; Murray et al., 1996). Prostaglandins act as
local hormones, having important physiological and pharmacologic activities (Murray et
al., 1996). There are many stimuli (hormonal, nervous, other chemical, mechanical
stimuli) known to activate phospholipase and initiate prostaglandin synthesis (Granstrom,
1981). The products formed and the amounts produced will vary within the same tissue
under different conditions (Granstrom, 1981).
Prostaglandins are C20 carboxylic acids. They have a central five-membrane ring
with two side chains, 7 and 8 carbon in length, respectively, attached to adjacent
positions on the ring. Depending on the number of double bonds (one, two or three
double bonds) on the side chain they are designated to the '1', '2' or '3' series
(Granstrom, 1981). The type of prostaglandin (A, B, C, .. .1) depends on the arrangement
of functional groups in the molecule (Champe and Harvey, 1994). The substituents in the
molecule determine its biological activity, most importantly those at position C-9 and C-
11 in the ring and C-15 in the side chain (Granstrom, 1981). The prostaglandins that are
biologically active compounds have a hydroxyl group (-OH group) at C-15 and a double
bond at C-13 (Granstrom, 1981).
Prostaglandins belong to a group of unsaturated fatty acids called eicosanoids.
Eicosanoids are not stored in cells, but are released upon synthesis and their biosynthesis
is limited by the availability of free precursor fatty acid (Katzung, 1995). Eicosanoids
include the prostanoids, leukotrienes and lipoxins (Murray et al., 1996).
Eicosanoids are produced through two different pathways. Leukotrienes and
lipoxins are produced through the lipoxygenase pathway. Prostaglandins and
thromboxanes are produced through the cyclooxygenase pathway, and each pathway
competes with the other for arachidonic acid (Katzung, 1995). Not all cell types make all
of these products (Katzung, 1995). The prostanoids consists of prostaglandins,
prostacyclins and thromboxanes. They act as local hormones and function through a G-
protein to elicit their biological effects (Katzung, 1995).
The precursor for prostaglandins is arachidonic acid (AA), which is an essential
fatty acid that forms part of the glycerophospholipids found in the lipid bilayer of the cell
membrane. Cleavage of arachidonic acid from membrane lipids and other lipid esters by
phospholipase is activated by both specific and nonspecific stimuli (Katzung, 1995).
Arachidonic acid is produced from the interaction of phospholipase A2 (PLA2) with
membrane phospholipids (Katzung, 1995). Phospholipase A2 and other phospholipases
cleave esterified arachidonic acid from the 2- position of glycerophospholipids (Dennis,
Free AA is converted to PGG2, an unstable product, by oxygenation and cyclization
of the pentane ring catalyzed by the enzyme prostaglandin endoperoxide synthase
(prostaglandin G/H synthase or cyclooxygenase; COX). Cyclooxygenase is a membrane-
bound hemoprotein (Katzung, 1995). The initial unstable product (PPG2) is quickly
reduced to PGH2 by COX. Microsomes from cow uteri have been shown to possess
strong endoperoxide F2a reductase activity (Wlodawer et al., 1976).
The next substance produced from PGH2 depends on the cell in which the reaction
occurs (Sun et al., 1977). In tissues containing the cytosolic enzyme, prostaglandin D
synthase, PGH2 is converted to PGD2. However, PGE2 is produced if the tissue contains
the membrane bound enzyme, prostaglandin E synthase. Reduced glutathione is required
for both processes. Prostaglandin E 9-ketoreductase and 15-hydroxyprostaglandin
dehydrogenase convert PGE2 into PGF2c, (Sun et al., 1977; Katzung, 1995).
There are two cyclooxygenases: (I and II) that have a 60% homology and which are
pharmacologically different (Katzung, 1995). Cyclooxygenase I is a constitutive form
and synthesizes basal levels of prostaglandin. Cyclooxygenase II (COX II) is an inducible
form of the enzyme. Cyclooxygenase II is induced by a variety of ligands (Herschman,
1994), and is regulated at transcriptional and posttranscriptional levels (Sirois and
Richards, 1992). Its induction is inhibited by glucocorticoids (Herschman, 1994).
Cyclooxygenase II is rapidly and transiently expressed, and leads to a bolus of
prostaglandin production in response to stimulation (Herschman, 1994).
In response to pituitary glycoprotein hormones ovarian granulosa cells induced
COX II (Herschman, 1994). Exposure of preovulatory follicles to FSH and LH rapidly
and transiently induced the COX II message (Herschman, 1994). Prostaglandin synthesis
in the female reproductive cycle depends on the expression of COX II in response to
pituitary glycoprotein hormones (Herschman, 1994).
Prostaglandins are rapidly inactivated in the body. Enzymes that catabolize
prostaglandins are found in the lung, kidney, spleen, adipose tissue and intestines
(Katzung, 1995). Oxidation of the secondary alcohol group at C-15 is catalyzed by the
enzyme, 15-hydroxyprostanoate dehydrogenase (PGDH). The main sources of PGDH are
the lungs, spleen and kidney (Kindahl, 1980). The lungs have the highest enzyme activity
and with its vast vascular bed can render large amounts of prostaglandins biologically
inactive (Katzung, 1995; Davis et al., 1980). After oxidation at C-15, the A13 double bond
is reduced by A13 reductase resulting in 15-keto-13, 14-dihydro compounds, which are
the main plasma metabolites (Kindahl, 1980). The main plasma metabolite of PGF2a in
male calves is 15-keto-13, 14-dihydro PGF2a. Urinary excretion was completed in
approximately 6 hours, with recovery of 80% of the injected PGF2a (Kindahl, 1980). In
heifers the main metabolite of PGF2a is 15-keto-13, 14-dihydro PGF2a (Kindahl et al.,
Mechanism of Action
The eicosanoids are short-lived, highly potent local mediators that produce an
astonishing array of biological effects by binding to specific cell surface receptors
(Katzung, 1995). All binding appears to involve a G-protein linkage (Katzung, 1995).
Receptor binding initiates a signal transduction pathway, which links the regulatory
substance (PGF2ca) with its intracellular effectss.
Prostaglandin F2ca is released from the uterus, and transferred from the utero-
ovarian vein to the ovarian artery by a countercurrent mechanism. On reaching the ovary,
PGF2ca binds to high and low affinity-binding sites (receptors) located in the plasma
membrane of the corpus luteum (Samuelsson et al., 1978). These receptors are G protein-
coupled receptors designated FP and subtyped into FPA and FPB. The high affinity-
binding site requires calcium ions in order to be detected (Samuelsson et al., 1978).
Prostaglandin F2ca receptors are coupled to phospholipase C (PLC). Receptor
binding activates PLC and induces hydrolysis of phosphatidylinositol 4, 5-bisphosphate
to generate two second messengers, inositol 1,4,5-triphosphate (IP3) and 1,2-
diacylglycerol (DAG). Inositol 1,4,5-triphosphate diffuses into the cytoplasm and is
involved in the liberation of intracellular calcium (Ca2+) from the endoplasmic
Calcium is required for the activation of PKC and DAG increasing PKC's affinity
for Ca2+. Activation of PKC leads to the opening of calcium channels. Calcium and PKC
promote protein phosphorylation and this eventually leads to the inhibition of
progesterone secretion and regression of the corpus luteum (Samuelsson et al., 1978).
Function in the Reproductive Tract of the Cow
During the bovine estrous cycle, PGF2ca is released for 2 to 3 days as rapid pulses
with duration of 1 to 5 hours prior to and during luteolysis (Kindahl et al., 1976; Kindahl,
1980). A 9-keto-reductase enzyme that reduces PGE2 to PGF2ca can also form PGF2ca.
However, this enzyme is not present in the bovine uterus (Kindahl, 1980).
The precise release of PGF2ca throughout the bovine estrous cycle presupposes that
there is an inhibiting factor in the uterus. This inhibiting factor is important for the
regulation of the physiologic PG biosynthesis and thus regulates its production to prevent
premature lysis of the corpus luteum. Wlodawer et al. (1976) noted that an inhibiting
factor was found in bovine uterine preparations that suppressed the fatty acid
cyclooxygenase. Knickerbocker et al. (1986) noted that the bovine concepts suppressed
uterine production of PGF2ca production during pregnancy recognition by what was then
called bovine concepts secretary proteins (CPS) and is now known as interferon tau
(INF-'c). However, the suppression of PGF2ca release from the endometrium is regulated
by a number of hormones; estrogen, progesterone (Salamonsen et al., 1990; Xiao et al.
1998), oxytocin and endothelin-1 (ET-1).
The bovine endometrium contains large quantities of AA and has the ability to
metabolize AA into a variety of products (Salamonsen and Findlay, 1990). Ovarian
steroids influence the expression of the COX gene in bovine endometrial cells (Xiao et al.
1998). Progesterone directly influenced the basal secretion of PGF2ca by the
endometrium (Xiao et al. 1998). Progesterone has been shown to stimulate basal PGF2ca
secretion by bovine endometrial cells and tissues. However, it inhibits oxytocin-induced
PGF2ca secretion while in luteal cell culture while estrogen stimulated only PGF2ca
Prostaglandins have also been shown to have a direct effect on the bovine
myometrium. (Patil et al., 1980). In vitro studies carried out on the bovine myometrium
indicate that PGF2ca has the ability to increase uterine tone and motility (Patil et al.,
1980). However, the extent to which motility is enhanced is dependent on the stage of the
estrous cycle. During the follicular phase, the myometrium is more active and
contractions are more frequent and stronger than in the luteal phase. Contractions occur
on average 9 times per 10 minutes, with mean amplitude of 12 mm. During the luteal
phase, the frequency of contractions is less, on average 6 per 10 minutes, with mean
amplitude of 5 mm. Under the influence of PGF2ca, there is a general increase in mean
contraction and strength in both stages of the estrous cycle (Patil et al., 1980). Zetler et
al. (1969) demonstrated that PFG2ca had a similar effect on the fallopian tubes, increasing
Prostaglandin F2a and its Uses in the Dairy Cow
Prostaglandins are widely used in herd management due to their luteolytic
properties. Prostaglandin F2a has been use in cattle as an abortifacient (Johnson, 1981).
Vandeplassche et al. (1974) induced abortion in cases of pathological gestation in cattle
by intrauterine infusion (into the horn ipsilateral to the ovary with the CL) of 10.5 to 45
mg of PGF2ca. In cases where the cows carried a mummified fetus, 3 of 5 expelled the
fetus. However, in cases of maceration, the fetus was not expelled. There was rapid
regression of the CL within 4 to 5 day of intrauterine infusion, followed by estrus within
6 to 7 days.
Prostaglandin F2a has also been use in cattle to induce parturition usually from 240
days gestation or within 20 days of predicted term (Johnson, 1981; Schultz and Copeland,
1981). Bosc et al. (1975) compared the use of dexamethasone to an analogue of PGF2ca
(ICI 79939) for the induction of parturition in cattle. Parturition was successfully induced
when prostaglandin was administered in 2 mg intramuscular doses five-hours apart using
a dosage which ranged from 4 to 10 mg. Plasma progesterone decreased to a low level
before parturition following two successive injections. This decrease in progesterone
concentration was attributed to lysis of the CL of pregnancy. However, cows in which
parturition was induced had a higher incidence of retained fetal membranes.
Prostaglandin F2a has been used to treat unobserved estrus in lactating dairy cattle
with a corpus luteum (Eddy, 1977; Seguin et al., 1978; Seguin, 1981) and for estrus
synchronization. Intramuscular administration of the PGF2a analogue, clorprostenol (ICI
80, 996) in dairy cattle with unobserved estrus and a palpable mature CL resulted in cows
exhibiting estrus and being inseminated sooner than those treated with saline. In one
study (Eddy, 1977), intramuscular administration of 500g clorprostenol resulted in 69%
of unobserved estrus cows coming into heat within 8 days of treatment.
Prostaglandin F2a has been use in cattle for postpartum infections: pyometra,
metritis and endometritis (Ott and Gustafsson, 1981a, 1981b). Pyometra is defined as a
condition associated with accumulation of purulent material in the uterus, persistence of a
CL and anestrus (Roberts, 1971). The mode of action of PGF2a in the treatment of cows
with postpartum infection is based on its luteolytic activity. In cases of pyometra,
treatment leads to the regression of the CL resulting in emptying of the uterus.
Prostaglandin F2a has been shown to stimulate the myometrium and may aid in the
physical evacuation of purulent material from the uterus (Ott and Gustafsson, 1981 a,
1981b). Gustafsson et al. (1976) demonstrated that PGF2ac administered both
intramuscularly and intravenously at various dosages will effect resolution of postpartum
and post insemination pyometra in dairy cows not previously treated with antibiotics
systemicallyy or intrauterine) or any other drug. Gustafsson et al. (1976) treated 26 cows
(23 with postpartum pyometra and 3 with post insemination pyometra). Eighty-five
percent responded by emptying the uterus and exhibited estrus 3 to 4 day following
treatment. Sixty-five percent became pregnant following insemination beginning at the
second estrus after treatment. Several other studies have shown the beneficial effect of
using PGF2ca in cows with pyometra (Jackson, 1977; Fazeli et al., 1980; Ott et al, 1981a;
Paisley et al., 1986; Gilbert et al., 1992). Prostaglandin F2a has also been use in cattle
with acute and chronic endometritis (Jackson, 1977;Ott et al., 1981a, 1981b;Paisley et al.,
1986; Pepper et al., 1987;Gilbert et al., 1992; Sheldon et al., 1998; Heuwieser et al.,
2000; Knutti et al., 2000).
Estrous Cycle of the Cow
There are four stages of the estrous cycle: estrus, metestrus, diestrus and
proestrus. The estrous cycle can also be classified as luteal or follicular depending on the
dominant structures on the ovaries. In the luteal phase, the CL is the dominant structure,
and in the follicular phase the preovulatory follicle is the dominant structure. The average
length of the estrous cycle in the heifer is 20 days with a range of 18 to 22 days, and in
the cow is 21 days with a range of 18 to 24 days (Roberts, 1971).
The follicular phase consists of proestrus and estrus. It starts with corpus luteum
regression and ends with ovulation of the dominant follicle. The follicular phase is
approximately 4 to 5 days in the cow.
In the event that pregnancy does not occur, the uterus releases prostaglandin F2ca
(PF2ca; Kindahl et al., 1976). Following release of PF2ca from the endometrium, the
corpus luteum of the luteal phase undergoes functional and structural regression. At this
time there is a sharp decline in the blood progesterone (P4) concentration (Kindahl et al.,
1976). The sudden fall in P4 removes the negative feedback on the developing follicles
leading to the selection and acceleration of preovulatory follicular growth (Savio et al.,
The preovulatory follicle produces increasing concentrations of estradiol-170 and
this influences the sex centers in the brain to induce estrus. Peak concentrations of
estradiol-170 coincide with estrus. At the level of the hypothalamus, estradiol-170
induces a GnRH surge, which induces the preovulatory LH surge and FSH release from
the anterior pituitary. There is considerably less estradiol-170 during the 18 to 20 hour
period of estrus as compare to proestrus. At the level of the pituitary, estradiol increases
the responsiveness of gonadotrophs to GnRH, which results in an increase in the LH
The duration of estrus is approximately 18 to 20 hours, and ovulation occurs 10 to
12 hours after estrus. During estrus, the cow or heifer will stand to be mounted by the
bull or by herd-mates. During estrus, the vulva of the cow sometimes becomes swollen,
the mucous membranes of the vagina are hyperemic, and a clear, viscous, mucous
discharge may be seen hanging from the vulva.
Ovulation is spontaneous and occurs 10 to 12 hours after estrus in cows and 3 hours
earlier in heifers (Roberts, 1971). The ovulatory process consists of the following:
cytoplasmic and nuclear maturation of the oocyte; disruption of the cumulus cell
cohesiveness among the cells of the granulose layer and thinning and rupture of the
external follicular wall, with expulsion of the mature oocyte (Hafez et al., 2000).
The luteal phase is the period following ovulation and involves luteinization and
development of the CL. It consists of metestrus (Days 1 to 4) and diestrus (Days 5 to 18).
In this stage, the corpus luteum develops from the remnants of the pre-ovulatory follicle
and produces increasing quantities of progesterone. Progesterone is usually detectable in
blood by day 5 after ovulation and CL formation. The CL also produces oxytocin and
In the non-pregnant cow, the CL undergoes regression by day 17 after ovulation.
Luteolysis occurs as a consequence of the interaction between the CL and prostaglandin
F2ca (PGF2ca) released from the uterus. If pregnancy occurs, uterine production of
PGF2ca is blocked by the action of interferon tau (INF--T) on the uterus. The up-regulation
of oxytocin receptors in the endometrium is inhibited by the secretion of INF-T from the
trophodectoderm from days 12 to 25 in cattle (Farin et al. 1990). The CL is required for
the maintenance of pregnancy in the cow.
During the luteal phase, the quantity of progesterone produced by the CL increases.
The concentration of progesterone impacts LH secretion patterns. When progesterone is
low, the LH pulse is characterized by high frequency and low amplitude. When
progesterone is high, the LH pattern is characterized by a low frequency and high
Hormonal Control of the Estrous Cycle
Hormones secreted by the hypothalamus, anterior pituitary, ovary and uterus
regulate the estrous cycle. The hypothalamus integrates information from higher brain
centers, involving internal and external signals, such as nutritional status, season,
photoperiod, presence of a male and others (Garverick and Smith, 1993).
Gonadotropin-releasing hormone (GnRH) is produced by the neurosecretory cells
within the hypothalamus and regulates the release of gonadotropins luteinizingg hormone,
LH and follicle stimulating hormone, FSH) from the anterior pituitary (Smith and Jennes,
2001). GnRH is produced in the cell bodies of neurosecretory cells located in the arcuate,
suprachiasmatic and preoptic nuclei of the hypothalamus. GnRH is then transported along
the axons to the median eminence, where it is released into the hypothalamo-hypophyseal
portal circulation and travels to the anterior pituitary (Garverick and Smith, 1993). GnRH
is released in a pulsatile manner in response to appropriate physiological cues.
Luteinizing hormone and FSH are secreted in a pulsatile manner in response to GnRH
secretion (Moenter et al., 1991).
Luteinizing hormone is a glycoprotein hormone produced by the gonadotrophs in
the anterior pituitary. It consists of alpha- and beta-subunits. The alpha-subunit is
common to several hormones, including FSH and thyroid stimulating hormone (TSH),
and the beta-subunit is unique to the particular hormone. LH is a short-lived molecule,
having a half-life of approximately 30 minutes.
Follicle-stimulating hormone is a glycoprotein produced in the anterior pituitary by
gonadotrophs. It is composed of a common alpha-subunit and specific beta-subunit. FSH
stimulates the growth and maturation of ovarian follicles and in the presence of LH
stimulates secretion of estrogen from ovarian follicles.
Luteinizing hormone and FSH are secreted in a pulsatile manner from the anterior
pituitary in response to the pulsatile release of GnRH. Tonic levels are controlled by the
negative feedback mechanism from the ovaries.
The pulsatile secretion of LH varies throughout the estrous cycle (Rahe et al., 1980;
Walters et al., 1984a, 1984b; Schallenberger et al., 1985). Rahe et al. (1980) suggests that
the LH pattern throughout the estrous cycle is dependent on the stage of the cycle and
ovarian steroids influence the LH pattern.
Studies have been done in ovariectomized animals to investigate the effect of
steroids on the LH release. Ovariectomy in the cow results in increases in the pulse
frequency and amplitude of LH and FSH (Schallenberger and Peterson, 1982). The
pattern of secretion is related to circulating concentrations of progesterone. In the early
luteal phase when progesterone concentrations are low, the LH pulse frequency is much
higher than that seen in the mid-luteal phase; 8.0 pulses/12 hours and 3.6 pulses/12 hours
respectively (Walters et al., 1984b). In the early luteal phase the pulse frequency of LH
and FSH are similar. However, as the luteal phase progresses, the FSH pulse frequency
becomes greater than the LH pulse frequency (Walters et al., 1984b).
Luteinizing hormone release is suppressed by exogenous progesterone (Beck et
al., 1976). In the luteal phase, progesterone and estrogen are the main factors, which
decrease the frequency of the LH pulses in cattle. Individually these hormones can
suppress the LH pulse, but when administered together there is a more profound
inhibiting effect on LH pulse frequency. Stumpf et al. (1993) showed that when estradiol-
170 was administered to ovariectomized cows the LH pulse frequency was 0.97 0.07
pulses/hour and when progesterone was administered the LH pulse frequency was 0.52 +
0.08 pulses/hour. However, when both estradiol-170 and progesterone were give
concomitantly the LH pulse frequency increased to 14 0.07 pulses/hour.
Effect of Season
The effect of season or time of year on the bovine estrous cycle may be thought of
as two fold. There is the effect of photoperiod on the estrous cycle and the effect of "heat
stress" or high environmental temperatures on the estrous cycle.
High environmental temperatures reduce estrus behavior. Abilay et al. (1975)
demonstrated that the duration of estrus was reduced on average by 5.5 hours in
Guernsey heifers maintained at 18.2 C. It is thought that the reduction in estrus behavior
is as a consequence of the decrease in circulating estradiol (Wolfsenson et al., 1997; Roth
et al., 2001) noted during periods of heat stress. There is a reduction in the numbers of
mounting episodes per estrus during heat stress (Nebel et al, 1997). Badinga et al. (1985)
demonstrated that in Florida there is a decrease in conception rate in from 48% in March
to 18% in July with recovery occurring in November. Imtiaz Hussain et al. (1992) noted
that only 36.8% of cows expressed estrus, even though all cows were cycling based on
High environmental temperatures have been shown in affect cortisol levels. There
is an increase in cortisol levels in dairy cows that experience heat stress (Wise et al.,
1988; Imtiaz Hussain et al., 1992) However, Abilay et al. (1975) demonstrated that there
was a decrease in plasma cortisol levels in heat stressed heifers. Cortisol levels have been
linked to GnRH release. Increased cortisol levels inhibit GnRH secretion, which in turn
decreases LH secretions (Dobson and Smith, 2000). Cortisol has been shown to have a
direct effect on the pituitary gland to depress both basal and GnRH-stimulated LH release
in the cow (Li and Wagner, 1983; Padmanabhan et al., 1983).
High environmental temperatures have been shown to have an effect on the
gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The
effect of high environmental temperatures on LH is inconsistent. Vaught et al. (1977)
found no difference in the frequency of preovulatory increase of luteinizing hormone or
in the interval between the preovulatory increase and ovulation in either lactating or non-
lactating cows exposed to summer heat stress. This is in contrast to findings by Wise et
al. (1988). There was a decrease in the number of LH pulses on Day 5 of the estrous
cycle in heat stressed cows compared to controls (Wise et al., 1988).
Ovarian follicles are susceptible to high environmental temperatures. Roth et al.
(2000) demonstrated that there are alterations in the pattern of growth and development
of medium-sized follicles associated with an increase in plasma FSH concentration in the
first follicular wave following acute heat stress. Wolfsenson et al. (1997) demonstrated
that estradiol concentration in follicular fluid and androstenedione production by thecal
cells were lower in dominant follicles collected in autumn than those collected in winter.
This finding by Wolfenson et al. (1997), indicate the carry over effect of heat stress. Roth
et al. (2001) investigated the delayed effect of heat stress on medium-sized and
preovulatory follicles at 20 and 26 days after acute heat exposure. It was noted that the
number of medium-sized follicles that emerged during the first follicular wave after heat
stress was the same in both control and heat-stressed cows. However, there were more
healthy medium-sized follicles in the control than heat-stressed cows (56% vs 38%) but
this was not significantly different. In healthy medium-sized follicles, estradiol
production by granulosa cells and androstenedione production by thecal cells were lower
and follicular fluid progesterone concentration was higher in heat-stressed than in control
cows. In preovulatory follicles the viability of the granulose cells, concentration of
androstenedione in follicular fluid and androstenedione production by thecal cells were
lower in heat-stressed than in control cows (Roth et al., 2001).
Guzeloglu et al. (2001) investigated the effect of acute heat stress on long-term
follicular dynamics and biochemical characteristics of dominant follicles in non-lactating
dairy cows. They found that there was no difference in estradiol or progesterone
concentration in the follicular fluid of the dominant follicle between heat stressed and
control cows. However, there was a significant difference in the size of the dominant
follicle between the control and heat stressed cows. The dominant follicle size was
greater in control cows than in heat stressed cows. Guzeloglu et al. (2001) noted that heat
stress reduces follicular dominance during a follicular wave based on the increase in the
number of class 3 follicles on Days 7 and 8. Roth et al. (2000) investigated the immediate
effect of heat stress on plasma FSH and inhibin in Holstein dairy cows. It was
demonstrated that there was a larger cohort of medium sized follicles (6-9 mm) during
the second follicular wave of the estrous cycle of heat stressed cows compare to controls.
This increase growth was associated with higher plasma concentrations of FSH, which
lasted for 4 more days in heat stressed cows than in controls. The increase in plasma FSH
was also associated with a decrease in plasma concentrations of inhibin.
There is an increase in the length of the estrous cycle in cows experiencing heat
stress. Wilson et al. (1998a) noted that the second wave dominant follicle was less likely
to ovulate in heat stressed lactating dairy cows. In heat stressed cow, 18% ovulated
compared to controls 91%. The average day of luteolysis was delayed by 9 days in heat
stressed cows. Similar finding were found in heifers. Heat stress inhibited the growth and
function of the dominant follicle, such that heat stressed heifers had three follicular waves
and there was a delay in corpus luteum regression (Wilson et al., 1998b).
High environmental temperatures have been shown to affect corpus luteum
function. These observations were made based on progesterone concentrations. In some
studies progesterone concentration was elevated (Abilay et al, 1975; Vaught et al., 1977)
in cows experiencing heat stress. However, in other studies progesterone concentration
was unchanged (Wise et al., 1988) and yet in other studied progesterone concentrations
were lowered during heat stress (Howell et al., 1994). Wilson et al. (1998a, 1998b)
demonstrated that there was delayed regression of the corpus luteum in lactating dairy
cows and heifer subjected to controlled heat stress.
Heat stress reduced feed intake (Fuquay, 1981; Rensis and Scaramuzzi, 2003).
Reduced dry matter intake will prolong the period of negative energy balance in the
postpartum dairy cow. Negative energy balance leads to decreased plasma insulin,
glucose and insulin-like growth factor I (IGF-I; Rensis and Scaramuzzi, 2003). These
factors are required for the normal growth and function of ovarian follicles and thus the
Folliculogenesis and Ovarian Dynamics in the Dairy Cow
An ovarian follicle is a spherical aggregation of cells that contain the developing
gamete (Banks, 1986). Oogenesis begins during embryologic development and continues
throughout fetal development until shortly after birth. Multiplication halts in meiotic
prophase. At this stage the germ cell is termed a primary oocyte (Noden, 1985).
Follicular development proceeds through further development of the primordial follicle
into a primary follicle; primary to secondary follicle and finally a mature follicle.
Follicular growth and maturation is under the influence of gonadotropins from the
pituitary (Noden, 1985).
The primordial follicle contains the primary oocyte. At this stage the primary
oocyte has paired homologous chromosomes and each has replicated to form two
chromatids (Noden, 1985). At the time the oocyte is undergoing the first stages of
meiosis follicular cells surround the primary oocyte to form a primordial follicle. Once
the primordial follicle is activated it becomes the primary follicle. Histologically, the
primordial follicle appears as a single layer of flattened follicular cells surrounding the
primary oocyte (Banks, 1986)
Activation of the primordial follicle results in the development of the primary
follicle, involving alterations in the primary oocyte, follicular cells and stromal elements.
Histologically, the primary follicle appears as single layer of cuboidal or columnar
follicular cells surrounding the primary oocyte. At the stage, accumulation of yolk
granules can been seen in the primary oocyte (Banks, 1985).
The secondary follicle consists of the primary oocyte separated from the follicular
cells by the zona pellucida. The follicular cells are actively dividing and are now know as
the membrana granulosa. As the secondary follicle continues to develop, the stromal cells
differentiate into theca internal and theca externa, separated from the membrana granulosa
by a basement membrane. The theca internal consists of large, epitheliod cells and an
extensive vascular network. The theca externa consists of a fibroblastic layer of cells
The tertiary follicle develops from the secondary follicle. Histologically, the
tertiary follicle consists of cuboidal and columnar follicular cells surrounding the primary
oocyte. Within the layers of follicular cells small, fluid filled periodic-acid-Schiff-
positive spaces can been seen. Further secretion of follicular fluid by the granulosa cells
leads to the formation of a follicular antrum seen in the mature follicle. The mature
follicle is also known as the Graafian follicle or preovulatory follicle. The preovulatory
follicle ranges form 15 to 17 mm in diameter in the bovine ovary.
Follicles protrude from the ovary and are recognized as a smooth fluctuant
structures on palpation. The size of the follicle increases to approximately 20 to 25 mm in
diameter by the middle of the estrous cycle. The increase in follicular fluid within the
antrum is reflected by the tension in the palpable follicle, which increases up to six to
twelve hours before ovulation (Zemjanis, 1970).
In terms of the endocrinology, the development of the follicle can be divided into
four stages. The initial stage of development is independent of the gonadotropins, follicle
stimulating hormone (FSH) and luteinizing hormone (LH). In this stage follicles are
usually < 3 mm in size. Next follows a stage in which the follicle is dependent on FSH,
the follicles range from 3 to 10 mm in diameter. In the thirds stage the follicle is
dependent on pulsatile LH and ranges from 10 mm to preovulatory size. The final stage is
dependent on the preovulatory LH surge for ovulation (Thatcher et al., 2002).
Ovarian follicles develop in waves. Rajakoski (1960) first put this theory forward
after he conducted extensive studies of ovaries taken at slaughter. Pierson and Ginther
(1984) later confirmed the existence of follicular waves using ultrasonography.
Cattle either have two or three follicular waves during a single estrous cycle. In
cows that have 2 follicular waves, recruitment occurs on Day 0 (day of ovulation) and
day 10 of the estrous cycle. In cows with 3 follicular wave cycles, recruitment occurs on
Days 0, 9 and 16 of the estrous cycle (Ginther et al., 1989a; Ginther et al., 1989b).
Follicular waves occur in cyclic cows and heifers and in dairy and beef heifers prior to
the onset of cyclicity, in prepubertal calves as early as 2 weeks of age (Evans et al. 1994)
and during most of pregnancy (Ginther et al. 1996). Follicular growth begins in the fetal
ovaries, with the final maturation stages occurring days prior to ovulation.
Follicular dynamics in the cow consists of three distinct phases: recruitment,
selection and dominance. Recruitment is the process by which a group of follicles begins
to mature and grow under the influence of gonadotropins. In selection, a single follicle is
chosen and avoids atresia with the potential to ovulate. In dominance, the selected
follicle inhibits the recruitment of a new cohort of follicles (Lucy et al., 1992).
Hoak and Schwarts (1980) showed that the secondary increase in FSH levels
following the preovulatory LH surge recruits follicles for the next estrous cycle in the rat.
Adams et al., (1992) showed that the FSH surge in heifers began 2-4 days before
ultrasonically detectable emergence of a follicular wave (follicles <5 mm). FSH peaked 1
or 2 days before emergence and began to decrease at the time of deviation, when the
follicles of a wave begin to diverge into a dominant follicle and subordinate follicles
(follicle 6-7 mm). One of these follicles becomes dominant and has the potential to
In a two-wave estrous cycle, a group of small follicles (<5 mm) is recruited and
begins to grow under the influence of follicle-stimulating hormone (FSH; Adams, et al
1992). Later a single follicle becomes dominant and continues to grow while subordinate
follicles will eventually undergo atresia. In the presence of the CL and high progesterone
concentration, the dominant follicle of the first wave does not ovulate and regresses. A
second follicular wave develops with recruitment of another group of follicles around
midcycle. The dominant follicle of this second wave is functional at the time of CL
regression and decreasing progesterone concentration and this follicle ovulates after
In a three-wave cycle the second dominant follicle fails to ovulate and a third
follicular wave develops. The dominant follicle of the second wave is usually smaller
than the first and third dominant follicle (Lucy et al., 1992). Cows with a three-wave
cycle have a longer estrous cycle, as the third dominant follicle requires time to develop
prior to ovulation (Lucy et al., 1992).
Regulation of Follicular Growth by FSH and LH
Follicular deviation or selection has been defined as the beginning of the greatest
difference in the growth rates between the largest follicle (dominant follicle) and the
second largest follicle (largest subordinate follicle) at or before the largest subordinate
follicle reaches it maximum diameter (Ginther et al., 1996). The follicle that becomes
dominant develops receptors for luteinizing hormone (LH) around the time of deviation,
while the subordinate follicles do not. Under the influence of declining FSH
concentrations and increasing LH concentrations, the subordinate follicles regress and the
dominant follicle continues to develop. In the event that luteolysis does not occur with a
resultant fall in progesterone concentrations, the dominant follicle will not ovulate and
The dominant follicle secretes both inhibin and estradiol. Inhibin is also secreted by
follicle greater than 3 mm and also by atretic follicles. Estradiol and inhibin feed back on
the pituitary and hypothalamus and decreases the secretion of FSH from the pituitary.
Estradiol biosynthesis is dependent on a two-cell system. Theca cells and granulosa
cells individually are not equipped with the enzymes necessary for biosynthesis of
estradiol from cholesterol. However, together these cells are capable of producing
estradiol. Theca cells are the sites of androgen synthesis in follicles. Under the influence
of LH androgen secretion is increased. The end product in steroidogenesis in theca cells
is progesterone. Androstenedione produced by the theca cells is transported into the
granulosa cells where it is converted into testosterone. Under the influence of FSH,
testosterone is converted into estradiol within the granulosa cells (Hansel and Convey,
Production of Estrogen
Prior to the LH surge and luteinization of the granulosa and theca cells, the major
hormone produced by the ovary is estrogen, estradiol-17P3. The granulosa and theca cells
individually are unable to produce estradiol, since both lack the enzymatic pathways to
do so. In order to produce estradiol, cholesterol must be converted to androgens and
androgens to estradiol. Thecal cells are capable of the former, but not the latter, while
granulosa cells are capable of the latter (Niswender et al., 2000).
Corpus Luteum in the Cow
The corpus luteum (CL) is a transient endocrine structure, which secretes
progesterone, oxytocin (Fields et al., 1992), neurophysin, relaxin and other substances,
which act in an autocrine and/or paracrine manner. There is some evidence that the
bovine CL may produce estrogen. Kimbal and Hansel (1974) used a dextrand-charcoal
adsorption method to show that estrogen-binding proteins (estrogen receptor, ER) were
present in the bovine corpus luteum. Recent work by Okuda et al. (2001) demonstrated
that estradiol 17P3 is produced in the bovine CL.
The major secretary hormone is progesterone, which is essential for the
establishment and maintenance of pregnancy. In the non-pregnant animal, the life span of
the CL determines the length of the estrous cycle.
Corpus Luteum Development
The corpus luteum is formed from the remnants of the ovarian follicle following
ovulation. Simmons and Hansel (1964) postulated that any hormone that could stop
oxytocin from shortening the bovine estrous cycle had luteotropic properties. The
luteotropic property of luteinizing hormone (LH) was later confirmed by Donaldson et
al., (1965b, 1965c).
Ovulation is initiated by a preovulatory LH surge. Rupture of the follicular wall is
believed to involve enzymatic hydrolyzation of connective tissue by LH-induced
collagenases, proteases and plasmins (Banks, 1986). The oocyte and its surrounding
follicular fluid are simultaneously ejected from the ruptured follicle. The granulosa and
theca cells collapse into the cavity. The hemorrhage associated with ovulation clots,
forming the corpus hemorragicum. This stage is a transitory stage.
The granulosa and thecal cells undergo hypertrophy and division to become the
large and small luteal cells of the corpus luteum, respectively. The production of highly
specific antibodies against granulosa and thecal surface antigens proved that as the CL
ages the small luteal cells undergo structural and morphological changes to become large
luteal cells (Alila et al., 1984). This substantiates earlier work by Donaldson and Hansel
(1965a) using histology and histochemical staining techniques. They demonstrated that
granulosa cells stop dividing by Day 4 of the estrous cycle and small luteal cells derived
from thecal cells continue to respond to luteotropins grow and develop into large luteal
cells (Donaldson and Hansel, 1965a).
Ursely and Leymarie (1979) identified three types of cells in collagenase
dispersed luteal preparations. These included small (10-20[tm in diameter) steroidogenic
cells, large (>25 |tm diameter) steroidogenic cells and numerous small (<10 [tm
diameter) non-steroidogenic cells consisting mainly of vascular cells (endothelial cells,
erythrocytes leukocytes) and connective tissue cells (Hansel and Blair, 1996).
The non-steroidogenic cells of the bovine corpus luteum consist of macrophages
and endothelial cells which account for approximately 14% of the volume and
approximately 53% of the cells of the mature CL (Parry et al., 1980;O'Shea et al., 1989).
Macrophages are important for their phagocytic activity and for participation in the
immune response involved in the regression of the CL (Pate, 1994). Endothelial cells are
those that line the microvasculature and are thought to secrete substances which are
involved in both luteotropic and luteolytic processes. Fields and Fields (1996) reported
the presence of five different phenotypes of endothelial cells from the bovine CL. Type 1
cells are believed to be true endothelial cells, while Type 5 cells displays characteristic of
immature granulosa cell, and it has been suggested to be a putative stem cell for renewal
of luteal cells (Fields and Fields 1996). Fibroblasts comprise approximately 10% of all
luteal cells and approximately 6% of the total volume of the bovine CL (Fields and Fields
The steroidogenic luteal cells make up the majority of the bovine CL. The small
luteal cells and the large luteal cells together account for approximately 70% of the
volume of the bovine CL (Parry et al., 1980; O'Shea et al., 1989). The small luteal cells
make up approximately 28% of the volume of the CL and are known for its low basal
production of progesterone. When stimulated with LH, these cells increase their
production of progesterone. The large luteal cells comprise 3% of the luteal cells.
However, they comprise approximately 40% of the volume of the CL (O'Shea et al.,
1989). The large luteal cells do not respond to LH. However, they do secrete oxytocin
and are the steroidogenic cells, which produce the majority of basal progesterone.
During the early development of the bovine CL, there is rapid growth and increase
in size. The CL has a six-fold increase in size during early luteal phase of the estrous
cycle (Zheng et al., 1994). Along with an increase in weight, there is also an increase in
the amount of progesterone produced and secreted.
Function of the Corpus Luteum
The major hormone produced by the CL is progesterone. Progesterone functions in
determining the length of the estrous cycle and is required for the maintenance of
pregnancy in the cow.
The preovulatory LH surge results in luteinization of the granulosa and thecal cells
to large and small luteal cells, respectively. The luteinization process results in a shifting
of the steroidogenic pathway such that the major secretary product is progesterone. This
process includes increased expression of enzymes required for conversion of cholesterol
into progesterone. There is an increase in cytochrome P-450 side chain cleavage (P-
450soc) enzyme and 3p3-hydroxysteroid dehydrogenase/A ,A4 isomerase (3P3-HSD) and
decrease expression in the enzymes that convert cholesterol to estradiol-17a-hydroxylase
cytochrome P-450 and aromatase cytochrome (Niswender et al., 2000).
In the female reproductive tract, prostaglandin E2 (PGE2) is considered
luteotrophic (Pratt et al., 1977). Intrauterine administration of PGE2 protects the CL
against induced and spontaneous luteolysis (Pratt et al., 1977; Henderson et al., 1977).
Prostaglandin E2 stimulates the production of progesterone (P4) through the activation of
cyclic-AMP protein kinase A (PKA) pathway (Kotwica et al., 2003).
Prostaglandin E2 binds to its receptor, a G protein-coupled receptor, (EP) located in
the cell membrane. There are four receptor subtypes, EP1, EP2, EP3 and EP4. Only EP2
and EP4 are coupled to adenylate cyclase. Activation of adenylate cyclase leads to an
increase in cAMP that in turn activates PKA leasing to a cascade of intercellular signals
with the eventual activation of genes for progesterone syntheses. Prostaglandin E2 EP2
receptor is highly expressed in the large luteal cells. The EP2 receptor is the major
cAMP-generating PGE2 receptor in the bovine CL (Arosh et al. 2004) and the large
luteal cells produce 80% of progesterone (Diaz et al., 2002)
The Corpus Luteum and Pregnancy
In the event of pregnancy, the secretion of PGF2ua from the uterus is blocked,
luteolysis does not occur and progesterone concentration is maintained. Between Day 15
and 17 of the estrous cycle, the bovine concepts produces a signal, which prevents
luteolysis induced by the pulsatile release of PGF2ua (Kindahl et al., 1981; Asselin et al.
1996). The signal molecule, for the recognition of pregnancy in cows was first identified
as bovine trophoblast protein-1 (bTP-1), which was later called interferon-'c (INF-'c)
Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon. (Klemann et al.,
In the cow, INF-'c prevents the luteolysis by down-regulation of oxytocin receptors
(Meyer et al., 1996). Reducing the number of oxytocin-receptors available for oxytocin
binding attenuates oxytocin-stimulated secretion of PGF2a (Meyer et al., 1996). This
prevents oxytocin-stimulated PGF2a release and subsequent luteolysis.
Recombinant bovine interferon-'c (rblNF-'c) also causes a decrease in the expression
of cyclooxygenase II (COX II) and prostaglandin F synthase (PGFS; Xiao et al., 1998).
Cyclooxygenase II is an inducible rate-limiting enzyme for the conversion of arachidonic
acid to PGG2 and PGH2, the precursors of PGF2 and PGE2. Prostaglandin F synthase
(PGFS) is responsible for the reduction of PGH2 to PGF2 and PGD2 to 9ac, 11 3-PGF2 (a
stereoisomer of PGF2a). Recombinant bINF-'c has been shown to decrease COX II
mRNA in epithelial cells (the primary source of PGF2a) and to increases COX II mRNA
and prostaglandin synthesis in stromal cells (the primary source of PGE2; Xiao et al.,
1998). Xiao et al. (1998) also demonstrated that there was a reduction in PGFS mRNA in
both epithelial and stromal cells, and this was associated with an increase in PGE2: PGF2a
Bovine interferon-'c has been shown to shift the primary prostaglandin (PG)
produced by the endometrium from PGF2a to PGE2 (Asselin et al., 1997: Xiao et al.,
1998). Prostaglandin E is thought to be a luteotrophic agent (Pratt et al., 1977). Asselin et
al (1997) showed that cultured bovine endometrial cells treated with bovine recombinant
INF-'c (rINF-'c), in the presence and absence of oxytocin had a net PGE2: PGF2a ratio of
3.8 and 7.7 respectively. The conclusions made by Asselin et al. (1997) were rINF-'c
regulates PGs by stimulating PGE2 preferentially and rINF-'c transforms the response to
OT from stimulation of PGF2a to stimulation of PGE2.
Progesterone is necessary for the maintenance of pregnancy in the bovine. The
normal CL produces more progesterone than required to maintain the embryo until Day
15 of pregnancy. Normal embryo development proceeds to Day 15 when the levels of
total progesterone production exceed a threshold value by approximately 100 |tg of total
luteal progesterone; 15-day CL normally contain nearly 300 |tg of progesterone (Hansel
and Blair, 1996).
Luteal regression is caused by a pulsatile release of prostaglandin F2ca primarily
from the intercaruncular region of the surface epithelium of the uterus (Asselin et al.
1996) in the late luteal phase. It is thought that the pulsatile secretion of PGF2ca is
generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and
uterine PGF2ca. Regression of the CL is essential for normal cyclicity, and allows the
development of a new ovulatory follicle. However, prevention of luteolysis is necessary
for establishment and maintenance of pregnancy (Okuda et al., 2002).
Prostaglandin F2ca secretion from the bovine endometrium varies during the estrous
cycle. During the follicular phase and at estrus, Prostaglandin F2ca is at its highest and
then declines at early to mid-luteal phase of the estrous cycle (Kindahl et al., 1981).
PGF2au is released from the uterus in a series of short pulses 2 to 3 days during and after
luteolysis (Kindahl et al., 1981). Schramm et al. (1983) demonstrated that the CL is
sensitive to pulsatile administration of PGF2ca resulting in luteolysis. Skarzynski and
Okuda (1999) demonstrated that long-lasting stimulation with PGF2ca desensitizes luteal
PGF2au receptors in the cow and luteolysis fails to occur.
Oxytocin released from the corpus luteum acts upon the uterus to stimulate
production of PGF2ca, which in turn causes luteal regression. Prostaglandin F2ca has the
ability to cause further release of oxytocin from the ovine corpus luteum. Oxytocin has
been proven to be essential for the initiation of luteolysis in the ewe (Silvia et al., 1991).
It has been shown that oxytocin administered during the early stages of the estrous cycle
(Days 2-6) causes PGF2ca release, which is measurable in uterine venous blood (Milvae
et al., 1980). However, the level of oxytocin in the CL, plasma and oxytocin mRNA in
the CL are all low during normal luteolysis (Hansel and Blair, 1996).
It has been shown that exogenous administration of PGF2ca stimulates the utero-
ovarian release of PGF2ca in the ewe (Wade and Lewis, 1996). Similarly, Kotwica et al.
(1997) and Okuda et al. (2002) reported that administration of exogenous PGF2ca
increased PGF2ca release from the uterus on day 18 of the estrous cycle. It has been
demonstrated by Skarzynski and Okuda (1999) that PGF2ca activates protein kinase C
(PKC) and increases intracellular calcium mobilization, which may in turn stimulate
PGF2ca production in the endometruim.
It has been reported by Schams and Berisha (2002) that exposure to progesterone or
inhibition of progesterone action by a progesterone antagonist in early to mid-diestrus
regulates the onset of uterine release of PGF2ca from endometrium causing shortening or
extension of the interestrous interval in sheep and cows. Sheep that were treated with
onapristone (progesterone antagonsist) on day 4, 6 and 8 had a longer estrous cycle. The
controls had an estrous cycle length of 17.0 0.5 days while those treated with
onapristone had a cycle length of 22.0 0.8 days.
Endothelin-1 (ET-1), a 21 amino acid peptide is a potent vasoconstrictor originally
isolated from porcine aortic endothelial cells (Yanagisawa et al, 1988). Kisanuki et al.
(2001) demonstrated using gene-knockout technology in mice that ET-1 is produced
primarily by endothelial cells. It has been shown to be a modulator in female
reproduction where it serves to inhibit premature luteinization of follicular cells in
porcine ovaries (Flores, J.A., 2000) and in the propagation of the luteolytic process in the
ewe (Hinckley and Milvae, 2001).
The ET gene is transcribed to produce the preprohormone, preproET-1 (ppET-1).
PreproET-1 undergoes posttranslational processing to produce big ET-1, which is further
processed to mature ET-1. Big ET has little biologically active while ET-1 is the
biologically active form. The final processing stage from big ET-1 to the active form, ET-
1 is carried out by endothelin-converting enzyme-1 (ECE-1; Xu et al, 1994). Endothelin-
converting enzyme-1 is a membrane bound protein belonging to the zinc-binding
metalloendopeptidases (Xu et al, 1994), and has several isoforms (Meidan and Levy,
2002). Endothelin-converting enzyme-1 mRNA is highly expressed in steroidogenic
tissues, especially in the ovaries (Xu et al, 1994).
Endothelin-1 binds two receptors subtypes, ETA (for aorta) and ETB (type B
receptors; for bronchus; Meidan and Levy, 2002). These receptors belong to the seven-
transmembrane G protein-coupled receptor superfamily. The ETA receptor has a higher
affinity for ET-1, while ETB binds all three endothelins (ET-1, -2 and -3) equally
(Meidan and Levy, 2002).
Hinckley and Milvae (2001) demonstrated that ET-1 has an inhibitory effect of on
progesterone synthesis by luteal cells in the ewe. They demonstrated that ET-1 inhibited
basal and LH-stimulated progesterone production by dispersed ovine cells and that
inhibition was removed by pre-incubation with BQ123 (a highly specific endothelin ETA
receptor antagonist). They also showed that intramuscular administration of ET-1 at
midcycle reduced plasma progesterone concentrations for the remainder of the estrous
cycle. It has been shown that administration of a luteolytic dose of PGF2Uc stimulates
gene expression of ET-1 in ovine CL collected at mid-cycle, and intra-luteal
administration of BQ123 on Days 8 and 9 of the estrous cycle removes the luteolytic
effect of PGF2ca (Hinckley and Milvae, 2001).
Hinckley and Milvae (2001), working with ewes, supported the hypothesis that ET-
1 plays an integral part in PGF2a-mediated luteolysis. Following pretreatment with a
subluteolytic dose of PGF2ca, intramuscular administration of ET-1 caused a rapid
decline in plasma progesterone concentrations and shortened the length of the estrous
cycle in the ewe (Hinckley and Milvae, 2001).
The ETA receptor gene is expressed in the two luteal cell types enriched from
bovine CL and the thecal- and granulosa-derived luteinized cells of the CL in vivo.
However, the endothelial cells of the CL have higher levels of ETA receptor mRNA
levels than in each of the steroidogenic luteal cell types (Mamluk et al, 1999). Little is
known about the ETB receptor in the CL.
In theca-derived luteal cells, LH and forskolin downregulate ETA receptors, while
in granulosa-derived luteal cells, insulin-like growth factor-I (IGF-I) inhibits the ETA
receptor mRNA levels (Mamluk et al, 1999). This suggests that during the early stages of
luteinization, when both IGF-I and LH are at their peak levels, the expression of ETA
receptor is suppressed (Meidan and Levy, 2002).
Levy et al. (2003) showed that the ECE-1 gene is expressed by both bovine ovarian
endothelial and steroidogenic cells using enriched follicular and luteal cell
subpopulations and in situ hybridization techniques. Different ECE-1 isoforms exist
based on the different N-terminal cytoplasmic tails, and are located in different
intracellular areas. The intracellular ECE-la isoform is present only in ET-1-expressing
endothelial cells (Levy et al. 2003). The membrane-bound ECE-lb isoform is expressed
in both steroidogenic and endothelial cells of the preovulatory follicle and the CL. Insulin
and IGF-I upregulate ECE-1 expression when cultured with granulosa cells with
concomitant increase in progesterone production while ET-1 and LH down regulate ECE-
1 levels in steroidogenic cells.
The levels of ECE-1 mRNA are lowest in the bovine CL during the early stages of
CL development (Days 2-4). As the CL matures (Days 7-12), ECE-1 levels increase and
remain elevated in the late CL (Daysl3-16). ECE-1 levels decline in the regressed CL
(Days 20+; Levy et al. 2003). Levy et al. (2003) suggested the following model for ET-1
biosynthesis in the ovaries. Endothelial cells expressing ppET-1 and the two endothelin-
converting enzyme-1 (ECE-1) isoforms are capable of secreting both the precursor, big
ET-1 and mature ET-1. Steroidogenic cells only express the cell surface form of ECE-1,
ECE-lb, without ppET-1. In order to produce ET-1, steroidogenic cells are dependent on
the extracellular supply of big ET-1. Endothelial cells provide the precursor (big ET-1)
and the steroid-secreting cells convert it into mature ET-1, ensuring that ET-1 is
generated near its site of action, the ETA receptor, to ensure that the short lived ET-1 is
Involution of the Bovine Uterus
The postpartum interval is the period from parturition to the first postpartum
estrus accompanied by ovulation. During this period, several processes take place. The
uterus involutes to its previously non-gravid state, the endometrium regenerates, bacterial
contaminants are cleared from the reproductive tract and ovarian cyclicity resumes.
During the process of uterine involution, there is a reduction in the size of the
uterus accompanied by loss of tissue and regeneration of the endometrium. The reduction
in the size of the uterus is contributed to by strong muscular contractions that occurs
every three to four minutes during the first 24 hours post partum (Moller 1970b). After
calving, the uterus decreases in a logarithmic fashion in weight, length and diameter of
the previously pregnant horn. The greatest changes in these parameters occur rapidly in
the first few days after parturition. At calving, the uterus of a cow weighs approximately
9 kg. At Day 4 to Day 7 post partum, the uterine horns can be palpated cranial and ventral
to the pelvis (Morrow et al., 1969c). Over a period of 10 days the uterus rapidly involutes
to approximately 3 kg (Gier and Marion, 1968). The rapid reduction in size is
accompanied by an increase in uterine tone from Day 10 to Day 14 post partum and the
previously pregnant horn decreases from approximately 12 cm to 7 cm in diameter
(Morrow et al., 1969c). The period of rapid involution coincided with the first estrus in
normal cows and is associated with discharge of uterine lochia (Morrow et al., 1969c).
Following the rapid reduction in the size of the uterus, the rate of involution
decreases. The rate of involution decreases such that the uterus weighs about 1 kg by 20
to 30 days post partum and 750 g by 50 days post partum (Gier and Marion, 1968). The
length of the previously gravid horn decreases to half it length at parturition by Day 15
post partum and by Day 30 it is a third its size at parturition (Gier and Marion, 1968). The
postpartum uterus decreases to half its gravid diameter by Day 5 postpartum.
Gier and Marion (1968) noted that the cervix in the postpartum cow decreases in
diameter from 15 cm on Day 2 postpartum, 9-11 cm by 10 days post partum; 7-8 cm on
Day 30 post partum to 5-6 cm 60 days post partum. Morrow et al. (1969c) noted that at
Day 4 to Day 7 post partum the cervix was palpated cranial to the pelvis. There is an
initial decrease in diameter between days 5 and 10 followed by relaxation of the cervix at
day 10 post partum when final sloughing of the caruncular masses occur (Gier and
Marion, 1968). The relaxation of the cervix is interpreted on palpation and a slight
enlargement and is associated with discharge of lochia from the uterus and first
postpartum estrus (Morrow et al., 1969c). Involution of the cervix continues gradually
until Day 30 post partum in normal cows and until Day 35 post partum in abnormal cows
(Morrow et al., 1969c). Any other changes taking place after Day 30 to Day 35 post
partum could not be detected by transrectal palpation.
There is a difference in the rate of reduction between the myometrium and the
endometrium. During the first few days post partum, the myometrium reduces in size,
while the endometrium becomes edematous during the first day post partum. Endometrial
edema then resolves slowly over the next 5 days and by Days 6 to 8 post partum it has
disappeared and the endometrium regresses rapidly (Gier and Marion, 1968).
Further tissue losses after Day 19 consist of reduction of blood vessels, regression
of uterine glands, and contraction of tissue with reduction in cell numbers and cell
volume. The caruncles further reduce in size. On Day 19 the caruncles are 15 to 20 mm
in diameter. By Day 39 the caruncles appear as smooth knobs 10 to 15 mm in diameter
and by Days 50 to 60 the caruncles are reduced to circular cratered cones 8 to 10 mm in
diameter across the base and 4 to 6 mm across the crown (Gier and Marion, 1968). At the
end of the regression changes the caruncles appear as rows of white disks in a pink
endometrium (Gier and Marion, 1968). Involution of the uterus is considered to be
complete by 40 to 60 days post partum, when caruncles have regressed to a smooth,
oblong, epithelium-covered, avascular knob (Gier and Marion, 1968). Garcia and Larsson
(1982) reported that complete uterine involution occurred between Days 41 to 50 post
Uterine lochia is a mixture of normal and degenerate cells from the mucosa,
maternal placenta and blood from the shedding of the fetal placenta (Moller, 1970b). A
considerable amount of blood is found in the lumen of the uterus following parturition.
By Day 4 post partum the blood becomes mixed with sloughed caruncular material. By
Day 12 post partum, however, the luminal content changes to a lymph-like fluid that
decreases in quantity as the postpartum period progresses (Gier and Marion, 1968). This
fluid (lochia) is palpable in the uterus from Days 8 to 13 post partum. Uterine lochial
fluid diminishes from 1,400 to 1,600 ml at Day 2 to naught by Day 21 to 25 post partum.
The amount that is seen as a vaginal discharge varies from animal to animal and is
usually seen between Days 5 to 10 post partum (Moller, 1970b).
In the cow, fetal cotyledons fuse with maternal caruncles of the uterine mucosa to
form placentomes (Hafez, 1993). The fusing of the two tissues forms the primary
anchoring system of the placenta, keeping the maternal and fetal tissues in apposition
(Eiler, 1997). The secondary anchoring system consists of root-like penetration of
caruncle crypts by cotyledon villi and adhesive fluid between fetal and maternal
epithelium. The secondary anchoring system functions to hold fetal and maternal
epithelia together for physiologic exchange (Eiler, 1997).
After parturition, the septa and crypts of the caruncles contain remnants of
chorionic villi (Gier and Marion, 1968; Archbald, et al 1972). The remnants of
chorioallantoic cells in the maternal crypts undergo necrosis and mineralization and are
phagocytized or expelled in the lochia and are not observed in the caruncles after Day 11
post partum (Archbald et al., 1972).
The medium-sized and small arteries of the caruncule undergo progressive
vascular degeneration from Days 1 to 19 post partum (Archbald et al., 1972). Gier and
Marion (1968) found that within the first 2 days post partum, caruncular blood vessels
constrict and the septum and crypts undergo necrosis. The smooth muscle cells of the
tunica media undergo hydropic degeneration and pyknosis and the tunica media
undergoes fibrinoid necrosis (Archbald et al., 1972).
Archbald et al. (1972) noted that sloughing of the superficial areas of the caruncles
began on Day 1 post partum and necrotic changes in the stratum compactum were noted
on Day 5 post partum with sloughing of the superficial layer occurring by Days 6 and 7
post partum. The stratum compactum is reduced almost to the level of the inter-
caruncular area by Day 15 post partum (Archbald et al., 1972). By Day 5 post partum,
there is a necrotic layer 1 to 2 mm thick over the stratum compactum and cellular
organization is lost leaving only blood vessels and clumps of leukocytes (Gier and
By Day 10 post partum, most of the necrotic material is removed, and, all of the
caruncular mass involved in the placentomes sloughs by Day 15 post partum, leaving
stubs of blood vessels extending beyond the stratum compactum. At 19 days postpartum,
the arterioles within the stratum compactum and beyond disappear, leaving the caruncle
relatively smooth (Gier and Marion, 1968).
The leukocytes found in clinically normal cows consist of lymphocytes and
plasmacytes. Histiocytes and occasional polycytes are also found in the clinically normal
cow. In cows that have a uterine infection, organized lymphocytic nodules are found in
the stratum compactum.
Tissue regeneration occurs in all areas of the endometrium. However, the earliest
to show full regeneration is the inter-caruncular epithelium, which occurs by 8 days post
partum (Gier and Marion, 1968). Archbald et al. (1972) reported that at no time during
uterine involution was the inter-caruncular area devoid of an epithelial layer.
Tissue regeneration of the uterine epithelium begins just after parturition (Gier and
Marion, 1968) with focal areas of epithelial cells found on the caruncular surface on Day
1 post partum (Archbald et al., 1972). These epithelial cells seen on Day 3 and 5 are later
sloughed along with the superficial layer of the caruncle due to necrosis of the stratum
compactum of the caruncle between Days 5 and 6 (Archbald et al., 1972). The epithelial
cells are pleomorphic, and contain large hyperchromatic nuclei and granular cytoplasm.
Archbald et al. (1972) observed degenerated and regenerated epithelial cells in the
inter-caruncular epithelium on Day 1 post partum. The degenerative cells were confined
to the basal area of the epithelium and characterized by pyknosis and vacuolation of the
cytoplasm. Regenerated cells were interspersed among degenerated cells and
characterized by large, hyperchromatic nuclei and granular cytoplasm. Degeneration and
regeneration of the epithelial cells occurred simultaneously until Day 15 when
degenerative cells were no longer observed (Archbald et al., 1972).
In the event of bacterial contamination, the uterine epithelium may be totally or
partially destroyed (Gier and Marion, 1968). Re-epithelialization of the caruncle is
complete by Day 25 post partum, 10 days after obvious sloughing is complete (Gier and
Marion, 1968). Archbald et al. (1972) found that a layer of epithelial cells covered the
entire caruncular surface by Day 19 post partum.
The epithelial cells of the uterine endometrial glands exhibit a pattern of
simultaneous degeneration and regeneration similar to that observed in the inter-
caruncular area (Archbald et al., 1972). Microscopic changes in the myometrium begin
on Day 3 post partum and progress to Day 27 post partum. These changes include
granular degeneration of the sarcoplam, vacuolation of the muscle cell and atrophy of the
nucleus. By Day 31, the fibers appear normal and the myometrium is greatly reduced in
size in comparison to that in the early postpartum period. Necrosis of the muscle fibers
was not observed during postpartum myometrial regression (Archbald et al., 1972). Prior
to Day 3 post partum the muscle fibers of the uterus contract from 750-800 tm to 400
lm and by Day 3 are approximately 200 [im (Moller, 1970b). These changes in the size
of the muscle fibers have been attributed to glycogen formation leading to degeneration
and absorption (Moller, 1970b).
Factors Affecting Uterine Involution
Parity has been show to influence the interval from parturition to complete uterine
involution. Marion et al. (1968) found that the average interval from parturition to uterine
involution was significantly shorter in primiparous cows (34.0 days) compared to
pluriparous cows (40.6 days). Based on rectal palpation, Morrow et al. (1969c) noted that
multiparous cows of six lactations or more had larger uteri than primiparous cows, and
took a longer time to involute. However, Moller (1970a) and Miettinen (1990) found no
difference in the rate of uterine involution between primiparous and multiparous cows.
Cervical involution is influenced by parity. Miettinen (1990) found that there was a
difference in the size of the cervix with the cervix being larger in multiparous cows.
However, there was no difference in the time the cervix took to involute. In contrast,
Otlenacu et al. (1983) found that involution of the cervix occurred earlier in primiparous
than in multiparous cows. Based on rectal palpation Morrow et al. (1969c) noted that
multiparous cows of six lactations or more had a larger cervix than primiparous cows,
and took a longer time to involute. On Day 10 and Day 20 post partum there was a
significant difference of 1.2 and 1.0 cm in the diameter of the cervix. However, by Day
30 post partum the cervix was of similar sizes between 3.2 and 4.1 cm in diameter and by
Day 50 post partum the cervix was between 2.9 and 3.3 cm in diameter.
Otlenacu et al. (1983) noted that the time for complete involution of the cervix in
cows with a normal discharge was less than that for cows with an abnormal uterine
discharge. The greatest difference in cervix diameter between cows with normal and
abnormal discharge was 10 mm at three weeks postpartum (Otlenacu et al. 1983).
Time of year, or season, has been shown to influence the rate of uterine involution.
Cows calving in the spring and summer have shorter intervals from parturition to uterine
involution than cows which calve in the winter, regardless of parity (Marion et al., 1968).
Cows calving in the summer have variable intervals from parturition to uterine
involution. Marion et al. (1968) found that in cows calving in the summer with an
ambient temperature of > 380C, the heat stress increased the interval from parturition to
Retained fetal membranes have been shown to increase the interval from
parturition to the completion of uterine involution. Marion et al. (1968) found that it
required 50.1 4.9 days for the uterus to involute in pluriparous cows with retained fetal
membranes, and 45.1 3.2 days for primiparous cows. It was also noted that the rate of
uterine involution was affected more in primiparous cows than in pluriparous cows with
retained fetal membranes (Marion et al, 1968).
Morrow et al. (1969c) defined an abnormal cow as a cow that experienced any of
the following: abortion, dystocia, twins, retained fetal membranes, metritis, milk fever,
acute mastitis, ketosis or other debilitating disease. Abnormal cows had larger uteri in the
early postpartum period, especially cows with retained fetal membranes and metritis. At
Day 10 and Day 20 post partum, there was a significant difference of 1.3 cm and 0.8 cm
respectively between normal and abnormal cows. Abnormal cows had a combined
diameter of both uterine horns on rectal palpation of 14.9 cm on Day 10 and 8.4 cm on
Day 20 post partum, respectively. The gross palpable involution of the uterus was notable
up to Day 25 post partum in normal cows, and Day 30 post partum in abnormal cows.
Any changes that occurred after Day 25 and Day 30 post partum could not be felt on
transrectal palpation (Morrow et al., 1969c).
Metabolic disturbances can have a negative effect on uterine involution. Cows or
heifers that experience hypocalcaemia have a reduction in uterine contractions (Roberts,
1989; Al-Eknah and Noakes, 1989) and rate of uterine and cervical involution (Fonseca
et al., 1983). Kamgarpour et al. (1999) investigated subclinical hypocalcaemia in Friesian
cows. Subclinical hypocalcaemia was defined as occurring if the plasma total calcium
(PTCa) fell below 2.0 mmol/L. Cows calving in the winter and experiencing subclinical
hypocalcaemia took longer to complete uterine and cervical involution in comparison to
normocalcaemic cows, with hypocalcaemic cows taking 6 more days to reach a mean
size. Similar findings were noted in cows that calved in the summer and experienced
The bovine uterus is invaded and colonized by bacteria within the first two weeks
postpartum (Elliot et al., 1968; Griffin et al., 1974). In most cases the uterus clears this
infection by the third or fourth week post partum. Not all cows clear the bacterial
infection and subsequently develop endometritis. Endometritis has been shown to delay
uterine and cervical involution. Endometritis delays the resumption of ovarian cyclicity,
increases the interval from calving to ovulation and delays the return of prostaglandin F2a
metabolite (PGFM) to basal levels (Lindell et al., 1985; Del Vecchio et al., 1992).
Endometritis accounts for approximately 20% of reproductive disorders in postpartum
dairy cows (Coleman et al., 1985). Moller (1970a) examined the rate of uterine involution
between milked cows and cows suckling three or four calves and found that there was no
difference in the rate of involution between milked and suckled cows.
Steroid hormones have variable effects on the interval from parturition to uterine
involution. In cattle, the first postpartum dominant follicle develops on the ovary
contralateral to the previously gravid uterine horn. However, the presence of an estradiol-
secreting dominant follicle in the ipsilateral ovary is a marker of subsequent fertility
(Sheldon et al., 2000a). Based on their findings, Sheldon et al. (2000b) attempted to
promote a dominant follicle on the ipsilateral ovary using equine chorionic
gonadotrophin (eCG). Cows treated intramuscularly with 250 iu eCG or 750 iu eCG on
Day 14 post partum showed no difference in the rate of uterine involution compared with
those treated with a placebo. Treatment with eCG, or the presence of a follicle > 8 mm in
diameter in the ovary ipsilateral to the previously gravid uterine horn, did not affect the
rate of uterine involution (Sheldon et al. 2000b). The intramuscular administration of
estradiol benzoate approximately 48 hours after calving had no effect on the intervals
from calving to the completion of involution or between the intervals from calving to the
first ovulation (Tian and Noakes, 1991).
Sheldon et al. (2003) infused estradiol benzoate into the previously gravid uterine
horn on Days 7 and 10 postpartum, and monitored uterine involution by ultrasonography.
There was no effect of estradiol treatment on the diameter of the previously gravid or
nongravid uterine horns. They concluded that utero-ovarian signaling in the direction
from the uterus to the ovary may be more important during the postpartum period.
Administration of oestradiol into the uterine lumen increased uterine pathogenic
anaerobic bacterial contamination (Sheldon et al., 2004). Estradiol benzoate was infused
into the previously gravid uterine horn on Days 7 and 10 postpartum. Animals treated
with estradiol benzoate had higher bacterial load on Day 14, than on Days 7 or 21,
attributable to pathogens associated with endometritis, Prevotella melaninogenicus and
These finding by Sheldon et al. (2000a, 2000b, 2003, 2004) and others indicate that
estradiol administration in the early postpartum period had no effect on uterine involution
(Tin and Noakes, 1991) and may be detrimental by promoting endometritis (Sheldon et al
2004). More first postpartum dominant follicles are selected in the ipsilateral ovary when
there is a lower uterine bacterial load (Sheldon et al., 2002).
Exogenous progestogens have been administered to cows in an attempt to improve
uterine involution. Melengestrol acetate (MGA) was feed to Holstein cows at 1 mg per
cow per day for 14 days beginning 14 to 18 days post partum in one group of cows. In a
second group MGA was fed in a similar dosage and 500 micrograms of estradiol
benzoate was administered intramuscularly 48 hours after the last feeding of MGA. The
third group consisted of control cows that received no treatment. Based on rectal
palpation, there was no significant difference in the rate of involution (Britt et al., 1974).
Subcutaneous administration of 0.05 mg and 1.0 mg estradiol 17P3 given every
other day from Day 3 post partum was shown to have no effect on the rate of involution.
The oral administration of 300 mg medroxyprogesterone acetate every other day from
Day 3 postpartum had no effect on the rate of uterine involution. However, daily
administration of 30 mg of progesterone has been shown to increase the time for the
uterus to involute completely in both intact and ovariectomized cows (Marion et al.,
1968). Removal of ovarian hormone by ovariectomy has been shown to significantly
reduce the time for uterine involution in pluriparous cows (Marion et al., 1968).
The administration of prostaglandin F2a has been shown to decrease the time for
complete involution of the uterus as detected by rectal palpation (Lindell and Kindahl,
1983). Lindell (1982) demonstrated that there is a massive release of PGF2a postpartum,
which continues for 2 to 3 weeks. It was also deduced from this study that cows that had
a shorter interval from parturition to uterine involution had a longer period of postpartum
In a study by Tian and Noakes (1991) five groups of five cows were treated
intramuscularly with a single dose of either 100mg progesterone in oil, 25 mg dinoprost
trmethamine (PGF2ac tromethamine), 5 mg oestradiol benzoate, 1.2 mg long-acting
oxytocin analogue, carbonectin or 5 ml sterile water 48 hours after parturition. There was
no statistical difference in the rate of uterine involution.
Methods to Assess Uterine Involution
Uterine involution may be studied by observing the reduction in the size of the
vulva, vagina, cervix and the uterus (body and horns). These observations may be made
using the following: 1) palpation per rectum of the cow's reproductive tract from calving
to completion of uterine involution (Marion et al., 1968; Moller, 1970a; Garcia and
Larsson 1982); 2) removal of the reproductive tracts at slaughter at predetermined
intervals from calving (Gier and Marion, 1968; Moller 1970a); 3) transrectal
ultrasonography of the reproductive tract (Mateus et al., 2002; Wehrend et al., 2003; 4)
cervical forceps (Wehrend et al., 2003)
Garcia and Larsson (1982) performed rectal palpation of the cervix, uterus and
ovaries. They assessed uterine size, tone, symmetry and location of the uterus in pelvic
cavity as indicators of uterine involution. Uterine involution was considered complete
when the uterus was in the normal position in the pelvic cavity, the uterine horns were
equal or almost equal in size and there was no enlargement in the thickness of the uterine
wall. Marion et al., (1968) used rectal palpation twice weekly to monitor uterine
involution, using criteria set by Gier and Marion (1968) as a marker for complete uterine
Moller (1970b) refers to Rasbech's (1950) four-stage sequence in involution, which
can be followed by rectal palpation. During the first stage (1-8 days post partum), the
vagina can be palpated as a band approximately 8 cm in width within 24 hours post
partum. The cervix is not distinguishable until Day 3 and by Days 4 and 5 has enough
tone to be distinguished from the uterus and is usually located at the anterior edge of the
pelvic floor. The surface of the uterus feels hard and corrugated and the uterine caruncles
can only be felt through the uterine wall when it is relaxed. In the second stage (8-10 days
post partum) the entire uterus can be palpated. The surface is smooth and soft with
fluctuation in the post-gravid horn. The caruncles are palpable as hazel-nut shaped
structures and the cervix is firm and lies within the pelvic cavity in younger animals.
During the third stage (10-18 days post partum) the uterus feels like a soft plastic body;
caruncles and fluctuations are less pronounced; the cervix is firm and continues to
decrease in size until it is similar to that of the post-gravid uterine horn. During the fourth
stage (18-25 days post partum) there is an increase in uterine tone and the previously
gravid horn reduces to a similar size to the non-gravid uterine horn.
Moller (1970b) reports that authors on the subject of uterine involution agree that
the previously pregnant horn rarely returns to its pre-gravid size. Although the uterus
continues to undergo involutional changes after 25-30 days (Gier and Marion, 1968),
these changes are small and difficult to detect by rectal palpation (Moller, 1970b).
However, Marion et al. (1968) concluded that uterine involution monitored by rectal
palpation take 40 days.
Gier and Marion (1968) used reproductive tracts collected from slaughtered cows
to assess involutional changes. They removed the excess fat and vagina from the cows to
access the changes that occur during the process of involution. The remainder of the tract
was then measured. The weight, length and diameter of the previously pregnant uterine
horn were used to access the rate of regression. The diameter of the cervix was measured
to assess uterine involution.
Wehrend et al (2003) used a combination of ultrasonography and cervical forceps
to measure the rate of uterine and cervical involution. Cervical forceps were positioned in
the cervical canal and the position verified by ultrasonography. The distance of the legs
of the forceps was proportional to the opening of the tip of the forceps. A gauge table was
used to determine the degree of the opening of the intracervical tip of the forceps by
measuring the distance of the extracorporeal legs of the forceps.
Endocrinology of the Immediate Postpartum Period in the Cow
The corpus luteum of pregnancy secretes progesterone throughout pregnancy and
regresses about 2 days prior to parturition (Garverick and Smith, 1993). The placenta also
contributes progesterone in the latter part of pregnancy. At the end of pregnancy there is
a rapid increase in the secretion of estrogen (Hoffmann and Schuler, 2002). Prior to
calving, progesterone and estrogen concentrations are high (Hoffmann and Schuler
The progesterone concentration begins to decrease about two days prior to
parturition and is low following calving. Following parturition, there is a rapid decline in
circulating concentrations of estrogen. The fall in progesterone and estrogen removes the
inhibitory block on the hypothalamic-pituitary axis (Garverick and Smith, 1993; Noakes,
1996) The inhibitory effects of estradiol are thought to operate through inhibition of the
expression of mRNA coding for the common ac-subunit and specific P-subunit of FSH
and LH (Garverick and Smith, 1993). Messenger RNA expression of the subunits is low
following parturition and increases thereafter.
During gestation, LH is secreted in a pulsatile manner. However, the pulse
frequency and amplitude decrease as gestation progresses. Pituitary LH content and
plasma LH concentrations are low shortly after calving and generally increase during the
postpartum period (Schallenberger et al., 1982; Garverick and Smith, 1993). Short-term
episodic surges of LH increase as time post partum increases and LH responsiveness to
GnRH is low at parturition and increases as time post partum increases (Garverick and
Smith, 1993). The frequency of the pulsatile LH secretary pattern increases just prior to
the first ovulatory surge ofLH (Garverick and Smith, 1993).
Ovariectomy in the early postpartum cow has a profound effect on the tonic
secretion of gonadotropin from the pituitary. Schallenberger et al. (1982) showed that
ovariectomy performed in the early postpartum period caused a two-fold increase in
mean LH values as well as the amplitude and frequency of pulsatile release. A similar
response was seen for FSH. However, the increase in amplitude was less pronounced
than that seen for LH.
Following parturition, FSH concentration is within normal range (Garverick and
Smith, 1993). Schallenberger et al. (1982) noted pulsatile FSH release during the first day
post partum. The frequency of pulsatile LH release coincided with FSH pulses. However,
there were additional pulses between the FSH and LH pulses that coincided during the
early postpartum period (Schallenberger et al., 1982). Increasing progesterone
concentrations caused an immediate suppression of frequency and basal LH, but not FSH
secretion. This is similar to that seen during normal cyclicity (Schallenberger et al.,
The administration of estrogen has no effect on LH secretion on Days 0 and 5 post
partum. However, by Day 10 post partum and beyond, administration of estradiol elicited
an LH surge with increasing magnitudes within 12 to 24 hours. Cows possessing a
functional CL did not respond to estrogen with an LH surge. However, this was not the
case for FSH. FSH surges also occurred in cows at 5 days post partum (Schallenberger et
Schallenberger et al. (1982) showed that first calf heifers could be induced to cycle
early in the post partum period when GnRH is administered on an hourly basis at 500
micrograms followed by challenge with lmg estradiol-benzoate.
It has been hypothesized that the following sequence of endocrine events occur in
the normal cow after calving based on studies carried out by themselves and others
(Peters and Lamming, 1986). Gonadotropin releasing hormone (GnRH) is secreted from
the hypothalamus immediately after calving. However, the quantity or frequency of
secretion is inadequate to cause sufficient release of LH and FSH from the pituitary to
restore normal ovarian cyclicity. The concentration of FSH rises quickly after parturition
and stimulates follicular development. The plasma concentration of LH and the frequency
of LH pulses increase gradually with time post partum. Secretion of LH and FSH
stimulates follicular growth and estradiol production. There is a gradual recovery in the
positive feedback mechanism such that by two weeks postpartum normal ovarian
Resumption of Ovarian Cyclicity Post Partum
The interval from parturition to first observed estrus ranges from 30 to 76 days in
the dairy cow (Roberts, 1971). However, observation of estrus may not be a reliable
method to estimate the resumption of ovarian activity since some cows experience
"silent" estrus. As time progresses post partum, the percentage of cows in which silent
estrus occurs decreases (Roberts, 1971). Other methods of determining the onset of
cyclicity have evolved which are more accurate. The milk progesterone assay determines
the resumption of ovarian cyclicity by the presence of elevated progesterone
concentrations (Noakes, 1996). Using milk progesterone concentrations, Bulman and
Wood (1980) determined that approximately 50 percent of cows resumed normal
cyclicity by 20 days postpartum and greater than 90 percent by 40 days post partum
Ultrasonography has been used to follow the development of ovarian follicles in
the post partum cow. Sheldon et al. (2002) detected follicular development by 7 to 10
days post partum with emergence of a dominant follicle within 14 days of parturition and
ovulation of this dominant follicle by 17 to 18 days post partum. This interval was
increased in cows with high milk production, in cows nursing calves or being milked four
times a day, in cows on a poor or on low plane of nutrition and in older cows with greater
than four parturitions (Roberts, 1971).
In cows with a normal postpartum period, a mature follicle develops and ovulates
with subsequent development of a CL by Days 13 to 15 after calving (Roberts, 1971).
The first estrous cycle is usually shorter than the normal 20 to 21 days (Roberts 1971).
Terqui et al. (1982) found that the earlier ovarian activity occurs the more likely cows
will experience a short luteal phase. The CL associated with the short estrous cycle has a
short life span as a result of lack of luteotropic support, failure of the luteal tissue to
recognize a luteotropin, or enhanced secretion of a luteolytic agent (Hafez, 1993).
The first ovulation post partum usually occurs on the contralateral ovary.
Transrectal palpation of the ovaries has shown that approximately 90 percent of corpora
lutea formed within 15 days post partum occur on the ovary opposite the previously
pregnant horn and 60 percent in cows ovulating between 15 and 20 days after parturition
(Roberts, 1971). This has been substantiated by sequential transrectal ultrasonography.
The first postpartum dominant follicle is usually found on the ovary contralateral to
the previously pregnant uterine horn, that is, opposite the ovary containing the CL of
pregnancy (Foote et al., 1968; Kamimura et al., 1993; Nation et al., 1999). This led many
to speculate that the CL of pregnancy may have a local inhibitory effect on
folliculogenesis. Dufour et al. (1985) concluded that the CL of pregnancy and/or the
concepts have a carry-over effect on the rate of growth of the antral follicles even after
parturition. Bellin et al. (1984) found that the ovary with the CL of pregnancy had
smaller follicles and lower follicular estrogen concentrations than those of the ovary
without a CL. It was also noted that only 18% of the follicles on the ovary with a CL
were healthy compared to 48% of follicles in the ovary without a CL (Bellin et al., 1984).
However, recent work done by Sheldon et al. (2002) suggested that the CL of pregnancy
does not have a local inhibitory effect on postpartum folliculogenesis. It was further
suggested that the previously pregnant uterine horn shortly after parturition may play
more of a role.
It has been shown that the presence of a large follicle on the ovary ipsilateral to the
previously pregnant uterine horn within 4 weeks of parturition was associated with
improved subsequent fertility (Sheldon et al., 2000). Bridges et al. (2000a) reported
similar findings in beef cows, where less cows ovulated from the ipsilateral ovary.
However, ovulation from the ipsilateral ovary tended to increase fertility (Sheldon et al.,
There are several factors that have a negative impact on the resumption of ovarian
cyclicity in dairy cows. The interval from calving to first ovulation increased in cows
with high milk production, cows nursing calves or being milked four times per day, cows
on a poor or on low plane of nutrition, and in older cows with greater than four
parturitions (Roberts, 1971). Bellin et al. (1984) found that suckled cows had smaller and
fewer follicles and lower follicular concentrations than nonsuckled cows.
There is a close association between the time to first ovulation and negative energy
balance in dairy cows (Zurek et al., 1995). The exact mechanism by which energy
balance influences reproduction is not completely understood. However, one mechanism
may be through the suppression of GnRH and the LH pulse frequency required for
follicles to grow to the preovulatory stage (Schillo, 1992).
Cystic Ovarian Disease in the Dairy Cow
Cystic ovarian degeneration, ovarian follicular cysts, cystic ovarian disease, ovarian
cysts and cystic ovaries are terms used to describe the same condition. Ovarian cysts are
follicles that fail to ovulate at the time of estrus (Garverick, 1999), and commonly occur
in lactating dairy cows. They can also occur in beef cows and dairy heifers. Ovarian cysts
have been defined as follicular structures 2.5 cm or greater in diameter that persists for an
extended period of time in the absence of a CL (Garverick, 1999). Ovarian cysts can be
classified as follicular cysts or luteal cysts. Follicular cysts are thin walled, may be single
or multiple and affect one or both ovaries (Elmore, 1986). Higher concentrations of
estradiol are usually found in follicular cyst fluid than in fluid from luteal cysts and
normal follicles (Odore et al, 1999). Luteal cysts are thick-walled, usually occurring
singly, and affect one ovary and have higher concentrations of progesterone in their
cystic fluid than follicular cysts and normal follicles (Odore et al, 1999).
Bane (1964), in a review of fertility and reproductive disorders in Swedish cattle,
quoted Henricson reporting that the risk of ovarian cysts increased from 0.3% for heifers
to 8-10% for cows four to five years old and that Henricson found the incidence of
ovarian cysts was higher in the winter months than in the summer months. Bane analyzed
the frequency of ovarian cysts in relation to the month of calving and found that cows
calving in the winter had a higher risk of developing ovarian cysts in the following
reproductive period (Bane, 1964). Lopez-Gatius et al. (2002) indicated that ovarian cysts
occur most commonly in cows calving in the summer. Garverick (1999) estimated the
incidence of ovarian follicular cysts between 10 -13% in the United States.
The calving date influences the frequency of ovarian cysts. Cows that calved in
April-May were less likely (2.2%) to develop ovarian cysts while cows calving in
October had a higher frequency (9%) of ovarian cysts (Bane, 1964).
In Sweden between 1954 and 1961, the frequency of animals with ovarian cysts in
five-year-old cows fell from 10.8 % to 5.1%. This was brought about by the culling of
bulls with a high frequency of cystic ovarian degeneration among their daughter (Bane,
1964) suggesting that there may be a hereditary aspect.
Spontaneous recovery occurs in some cows that develop ovarian cysts. L6pez-
Gatius et al. (2002) found that in cows diagnosed with cysts 43-49 days postpartum, only
38.8% recovered spontaneously while in cows diagnosed with cysts 57-63 days
postpartum, 71.4% recovered spontaneously. Erb and White (1981) reported that the
highest incidence of ovarian cysts occurs in the first 60 days of lactation, while Morrow
et al. (1969b) reported that there was a peak in incidence between 14 and 40 days post
L6pez-Gatius et al. (2002) reported that ovarian cysts occur most commonly in
cows calving in the summer, in high milk producers, in older cows, and in cows that body
condition score increased during the prepartum period. The major risk factor for the
presence of cysts at the time of insemination was the development of cysts early (43-49
days postpartum) in the postpartum period (L6pez-Gatius et al., 2002). The risk of having
a cyst at the time of insemination was 36.6 times higher in cows with early cysts (L6pez-
Gatius et al., 2002).
Postpartum uterine infections may increase secretion of PGF2a and cortisol
associated with the formation of cystic ovaries in dairy cows (Bosu and Peter, 1987; Peter
et al., 1989). In a study carried out on cows that calved normally but subsequently
developed ovarian cysts, a correlation between postpartum uterine infections and the
development of ovarian cysts was made. In 88.9% and 11.1% of cows that subsequently
developed ovarian cysts endometrial swabs yielded bacterial growth densities of +3 and
+2 respectively, while 35.1% of cows that did not develop ovarian cysts yielded bacterial
growth densities of +1 and +2 (5.4%) (Bosu and Peter, 1987). Further, consistently high
cortisol levels were detected prior to cyst detection and in association with high bacterial
growth densities in the uterus prior to cyst detection (Bosu and Peter, 1987).
Cysts have been experimentally induced using anti-LH serum, estradiol valerate
and adrenocorticotropic hormone (ACTH) administration. Cysts have also been induced
by daily exogenous injection of 30 mg of estradiol-17P3 and 150 mg of progesterone
dissolved in alcohol for 7 days (Cook et al., 1990; Garverick, 1999).
Ovarian cysts have been induced by ACTH administration in the preovulatory
period. Adrenocorticotropic hormone has been shown to block the preovulatory LH
surge, possibly through cortisol-mediated inhibition of LH release (Refsal et al, 1987).
The mode of action of opioids is by inhibiting the release of hypothalamic GnRH.
Through their action on GnRH, they are thought to suppress LH secretion from the
pituitary in the prepubertal period and modulate LH during the estrous cycle. It has been
established that gonadal steroids suppress LH secretion by negative feedback on the
hypothalamic-pituitary axis, and this action may be brought in part by intermediate
opioidergic neurons (Brooks et al., 1986).
It has been demonstrated that follicular cysts can be induced using estradiol
benzoate by stimulating a GnRH/LH surge in the absence of a preovulatory follicle.
Subsequent exposure of the cystic cow to progesterone can resolve the cystic condition
by reinitiation of GnRH/LH surges in response to estradiol (Gumen et al, 2002).
Gumen and Wiltbank (2002) demonstrated in cows that an initial GnRH/LH surge
and subsequent ovulation could be induced with high levels of estradiol, but estradiol
induction of a subsequent GnRH/LH surge required exposure to progesterone. Follicular
cysts were induced using an intravaginal progesterone insert (IPI)/PGF2a/estradiol
benzoate (EB) protocol, and indicated that the effect is mediated at the level of the
hypothalamus (Gumen and Wiltbank, 2002).
Odore et al., (1999) suggested that ovarian cysts might be due to alterations in the
feedback regulatory mechanism. This hypothesis was based on findings that indicated a
difference in the concentration of luteinizing hormone receptors and follicle stimulating
receptors in the ovary and pituitary between normal and cystic cows.
Silvia et al. (2002) proposed the following model for how intermediate levels of
progesterone could lead to the development of ovarian follicular cysts in the dairy cow.
During the follicular stage in cows without ovarian cysts, the tonic center of the
hypothalamus secretes GnRH at a high frequency stimulating a frequency mode of LH
secretion by the anterior pituitary. The high frequency LH secretion promotes maturation
of the dominant follicle. This follicle in turn secretes increasing concentrations of
estradiol that eventually reach a threshold adequate to trigger the surge center of the
hypothalamus to release GnRH in quantities that will trigger a preovulatory LH surge and
induce ovulation of the dominant follicle. However, in cows with ovarian cysts, the
intermediate progesterone levels make the surge center of the hypothalamus insensitive to
estradiol. The preovulatory GnRH/LH surge is blocked and ovulation fails to occur.
However, the tonic center of the hypothalamus is unaffected by intermediate
progesterone concentrations and thus the high frequency, tonic pattern of LH is
maintained. This tonic pattern of LH provides the cysts with the gonadotropic support it
requires to function (Silvia et al., 2002).
Ovarian cysts are dynamic structures. They regress and are replaced with other
cystic structures (turnover), or spontaneous recovery occurs characterized by ovulation of
a new follicle at a site different from that of the original cysts (Cook et al. 1990). Cook et
al. (1990), induced ovarian cysts with twice daily subcutaneous injections of 15 mg
estradiol-17P3 and 37.5 mg of progesterone for 7 days starting on Day 15 post partum. Of
the 23 cows that became cystic, 7 ovulated (spontaneous recovery), 13 showed cystic
turnover and 3 persisted (Cook et al. 1990). In no case did the marked cyst ovulate. In
another study carried out on cows that calved normally over a one-year period, 12 out of
47 cows were found to be cystic. Follicular cysts were detected on the ovary as early as 8
days post partum. In 10 cows, serial rectal and ultrasound examination of the cysts
indicated an increase in size confirming the dynamic nature of ovarian cysts (Bosu and
Peter, 1987). In 4/12 cows (33.3 %) the cyst regressed before Day 28 post partum and in
the remaining 8/12 cows (66.6%) the cyst regressed between Days 29 and 57 post partum
(Bosu and Peter, 1987). In a study of 29 heifers undergoing daily transrectal ultrasound in
order to follow follicular dynamics, one heifer developed follicular cysts. This heifer
regressed the corpus luteum in a normal manner, developed a normal preovulatory size
follicle on the ovary, exhibited standing heat, but the follicle failed to ovulate. This
follicle lost follicular dominance and a new follicular wave emerged. Two dominant
follicles emerged from this wave. When one reached preovulatory size, the heifer once
again exhibited estrus but did not ovulate. This process continued through a third
follicular wave and finally the heifer was successfully treated using the Ovsynch protocol
after emergence of the fourth follicular wave (Wiltbank et al., 2002).
Due to the dynamic nature of ovarian cysts, weekly rectal palpation and/or
ultrasonic observations will detect those cows with cysts more readily than monthly or
bimonthly examinations. The diagnosis of ovarian cysts is usually based on rectal
palpation of a fluid filled structure greater than 2.5 cm in diameter on one or both ovaries
in the absence of a CL. The diagnosis may be confirmed using transrectal
ultrasonography and the type of cysts (luteal or follicular cysts) determined. Luteal cysts
may also be confirmed by progesterone assay of milk or serum. The most common sign
of cows that have been diagnosed as cystic is a lack of estrus (Farin and Estill, 1993).
Nymphomania has also been noted in cows that have follicular cysts (Farin and Estill,
In order to differentiate between cystic follicles and a large preovulatory follicle,
the characteristic of the uterus must be evaluated. On rectal palpation a diagnosis of
cystic ovaries will be made when the uterus has no tone and is unresponsive to
manipulation. The uterus of a cow in estrus has tone and responds to manipulation by
increase coiling of the horns.
Although cows with ovarian cysts have been shown to recover spontaneously,
only a portion of cows affected with cystic ovarian disease recovers spontaneously and
within a time frame that is economical to the dairy farmer. Morrow et al (1966) reported
that only 48% of cows affected with follicular cysts recovered spontaneously. Kesler and
Garverick (1982) estimated that even less cows (approximately 20%) with cystic ovaries
will spontaneously recover and return to normal cyclicity.
One of the earliest methods of resolving ovarian cysts was manual rupture via
rectal palpation. Repeated manual rupture of cysts at 6 to 10 day intervals, especially
follicular cysts in the early postpartum period, has produced 37% (Roberts, 1971) to 45%
recovery rates (Kesler and Garverick, 1982; Ijaz et al., 1987). However, manual rupture
can cause injury to the tissue of the ovary and its surrounding structures, causing
hemorrhage, promoting adhesions and infertility (Roberts, 1989; Elmore, 1986).
Gonadotropin-releasing hormone (GnRH) is commonly used to treat ovarian cysts
regardless of the type of cysts. Exogenous administration of GnRH triggers an LH surge
that results in ovulation of a LH-responsive follicle at the time of treatment or
luteinization of the cyst (Kittok, et al. 1973; Cantley et al. 1975). Repeated use of GnRH
intravenously 120 minutes apart has been shown to cause a LH surge similar to the
preovulatory LH surge in normal cycling cows; the second dose corresponding to peak
LH serum concentrations in cows with cystic follicles (Kittok et al. 1973). The LH surge
is induced with 30 minutes of GnRH administration and remains elevated for 4 hours
(Cantley et al. 1975). However, the response of the cow with ovarian cysts differs from
that of cows without ovarian cysts. that have an elevated LH concentration for up to 10
hours which may due to luteinization of the cystic structure as a result of ovarian
response to GnRH induced LH release (Cantley et al. 1975). Cow treated with GnRH
have been shown to return to estrus within 20 to 24 days (Kittok et al. 1973) after
treatment. Cows with ovarian cysts treated with a single intramuscular injection of either
50, 100 or 250 [tg GnRH exhibited estrus by 22 days and most cows exhibited estrus 18
to 23 days post-treatment (Bierschwal et al. 1975). The 100 tg dosage had the best
response of the three dosages with 82% returning to estrus and 87% of those that returned
to estrus conceiving (Bierschwal et al. 1975).
In order to shorten the interval between GnRH treatment and estrus, PGF2ca has
been used 9-14 days after GnRH treatment (Neil, 1991). However, the need for estrus
detection has been eliminated with the introduction of the Ovsynch protocol (Pursley,
1995). This protocol uses a GnRH/PGF2a/GnRH treatment scheme which has proven
successful in treating cystic ovarian degeneration (Bartolome et al., 2000; Gumen et al.,
Human chorionic gonadotropin (hCG) is used for its high luteinizing hormone
activity (Kesler and Garverick, 1982). It has the same effect as GnRH. However, hCG
has the ability to induce antibody production where as GnRH because of its small
molecular size is not likely to induce an immune response (Bierschwal, et al. 1975).A
dose of hCH between 2,500 and 10,000 IU, given intramuscularly or intravenously will
have the same effect as GnRH (treatment response and post treatment fertility). However
it can cause undesirable effects (Neil, 1991)
Progesterone has been shown to resolve ovarian cysts. Methods of administering
progesterone have included injections, intravaginal devices or ear implants. Zulu et al.
(2003) investigated the use of a progesterone releasing intravaginal device (PRID) in the
treatment of cows with ovarian cysts. Their study confirmed that follicular and luteal
cysts could be successfully treated with PRID; resulting in ovulation 2-4 days after
removal of the device with subsequent formation of a CL.
Prostaglandin F2a (PGF2a) has been used alone in the treatment of ovarian cysts
(Chavatte et al., 1993). It is the treatment of choice for luteal cysts or cysts containing
luteinized tissue. Luteal cysts respond by regression with subsequent follicular
development and estrus in 2-5 days (Kesler and Garverick, 1982; Neil, 1991). It has been
shown that there is a higher PGF2ca receptor concentration in luteal cysts than in
follicular cysts (Odore et al., 1999) and this may explain the success of prostaglandin
treatment. Prostaglandin F2a is generally believed to be ineffective in follicular cysts, but
some cows with progesterone as low as 0.5 ng/ml will respond positively (like luteal
cysts; Neil, 1991).
Timed Artificial Insemination Programs in the Dairy Cow
One of the factors affecting pregnancy rate in dairy herds is the detection of
estrus. In some instances estrous behavior is reduced. Reduced estrus behavior has been
noted in cows treated with bovine somatotropin (bST) (Kirby et al., 1997). It has been
shown that the physiological state of lactation is associated with lower concentrations of
estradiol during proestrus than levels found in non-lactating dairy cows and thus
behavioral estrus is reduced in these cows. During lactation progesterone levels are also
lower than in non-lactating dairy cows. In addition it has been shown that cows in
lactation have a lower reproductive rate than do heifers.
Heat stress is another factor that decreases the expression of estrus behavior
(Abilay et al., 1975; Nebel et al., 1997). However, lack of heat detection can play a major
role in reducing pregnancy rate. The development of programs for estrus synchronization
and timed insemination have eliminated the need for estrus detection, and have been
shown to increase conception rates to artificial insemination programs. It should be noted
however, that pregnancy rate is affected by a number of factors, such as anestrus, low
conception rates and increased embryo mortality. In many regions of the world another
factor which influences the dairy cow, especially high producing dairy cows is heat
In an attempt to return the high producing lactating dairy cow to normal
reproductive function, various pharmacological programs have been devised. These
programs manipulate ovarian function. The primary goal of estrous synchronization
programs is to synchronize estrus and ovulation effectively, such that cows may be
inseminated at a predetermined time without estrus detection and reduction in fertility
(Rathbone et al., 2001).
Several synchronization protocols have developed over the years for
synchronization of estrus. The basis of these protocols rely on one of the following: 1)
regression of the CL with PGF2a or its analogue with the cows returning to estrus and
ovulating within 2 to 4 days of the (final) injection of PGF2a; 2) the use of exogenous
progesterone or synthetic progestins to prevent estrus and ovulation for a long enough
period to allow for regression of the CL; upon removal of the exogenous progesterone or
synthetic progestins the cows should return to estrus and ovulate (Rathbone et al., 2001).
Although these pharmacological substances can be used alone, the conception rates were
found to be low and thus they have been used in combination with other reproductive
drugs to increase conception rates to timed insemination.
Prostaglandin F2a given randomly during the estrous cycle between Days 5 and 16
will cause luteolysis of the CL and cows should exhibit estrus between 2 and 4 days post
treatment. The variability in return to estrus and ovulation depends on the stage of the
dominant follicle at the time of luteolysis. If luteolysis occurs when the dominant follicle
is viable, then estrus and ovulation will occur in a relatively short period from treatment.
However, if luteolysis occurs when the dominant follicle is nonresponsive, then the
dominant follicle of the following wave will grow and become the ovulatory follicle and
the interval between treatment and ovulation will be longer (Kastelic et al., 1990). For
these reasons the use of one luteolytic dose of PGF2a with insemination at a fixed time
yielded low conception rates.
Combination of Gonadotropin-releasing Hormone (GnRH) and PGF2a
Pursley et al. (1995) introduced a new method for synchronizing the time of
ovulation in cattle using GnRH and PGF2a (Ovsynch program). In this protocol, GnRH
is given at a random stage of the estrous cycle and PGF2a is given seven days later. A
second GnRH injection is administered 2 days after PGF2a and the cow or heifer was
bred 24 hours later. The first GnRH injection either caused ovulation of the dominant
follicle or had no effect. Next, PGF2a caused regression of the CL, which resulted from
the initial injection of GnRH. The seven-day waiting period between the initial GnRH
and PGF2ca injection gives the CL enough time to mature and become responsive to
PGF2ca. The final GnRH injection caused ovulation of the dominant follicle. The period of
48 hours between PGF2ca and GnRH allowed for a new follicle to emerge, grow to
preovulatory size and become sensitive to the LH surge induced by GnRH to cause
This study demonstrated that the Ovsynch protocol was more suited to lactating
dairy cows than heifers. Ninety percent of lactating dairy cows ovulated to the first
GnRH injection in comparison to fifty percent of heifers.
There are stages within the estrous cycle when the initiation of an Ovsynch
protocol will cause a reduction in pregnancy rates. Initiation of the Ovsynch protocol
during late luteal phase of the estrous cycle, between Days 13 to 17, may lead to
premature lysis and regression of the CL. These cows are asynchronous and exhibit estrus
prior to the second GnRH injection. In these cases insemination within 16 to 20 hours
after the last GnRH injection will be ineffective and conception is not likely (Moreira et
al., 2000a). In the early stages of the estrous cycle, Days 2 to 4, the dominant follicle is
not yet sensitive to LH and will not respond to GnRH. These follicles will ovulate to the
second GnRH injection. Follicles that fall into this category will range in age from 11 to
13 days, and have been shown to be less fertile.
The early luteal phase, between Days 5 and 11 of the estrous cycle, is the optimal
time to initiate the Ovsynch protocol to achieve acceptable pregnancy rates. As a result it
becomes important to presynchronize lactating dairy cows to an optimal stage in the
estrous cycle at which time initiation of the Ovsynch protocol is most effective.
In this presynchronization program PGF2ca is given twice, 14 days apart, and the
Ovsynch protocol is initiated 12 days after the last PGF2ca injection. Presynchronization
programs have been shown to increase the first-service pregnancy rates in comparison
with initiation of the Ovsynch protocol at random stages of the estrous cycle in lactating
dairy cows (Moreira et al, 2001). The increase in the pregnancy rate has been attributed
to the initiation of the Ovsynch protocol in the most favorable stage of the estrous cycle.
Presynchronization beginning on Day 22 post partum with a second injection 14
days later has been shown to lower the incidence of metritis-pyometra, ovarian cysts,
improve luteal activity rate on Day 50 post partum, improve estrus detection rate,
increase ovulation rate and increase pregnancy rate (L6pez-Gatius et al., 2003).
Endometritis is defined as inflammation of the endometrium. The term is
descriptive and refers to the extent and anatomical distribution of the inflammatory
process. There are a plethora of papers published on the topic of postpartum uterine
infection. However, the definition of uterine infection varies and the terms endometritis
and metritis have been used interchangeably in the literature (Bretzlaff, 1987; Lewis,
1997). The following terms have been used to define uterine infection: metritis,
endometritis, and pyometra.
Postpartum endometritis is a pathologic condition usually diagnosed during the
intermediate postpartum period (Ball et al., 1984; Olson et al 1984) during routine
postpartum examination of the cow or heifer. It is commonly characterized by the
absence of estrus, a vaginal discharge of creamy-white or yellow pus and a large doughy
uterus that fails to involute. Postpartum endometritis is the most common cause of
infertility in cows. It delays uterine involution (Tennant and Peddicord, 1968; Griffin et
al., 1974), prolongs the time to first estrus, increases the number of services per
conception and prolongs the interval to calving (Griffin et al., 1974). Lewis (1997) stated
that as many as 40% of postpartum cows within a herd may be diagnosed and treated for
uterine infections. Coleman et al. (1985) stated that endometritis was a common
reproductive disorder accounting for 20% of the reproductive disorders in dairy cows.
However, Ruder et al. (1981) found that the percentage of cows with endometritis could
be as high as 67%. It has been reported that each lactating dairy cow with a uterine
infection can cost a farmer up to $106. This costs consists of treatment, loss in milk
production (associated with the infection and as a consequence of milk discarded due to
antimicrobial therapy) and the loss of cows due to culling for reproductive disorders
associated with and as a direct cause of uterine infection (Lewis, 1997).
Ball et al. (1984) defined the postpartum period as the period from parturition to
complete involution. This period was further divided into the 'early postpartum period',
'intermediate period' and 'postovulatory period'.
The 'early postpartum period' is the period following parturition until the pituitary
becomes sensitive to GnRH, and usually lasts 8 to 14 days. The 'intermediate period'
begins when the pituitary becomes responsive to GnRH and ends with the first
postpartum ovulation. The 'postovulatory period' starts with the first postpartum
ovulation and ends with the completion of involution.
During the early postpartum period bacteria invade the uterus (Elliot et al., 1968;
Griffin et al., 1974; Peter and Bosu, 1987). Elliot et al., (1968) recovered bacteria from
93% of uteri examined between 3 to 15 days post partum. The major groups of bacteria
isolated were Streptococcus, Staphylococcus, Micrococcus, Archanobacteria formally
known as Corynebacteria, Pseudomonas and Escherichia (Elliot et al., 1968). Griffin et
al. (1974) found 92% of uteri sampled infected with bacteria between Days 1 and 7
postpartum, and 96% infected between Days 8 and 14 post partum. The fertility of cows
were not affected by the variable uterine flora or endometritis found within the first two
weeks post partum. However, cows that had a uterine infection with Archanobacterium
pyogenes following Day 21 postpartum developed severe endometritis and were infertile
to the first service (Griffin et al., 1974).
During the intermediate period and into the postovulatory period, spontaneous
clearance of the bacterial contaminant and recontamination occurs. It was noted that the
composition of uterine flora changed throughout the first 7 weeks post partum as a result
of spontaneous contamination, clearance and recontamination (Griffin et al., 1974). It is
likely that as the cow or heifer begins to cycle and the uterus comes under the influence
of high concentrations of estrogen, that infections are cleared and there is an increased
rate of repair of the endometrium. Rowson et al. (1953) demonstrated that the bovine
uterus is more susceptible to bacterial infection during the luteal phase of the estrous
cycle when progesterone is the dominant hormone. It was also noted that under the
influence of estrogen during the follicular stage of the cycle, the uterus was less
susceptible to bacterial infection. Hawk et al. (1960; 1964) demonstrated that the uterus
of heifers differed in their leukocytic response during the estrous cycle. The leukocytic
response to an inoculum of Escherichia coli was more pronounced and occurred much
faster during estrus than in the luteal phase.
During intermediate period and postovulatory period, intrauterine bacterial
infection has been associated with short estrous cycles. Peter and Bosu (1987)
demonstrated the temporal relationship between infection patterns and serum
concentrations of LH, P4 and PGF2ca metabolite (PGFM). They noted that cows with a
short first estrous cycle length were those with uterine infections. High infection rates
were associated with higher concentrations of PGFM before the first postpartum LH
surge and ovulation. Following the LH surge, increasing concentration of PGFM was
associated with increasing intensity of uterine infection. There was a further increase in
PGFM prior to the decrease in P4 and lysis of the CL. However, in cows with a normal
estrous cycle length, low PGFM was noted prior to and following the LH surge and only
prior to onset of luteolysis (Peter and Bosu, 1987).
Endometritis is a localized inflammation of the endometrium of the uterus
associated with chronic postpartum infection with pathogenic bacteria. The primary
bacterium associated with endometritis is Archanobacterium pyogenes (Elliot et al.,
1968; Griffin et al., 1974; Peter and Bosu, 1987; Bonnett et al., 1991; Dohmen et al.,
1995; 2000). Griffin et al. (1974) noted that 80% of the time Archanobacterium pyogenes
was isolated form uteri with moderate or severe endometritis. Classification of the
severity of endometritis was based on the number of neutrophils, histiocytes, plasma cells
and lymphocytes in the biopsy specimen. Moderate endometritis was defined as medium
infiltration of the stratum compactum and upper part of the stratum spongiosum and/or
three to four foci of cellular reaction per section in a number of sections. Severe
endometritis was defined as dense infiltration in the stratum compactum and stratum
spongiosum and/or five or more foci of cellular reaction per section in a number of
sections (Griffin et al., 1974).
Dohmen et al. (2000) investigated the relationship between intra-uterine bacterial
contamination, endotoxin levels and the development of endometritis in postpartum cows
with dystocia or retained fetal membranes. They found that the presence of an abnormal
cervical discharge at Day 14 post partum was higher in cows with retained fetal
membranes compared to cows that experiences dystocia. However, Archanobacterium
pyogenes was isolated more often in cows with retained fetal membranes on Day 14 post
partum than dystocia cows and was positively associated with an abnormal discharge on
Days 14 and 28 post partum. Dohmen et al. (2000) suggested that the presence of
Escherichia coli and lipopolysaccharides (endotoxins) in lochia early in the postpartum
period predisposed the development of uterine infections by A. pyogenes and Gram-
Reduced neutrophil function plays a role in the development of endometritis.
Neutrophils function in phagocytosis and elimination of bacterial contaminants in the
uterus. In cows with retained fetal membranes, neutrophil functions such as chemotaxis
and bacterial ingestion were reduced in comparison to health cows (Cia et al., 1994). The
reduction in neutrophil function may increase the susceptibility of the postpartum dairy
cow to infection.
Diagnosis of endometritis may be made based using a combination of visual
inspection of the vulva and tail, vaginoscopy, rectal palpation, uterine culture or biopsy
and/or ultrasonography. Visual inspection may reveal crusts formed on the vulva and/or
tail. There may be discharge attached to the ventral commissure of the vulva (Zemjanis,
1970). Visual inspection of the vulva and tail alone is not adequate to diagnose
Rectal palpation is the most common method used for diagnosing endometritis.
However, this has been noted as an insensitive and non-specific method (Bretzlaff, 1987;
Lewis, 1997). The results obtained through this method depend on the skill and
experience of the individual. The size and consistency of the uterus and cervix along with
the fluid content of the uterus are used to ascertain the presence of uterine infection
compared to the "normal" finding for a given time postpartum.
Vaginoscopy is seldom used routinely as a diagnostic technique in dairy cows
(Zemjanis 1970; Bretzlaff, 1987). Vaginoscopy, visual examination of the vagina,
luminal content and external cervical os have been shown to be a superior means of
diagnosing endometritis. LeBlanc et al. (2002) found that the prevalence of clinical
endometritis was 16.9% and vaginoscopy was required to identify 44% of these cases.
Visual and transrectal palpation of the uterus was not sufficient to identify cases oc
Le Blanc (2002) suggested that the diagnosis of clinical endometritis should be
made by the presence of purulent uterine discharge or cervical diameter > 7.5 cm after 20
DIM, or mucopurulent discharge after 26 days in milk. This definition of endometritis is
based on the negative effect these criteria have on subsequent fertility. In the absence of
vaginoscopy a combination of history, inspection and palpation may be used to diagnose
clinical endometritis. In the absence of vaginoscopy the following criteria can be used to
classify clinical endometritis (LeBlanc et al., 2002): presence of mucopurulent or
purulent discharge on the perineum, cervical diameter >7.5 cm, and presence of a uterine
horn >8 cm in diameter.
Griffin et al. (1974) identified cases of endometritis using uterine biopsy and
uterine culture. The severity of endometritis was determined on histological findings and
the predominant bacterial flora evaluated by uterine culture. Recently, endometrial
cytology has been used to identify cows with subclinical endometritis in the postpartum
dairy cow (Kasimanickam et al., 2004). Endometrial cytology was performed on
clinically normal cows, cows without evidence of an abnormal discharge on visual
inspection of the vulva, tail and surrounding areas and vaginoscopy between Days 20 and
Day 33 post partum. Clinical endometritis was defined as > 18% polymorphonuclear
cells seen in endometrial cytology samples or fluid in the uterus at the first visit (Days 20
and Day 33 post partum) and as > 10% polymorphonuclear cells seen in endometrial
cytology samples or fluid in the uterus at the first visit (Days 34 and Day 47 post partum).
In a similar fashion to LeBlanc et al (2002) and Kasimanickam et al (2004) defined
clinical endometritis based on its impact on subsequent pregnancy rates. Although there
was no evidence of abnormal discharge on the day of enrollment, 9.1% had a discharge
within 24 hours. This indicated that evaluation of endometritis should be done on more
than one occasion. It is interesting to note that 35.1 % of cows were diagnosed with
subclinical endometritis by endometrial biopsy on the first visit and 34% on the second
visit 2 weeks later. However, 48% were diagnosed with subclinical endometritis by the
presence of fluid in the uterus on the first visit and 20.5% on the second visit 2 weeks
later. Cows with subclinical endometritis were less like to become pregnant than those
without subclinical endometritis (Kasimanickam et al., 2004).
Ultrasonography may be used to aid in the diagnosis of endometritis. One of the
sonographic indications of endometritis is the appearance of fluid in the uterine lumen.
This fluid accumulation must be distinguished from that which is seen with estrous
secretions or the embryonic fluid of the early concepts. Fluids that accumulate during
estrus and early pregnancy are less echogenic than that of endometritis (Fissore et al.,
In mild endometritis, there may be no fluid present or few fluid filled pockets found
when scanning the uterus (Kahn et al., 1989). In cases of severe endometritis, the uterus
may be distended with fluid and involve both horns (Kahn et al., 1989). The echogenicity
of the fluid that accumulated during endometritis varies with the products of
inflammation. Kahn et al. (1989) described the echogenic pattern displayed by the
products of inflammation. Echogenic patterns range from small bright spots in mild cases
to a "snow-storm effect" or, in severe cases, to almost bright white images on the screen.
Real-time-scanning may reveal turbulences within the larger collections of fluid.
Since plasma levels of 13,14-dihydro,15-keto-PGF2a (PGFM), the stable
metabolite of PGF2a, are elevated in spontaneous uterine infection, it has been suggested
that these levels may aid in the diagnosis of endometritis in the postpartum cow (Del
Vecchio et al., 1994). However, Archbald et al. (1998) were unable to correlate plasma
levels of PGFM with abnormal uteri identified by per rectum palpation during Days 24 to
29 post partum in lactating dairy cows. In addition, Archbald et al. (1998) demonstrated
that the postpartum rate of decline of plasma levels of PGFM was influenced by the
presence of a corpus luteum (CL). In cows which had a CL, the rate of decline of PGFM
was slower compared to cows without a CL. It has been reported that progesterone plays
an extremely important role in regulating pulsatile secretion of PGF2a from the bovine
uterus (Mann et al., 1995) since it can completely restore the number of pulses and
partially restore pulse magnitude when administered to ovariectomized ewes. However, a
decrease in progesterone appeared to be essential for pulses of PGF2a to reach a
maximum magnitude (Silvia et al., 1991).
EXPERIMENT 1: AN EVALUATION OF PRETREATMENT WITH GnRH ON
OVARIAN RESPONSE AND PREGNANCY RATE OF LACTATING DAIRY COWS
WITH OVARIAN CYSTS SUBJECTED TO THE OVSYNCH PROTOCOL
Bovine ovarian cysts are follicles that fail to ovulate at the time of estrus
(Garverick, 1997; 1999). They are an economic problem in the dairy cow because these
cows are infertile as long as the condition persists (Kesler and Garverick, 1982). The
exact cause of ovarian cysts is not presently known, but some predisposing factors
include age, stress, high milk production and genetics. It appears that an important
component in the pathogenesis of this condition is the inappropriate, or lack of, release of
hypothalamic gonadotropin-releasing hormone (GnRH) at the time of estrus (Refsal et
al., 1987; Giimen et al., 2002; Silvia et al., 2002). One therapeutic approach involves the
use of exogenous GnRH, which releases LH from the anterior pituitary and causes
ovulation of an ovarian follicle, and/or luteinization of the ovarian cysts (Garverick,
The occurrence of ovarian follicular waves has been adequately demonstrated in
the dairy cow without ovarian cysts (Rajakoski, 1960; Pierson and Ginther, 1984). It has
been suggested that ovarian follicular waves also occur in cows with ovarian cysts
(Garverick, 1999). The difference is that ovulation of follicles occur in cows without
ovarian cysts, but does not occur in cows with ovarian cysts. It has further been suggested
that the administration of GnRH to cows with ovarian cysts causes ovulation of a
functionally mature follicle of an ovarian follicular wave (Garverick, 1999). An injection
of GnRH at random during the estrous cycle of cows without ovarian cysts will either
cause ovulation or luteinization of large follicles present in the ovary and synchronize the
recruitment of a new follicular wave (Thatcher et al., 1989; Macmillan and Thatcher,
1991). Gonadotropin releasing hormone can advance the follicular wave by increasing
the rate of atresia in cows without ovarian cysts (Thatcher et al., 1989; Macmillan and
In cows without ovarian cysts, the initiation of a protocol for synchronization of
ovulation and timed insemination (Ovsynch) at a specific stage of the estrous cycle can
influence the reproductive responses to this protocol. The stage of the estrous cycle that
appears to be the most appropriate for increased pregnancy rates using this protocol is the
early luteal phase of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000a).
The hypothesis of this study was that GnRH administered to cows with ovarian
cysts at the time of diagnosis will induce an early luteal phase of the estrous cycle which
will be conducive to an increased pregnancy rate to a protocol for synchronization of
ovulation and timed insemination. The objectives of this study were: i) to compare the
ovarian response of cows with ovarian cysts to no treatment and ovarian response 7 days
after treatment with GnRH; and, ii) to determine the pregnancy rate of cows with ovarian
cysts subjected to Ovsynch and pretreatment with GnRH 7 days prior to the initiation of
the Ovsynch protocol.
Materials and Methods
The study was conducted during the period of January 2002 to May 2003 in a dairy
herd (approximately 700 milking cows) in north-east Florida. Cows were milked three
times per day and were kept in covered, open-sided barns between milking. They were
fed a total mixed ration. The herd was visited weekly, and all reproductive health and
management records were computerized.
At the time of diagnosis, cows with ovarian cysts were sequentially allocated to one
of three groups (Day 0). The diagnosis of ovarian cysts was based on palpation of the
ovaries and uterus per rectum, and by ultrasonographic examination of the ovaries. The
criteria used on rectal palpation were the presence of multiple follicles on the ovary with
at least one follicle being > 17 mm diameter (Hatler et al., 2003), the absence of a corpus
luteum (CL) on either ovary, and the lack of tonicity of the uterus (Archbald et al., 1991;
Bartolome et al., 2000; Bartolome et al., 2002). On ultrasonographic examination of the
ovaries, ovarian cysts were recognized by the hypoechogenicity of the structure, and the
absence of a CL on either ovary (Pierson and Ginther, 1984). The size of ovarian cysts
was determined using ultrasonography and the recorded size was based on the largest
diameter observed on ultrasonography.
Cows in Group 1 were treated with GnRH (100 rtg, im; Cystorelin, Rhode Merieux
Inc., Athens, GA) on Day 0, and Day 7, PGF2ca (25 mg, im; Lutalyse, Pharmacia Upjohn,
Kalamazoo, MI) on Day 14, GnRH (100 ug, im) on Day 16, and timed inseminated 16-20
h later. Cows in Group 2 were treated with GnRH (100 ug, im) on Day 0, PGF2ca (25 mg,
im) on Day 7, GnRH (100 ug, im) on Day 9, and timed inseminated 16-20 h later. Cows
in Group 3 were not treated with GnRH on Day 0, but were treated with GnRH (100 ug,
im) on Day 7, PGF2ca (25 mg, im) on Day 14, GnRH (100 ug, im) on Day 16, and timed
inseminated 16-20 h later (Figure 3-1). Pregnancy was determined by rectal palpation of
the uterus between 45-50 d after timed insemination using previously described
techniques (Zemjanis, 1970).
Group I (GnRH + Ovsynch)
GnRH GnRH PGF2a GnRH TAI Pregnancy Diagnosis
Day 7 14 16 17/18 62-68
Group 2 (Ovsynch)
GnRH PGF2a GnRH TAI Pregnancy Diagnosis
Day 0 7 9 10/11 54-61
Group 1 (No Tx + Ovsynch)
No Tx GnRH PGF2a GnRH TAI Pregnancy Diagnosis
Day 0 7 14 16 17/18 62-68
Figure 3-1. Experiment 1 Experimental Design
On both Days 0 and 7, cows in all groups were subjected to ultrasonographic
examination of the ovaries. On both Day 0 and Day 7, blood samples for progesterone
(P4) concentration were obtained from all cows and placed on ice immediately after
collection. Samples were centrifuged at 5000 x g for 15 minutes, and serum was stored at
-200C until assayed for progesterone using a solid-phase, no-extraction RIA previously
described (Srikandakumar et al., 1986). Serum progesterone concentration on Day 0 was
classified as high (> 0.5 ng/ml) or low (< 0.5 ng/ml). A serum progesterone concentration
>1 ng/ml on Day 7 was used to confirm the presence of a functional corpus luteum.
Baseline data for parity, DIM, time of year and P4 on Day 0 were compared using
Chi-square (P<0.05). The outcomes of interest for this experiment were ovarian response
7 days after treatment with GnRH, and pregnancy rate. Data for ovarian response on Day