CYSTIC OVARIAN DISEASE
IN CATTLE ON DAIRIES IN CENTRAL AND WESTERN OHIO: ULTRASONIC,
HORMONAL, HISTOLOGIC, AND METABOLIC ASSESSMENTS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Cynthia J. Johnson, M.S.
*****
The Ohio State University
2004
Dissertation Committee:
Approved by
Professor Joseph S. Ottobre, Adviser
Professor William P. Shulaw
Professor Thomas E. Wittum
Asst. Professor Richard W. Meiring
___________________________
Adviser
Veterinary Preventive Medicine
Graduate Program
ABSTRACT
Cystic ovarian disease (COD) has been recognized as a managerial and
financial problem on dairies for almost two centuries. In spite of the large
body of literature regarding COD, the etiology of COD is not entirely
understood. Additionally, preventive management is not yet perfected.
This dissertation examines the dynamics of spontaneously occurring
cystic ovarian disease (COD). Ultrasonography was used to monitor cows with
COD on six dairies in western and central Ohio every Monday, Wednesday, and
Friday for five weeks. The specific objectives for this study were to determine:
1) if the use of ultrasonography to select a treatment based on cyst type and
/or presence of luteal-like tissue (LLT) would increase estrous expression and
pregnancy in cows with spontaneously occurring COD, 2) the biochemical
profiles of cows with spontaneous COD, 3) the ovarian dynamics and
progesterone profiles of cows following treatment for COD, 4) the association
between COD and length of the previous dry period and between COD and the
number of days open, and 5) if luteal-like structures induced with gonadotropin
releasing hormone (GnRH) in cystic cows are steroidogenically and
ii
histologically similar to CL induced in cycling cows and to spontaneously
occurring CL in cows with normal estrous cycles.The major results of this
research were that when luteal cysts and other LLT are identified via
ultrasonography, using prostaglandin F as the treatment may allow an earlier
opportunity for breeding and thus potential pregnancy. Biochemical profiles of
cows for five weeks following treatment were reported and provided an initial
assessment of possible associations between concentrations of clinically
important metabolites and COD. The dry period after the preceding lactation
was longer in cows that had ovarian cysts than those that did not. Cystic cows
appeared to achieve pregnancy at a similar rate to non-cystic cows in the early
postpartum period. It was observed that 2 cows with follicular cysts became
pregnant. Luteal-like structures that were induced by GnRH treatment in cystic
cows are structurally similar to GnRH-induced accessory CL in noncystic cows
with normal estrous cycles and were steroidogenically active in vivo and in
vitro.
iii
Dedicated to the loving memory of
Florence B. Buhrows
April 9, 1911 – January 1, 2000
She loved to learn.
iv
ACKNOWLEDGMENTS
I am honored to acknowledge those whose direct or indirect influence helped
support my development as a researcher, as a veterinarian, and as a person.
My special thanks to:
•
Joe Ottobre, my adviser, for his constant support throughout the trials
and tribulations of my research program. His belief in me helped me to
achieve more than I thought possible at times.
•
The rest of my committee, Rich Meiring, Tom Wittum, and William
Shulaw for their never ending encouragement, support, and friendship
have been invaluable.
•
My family and friends: Carole, Tom, and Kris Johnson, Helen May, Holly
Monke, Elaine Hughes, Sue Ault, Helen Bateman, Andrea Musser, Nick
Wagner, Mike Brothers, Cassia Orlandi, Ann Auer, Christie Haines, Cathy
Bremer, Sue Skorupski, and Diane Gross without their love, friendship,
laughter, and advice the successful completion of this project would not
have been possible.
•
Paivi Rajala-Schultz for her invaluable advice and encouragement.
v
•
Grant Frazer for his role as my adviser during a portion of my doctoral
training and for providing me with some unique opportunities
throughout my graduate program.
•
Armando Hoet, for inspiring me and keeping me focused on the goal. I
am grateful for your friendship.
•
Randy Lewis, for being a source of strength and encouragement for me
even over the miles between us. He is truly a gifted person and I am
honored to be his friend. He kept my head in the sky and my feet in a
cowpie.
•
Sheryl Hansen. I am forever grateful for her friendship, advice, wisdom,
and eternal encouragement. She is one of the greatest people I have
had the privilege to know or will ever know in my life.
•
We appreciate research funding and support from the following
sponsors: Purina Mills, Inc., Country Mark, Inc., Select Sire, Inc., RhoneMerieux, Inc., Ohio Dairy Research Fund, Michigan State UniversityAniaml Health Diagnostic Lab.
My eternal thanks to all of you!
vi
VITA
August 10, 1970…………………………..Born – Raleigh, North Carolina
1993……………………………………………B.S. Animal Science
North Carolina State University
Raleigh, North Carolina
1996……………………………………………M.S. Reproductive Physiology
The Ohio State University
Columbus, Ohio
1996 – 2000…………………………………Graduate Research Associate
Department of Veterinary Preventive
Medicine, The Ohio State University
1996 – present……………………………..Graduate Student
Department of Veterinary Preventive
Medicine, The Ohio State University
2001 – 2005…………………………………Veterinary Student
College of Veterinary Medicine,
The Ohio State University
PUBLICATION
Townson, D.H., P.C.W.Tsang, W.R. Butler, M. Frajblat, L.C. Griel, C.J. Johnson,
R.A. Milvae, G.M. Niksic, and J.L.Pate. Relationship of Fertility to
Ovarian Follicular Waves before Breeding in Dairy Cows. 2002 J Anim
Sci 80(4): 1053-58.
FIELDS OF STUDY
Major Field: Veterinary Preventive Medicine
Reproductive Physiology
vii
TABLE OF CONTENTS
Page
Abstract………………………………………………………………… ............................. ii
Dedication………………………………………………………………….......................... iv
Acknowledgments……………………………………………….................................. v
Vita………………………………………………………………………… ........................... vii
List of Tables……………………………………………………… ................................ xi
List of Figures……………………………………………………… ............................... xiii
Chapters:
1. Introduction……………………………………………… ................................ 1
Terminology……………………………………………................................... 2
Incidence……………………………………………… .................................... 5
Clinical Signs………………………………………… .................................... 8
Cysts and Cyst Type………………………............................................. 10
Diagnosis……………………………………… ............................................ 14
Therapeutic Strategies……………………………… ................................. 17
Etiology…………………………………………… ......................................... 24
Associations……………………………………… ........................................ 28
Research Models…………………………………………………....................... 32
Other Species Affected………………………………… .............................. 34
2. Research Objectives……………………………………………………… ............ 36
3. Use of Ultrasonography for Determining Cyst Type, Treatment, and
Outcomes for Dairy Cattle Exhibiting Spontaneous Cystic Ovarian
Disease…………………………………………………………… ......................... 38
Introduction……………………………………………................................... 38
viii
Materials and Methods…………………………………………….................... 41
Ultrasound Diagnosis Study……………………………............................. 41
Presence of Luteal-like Tissue Study
(Retrospective Analysis)…........................................................... 43
Statistical Analysis………………………………… ..................................... 45
Results……………………………………………………… ............................... 48
Ultrasound Diagnosis Analysis………………… .................................... 48
Retrospective Analysis…………………………………………....................... 48
Progesterone Analysis…………………………........................................ 50
Discussion………………………………………………................................... 51
4. Biochemical Profiles of Cows with Cystic Ovarian Disease in
Central and Western Ohio……………………………………………………....... 61
Introduction……………………………………………………........................... 61
Materials and Methods……………………………………............................ 63
Statistical Analysis……………………………………… ............................... 64
Results………………………………………… ............................................. 66
Discussion………………………………………………................................... 68
5. Differences in Length of Dry Period and Time to Pregnancy
(Days Open) Between Cystic and Noncystic Cows on Central and
Western Ohio Dairies……………………………………… ........................... 90
Introduction…………………………………… ........................................... 90
Materials and Methods……………………… ......................................... 92
Statistical Analysis……………………………………………… ....................... 93
Days Dry…………………………………………… ....................................... 93
Days Open……………………………………………………............................. 94
Results………………………………………………........................................ 95
Discussion………………………………… ................................................ 96
6. Case Reports of Two Cows that Became Pregnant with Persistent
Ovarian Cysts Present and Four Cows Observed to have
Hyperechogenicity within Follicular
Cysts……………………………………………………………………………….......... 100
Introduction………………………………….............................................. 100
Descriptive Statistics………………… .................................................. 100
Results…………………………………………………… .................................. 102
Cows with Follicular Cysts……………………........................................ 102
Cows with Luteal Cysts………………………......................................... 102
ix
Case Report 1: Two Cows with Persistent Follicular
Cysts Present that Became Pregnant……………… ......................... 103
Cow 159…………………………………………… ........................................ 103
Cow 168………………………………...................................................... 104
Case Report 2: Observation of Hyperechogenicity within
Follicular Cysts in Four Cows…………………………………… ............... 106
Discussion……………………………………… ........................................... 107
7. Induced Luteal-like Structures Following GnRH Treatment of
Dairy Cattle with Cystic Ovarian Disease: Assessment of
Histological Characteristics and Steroidogenic Capability…… ............ 118
Introduction……………………………………………… ................................ 118
Materials and Methods……………………… ......................................... 120
General…………………………………………… ......................................... 120
In vitro CL Short-term Incubation……............................................. 121
Histology…………………………………………… ....................................... 122
Radioimmunoassay……………………………… ...................................... 123
Statistical Analysis………………………................................................ 124
Results………………………………………………........................................ 125
Concentrations of P4 in Plasma……… ............................................. 125
Concentrations of P4 in Culture Medium……………….. ...................... 126
Luteal Cell Measurements……………………........................................ 127
Discussion………………………………………………................................... 127
8. Key Points………………………………………………… ................................ 136
Bibliography………………………………………………………… .......................... 138
x
LIST OF TABLES
Table
Page
3.1
Retrospective analysis of responses (displayed estrus and was
bred, ovulated, developed new LLT, and diagnosed pregnant)
of cows with follicular cysts divided into groups treated according
to the presence of LLT…………………………………………….................... 60
4.1
Multivariate ANOVA model for predicting mean magnesium
concentrations for cystic and noncystic-control cows…………............ 73
4.2
Multivariate ANOVA model for predicting mean calcium
concentrations for cystic and noncystic-control cows……… .............. 74
4.3
Multivariate ANOVA model for predicting mean GGT
concentrations for cystic and noncystic-control cows… .................... 75
4.4
Multivariate ANOVA model for predicting mean AST
concentrations for cystic and noncystic-control cows…… ................. 76
4.5
Multivariate ANOVA model for predicting mean albumin
concentrations for cystic and noncystic-control cows……… .............. 78
4.6
Multivariate ANOVA model for predicting mean total protein
concentrations for cystic and noncystic-control cows……… .............. 80
4.7
Multivariate ANOVA model for predicting mean phosphorus
concentrations for cystic and noncystic-control cows…………............ 82
4.8
Multivariate ANOVA model for predicting mean cholesterol
concentrations for cystic and noncystic-control cows…………............ 84
4.9
Multivariate ANOVA model for predicting mean β-hydroxybuterate
concentrations for cystic and noncystic-control cows……… .............. 86
xi
4.10
Multivariate ANOVA model for predicting mean creatinine
concentrations for cystic and noncystic-control cows……… .............. 87
4.11
Multivariate ANOVA model for predicting mean blood urea nitrogen
concentrations for cystic and noncystic-control cows……… .............. 89
7.1
Means and standard errors for the plasma samples collected at the
time of CL collection for all three treatment groups… ...................... 131
7.2
Means and standard errors for P4 concentrations in medium (ng/ml)
for the three treatment groups ………………………….......................... 131
7.3
Means and standard deviations for area and perimeter of luteal
cells from each of the three treatments and the primary CL for the
GnRH-induced group ………………………………………........................... 132
xii
LIST OF FIGURES
Figure
Page
3.1
Ultrasound-based treatment protocol for cows with COD……… ........ 46
3.2
Combined groups for day 14 retrospective analysis of cows with
follicular cysts (with and without LLT) that were treated with GnRH
or PGF…………………………………………………………………………… .......... 47
3.3
The percentage of cows with luteal cysts that were inseminated
(P=0.003) and that became pregnant (P=0.64) within the first two
weeks following the initial treatment compared to cows with
follicular cysts……………………………………………… ............................. 57
3.4
The number of cows with follicular cysts (n=55) that have high and
low P4 concentrations with luteal tissue (LT) present or not present
on d1 of the study…………………………………………….......................... 58
3.5
Number of cows with luteal cysts (n= 13) that have high and low
P4 concentrations with luteal tissue (LT) present or not present on
d1 of the study……………………………………… .................................... 59
4.1
Plasma concentrations of albumin (diamonds, mean +SE) over time
(i.e., sample number) in cystic cows compared to the pooled
noncystic cow samples (thick line; P<0.01)…………… ...................... 77
4.2
Plasma concentrations of total protein (diamonds, mean +SE) in
cystic cows over time (i.e., sample number) compared to the pooled
noncystic cow samples (thick line; P<0.01)…………… ...................... 79
4.3
Plasma concentrations of phosphorus (diamonds, mean +SE) over
time (i.e., sample number) in cystic cows compared to the pooled
noncystic cow samples (thick line; P=0.01)………………… ................. 81
xiii
4.4
Plasma concentrations of cholesterol (diamonds, mean +SE) in
cystic cows over time (i.e., sample number) compared to the
pooled noncystic cow samples (thick line; P=0.01)……… ................. 83
4.5
Plasma concentrations of β-hydroxybuterate (diamonds,
mean +SE) in cystic cows over time (i.e., sample number) compared
to the pooled noncystic cow samples (thick line; P=0.01)…… .......... 85
4.6
Change from D1 concentration of blood urea nitrogen in cystic
cows over the five week study period (illustrated by sample number;
P=0.94)………………………………………………………… ........................... 88
5.1
The percentage of non-pregnant cystic and non-cystic cows
over time… .................................................................................. 99
6.1
Follicular and cyst patterns observed via ultrasonography for
Cow 159.. .................................................................................... 111
6.2
Concentration of Progesterone (ng/ml) over time in Cow 159.......... 112
6.3
Follicular and cyst patterns observed via ultrasonography for
Cow 168.. .................................................................................... 113
6.4
Concentration of Progesterone (ng/ml) over time in cow 168…………114
6.5
Concentration of plasma progesterone (ng/ml) over the study
period (36 days) for Cow 21………………………………………………… ...... 115
6.6
Concentration of plasma progesterone (ng/ml) over the study period
(36 days) for Cow 77…………………………………………………………… ..... 116
6.7
Concentration of plasma progesterone (ng/ml) over the study period
(36 days) for cow 727………………………………………………………. ....... 117
7.1
Example of normal D6 CL used to for obtaining luteal cell area and
perimeter data for control-N group (20x)……………………………. ........ 133
7.2
Example of GnRH-induced D6 CL used to obtain area and perimeter
data for control-G group (20x)………………………………………….. ......... 134
7.3
Example of GnRH-induced CL in a cystic cow used to obtain area
and perimeter data (20x)………………………………………………… .......... 135
xiv
CHAPTER 1
INTRODUCTION
Cystic ovarian disease (COD) has been recognized as a managerial and
financial problem on dairies for almost two centuries, and there has been a
considerable amount of research published on the disease. In spite of this
large body of literature, the etiology of COD is not entirely understood. In
addition, preventive management is not yet perfected. Fortunately, our
knowledge base of COD has increased over the past thirty years. Much of the
knowledge has been gained due to the advent of better technology.
Radio/enzymeimmunoassay and ultrasonography, for example, have
contributed to improvements in monitoring hormones and ovarian dynamics.
My doctoral research combines the use of transrectal ultrasonography,
radioimmunoassay techniques, biochemical analyses, and assessment of Dairy
Herd Improvement records to help dairy practitioners and producers better
understand the response to treatments by dairy cows with spontaneously
occurring COD. This literature review will cover the diagnosis, etiology,
therapeutic strategies, clinical signs, and research models for COD in dairy
1
cattle. Some of the research pertaining to the adrenal glands and anterior
pituitary are not summarized in this literature review. While data from those
studies are important to determining the causative factors of COD, they are
not directly relevant to the understanding of the doctoral research presented.
TERMINOLOGY
Cystic ovarian disease has been referred to by many names over the
past century including adrenal virilism, nymphomania, cystic ovarian
degeneration, cystic ovaries, and ovarian cysts (Garverick, 1997). Below is an
attempt to explain how these terms came to be associated with COD, and
whether or not they are still considered valid synonyms of COD.
Historically, adrenal virilism was thought to be one of the causes of
cystic ovaries and was specifically associated with luteal cysts. Virilism is the
acquisition of secondary male sex characteristics. Adrenal virilism is the
masculinization of the female due to “hyperproduction of androgenic
substances in the adrenals” (Garm, 1949a). Many of the criteria associated
with adrenal virilism were a catchall for irregular estrous behavior. Some of
the obvious signs of adrenal virilism include: bellowing with deepened
vocalizations, hypertrophy of the clitoris, thickened neck, deepening of
vocalizations, and increased hair on the vulva with a noticeable coarseness of
2
this hair (Garm, 1949a; Dawson, 1957). A “buller” cow is defined as a cow
becoming bull-like in appearance and behavior (Garm, 1949a).
Enlarged adrenal glands in the human female had been previously
related to an increased urinary steroid output (Garm, 1949b). Similarly,
urinary androgens were thought to be secreted in greater amounts in cows
with adrenal virilism than in normal cows (Garm, 1949a). Conflicting evidence
was later observed, however, when no difference in the size of adrenal glands
and less steroid production was found to occur in a small group of cows with
adrenal virilism (Cupps et al., 1959). Few of these studies actually confirmed
the presence of ovarian cysts in cows with adrenal virilism and of these, most
did not confirm the presence of ovarian cysts in the entire study group (Garm,
1949a; Cupps et al., 1959; Dawson, 1957).
Another problem was that concentrations of urinary 17-ketosteroids
were assumed to reflect concentrations of circulating androgens. Short (1961)
cast doubt on the adrenal virilism theory for cystic cows, when he pointed out
that there are several renal metabolites that cross-reacted with the 17ketosteroid urinary analysis. For example, 17β-estradiol metabolites that are
excreted in the urine of many mammals contributed to urinary 17-ketosteroids.
Consequently, adrenal glands were not considered the major source of rapidly
measurable reproductive androgens in the cow. Eventually, adrenal virilism
was no longer considered the primary cause of cystic ovaries.
3
Cystic ovaries and ovarian cysts are commonly used as synonyms for
COD in ruminants, companion animals, and exotic species. These species
typically have only one cyst on one ovary, however, more than one cyst is not
rare. Polycystic ovarian disease (PCOD) is used when discussing a cystic
condition that occurs in women, nonhuman primates, and swine. This
condition is characterized by multiple cysts present at any time. PCOD is a
more appropriate term for use with swine since they are litter-bearing species
and form multiple follicles during a normal estrous cycle. So the traditional
term of COD is more appropriately used with cattle and in an animal science
forum. PCOD is commonly used with women in the human medicine forum as
well as with pigs in the animal science setting.
Cystic ovarian degeneration is misleading in that the ovary containing
the cyst has not degenerated. In fact, both ovaries are still capable of
supporting follicular development in the presence of a cyst (Hamilton et al.,
1995). Also, ovulation of follicles subordinate to the cyst (based on size) can
occur naturally (Cook et al., 1990) or can be induced by GnRH (Lopez-Gatius
and Lopez-Bejar, 2002). Limited evidence exists cows can even become
pregnant when they have follicular cysts. Assey and colleagues (1997), using
data collected in the abattoir, reported that a pregnant East African zebu cow
had multiple ovarian cysts on both ovaries and a single CL. In addition, a
Japanese research group found no difference in oocyte morphology or
maturation rate in oocytes collected from subordinate follicles in cystic and
4
noncystic cows (Takagi et al., 1998). Therefore, it is unlikely that the ovary
containing the cyst and the contralateral ovary are degenerate.
The cyst proper, however, is an anovulatory follicle and is degenerate in
that the basement membrane is missing or has disintegrated in the majority of
cysts (Al-Dahash and David, 1977b; Brown et al., 1982). Cysts may or may
not contain luteal tissue. Depending on the stage or type of cyst, granulosa
cells may be absent (Yamauchi, 1955; Al-Dahash and David, 1977b; Brown et
al., 1982). Therefore, the name cystic ovarian disease may be the most
accurate name to describe this diversion from the normal ovulatory course of
the dominant follicle.
INCIDENCE
An anovulatory, cystic follicle has been reported to occur in 3-32% of
lactating dairy cattle worldwide (Eicker et al., 1996; Morrow et al., 1969).
Average occurrence is estimated to be 10% (Garverick, 1999). This would
translate into approximately one million dairy cattle becoming cystic each year
in the United States (Garverick, 1999). Roughly one out of every ten lactating
dairy cows will at sometime during their lactation have COD (1997 Census of
Agriculture). In actuality, the true incidence of COD may be 32% or higher
due to the high incidence of spontaneous resolution in cows less than 60 days
postpartum (Kesler and Garverick, 1982; Carroll et al., 1990). A greater
5
percentage (60%) of cows with COD prior to 30 days have been reported to
spontaneously correct (Youngquist, 1986) than cows less than 60 days (50%;
Morrow et al., 1966). Many of the cows with COD occurring during the 60 day
postpartum period have a delay in returning to estrus, whereas cows
diagnosed later in lactation have an extended calving interval due primarily to
an anestrus period after having previously had regular estrous cycles (Morrow
et al., 1966; Kesler et al., 1979). This translates into a greater financial loss
to the dairy industry when cows with COD are at the height of their lactation
and in their best years of production.
Hardie and Ax (1981) reported that among the different dairy breeds,
Holsteins had the highest incidence of COD followed by Guernseys, Jerseys,
and Ayrshires. Brown Swiss were the least likely to have COD. Menge and
colleagues (1962) reported no difference in the incidence of COD in inbred and
outbred Holstein-Fresian cows. However, it is clear that genetics do play a role
in COD. Besides the data above of varying incidence among breeds, Sweden
was able to decrease the incidence of COD from 10.8% to 3.0% over 23 years
by culling bulls with daughters that had COD (Kesler and Garverick, 1982).
In an abattoir survey, Herenda (1987) found COD affected ovaries in
approximately 15% of 5800 beef heifers. Cystic ovarian disease has seldom
been detected in beef cattle. This is possibly due to managerial differences
between dairy and beef cattle and the different intended uses. Cystic ovarian
disease is more of a problem in dairy cattle partly due to the more intensive
6
production and reproduction management practices. Additionally, the typical
cow-calf operation owner does not rely on profit from calf production as their
sole source of income. Dairy producers usually depend on milk production
from the cows as their primary source of income and without producing a calf,
there is no milk production. Therefore, the incidence of COD in beef heifers
and cows would potentially have less of an impact on production and
profitability than the incidence of COD in dairy cattle.
Conventionally, it has been accepted that cows with COD were infertile
until the condition resolved (Garverick, 1999). This would mean that a cow
with COD would have a longer calving interval (or days open) compared to
noncystic herdmates (Garverick, 1999). Bartlett and colleagues (1986)
estimated that each episode of COD costs the producer $137 per lactation.
This estimate included additional days open, semen, treatment and culling
costs, as well as, an estimate for labor and management. However, a price
estimate of $98 was made based on the estimated additional milk produced by
the cystic cow (Bartlett et al., 1986). (Increased milk production from cows
with COD has been a controversial association and is discussed later in this
chapter. See Associations.) Depending on many factors, the overall loss due
to COD per cow per lactation would range from approximately $40$137. Therefore, based on the number of cows potentially affected, cystic
ovarian disease can translate into a great financial loss for the individual
producer and the industry as a whole.
7
CLINICAL SIGNS
Cystic ovarian disease has been a concern for producers and
veterinarians for almost two centuries because a diagnosis of COD usually
means the cow is not pregnant. A German scientist in 1831 (Gurlt, as cited by
Garm, 1949a) wrote one of the earliest records of COD noting its relationship
to infertility. Williams and Williams (1923b) documented some of the physical
conditions associated with COD. These symptoms included:
1) Loss of tone throughout the female genital tract.
2) Relaxation or stretching of the sacrosciatic and sacroiliac ligaments
giving the raised tailhead appearance.
3) Formation of a cyst or cysts on the ovary or ovaries.
4) Behavioral changes occurred (buller cow) which are characteristic of
nymphomania (i.e., excessive mounting, standing, and bawling
with noticeably deeper tone).
5) Erratic milk production
Nymphomaniacal behavior has been so extreme that some cows have
been reported to “mount a man, especially while being led” (Williams and
Williams, 1923a). Most of these symptoms are still recognized in cows.
However, with earlier detection, only extreme cases tend to exhibit the relaxed
ligaments, bulling and a deepening of the vocalizations. The main symptom
8
observed in modern day dairy cattle is anestrus (Garverick, 1999).
Nymphomaniais a term that is better used as a descriptive word for a symptom
in certain cases of COD, rather than a synonym for the overall condition.
Historically, the producer has recognized COD primarily on
nymphomaniacal behavior. Through increased veterinary involvement with
herd management and production and producer education, a strong focus has
been placed on increasing milk production and reproductive efficiency of dairy
cattle. With these changes in management and breeding strategy, a greater
awareness of COD has occurred and anestrus is recognized as the key clinical
sign.
For many years cysts have been determined by behavioral and
palpation criteria. Generally, a cyst is defined as a palpable structure on one
or both of the ovaries greater than or equal to 2.5cm (approximately one inch)
irrespective of other ovarian structures (Farin et al., 1982). In practice, a
veterinarian seldom has the opportunity to recheck a cow in 10 days. Thus,
most diagnoses are based on the one time detection of a large fluctuant
structure meeting most of the previous criteria and treatment is usually
administered at the time of diagnosis.
9
CYSTS AND CYST TYPE
There are three types of cysts that occur on the bovine ovary: follicular
cysts, luteal cysts and cystic corpora lutea (CL). Follicular and luteal cysts are
the only two true types of cysts that are associated with an abnormal condition
in cows. Cystic CL are considered to be nonpathological (McEntee, 1958;
Morrow et al., 1966; Kesler and Garverick, 1982; Garverick, 1999).
An ovarian cyst is typically detected based on its diameter. The cut-off
diameter of 25mm is reported by a majority of studies throughout the
literature (Isobe and Yoshimura, 2000; Jou et al., 1999; Borromeo et al., 1996;
OHNAMI et al., 1995; Dobson and Nanda, 1992). However, with the
incorporation of ultrasound technology during the early 1980s in research and
veterinarian practice, the minimum cut-off has become more variable. Carriere
and colleagues (1995) used novel size criteria. They defined a follicular cyst as
being “larger than the average maximal diameter [of follicles of the control
group] plus two standard deviations. Ohnami and colleagues (1995) used the
25mm diameter cut-off, but also included 20mm follicles provided there were
multiple follicles (cysts) present on the ovaries. The area of the cyst was used
as a cut-off in one study. In this study, it was shown that the average area of
4.9 cm2 corresponded with a 25mm diameter (Carroll et al., 1990). Several
studies used 20mm as a cut-off and some of these studies did not confirm or
measure the “cyst” using ultrasonography (Hamilton et al., 1995; Chavatte et
10
al., 1993; Cook et al., 1991). Overall there is some variability in the literature
as to the minimum size a follicle has to be before it is termed a cyst. A range
of 20-25 mm as the minimum diameter of an anovulatory follicle on one or
both ovaries and reproductive history of infertility or ovarian dysfunction
indicate a cow with COD (Farin et al., 1992).
Follicular cysts are defined as thin-walled, fluid filled structures on one
or both ovaries (Kesler et al., 1981) and may be associated with high plasma
estradiol. Farin and colleagues (1992) described the appearance of follicular
cysts on ultrasound screens as “an anechoic antrum and a smooth, thin wall
with few or no gray patches.” The gray patches that Farin and colleagues
(1992) referred to are areas of luteinization of granulosa cells within the cyst
or cyst wall. Garm (1949a) describes most of the follicular cysts in his study as
thin-walled with some containing a small amount of luteal tissue towards the
ovary side or surrounding the wall of the cyst.
Luteal cysts are thick-walled structures on the ovary that do not have
an ovulation papilla, so the surface of the luteal cysts is smooth. They
typically occur as a single dominant cyst, but have been reported to occur
along with a follicular cyst (Brown et al., 1982). On ultrasonography, they
have a gray rim or “gray echogenic patches along the inner cyst wall or within
the antrum of the cyst” (Farin et al., 1992). Another criterion used to
determine a luteal cyst is the plasma or milk P4 concentration. To differentiate
follicular cysts from luteal cysts, follicular cysts are typically associated with a
11
plasma progesterone (P4) concentration of less than 1 ng/ml. However,
plasma P4 concentrations ranging from 0.5 to 5.0ng/ml have been used as the
maximum P4 concentration (cut-off) for a follicular cyst (Farin et al., 1992;
Ribadu et al., 1994a; Sprecher et al., 1988). Many researchers have used a
plasma P4 concentration greater than 1 ng/ml as the minimum P4
concentration for a cyst to be considered luteal (Jou et al., 1999; Santos et al.,
2000; Nakao et al., 1983; Ax et al., 1986; Dinsmore et al., 1989). However,
P4 limits of greater than 0 ng/ml (plasma) up to 10 ng/ml (milk) have been
used (Kasari et al., 1996; Chavatte et al., 1993; Farin et al., 1990; Ribadu et
al., 1994b; Nanda et al., 1991).
Cystic CL have been defined as the post-ovulatory formation of a CL
containing a fluid-filled cavity of 10mm or greater in diameter (Morrow et al.,
1966). Clinically, a cystic CL is round and fluctuant sometimes with an
ovulation papilla. Cystic CL are reported to fluctuate when palpated “more
than normal luteal tissue, but not so much as with a cystic follicle” (McEntee
and Jubb, 1957; Morrow et al., 1966). Generally, it is larger than a CL with
out a large, fluid-filled cavity in a cow during diestrus (Morrow et al., 1966).
The maximum size of the internal cavity or thickness of luteal tissue rim in a
cystic CL appears to be subjective and determined by each research group or
ultrasonographer (Nakao et al., 1983). There have been no established
criteria for thickness of a luteal rim of tissue or antrum size to distinguish
between cystic CL and luteal cyst. Presently, the differentiation is based on
12
subjective judgment. In an abattoir survey, Al-Dahash and David (1977a)
found cavities in cystic CL that ranged from 0.5-3.7cm in diameter. One main
difference between a cystic CL and a luteal cyst is that the cystic CL is formed
after ovulation. Generally, the cystic CL is determined to have a “thick” luteal
rim when observed via ultrasonography, although this judgment is subjective.
As the ultrasonographer becomes skilled, consistency in the differentiation
between cystic CL and luteal cysts develops.
In a study looking at the differences in normal CL versus cystic CL,
Labhsetwar and colleagues (1964) found that all CL that formed following
oxytocin treatment had a cavity and were smaller CL. These cavities were
reported to consist of blood clots. However, “clear fluid“ similar to plasma was
contained inside of the control CL that formed cavities. In a study by Simmons
and Hansel (1964), 11 of 16 control cows were diagnosed with cystic CL. The
fluid within the cavities contained 3 µg P4/ml. The weight and P4
concentration of the surrounding luteal tissue were comparable to the weight
and P4 concentration of the CL in the remaining 5 control cows. In contrast,
Donaldson and Hansel (1968) found that cystic CL contained higher P4
concentration on day 3 and a lower P4 concentration on day 7 of the estrous
cycle than normal CL. They only found blood clots in CL following palpation
and suggested a cause and effect relationship (Donaldson and Hansel, 1968)
13
in that the blood clots were thought to be a result of the palpation.
Additionally, they did not find an increase in cystic CL following oxytocin
treatment.
A previous study indicated that cystic CL did not have any detrimental
effect on pregnancy (Morrow et al., 1966). Another study showed that cystic
CL did not appear to alter the estrous cycle (McEntee, 1958). Cows with cystic
CL were found to have similar plasma P4 concentrations and luteal tissue P4
concentrations regardless of the size of the cavity (Kastelic et al., 1990; Okuda
et al., 1988). In an abattoir study, cystic CL had higher P4 concentrations per
gram of luteal tissue and contained higher overall progesterone in the luteal
tissue than noncystic CL (Okuda et al., 1988). Generally, cystic CL have not
been considered an anomaly that requires treatment (McEntee, 1958; Morrow
et al., 1966; Garverick, 1999).
DIAGNOSIS
Accuracy of diagnosing COD based on palpation alone has been
controversial since before 1917 (Albrechtsen, 1917). Morrow (1968) stated,
“luteal cysts, anovulatory cysts or luteinized follicles were hard to differentiate
from cystic follicles on rectal examination”. A source of error in diagnosing
cyst type may be the lack of experience of the palpator. Dinsmore and
colleagues (1989) did a comparison study between staff veterinarians and
14
interns with less than 3 years palpating experience. Staff veterinarians
correctly identified 48% of the follicular cysts based on milk P4 concentrations.
Interns correctly identified 35% though the percentages were not different
statistically (Dinsmore et al., 1989). Some luteal tissue that is present inside
the cyst cannot be felt and some luteal tissue present on the ovary that is
palpable, may not be steroidogenically active (Farin et al., 1992).
In differentiating the type of cyst, Nakao and colleagues (1983) found
that rectal palpation was highly inaccurate in diagnosing cyst type (follicular,
luteal, cystic CL) when compared to milk P4 enzyme immunoassay (EIA). Of
the 160 cows diagnosed as having follicular cysts, only 65% actually had
follicular cysts based on milk P4 EIA (Nakao et al., 1983). Luteal cysts made
up 19% and cystic CL made up the remaining 16%. In another study, 42% of
the follicular cysts diagnosed via rectal palpation were retrospectively
considered to be luteal cysts based on milk P4 (Ax et al., 1986). This means
that 42% of the treatments were given in error based on cyst type. Recently,
33% of 40 cystic cases were incorrectly called follicular cysts by a group of 13
veterinarians (Dobson and Nanda, 1992).
Some of the variability in diagnosing cyst type may be due to the
inconsistent cut-off point for the milk or plasma P4 concentration. For
example, using 2 ng/ml milk P4 as a cutoff for follicular cysts, 84% of the
follicular cysts and 46% of the luteal cysts were correctly diagnosed using
palpation (Booth, 1988). When 5 ng/ml milk and plasma P4 was used as the
15
minimum P4 concentration to be considered a luteal cyst, palpation had a 75%
predictive value for follicular cysts and 35% predictive value for luteal cysts
(Sprecher et al., 1988). Accurate identification of cyst type may be helpful for
faster resolution of the cyst problem. Bajema and colleagues (1994)
demonstrated that cowside milk P4 EIA tests reduce days open and financial
loss. They found increased pregnancy rates when the cowside tests were used
to monitor herd cyclicity and to identify cyst type for treatment determination
(Bajema et al., 1994).
With the introduction of ultrasonography it has been possible to
visualize the female reproductive tract in real-time. Recently, ultrasonography
has been used to differentiate between cyst type for cows with COD. Farin
and colleagues (1990) found that the ultrasound was better at detecting luteal
cysts (92% sensitivity) than follicular cysts (70% specificity). This accuracy
was determined using 0.5 ng/ml P4 as the cut-off concentration. Farin and
colleagues (1990) noted that some of the incorrect diagnoses of luteal cysts
were due to patches of luteal tissue spread along the cyst wall. Also, they
reported that hormones might vary and contradict the ultrasound images when
trying to classify cysts from palpation or ultrasound. Carroll and colleagues
(1990) thought that the hormonal contradiction may be due to missed CL, age
of the cyst and age of the luteal tissue present. In general, ultrasound was
found to have a higher sensitivity and specificity (87% and 82%, respectively)
than palpation (43% and 65%, respectively) for detecting cysts in cattle (Farin
16
et al., 1992). In a follow-up study, ultrasound was found to correctly diagnose
cyst type 85% of the time when based on serum P4 concentrations with a cutoff of 0.5 ng/ml P4. In this same study, palpation was reported to be correct
51% of the time (Farin et al., 1992). A Japanese study subjectively
demonstrated that ultrasonography was more accurate at detecting luteal
tissue than rectal palpation (OHNAMI et al., 1995). Ohnami and colleagues
(1995) claimed that palpation would have missed luteal tissue that formed in 4
out of 9 cows with follicular cysts. Ultrasound has been a better diagnostic aid
in determining cyst type than palpation, but the gold standard remains P4
radio/enzymeimmunoassay.
THERAPEUTIC STRATEGIES
Many techniques and therapeutic strategies have been used to treat
COD in dairy cattle. Some of the earliest treatments included: ovariectomy,
injection of ovarian extract, injection of CL extract, uterine infusions of
antibiotics or antiseptics, and injections of adrenaline chloride and pituitrin
(Albrechtsen, 1917; Williams and Williams, 1923b; Tutt, 1932; Casida et al.,
1944). There is only an empirical statement by Roberts (1986) that refutes
ovariectomy as a treatment option. He states that “spaying will correct
17
nymphomania but removing only one ovary if it is affected with cysts is
useless, since the remaining ovary will promptly develop cysts” (Roberts,
1986).
The first widely accepted treatment for COD is still used today. As early
as 1874, manual rupture of the cysts has been advocated as a treatment for
COD (Zschokke, 1874 in German as cited by Williams and Williams, 1923a).
Williams and Williams (1923b) supported this treatment so strongly that they
recommended that if cysts did not rupture on normal palpation, the “full
exertion of a powerful man” might be required. They also noted that if this did
not work, the cyst might need to be ruptured with a scalpel (Williams and
Williams, 1923b). In a study conducted at Cornell University, Roberts (1955)
reported that when comparing manual rupture to hormonal therapy, “there is
no advantage in manual rupture of the cysts and there may be some
contraindications from the standpoint of producing trauma and adhesions.”
Dawson (1957) recommended a combination of manual rupture of the cyst
and hormonal therapy. Another variation was repeated rupture of the cyst until
it no longer reappeared (Schjerven, 1965). Schjerven (1973) reported that
combining manual rupture and hormonal therapy did not provide any
advantage over manual rupture of the cyst alone. Manual rupture was not
used in one study due to the author’s prior experience with severe and fatal
bleeding that occurred in some cows (Dobson et al., 1977). Throughout the
literature there are conflicting reports (some have data to support their claims)
18
about the best method for COD treatment. Roberts (1986) suggests rupture
of the cyst as a treatment. However, he does indicate that “repeated
rupturing of the cyst may become costly and could potentially do harm to the
ovary.”
Hormonal therapy seems to be the more widely used treatment for COD
in the present day. Unfractionated pituitary extract was noted to caused CL
formation in some COD cows (Casida et al., 1944) and the route of treatment
given seemed to vary the results (intravenously versus subcutaneously).
However, this treatment caused an increase in the number of follicles that
developed post-treatment (Casida et al., 1944). They were in essence
hyperstimulating the ovaries of the cystic cows due to the combination of
luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the pituitary
extract.
Human chorionic gonadotropin (hCG) has high LH activity and is a
protein hormone. Human chorionic gonadotropin induces luteinization either of
the cyst or other follicles present (Yamauchi, 1955; Roberts, 1955).
Subsequently, endogenous or exogenous prostaglandin can induce regression
of functional luteal structures and reinitiate estrous cycles. Human chorionic
gonadotropin is an expensive treatment and due to its protein structure can
have complications following treatment. One of the disadvantages of hCG, is
that cows can build up immunity against the hCG protein. Anaphylaxis can
occur following treatment or cows may become refractory to the therapy
19
(Roberts, 1957; Roberts, 1986). This refractoriness to hCG has also been
demonstrated in rabbits (Greenwald, 1970). The percentage of cows
developing luteal tissue in response to hCG treatment ranged from 58-86%
(Morrow et al., 1966; Roberts, 1955; Nakao et al., 1978; Trainin and Adler,
1962). These results were obtained using a variety of routes of administration
from intravenously to intracystically and various combinations of these routes
(Morrow et al., 1966; Roberts, 1955; Roberts, 1957; Pepper, 1973; Nakao et
al., 1978; Alanko and Katila, 1980). In several studies, hCG was given in
combination with or following progesterone treatment. This produced
responses ranging from softening of the cyst for manual rupture to “induced
resorption of the ovarian cysts” (Daniel and Sulman, 1964) to ovulation of
subordinate follicles and subsequent conception to the insemination (Nakao et
al., 1978; Trainin and Adler, 1960; Trainin and Adler, 1962). Human chorionic
gonadotropin is primarily used in cystic cows that do not respond to their initial
therapy or that have reoccurring cysts.
Luteinizing hormone releasing hormone (LHRH) is also known as
gonadotropin releasing hormone (GnRH). Kittock and colleagues (1973) were
the first to report on using exogenous GnRH to treat cysts in cattle. They
found that all five cystic cows that were treated with GnRH were in standing
estrus within 20-24 days post treatment (Kittok et al., 1973). LH reached the
highest concentration in the plasma following the first injection of GnRH.
Subsequent injections of GnRH stimulated a less robust response. Plasma P4
20
concentrations were similar in cystic cows receiving GnRH therapy compared to
regular cyclic cows (Kittock et al., 1973; Garverick et al., 1976). Nessan and
colleagues (1977) performed a field trial to compare hCG and manual rupture
to GnRH and manual rupture or only GnRH in cystic cows on commercial
dairies in Canada. The three groups had a pregnancy rate ranging from 4047% and were not statistically different (Nessan et al., 1977). Therefore, hCG
and manual rupture were not necessary when GnRH was used. A previous
study found no difference when comparing GnRH to hCG in days to first estrus,
conception rate, conception on first service, services per conception, and days
to conception. No differences were found in comparing synthetic GnRH to
GnRH analogs and among various concentrations of GnRH or LHRH (Pedersen,
1983; Dinsmore et al., 1987; Osawa et al., 1995). Therefore, many of the
GnRH products may provide reasonable resolution or results in cows with COD.
Varying concentrations of GnRH were tested and compared to 0 µg
GnRH or saline. Earlier studies reported that GnRH improved days to estrus,
time to detection of a luteal structure, or increased plasma P4 concentration
(Edquist et al., 1974; Bierschwal et al., 1975; Seguin et al., 1976; Kesler et al.,
1978b; Ax et al., 1986). Ax and colleagues (1986) found that Procystin, a
GnRH product, decreased days to estrus; however, there was no effect on
first, second, and third service conception rate, percentage of cows pregnant
by 60 days, and days to conception. More recent studies, however, do not
support a benefit in using GnRH over saline (Dailey et al., 1983; Jou et al.,
21
1994; Jou et al., 1999). The difference in the studies may be that the more
recent studies preferentially included follicular cysts whereas the earlier studies
did not discriminate in cyst type.
Another hormonal therapy used has been GnRH followed by
prostaglandin F2α (PGF). This therapy did not improve time to conception,
pregnancy rate, first-service conception rate, or days open when compared to
GnRH alone (Archbald et al., 1991). Archbald and colleagues (1991)
concluded that neither treatment worked sufficiently. Therefore, they suggest
using the cheaper of the two treatments, a single injection of GnRH. Also, no
advantage was found when GnRH and Cloprostenol (PGF analog) were given
simultaneously compared to GnRH alone (Dinsmore et al., 1990). A recent
study compared noncystic cows on the Ovsynch protocol (injection of GnRH
followed seven days later by injection of PGF followed two days later by GnRH
and then timed inseminated 16 hours later) to cystic cows on Ovsynch and
cystic cows treated with GnRH followed by PGF seven days later and then bred
on observed estrus as possible treatments for cows with COD (Bartolome et
al., 2000). They found that there was no difference in pregnancy rate
between the two cystic cow groups. However, cows with COD treated with
Ovsynch and the GnRH/PGF treatment combined, had a lower pregnancy rate
than noncystic cows treated using the Ovsynch protocol (Bartolome et al.,
2000).
22
When cysts were classified according to type as determined via rectal
palpation, cows with follicular or luteal cysts received GnRH or PGF,
respectively. In a retrospective analysis of 227 cystic cows, Nanda and
colleagues (1988) found that cows with luteal cysts had a shorter time to
recovery than those with follicular cysts. About 65% of the cows with luteal
cysts recovered within an average of 5 days, while 53% of the cows with
follicular cysts recovered in an average of 10 days. Booth (1988) performed a
similar study using 200 cows with COD, but did not find any treatment
differences when the two groups were compared. When treatments were
based on P4 concentration, Chavatte and colleagues (1993) found that cows
treated with PGF had a higher incidence of estrus and pregnancy rate by days
7-14. However, there was no difference in pregnancy rate between the two
groups by day 35 (Chavatte et al., 1993). In another study using P4
concentration to determine cyst type, there was no difference between the two
treatment groups compared to a GnRH treated control group composed of
cows with both types of cysts (Sprecher et al., 1990).
Progesterone has been used to treat cows with COD. Norgestomet
implants, CIDRs (Controlled Internal Drug Release) and PRIDs (Progesterone
Releasing Intravaginal Device) have been used to deliver P4 at a reasonably
constant rate in cystic and chronically cystic cows. The premise for using
exogenous P4 therapy in cows with COD is to inhibit LH release and allow time
for LH to be replenished in the pituitary. Therefore, when P4 is removed,
23
GnRH may stimulate a release of LH that would induce ovulation in a
responsive follicle (Nakao et al., 1978). Sixty-eight percent (17/25) of the
cystic cows that received a PRID had resolution of the cyst with or without a
display of estrus (Nanda et al., 1988). Of these 25 cows treated with a PRID,
15 (60%) became pregnant to an average of 1.5 services per conception
(Nanda et al., 1988). Todoroki and colleagues (1998) used CIDRs as a
treatment for COD. Normal cyclicity was established for at least three cycles
and normal follicular waves occurred while the CIDR was in place (Todoroki et
al., 1998).
Many other hormones have been used to treat cows with COD.
Prostaglandin F2α and PGF analogs have been used with varying responses
(Eddy, 1977; Leslie and Bosu, 1983). Leslie and Bosu (1983) treated cystic
cows with PGF regardless of cyst type and reported poor responses in cows
with low plasma P4 concentrations. Also, these cows had a higher
reoccurrence of cysts than cows with high P4 concentrations. Estradiol
benzoate (EB) has been used at various times and in combination with other
hormones with a diverse range of results (Nanda et al., 1991). Corticosteroids
and FSH have also been used to treat cows with COD (Nakao and Miyake,
1977; Brown et al., 1986). The response to corticosteroids was comparable to
treatment with hCG and P4 (Nakao and Miyake, 1977).
24
ETIOLOGY
At one time, cystic ovaries were thought to be a secondary symptom
(Garm, 1949a) to nymphomania and adrenal virilism. Nymphomania and
adrenal virilism were considered the actual diseases. Early treatments focused
on “crushing” the cyst or manual rupture because it was believed that getting
rid of the follicle or cyst would alleviate the nymphomania (Albrechtsen, 1917).
The ovary was also suspected of being the cause of nymphomania. Bilateral
ovariectomies were performed and the symptoms subsided. To retest the
theory that the ovary is responsible for nymphomaniacal behavior, whole
ovarian and corpus luteum extracts were given to cows with COD. These
treatments intensified the nymphomaniacal symptoms (Williams and Williams,
1923b). However, none of this evidence conclusively proved that a problem
with the ovary was the sole cause of nymphomania and knowledge was
expanding on the function and role of other organs in the ovulatory process.
One group of scientists considered ovarian cysts to be “nodular venereal
disease” or a form of genital tuberculosis. However, no bacteria were isolated
from the cyst or ovary (Garm, 1949a). Nymphomaniacal symptoms were also
thought to occur as a result of an abnormality of the ovary (Albrechtsen, 1917)
or possibly as a result of a mutated gene (Dawson, 1957).
One theory that has had repercussions for many years was that
nymphomania was the result of endometritis (Williams and Williams, 1923b).
25
This statement, without substantiation, has had producers and veterinarians
infusing many obscure treatments in the uterus for treatment of COD. Casida
and colleagues (1944) described COD in cattle as a potential consequence of
endometritis. It was believed that cystic ovaries occurred “as a result of
extension of infection from the uterus through the fallopian tubes to the ovary”
(Casida et al., 1944). As a result, cows with endometritis and cystic ovaries
were considered to be sterile. Disinfecting of the uterus to correct the
endometritis and ovarian cysts became the ascribed to therapy (Williams and
Williams, 1923a; Casida et al., 1944). However, few cows with endometritis
were reported to also have had ovarian cysts and vice versa (Garm, 1949a;
Roberts, 1955; Dawson, 1958). Again, no bacteria were isolated from the
cyst. More recently, (Singh et al., 1982) five cows diagnosed with COD and a
concurrent uterine infection were treated with 300 µg of GnRH and had uterine
infusions with the drug of choice based on in vitro sensitivity tests. Three of
the five cows became pregnant within 23 days and all five showed heats.
Bosu and Peters (1987) found that cystic cows predominantly had E. Coli as
the major gram-negative pathogen in their uterus compared to noncystic cows.
Also, cows that became cystic had high colony counts of uterine bacteria
before detection of cysts (Bosu and Peter, 1987). Presently, this theory for
COD etiology is believed to be more coincidental than causative.
Another theory was that a disruption in the endocrine or ovulatory
system was causing COD in cattle (Garm, 1949a; Nadaraja and Hansel, 1976;
26
Eyestone and Ax, 1984). It was proposed that the amount of LH released
from the anterior pituitary dictates the degree of luteinization, lack of ovulation
and formation of cysts (McEntee and Jubb, 1957). Nadaraja and Hansel
(1976) inferred from their work and that of others that COD may be related to
an insufficient release of LH or the lack of an LH surge. This theory is
currently the basis for determining the etiology of COD. The actual cause of
COD in cattle is still unknown.
The major emphasis of research is looking at the cow’s response to
different stressors and other factors associated with cattle that have COD. It is
known that stress induces the release of cortisol and cortisol inhibits LH. The
actual time that the stress or inhibition of the LH surge has to happen to
disrupt the LH surge has largely been unpredictable. Also, the amount of
stress needed to induce this response is unknown. Cook and colleagues
(1991) could not prove that higher LH pulse frequency and amplitude in
steroid-induced cystic cows was the cause or the effect of the ovarian cyst.
One of the main problems in determining the etiology of COD has been that
most studies use cows that have been diagnosed cystic and the condition
already exists. In the studies where COD is induced, it is controversial as to
whether these cows respond and act similar to cows with naturally occurring
cysts.
The anterior pituitary is known to release the gonadotropins, LH and
FSH. They are released in response to the hypothalamic release of
27
gonadotropin releasing hormone (GnRH). In turn, a series of events occurs
preceding the release of GnRH. In summary, progesterone decreases allowing
the surge of estradiol and thus the release of GnRH followed by the LH surge.
These hormones act so that ovulation can occur. Any alteration outside of the
normal for these hormones or functions at the cellular level (steroid passing
through cell membranes, binding of hormone to receptors, etc.) may be the
stimulus for the formation of cystic ovaries.
ASSOCIATIONS
In trying to separate out the true cause of COD, there are many factors
that have been associated with its occurrence. Cystic ovaries have been
associated with high milk production as early as 1934 (Clapp, 1934). Cows
with COD tended to have higher milk production compared to their noncystic
herdmates (Clapp, 1934). Similarly, in 1968, more cases of COD were found
to occur in high producing cows than cows in medium and low milking groups
(Marion and Gier, 1968). Johnson and colleagues (1966) found that cystic
cows had higher 90 day and 305 day mature equivalent (ME) production than
their noncystic herdmates. Erb (1984) summarized four previously reported
studies that determined the association between high milk production and
COD. He stated that for a proper causal relationship, the “cause” must be
present prior to observing the “effect.” He introduced a current study that
28
compared milk production and COD and concluded that high milk production
does not cause COD (Erb, 1984). A later study found that as milk production
increased the incidence of COD increased. The authors claimed that this also
does not indicate a causative relationship (Emanuelson and Bendixen, 1991).
In contrast, a number of investigators did not find an association
between COD and milk production. Whitmore and colleagues (1974b) did not
find a difference in the percent of high producing cows having COD versus low
producing cows. Over a 3 year period, cystic cows did not consistently
produce more milk than noncystic cows (Hackett and Batra, 1985). In a study
using 200 cystic cows, no difference was seen in milk production when cows
with follicular cysts and luteal cysts were treated as separate groups or as one
group compared to noncystic counterparts (Booth, 1988). In another study,
305 day ME were not different between cystic and noncystic herdmates (Zulu
and Penny, 1998). Perhaps it is not surprising that some investigators have
not found a significant association between COD and milk production.
Considering the individual variability involved and different definitions of high
and low milk production, it should not be expected that all experiments would
discover the association. However, since several studies have identified such
an association, and none of the studies found the opposite association (COD
and low milk production), it seems most likely that there is an association
between COD and increased milk production. Whether there is a cause and
effect relationship is still unknown.
29
A related issue is that cystic cows exhibiting nymphomania had lower 90
day and 305 day ME than cystic cows that did not show any signs of estrus
(Johnson et al., 1966). This should not be surprising since milk production is
known to decrease during estrus due to increased activity.
Two experiments were designed to investigate the possible relationship
between percent milk fat and COD. In the first experiment, there was no
increase in butterfat associated with cystic cows even though milk production
was greater (Casida and Chapman, 1951). In another experiment, when the
data were adjusted for calving interval and standardized to the second and
third year production in herds of similar overall farm production, no differences
in butterfat or milk production were detected for cystic versus non-cystic cows
(Henricson, 1957).
A study by Monsanto Co. found no association between treatment with
bovine somatotropin (bST) and an increase in the occurrence of COD in dairy
cattle (Cole et al., 1992). Fewer heifers treated with bST (as part of a
superovulation protocol) developed cystic ovaries than non-bST treated heifers
(Gong et al., 1993). Also, cows that received bST did not have a higher
lactational incidence of COD than untreated control cows (Judge et al., 1999).
Twinning has also been a major concern to the dairy producer whether
it is caused in response to treating a disease or a hereditary condition. Clapp
(1934) reported that cystic cows had a higher percentage of twins than their
noncystic herdmates (9/24 versus 21/126). In a case referent study by a
30
Swedish group, it was not conclusively determined that cows with an episode
of COD prior to conception had a higher risk of having twins (Bendixen et al.,
1989). In a later study by this same group, it was found that cows having had
twins were 2.0-2.7 times more likely to have COD than cows that did not have
twins (Emanuelson and Bendixen, 1991). Therefore, cows that had an episode
of COD and conceived did not necessarily have more sets of twins than cows
with out COD. However, having twins seems to predispose the cow to COD
during the following lactation.
Genetics has also been associated with COD in dairy cattle. Casida and
Chapman (1951) studied a Wisconsin dairy herd and found that COD occurred
in a higher percentage of mother daughter pairs when the mother had COD
compared to mother daughter pairs when the mother did not have COD. Sire
line was focused on in another study. A three-year study found that cows
bred to certain sires had different percentages of cows with COD (Wiltbank et
al., 1953). These percentages ranged from 0-23% (Wiltbank et al., 1953).
Even though COD has been associated with high milk production, it was
theorized that COD was not directly linked to the gene for milk production
since there were so many factors that influenced milk production (Henricson,
1957). Therefore, focusing efforts to control COD would be better
concentrated on the sires. Sires could be selectively used based on their
production of daughters with high milk production and low incidence of COD
(Henricson, 1957). Menge and colleagues (1962) demonstrated that sire line
31
had an effect on the percentage of cows that become cystic. However, there
was no difference between the percentage of outbred cows that became cystic
(18%) compared to inbred cows using the same two Holstein-Fresian sire lines
(15%; Menge et al., 1962). A study in India conducted on Gir and Gir-crosses
reported no difference in the inbred versus outbred groups (Pandit and Parekh,
1982).
From 1954-61, overall occurrence of COD cases diagnosed in Sweden
decreased from 11% to 5% due to the culling of bulls with a high incidence of
COD in their offspring (Bane, 1964). More recently, heritability of COD was
low for first and second lactation cows (0.15 and 0.11; Ashmawy et al., 1990).
In another study, Van Dorp and colleagues (1998) estimated the heritability of
COD to be 0.02 (very low). These low heritability estimates indicate that it
would take a great amount of time to decrease the incidence of COD by culling
cows based on the occurrence of COD (Ashmawy et al., 1990).
Various other factors have been associated with the occurrence of COD
in dairy cattle. The use of electric cow trainers (Oltenacu et al., 1998) and
frequent use of PGF (Jasko et al., 1984) were not found to increase the
incidence of COD. Heat stress (Wolfenson et al., 1988; Marion and Gier,
1968), melengesterol acetate use in beef heifers (Herenda, 1987), estrogen
induced lactations (Hammond and Day, 1944), and overcrowding of barns
(Arthur, 1975) all were associated with the higher occurrence of COD.
However, no causal relationships were established.
32
RESEARCH MODELS
Most of the research on COD in cattle has been done following cyst
diagnosis or after the condition already existed. Consequently, the conditions
that led up to cyst formation may have been altered over the time it took to
select the cow as suspect and determine she had COD. Trying to determine
the initial cause of COD becomes a challenge. Some research groups have
tried to mimic cyst development by inducing cysts with exogenous hormones.
Prior to 1957, diethyl stilbesterol (DES) was used to induce cysts and
when used for therapy of other medical conditions, tended to induce cysts
(Dawson, 1957). Results of inducing cysts using DES were not predictably
repeatable. Hansel and Wagner (1960) reported the formation of small,
subnormal CL, ovarian cysts, and cystic CL following oxytocin treatment at
various times during the estrous cycle. Estradiol valerate (EV) on day 15 to 16
of the estrous cycle also induced cysts. When it was given at other times
during the estrous cycle, cysts did not form or very few were induced
(Wiltbank et al., 1961). Rajamahendran and Walton (1990) unsuccessfully
used EV on day 16 of the estrous cycle. Luteolysis occurred following EV
injection and subsequent follicular development occurred without ovulation
(Rajamahendran and Walton, 1990). Nadaraja and Hansel (1976) induced
cysts in heifers using estradiol benzoate (EB) and bovine LH antiserum,
33
separately. They found that EB produced cysts 2.5 to 3.0 cm in diameter that
were “soft”. These heifers had higher plasma estradiol concentrations than
heifers that received the LH antiserum. Also, heifers that received the LH
antiserum had “harder” cysts that were larger (5-6 cm; Nadaraja and Hansel,
1976).
Fathalla and colleagues (1978) induced cysts in five cows using
testosterone proprionate in increasing doses up to 250mg. Cysts induced by
testosterone underwent atresia by approximately 14 days after formation.
Estradiol-17β and adrenal corticotropic hormone (ACTH) were used separately
to induce cysts in nonlactating dairy cattle (Refsal et al., 1987). The group
that received ACTH had higher serum cortisol concentrations. Also,
combinations of estradiol-17β and P4 have been used to induce cysts (Cook et
al., 1990; Cook et al., 1991). All combinations resulted in varying outcomes of
cyst formation.
Carriere and colleagues (1995) induced an abnormal ovarian response
in six of seven heifers treated with EV and cloprostenol on day 17, 18 or 19 of
the estrous cycle. Two heifers had persistent CL and two heifers had large
follicles ovulate and large CL form in both cases. One heifer developed a
luteinized cyst and one developed multiple large follicular cysts. Estradiol
valerate induced a wide, unpredictable range of cystic episodes (Carriere et al.,
1995).
34
Induced cysts in dairy cattle may provide some insight into cyst
development. However, the cows with induced cysts do not seem to respond
to treatment or nontreatment in the same manner as cows with naturally
occurring cysts.
OTHER SPECIES AFFECTED
Other species have been reported to have COD. Some clues to the
formation and development of COD in cattle may be gained from these other
species. Women, swine, rats, mice and guinea pigs provide the greatest
amount of research data and information regarding PCOD. However, there are
species differences and different actions involved in PCOD versus COD in
cattle. Cystic ovarian disease has been reported to occur in lions, dolphins,
domestic cats and camels among other species.
35
CHAPTER 2
RESEARCH OBJECTIVES
This dissertation examines the dynamics of spontaneously occurring
cystic ovarian disease. Ultrasonography was used to monitor cows with COD
on six dairies in western and central Ohio. Two farms used bulls for breeding;
the rest used artificial insemination. The herd veterinarian discovered cows
with COD during routine farm visits. All COD cows were monitored via
ultrasonography every Monday, Wednesday, and Friday for five weeks. Blood
was collected for blood chemistry analysis and progesterone concentration at
each farm visit. No managerial changes (feeding, housing, milking, etc.) were
made after cows were diagnosed with COD. All cows were bred according to
the am/pm rule if artificial insemination was used, and farm personnel
recorded any breeding by bulls. The specific objectives for this study were to
determine:
1) whether the use of ultrasonography to determine cyst type
and consequently base the initial treatment was economical
compared to palpation and a traditional treatment regime
36
2) the effect of basing the follow-up (secondary) treatment 14
days after initial treatment on the presence of luteal tissue
3) the biochemical profiles of cows with spontaneous COD
4) the ovarian dynamics and progesterone profiles of cows
following treatment for COD from frequent observation and
blood sampling
5) retrospectively, the effects of various management practices
(length of dry period and days open) on COD cows compared
to normal herdmates using DHI records.
In a final study, luteal-like structures observed on the ovaries of COD cows
treated with GnRH were compared to naturally occurring day 6 (estrus =day 0)
CL and GnRH-induced CL in the presence of luteal tissue. Tissues were
examined for gross/histologic morphology differences and compared with
blood and tissue P4 production. After recording gross morphologic features
(color, weight), pieces of all tissues were preserved for histological analysis.
All tissues were then dissociated and short-term incubated in the presence or
absence of LH. The main objective for this study was to determine if luteal-like
structures induced in cystic cows are steroidogenically and histologically similar
to CL induced in cycling cows and to spontaneously occurring CL in cows with
normal estrous cycles.
37
CHAPTER 3
USE OF ULTRASONOGRAPHY FOR DETERMINING CYST TYPE, TREATMENT,
AND OUTCOMES FOR DAIRY CATTLE EXHIBITING SPONTANEOUS CYSTIC
OVARIAN DISEASE
INTRODUCTION
Cystic ovarian disease (COD) is a multifaceted condition affecting almost
a million dairy cattle in the United States each year (Garverick, 1997). As a
result of this condition, the dairy industry has suffered substantial financial and
reproductive losses. The pathology of COD manifests itself most commonly as
a failure to ovulate and is characterized by two types of cysts, luteal and
follicular.
As far back as 1917, the accuracy of palpation for differentiating cysts
and the different cyst types from normal ovarian structures has been
questioned (Albrechtsen, 1917). Several studies have found that palpation
correctly diagnosed follicular cysts 58-84% of the time when compared to milk
or plasma progesterone (P4) concentrations (Ax et al., 1986; Booth, 1988;
Dobson and Nanda, 1992; Nakao et al., 1983; Sprecher et al., 1990).
However, the accuracy of the diagnosis may have depended upon the cutoff
38
value of P4 used to differentiate luteal from follicular cysts. In these studies,
the cutoff concentrations for diagnosis of luteal cysts ranged from >1.0 ng/ml
to >5.0 ng/ml (Ax et al., 1986; Booth, 1988; Dobson and Nanda, 1992; Nakao
et al., 1983; Sprecher et al., 1990). One study used histology to verify the
diagnosis of cyst type and (Marion and Gier, 1968) concluded that palpation of
cystic structures was accurate based on 600 pairs of ovaries collected at
slaughter. Recently, ultrasonography has been compared to serum P4
concentration and used to diagnose cyst type. Ultrasound (5 MHz) was 92%
sensitive in detecting luteal cysts and had 70% specificity in detecting follicular
cysts using 0.5 ng/ml P4 as a cutoff value (Farin et al., 1990). Specificity is
defined as an animal that is identified by a test as not having a condition when
they truly do not have the condition. Sensitivity is defined as an animal that is
positively identified by a test as having a given condition and the animal does
have the condition. Farin and colleagues (1992) compared ultrasound (U/S) to
palpation in the diagnosis of cyst type using 0.5ng/ml serum P4 as the gold
standard cutoff. Ultrasound was found to have a higher sensitivity and
specificity (87%; 82%) than palpation (43%; 65%) for diagnosing cysts.
Ultrasound provided a higher percentage of correct diagnoses (85%)
compared to palpation (51%; Farin et al., 1992) based on plasma P4
concentrations.
Bajema and colleagues (1994) used cowside milk P4 ELISA tests to
monitor herd cyclicity and determine treatments for cows with cystic ovaries in
39
a herd of 82 cows. They found that days open decreased, monetary loss was
reduced, and pregnancy rates increased when P4 concentrations were used to
determine treatments and reproductive status for cows with COD. Several
studies have reported that using some type of assay (RIA or EIA or Latex
agglutination) to determine P4 concentration provided the practitioner with a
more accurate diagnosis of cyst type (Booth, 1988; Sprecher et al., 1988).
However, Ruiz and colleagues (1992) reported that treating cows for COD
based on milk P4 concentrations was not economically beneficial due to the
low occurrence and the highly variable individual animal response to COD
treatments. Carroll and colleagues (1990) found that there was great
variability in ovarian cyst determination via ultrasonography and plasma P4
concentrations. They (Carroll et al., 1990) suggested that this was due to
undetected corpora lutea (CL) and imprecise determination of the age of any
luteinized tissue (cyst or CL).
The present study was conducted to evaluate whether the use of
ultrasonography to determine treatment based on cyst type and /or presence
of luteal-like tissue (LLT) would increase estrous expression and pregnancy in
cows with spontaneously occurring COD. On d1, cyst treatment was based on
the diagnosis of cyst type via ultrasonography. Ultrasound determination of
the presence of LLT on d14 of the study was used to decide the follow-up
treatment. Treating cows according to cyst type or presence of LLT was
40
expected to improve treatment outcomes compared to control cows receiving
a standard COD treatment (Kesler and Garverick, 1982; Kesler et al., 1978b).
MATERIALS AND METHODS
Ultrasound Diagnosis Study. This study was conducted from September 199798 on six average size commercial dairies (180-300 milking cows) located in
central and western Ohio. Throughout the year, 68 cows (Holstein, Jersey,
and Guernsey) were classified as being cystic by the respective herd
veterinarian. Before entering the study, a second reproductive exam was
performed using an ultrasound machine (Aloka 500) with a 5 MHz linear array
transducer (Aloka, Wallingford, CT). The initial criterion for inclusion in the
study was ultrasonographic determination of a cystic ovarian structure with a
diameter of at least 25mm. However, five cows were included in the follicular
cyst and control groups even though the original large cyst had ruptured on
palpation. These cows were treated as if they had follicular cysts. In addition,
four cows were included that had follicular cysts ranging from 23-24mm since
they had exhibited symptoms of COD within the previous month (i.e.,
anestrous or excessive mounting). All cows were a minimum of 32 days
postpartum with a range of 32-382, except for one cow that was 526 days
postpartum. This cow had been used as part of the farm’s superovulation
program, and the decision was made to include her in the breeding herd.
41
Cows were treated according to cyst type as determined via U/S (day
1=day of ultrasound diagnosis; d1). On d1, cows with luteal cysts (LC; n=12)
were treated with prostaglandin F2α (25mg; PGF) and cows with follicular cysts
(FC; n=38) received gonadotropin-releasing hormone (100µg; GnRH)
regardless of other ovarian structures (Fig. 3.1). If cows in the LC and FC
groups were not inseminated prior to d14, they were treated again. The d14
treatment (PGF or GnRH) was based on the presence of LLT on either ovary,
irrespective of cyst resolution or presence. Every fourth cow with a palpable
cyst was allocated to a cystic cow control (CCC; n=18) group prior to U/S
determination of cyst type. The control group was formed to mimic
therapeutic procedures based on palpation and received a standard COD
treatment of GnRH on d1. Each CCC animal, regardless of the type of cyst
(based on U/S) received PGF on d14 unless priorly inseminated.
All cows were inseminated at every standing estrus. Two farms on the
study had bull-bred herds. These farms contributed five total cows to the
study and were included because the breeding event was observed and
recorded by the producer.
Administration of the second treatment was based on ultrasonographic
evidence of luteal-like tissue (LLT) on either ovary irrespective of the presence
of a cyst. Due to the assumed aberrant hormonal milieu and diversity of
ovarian structures in cystic cows, apparent luteal tissue in the ovary or within
42
the cyst as viewed on the ultrasound screen was termed luteal-like tissue.
Cows with LLT present received PGF, whereas cows without LLT received
GnRH.
Ultrasound exams were repeated every Monday, Wednesday and Friday
for five weeks. All ovarian structures greater than or equal to 5mm in
diameter were recorded on ovarian maps. The image of the maximum size of
all structures was frozen on the ultrasound machine and elliptic calipers were
used to record the largest diameter in two perpendicular directions. The
diameter of the cyst was reported as the average of these two measurements.
Pregnancy status was confirmed at routine herd checks. Ovulation was
defined as the presence of new LLT as observed via ultrasonography within
seven days of an observed estrus. New LLT was considered to be any new
luteal-like structure that formed within 14 days following treatment, as
observed via ultrasonography.
Blood samples were collected in heparinized vacutainer tubes every
Monday, Wednesday and Friday. These samples were kept on ice until
centrifuged and plasma was aspirated and frozen for analysis of P4
concentration. Analysis of P4 was performed using a radioimmunoassay (RIA)
previously validated for bovine plasma (Clapper et al., 1990). Coefficients of
variation within and between assays were 7.74% and 10.71%, respectively.
43
Presence of Luteal-like Tissue Study (Retrospective Analysis). A retrospective
analysis was performed on cows with follicular cysts. Data from all cows in the
FC treatment group (n=38) and cows with follicular cysts in the CCC group
(n=17) on d1 were pooled. All of these cows had received GnRH as their
initial treatment irrespective of the presence of LLT. Data from cows where
the initial cyst had been ruptured (n=6) were not included in this retrospective
analysis. Additionally, one cow received the incorrect treatment on d14 and
was not included in the d14 analysis.
For the retrospective analysis, data from the cows with follicular cysts
that were treated with GnRH on d1 (n=49) were divided into two groups.
Group 1 (n=36) was composed of cows with a follicular cyst and no LLT
present on either ovary. Group 2 (n=13) were cows with a follicular cyst that
had LLT present on one or both ovaries. On d14, cows that still had a follicular
cyst and that had received PGF if LLT was present or GnRH if LLT was not
present (n=20) were divided into two groups (Fig. 3.2). Cows that were bred,
treated incorrectly, had a luteal cyst, or had resolved their cystic condition
were not included in the d14 retrospective analysis. Group 3 (n=7) included
data from cows with unresolved follicular cysts that had no LLT present and
had received GnRH on d14. Group 4 (n=13) included data from cows with
unresolved follicular cysts that had LLT present on d14 and had received PGF.
Estrus, ovulation and pregnancy were used as end points for the
comparisons between:
44
i) FC cows without LLT (group 1) versus FC cows with LLT (group 2)
that both received GnRH,
ii) FC cows without LLT (group 3) that received GnRH versus FC cows
with LLT that received PGF (group 4),
iii) FC cows with LLT that received GnRH (group 2) versus FC cows with
LLT that received PGF (group 4).
The observation of estrus was compared between retrospective treatment
groups for the two weeks following each treatment. Ovulation was defined as
the observation of new luteal tissue via ultrasonography other than within the
cyst within 7 days from reported estrus. If signs of estrus were not observed
and appearance of new LLT was noted, then this episode was not recorded as
being an ovulation. Pregnancy was determined at 21 days via ultrasonography
and confirmed at 35-40 days via rectal palpation for cows that displayed estrus
within the two weeks post treatment.
Statistical Analysis. Effect of cyst type and presence of LLT at the time of
treatment on display of estrus and pregnancy were compared using Fisher’s
exact test. Associations between cyst type and ultrasound results for time to
pregnancy were analyzed using Proportional Hazards Regression. All
comparisons made between the cows with follicular cysts (Groups 1-4) in the
retrospective analysis were made using Fisher’s exact test. All statistics were
run using SAS version 8.0.
45
Ultrasound-based Treatment Protocol
Day 14
Day 1
Follicular Cysts (FC)
GnRH
Estrus
No Tx (n=6)
PGF2α (LLT; n=17)
GnRH (No LLT; n=8)
Estrus
No Tx (n=7)
PGF2α (LLT; n=4)
GnRH (No LLT; n=0)
Estrus
No Tx
(n=38*)
Luteal Cysts (LC)
PGF2α
(n=12**)
Control (CCC)
GnRH
(n=4)
PGF2α
(Fol. n=17 or Lut. n=1)
(Lut. n=13; Non-Lut. n=1)
Figure 3.1: Ultrasound-based treatment protocol for cows with COD. (*Seven
cows were dropped from d14 analysis due to incorrect treatment. **One cow
received treatment out of sequence and was dropped from d14 analysis.)
46
Grouping of Follicular Cyst Cows (Day 14) Based on LLT for
Retrospective Analysis
DAY 1
DAY 14
LLT (n=4)
Follicular Cyst
(LLT; n=5)
Group 2
(LLT/GnRH; n=13)
Group 3
(No LLT/GnRH; n=7)
No LLT (n=1)
Not Included
(n=8)
LLT (n=9)
Follicular Cyst
(No LLT; n=15)
Group 4
(LLT/PGF2α; n= 13)
No LLT (n=6)
Group 1
(No LLT/GnRH; n=36)
Not Included
(n=21)
Figure 3.2. Combined groups for day 14 retrospective analysis of cows with
follicular cysts (with and without LLT) that were treated with GnRH or PGF.
47
RESULTS
Ultrasound Diagnosis Analysis. Due to the fact that only 1/18 cows had a
luteal cyst in the control group (CCC; every fourth COD cow on study), there
were, in effect, two FC groups. Since, all follicular cyst cows in both the FC
and CCC groups had received GnRH on d1, they were combined as one group
that was compared to the luteal cyst (LC) group which received PGF. More
(P<0.05) cows with luteal cysts exhibited estrus and were bred within the first
two weeks after initial PGF treatment (62%, 8/13) compared to cows with
GnRH treated follicular cysts (18%, 10/55; Figure 3.3). Cows with luteal cysts
were 7.2 (Odds Ratio) times more likely to be bred before d14 than cows with
follicular cysts. No statistical difference was found in the percentage of cows
pregnant to the first service following treatment between cows with luteal
cysts (25%; 2/8) and cows with follicular cysts (40%; 4/10; Figure 3.3).
Overall pregnancy rate to insemination within the first two weeks following
treatment for COD was 33% (6/18). All cows with LC responded to d1 PGF
treatment with either an observed estrus and/or the development of new LLT,
and thus none were treated with GnRH on d14.
Retrospective Analysis. All d1 cows with follicular cysts were divided into two
groups based on the presence of LLT (group 1; no LLT and group 2;LLT). All
48
cows with a follicular cyst had received GnRH as a treatment for COD on d1.
Estrus (17% vs 23%; 6/36 vs 3/13), ovulation (83% vs 66%; 5/6 vs 2/3), and
formation of new LLT (81% vs 85%; 29/36 vs 11/13) were similar between
groups 1 (LLT present) and 2 (No LLT; Table 3.1). Two pregnancies were
detected in cows from group 1 (n=36) and group 2 (n=13) within the two
weeks after the initial GnRH treatment (Table 3.1).
On d14, cows with unresolved follicular cysts present were divided into
two groups, again based on LLT absent (group 3; n=7) and LLT present
(group 4; n=13). Cows in these two groups were treated with GnRH and PGF
respectively (Fig. 3.2). Forty-three percent (3/7) of cows with a follicular cyst
and no LLT present treated with GnRH (group 3) displayed estrus. Seventyseven percent (10/13) of cows with a follicular cyst and LLT present (group 4)
displayed estrus after PGF treatment. Percentages of cows in group 3 and 4
that ovulated (66% vs 70%; 2/3 vs 7/10) and that formed new LLT (57% vs
77%; 4/7 vs 10/13) were similar (Table 3.1).
When groups 2 and 4 were compared, significantly more cows with
follicular cysts and LLT present that received PGF (group 4; 77%; 10/13)
displayed estrus than cows with follicular cysts and LLT present that received
GnRH (group 2; 23%; 3/13; P=0.02). There was no difference in the number
of cows that ovulated following an observed estrus in group 2 (2/3) and group
4 (7/10). Formation of new LLT was also similar between the two groups
(85% vs 77%; 11/13 vs 10/13; Table 3.1).
49
Progesterone Analysis. Progesterone concentrations from serum samples
collected on d1 were compared to the initial ultrasound diagnosis of ovarian
structures. Active luteal tissue was assumed to be present when the P4
concentration was >1.0 ng/ml. This active luteal tissue may have been within
the LC or present as LLT on one or both ovaries. The protocol for this study
stated that treatment on d1 was dictated by the type of cyst as seen via
ultrasonography regardless of other ovarian structures. The presence of luteal
tissue was evaluated retrospectively based on ovarian maps created at the
time of each ultrasound exam. Results are presented in Figure 3.4.
Thirty-six percent of all of the cows with FC (20/55) had P4
concentrations > 1.0 ng/ml (high P4) on d1. Of the 20 cows with follicular
cysts and high progesterone, 6 cows had no observed LLT on either ovary as
determined via ultrasonography. In the remaining 35 cows that were classified
as having follicular cysts and low P4 concentrations, 6 cows had a luteal-like
structure present on d1.
Eighty-five percent of the cows with LC (11/13) had P4 concentrations
> 1.0 ng/ml (high P4) on d1. Of these 11 cows, 1 cow had an additional
luteal-like structure on her ovaries in addition to the luteal cyst. Only two
cows had luteal cysts with no other observed LLT and low plasma P4
concentrations (Figure 3.5).
50
DISCUSSION
This study was conducted on six commercial dairy farms in central and
western Ohio. All cows used in the study had naturally occurring ovarian
cysts. The control group (CCC) was established such that it would be a group
of cystic cows that received a standard treatment for ovarian cysts, which
consisted of GnRH administered at the time of diagnosis and PGF at the
subsequent herd check, if needed (Youngquist, 1986). Timing of the standard
treatment used in this study was based on herd visits conducted every other
week by the attending veterinarian.
Due to the inherently difficult nature of a field study, all factors that
created a bias in the data could not be accounted for in this study design.
Time constraints meant that it was not possible to wait for every fourth cow
with a follicular or luteal cyst in order to create two separate control groups
based on cyst type. The cows were allocated to the CCC group on the basis of
every fourth cow diagnosed as being cystic regardless of cyst type. The
composition of the CCC group ended up consisting primarily of cows with
follicular cysts, with only one luteal cyst cow being randomly allocated to this
group. The high number of cows with follicular cysts in the CCC group made
comparisons between the CCC group and other groups meaningless.
There were 81% follicular cysts (as diagnosed with ultrasonography)
and 19% luteal cysts in the study population. That is, approximately four out
51
of every five cows diagnosed cystic had a follicular cyst. The proportion of FC
and LC cows in this study is similar to that reported by other authors. Several
studies have reported that the percent of follicular cysts in their study
population was about 70% and percent of luteal cysts was 30% (Booth, 1988;
Sprecher et al., 1988; Al-Dahash and David, 1977c). Early detection of these
cysts and intervention may have precluded them from becoming luteinized at a
later stage of development (Garverick, 1997; Carroll et al., 1990; Zemjanis,
1970).
The term luteal-like tissue (LLT) was used because there was not
always an observed estrus prior to seeing a new luteal-like structure on one or
both ovaries via ultrasonography. Also, an ovulation papilla was not always
observable via ultrasonography or apparent with palpation. Ovulation papilla
are not always observed via ultrasonography and some cystic cows have luteal
tissue deeply embedded in the ovarian stroma (Kesler et al., 1981). Kesler
and colleagues (1981) also observed small hemorrhagic spots on the ovaries
and speculated that these may have been small corpora hemorrhagica or
hemorrhagic follicles. Ultrasonic images of luteal tissues appeared as
homogenous gray structures sometimes mottled with hyperechoic specs visible
randomly throughout the structure. Others have made similar observations
(Hanzen et al., 2000). Cause and effect was not accepted by the mere
detection of new luteal tissue within two weeks of treatment. In some cases,
LLT was observed in the ovaries of some cystic cows, but it remains unknown
52
if these structures are capable of producing P4. Additionally, LLT may have
formed as a result of the cow spontaneously resolving the problem, or natural
turnover of ovarian structures may have been occurring (Kesler et al., 1980;
Cook et al., 1990). Therefore, the term LLT was used in this study.
When the cystic cows were divided into treatment groups based on cyst
type by use of ultrasonography, more cows with luteal cysts that received PGF
on d1 responded with displays of estrus (62%) compared to the FC cows that
received GnRH (18%). This was not unexpected. The GnRH treatment was
not used to induce estrus but was intended to induce ovulation of a
subordinate follicle or to increase steroidogenically active luteal tissue. In a
similar study, Sprecher and colleagues (1990) reported that 75% of the cystic
cows with high milk P4 concentrations responded to PGF treatment with a
decrease in P4 concentraion 4 days post-treatment. Another study (Dobson et
al., 1977) reported that cystic cows with high P4 concentrations were the only
ones that displayed estrus following PGF treatment.
The overall pregnancy rate for cows with either type of cyst within two
weeks of treatment was 33%. Similar results have been reported by Chavatte
and colleagues (1993). They found that cows with luteal cysts had a 30%
pregnancy rate within the first two weeks following PGF treatment and cows
with follicular cysts that were treated with GnRH had a less than 12%
pregnancy rate with an overall herd pregnancy rate of 33%. Other studies
have reported higher pregnancy rates (60-80%) following treatment with hCG
53
and GnRH (Bierschwal et al., 1975; Dinsmore et al., 1989; Elmore et al., 1975;
Nanda et al., 1988). However, these studies used a longer interval than two
weeks post treatment for determining pregnancy rate and several included
multiple inseminations.
In the current study, there were no cows with LC that were initially
treated with PGF that were found to have a follicular cyst by d14 and all of the
LC cows showed estrus and were bred. In this study, cows with LC that
received PGF were 7.2 times more likely to be bred within two weeks of
treatment than cows with FC that received GnRH. Not all of these cows
conceived, but all had the opportunity to conceive, in that they were observed
in estrus. If estrus detection is a problem, Ovsynch may be a reasonable
alternative, but as evidenced by this study, cows can be fertile on the first
estrus following treatment for COD. The Ovsynch protocol has been used as
an alternate treatment for COD and pregnancy rates were reported to be
similar between cystic cows and reproductively normal cows (Bartolome et al.,
2000).
A potential benefit to the producer and veterinarian may be the ability
of the ultrasound to detect LLT at the time of diagnosis with greater accuracy
than palpation, even if functionality of the LLT is not determined. Farin and
colleagues (1990; 1992) reported that the use of ultrasonography was more
accurate for determining luteal cysts compared to palpation. In this study, the
retrospective analysis of cows with follicular cysts demonstrated a greater
54
tendency for cows diagnosed ultrasonographically to have LLT present to
respond to PGF treatment by exhibiting signs of estrus and ovulating compared
to cows that received GnRH. By using the ultrasound to detect the presence
of LLT, it may not be necessary to give GnRH and wait 9-14 days before giving
PGF. The cyst may not be an issue once the cow returns to a more normal
hormonal pattern (Kesler et al., 1980) or if active luteal tissue can be detected
or determined (Zaied et al., 1981).
In this study, 6 cows that had follicular cysts and high P4
concentrations had no observable luteal-like tissue present. It is likely that
luteal tissue is not always detectable using ultrasound (Farin et al., 1992).
Perhaps there was luteinization of the granulosa layer of cells, but not thecal
cells, within the follicular cysts in these cows.
In this study, luteal–like structures appeared to have low steroidogenic
activity in 6 cows with follicular cysts that had low P4 concentrations. Two
cows with luteal cysts had low P4 concentrations. This may have occurred
because the luteal rim observed via ultrasonography within the cyst had
limited steroidogenic activity. Douthwaite and Dobson (2000) reported similar
results in that 5 of 19 cows diagnosed with luteal cysts had low P4
concentrations.
In summary, data presented in this study suggest that when luteal cysts
and other LLT are identified via ultrasonography, using PGF as the treatment
may allow an earlier opportunity for breeding and thus potential pregnancy.
55
Using ultrasonography to determine cyst type and to detect the presence of
luteal tissue may provide the bovine practitioner with a means to correctly
treat more COD cases compared to a standard treatment based on palpation.
56
Percentage of Cows (%)
70
60
62%
50
40%
40
30
25%
20
10
0
Luteal
Follicular
18%
8/13
10/55
2/8
Estrus
4/10
Pregnancy
Figure 3.3. The percentage of cows with luteal cysts that were
inseminated (P=0.003) and that became pregnant (P=0.64) within the
first two weeks following the initial treatment compared to cows with
follicular cysts.
57
Percentage of cows (%)
90
83%
80
70
70%
60
50
LT present
40
LT not
30
20
10
0
present
30%
17%
14/20 6/20
High P4
6/35 29/35
Low P4
Figure 3.4. The number of cows with follicular cysts (n=55) that have high
and low P4 concentrations with luteal tissue (LT) present or not present on
d1 of the study.
58
Percentage of cows (%)
100
90
80
70
60
50
40
30
20
10
0
100%
91%
Accessory LT
present
Accessory LT not
present
9%
1/11 10/11
High P4
0%
2/2
Low P4
Figure 3.5. Number of cows with luteal cysts (n= 13) that have high and low
P4 concentrations with luteal tissue (LT) present or not present on d1 of the
study.
59
Effect of LLT and Treatment on Estrus, Ovulation, and
Development of New LLT in cows with Follicular Cysts
Estrus
(%)
n
Treatment
Comparisons
Group 1
17
6
(n=36; GnRH; No LLT)
Group 2
23
3
(n=13; GnRH; LLT)
Group 3
43
3
(n=7; GnRH; No LLT)
Group 4
77
10
(n=13; PGF; LLT)
Group 2 (n=13)
23*
3
Group 4 (n=13)
77* 10
*P=0.02
Comparison Variables
Ovulation
New LLT
(%)
n (%)
n
Pregnant
(n)
83
5
81
29
2
66
2
85
11
2
66
2
57
4
1
70
7
77
10
1
66
70
2
7
85
77
11
10
2
1
Table 3.1. Retrospective analysis of responses (displayed estrus and was
bred, ovulated, developed new LLT, and diagnosed pregnant) of cows with
follicular cysts divided into groups treated according to the presence of LLT.
60
CHAPTER 4
BIOCHEMICAL PROFILES OF COWS WITH CYSTIC OVARIAN DISEASE IN
CENTRAL AND WESTERN OHIO
INTRODUCTION
Formation of ovarian cysts in dairy cattle is probably a multifaceted
process. It has been proposed that COD occurs following a diminished surge
of luteinizing hormone (LH) at the expected time of ovulation in the cow
(Nadaraja and Hansel, 1976). Exogenous cortisol has been used to inhibit the
LH surge and behavioral estrus (Stoebel and Moberg, 1982). Cortisol has been
associated with stress in cattle. Another stress hormone, adrenocorticotropin
(ACTH), has been used to induce ovarian cysts in dairy cattle (Liptrap and
McNally, 1976; Ribadu et al., 2000; Ribadu et al., 1999). Still, the connection
between stress and COD is unclear.
Inadequate nutrition is linked to stress and can be the cause of
infertility in dairy cows. Mwaanga and Janowski (2000) suggested that there
might be a metabolic signal that is interrupted as a result of poor nutrition.
The theory is that this metabolic signal is necessary for a proper LH surge.
Inadequate nutrition (poor quality protein and/or inadequate dry matter
intake) during the early postpartum period can increase the severity
61
of negative energy balance and has been proven to suppress the LH surge in
normal lactating dairy cows (Butler, 2000). Inadequate protein during the
early postpartum period in dairy cows has been shown to decrease plasma
concentrations of P4 and alter the uterine environment (Butler, 1998). Poor
dry matter intake has been directly related to increased severity of the
negative energy balance nadir (Butler, 2000).
Beam and Butler (1997) reported that the first ovulatory-size follicle
postpartum did one of three things: ovulated, was anovulatory and turned
over, or became cystic. They also reported that time to first ovulation was
directly related to the number of days required to reach the cow’s negative
energy balance nadir (Beam and Butler, 1997). Thus, a number of studies
indicate that nutrition can affect the LH surge. However, there is little
information regarding the specific nutritional state of cows with COD.
Associations have been made between some nutrients and metabolic
byproducts and the occurrence of COD.
Harrison and colleagues (1984)
reported that selenium injections given during the dry period decreased the
incidence of COD. Mohammed and colleagues (1991a) found no difference in
the blood concentration of selenium between cystic and noncystic cows;
however, cows with the highest concentrations had the highest risk of
developing COD. Few causative associations have been determined between
nutrients and the occurrence of COD. Vitamin E fed orally did not alter the
incidence of COD (Harrison et al., 1984). Vitamin A and β-carotene were
62
found to be lower in cystic cows compared to noncystic cows with no causal
relationship established (Inaba et al., 1986). Also, high acetone
concentrations increased the risk of COD in first-calf heifers on Swedish dairies
with a high prevalence of ketosis (Jukola et al., 1996). The objectives of this
study were: 1) to determine if blood chemistry profiles differ between cystic
and non-cystic lactating dairy cattle, and 2) to determine if blood chemistry
profiles change over time in cystic lactating dairy cattle.
MATERIALS AND METHODS
This study was conducted on six dairies in central and western Ohio
from September 1997 to September 1998. These dairies ranged in size from
180 to 300 milking cows. Throughout the study year, 72 cows were diagnosed
cystic by the respective herd veterinarian. To be considered cystic, a cow had
to have an ovarian structure with a diameter of at least 25mm. Four cows
were included that had follicular cysts ranging from 23-24mm and had
exhibited symptoms of COD within the last month.
Blood samples were collected and ultrasonography was repeated every
Monday, Wednesday and Friday for five weeks (samples 1 through 16) on the
cystic cows only. Day 1 of the study was not the same day of the week for all
cows. Ultrasonography was part of another study (Chapter 3 and 6). Blood
was collected from control cows (n=258) once a week for five weeks. Control
63
cows were from the same feed group as cystic cows, and thus were at a
similar stage of lactation. The same cows were not used as controls each
week due to varying management practices. Therefore, different noncystic
herdmates on the respective farms were used each week as controls.
All blood samples were collected in heparinized vacutainer tubes. These
samples were kept on ice until centrifuged and plasma was aspirated and
frozen for analysis. Frozen plasma was sent to Purina Veterinary Services
Laboratory in Gray Summit, MO for analysis. The first vitamin E sample for
each cow was analyzed at Purina Veterinary Services Laboratory. The last
vitamin E sample for each cow was analyzed at Michigan State Veterinary
Diagnostic Laboratory, East Lansing, MI. Vitamin E concentration of samples 2
– 15 were cost prohibitive to have analyzed, so only the first and last sample
were analyzed.
Cystic cows were treated according to a protocol from another study
(Chapter 3) on d1 after blood collection. Blood samples were collected from
cystic cows three times a week for five weeks (Monday, Wednesday, and
Friday). All cows were inseminated at every standing estrus. Pregnancy
status was confirmed at routine herd checks.
Statistical Analysis
Plasma chemistry variables in cystic cows (albumin, cholesterol, blood
urea nitrogen [BUN], magnesium, phosphorus, creatinine, calcium, gamma
64
glutamyltransferase [GGT], aspartate aminotransferase [AST], total protein,
vitamin E, and β-HBA) were compared to the control group and to d1 to
determine change over time. PROC MIXED procedure for repeated measures
analysis of variance was used to analyze these data. (Magnesium was not
measured on d1.) The initial F-test was performed using PROC MIXED. Farm
and sample number were included as fixed effects in all analyses. The sample
date was included as a fixed effect when significant in the model so as to
remove any known variability in sampling from the model. The optimum
covariate structure was determined for each variable. Least squares means
were used to detect individual day differences between control and cystic cows
and changes over time in cystic cows. Independent variables included in the
multivariate ANOVA model for each chemical variable in the biochemistry panel
are presented in table format. Vitamin E concentrations for sample 1 and
sample 16 compared to control were analyzed by multivariate linear
regression.
Due to the varying starting times throughout the year and because
individual control cows were not consistently sampled, control cow data were
pooled for comparison to cystic cow data over time (samples 1-16). All
statistics were run using SAS version 8.0.
65
RESULTS
Initially, the values for each parameter for the cystic cows over all times
were compared to those for the reference controls. We then examined the
changes over time within the cystic group. There were no differences in
plasma concentrations of magnesium, calcium, AST, and GGT between groups
or over time within the cystic cows (Tables 4.1, 4.2, 4.3, and 4.4,
respectively). These four variables were not included in any further analyses.
Plasma concentrations of albumin, total protein, creatinine, phosphorus,
cholesterol, blood urea nitrogen, and β-HBA were different between cystic and
noncystic cows (P=0.01).
Since there was a significant F-test, plasma concentrations of albumin
on specific days in cystic cows were compared to pooled control cow samples.
This analysis revealed that cystic cows had statistically higher plasma albumin
concentrations than the reference control (P<0.05) for five (d4, 9, 11, 13, and
15) of the sixteen samples (Figure 4.1). Concentration of albumin was lower
(P<0.01) on d1 than on other sample days over the five week study period.
Multivariate ANOVA model for predicting mean plasma albumin concentrations
for cystic and noncystic cows is presented in Table 4.5.
Total plasma protein concentration in cystic cows was higher than the
reference control concentration for eight (d2-4, 6, 7, 9, 13, and 15) of the
sixteen sample days (P<0.05, Figure 4.2). Total protein was lower on d1 than
66
on any other day over the five week study period (P<0.01). Multivariate
ANOVA model for predicting mean plasma total protein concentrations for
cystic and noncystic cows is presented in Table 4.6.
Plasma phosphorus was higher in cystic cows compared to the
reference control only on day 1 (Figure 4.3). Phosphorus concentration on d1
was higher than any other day sampled in the study (P<0.01). Multivariate
ANOVA model for predicting mean plasma phosphorus concentrations for cystic
and noncystic cows is presented in Table 4.7.
Cholesterol was higher in cystic than noncystic cows for the entire study
period (P<0.01, Figure 4.4). Cholesterol concentration did not differ over time
in the cystic cows. Multivariate ANOVA model for predicting mean plasma
cholesterol concentrations for cystic and noncystic cows is presented in Table
4.8.
β-hydroxybutyrate was lower in cystic than control cows on five (d2, 4,
9, 13, and 16) of the sixteen sample days (P<0.05, Figure 4.5). βhydroxybutyrate was lower in samples 2 to 16 compared to sample 1
(P<0.01). Multivariate ANOVA model for predicting plasma β-hydroxybutyrate
in cystic and noncystic cows is presented in Table 4.9.
Creatinine and blood urea nitrogen were different between cystic and
noncystic cows, however on individual pairwise comparisons, no differences
were detected (Table 4.10 and Table 4.11). Blood urea nitrogen in cystic cows
changed over time. On d1 BUN (sample 1; 15.25 mg/dL+0.41) was lower
67
than it was in samples collected on d12, 14, and 16 (17.07+0.41 to
17.38+0.41 mg/dL, P<0.05; Figure 4.6).
On d1 of the study, vitamin E concentrations were greater in cystic
cows (n=72; 5.28 µg/ml) compared to noncystic cows (n=32; 3.71 µg/ml;
P<0.001).
Also, on the last day of the study, vitamin E concentrations were
greater in cystic cows (n=72; 5.06 µg/ml) compared to noncystic cows (n=41;
3.65 µg/ml; P=0.0063).
DISCUSSION
Blood chemistry profiles for cows with COD were presented in the
results for the time of diagnosis and for five weeks following treatment. To
the authors’ knowledge, biochemical profiles of cystic cows have not been
reported in the literature. It was of interest to determine if there were
differences between cystic and non-cystic cows in any of the parameters that
are typically examined. In comparing the nutrient and metabolic byproduct
concentrations of cystic cows with noncystic herdmates, there were several
statistically significant differences. However, most of these differences were
not out of the normal physiological range (albumin, total protein, phosphorus,
BUN, and β-HBA).
Plasma albumin concentrations in cystic cows increased from the first to
the second sample and were then sustained over time. Although plasma
68
albumin in the first sample was similar to that in control cows, subsequent
samples from cystic cows had higher concentrations of albumin than controls.
Since plasma albumin concentrations were higher in cystic cows, there may be
an association between the presence of cysts and elevated plasma albumin.
Since plasma albumin increased from day one to subsequent days, the
increase in albumin appears to develop after the cyst occurs. As such, the
higher albumin levels may be a consequence rather than a cause of the cyst.
Few authors have examined plasma albumin patterns over time in cows in
relation to reproduction. Jordan and Swanson (1979) investigated plasma
albumin in relation to stage of lactation and protein level in the diet. However,
it was difficult to relate these data to the data in the current study with respect
to cystic cows.
It was of interest to note that the concentrations of albumin and total
protein in the first sample were lower than those in the following fifteen
samples. Since albumin is one component of total protein, the increase in
albumin is most likely reflected in the total protein. Additionally, the
concentrations of phosphorus and β-hydroxybutyrate were higher in the first
sample than they were in any of the following samples.
It is a curious observation that the concentrations of these four plasma
chemistry components were altered after sample one. To help rule out
processing error, all 16 samples of each individual cow were sent to the
69
laboratory to be included in one analysis. Therefore, all of day one samples
were processed in conjunction with the rest of the samples of an individual
cow.
One common denominator in trying to explain the pattern of
concentrations is that all cystic cows were treated within 30 minutes of the
first blood sample being collected. But, not all of the cystic cows received the
same treatment. Cows with follicular cysts received GnRH and cows with
luteal cysts received PGF. Therefore, it is difficult to attribute the change in
concentration of biochemical parameters to one specific treatment. Although
the pattern of plasma concentrations for albumin, total protein, phosphorus,
and β-hydroxybutyrate is an intriguing observation, the explanation for these
patterns is unknown.
Plasma cholesterol was high in the cystic cows compared to noncystic
cows and these concentrations were above the normal physiological range.
Over the study period, there was no significant decrease in these values. High
cholesterol is a characteristic of polycystic ovarian disease (PCOD) in women
(Wild et al., 1985). In contrast, high plasma cholesterol has been associated
with a shorter time from calving to conception, increased expression of estrus
at the time of first ovulation post-partum, and the increased likelihood of
conception and pregnancy in dairy cows fed a diet low in rumen-degradableprotein compared to dairy cows fed a high rumen-degradable-protein diet
(Westwood et al., 2002). Dietary fat supplementation has been associated
70
with increased numbers of large follicles in dairy cattle (Lucy et al., 1991).
Additionally, low blood cholesterol and blood glucose concentrations postpartum in dairy cows have been associated with an increase in time from
calving to conception (Kappel et al., 1984). Due to the association between
plasma cholesterol and increased fertility in post-partum dairy cows, it is
unlikely that a causative relationship between high cholesterol and COD exists.
However, the current study included cystic cows over a wide range of days in
milk (not just early post-partum cows) and plasma cholesterol was high in this
group as a whole when compared to their peers. Therefore, it is difficult to
completely rule out that any association between high cholesterol and COD
might exist.
Cystic cows on this study had higher concentrations of vitamin E than
their noncystic herdmates. Vitamin E supplementation has been used to aid
the immune system in moderating infection and it is controversial in its
usefulness of preventing retained placenta. Batra and colleagues (1992)
reported higher vitamin E concentrations in herds with low fertility. In
contrast, Jukola and colleagues (1996) did not find any difference in fertility of
cows based on the vitamin E concentration at the time of artificial
insemination. Harrison and colleagues (1984) found that supplementation of
vitamin E did not affect the incidence of COD. As such, even though vitamin E
concentrations in plasma were higher in cystic cows, a relationship between
vitamin E and COD has not been established.
71
In summary, biochemical profiles of cows at the time of cyst diagnosis
and for five weeks following treatment have not been previously reported.
The concentrations of plasma and total protein increased while phosphorus
and β-hydroxybutyrate decreased from the first sample to the following
samples and this level was sustained throughout the remainder of the study.
Although the concentrations of these metabolites were not outside of the
physiological ranges, the patterns of concentrations observed may reflect
clinically significant relationships with COD. Cholesterol and vitamin E were
higher in cystic cows than their noncystic herdmates, however the nature of
this apparent association with COD is unclear at this time. This experiment
represents an initial assessment of possible associations between
concentrations of clinically important metabolites and cystic ovarian disease.
Further research may provide causative relationships between these
metabolites and COD.
72
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
0.036
0.180
0.203
-0.014
0.323
.
0.077
0.053
0.079
0.115
0.059
.
0.6370
0.0007
0.0104
0.9034
0.0001
.
2.45
2.59
2.61
2.40
2.73
2.41
0.0925
-0.355
-0.017
-0.031
-0.027
-0.039
-0.018
-0.019
0.011
-0.011
-0.024
-0.012
-0.058
-0.010
-0.002
0.010
.
-0.0004
0.043
0.245
0.040
0.040
0.040
0.040
0.040
0.040
0.040
0.040
0.040
0.040
0.039
0.039
0.039
0.039
.
0.0002
0.0297
0.1473
0.6715
0.4370
0.4934
0.3278
0.6488
0.6373
0.7901
0.7877
0.5439
0.7661
0.1414
0.8011
0.9608
0.7957
.
0.0176
2.65
2.21
2.55
2.53
2.54
2.52
2.54
2.54
2.57
2.55
2.54
2.55
2.50
2.55
2.56
2.57
2.56
Table 4.1. Multivariate ANOVA model for predicting mean magnesium
concentrations for cystic and noncystic-control cows.
β represents the regression coefficient for the various levels of the
independent variables used to predict the outcome variable (variable from the
biochemistry panel). SE (β) is the standard error of the regression coefficient.
The P-value listed in the table represents the comparison of each independent
variable (contrast variable) to the reference variable. Least squares mean is
the expected value of the outcome variable from the model. Herd 6 and
Sample 16 are the reference variables. Date is a continuous variable.
73
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
β
SE (β)
P value
LS mean
0.201
0.146
0.477
-0.275
0.738
.
0.151
0.107
0.158
0.229
0.121
.
0.1833
0.1733
0.0026
0.2310
0.0001
.
10.00
9.90
10.29
9.54
10.48
9.74
-0.132
-0.283
-0.110
-0.064
-0.008
-0.143
-0.030
-0.015
-0.078
-0.077
-0.035
-0.047
-0.274
-0.061
-0.125
-0.065
.
0.097
0.100
0.100
0.100
0.100
0.099
0.100
0.100
0.100
0.100
0.100
0.100
0.099
0.100
0.100
0.100
.
0.1739
0.0047
0.2701
0.5211
0.9333
0.1486
0.7664
0.8780
0.4348
0.4437
0.7286
0.6385
0.0058
0.5394
0.2095
0.5121
.
9.93
9.78
9.95
10.00
10.06
9.92
10.03
10.05
9.99
9.99
10.03
10.02
9.79
10.00
9.94
10.00
10.06
Table 4.2. Multivariate ANOVA model for predicting mean calcium
concentrations for cystic and noncystic-control cows.
74
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
β
SE (β)
P value
LS mean
-15.347
-13.147
-14.345
-14.691
-12.519
.
1.823
1.380
1.544
2.334
1.555
.
0.0001
0.0001
0.0001
0.0001
0.0001
.
23.06
24.69
24.42
24.11
24.98
37.64
1.604
0.293
0.765
0.556
0.556
-0.033
0.267
0.264
0.375
0.176
0.010
0.194
0.557
0.500
0.375
0.528
.
0.918
0.648
0.648
0.645
0.645
0.643
0.648
0.645
0.645
0.648
0.649
0.649
0.643
0.645
0.645
0.645
.
0.0808
0.6516
0.2381
0.3896
0.3896
0.9588
0.6807
0.6827
0.5614
0.7863
0.9874
0.7654
0.3872
0.4387
0.5614
0.4137
.
27.29
25.97
26.45
26.24
26.24
25.65
25.95
25.94
26.06
25.86
25.69
25.87
26.24
26.18
26.06
26.21
25.68
Table 4.3. Multivariate ANOVA model for predicting mean GGT concentrations
for cystic and noncystic-control cows.
75
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
-18.06
-21.65
-21.22
-24.36
-29.97
.
4.26
3.16
4.71
5.56
3.48
.
0.0001
0.0001
0.0001
0.0001
0.0001
.
75.28
70.61
92.51
68.28
63.68
100.86
-4.102
-4.076
-6.266
-8.059
-8.186
-8.611
-6.298
-6.217
-6.270
-4.885
-4.354
-2.602
-2.964
-2.203
-2.161
-1.469
.
0.0402
2.47
2.97
3.02
3.04
3.05
3.03
3.00
2.95
2.89
2.80
2.70
2.58
2.39
2.16
1.84
1.36
.
0.009
0.0964
0.1697
0.0381
0.0082
0.0073
0.0046
0.0362
0.0354
0.0302
0.0816
0.1076
0.3127
0.2151
0.3078
0.2407
0.2808
.
0.0001
75.02
75.04
72.85
71.06
70.93
70.51
72.82
72.90
72.85
74.24
74.77
76.52
76.16
76.92
76.96
77.65
79.12
Table 4.4. Multivariate ANOVA model for predicting mean AST concentrations
for cystic and noncystic-control cows.
76
Albumin (g/dL)
3.8
3.7
3.6
3.5
3.4
3.3
3.2
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Sample
Figure 4.1. Plasma concentrations of albumin (diamonds, mean +SE) over
time (i.e., sample number) in cystic cows compared to the pooled noncystic
cow samples (thick line; P<0.01). Standard error of the mean for the
concentration of albumin in the control cows was 0.03 g/dL. Sample one was
taken prior to initial treatment on d1 of the study.
77
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
0.459
0.431
0.154
0.477
0.395
.
0.081
0.058
0.077
0.110
0.065
.
0.0001
0.0001
0.0458
0.0001
0.0001
.
3.77
3.74
3.47
3.79
3.71
3.31
-0.123
-0.169
0.009
-0.006
0.022
-0.039
-0.011
-0.001
-0.027
0.036
-0.005
0.021
-0.032
0.047
0.017
0.047
.
0.0004
0.042
0.033
0.033
0.033
0.033
0.033
0.033
0.033
0.033
0.033
0.033
0.033
0.032
0.033
0.033
0.033
.
0.0002
0.0032
0.0001
0.7769
0.8523
0.4981
0.2320
0.7258
0.9652
0.4162
0.2661
0.8774
0.5120
0.3294
0.1463
0.5989
0.1504
.
0.0298
3.52
3.48
3.65
3.64
3.67
3.60
3.63
3.64
3.62
3.68
3.64
3.66
3.61
3.69
3.66
3.69
3.64
Table 4.5. Multivariate ANOVA model for predicting mean albumin
concentrations for cystic and noncystic-control cows.
78
8.8
Total Protein (g/dL)
8.7
8.6
8.5
8.4
8.3
8.2
8.1
8
7.9
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Sample
Figure 4.2. Plasma concentrations of total protein (diamonds, mean +SE) in
cystic cows over time (i.e., sample number) compared to the pooled
noncystic cow samples (thick line; P<0.01). Standard error of the mean for
the concentration of total protein for control cows was 0.06 g/dL. Sample
one was taken prior to initial treatment on d1 of the study.
79
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
-0.561
-0.481
-0.281
0.134
-0.078
.
0.154
0.111
0.145
0.206
0.124
.
0.0003
0.0001
0.0519
0.5163
0.5277
.
8.12
8.20
8.40
8.81
8.60
8.68
-0.265
-0.210
0.021
0.029
0.107
-0.016
0.009
0.013
-0.030
0.037
-0.058
-0.026
-0.127
0.013
-0.045
0.035
.
0.0009
0.078
0.061
0.061
0.060
0.060
0.060
0.060
0.060
0.060
0.060
0.060
0.060
0.059
0.059
0.059
0.059
.
0.0003
0.0007
0.0005
0.7305
0.6349
0.0745
0.7871
0.8758
0.8229
0.6212
0.5336
0.3355
0.6613
0.0325
0.8233
0.4530
0.5516
.
0.0086
8.23
8.29
8.52
8.52
8.60
8.48
8.51
8.51
8.47
8.53
8.44
8.47
8.37
8.51
8.45
8.53
8.50
Table 4.6. Multivariate ANOVA model for predicting mean total protein
concentrations for cystic and noncystic-control cows.
80
Phosphorus (mg/dL)
7.5
7
6.5
6
5.5
5
4.5
4
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Sample
Figure 4.3. Plasma concentrations of phosphorus (diamonds, mean +SE)
over time (i.e., sample number) in cystic cows compared to the pooled
noncystic cow samples (thick line; P=0.01). Standard error of the mean for
the concentration of phosphorus in control cows was 0.08 mg/dL. Sample
one was taken prior to initial treatment on d1 of the study.
81
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
β
SE (β)
P value
LS mean
-0.291
0.158
-0.216
0.129
-0.271
.
0.217
0.155
0.223
0.325
0.175
.
0.1787
0.3093
0.3344
0.6907
0.1226
.
5.75
6.16
5.85
6.19
5.72
5.99
0.095
0.926
-0.062
-0.282
-0.104
-0.167
-0.108
-0.069
-0.065
0.117
-0.060
-0.029
-0.004
-0.004
0.028
-0.199
.
0.135
0.135
0.135
0.134
0.134
0.134
0.135
0.134
0.134
0.135
0.135
0.135
0.134
0.134
0.134
0.134
.
0.4853
0.0001
0.6455
0.0358
0.4377
0.2109
0.4244
0.6049
0.7250
0.6285
0.3851
0.6551
0.8305
0.9752
0.8360
0.1391
.
6.02
6.85
5.86
5.64
5.82
5.76
5.82
5.85
5.88
5.86
6.04
5.86
5.90
5.92
5.95
5.73
5.92
Table 4.7. Multivariate ANOVA model for predicting mean phosphorus
concentrations for cystic and noncystic-control cows.
82
Cholesterol (mg/dL)
270
260
250
240
230
220
210
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Sample
Figure 4.4. Plasma concentrations of cholesterol (diamonds, mean +SE) in
cystic cows over time (i.e., sample number) compared to the pooled
noncystic cow samples (thick line; P=0.01). Standard error of the mean for
the concentration of cholesterol in control cows was 4.31 mg/dL. Sample
one was taken prior to initial treatment on d1 of the study.
83
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
-12.35
8.52
-42.25
-22.64
-25.48
.
9.87
7.88
9.95
12.10
8.61
.
0.2110
0.2798
0.0001
0.0617
0.0032
.
248.52
262.90
263.48
235.72
246.01
275.96
-32.98
-11.86
-7.92
-7.23
-3.34
-6.00
-3.42
-3.41
-3.08
1.00
-1.44
2.28
0.04
3.52
0.77
3.60
.
0.078
5.35
6.12
6.18
6.19
6.16
6.10
6.01
5.87
5.70
5.48
5.24
4.94
4.53
4.03
3.38
2.46
.
0.0201
0.0001
0.0530
0.1999
0.2427
0.5879
0.3256
0.5695
0.5607
0.5892
0.8558
0.7835
0.6452
0.9938
0.3824
0.8196
0.1433
.
0.0001
213.00
239.11
243.05
243.74
247.63
244.97
247.55
247.56
247.89
251.97
249.53
253.25
251.01
254.49
251.74
254.57
250.97
Table 4.8. Multivariate ANOVA model for predicting mean cholesterol
concentrations for cystic and noncystic-control cows.
84
Beta-hydroxybutyrate
(mg/dL)
6
5.5
5
4.5
4
3.5
3
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
Sample
Figure 4.5. Plasma concentrations of β-hydroxybuterate (diamonds, mean
+SE) in cystic cows over time (i.e., sample number) compared to the pooled
noncystic cow samples (thick line; P=0.01). Standard error of the mean for
the concentration of β-hydroxybuterate in control cows was 0.19 mg/dL.
Sample one was taken prior to initial treatment on d1 of the study.
85
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
0.989
1.141
-3.256
-1.026
0.659
.
0.498
0.343
0.525
0.733
0.384
.
0.0473
0.0009
0.0001
0.1620
0.0867
.
5.46
5.61
1.21
3.44
5.13
4.47
1.080
1.453
-0.065
0.279
0.010
0.237
0.251
0.118
0.102
0.059
0.372
0.416
0.387
-0.039
0.441
0.255
.
0.002
0.289
0.281
0.281
0.279
0.279
0.278
0.279
0.278
0.279
0.279
0.279
0.279
0.276
0.277
0.277
0.277
.
0.001
0.0002
0.0001
0.8184
0.3183
0.9719
0.3945
0.3692
0.6702
0.7148
0.8321
0.1831
0.1362
0.1622
0.8875
0.1116
0.3573
.
0.0470
4.98
5.36
3.84
4.18
3.92
4.14
4.16
4.02
4.01
3.96
4.28
4.32
4.29
3.87
4.35
4.16
3.91
Table 4.9. Multivariate ANOVA model for predicting mean β-hydroxybuterate
concentrations for cystic and noncystic-control cows.
86
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Date
β
SE (β)
P value
LS mean
0.151
0.068
0.289
0.204
0.086
.
0.024
0.017
0.024
0.034
0.019
.
0.0001
0.0001
0.0001
0.0001
0.0001
.
1.09
1.01
1.23
1.15
1.03
0.94
-0.004
0.019
0.018
0.009
0.010
-0.018
-0.012
-0.012
-0.016
-0.004
-0.016
-0.015
-0.013
-0.004
-0.007
-0.002
.
0.0002
0.013
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.012
0.011
0.012
0.012
0.012
.
0.0001
0.7638
0.0970
0.1199
0.4520
0.4080
0.1087
0.3129
0.3157
0.1755
0.7379
0.1609
0.2004
0.2457
0.7368
0.5286
0.8446
.
0.0001
1.07
1.10
1.10
1.09
1.09
1.06
1.07
1.07
1.06
1.08
1.06
1.06
1.07
1.08
1.07
1.08
1.08
Table 4.10. Multivariate ANOVA model for predicting mean creatinine
concentrations for cystic and noncystic-control cows.
87
Blood Urea Nitrogen
(mg/dL)
19
18
17
16
15
14
13
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16
Sample
Figure 4.6. Change from D1 concentration of blood urea nitrogen in cystic
cows over the five week study period (illustrated by sample number;
P=0.94). Sample one was taken prior to initial treatment on d1 of the study.
88
Variable
Herd
1
2
3
4
5
6
Sample
Control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
β
SE (β)
P value
LS mean
1.042
0.266
-3.000
-3.763
-0.051
.
0.574
0.396
0.661
0.866
0.452
.
0.0696
0.5020
0.0001
0.0001
0.9104
.
17.31
17.58
13.21
12.44
17.32
17.31
-1.18
-1.93
-0.87
-0.84
-1.08
-1.24
-1.20
-1.14
-0.94
-0.92
-0.55
-0.16
-0.11
-0.22
0.20
-0.51
.
0.416
0.510
0.519
0.521
0.523
0.523
0.524
0.522
0.520
0.517
0.513
0.506
0.490
0.467
0.423
0.336
.
0.0045
0.0002
0.0878
0.1080
0.0390
0.0182
0.0220
0.0286
0.0697
0.0760
0.2880
0.7585
0.8209
0.6364
0.6454
0.1315
.
16.00
15.25
16.30
16.34
16.10
15.95
15.98
16.04
16.24
16.26
16.64
17.03
17.07
16.96
17.38
16.67
17.18
Table 4.11. Multivariate ANOVA model for predicting mean blood urea
nitrogen concentrations for cystic and noncystic-control cows.
89
CHAPTER 5
DIFFERENCES IN LENGTH OF DRY PERIOD AND TIME TO PREGNANCY (DAYS
OPEN) BETWEEN CYSTIC AND NONCYSTIC COWS ON CENTRAL AND
WESTERN OHIO DAIRIES
INTRODUCTION
Cystic ovarian disease (COD) tends to occur most frequently in early
lactation (Erb et al., 1981; Bartlett et al., 1986) and prior to the first ovulation
postpartum (Whitmore et al., 1974a; Kesler and Garverick, 1982). Some cows
with COD will spontaneously recover and others will require intervention
(Kesler and Garverick, 1982). It is generally believed that cows with COD will
have longer calving intervals (Garverick, 1997; Bartlett et al., 1986).
However, it is controversial whether COD actually causes an increase in the
calving interval (Morrow et al., 1966; Mohammed et al., 1991b; Mellado and
Reyes, 1994; Zulu and Penny, 1998; Kinsel and Etherington, 1998; Fourichon
et al., 2000).
Unfortunately, the etiology of COD is still unknown. The majority of
cysts are detected early in lactation (before d50), and there is another period
when cysts are prevalent later in lactation (d190-220; Garverick, 1997).
Detection of cysts prior to the first ovulation coincides with metabolic distress
90
and negative energy balance (Opsomer and de Kruif, 1999). Garverick (1997)
reported that detection of COD in dairy cows later in lactation may be a
reflection of management practices.
Management practices on a dairy involve a broad range of topics. If
management is a causative factor of COD, nutrition may be a key component.
Results of management prior to the cystic episode may be reflected in cows
that develop COD in early lactation. Dry period management and feeding
practices of dairy cows are critical for decreasing retained placentas and
decreasing negative energy balance in high producing cows, and may also be
important in prevention of COD.
Opsomer and colleagues (2000) found that a longer dry period directly
increased the likelihood of anestrous or ovarian inactivity. They suggested
that this increased likelihood of anestrous might be due to cows gaining more
weight prior to calving if they have a long dry period. Excessive body
condition gained during the dry period has been associated with greater
negative energy balance postpartum (Butler, 2000). Severe negative energy
balance has been directly correlated to increased time to first ovulation (Butler,
2000). Severe negative energy balance has also been associated with
impaired luteinizing hormone release and anovulation in the early postpartum
period (JOLLY et al., 1995). The length of the dry period may be a factor that
influences the occurrence of COD with the main symptom being anestrous.
91
The first objective of this study was to determine if length of the dry
period prior to the onset of estrous cycling or cyst formation was different
between cystic cows and non-cystic cows. The second objective was to
determine if the number of days open differed between cystic and noncystic
herdmates on farms in central and western Ohio.
MATERIALS AND METHODS
Dairy Herd Improvement (DHI) records were obtained for 5,554
lactations for cows on six dairy herds located in central and western Ohio.
Sixty-eight cystic cows were used in the original cystic cow data set (see
Chapter 3), so the DHI records included these cows. The lactation that
corresponded to the cystic episode was used in the analysis for days open.
The length of the dry period (days) was used from the previous lactation. First
lactation heifers were not included in the analysis for length of dry period.
Cows were initially determined to be cystic by the respective herd
veterinarian. To be considered cystic, a cow had to have an ovarian structure
with a diameter of at least 25mm and/or abnormal estrous cycles (see Chapter
3 for more details). Cows were confirmed cystic within 24-72hrs by transrectal
ultrasonography (Aloka 500v, Wallingford, CT) using a 5 MHz linear array
transducer. The ultrasonography study period was from September 1997 to
September 1998 for cystic cows. These cows were monitored by
92
ultrasonography every Monday, Wednesday, and Friday for five weeks. Cows
entered the study on arbitrary days of the week depending on their day of
diagnosis.
Statistical Analysis
Days Dry
The first analysis was done to determine if length of dry period differed
between cystic and noncystic herdmates on farms in central and western Ohio.
Cows were included in the data set if there was a record of their previous dry
date and following fresh date from January 1997 through May 1998. This time
period was chosen so that the control cows included in the data set would
have had the possibility of being included in the cystic study reported in
Chapter 3, otherwise there was no way of knowing whether they would have
been cystic or not. Two farms were not included in the analysis, because none
of the cystic cows met the criteria (i.e., missing previous dry date, fresh date,
or were first-calf heifers). Forty-seven cystic cows had complete information
and were included in the data set. Seven hundred and ninety noncystic
herdmates were used for comparison.
Lactation was used as a categorical variable and cows were divided into
three groups based on lactation status at the time they were dried off: lact2
(second lactation), lact3 (third lactation), lact4 (fourth lactation or greater).
Herd and lactation number were used as independent variables in the model to
93
account for confounding effects. Type III Anova (Proc GLM in SAS) was used
to compare the mean number of days dry between cystic and noncystic cows.
Days Open
The second analysis was done to compare the likelihood that cystic
cows and non-cystic cows will become pregnant. In addition, it was of interest
to assess time to pregnancy in both of these groups using a hazard analysis.
Cows were included in the days open data set if they calved between
September 1, 1996 and May 30, 1998. This time range was used since the
most of the complete records spanned this time, and the time range
overlapped with our study period. Complete records consisted of lactation
number, last fresh date, times bred, and date pregnant, sold or died. Fourteen
cystic cows were dropped from the analysis due to incomplete data sets.
Since there were no complete data sets for cystic cows on one farm, this farm
was excluded from the analysis. Fifty-eight cystic cows and 1927 noncystic
herdmates were included in the analysis.
Information for the lactation in
which the cystic episode occurred was used for comparison of cows with COD
to noncystic herdmates. Information for only one lactation was used for each
cow.
Herd, lactation number, season, and times bred were used as
categorical variables. Lactation number was divided into first lactation, second
lactation, third lactation and fourth or higher. Season was divided into
94
quartiles: summer (June, July, August), spring (March, April, May), fall
(September, October, November) and winter (December, January, February).
Times bred was divided into four categories: xbred1 (bred once), xbred2 (bred
twice), xbred3 (bred three times) and xbred4 (bred four or more times).
Proportional hazards regression was used to compare days open between
cystic and noncystic cows. Pregnancy was used as the censoring variable.
RESULTS
Cystic cows had a longer dry period at the end of the previous lactation
(67+3 days) than noncystic cows (62+0.5 days, P=0.065).
Seventy six percent (75.9%) of the cystic cows (n=58) in the time-topregnancy data set became pregnant and 71.5% of the noncystic herdmates
(n=1927) became pregnant. The probability that cystic cows would become
pregnant was 84% of the probability that non-cystic cows would become
pregnant (P=0.31). In other words, normal noncystic cows were 1.19 times
more likely to become pregnant than cystic cows. However, these results
were not statistically significant (P=0.31).
The proportion of cows that became pregnant naturally increased as
time progressed postpartum. The rate at which this increase occurred seemed
to be faster for noncystic cows than it was for cystic cows. To examine these
relationships, we used a graphic representation of the percentage of non95
pregnant cows over time in a survival analysis curve (Figure 5.1). The
percentage of non-pregnant cows began at 100% and was reduced over time
as more cows became pregnant. Using this approach, we visually compared
the rate at which groups of cystic and non-cystic cows achieved pregnancy.
Although these curves deviated from one another at times, the patterns did
not differ significantly (P>0.05).
DISCUSSION
Cows with longer dry periods tend to have more metabolic problems
(i.e., ketosis, severe negative energy balance, etc.) after calving. In this
study, it was hypothesized that dry period may have some influence on cows
becoming cystic.
The longer the dry period is, the more time cows have to
consume feedstuffs and gain weight. Excess body condition at the time of
calving has been associated with increased negative energy balance due to
reduced feed consumption in early lactation and the sudden increase in
demand for body energy stores due to lactation (Garnsworthy and Topps,
1982). Body condition score at the time of calving has been associated with
increased metabolic problems during the postpartum period (Butler, 2000).
The greater the negative energy balance nadir, the longer time required to
first ovulation (Beam and Butler, 1999). There is a growing body of evidence
that advocates increased management of dairy cows in the periparturient
96
period in order to increase fertility in the postpartum period and decrease
potential hormonal and reproductive problems.
It is possible that length of dry period and the occurrence of COD are
associated. The majority of cysts are detected early postpartum, a time close
to the preceding dry period. Dry period and dry cow management affect the
reproductive status of a cow. The current data provide some idea of when the
stressors or factors are taking place that may influence the occurrence of COD.
Based on these data, dry period may have an influence on a cow becoming
cystic. Future research may be able to focus more closely on the influence of
specific factors or management during the dry period on the occurrence of
COD in early postpartum cows.
The majority of studies support that COD increases the calving interval
and cows with COD require more services per pregnancy. In survival analysis,
incomplete or censored data can be used, however, this study used a data set
that contained only complete records. No difference was detected in the time
to pregnancy or days open between cystic and noncystic cows.
Examination of Figure 5.1 revealed a very similar rate of decrease in the
proportion of cows that were not pregnant from day 0 to 130. The difference
between the percentages of cystic and non-cystic cows that were not pregnant
was not more than 10 percentage points at all times before day 130 except
one (i.e., On day 90 the difference was ~15 percentage points). After day 140
these differences increased to 20 percentage points at many of the days
97
examined postpartum. This dichotomy was maintained until about day 330,
when the differences between groups decreased to less than 10 percentage
points.
In the early postpartum period, it appeared that cystic cows achieved
pregnancy at a similar rate to non-cystic cows. Perhaps the cows that are
cystic in the early postpartum period are less likely to have an increased
calving interval, whereas the cows that become or remain cystic later
postpartum may have a greater risk of delays in achieving pregnancy. Most
research has shown that cows with COD have increased days open. This may
be because most experiments include cows at all stages postpartum, including
the later stages where the differences that we see are greatest.
In summary, the dry period after the preceding lactation was longer in cows
that had ovarian cysts than those that did not. As such, the length of the dry
period and management of cows during the dry period may have an influence
on a cow becoming cystic. To the author’s knowledge, this is the first study to
specifically look for an association between the dry period and COD in dairy
cattle. In addition, cystic cows appeared to achieve pregnancy at a similar
rate to non-cystic cows in the early postpartum period. It is possible that
cystic cows in the early postpartum period are less likely to have increased
days open, whereas the late postpartum cystic cows may have a greater risk of
delays in achieving pregnancy.
98
nep Os w
oCf o )
%
( t necr eP
99
400
350
250
200
150
100
50
0
Figure 5.1. The percentage of non-pregnant cystic and non-cystic cows over time. The percentage
of non-pregnant cows that do not have a cyst seems to drop more rapidly than the percentage of
non-pregnant cows with a cyst, but these patterns were not significantly different (P>0.05).
0
10
20
30
40
50
60
70
80
450
90
Cystic Cows
Noncystic Cows
500
100
300
m
ut
ra
pt
so
P
sy
aD
CHAPTER 6
CASE REPORTS OF TWO COWS THAT BECAME PREGNANT WITH
PERSISTENT OVARIAN CYSTS PRESENT AND FOUR COWS OBSERVED TO
HAVE HYPERECHOGENICITY WITHIN FOLICULAR CYSTS
INTRODUCTION
Cystic ovarian disease (COD) occurs in over one million dairy cows per
year based on the 1997 Agricultural Census (Garverick, 1997). This number
translates into a great financial loss for the dairy industry each lactation due to
the increased days open or longer calving interval (Borsberry and Dobson,
1989). Additionally, COD is noted as a condition of infertility until resolution
(Garverick, 1997).
DESCRIPTIVE STATISTICS
Sixty-eight cystic cows from six dairies in Ohio were used in this study.
Data were collected on the sixty-eight cows as part of a prior study (Chapter
3). The following is a brief summary of the previous study. Cows were initially
diagnosed cystic by the herd veterinarian. This diagnosis was confirmed using
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transrectal ultrasonography (Aloka 500v, Wallingford, CT) within three days of
the initial diagnosis. Day of ultrasound diagnosis is considered d1 of the study.
Cows were treated according to cyst type as determined via U/S. On
d1, cows with luteal cysts (LC; n=12) were treated with prostaglandin F2α
(25mg; PGF) and cows with follicular cysts (FC; n=38) received gonadotropinreleasing hormone (100µg; GnRH) regardless of other ovarian structures.
Every fourth cow with a palpable cyst was allocated to a cystic cow control
(CCC; n=18) group prior to U/S determination of cyst type. The control group
was formed to mimic therapeutic procedures based on palpation and received
a standard COD treatment of GnRH on d1. Each CCC animal, regardless of the
type of cyst (based on U/S) received PGF on d14 unless priorly inseminated. If
treatment group cows were not inseminated prior to d14, they were treated
again. The d14 treatment (PGF or GnRH) was based on the presence of
obvious luteal tissue on either ovary, irrespective of cyst resolution or
presence. Cows were to be bred at all observed standing estrus.
The previous study monitored ovarian activity via transrectal
ultrasonography every Monday, Wednesday, and Friday for 5 weeks. Follicles
less than 5mm in average diameter were not recorded. Blood samples for
progesterone (P4) analysis were collected prior to each ultrasound session.
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RESULTS
Cows with Follicular Cysts
Fifty-five cows had follicular cysts (81%) and 13 cows had luteal cysts
(19%). Of the 55 cows with follicular cysts, 4 cows became pregnant (40%)
after being bred within 14 days from the initial treatment (10 cows were bred).
Thirty-one cows with follicular cysts were bred after d15 (2nd treatment) and
10 of these cows became pregnant to that breeding (32%). One cow had
previously been bred during the first 2 weeks of the study and became
pregnant to the second breeding. At 6 months from the start of the study, 39
of 55 (71%) cows with follicular cysts were diagnosed pregnant. One cow
died and three were sold before pregnancy status could be determined.
Cows with Luteal Cysts
Of the 13 cows with luteal cysts, two cows became pregnant (25%)
within 15 days of initial treatment (8 cows were bred). Three of seven (43%)
cows with luteal cysts became pregnant during days 16-36 of the study. Two
cows bred during this time period had been bred during the first two weeks of
the study. At 6 months from the start of the study, 9 of 13 (69%) cows
initially diagnosed with luteal cysts were pregnant. One cow died prior to
confirming pregnancy status.
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CASE REPORT 1: TWO COWS WITH FOLLICULAR CYSTS PRESENT BECAME
PREGNANT
Cow 159
Two cows from the same dairy became pregnant while maintaining a
large follicular cyst for at least 36 days. Neither cow had a luteal structure or
any observable luteal tissue present at diagnosis. Plasma P4 concentration on
d1 in cow 159 was less than 1 ng/ml and in cow 168 was 1.06 ng/ml. Cow
159 was first diagnosed as cystic at 87 days in milk (DIM) with one large
follicular cyst on each ovary. The average diameter of these two cysts
measured 36.0mm and 41.5mm. Cow 159 also had three large follicles
present and the average diameter of these measured 14.5mm, 17.5mm, and
19.5mm.
On d4 following the GnRH treatment, the largest cyst on the ovaries of
cow 159, decreased in size from 41.5mm to 17mm – a decrease of 59%
(Figure 6.1). This cyst also appeared ultrasonographically to have hyperechoic
lines transversing the cyst (discussed below in Case Report 2). These
hyperechoic lines may be associated with P4 production in this cow (Figure
6.2). The plasma progesterone concentration was less than 1 ng/ml on d4 and
increased to almost 4 ng/ml by d15. The cyst then appeared to enlarge
beginning on d6 and reached greater than original size on d11. This cyst
continued to appear as a follicular rather than luteal cyst throughout the 36
day experimental period.
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On d15, cow 159 was given 100µg GnRH. According to the
experimental criteria, cows without obvious luteal tissue were to be injected
with GnRH at this time. Progesterone concentration decreased from d15 to
d22. No obvious luteal tissue formed within seven days following GnRH
treatment on d15. On d22 there were two follicular structures that were
cystic. The largest follicular structures were 31mm and 33mm. These cystic
structures remained greater than or equal to 25mm through day 36 (end of
study). Cow 159 was observed in estrus on d22 and was bred that evening.
The cow became pregnant from this breeding. No luteal structures were
observed via ultrasonography until d32, which was 10 days after the breeding
date. Circulating progesterone increased from < 0.5 ng/ml on d22 to ~2
ng/ml on days 32-36 (Figure 6.2). Based on these progesterone data, luteal
tissue was present. Perhaps it was difficult to observe the luteal tissue using
ultrasonography because there were a number of follicular structures present
on both ovaries that may have obstructed or interfered with our interpretation
of the images.
Cow 168
Cow 168 was diagnosed at 64 DIM with two large follicular cysts on one
ovary (Figure 6.3). The average diameter of the two cysts was 32.5mm and
41.5mm. This same ovary also had one follicle whose diameter averaged
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12.5mm. Cow 168 was treated with 100µg GnRH (d1). Both cysts increased
in size on d4 and d6 post treatment. On d8, there were two CL observed via
ultrasonography on the contralateral ovary and two follicles whose average
diameters measured 9mm each. Plasma progesterone concentration increased
from d4 through d8 to >2 ng/ml (Figure 6.4). On d11 after GnRH treatment,
cow 168 was observed in estrus and bred. At this time, she still had two
follicular cysts with an average diameter of 46.5mm and 30mm. A single CL
was detected on d13. Circulating progesterone increased from d15 after GnRH
treatment (=day 4 after estrus) through d29 to a concentration greater than
12 ng/ml. On d15 it was noted with ultrasonography that there was a
hyperechoic mass in the center of the CL. This mass was observed through
d20.
Cow 168 was diagnosed pregnant to the d11 breeding. The two
follicular cysts were observed up to d27. On d27, one of the cysts decreased
to 24.5mm and continued to decrease through the end of the study (d36).
The remaining cyst measured 38.5mm on d36. Progesterone concentration
decreased from d29 to d36 and remained ~7 ng/ml.
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CASE REPORT 2: OBSERVATION OF HYPERECHOGENICITY WITHIN
FOLICULAR CYSTS IN FOUR COWS
Four cows diagnosed with follicular cysts and no other observable
luteal tissue, were observed to develop hyperechoic lines within the center of
the follicular cyst present on ultrasound examination. These hyperechogenic
lines were first noticed within three days following GnRH treatment in three
of the four cows. These lines were observed 10 days post-GnRH treatment
in the fourth cow. In all four cases, these hyperechogenic lines were initially
seen as plasma P4 concentrations were increasing. As plasma P4
concentrations increased, there was no observable luteal tissue present on
either of the ovaries in each cow.
In cow 159 (Figure 6.2), these hyperechogenic lines remained for 21
days (d4-d25), although they were not obvious on d18 and d22. On d13, the
hyperechoic lines appeared to conglomerate in the center of the cyst with
possibly small areas of luteinized tissue within the cyst.
Cow 21 (Figure 6.5) was observed to have these hyperechogenic lines
present on d18 through d25. Plasma P4 concentration continued to increase
through d32 even though the hyperechoic lines were no longer observed
from d27 –36.
In cow 77 (Figure 6.6), plasma P4 concentration had already begun to
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increase before the hyperechoic lines were observed on d11. Plasma P4
continued to increase from d11-15. The hyperechoic lines were present from
d11 through d25.
Cow 727 (Figure 6.7) developed hyperechoic lines on d4 and they
were observed through d20. Plasma P4 increased from d4 through d13.
This cow was bred on d13 based on signs of estrus, at the peak of plasma P4
concentration. However, based on plasma P4 concentration and ultrasound
recordings her true date of estrus was probably d20. This was also
evidenced by a red Kamar present, but the producer did not observe any
signs of estrus during this time.
DISCUSSION
The largest cyst on the ovaries of cow 159 decreased in size after the
cow was treated with GnRH. This was initially thought to be the start of
luteinization of the cyst. Over the next seven days, the cyst increased in size
and the hyperechoic area became centrally located within the cyst. Several
studies have reported cyst turnover over various periods of time (Kesler et al.,
1980; Cook et al., 1990; Hamilton et al., 1995). However, this cyst did not
turnover but regained its size and remained for the entire 36 days of the
study. Similarly, Cook and colleagues (1990) reported that a small number of
cysts were not replaced by new cysts, but actually persisted for 40 days.
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Cow 159 was treated with GnRH on d1 and responded with an increase
in circulating P4. In spite of the presence of two follicular cysts, the pattern of
plasma progesterone resembled that of a normal estrous cycle. This pattern
was consistent with luteal development from days 4-15 and luteal regression
from days 15 through 20. Again, despite the follicular cysts that were present,
on d22 estrus was observed, the cow was bred, and pregnancy was achieved.
A similar finding has been reported by a research group in Tanzania (Assey et
al., 1997). In an abattoir study, they found a pregnant reproductive tract from
a Zebu cow that had multiple follicular cysts on both ovaries.
As previously reported, the cystic structure has not been observed to
ovulate (Cook et al., 1990; Hamilton et al., 1995). In the case of cow 159 as
well, the cyst did not ovulate, but, based on plasma progesterone, the luteal
tissue was lysed. Although the structure of the cyst remained after plasma
progesterone fell, the hyperechogenicity disappeared within 4 days after
estrus. As previously stated by Jou and colleagues (1999), the cyst may not
interfere with normal ovarian function. In this example, although follicular
cysts are present, they were not the functionally dominant structures on the
ovaries.
On d1 in cow 168, plasma progesterone concentrations were 1.06
ng/ml. Although we did not detect luteal tissue on this day, this level of
circulating progesterone suggests that functional luteal tissue was present.
Not all luteal tissue is readily apparent, in that, the cyst itself may have had a
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layer of luteinized theca cells undetectable via ultrasonography (Cook et al.,
1990). Plasma progesterone was greater than 2 ng/ml from d4 through d8,
however, during this time frame luteal tissue (2 CL) was only detected on d8.
Additionally, no hyperechoic areas were observed in the follicles of this cow.
Estrus was displayed on d11 and the cow was bred and became
pregnant. Based on the pattern of decreasing plasma P4, the two CL
appeared to be regressing from day 8 to 11. As in cow 159, cow 168
displayed a fertile estrus even though two follicular cysts were present. In cow
168, neither of the cysts ovulated. In fact, ovulation occurred on the ovary
contralateral to the ovary with the two follicular cysts.
Hyperechogenic lines were reported in four cows in this paper. These
hyperechoic lines/areas within the cyst may have been areas of luteinization.
This is consistent with the observation of increasing concentrations of
circulating progesterone concentration while the hyperechoic lines were
present. In all four cows, there was no observed luteal tissue or luteal-like
tissue present on either of their ovaries while the hyperechogenic lines were
detected. Therefore, the increased plasma P4 concentration does not appear
to be a result of production from a CL. However, it is likely that there was
some luteal tissue present, but was undetected within the cyst or the
hyperechoic lines may indicate its presence within the cyst.
In summary, cows with follicular cysts can become pregnant. The cyst
may in fact be a static structure on the ovaries, so endocrine profiles may be a
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better estimate of fertility versus one-time palpation or ultrasonographic
observation. Endocrine profiles in cows diagnosed and treated for COD ideally
should be determined over several days versus a one-time observation as well.
Cows with large persistent follicular cysts that have been treated without cyst
resolution can possibly become pregnant. Furthermore, hyperechoic lines or
areas observed within follicles or cysts may represent functional luteal tissue
within that structure.
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45
40
35
30
25
20
15
10
5
0
P
1
4
6
8
11
13
15
18
20
22
25
27
29
32
34
36
Days from Treatment
Figure 6.1. Follicular and cyst patterns observed via ultrasonography for
Cow 159. P=breeding that the cow became pregnant. Open circles
represent follicles. Closed circles represent luteal tissue or CL.
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Concentration of Progesterone (ng/ml)
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
P
1
4
6
8
11
13
15
18
20
22
25
27
29
32
34
36
Days from Treatment
Figure 6.2. Concentration of Progesterone (ng/ml) over time in cow 159.
P=pregnant to breeding on this day. Down arrow indicates day that
hyperechoic lines inside the follicular cyst were observed.
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Size of Ovarian Structures
(mm)
55
50
45
40
35
30
25
20
15
10
5
0
P
1
4
6
8
11 13 15 18 20 22 25 27 29 32 34 36
Days from Initial Treatment
Figure 6.3. Follicular and cyst patterns observed via ultrasonography
for Cow 168. P=breeding that the cow became pregnant. Open
circles represent follicles. Closed circles represent luteal tissue or CL.
113
15
14
)l 13
m
/g12
n(
en11
or 10
et 9
se
go 8
rP 7
fo
no 6
it 5
ar 4
tn
ec 3
no 2
C
P
1
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
Days from Initial Treatment (d1)
Figure 6.4. Concentration of Progesterone (ng/ml) over time in cow 168.
P=pregnant to this breeding.
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Concentration of Progesterone (ng/ml)
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
1
4
6
8
11
13
15
18
20
22
25
Days from Initial Treatment (d1)
27
29
32
34
36
Figure 6.5. Concentration of plasma progesterone (ng/ml) over the study
period (36 days) for cow 21. Data for d22 is missing. Arrows indicate day of
observed herperchoic lines within the cyst.
115
e
n
o
r
e
t
s
e
g
o
r
P
f
o
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o
i
t
a
r
t
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o
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15
14
13
12
11
10
l) 9
m
/g 87
n( 6
5
4
3
2
1
0
1
4
6
8
11
13
15
18
20
22
25
27
Days form Initial Treatment (d1)
29
32
34
36
Figure 6.6. Concentration of plasma progesterone (ng/ml) over the study
period (36 days) for cow 77. Arrows indicate day of observed herperchoic
lines within the cyst.
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)l 15
14
m
/g13
n( 12
en11
or 10
et
se 9
go 8
rP 7
fo 6
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it
ar 4
tn 3
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C1
0
1
4
6
8
11
13
15
18
20
22
25
Days from Initial Treatment (d1)
27
29
32
34
36
Figure 6.7. Concentration of plasma progesterone (ng/ml) over the study
period (36 days) for cow 727. Arrows indicate day of observed herperchoic
lines within the cyst.
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CHAPTER 7
INDUCED LUTEAL-LIKE STRUCTURES FOLLOWING GnRH TREATMENT OF
DAIRY CATTLE WITH CYSTIC OVARIAN DISEASE: ASSESSMENT OF
HISTOLOGICAL CHARACTERISTICS AND STEROIDOGENIC CAPABILITY
INTRODUCTION
Dairy cows with cystic ovarian disease (COD) have one of two kinds of
cysts, follicular or luteal. For the past three decades, GnRH has been used to
treat cystic ovarian disease (COD; Lopez-Gatius et al., 2002). GnRH is a useful
treatment to eliminate follicular cysts, but it has little effect on luteal cysts
(Kesler et al., 1978b). Prostaglandin F2α (PGF2α) has also been used to treat
COD, but it induces regression of luteal cysts and has little effect on follicular
cysts (Sprecher et al., 1990; Kesler et al., 1978a). GnRH and PGF2α have been
used simultaneously to treat COD (Dinsmore et al., 1990). The strategy was
to provide treatments for both types of cysts so that treatment would be
successful no matter what type of cyst was actually present. Unfortunately,
the outcome of this experiment ended with poor pregnancy rates. Another
formula for treating COD has been GnRH followed 9-14 days later by PGF2α.
GnRH stimulates the pituitary gland to release luteinizing hormone (LH), and
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LH induces luteinization of the cyst. PGF2α can then cause lysis of the
luteinized tissue. Following regression of the luteinized tissue, normal estrous
cycles can resume.
This treatment regimen is thought to reduce time to estrus in COD cows
regardless of cyst type, but the regimen necessitates heat detection
(Garverick, 1997). Recently, these same two hormones have been used in
combination to synchronize ovulation allowing for a timed-insemination in
normally cycling cows (Ovsynch; Pursley et al., 1995). The Ovsynch protocol
consists of: GnRH injection; PGF2α injection 7 days later; GnRH injection 2 days
later; timed insemination 12-48h later. Regardless of cyst type, when Ovsynch
was compared to GnRH followed 7 days later with PGF2α, similar pregnancy
rates were observed (Bartolome et al., 2000). Both treatments resulted in
lower pregnancy rates compared to those in normally cyclic cows treated with
the Ovsynch protocol.
As mentioned above, exogenous GnRH in cows with COD usually results
in luteinization of the cyst, but it also may cause luteinization or ovulation of
subordinate follicles (Kesler et al., 1981). Accessory corpora lutea may be
induced if there is already a corpus luteum (CL) present and depending on
when in the wave of follicular development GnRH is given. This results in
increased plasma progesterone (P4) concentrations, possibly due to the
presence of another functional CL (Schmitt et al., 1996). In addition, the LH
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released in response to GnRH may stimulate an increase in P4 production by
the luteal tissue that is already present.
Thus, the most popular and effective strategies to treat follicular cysts
in cows involve GnRH. To gain an improved understanding of the
endocrinology of the cystic cow that is treated with GnRH, it is important to
learn the nature of the ovarian structures that develop as a consequence of
this treatment. Currently, it is not known if the luteal-like structures induced
by GnRH are steroidogenically active and histologically similar to normal CL.
Thus, the hypothesis was tested that luteal-like structures induced in cystic
cows are steroidogenically and histologically similar to CL induced in cycling
cows and to spontaneously occurring CL in cows with normal estrous cycles.
MATERIALS AND METHODS
General
This experiment was performed using 10 Holstein and 2 Jersey cows.
Ten cows were from the Ohio State University Waterman Dairy, one cow was
from the OSU College Veterinary Medicine’s research herd, and one cow was
from a dairy in northwestern Ohio. Cystic cows (n=5) were anestrus with
cysts > 25mm in diameter. Cystic cows were treated with 100µg GnRH
(Cytsorelin, i.m.) on the day of diagnosis (d0). Based on ultrasonography,
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none of the cystic cows had a CL at the time of GnRH treatment. Luteal-like
structures resulting from the GnRH injection were collected on d6.
Two different control groups were used in this study:
1) Cows with normal estrous cycles (Control-N). These cows
were nonlactating cows that exhibited estrus at regular intervals (i.e.,
Estrous cycles were 18-24 days in length). Estrus was considered d0,
and the CL were collected on d6 of the cycle.
2) GnRH-induced control cows (Control-G). This group included
nonlactating cows with normal estrous cycles that received one injection
of 100µg GnRH (i.m.) between d6-10 of the estrous cycle in the
presence of a functional CL. The CL, both primary and induced, were
collected on d6 relative to GnRH injection.
Two cows, one in each control group, were used twice with a minimum of four
normal estrous cycles prior to tissue collection. Blood samples for P4 analysis
were collected in EDTA-coated evacuated tubes (Vaccutainer, BectonDickinson, Franklin Lakes, NJ) on cows via coccygeal vessels prior to GnRH
treatment and at the time of CL or ovary collection. Cyst diagnosis was made
and luteal-like structure formation was determined by transrectal
ultrasonography (Aloka 500v, Wallingford, CT) using a 5 MHz linear array
transducer.
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In vitro CL short-term incubation
Luteal-like structures and CL were removed via transvaginal luteectomy
(Pate and Condon, 1982) except for one cow in the control-G group and one
cow in the cystic group. The CL was collected from one cow in the control-G
group by a standing paralumbar endoscopic ovariectomy; primary CL and
luteal tissue were removed from ovaries once in the laboratory. The CL was
collected from one cow in the cystic group cow by harvesting the ovaries at
the time of slaughter; luteal tissue was isolated in the laboratory.
Primary CL from control-G cows were used in the histomorphologic
analysis, but they were not used for short-term incubation studies. Following
surgery, luteal tissue was kept on ice in sterile Ham’s F-10 medium while
transported to the laboratory. Luteal tissue was weighed and an approximately
0.2g center cross-section was removed and preserved in 10% formalin for
histopathologic assessment. Remaining luteal tissue was weighed and
dissociated as described by Pate and Condon (1982). Eight plastic 12 x 75 mm
test tubes were seeded with 100,000 cells/250 µl. Cells were incubated in the
presence and absence of bovine luteinizing hormone (15 ng bLH) for 0hr or
2hrs in duplicate in 5% CO2. Bovine LH (AFP-11743B) was kindly provided by
Dr. A.F. Parlow, Scientific Director of the National Hormone and Pituitary
Program. At each time point, samples were frozen until radioimmunoassay for
P4.
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Histology
Approximately 0.2g of luteal-like tissue was randomly collected as
center cross-sections from each CL and CL-like structures. These samples
were preserved in 10% formalin. Tissue was processed by the Ohio State
University Pathology Laboratory. All tissue slides were stained with
hematoxylin and eosin. Bioquant image analysis system as described by Lei
and colleagues (1991) was used to analyze the tissue slides. Area and
perimeter of luteal cells were the only parameters measured by the traced
images in the Bioquant system. Criteria used for luteal cell verification was
described by Wiltbank (1994). Large and small cell criteria were used for
identification. Measurements for normal CL (control-N), GnRH-induced CL
(control-G), primary CL from the control-G cows, and luteal-like structures
collected from the cystic cows were saved in a Microsoft Excel spread sheet.
Radioimmunoassay
Plasma P4 was quantified by radioimmunoassay (RIA) as previously
described and validated by Clapper and colleagues (1990). All plasma samples
were analyzed in one assay except for one cow (cystic) that was analyzed at a
later time using an I-125 kit assay. The coefficient of variation within the RIA
was 16.22%. The coefficient of variation within the I-125 kit assay was
4.32%.
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Concentrations of P4 in culture medium were quantified as described by
Johnson and colleagues (1988). All samples were analyzed in one assay
except the samples for one cystic cow. These samples were analyzed using an
I-125 kit assay. The coefficient of variation within the RIA was 4.44%. The
coefficient of variation within the I-125 kit assay was 5.0%.
Statistical analyses
Nonparametric one-way analyses using Wilcoxon Rank Sum Test (SAS,
v.8) was used to make the following comparisons. Pre-treatment samples for
cystic cows were used to determine if there was functional luteal tissue
present. One cystic cow produced two luteal-like structures which were
collected, dissociated, and incubated separately, but final values were
averaged because cow was used as the experimental unit. Cows with plasma
progesterone concentrations > 1.0 ng/ml were considered to have
steroidogenically active luteal tissue present. Plasma progesterone
concentrations at the time of tissue collection were compared between cystic
and control-G cows, and cystic and control-N cows. Comparisons were not
made between control-N and control-G cows.
Progesterone concentrations in culture medium were compared
between luteal-like tissue collected from cystic cows and CL collected from the
two control groups (control-N and control-G). Cells were incubated for 0h and
2h with and without LH. Basal production of P4 was calculated by subtracting
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the P4 concentration at 0h from the P4 concentration at 2h. Stimulation of P4
production by LH was examined in two ways: first by determining
progesterone production in the presence of LH in 2h (i.e., relative to the
concentration of progesterone at Oh) and then by determining the stimulation
by LH over basal production.
Histology results included cell area and cell perimeter measurements as
calculated from luteal cell tracings performed in the Bioquant system.
Nonparametric one-way analyses using Wilcoxon Rank Sum Test (SAS, v.8)
was used to make the following comparisons. All possible comparisons were
not made due to the nonparametric analyses used. Area and perimeter
averaged measurements were compared between luteal-like tissue collected
from cystic cows and the two control groups (d6 CL and CL from GnRH-treated
cows).
RESULTS
Concentrations of P4 in Plasma
Blood samples were collected for P4 analysis from all cows that received
GnRH and at the time of tissue collection for all groups. These blood samples
were used to determine if functional luteal tissue was present. Pre-treatment,
two cystic cows had concentrations of P4 < 1.0 ng/ml, one cow had a P4
concentration of 2.75 ng/ml, and one cystic cow did not have a pre-treatment
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blood sample drawn. Based on ultrasound, all control-G cows (n=5) had a
corpus luteum present at the time of GnRH treatment. Pre-treatment plasma
P4 concentrations were > 1.0 ng/ml in all of the control-G cows that were
sampled (n=4).
At the time of tissue collection, plasma P4 concentrations in control-N
cows ranged from 1.28-2.97 ng/ml and in control-G cows ranged from 3.3-5.5
ng/ml. Plasma P4 concentrations in cystic cows ranged from 1.55-4.72 ng/ml
at the time of tissue collection. Cystic cows had higher P4 concentrations than
Control-N cows (Table 7.1, P=0.014). Cystic cows had plasma P4
concentrations similar to Control-G cows (P=0.221).
Concentrations of P4 in Culture Medium
The two CL produced by one cystic cow were dissociated separately and
P4 concentrations following short-term incubation were averaged together.
Mean P4 values (ng/ml) for the three treatment groups are reported in table
7.2. The content or initial P4 concentration in luteal cells is reflected by the
progesterone concentration at 0h. Initial P4 concentration in luteal cells
collected from cystic cows did not significantly differ from that in luteal cells
from either control-N or control-G groups (P=0.33, P=0.33, respectively).
Basal progesterone production during the 2h incubation did not differ between
cystic cows and control-N or control-G groups (P=0.62, P=0.62, respectively).
Also, LH-stimulated P4 production relative to content was not different
126
between cystic cows and the control-N or control-G groups (P=0.18, P=0.22,
respectively). However, P4 production was stimulated by LH over basal
production to a greater degree in control-N luteal cells than it was in luteal
cells from the cystic group (P=0.086). There was no difference between LHstimulated P4 production over basal production in the control-G and cystic cow
groups (P=0.33).
Luteal Cell Measurements
The two CL produced by one cystic cow in response to GnRH treatment
were measured as separate structures and all cell measurements were
averaged for analysis. Mean area and perimeter values are reported in table
7.3. There was no difference in cell area (P=0.327) or cell perimeter
(P=0.327) between luteal-like structures produced in cystic cows and primary
CL in mid estrous cycle cows treated with GnRH. Cell areas and perimeters in
luteal-like structures produced in cystic cows were smaller than in the controlG CL (P=0.028, P=0.028, respectively) and in the control-N CL (P=0.014,
P=0.014, respectively). Examples of the luteal tissue views used to generate
the area and perimeter data for control-N, control-G, and cyst cow groups are
presented in figures 7.1, 7.2, and 7.3, respectively.
127
DISCUSSION
Based on the pre-treatment plasma P4 concentrations and
ultrasonography, cows in the cystic group had either a follicular cyst or a luteal
cyst. Thus, luteal-like tissue that formed in cystic cows in response to GnRH
developed either in the presence or absence of P4. As planned, all control-G
cows had a CL present at the time of GnRH treatment. Since control-N cows
ovulated spontaneously, they did not have a CL present at the time of the LH
surge. Therefore, GnRH was not overriding an innate negative feedback
system of P4 to the hypothalamus to inhibit GnRH release that stimulates LH
release in the normal estrous cycle of a cow. Luteal-like tissue that developed
in cystic cows in response to GnRH can be compared to CL that developed in
the control-N group in the presence of low plasma P4 and in the control-G
group in the presence of high plasma P4.
One of the cystic cows had a plasma P4 concentration > 1 ng at the
time of GnRH treatment. This mostly likely meant that this cow had a luteal
cyst or some functional luteal tissue present. Two cystic cows had no
functional luteal tissue present prior to GnRH treatment and were assumed to
have follicular cysts. Since luteal-like structures were induced in all of the
cystic cows, GnRH was able to stimulate LH release and induce luteal-like
tissue in cows with a follicular or luteal cyst. Additionally, based on the precollection plasma P4 concentrations these induced luteal-like structures were
128
competent to produce serum P4 at an average concentration comparable to
the luteal structures in control-G cows.
With respect to luteal P4 in vitro, initial P4 concentrations and basal P4
production were similar among all three treatment groups. Similar results
have been reported using luteal slices from induced luteal structures from hCG
and a GnRH agonist in that no treatment differences were detected in basal P4
(Schmitt et al., 1996). In this study, LH increased P4 production from luteal
cells in all three treatment groups relative to the initial P4 concentration. Basal
P4 production was stimulated by LH to a greater degree in luteal cells from
control-N cows when compared to luteal cells from cystic cows. Luteal cells
from the control-G and cystic groups produced similar increases in P4
production to LH-stimulation over basal P4 production. The follicle that was
induced to luteinized or ovulate was in an unknown phase of development or
dominance and had been present for an unknown length of time in the cystic
cows. Whereas the follicle in the control-G group was in a more defined phase
(growing/dominant) and was from cows that were known to be coming in to
estrus at regular intervals.
One study reported that P4 secretion did not increase in cultures of
large luteal cells that were stimulated by LH although small bovine luteal cells
were responsive to LH stimulation at low doses in vitro (Hansel et al., 1991).
In this study, cell area and perimeter were used as measurements for all luteal
cells and large and small cells were not differentiated. The size difference
129
between luteal cells in the cystic group compared to the control-N and controlG groups might explain the difference in P4 response to LH stimulation in
short-term incubated luteal cells, except that luteal cells from induced CL in
the cystic cow group were on average smaller than the control-N group.
However, this does not necessarily mean that the control group had more
large luteal cells since they were not differentiated in this study. There may be
more mature or larger “small luteal cells” in the control-N group and they are
more prepared to respond to LH stimulation.
In conclusion, luteal-like structures that were induced by GnRH
treatment in cystic cows are structurally similar to GnRH-induced accessory CL
in noncystic cows with normal estrous cycles. Luteal tissue induced by GnRH
in cystic cows had an in vitro basal concentration of P4 and was capable of
producing P4 during short–term incubation and in response to LH stimulation.
The induced luteal structures in cystic cows were able to produce as much
serum P4, on average, in vivo as the luteal structures that were present and
induced in control-G cows. Therefore, the induced luteal structures in cystic
cows that had a follicular cyst or functional luteal tissue already present, were
steroidogenically active in vivo and in vitro.
130
Control-N
Cystic
Control-G
n
5
4
5
Progesterone (ng/ml)
2.23+0.29*
4.38+0.45*
3.26+0.53
*P=0.014, control-N vs cystic group.
Table 7.1. Means and standard errors for the plasma samples collected at the
time of CL collection for all three treatment groups.
Control-N
Cystic
Control-G
P4 content
(0h)
10.6+3.0
21.4+10.1
31.1+9.5
Basal P4
production
(2h-0h)
60.9+20.4
61.8+23.4
72.9+20.4
LH-stimulated P4
production
relative to content
(2h w/LH-0h)
295.2+70.4
183.6+60.4
242.1+30.4
LH-stimulated P4
production over
basal production
(2h w/LH-2h)
234.3+52.1*
121.9+48.9*
169.2+20.6
*indicates means that are different P=0.086.
Table 7.2. Means and standard errors for P4 concentrations in medium
(ng/ml) for the three treatment groups. Data are presented for initial P4
concentration (0h), basal production of P4 (increase in P4 concentration after
2h incubation), LH-stimulated P4 production (increase in P4 concentration
during a 2h incubation with LH), and LH-stimulated P4 production over basal
production (comparison of P4 production in 2h in the presence and absence of
LH).
131
Control-N
Control-G
Primary CL
Cystic
Area + s.d.
240.1 +16.0*
227.7 +27,0**
247.8+63.7
187.3+15.2
Perimeter + s.d.
62.1+2.4*
60.8+3.1**
61.6+7.9
55.2+2.2
*P=0.014, control-N vs cystic group.
**P=0.028, control-G vs cystic group.
Table 7.3. Means and standard deviations for area and perimeter
of luteal cells from each of the three treatments and the primary
CL for the GnRH-induced group.
132
Figure 7.1. Example of normal D6 CL used to for obtaining luteal cell area and
perimeter data for control-N group (20x).
133
Figure 7.2. Example of GnRH-induced D6 CL used to obtain area and perimeter
data for control-G group (20x).
134
Figure 7.3. Example of GnRH-induced CL in a cystic cow used to obtain area
and perimeter data (20x).
135
CHAPTER 8
KEY POINTS
Chapter
3.1
When luteal cysts and other LLT are identified via
ultrasonography, using PGF as the treatment may allow an earlier
opportunity for breeding and thus potential pregnancy.
4.1
Biochemical profiles of cows at the time of cyst diagnosis and for
five weeks following treatment have not been previously reported
in the literature.
4.2
This experiment represents an initial assessment of possible
associations between concentrations of clinically important
metabolites and cystic ovarian disease.
5.1
The dry period after the preceding lactation was longer in cows
that had ovarian cysts than those that did not. As such, the
length of the dry period and management of cows during the dry
period may have an influence on a cow becoming cystic. To the
author’s knowledge, this is the first study to specifically look for
an association between the dry period and COD in dairy cattle.
5.2
Cystic cows appeared to achieve pregnancy at a similar rate to
non-cystic cows in the early postpartum period. It is possible
that cystic cows in the early postpartum period are less likely to
have increased days open, whereas the late postpartum cystic
cows may have a greater risk of delays in achieving pregnancy.
6.1
Cows with follicular cysts can become pregnant. The cyst may in
fact be a static structure on the ovaries, so endocrine profiles
may be a better estimate of fertility versus one-time palpation or
ultrasonographic observation.
136
6.2
Hyperechoic lines or areas observed within follicles or cysts may
represent functional luteal tissue within that structure.
7.1
Luteal-like structures that were induced by GnRH treatment in
cystic cows are structurally similar to GnRH-induced accessory CL
in noncystic cows with normal estrous cycles.
7.2
Induced luteal structures in cystic cows that had a follicular cyst
or functional luteal tissue already present, were steroidogenically
active in vivo and in vitro.
137
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