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This is to certify that the
thesis entitled

REPEATABILITY OF NUMBERS OF ANTRAL
FOLLICLES IN FOLLICULAR WAVES IN DAIRY
CATTLE AND ASSOCIATION WITH SECRETION OF
FSH, ESTRADIOL AND INHIBlN-A

presented by
David 8. Burns

has been accepted towards fulfillment
of the requirements for the

MS. degree in Animal Science

 

 

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6/01 c;/ClRC/DateDue.p65-p.15

 

REPEATABILITY OF NUMBERS OF ANTRAL FOLLICLES IN
FOLLICULAR WAVES IN DAIRY CATTLE AND ASSOCIATION
WITH SECRETION OF FSH, ESTRADIOL AND INHIBIN-A
By

David S. Burns

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2003

ABSTRACT
REPEATABILITY OF NUMBERS OF ANTRAL FOLLICLES IN
FOLLICULAR WAVES IN DAIRY CATTLE AND ASSOCIATION
WITH SECRETION 0F FSH, ESTRADIOL AND INHIBIN-A
By

David S. Burns

The objective of this study is to examine the variation in antral follicle numbers
that develop during follicular waves in cattle, and to determine if the variation in numbers
of follicles per wave is associated with serum FSH, estradiol and inhibin-A
concentrations. Cattle were subjected to twice daily ultrasound analysis to count number
of antral follicles during different follicular waves in estrous cycles. The results
demonstrated that maximal numbers of antral follicles in different follicular waves were
highly variable amongst cattle, but remarkably highly repeatable (0.95) within
individuals. In another experiment, cows that consistently had very high or low numbers
of antral follicles per wave were identified and blood samples were taken. Serum was
subjected to FSH, estradiol and inhibin-A immunoassays. The results demonstrated that
serum FSH concentrations were 50% lower in cows with very high numbers of antral
follicles per wave vs cows with low numbers. However, estradiol and inhibin-A were
similar between the two groups of cows. These results led us to conclude that: a) a
chronic compensatory mechanism must exist in ovaries to maintain growth of a constant
number of antral follicles in follicular waves, and b) the variability in numbers of follicles
per wave is probably not explained by the interaction of FSH, with estradiol and inhibin-

A concentrations-

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. v
LIST OF FIGURES ................................................................................. vi
LIST OF ABBREVIATIONS ..................................................................... vii
INTRODUCTION ..................................................................................... 1

Abstract ...................................................................................... 14
Introduction ................................................................................... 15
Materials and Methods ...................................................................... 17
Animals .............................................................................. 17
Ultrasound scanning procedure to count
antral follicles ........................................................................ 17
Study 1: Validation of ultrasound scanning
to count antral follicles ................................................ 18
Part A. Repeatability of counting number of
antral follicles by one technician .............................. 18
Part B. Repeatability of counting number of antral
follicles between two technicians ........................... 18
Study 2: Variability and repeatability of numbers of antral
follicles per wave ...................................................... 19

Part A. Repeatability of numbers of antral follicles
in different follicular waves during the
same or consecutive estrous cycles ........................... 19
Part B. Repeatability of number of antral follicles
in follicular waves of individual cattle
using a different ultrasound operator. 19
Part C. Repeatability of number of antral follicles
in follicular waves that occurred in estrous
cycles spaced3months apart... 19
Study 3: Relationship of variation in maximal numbers Of
antral follicles per wave with the dynamics of
development of dominant and subordinate
follicles .................................................................. 20
Study 4: Association of patterns of FSH, estradiol and
inhibin-A secretion with nmnbers of antral follicles

that grow during follicular waves ................................... 21
Part A. Identification of cows with low or very high

numbers of antral follicles per wave ......................... 21

Part B. Blood collection ................................................. 22

iii

Immunoassays .................................................................... 22

Statistical Analysis ............................................................... 23
Results ....................................................................................... 25
Study 1: Validation of ultrasound scanning
to count antral follicles ............................................. 25
Part A. Repeatability of counting number of
annal follicles by one technician ............................ 25
Part B. Repeatability of counting number of antral
follicles between two technicians ........................... 25
Study 2: Variability and repeatability of numbers of antral
follicles per wave ..................................................... 26

Part A. Repeatability of numbers of antral follicles
in different follicular waves during the
same or consecutive estrous cycles .......................... 26
Part B. Repeatability of number of antral follicles
in follicular waves of individual cattle
using a different ultrasound operator ......................... 27
Part C. Repeatability of number of antral follicles
in follicular waves that occurred in estrous
cycles spaced 3 months apart .................................. 27
Overall follicular dynamics and variability and
repeatability of maximal numbers of antral
follicles per follicular wave ................................... 27
Study 3: Relationship of variation in maximal numbers of
antral follicles per wave with the dynamics of
development of dominant and subordinate
follicles .................................................................. 28
Study 4: Association of patterns of FSH, estradiol and
inhibin-A secretion with numbers of antral follicles
that grow during follicular waves ................................... 29
Discussion .................................................................................... 30

CHAPTER 2 (Appendix of data for individual animals in Studies 2 and 4).... . 46
Study 2: Variability and repeatability of numbers of antral
follicles per wave for each animal ..................................... 57
Part A. Repeatability of numbers of antral follicles
in different follicular waves during the
same or consecutive estrous cycles ........................... 57
Part B. Repeatability of number of antral follicles
in follicular waves of individual cattle
using a different ultrasound operator........................ 119
Study 4: Association of patterns of FSH, estradiol and
inhibin-A secretion with numbers of antral follicles
that grow during follicular waves ........................................... 126

BIBLIOGRAPHY ................................................................................. 143

iv

Table 1

Table 2

Table 3

CHAPTER 1
LIST OF TABLES

Effect of age, season, number of follicular waves per estrous
cycle, lactational status, and technician on repeatability of
maximal numbers of antral follicles per follicular wave ............ 42

Distribution of numbers of heifers or cows with two or three
follicular waves per estrous cycle in the low, intermediate, high,

or very high numbers of antral follicles per follicular wave
classifications .................................................................................. 43

Distribution of maximal numbers of antral follicles per follicular
wave in different follicle size categories for cattle that consistently
had low, intermediate, high, or very high numbers of antral

follicles per follicular wave ............................................. 44

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4

INTRODUCTION AND CHAPTER 1

LIST OF FIGURES
The dynamics of ovarian follicular waves and antral
follicle turnover during the estrous cycle of cattle ........................ 4
Hormonal patterns associated with follicular waves ....................... 4

A hypothetical model depicting the association of a cow with high vs
low number of antral follicles ................................................ 8

Alterations in total numbers of antral follicles (2 3mm) during the

first and ovulatory follicular waves of an estrous cycle and during

the early stages of the first follicular wave of the next estrous cycle

of Holstein cows with low, intermediate, high or very high numbers
of antral follicles per follicular wave ...................................... 37

Serum hormone profiles, dominant follicle growth, and numbers
of antral follicles ............................................................. 39

vi

F SH
LH

PGqu
DF

NA

DF Dev
Sub Dev
DF Peak
Sub Peak
GnRH

LIST OF ABBREVIATIONS

follicle stimulating hormone

luteinizing hormone

estradiol 17-13

progesterone

prostaglandin F2.ll

dominant follicle

non-lactating

lactating

not available

size of the dominant follicle at deviation

size of the largest subordinate follicle at deviation
largest diameter of the dominant follicle

largest diameter of the largest subordinate follicle
gonadotropin releasing hormone

vii

Introduction

Background. The development of ovarian follicles begins during embryonic
development [1]. In sheep and cattle, “nests” of primordial follicles are first observed in
ovaries of 2 to 3-month old embryos [1, 4]. Primordial follicles contain immature oocytes
arrested in meiosis I and surrounded by a single layer of flattened pre-granulosa cells.
Unlike males that produce germ cells (sperm) throughout most of life, the final size of the
pool of germ cells (oocytes) in females is established at birth. Although highly variable
(0 in freemartins to 800,000), average number of primordial follicles in calves at birth is
100,000 per pair of ovaries [5]. Follicle recruitment, which is the process whereby a
cohort of primordial follicles enters into a trajectory of growth [6], begins in embryos and
continues until the primordial follicle pool is depleted [l ]. Recruitment is initiated by the
transformation of a cohort of primordial follicles into primary follicles. Primary are
distinguished from primordial follicles because they have an oocytes surrounded by one
complete layer of cubodial rather than squamous granulosa cells. The hormonal or
growth factors that regulate recruitment are unknown. As primary follicles grow, they
develop into secondary or preantral follicles. Secondary follicles consist of an oocyte
serially surrounded by >2 layers of granulosa cells, a basement membrane and theca
layers of cells. As secondary follicles grow, they become antral or tertiary follicles.
Antral follicles are similar to secondary follicles, except they are larger and have antral
cavities filled with follicular fluid. In cattle, antral follicles begin to form as secondary

follicles reach from 0.14 to 0.75 mm in diameter [7, 8]. Some antral follicles continue to

grow until they reach ovulatory size, which is usually 15 to 20 mm in diameter [9].
Understanding the mechanisms that regulate development of primordial into ovulatory
follicles is fundamental to the design of better therapeutic methods to improve fertility in

cattle.

In cattle, ultrasound, coupled with follicle mapping, has been widely used to
monitor the patterns of grth of antral follicles that are 2 3 mm in diameter, and to
characterize development of “dominant” follicles during the estrous cycle. A dominant
follicle is defined as a follicle that survives in a hormonal milieu unfit for growth of other
follicles, and (or) a follicle that prevents growth of other follicles during a follicular wave
[2, 10]. Based on results of ultrasound analysis from numerous laboratories, growth of
antral follicles occurs in a cyclic or wave-like fashion every 7 to 10 days beginning
before puberty and continuing unabated throughout most of life [2, 9, 11-13]. During an
estrous cycle, there are usually two or three waves of antral follicle growth with the final
or ovulatory wave ending in ovulation of the dominant follicle on day 1 of the estrous
cycle (Figure 1A). During emergence (first day of wave), a cohort of several dozen
antral follicles 3 to 4 mm in diameter are stimulated to continue to grow by a transient
rise in serum concentrations of follicle stimulating hormone (FSH) (Figure 1A; [11, 14].
As F SH declines during the first 1 to 2 days of a wave, the growing FSH-dependent
follicles in the wave undergo selection [15]. Selection is the reduction in size of a cohort
of growing follicles in a wave to the species-specific ovulatory quota [16], which is one
in cattle. The end of selection during a wave occurs 2 to 3 days afier emergence and is
associated with the following well characterized physiological and morphological

phenomena: a gradual decline in serum concentrations of FSH (Figure 1B; [14, 15]),

 

deviation in rate of growth of a single dominant compared with other subordinate
follicles (Figure 1A; [13]), enhanced estradiol production and diminished production of
the small molecular weight insulin-like growth factor binding proteins (IGFBPS) by the
dominant compared with other subordinate follicles [17], onset of dominance (Figure 1A;
[10]), and atresia (death) of all remaining F SH-dependent subordinate follicles (Figure
1A; [15]). During dominance, while serum FSH concentrations are low, the dominant
follicle, which has enhanced numbers of LH receptors compared with subordinate
follicles [18, 19], becomes primarily dependent on luteinizing hormone (LH) for its
continued survival [20, 21]. Based on ultrasound analysis, deviation occurs as the growth
rate of the dominant follicle exceeds that of all other subordinate follicles in the wave
(Figure 1A; [13]), whereas onset of dominance commences when a single dominant
follicle reaches a diameter 1 to 2 mm larger than the next largest subordinate follicle, and
the growth of all subordinate follicles in a wave ceases (Figure 1A; [15, 22]). AS
dominant follicles continue to develop during a wave, the frequency of episodic LH
secretion is altered (Figure lB;[20, 21]). A decrease in the frequency of LH secretion,
which occurs during the luteal phase of an estrous cycle, leads to loss of dominance and
eventual regression of non-ovulatory dominant follicles (Figure lA;[23]). In contrast, an
increase in episodic secretion of LH, which occurs coincident with the reduction in
progesterone concentrations during regression of the corpus luteum, leads to development
of an ovulatory dominant follicle during the follicular phase of an estrous cycle (Figure
1A; [23]). Loss of dominance not only marks the end Of a follicular wave, but also
coincides with emergence of the next follicular wave, and usually occurs at the time the

dominant follicle ceases to grow (Figure 1A).

Figure 1. A. The dynamics of ovarian follicular waves and antral follicle turnover
during the estrous cycle of cattle [2]. B. Hormonal patterns associated with
follicular waves [3]. E = Estradiol. LH = Lutenizing Hormone. FSH = Follilcle
Stimulating Hormone. P = Progesterone. Detailed explanation provided in the

Introduction.

A. Folicular Wave Dynanics

First wave Second wave Ovulatory wave
Loss of Loss of

15-O_\_I-ulation , domi‘p'ance , domancs ,

Diameter (mm)

 

 

   

   

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Growth of a dominant follicle can be classified into three phases based on
ultrasound analysis [13]: growing, static, and regressing (Figure 1A). The growth phase
begins at emergence and ends on the day the dominant follicle ceases to increase in size.
The static phase is when growth of the dominant follicle ceases until a day before
dominant follicle diameter begins to decrease. The static phase usually coincides with
loss of dominance and emergence of a new follicle wave. The regressing phase is the last
day of the static phase until the dominant follicle is no longer detectable by ultrasound.

Statement of problem. Although a Single dominant follicle usually develops to
ovulatory size during a follicular wave, numerous smaller antral follicles also typically
develop and die during waves (Figure 1A; [13]). However, the variation in numbers of
antral follicles in different follicular waves has not been carefully examined during
estrous cycles of dairy heifers and cows. In addition, the physiological significance and
the factors that regulate the variation in numbers of antral follicles growing during
follicular waves have not been elucidated.

Speculations on the potential physiological significance of the variation in
numbers of antral follicles during follicular waves in cattle. Despite cattle being single-
ovulators, it is reasonable to postulate that the number of follicles in waves influences
many economically important reproductive traits (Figure 2). For example, variation in
number of antral follicles in waves may be associated with the variation in size of the
primordial follicle pool [5, 24-26]. This finding could indicate that number of antral
follicles in waves is a physiological indicator of the potential length of the reproductive

lifespan of a cow (Figure 2). Perhaps, a heifer identified by ultrasound as having a

relatively high number of antral follicles in a wave also has a large primordial follicle
pool, which enables her to produce more calves in her lifetime compared with cows with
small pools of primordial follicles. The number of antral follicles in a wave may
influence, and thus at least partially explain, the variability in responses of cattle to
ovulation synchrony or superovulation protocols for embryo transfer [27-30]. For
example, cattle with greater numbers of antral follicles in waves may produce more
estradiol [17], which in turn may cause dominant follicles to grow faster [31] and
produce more estradiol during follicular waves compared with cattle with low numbers of
antral follicles in waves (Figure 2). If so, differences in number of antral follicles in
waves would be expected to contribute to the variation not only in intensity of estrus
expression [32], but also in the rate of development of dominant follicles and thus stage
of maturation and time to ovulation after cattle are subjected to ovulation synchrony or
superovulation protocols. Cattle with high numbers of antral follicles in waves may
respond greater to superovulation protocols and thus produce more high quality oocytes
for embryo transfer compared with cattle with low numbers of antral follicles in waves
(Figure 2). Number of antral follicles in waves may also contribute to the high variability
usually associated with responses of cattle to superovulation [28, 30, 33, 34]. It is highly
likely that each follicle in a wave contributes to the overall hormonal and growth factor
milieu both in the peripheral circulation and locally within ovaries [35]. Therefore, the
hormones and growth factors produced by subordinate follicles during waves may
influence not only dominant follicle development, estrus expression, and responses to
superovulation, but also embryo development and overall fertility of the cow. Most

important, if the variation in antral follicle number in waves is indeed linked to

Figure 2: A hypothetical model depicting the association of a high vs low
number of antral follicles per wave with size of the primordial follicle pool, reproductive
lifespan, number of calves, superovulation, estrus expression, dominant follicle growth,

and fertility. E = Estradiol. DF = Dominant Follicle.

fiEstmsExpression
Ir IIFertilty

[l EslrusExpressbn
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economically important reproductive traits, then number of antral follicles in a wave
could be used as a novel criterion to improve existing and perhaps develop new
reproductive management systems. In addition, should number of antral follicles in
waves be linked to important traits and controlled genetically, then ultrasound
determination of number of antral follicles per wave would be a simple, novel criteria to
improve breeding schemes for selection of high fertility cows. The factors that regulate
the variation in number of antral follicles that develop in follicular waves are unknown,
but may most likely be associated with the following: differences in serum concentrations
of FSH that precede each follicular wave [14, 36-39], stage of estrous cycle when waves
occur [40], variability in size of the pool of primordial preantral or antral follicles in
ovaries [5, 41], environmental influences such as nutrition and temperature [42-45],
and(or) genetic components [46, 47].

Hypotheses. Because very little is known about the alterations in number of
antral follicles that develop in follicular waves during the bovine estrous cycle, I propose
to test the following two hypotheses to complete the research requirement for my
Master’s degree:

I. Number of antral follicles in different follicular waves is as variable

within individuals as it is amongst cattle.

11. Variation in numbers of antral follicles within follicular waves is

positively correlated with each wave’s corresponding F SH, inhibin-A and
estradiol secretion profiles.

The Specific reasons I chose these hypotheses to test are explained in Chapter I.

10

Approach. My general approach to address these hypotheses will be to use
ultrasound and follicle mapping to monitor number of antral follicles that develop in the
first, second and ovulatory waves of follicular development (Figure 1A) in heifers and
cows, and to use serial blood sampling and ultrasound analysis to establish whether
patterns of serum F SH concentrations are associated with number of antral follicles in
waves (Figure 1B).

My thesis is in two parts: In Chapter 1, I have prepared a manuscript for
submission to Biology of Reproduction describing the results of the studies in my thesis.
In Chapter 2, I have included an appendix of all the follicular wave and hormone

secretion data generated for each animal during my studies.

11

Chapter 1
Manuscript to be submitted to

Biology of Reproduction

12

Repeatability of Numbers of Antral Follicles in Follicular Waves in Cattle and

Association with Secretion of FSH, Estradiol and Inhihin-A

Short title: Repeatability of Antral Follicle Numbers during Follicular Waves

David 8. Burns], Fermin Jimenez-Krassel' , Janet L.H. Ireland', P.G. Knightz

and .I. .I. Ireland”

Keywords: number of antral follicles per wave, repeatability of follicle growth,

follicle waves, FSH, estradiol, inhibin-A

 

lMolecular Reproductive Endocrinology Laboratory, Department of Animal
Science, Michigan State University, East Lansing, MI 48824. Research supported
by the Agricultural Experiment Station at Michigan State University.

2School of Animal and Microbial Sciences, University of Reading, Reading R06
2AJ, United Kingdom

3To whom reprint requests should be addressed. FAX: 517-353-3149; email:

Ireland@msu.edu

13

Abstract

The objective of our study is to examine the variation in antral follicle numbers
that develop during follicular waves in cattle, and to determine if the variation in numbers
of follicles per wave is associated with serum FSH, estradiol and inhibin-A
concentrations. Non-pregnant cattle (10 months to 7 years old, n = 44) were subjected to
twice daily ultrasound analysis to count number of antral follicles _>_3 mm in diameter
during different follicular waves (n = 138 waves) in the same or consecutive estrous
cycles or in estrous cycles spaced several months apart. The results demonstrated that: a)
maximal numbers of antral follicles in different follicular waves were highly variable
amongst cattle (8 to 54), but remarkably highly repeatable (0.95) within individuals; b)
repeatability of the maximal number of follicles per wave in individuals was very high
(0.86 to 0.95) regardless of age, season, stage of lactation, two vs three follicular waves
per estrous cycle or technician; and c) most of the variation in maximal number of antral
follicles per wave amongst cattle was attributable to follicles 3 to 5 mm in diameter. In
another experiment, ultrasound analysis was used to identify cows (3 to 4 years old) that
consistently had very high (29 to 44 follicles, n = 4 cows) or low numbers (11 to 18
follicles, n = 3 cows) of antral follicles per wave. Blood samples were taken at frequent
intervals beginning 48 h after PGan and continuing until 4 d after ovulation. Serum was
subjected to FSH, estradiol and inhibin-Aimmunoassays. The results demonstrated that
serum F SH concentrations were 50% lower (P<0.03) in cows with very high numbers of
antral follicles per wave (34.9:1.1 follicles) vs cows with low numbers (15.1:09

follicles). However, serum concentrations of estradiol and inhibin-A were similar

14

between the two groups of cows. We concluded that: 3) despite the high variation in
antral follicle number per wave amongst cattle, a chronic compensatory mechanism must
exist in ovaries to maintain growth of a constant number of antral follicles in follicular
waves throughout estrous cycles of individuals, b) the variability in numbers of follicles
in waves amongst cattle is probably not explained by the interaction of serum FSH with
inhibin-A and estradiol concentrations, and c) factors in addition to estradiol and inhibin-
A from antral follicles may control FSH secretion fiom the pituitary gland during

follicular waves.

Introduction

Ultrasonography has firmly established that two or three follicular waves usually
occur during the human menstrual cycle [48, 49] and during the bovine estrous cycle [2,
13]. However, follicular waves are best characterized in cattle [2, 12, 13, 50-52]. In
cattle, follicular waves occur every 7 to 10 days beginning several months before first
ovulation at puberty and continuing throughout most of life [2, 13]. Each follicular wave
coincides with a transient rise in serum concentrations of follicle stimulating hormone
(FSH) [14, 52]. The increase in circulating concentrations of FSH stimulates grth of a
cohort of 3- to 4-mm antral follicles, which is followed by development of a single
dominant ovulatory-size (15-20 mm) follicle and atresia of the remaining follicles in the
original cohort [2, 13, 52]. Follicular waves synchronous with the follicular phase of an
estrous cycle culminate in development of a single dominant ovulatory follicle. In
contrast, follicular waves asychronous with a follicular phase are non-ovulatory [2, 13,

52]. Taken together, the wave-like growth and atresia of antral follicles, coincident with

15

a shrinking ovarian reserve (total number of follicles in ovaries) during aging in cattle [5,
53] and humans [25, 26], is reflective of a unique biological rhythm that may be critical
for successful reproduction. However, neither the physiological significance nor the
cause Of the high variation in the number of antral follicles that grow during follicular
waves is known. Elucidation of these questions may provide new insight into the
physiological importance of follicular waves to fertility.

The variation in numbers of antral follicles during follicular waves amongst dairy
heifers ranges from 11 to 42 [13]. However, the variation in maximal number of antral
follicles growing during follicular waves amongst and within individual cattle of different
ages has not been evaluated. We hypothesized that number of antral follicles in different
follicular waves is as variable within individuals as it is amongst cattle because: a) the
different follicular waves during an estrous cycle occur in markedly different hormonal
milieus [54]; and b) size of the ovarian reserve, which may influence follicular growth, is
highly variable amongst cattle and decreases as cattle age [5]. To test the aforementioned
hypothesis, our first study was designed to examine the variation in number of antral
follicles in different follicular waves amongst and within individual cattle of different
ages. Also in cattle, F SH is the primary hormone that induces follicular waves [14, 52,
55], and estradiol and inhibin-A are produced by antral follicles during waves [17].

Thus, we hypothesized that the variation in number of antral follicles during follicular
waves is positively associated with secretion of FSH, estradiol and inhibin-A. To test
this hypothesis, the second study examined whether the variation in numbers of antral
follicles per wave was positively associated with alterations in serum concentrations of

FSH, estradiol and inhibin-A during a follicular wave.

16

Materials and Methods
Animals
Holstein heifers and lactating and non-lactating cows utilized for our studies were
obtained from the Michigan State University Dairy Facility. Heifers were housed in free
stalls whereas cows were housed in tie stall barns. Animals were provided feed and
water ad libitum. All rations met NRC requirements, and studies were conducted during
the Spring, Summer or F all of 2001 and 2002. The All University Committee on Animal

Use and Care at Michigan State University sanctioned all procedures involving cattle.

Ultrasound scanning procedure to count antral follicles

Ovaries in each cow were scanned with an Aloka SSD-900 linear array trans-
rectal probe (Aloka Ultrasound, 7.5 MHz transducer, Wallingford, CT). To standardize
counting of follicles, the following ultrasound scanning procedure was adopted. Each
ovary was scanned from end to end to identify the position of the corpus luteum and
antral follicles in ovaries. Specifically, beginning at one end of the ovary, video images
for different ovarian sections were captured on a computer monitor. The location of the
corpus luteum and each antral follicle 2 3 mm in each section of ovary was drawn on an
ovarian map. Two separate measurements of diameter were made for each follicle and
recorded next to the appropriate follicle on each ovarian map. The final diameter of each
follicle was based on the average of the two separate diameter measurements. After all

ovarian structures were mapped and diameters of each follicle recorded, the scanning

17

procedure was repeated to confirm the locations of follicles on each ovarian map. Total
number of antral follicles per pair of ovaries for each animal was determined by counting

the number of follicles 2 3 mm in diameter on each map for each animal.

Study 1: Validation of ultrasound scanning to count antral follicles

This study examined the variability of using ultrasound scanning to count number
of antral follicles in both ovaries of individual cows within and among different
ultrasound Operators.

Part A. To determine the variability of counting follicles by a single ultrasound
operator, the repeatability of counting the total number of antral follicles _>_ 3 mm in
diameter in ovaries of five non-lactating 3- to 5-year-old cows on three separate
occasions (spaced 10 minutes apart) twice daily (0800 and 1800) for 11 consecutive days
beginning at random stages of the estrous cycle was determined. Repeatability is defined
below in Statistics.

Part B. To examine the variability of counting follicles amongst ultrasound
operators, the correlation of the total number of antral follicles 2 3 mm in diameter
counted in ovaries during ultrasound analysis on each of 11 randomly chosen days during
the estrous cycles of five non-lactating cows between two different ultrasound operators
was determined. The daily ultrasound session for each cow by each operator was
approximately 1 hour apart, and the results of counting follicles by each operator were

undisclosed until the study ended.

18

Study 2: Variability and repeatability of numbers of antral follicles per wave

This study determined if number of antral follicles z 3 mm in diameter that grow
in different follicular waves during the same or consecutive estrous cycles (Parts A, B),
or in estrous cycles several months apart (Part C) is repeatable in individuals.

Part A. To examine repeatability of numbers of antral follicles in different
follicular waves during the same or consecutive estrous cycles, fourteen 10- to 12-
month-Old heifers and sixteen 4- to 7-year-old lactating (n = 9 cows) or non-lactating (n =
7) cows were subjected to ultrasound analysis. All animals were treated with two
intramuscular injections of prostaglandin F20, (PGFZG, 25 mg/dose of Lutalyse®
Pharmacia-Upjohn Co., Kalamazoo, MI) spaced 11 days apart to induce luteolysis and
synchronize occurrence of estrus. Each animal was subjected to twice daily (0800 and
1800) ultrasound analysis by the same ultrasound operator (DB) beginning at the time of
the second PGan injection and continuing until 2 days after spontaneous ovulation of the
subsequent estrous cycle. At each ultrasound session, total number of antral follicles 2 3
mm in diameter in ovaries of each animal was determined, as described for Study 1.

Part B. This study determined repeatability of number of antral follicles in
follicular waves of individual cattle using a different ultrasound operator (F J). In this
study, three heifers (13 months of age) and two lactating Holstein cows (3-5 years of age)
were treated with PGan and subjected to twice daily ultrasound analysis to count number
of antral follicles 2 3 mm, as explained in Part A.

Part C. In this study, repeatability of number of antral follicles in follicular

waves that occurred in estrous cycles spaced 3 months apart for the same four non-

l9

lactating cows (4 to 7 years of age) was examined. Estrous cycles were either
spontaneous or synchronized with two or three injections of PGF;cl spaced 11 days apart
as explained above. Numbers of antral follicles 2 3 mm in diameter were counted twice
daily either throughout all the different follicular waves during each estrous cycle for two
cows or only during the first wave of three consecutive estrous cycles for the remaining

IWO COWS.

Study 3: Relationship of variation in maximal numbers of antral follicles per wave

with the dynamics of development of dominant and subordinate follicles

This study examined whether variation in numbers of antral follicles per wave
was associated with alterations in the dynamics of growth of dominant and subordinate
follicles. In this study, the animals used in Study 2 (Parts A, B) were classified arbitrarily
into four groups based on the maximal number of antral follicles 3 3 mm in diameter per
wave. Because maximal number of antral follicles was determined for 3 to 4 waves in
each animal in Study 2, individuals were classified based on their overall average for the
maximal number of follicles per wave as follows: low (315 follicles per wave; n = 30
waves, n = 9 animals), intermediate (16 to 20; n = 29 waves, n = 10 animals), high (21 to
25; n = 25 waves, n = 8 animals), and very high (>25; n = 30 waves, n = 8 animals). To
determine if numbers of antral follicles per wave were associated with alterations in the
dynamics of growth of dominant and subordinate follicles, we examined whether day of
emergence for each follicular wave, day of deviation, length of dominance, interval
between ovulations, and maximal diameter of the dominant or subordinate follicle at

deviation or during dominances differed for cattle with low, intermediate, high, and very

20

high numbers of follicles in waves. Definitions used to characterize dominant follicle
growth were: day of emergence = first day of new wave that a follicle 2 3 mm in
diameter is detected by ultrasound; day of deviation = first day of a new wave that the
largest or dominant follicle is consistently El 1 mm greater in diameter than the next
largest or subordinate follicle throughout the wave minus one day; length of dominance =
interval from first day of deviation in a new wave until emergence of the next wave.

To determine if basal numbers of antral follicles per wave differed amongst the
different groups of cattle, the overall average for the lowest number of antral follicles per
wave was determined for animals with low, intermediate, high or very high numbers per
wave.

To determine which size category of antral follicles was most variable during
waves for cattle with low, intermediate, high or very high numbers of follicles per wave,
the distribution of maximal numbers of follicles in each of the following different follicle
size categories (3 to 3.9, 4 to 4.9, 5 to 5.9, 6 to 7.9, 8 to 10, and >10 mm in diameter) was

determined for each group of animals.

Study 4: Association of patterns of FSH, estradiol and inhibin-A secretion with
numbers of antral follicles that grow during follicular waves

This study determined whether the serum concentrations of F SH, estradiol and
inhibin-A are positively associated with numbers of antral follicles 2 3 mm in diameter

that develop during follicular waves.

21

Identification of cows with low or very high numbers of antral follicles per
wave. A group (n = 25 cows) of 3- to 5-year-old lactating cows was subjected to
ultrasound analysis for 2 to 3 consecutive days chosen at random during an estrous cycle
to identify cows that consistently had low (5 15 follicles) or very high (> 25 follicles)
numbers of antral follicles 2 3 mm in diameter. Five animals in each group were
identified. To determine numbers of antral follicles in follicular waves, estrous cycles
for these ten animals were synchronized with three consecutive injections of PGF;,,
spaced 11 days apart. Each animal was subjected to daily (0800) ultrasound analysis
after each of the first two PGan injections and twice daily (0800 and 2000) ultrasound
analysis after the third PGan injection. Ultrasound analysis began at the time of each
injection Of PGFz.JI and ended 4 days afler ovulation. Number of antral follicles in the
first follicular wave that occurred after each PGqu injection was determined as explained
above. Day of ovulation after each PGan injection was determined based on inability of
the ultrasound operator to detect the dominant ovulatory follicle during scanning.

Blood collection. Blood samples (5 ml) were removed from each animal’s tail
vein beginning 48 h afier the third PGFZQ injection and continuing every 4 h until 24 h
after ovulation. Thereafier, blood was collected every 8 h until 96 h afier ovulation.
Based on previous results [54], this blood sampling regimen should span the days of an
estrous cycle that coincide with development of the first wave dominant follicle.
Immunoassays

Concentrations of FSH in duplicate IOO-ul serum samples for each cow were

determined using a previously validated heterologous RIA [54, 56, 57]. Results of the

FSH RIA correlate with in vitro bioassay of FSH bioactivity [58]. Ovine FSH (USDA

22

OF SH-l9-SIAFP-I-2) was used as radioiodinated tracer, bFSH (USDA bFSH-I-2) as
standard, and NIDDK anti-oFSH-l (AFP-C5288113; Rockville, MD) as antiserum. The
intra-assay coefficient of variation (CV) was 10.5%, and the sensitivity of the assay was
0.03 ng/ml. Estradiol 17-[3 concentrations were determined in duplicate 500-ul serum
samples previously extracted with ether using a modified version [59] of the commercial
MAIA Kit (Polyrnedco Inc., Courtlandt Manor, NY). Standard curves ranged from 0.195
to 50 pg/ml (or 0.039 to 10 pg/tube), and the mean _+_SEM EDso for standard curves (n =
4) averaged 4.8 10.8 pg/ml. Sensitivity of the assay was 0.04 i 0.01 pg/ml. Inter- and
intra-assay (n = 4 assays) coefficients of variation (CVs) were 17.5% and 9.6% for serum
samples that averaged 5.1 pg/ml, and 16.5% and 24.4% for samples (n = 4) that averaged
0.88 pg/ml. A recently validated non-radiometric two-site ELISA using highly purified
32-kDa bovine inhibin-A as standard [60] was used to determine concentrations of
inhibin-A in duplicate lOO-pl serum samples for each cow. Sensitivity of the inhibin
assay was 15 pg/ml, and within and between plate CVs were < 11%. The inhibin-A
assay does not cross react with the bovine inhibin or subunit proac, recombinant human
(rh) inhibin-B, rh-activin-A, rh-activin-B, or rh-follistatin-288[60].

Statistical Analysis

Repeatability (range = 0 to l, 1 = perfect) is defined as the proportion of the total
variance that could be attributed to animal variance, which is calculated as follows: (I2

animal/tr2 animal + C2 error [61-63]. Variance components were estimated using the

MIXED procedure of SAS [64].

23

In Study 1, repeatability or correlation analysis (range = -l to 1, 1 = perfect
positive correlation) was used to examine variability of a single or two different operators
using ultrasound scanning to count follicles.

In Study 2, repeatability of numbers of antral follicles in different follicular waves
during the same or consecutive estrous cycles, or in estrous cycles spaced three months
apart for individuals was determined, as explained above.

In Study 3, a mixed model, repeated measures approach was used to determine
whether growth of dominant and subordinate follicles during follicular waves was similar
for cattle that consistently had low, intermediate, high or very high numbers of antral
follicles per wave [64]. The cattle used in Parts A and B of Study 2 were used for this
analysis. Main effects included: technician, groups of cattle with low, intermediate, high
or very high maximal number of antral follicles per wave, lactation status, age of animals,
number of follicular waves per estrous cycle, ovulatory and non-ovulatory waves,
whereas dependent variables included: maximal follicle number per wave, length of
dominance, interval between ovulations, and diameter of the dominant or subordinate
follicles at deviation or during dominance. Also, a mixed model, repeated measures
approach was used to determine whether the distribution of maximal numbers of antral
follicles in different size categories was similar for cattle that consistently had low,
intermediate, high or very high numbers of antral follicles per wave. Number of waves
subjected to ultrasound analysis per animal was treated as a repeated measure across
individual cows for analyses. Least squares means were calculated for all main effects in
the model. When main effects were significant (P<0.05), Bonferroni t test was used to

determine whether statistical differences existed amongst individual means [64].

24

In Study 4, repeatability of maximal numbers of antral follicles during the three
synchronization periods was determined, as explained above. A mixed model, repeated
measures approach was used to determine if serum F SH, estradiol and inhibin-A
concentrations and numbers of antral follicles were different for cows with low vs very
high numbers of anual follicles per wave [64]. Main effects included: groups of cows
with low or very high numbers of antral follicles per wave, time, and an interaction of the
follicle groups with time. Serum FSH, estradiol and inhibin-A concentrations and
numbers of antral follicles were considered independent variables. Number of blood
samples and number of follicles per wave per animal were treated as a repeated measure
across individual cows. The data were aligned based on the first peak FSH concentration
following ovulation. Least squares means were calculated for all main effects in the
model. When main effects were significant (P<0.05), Bonferroni t test was used to

determine whether statistical differences existed amongst individual means [64].

Results
Study 1: Validation of ultrasound scanning to count antral follicles
Number of antral follicles (range = 4 to 22 follicles per cow) counted at three
separate ultrasound sessions spaced 10 min apart in individual animals was highly
repeatable (0.97, n= 330 counts) for a single ultrasound technician. In addition, daily
follicle counts (range = 6 to 25 follicles per cow) for each of the five cows during the 11-
day Observation period was highly correlated (correlation = 0.92, n=55 counts/operator)

between the two ultrasound operators.

25

Study 2: Variability and repeatability of numbers of antral follicles in follicular
waves amongst and within individuals

Part A: Numbers of antral follicles per wave for four representative cows that
consistently had low, intermediate, high, or very high numbers of antral follicles in the
different follicular waves during an estrous cycle are depicted in Figure 3. These results
typified the remarkable similarity in the maximal number of antral follicles in different
follicular waves of individual animals, and the great variation amongst animals. Despite
the high variability in maximal number of antral follicles in waves amongst cattle (range
= 11 to 54 follicles per wave), repeatability of the maximal number of antral follicles in
different follicular waves during the same or consecutive estrus cycles within each
individual was very high overall (0.95, n = 96 follicular waves for 30 total animals), and
very high for heifers (0.92, n = 14 heifers, n = 45 waves) or cows (0.96, n = 16 cows, 11 =
51 waves). In addition, repeatability of basal or minimal numbers of antral follicles per
wave in individuals (range = 2 to 31) was very high overall (0.85, n = 68 waves, n = 30
animals), and very high for heifers (0.84, n = 31 waves, n = 14 heifers) or cows (0.87, n =
37 waves, n = 16 cows). Note. The minimal number of follicles per wave was not
determined for all waves because number of follicles was not determined for the last 2 to
3 days of the last wave examined in each animal. Thus, the total numbers of waves
examined to determine minimal numbers of follicles per wave was less than total
numbers of waves examined per animal to determine maximal numbers of follicles per
wave.

The overall correlation for alterations in numbers of follicles in the right vs left

ovary during all follicular waves for 30 animals was 0.72 (P<0.01).

26

Part B: In a separate study by another ultrasound operator, the maximal number
of antral follicles per wave for five animals ranged from 9 to 39 (n = 18 waves), whereas
repeatability of numbers of follicles per wave for individuals was 0.86.

Part C: The maximal number of antral follicles per wave ranged from 9 to 33
amongst four animals (n = 21 waves), whereas repeatability of numbers of follicles per
wave in estrous cycles spaced three months apart was 0.90 in individuals.

Overall follicular dynamics and variability and repeatability of maximal
numbers of antral follicles per follicular wave: When the 35 animals in Parts A and
B of Study 2 were combined, seven (4 cows, 3 heifers) had three follicular waves while
28 (14 cows, 14 heifers) had two follicular waves during an estrous cycle. The maximal
number of antral follicles 2 3 mm in diameter in follicular waves averaged 21.5 -_I-_ 0.8
(range = 8 to 54 follicles per wave) and was similar for heifers (20 i 1.1, range = 8 to 42
follicles per wave, n = 17 animals) and cows (22.8 i 1.3, range = 11 to 54, n = 18).
Nevertheless, cows had longer (P<0.05) intervals between ovulations (23.7 i 0.6 vs. 21.3
i 0.8 d), larger (P<0.05) dominant follicles (15.8 i 0.3 vs 14.7 3; 0.5 mm), and longer
(P<0.05) periods of dominance (7.6 i 0.3 vs 6 i 0.6 (1) compared with heifers.

When animals in Parts A and B of Study 2 and Study 4 (described below) were
combined (11 = 44 animals, n = 138 waves), maximal number of antral follicles per wave
ranged from 8 to 54 amongst animals, whereas repeatability of maximal number of
follicles per wave for individuals was 0.95 (Table 1). In addition, repeatability of
maximal number of antral follicles per wave in individuals was very high (0.86 to 0.95)
regardless of age of cattle, season of year, numbers of follicular waves per estrous cycle,

stage of lactation, or technician (Table l).

27

Study 3: Relationship of number of antral follicles per wave and the dynamics of
development of dominant and subordinate follicles

Distribution of numbers of heifers or cows with two or three follicular waves per
estrous cycle in the low, intermediate, high or very high classifications for numbers of
antral follicles per wave is shown in Table 2. The overall distribution of heifers and cows
in each follicle number per wave classification was equally distributed (P > 0.10).

Ages of cattle, stage of lactation, length of dominance, interval between
ovulations, day of emergence, day of deviation, largest diameter of the dominant and
subordinate follicles during dominance, and diameter of the dominant and subordinate
follicles at deviation were similar (P > 0.10) for cattle with low, intermediate, high, or
very high numbers of follicles per wave (data not shown).

Basal numbers of antral follicles per wave were highest (P<0.05) in cattle with
very high numbers of follicles per wave (19.7:12), greater (P<0.05) in cattle with very
high or high (11.311) numbers of follicles per wave compared with cattle with
intermediate (7.3:1) or low (62:09) numbers of follicles per wave, and similar (P>0.lO)
for cattle with low or intermediate numbers of follicles per wave.

The distribution of maximal numbers of antral follicles per wave in the 5 to 5.9, 6
to 7.9, 8 to 9. 9 or >10 mm in diameter categories was sirrrilar (P > 0.10) for cattle with
different numbers of antral follicles per wave, except that cattle with very high numbers
of follicles per wave had greater (P<0.05) numbers of follicles 5 to 5.9 mm in diameter
compared with cattle with low numbers of follicles per wave (Table 3). In contrast,

numbers of follicles 3 to 3.9 and 4 to 4.9 mm in diameter were 2- to 3-fold higher

28

(P<0.05) in cattle with high or very high vs low or intermediate numbers of antral

follicles per wave.

Study 4: Association of patterns of FSH, estradiol and inhibin-A secretion with
numbers of antral follicles that grow during follicular waves

Of the 10 cows subjected to ultrasound analysis in Study 4, one cow predicted to
have a low number of antral follicles expired while another developed a large (>30 mm)
follicular cyst, and one cow predicted to have a very high number of antral follicles
developed leg problems. These three cows were excluded from blood sampling, but the
latter two cows were included in determination of repeatability. Despite high variability
in number of antral follicles in waves amongst cattle, repeatability of maximal number of
antral follicles in three consecutive estrous cycles was very high for individuals (0.93, n =
24 waves for 9 cows) and similar to the results of Study 2.

In the seven cows used to examine the association of serum F SH, estradiol and
inhibin-A concentrations to numbers of follicles per wave (Figure 4), the maximal
number of antral follicles 2 3 mm in diameter per follicular wave for the three cows
predicted to have a low number of antral follicles averaged 15.1 :1: 0.9 follicles per wave
(n = 10 follicular waves, range = 11 to 18, age = 4 years old), whereas the four cows
predicted to have a very high number of antral follicles averaged 34.9 i 1.1 follicles per
wave (n = 10 follicular waves, range = 29 to 44, ages = 3-4 years old).

Because of multiple large peaks Of FSH secretion for each cow during the blood-
sarnpling period (data not shown), FSH values were aligned for each cow for statistical
analysis based on the first peak F SH concentration after ovulation. Ovulation occurred

an average of 4 h before the first peak FSH concentration after ovulation. Follicle wave

29

emergence occurred coincident with ovulation, and deviation and development of
dominant follicles occurred an average of 63 h after the first peak FSH concentration

after ovulation.

Serum FSH concentrations varied (P<0.001) similarly during the first follicular
wave for cows with low vs very high numbers (Figure 4). However, serum FSH
concentrations were nearly 50% higher (P<0.03) throughout the treatment period in cows
with low (0.73 i 0.06 ng/ml) vs very high numbers of antral follicles (0.50 i 0.05 ng/ml).
Serum concentrations of estradiol and inhibin-A were unaltered (P>0.10) during the
treatment period (Figure 4). Also, in contrast to FSH, serum concentrations of estradiol
and inhibin-A were similar for cows with low vs very high numbers of antral follicles per

wave (Figure 4).

Discussion

The results of our study demonstrate for the first time that numbers of antral
follicles that grow during different follicular waves is very highly repeatable in individual
cattle. In support of repeatability of antral follicle growth in individuals, number of
antral follicles is similar in individual humans from one menstrual cycle to the next [41].
In addition, numbers of antral follicles that grow following serial ultrasound-guided
needle aspiration of antral follicles every 7 to 10 days is highly repeatable in individual
cattle [61, 65]. Although responsiveness to superovulation treatments is not repeatable in
cattle [28-30, 66], the negative effect of dominant follicles per se on follicular grth

[67, 68], coupled with timing of superovulation injections relative to emergence of

30

follicular waves, may explain the absence of repeatability of responsiveness to
superovulation in cattle. The high repeatability of follicle growth in individual cattle in
our study was maintained despite striking differences in hormonal rrrilieus associated
with the different waves during estrous cycles [54], ages of cattle, numbers of follicular
waves per estrous cycle, stages of lactation, and season of year. Repeatability of antral
follicle growth was also high when a different technician conducted the ultrasound
analyses, thus eliminating technician artifact as the cause of high repeatability.
Interpretation of our results, however, is somewhat limited because it was not established
definitively whether repeatability of antral follicle growth during waves remained high
throughout the reproductive lifespan of individual cattle. Nevertheless, the average
numbers of antral follicles per follicular wave were similar for 10- to 12-month-old
heifers compared with 3- to 7-year-old cows in our study. In addition, previous reports
indicate that despite the steep decrease in numbers of primordial follicles during aging,
numbers of antral follicles in ovaries remain relatively constant until cattle are 9 to 10
years old [5, 53] and humans are 35 to 38 years old [24, 41, 69-72]. Thereafier,
coincident with the marked exhaustion of primordial follicles, the numbers of antral
follicles decrease in cattle [5, 53] and humans [24, 41, 69-72]. These observations,
coupled with the remarkably high repeatability of antral follicle number per wave in
individuals observed in our study, imply that a chronic intraovarian compensatory
mechanism may exist to maintain a nearly constant rate of antral follicle growth as
numbers of primordial follicles decline during the lengthy reproductive lifespan of single-

ovulating Species such as cattle and perhaps humans. Whether this compensatory

31

mechanism is similar to the acute compensatory mechanism that maintains the species
specific ovulation rate after unilateral ovariectomy [73-75] is unknown.

Classical histological studies in cattle [5] and other species including sheep [76],
rats [77], mice [78], and humans [26] demonstrate that numbers of primordial follicles
are highly variable within Species at birth. Because the ovarian reserve, which is
comprised primarily of primordial follicles, is depleted during aging and never
replenished [5, 26, 69, 76, 78], numbers of the different follicle types in the ovarian
reserve vary greatly in individuals throughout reproductive life. In support of the high
variability in size of the ovarian reserve, histological, laparoscopic, and ultrasound
analyses of ovaries of animals of similar ages show that numbers of preantral and antral
follicles are highly variable in prepubertal or adult cattle [5, 13, 53, 79], sheep [76], rats
[77], mice [78], non human primates [80], and humans [26, 72]. The results of our study,
coupled with another report [13], clearly show that numbers of antral follicles that grow
during follicular waves in cattle are also highly variable. In cattle, higher numbers of
preantral or antral follicles are positively linked to fecundity [5, 81], shorter calving
intervals [82], and a higher percentage of normal oocytes [53] and number of transferable
embryos [79]. In cattle and humans, higher numbers of antral follicles are positively
associated with an increased responsiveness to gonadotropin treatments during
superovulation [30, 79, 83-85], a larger number of oocytes recovered for in vitro
fertilization [83, 85], and higher pregnancy rates following in vitro fertilization [79, 83,
85]. The high repeatability of numbers of antral follicles in waves of individuals,
coupled with the high variation in follicular growth amongst cattle, enable use of

ultrasound to identify reliably groups of cattle that consistently have low, intermediate,

32

high or very high numbers of antral follicles per wave. Consequently, future studies will
take advantage of this important finding and examine the relationship of the variation in
numbers of antral follicles in waves amongst cattle to size of the ovarian reserve,
reproductive lifespan, superovulation and fertility.

Cattle, like humans, are a single ovulating species. Consequently, the selection
process during follicular waves usually results in development of a single dominant
follicle regardless of numbers of antral follicles in waves. This raises the interesting
question of whether the variability in numbers of 3 to 5 mm antral follicles during waves,
which is the primary size category of follicles that varied during follicular waves in our
study, affects subsequent growth and function of dominant follicles. Results of our study,
however, show that serum estradiol and inhibin-A concentrations, and the dynamics of
deve10pment of dominant follicles during follicular waves, including emergence,
deviation, maximal diameter and length of dominance, were unaltered by variability in
numbers of antral follicles in waves. This surprising finding implies that numbers of
antral follicles per wave may not affect development or function of dominant follicles.
Nevertheless, our study focused primarily on the first half of the first follicular wave
during an estrous cycle, which is non-ovulatory. Thus, secretion of FSH, estradiol and
inhibin-A were not evaluated throughout the first follicular wave or during an ovulatory
wave when estradiol secretion is maximal [54]. Future studies will, therefore, be
necessary to determine if variation in numbers of antral follicles per wave alters dominant
follicle growth and function.

The mechanisms that regulate the variation in numbers of antral follicles that

grow during follicular waves are unknown. Moreover, the putative chronic

33

compensatory mechanism that maintains growth of a highly repeatable number of antral
follicles during waves as the ovarian reserve is depleted during aging in gonadally intact
individuals remains undefined. It is well established that F SH is a key hormone involved
in growth of antral follicles. In support of the role of F SH in antral follicle growth, serum
FSH concentrations peak prior to follicular waves in humans [49] and cattle [14, 54], and
inhibition of FSH secretion in cattle blocks occurrence of follicular waves [55, 86].
Studies in hypophysectomized rats [31] and genetic deletion studies in mice [87] show
clearly that FSH is required for development and differentiation of antral follicles in
laboratory species. Moreover, enhanced secretion of F SH afier removal of one ovary
causes acute compensatory ovarian hypertrophy in cattle [88-90] and other species,
including rats [77, 91], swine [92], rabbits [93], hamsters [94], and guinea pigs [95].
However, these results are controversial in cattle [96] and rats [97]. Nevertheless,
despite removal of half of the ovarian reserve after unilateral ovariectomy of the
aforementioned species, the species specific ovulation rate is maintained [73-75] or
enhanced [96, 98]. Taken together, these observations imply that FSH has important
roles not only in regulation of the variation in growth of antral follicles during follicular
waves, but also perhaps in the intraovarian compensatory mechanisms that maintain
growth of a constant number of antral follicles during follicular waves.

The reason several distinct peaks of serum F SH concentrations were observed in
our study and by others [38] during the first follicular wave Of the estrous cycle rather
than a single transient increase in FSH [14, 15, 54] is unknown. One possible
explanation for this disagreement among laboratories is that values in our study were

aligned relative to the first FSH peak after ovulation, while others align data relative to

34

the preovulatory LH surge [15], emergence of follicles [14, 99] or estrus [15, 54, 55,
100]. In addition, lactating cows rather than heifers were used in our study compared
with others [14, 15, 54, 55, 99]. '

Alterations in serum concentrations of inhibin-A and estradiol in our study were
similar to previous reports for changes in serum concentrations of inhibin-A [60, 101]
and estradiol [15, 54] during the first wave of follicular growth in cattle. Nevertheless, in
contrast to our expected results, serum F SH concentrations were inversely rather than
positively associated with numbers of antral follicles in follicular waves throughout the
blood-sampling period. Equally surprising was the finding that serum concentrations of
the negative feedback hormones, inhibin-A and estradiol, were similar for cows with low
vs very high numbers of antral follicles per wave. These observations imply that factors
other than F SH and its chief feedback hormones, inhibin-A and estradiol, could have a
role in regulation Of both FSH secretion and numbers of follicles growing during waves.
Because numbers of primordial, preantral and antral follicles in ovaries are positively
associated [5, 27, 53], it is likely that the variation in numbers of follicles per wave
reflects relative size of the ovarian reserve. Thus, the high serum FSH concentrations in
animals with low numbers of follicles per wave may be associated with ovarian aging. In
support of this possibility, serum FSH is higher in women [102] and cows [103]
compared with their younger counterparts. Factors that regulate variation in antral
follicle numbers during waves may not only include size of the ovarian reserve,
differential responsiveness of antral follicles to FSH [104], differential secretion of
hormones and growth factors that alter FSH responsiveness such as LH [31] or IGF-l

[105], and genetic mechanisms [46, 104], but also growth factors that regulate

35

recruitment and growth of preantral follicles such as anti-mullerian hormone [106-109],
kit ligand [110-112], activin [113], and GDF-9 [110, 114, 115]. Moreover, from a
neuroendocrine viewpoint, our findings imply that estradiol and inhibin-A may not be the
only feedback factors that regulate secretion of FSH during follicular waves. Rather,
control of FSH secretion during follicular waves may involve other follicular factors such
as activin, which stimulates FSH secretion; follistatin, which inhibits activin action; or
inhibin-B, which inhibits FSH secretion [116]. In addition, differential secretion of
GnRH could also explain our results. Therefore, classification of cattle based on
numbers of antral follicles per wave should provide a novel model to elucidate the role of
the aforementioned factors in regulation of F SH secretion and the dynamics of follicular
growth in future studies.

In summary, the most significant findings in our study demonstrated that: a)
numbers of antral follicles in different follicular waves are highly repeatable (0.95)
within individuals, but highly variable amongst animals, and b) serum F SH
concentrations are inversely associated whereas estradiol and inhibin-A are not associated
with numbers of antral follicles per wave. Based on these results, we conclude that: a)
despite the high variation in number of antral follicles in different waves amongst cattle,
a chronic compensatory mechanism must exist in ovaries to maintain growth of a
constant number of antral follicles in different follicular waves throughout estrous cycles
of individuals, b) the variability in numbers of follicles in waves amongst cattle is
probably not explained by the interaction of serum FSH with inhibin-A and estradiol
concentrations, and c) factors in addition to estradiol and inhibin-A may have a role in

regulation of FSH secretion from the pituitary gland during follicular waves.

36

Figure 3. Alterations in total numbers of antral follicles (2 3mm) during the first and
ovulatory follicular waves of an estrous cycle and during the early stages of the first
follicular wave of the next estrous cycle of four representative Holstein cows with low (<
15), intermediate (15 to 20), high (21 to 25) or very high (> 25) numbers of antral
follicles per follicular wave. Each bar represents the average for the total number of
antral follicles in both ovaries for a single cow determined at each of two daily ultrasound
sessions (0800, 1800). * Above bar = maximal number of follicles in each wave.

Arrows indicate ovulation. Lines represent changes in diameter of the dominant and the
largest subordinate follicle for the first and ovulatory wave. DNF = Dominant non-

ovulatory follicle, DOF = Dominant ovulatory follicle, SF = Largest subordinate follicle.

37

 

 

 

 

 

 

 

 

 

so- Ovrilon Low .1, .21
A 41 .. O. OO ... F18
O. O r O
E 32, no? 0120014 fnor -1s 9
E 60:0 \ O ” 12 m
ca 23‘ I \d O\ * ° 3
14- K
A " ”6 ‘D
v 5 ‘ - . . . . . . F"
60- ~ 21 (D
m ‘1 fl .1 Intermediate ,1, _ 18 q
a) Q: 0 0 - A
_ 32_ N ”“60 .0000 0Q o 15
.2 23‘ ..* .00 .0 5 r12 3
— Q -* I. .3. * l' 9
..6 14 1.85:}, ‘ ..." Q.T a
.. .. 1.. » - » a .. ~° -
— 6°- rrrrrrrrrrrrrr . rrrrr ‘l' rrrrrrrrr P21
to I High 1 _,, U
I. 411 0
4d 32- DC:- mlc: o '15
C °Oo’ * 9° 0’ 6* 00" .p’* - 12 11
(U 23~ .- O. o’ "
{O 9.88.. 1'9 U
h 14 " (5' I, ‘0
0‘1 o- t.” "6
o 54 a 0- (2‘0" ‘3‘ 0 Z
'1 fit IIIII J U U U IIIII
1.. so - 4,* Very ngh 1* - 21 '11
g 41- DNF9°°°*Q 1°“ ‘ ‘9 ..
E ,2. E9.“- Ooeoo .39 .1. m
I: DOF," 09’ - 12 '11
23 “ C5 '0 '1
3 é'p’q. [I a“ _ 9 v
14 ‘1 . " " “<2 ’3 "J.\ 3'
, . ,1; ~ I ; .
5 J 9' 5 o-Qb" L§ R. QC 6

 

 

 

 

"IUIUIIUTT‘YUVYTI'W UUUUUUUUU I Y

1 3 6 7 911131517192123252729

Days After PGF 2

38

Figure 4. Serum hormone profiles, dominant follicle growth, and numbers of antral
follicles for Holstein cows that consistently had low (< 15 follicles per wave, n = 3 cows,
open bars or circles) vs very high (>25 follicles per wave, n = 4 cows, solid bars or
circles) numbers of antral follicles during follicular waves. Cows with low or very high
numbers of antral follicles per wave were identified by ultrasound analysis, as explained
in detail in Methods. Blood sampling began 48 h after a PGFZQ injection and continued
every 4 h until 24 h after ovulation. Thereafter, blood was collected every 8 h until 96 h
after ovulation. Data (including ultrasound results) were aligned relative to the first peak
F SH concentration that occurred within 12 h after ovulation. In the upper panel, circles
represent average (:SEM) concentrations of FSH for the three cows with low (0) vs the
four cows with very high (0) numbers of follicles per wave. In the second panel, circles
represent average (:SEM) estradiol concentrations for cows with low (0) vs very high (0)
numbers of antral follicles per wave, whereas triangles represent average (:SEM)
inhibin-A concentrations for cows with low (13) vs very high (I) numbers Of follicles per
wave. In the third panel, circles represent the average (:SEM) size of the largest and
presumed dominant follicle for cows with low (0) vs very high (0) numbers of antral
follicles per wave. In the bottom panel, bars represent the average (:SEM) for the total
number of antral follicles 2 3 mm in diameter in both ovaries for the three cows with low
(:1) vs the four cows with high (I) numbers of antral follicles per wave. The arrow in the
first panel indicates that ovulation was detected an average of 4 h before the first peak

F SH concentration after ovulation. In the third panel, follicle wave emergence occurred

coincident with the day of ovulation, and the arrow depicts deviation in growth of

39

dominant vs the largest subordinate follicles (not shown), which occurred an average of

63 h after the first peak F SH concentration after ovulation.

40

FSH (nglml)

Diameterof lrhlbh-An |
DF(mm) ( 9"“)

Numbers of
antral follicles

1.20-

1.00:1

030"

0.60 r

0.40 r

2”-

 

--12 2436486072841~4

 

   

A121,: A «may?

L l
I I I I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.24-120 T224348607284
Hours from the first peak FSH
concentatlon after ovulalon

41

(iwlfid) 101921153

Table 1. Efi‘ect of age, season, number of follicular waves per estrous cycle, lactational
status, and technician on repeatability of maximal numbers of antral follicles ([1 3mm in

diameter) per follicular wave.A

 

Ramdrurberscf
Nrrberd‘ Nrrbasd stafclliclesper
airrds Waves fdliala'wave W
Season
Spring 5 18 8-3 0%
Surrrer 30 % 11-54 0%
Wirter 9 24 12-38 094
Nrrberdfcllicua
uavesper
meg/Clea
Two 28 86 8-54 093
Thee 7 28 11 -44 0%
LadaicrdStatus
Ladaing 20 58 12-44 094
hut-lactating 24 80 8-54 0.93
T I . .
DB 39 120 11-54 0%
FJ 5 18 8-39 0%
Age
Heifersc 19 56 8-42 0.92
Cows 25 82 11 -54 0%
Overdl 44 138 8-54 0%

 

A Cattle from Study 2 (Parts A and B) and Study 4 were used for this analysis.
8 Cattle from Study 2 (Parts A and B) were used for this analysis
C Heifers were 10 to 12 months old, whereas cows were 3 to 7 years old.

42

Table 2. Distribution of numbers of heifers or cows with two or three follicular waves
per estrous cycle in the low (< 15 follicles), intermediate (15-20 follicles), high (21-25
follicles), or very high (> 25 follicles) numbers of antral follicles (2 3 mm in diameter)

per follicular wave classifications'.

Classification of cattle with different
numbers of antral follicles per follicular wave

 

 

Low Intermediate High Very High
Number of cattle with
two follicular waves 6 9 7 6
per estrous cycle:
Heifers 3 4 3
Cows 1 6 3 3
Number of cattle with
three follicular waves 3 1 1 2
per estrous cycle:
Heifers l 1 0 l
Cows 2 0 1 1
Overall: 9 10 8 8
Heifers 6 4 4 4
Cows 3 5 4 5

 

' Holstein heifers (n = 18, 10 to 12 months of age) and cows (n = 17, 4 to 7 years of age)
were given two injections of PGan spaced 11 days apart to synchronize occurrence of
estrous cycles. Each animal was subjected to twice daily ultrasound analysis (0800 and
1800) beginning at the time of the second PGFZa injection and continuing until 2 days
after the spontaneous ovulation of the subsequent estrous cycle. At each ultrasound
session, total number of antral follicles 2 3 mm in diameter in ovaries of each animal was
determined. Each animal was classified based on the average of the maximal numbers of
antral follicles for 3 or 4 follicular waves.

43

Table 3. Distribution of maximal numbers of antral follicles per follicular wave in
different follicle size categories for cattle that consistently had low (< 15 follicles),
intermediate ( 15 to 20 follicles), high (21 to 25 follicles), or very high (> 25 follicles)

numbers of antral follicles (2 3 mm in diameter) per follicular wave'.

 

 

Nn'ba'sd‘a'tra 0‘35.de Smdftidstmj
folides N2 Widespam 113 3-39 4-49 5-59 6-7.9 8-9.9 >10
4 a5 a a a
Low 9 12811.5 3109106 29:03 31102 2910316102 1.5101

1mm 10 108:1.7b 29 14.6105" 4.1:03basxo2w30102 1.6202 1.6201
c b c d)
Hg» 8 2111.9 25 190107 5410.5 35103 2810215101 1.4102

VayHg1 6 30611.55d 30 2100126c 02:04c 4.1102b3410.41.710.2 1.6102

 

1 Holstein heifers (n = 18, 10 to 12 months of age) and cows (n = 17, 4 to 7 years of age)
were given two injections of PGF;Cl spaced 11 days apart to synchronize occurrence of
estrous cycles. Each animal was subjected to twice daily ultrasound analysis (0800 and
1800) beginning at the time of the second PGF2a injection and continuing until 2 days
after the spontaneous ovulation of the subsequent estrous cycle. At each ultrasound
session, total number of antral follicles 2 3 mm in diameter in ovaries of each animal was
determined. Each animal was classified based on the average of the maximal numbers of
antral follicles for 3 or 4 follicular waves.

2 N = number of animals

3 N = number of follicular waves.

4 Number of cattle and total number of waves examined by ultrasound for each antral
follicle growth classification follows: Low (n= 9 animals, n= 30 waves), Intermediate
(n= 10 animals, n= 29 waves), High (n= 8 animals, 25 waves), Very High (n= 8 animals,
n= 30 waves).

5 Means within a column with uncommon superscripts are significantly (P< 0.05)
different.

Chapter 2
Appendix of Data for Individual Animals

In Study 2 (Parts A and B) and Study 4

45

Table 4A
Table 43

Table 4C

Table 5A
Table SB

Table 5C

Table 6A
Table 63

Table 6C

Table 7A
Table 78

Table 7C

Table 8A
Table 8B

Table 8C

Table 9A
Table 9B

Table 9C

LIST OF TABLES

Animal information for cow ID 3547 ..................................... 59
Follicle wave dynamics for cow ID 3547 ................................. 59
Distribution of maximal number of antral follicles by size for cow

ID 3547 ........................................................................ 59
Animal information for cow ID 3612 ..................................... 61
Follicle wave dynamics for cow ID 3612 ................................. 61
Distribution of maximal number of antral follicles by size for cow

ID 3612 ........................................................................ 61
Animal information for cow ID 3713 ..................................... 63
Follicle wave dynamics for cow ID 3713 ................................. 63
Distribution of maximal number of antral follicles by size for cow

ID 3713 ........................................................................ 63
Animal information for heifer 3879 ........................................ 65
Follicle wave dynamics for heifer ID 3879 ............................... 65
Distribution of maximal number of antral follicles by size for

heifer ID 3879 ................................................................ 65
Animal information for heifer ID 3880 ................................... 67
Follicle wave dynamics for heifer ID 3880 .............................. 67
Distribution of maximal number of antral follicles by size for

heifer ID 3880 ................................................................ 67
Animal information for heifer 3887 ........................................ 69
Follicle wave dynamics for heifer ID 3887 ............................... 69

Distribution of maximal number of antral follicles by size for
heifer ID 3887 ................................................................ 69

46

Table 10A
Table 103

Table 10C

Table 11A
Table 11B

Table 11C

Table 12A
Table 123

Table 12C

Table 13A
Table 13B

Table 13C

Table 14A
Table 14B

Table 14C

Table 15A
Table ISB

Table 15C

Table 16A

Table 168

Animal information of heifer ID 3889 ..................................... 71

Follicle wave dynamics for heifer ID 3889 ............................... 71
Distribution of maximal number of antral follicles by size for

heifer ID 3889 ................................................................. 71
Animal information for heifer ID 3896 ................................... 73
Follicle wave dynamics for heifer ID 3896 ............................... 73
Distribution of maximal number of antral follicles by size for

heifer ID 3896 ................................................................. 73
Animal information for cow ID 3475 ...................................... 75
Follicle wave dynamics for cow II) 3475 ................................. 75
Distribution of maximal number of antral follicles by size for cow

ID 3475 ........................................................................ 75
Animal information for cow ID 3507 ...................................... 77
Follicle wave dynamics for cow ID 3507 ................................. 77
Distribution of maximal number of antral follicles by size for cow

ID 3507 ........................................................................ 77
Animal information for cow ID 3534 ..................................... 79
Follicle wave dynamics for cow ID 3534 ................................. 79
Distribution of maximal number of antral follicles by size for cow

ID 3534 ........................................................................ 79
Animal information for cow ID 3695 ...................................... 81
Follicle wave dynamics for cow ID 3695 ................................. 81
Distribution of maximal number of antral follicles by size for cow

ID 3695 ........................................................................ 81
Animal information for cow ID 3704 ..................................... 83
Follicle wave dynamics for cow ID 3704 ................................. 83

47

Table 16C

Table 17A
Table 173

Table 17C

Table 18A
Table 18B

Table 18C

Table 19A.

Table 193

Table 19C

Table 20A

Table 20B

Table 20C

Table 21A

Table 218

Table 21C

Table 22A

Table 22B

Table 22C

Table 23A

Distribution of maximal number of antral follicles by size for cow
ID 3704 ......................................................................... 83

Animal information for heifer ID 3894 ................................... 85
Follicle wave dynanrics for heifer ID 389485

Distribution of maximal number of antral follicles by size for

heifer ID 3894 ................................................................. 85
Animal information for heifer ID 3901 .................................... 87
Follicle wave dynamics for heifer ID 3901 .............................. 87
Distribution of maximal number of antral follicles by size for

heifer ID 3901 ................................................................ 87
Animal information for heifer ID 3903 ................................... 89
Follicle wave dynamics for heifer ID 3903 ............................... 89
Distribution of maximal number of antral follicles by size for

heifer ID 3903 ................................................................. 89
Animal information for cow ID 3160 ...................................... 91
Follicle wave dynamics for cow ID 3160 ................................. 91
Distribution of maximal number of antral follicles by size for cow

ID 3160 ........................................................................ 91
Animal information for cow ID 3459 ...................................... 93
Follicle wave dynamics for cow ID 3459 ................................. 93
Distribution of maximal number of antral follicles by size for cow

ID 3459 ........................................................................ 93
Animal information for cow ID 3533 ..................................... 95
Follicle wave dynamics for cow ID 3533 ................................. 95
Distribution of maximal number of antral follicles by size for cow

ID 3533 ........................................................................ 95
Animal information for cow ID 3785 ..................................... 97

48

Table 238

Table 23C

Table 24A
Table 248

Table 24C

Table 25A
Table 253

Table 25C

Table 26A
Table 26B

Table 26C

Table 27A
Table 278

Table 27C

Table 28A
Table 288

Table 28C

Table 29A
Table 298

Table 29C

Follicle wave dynamics for cow ID 3785 ................................. 97

Distribution of maximal number of antral follicles by size for cow

ID 3785 ........................................................................ 97
Animal information for heifer 3892 ....................................... 99
Follicle wave dynamics for heifer ID 3982 .............................. 99
Distribution of maximal number of antral follicles by size for

heifer ID 3892 ................................................................. 99
Animal information for heifer ID 3983 ................................. 101

Follicle wave dynamics for heifer ID 3983 ............................. 101

Distribution of maximal ntunber of antral follicles by size for

heifer ID 3893 ............................................................... 101
Animal information for heifer ID 3900 ................................. 103

Follicle wave dynamics for heifer ID 3900 ............................. 103
Distribution of maximal number of antral follicles by size for

heifer ID 3900 ............................................................... 103
Animal information for heifer 3905 ...................................... 105

Follicle wave dynamics for heifer ID 3905 ............................. 105

Distribution of maximal number of antral follicles by size for

heifer ID 3905 ............................................................... 105
Animal information for cow ID 3391 .................................... 107
Follicle wave dynamics for cow ID 3391 ............................... 107
Distribution of maximal number of antral follicles by size for cow

ID 3391 ....................................................................... 107
Animal information for cow ID 3434 .................................... 109
Follicle wave dynamics for cow ID 3434 ................................ 109

Distribution of maximal number of antral follicles by size for cow

49

Table 30A

Table 30B

Table 30C

Table 31A
Table 31B

Table 31C

Table 32A

Table 323

Table 32C

Table 33A
Table 333

Table 33C

Table 34

ID 3434 ....................................................................... 109

Animal information for cow ID 3500 .................................... 111
Follicle wave dynamics for cow ID 3500 ............................... 111
Distribution of maximal number of antral follicles by size for cow

ID 3500 ...................................................................... 111
Animal information for cow ID 3531 .................................... 113
Follicle wave dynamics for cow ID 3531 ............................... 113
Distribution of maximal number of antral follicles by size for cow

ID 3531 ...................................................................... 113
Animal information for heifer ID 3888 .................................. 1 15
Follicle wave dynamics for heifer ID 3888 ............................. 115
Distribution of maximal number of antral follicles by size for

heifer ID 3888 ............................................................... 115
Animal information for heifer ID 3897 ................................. 117
Follicle wave dynamics for heifer ID 3897 ............................. 117

Distribution of maximal number of antral follicles by size for
heifer ID 3897 ............................................................... 117

Ages and maximal numbers of antral follicles after ovulation for
three consecutive synchronizations ....................................... 127

50

LIST OF FIGURES

Figure 5 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3547 ............................................. 60

Figure 6 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3612 .............................................. 62

Figure 7 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3713 .............................................. 64

Figure 8 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3879 ............................................ 66

Figure 9 Alterations in total numbers of antral follicles (a 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3880 ............................................ 68

Figure 10 Alterations in total numbers of antral follicles (_>_ 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3887 ............................................ 70

Figure 11 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3889 ............................................ 72

Figure 12 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3896 ............................................ 74

Figure 13 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3475 .............................................. 76

51

Figure 14 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3507 .............................................. 78

Figure 15 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3534 .............................................. 80

Figure 16 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3695 .............................................. 82

Figure 17 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3704 .............................................. 84

Figure 18 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3894 ............................................ 86

Figure 19 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3901 ............................................ 88

Figure 20 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3903 ............................................ 90

Figure 21 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3160 ............................................. 92

Figure 22 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3459 ............................................. 94

52

Figure 23 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3533 ............................................. 96

Figure 24 Alterations in total ntunbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3785 ............................................. 98

Figure 25 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3892 ........................................... 100

Figure 26 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3893 .......................................... 102

Figure 27 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3900 ........................................... 104

Figure 28 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3905 .......................................... 106

Figure 29 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3391 ............................................ 108

Figure 30 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3434 ............................................ 110

Figure 31 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3500 ............................................ 112

Figure 32 Alterations in total numbers of antral follicles (2 3mm) during

53

the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3531 ............................................ 114

Figure 33 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3888 ........................................... 116

Figure 34 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3897 ........................................... 118

Figure 35 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3609 ............................................ 120

Figure 36 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for cow ID 3762 ............................................ 121

Figure 37 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3976 ........................................... 122

Figure 38 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3977 .......................................... 123

Figure 39 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next
estrous cycle for heifer ID 3980 .......................................... 124

Figure 40 Alterations in total numbers of antral follicles (2 3mm) during
the first and ovulatory follicular waves of an estrous cycle and
during the early stages of the first follicular wave of the next

estrous cycle for heifer ID 3981 .......................................... 125
Figure 41 Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3238 ..................... 128

54

Figure 42

Figure 43

Figure44

Figure 45

Figure 46

Figure 47

Figure 46

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

Figure 54

Figure 55

Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for
cow ID 3238 ............................................................. 129

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3495 ..................... 130

Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for
cow ID 3495 ............................................................. 131

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3531 ..................... 132

Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for
cow ID 3531 ............................................................. 133

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow II) 3674 ..................... 134

Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for
cow ID 3674 ............................................................. 135

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3539 ..................... 136

Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for
cow ID 3539 ............................................................. 137

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3456 ...................... 138

Inhibin-A, estradiol, and FSH profiles aligned to the first F SH surge for
cow ID 3456 ............................................................. 139

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3612 ..................... 140

Inhibin-A, estradiol, and F SH profiles aligned to the first FSH surge for
cow ID 3612 ............................................................. 141

Alterations in total numbers of antral follicles (2 3mm) during
the first wave of an estrous cycle of cow ID 3783 ..................... I42

55

Part A

Tables and figures for individual animals in Study 2A

Definitions and descriptions of tables for each animal in Study 2A.
‘ Table A shows the ages, milking status and genetic background for each animal, Table B
shows follicular wave dynamics, and Table C shows the distribution of maximal numbers

of antral follicles per follicular wave in different follicle size categories for each animal.

Table A. Definitions

NL non-lactating

L lactating

NA not available

Table B. Definitions

Classification

1 low

2 intermediate

3 high

4 very high

Dominant Follicle Growth

DF dominant follicle

DF Dev size of the dominant follicle at deviation

Sub Dev size of the largest subordinate follicle at deviation
DF Peak largest diameter of the dominant follicle

Sub Peak largest diameter of the largest subordinate follicle

Definitions and description for figures for each animal in Study 2A.

Alterations in total numbers of antral follicles (2 3mm) during the first and
ovulatory follicular waves of an estrous cycle and during the early stages of the first
follicular wave of the next estrous cycle of each animal in this study. Each bar represents
the average for the total number of antral follicles in both ovaries determined at each of
two daily ultrasound sessions (0800, 1800). * Above bar = maximal number of follicles

in each wave. Arrows indicate ovulation. Lines represent changes in diameter of the

56

dominant and the largest subordinate follicle for the first and ovulatory wave. DNF =
Dominant non-ovulatory follicle, DOF = Dominant ovulatory follicle, SF = Largest

subordinate follicle.

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Part B

Figures for individual animals in Study 23

Definitions and description for Figures from Study 23

Alterations in total numbers of antral follicles (2 3mm) during the first and
ovulatory follicular waves of an estrous cycle and during the early stages of the first
follicular wave of the next estrous cycle by a second ultrasound technician of each animal
in this study. Each bar represents the average for the total number of antral follicles in
both ovaries determined at each of two daily ultrasound sessions (0800, 1800). "' Above
bar = maximal number of follicles in each wave. Arrows indicate ovulation. Lines
represent changes in diameter of the dominant and the largest subordinate follicle for the
first and ovulatory wave. DNF = Dominant non-ovulatory follicle, DOF = Dominant

ovulatory follicle, SF = Largest subordinate follicle.

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Part [11
Figures and tables

from study 4

Definitions and description for Figures from study 23

l. Alterations in total numbers of antral follicles (_>_ 3mm) during the first wave of
an estrous cycle and bleeding period of each cow in this study. Each bar represents the
average for the total number of antral follicles in both ovaries determined at each of two
daily ultrasound sessions (0800, 1800). " Above bar = maximal number of follicles in
each wave. Arrows indicate ovulation. Lines represent changes in diameter of the
dominant and the largest subordinate follicle for the first and ovulatory wave. DNF =
Dominant non-ovulatory follicle, DOF = Dominant ovulatory follicle, SF = Largest
subordinate follicle.

2. Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for each
cow in this study. Lines represent changes in concentrations of inhibin-A, estradiol, and

F SH. Arrows indicate ovulation.

125

Table 35. Ages and maximal numbers of antral follicles afier ovulation for three

consecutive synchronizations.

 

 

Maximal number of antral follicles
Cow ID Age Synch 1 Synch 2 Bleed AVG]
3238 5y 2mo 34 37 44 38.3 e 2.9
3456 4y 101110 17 13 16 15.3.1.2
3495 4y 4mo 36 32 31 33.0 :h 1.5
3539 4y 3mo 15 2 18 14 15.61: 1.2
3531 4y 6mo 33 29 32 31.3 e 1.2
3612 4y 71110 13 13 11 12.3:O.6
3783 3y 9mo 14 16 23 3 17.6 2 2.7
3674 4y 9mo 35 38 37 36.6 e 0.9

 

‘ Average i standard error of the mean
2 Did not synchronize

3 Became cystic GnRH administered

126

 

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127

Figure 42. Inhibin-A, estradiol, and F SH profiles aligned to the first F SH surge for cow

ID 3238.

Estradiol (pg/ml) Inhibin-A (nglml)

FSH (nglml)

 

 

 

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Flgure 44. Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for cow

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Figure 46. Inhibin-A, estradiol, and FSH profiles aligned to the first FSH surge for cow

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Figure 48. Inhibin-A, estradiol, and F SH profiles aligned to the first FSH surge for cow

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Figure 52. Inhibin-A, estradiol, and F SH profiles aligned to the first FSH surge for cow

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