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HORMONAL MAN IPULATION OF ESTRUS AND OVULATION IN
THE GILT

presented by

RODRIGO MANJARIN ARREDONDO

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Large Animal Clinical Sciences

 

 

 

 

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5/08 K:IProj/Achres/ClRC/DateDuemdd

HORMONAL MAN IPULATION OF ESTRUS AND OVULATION IN THE GILT

BY

Rodrigo Manjarin Arredondo

A THESIS

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

MASTERS OF SCIENCE

Department of Large Animal Clinical Sciences

2008

ABSTRACT

HORMONAL MANIPULATION OF ESTRUS AND OVULATION IN THE GILT
By

Rodrigo Manjarin Arredondo

To meet weekly breeding targets, it is occasionally necessary to inject exogenous
gonadotrophins to induce estrus in prepubertal gilts. However, estrus and ovulation
responses to gonadotrophin treatment have often proven unpredictable. The objective of
the studies outlined in this thesis was to examine possible reasons for this unpredictability.
In the initial experiment, prepubertal gilts received an injection of either eCG (equine
chorionic gonadotrophin) or a combination of eCG and hCG (human chorionic
gonadotrophin), or were not injected controls. At the time of injection, the gilt ovaries had
1 to 2 mm follicles. The estrus and ovulation responses were consistently lower for eCG
than for the combination of eCG-hCG, and the failure to express estrus in eCG-hCG
treated gilts was not associated with a premature rise in progesterone. In the second
experiment, gilts were selected at 150 d or 180 d of age and assigned to injection of eCG-
hCG, eCG alone, pretreatment with follicular stimulating hormone (FSH) followed by
eCG, or controls. The estrus and ovulatory responses of gilts were consistently higher for
gilts treated with the combination eCG and hCG than for eCG groups. In the third
experiment, gilts were assigned to injection of hCG alone, pretreatment with FSH in
polyvinylpyrrolidinone (PVP) followed by hCG, or FSH pretreatment followed by eCG.
Pretreatment with FSH increased ovulatory response to hCG compared to hCG alone.

Pretreatment with FSH also increased ovulatory response to hCG compared to eCG.

DEDICATION

To the eternal care, to my parents and sister

iii

ACKNOWLEDGMENTS

I am extremely grateful to many people that have contributed to the thesis
presented here. In particular, I would like to thank Dr. Roy Kirkwood for providing me the
opportunity to undertake these studies and his continued support, advise and education. I
would also like to sincerely thank the other members of my committee, Dr. Carlos
Dominguez Tejerina, Dr. David Sprecher, Dr. Jose Cibelli, Dr. Robert Friendship and Dr.
Glen Cassar for their support and advise during my studies in the USA, Canada, and

Spain.

MJ. Castro and Evaristo Aguado, porque sin su inmensa ayuda nunca habria sido

posiblc realizar la investigacién en Espaﬁa.

I would also like to acknowledge Blas, Julio, Javier, Fernando, Victor and Chechu
who contributed to this thesis with their time and comprehension.
Last but not least, I would like to thank Faith Peterson for always being available

to solve problems and provide support.

TABLE OF CONTENTS

 

 

LIST OF TABLES ............................................................................................................ VII
LIST OF FIGURES ................. VIII
LIST OF ABREVIATIONS ............................................................................................... IX
CHAPTER 1
LITERATURE REVIEW: SWINE REPRODUCTIVE PHYSIOLOGY ....................... l
Oogenesis and follicular development in the pig ............................................................ 1
Hormonal proﬁles and ovarian development before puberty .......................................... 6
LH and FSH secretion ............................................................................................ 6
Regulation of gonadotrophin secretion (the gonadostat theory): ........................... 7
Ovarian dynamics before puberty: ......................................................................... 9
Hormonal proﬁles and ovarian development after puberty ........................................... 10
GnRH secretion .................................................................................................... 10
LH secretion ......................................................................................................... lO
FSH secretion and ovarian dynamics ................................................................... ll
Estrogen and progesterone secretion .................................................................... l3
Follicular recruitment and selection .............................................................................. 14
Recruitment .......................................................................................................... 14
Selection. .............................................................................................................. l6
Stimulating pubertal estrus ............................................................................................ 19
Boar exposure ....................................................................................................... l9
Exogenous hormones ........................................................................................... 21
References ...................................................................................................................... 24
CHAPTER 2
EFFECT OF ECG OR ECG PLUS HCG ON ESTRUS EXPRESSION AND
OVULATION IN PREPUBERTAL GILTS. - _ 28
Abstract. ......................................................................................................................... 28
Introduction .................................................................................................................... 29
Materials and Methods .................................................................................................. 30
Results ............................................................................................................................ 32
Discussion ...................................................................................................................... 34
Acknowledgements ........................................................................................................ 36
References ...................................................................................................................... 37

CHAPTER 3

EFFECT OF PRIOR TREATMENT ON THE ESTRUS AND OVULATION

RESPONSES TO ECG IN PREPUBERTAL GILTS - -- -
Abstract ..........................................................................................................................
Introduction ....................................................................................................................
Materials and Methods ..................................................................................................
Results ............................................................................................................................

 

Discussion ......................................................................................................................
Acknowledgements ........................................................................................................
References ......................................................................................................................
CHAPTER 4
EFFECT OF HCG TREATMENT ON THE ESTROUS AND OVULATION
RESPONSES TO FSH IN PREPUBERTAL GILTS
Abstract ..........................................................................................................................
Introduction ....................................................................................................................

 

Material and methods ....................................................................................................
Results ............................................................................................................................
Discussion ......................................................................................................................
Acknowledgements ........................................................................................................
References ......................................................................................................................

CHAPTER 5
SUMMARY AND CONCLUSIONS - --

 

vi

LIST OF TABLES

Table 1.1. Estrus responses of gilts to PG6OO injection ...................................................... 21
Table 1.2 Long-term performance of gilts bred at a PG600-induced or natural ﬁrst estrus 22

Table 2.1. Inﬂuence of eCG or eCG plus hCG on incidence of estrus by 7 days and
ovulation as indicated by elevated progesterone (+P4) or non-ovulation (-P4). 33

Table 2.2. Inﬂuence of gilt weight on the estrus response to eCG or eCG plus hCG ......... 33

Table 3.1. Inﬂuence of gilt age (150 vs. 180 d) on the estrus and ovulation responses to
eCG plus hCG combination, eCG alone, FSH pre-treatment then eCG, or no
treatment ............................................................................................................. 44

Table 4.1. Effect of injecting hCG, or FSH followed by eCG, or F SH followed by hCG,
into ISO-day gilts on the incidence of estrus by 7 days and ovulation as
indicated by elevated progesterone (+P4), or non-ovulation (-P4). .................... 54

vii

LIST OF FIGURES

Figure 1.1 Distribution of oogonia and oocytes in developing gilts (From Black and
Erickson 1968) ...................................................................................................... 2

Figure 1.2 Representative scheme of the structure of the ovary and the different phases in
the development of the follicle, corpus luteum and corpus albicans (From
Sacristan et al. 1996) ............................................................................................. 4

Figure 1.3 Mechanism involved in the development of an estrogen-active dominant follicle
(From Driancourt 2001). ....................................................................................... 5

Figure 1.4 Changes in mean luteinizing hormone (LH) and estrogen concentrations and
numbers of follicles greater than 3 mm in diameter in gilts during sexual
maturation (From Evans and Doherty 2001). ....................................................... 7

Figure 1.5 Different selection mechanisms between cattle and pigs (based on data from
Lucy 2007 and Driancourt 2001). ....................................................................... 17

Figure 1.6 Models for events during follicular selection. According to the Missouri model,
the initiating event is an increase in the LH receptor (LHR) in the granulosa cell
layer. According to the Cornell model, the initiating event is the increase in
pregnancy-associated plasma protein-A (PAPP-A). In each case, there is an
increase in estradiol (E) and inhibin (I) that feeds back negatively on FSH
secretion at the hypothalamo-pituitary axis. A = androgen; F SHR = F SH
receptor (From Lucy 2001). ................................................................................ 18

viii

LIST OF ABREVIATIONS

AI ........................................................................................................ artiﬁcial inseminations
Dpc ................................................................................................................ days post coitum
eCG ...................................................................................... equine chorionic gonadotrophin
F SH ............................................................................................ follicle stimulating hormone
GnRH ................................................................................ gonadotrophin releasing hormone
hCG ...................................................................................... human chorionic gonadotrophin
1M ...................................................................................................................... intramuscular
IU ................................................................................................................. international unit
LH ........................................................................................................... luteinizing hormone
NPD ........................................................................................................ non-productive days
PVP ..................................................................................................... polyvinylpyrrolidinone
SEM .............................................................................................. standard error of the mean
IGF-1 .................................................................................................. insulin growth factor-1
IGF BP ......................................................................... insulin growth factor binding protein
PAPP-A ................................................................... pregnancy-associated plasma protein-A

ix

CHAPTER 1
LITERATURE REVIEW: SWINE REPRODUCTIVE PHYSIOLOGY

Oogenesis and follicular development in the pig

In the early swine embryo (24 to 26 (I post coitum; dpc), primordial germ cells
migrate from the yolk sac into the undifferentiated gonad, the genital ridge, and organize
into the sex chords. The germ cells then undergo mitosis and their number increases from
5 x 103 at about 24 dpc to more than 1 x 106 at 50 dpc (Black and Erickson 1968). In the
absence of testis determining factor, the sex cords fragment into cellular clusters, each
enclosing a primitive germ cell. These clusters differentiate into primitive follicular cells
and the bulk of the genital ridge becomes the ovary. The germ cells change into oogonia
by successive mitotic divisions and then into primary oocytes through the ﬁrst meiotic
division. The oocytes are then arrested at the G2 and do not resume meiosis until
immediately before ovulation. The time needed for transformation of oogonia to oocytes
is longer in pigs than in other mammalian species (Black and Erickson 1968). The ﬁrst
oogonia enter meiotic prophase before day 40 of intrauterine development and their

transformation is not complete until approximately 30 days postpartum (Fig.1 .1).

Primary oocytes are surrounded by one or two layers of ﬂattened follicular cells
forming the ovarian primordial follicles. The ﬁrst primordial follicles are observed by 56
dpc and increase in number throughout prenatal life (Bielanska-Osuchowska 2006).

Approximately 500,000 primordial follicles are present in both ovaries by 10 (I post- --

partum in swine, and constitute the maximum number available throughout reproductive

life (Black and Erickson 1968).

 

100. HOOsonia
80.. O---000cytes
96 6""
40-1
204
0 I I I T W I I r k‘rﬁr‘

 

‘
30 4o 50 60 7o 80 go 100 no B’Il 10 20 30

Figure 1.1 Distribution of oogonia and oocytes in developing gilts (From Black and
Erickson 1968)

Within 10 d post partum some primordial follicles develop to the primary follicle
stage (0.12 mm), which is an oocyte with one to three layers of granulosa cells (Knox
2005). Initiation of growth of primordial follicles involves endocrine actions and
regulatory effects of local factors from the somatic cells of the follicle (Hirshﬁeld 1991)

and probably from the growing oocyte (Picton et al. 1998).

Follicular cells continue to proliferate to form several layers of granulosa] cells, as
well as the surrounding theca cells, becoming transformed into secondary follicles

(Sacristan et al. 1996; Fig. 1.2). This follicular development from the primary to

secondary follicle is independent of pituitary gonadotrophins, being observed also in
hypophysectomized animals. The follicle enters the secondary stage with 3 to 20 layers of
granulosa cells and a diameter of 0.14 to 0.40 mm. Growth beyond 0.4 mm is associated
with the formation of an antrum. In the antral (tertiary) class, most follicles range in size
from 0.4 to 1.5 mm with 10 to 30 layers of granulosa cells (Knox 2005). The follicular
ﬂuid is derived from the blood as well as substances synthesized by the theca and
granulosa cells. During antrum formation granulosa cells continue to proliferate and start
to develop F SH receptors. At the same time, theca cells appear completely differentiated
forming two concentric layers, the internal theca (with LH receptors) and external theca.
During maturation, cells of the theca and granulosa layers synthesize and secrete
signiﬁcant amounts of steroids that along with FSH and LH allow follicles to grow and

reach their ﬁnal development (Cardenas and Pope 2002).

As described by Richards (2001), once the follicle reaches a critical size, the
granulosa cells attached to the basal lamina become distanced from the oocyte-cumulus
complex and establish a second micro-environment in the follicle. This separation may
necessitate the regulation of mural granulosa cells by factors/hormones other than those
from the oocyte-cumulus complex. These include IGF-1 and F SH receptors in granulosa
cells, and LH receptors in theca cells. Locally, FSH dictates speciﬁc changes in granulosa
cell gene expression, enhanced proliferation, and the formation of the antrum that

separates the oocyte-cumulus component from the mural layer of endocrine cells.

ANTRAI. FOLLICLE
SECONDARY

 
 
      
   
      

 
 
 

  

    

 

   
     
  
   
   
    

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Figure 1.2 Representative scheme of the structure of the ovary and the different

phases in the development of the follicle, corpus luteum and corpus

albicans (From Sacristan 1996)

Ultimately, granulosa cells express genes for LH receptor, P450 aromatase,
activin, and inhibin. Theca cells differentiate to produce androgens via P45017u. All these
changes increase steroidogenesis that inhibits secretion of FSH, starving small follicles

into atresia and leading to the positive feedback of estradiol to trigger the ovulatory LH

surge.

 

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roteol
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-hishinhibln from

 

   

 

 

6
Cholesterol Testosterone Estrndiol
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Figure 1.3 Mechanism involved in the development of an estrogen-active dominant

follicle (From Driancourt 2001).

One controller of granulosa cell differentiation is FSH although IGF-1 and
estradiol exert supportive roles (Driancourt 2001, Fig. 1.3). For example, although IGF-1
and estradiol cannot induce expression of sterodigenic enzymes, they enhance the effect
of F SH to further increase aromatase activity. The FSH reduces amounts of IGF binding
protein (IGF BP) and stimulates production of follistatin and a protease (pregnancy-
associated plasma protein-A, PAPP-A) that also degrades IGF BP within the follicular

ﬂuid. The net decrease in IGF BP leads to an increase in free IGF-I that potentiates the

effect of LH and FSH. Also, the increased follistatin binds follicular activin changing the
inhibin/activin balance towards inhibin. A paracrine effect of inhibin produced within the

granulose cell layer also potentiates the action of LH in the theca layer.

Hormonal profiles and ovarian development before puberty

LH and FSH secretion: In prepubertal gilts, LH is a key hormone that appears to
control ovarian development and the age at puberty (Evans and O’Doherty 2001).
Concentrations of LH in blood decrease from birth to about 40 d of age, then increase to
120 d of age, then decline to a nadir by about 180 d of age (Colenbrander et al. 1977;
Pelletier et al. 1981; Diekman et al. 1983; Camous et al. 1985). From this nadir until ﬁrst
ovulation (puberty), LH concentrations increase (Pelletier et al. 1981; Prunier et al.
1993), as is typical in most species (Fig. 1.4). This peripubertal increase in LH secretion
is characterised by an increase in mean LH concentrations and pulse frequency (Pelletier
et al. 1981; Prunier et al. 1993), and is associated with ﬁnal maturation of ovarian

follicles (Beltranena et al. 1993) culminating in the preovulatory LH surge.

The FSH concentrations in blood are high early in life and then decrease after 70
to 125 days of age, and do not increase as puberty approaches (Diekman et al. 1983;
Camous et al. 1985; Prunier et al. 1993). Estradiol concentrations are low throughout the
majority of the prepubertal period but increase prior to puberty (Esbenshade et al. 1982;
Lutz et al. 1984; Camous et al. 1985). Progesterone concentrations only increase after

puberty with the formation of corpora lutea (Esbenshade et al. 1982; Prunier et al. 1993).

a S

l

LH

Concentration
8

(arbitrary units)
b
O

N
O
1

 

 

O
I

I I I I I I

80 120 160 200 240

O
A
O

204
15-

 
    

 

 

Number follicles
>3 mm In diameter
0 m 8

T I I I l

0 40 80 120 160 200 240

Days of age

Figure 1.4 Changes in mean luteinizing hormone (LH) and oestrogen concentrations
and numbers of follicles greater than 3 mm in diameter in gilts during

sexual maturation (From Evans et al. 2001).

Regulation of gonadotrophin secretion (the gonadostat theory): Regulation of
gonadotrophin secretion during the prepubertal period occurs via changes in GnRH
secretion. Synthesis of GnRH in the female is in neurosecretory cells located in two
separate areas in the hypothalamus, the tonic center and the surge center. Anatomically,
the surge center consists of three hypothalamic nuclei known as the preoptic nucleus, the
anterior hypothalamic area and the suprachiasmatic nucleus. The tonic center consists of

two nuclei, the ventromedial and arcuate nuclei. Axons from cells of the surge and tonic

7

centers extend into the pituitary stalk regions where the nerves terminate on a capillary
network called the hypothalamo-hypophyseal portal system. This system allows GnRH to
act immediately on the cells of the pituitary causing the release of the gonadotrophins

FSH and LH.

The tonic center is responsible for basal secretion of GnRH. The neurons in this
center release small pulses of GnRH over a prolonged period of time (tonic release of
GnRH occurs during the entire estrous cycle). The surge center is responsible for the
preovulatory release of GnRH. In contrast to the tonic release of GnRH, the preovulatory
surge occurs only once during the estrous cycle. Prior to puberty, the tonic centre has a
high sensitivity to negative feedback by estrogen and therefore GnRH secretion is
inhibited. During the immediate peripubertal period, the prevailing theory is that the
negative feedback sensitivity of the tonic centre to estradiol decreases allowing greater
GnRH secretion (gonadostat hypothesis). This permits LH secretion to increase (Pelletier
et al. 1981; Prunier et al. 1993) even in the face of increasing estradiol concentrations
(Lutz et al. 1984; Camous et al. 1985). When the estrogen concentrations in the blood
reach a high level the surge center becomes positively stimulated and releases larger
quantities of GnRH. This stimulates the surge of LH causing ovulation and triggering the
onset of the puberty. While there is evidence to support this theory (Berardinelli et al.
1984; Lutz et al. 1984), others have been unable to demonstrate a reduction in estradiol

negative feedback prior to ﬁrst ovulation (Elsaesser et al. 1991).

Ovarian dynamics before puberty: Antral follicles are ﬁrst observed
histologically at approximately 65 d of age and emerge from the ovarian surface at
approximately 80 d of age (Black and Erickson 1968). The formation and increase in
number of antral follicles around 100 d may be due to the early transient increase in LH
secretion (Evans and O’Doherty 2001) and FSH secretion (Camous et al. 1985).
Although these ﬁrst antral follicles formed during the prepubertal period potentially have
the ability to ovulate, all of them suffer atresia. The increased incidence of atresia after
120 d is coincident with a 60% decrease in mean plasma concentrations of F SH and LH
(Guthrie and Garret 2000; Fig. 1.6). Only during the last steps of the prepubertal stage

will these follicles complete ﬁnal development, establishing puberty.

Follicle turnover has been demonstrated in swine during the pre-pubertal period
between days 140 and 180 of age using laparoscopy repeated every 20 days (Grasso et al.
1988). Changes in the morphological aspects of the ovaries from “honeycomb type”
(with large numbers of small follicles) to “grape type” (with several large follicles) were
shown to occur within a few days (Bolamba et al. 1994). This may be taken as evidence
that follicular waves occur in gilts at these ages (Driancourt 2001), as has been shown in
prepubertal heifers (Evans et al. 1994; Melvin et al. 1999). However, in contrast with
prepubertal heifers, these changes in prepubertal gilts appear to be regulated by local
intraovarian factors other than by gonadotrophic and sex steroids hormones (Bolamba et
al. 1994), since asynchrony is observed between left and right ovaries in ovarian

morphology and changes.

Hormonal profiles and ovarian development after puberty

GnRH secretion: In the cycling female the tonic and surge centers are regulated
by the level of progesterone and estradiol in blood. Although progesterone in the female
exerts a negative feedback on both the tonic and the surge centers, it mostly exerts its
effects on the tonic center. In contrast, the surge center responds to estradiol with a
positive feedback. During the luteal phase (high circulating levels of progesterone) GnRH
episodes from the tonic center occur every 4 to 8 h (Senger 2003). Such basal secretion,
while allowing for some follicular growth, will not allow sufﬁcient follicular
development for the production of high levels of estradiol. Therefore, females under the

inﬂuence of progesterone do not cycle during the luteal phase or gestation.

During the early follicular phase, negative feedback by progesterone on the
hypothalamus is removed and GnRH is released by the tonic center at higher frequencies
than during the luteal phase (every 1.5 to 2 h). This stimulates increased release of FSH
and LH that in turn increases the secretion of estradiol by the follicles. During the late
follicular phase when estradiol reaches a high (threshold) level, the surge center is
positively stimulated and releases large quantities of GnRH causing the release of the

surge of LH that induces ovulation, as described earlier for puberty.

LH secretion: In sows, cows and the ewes, LH is secreted in a pulsatile manner
that changes from a low-frequency, high-amplitude mode of secretion during the luteal
phase to a high-frequency, low-amplitude mode of secretion during the follicular phase
(Clarke 1989). Studies on GnRH secretion in sheep show that the increase in plasma LH

10

pulse frequency may reﬂect the removal of a negative feedback inﬂuence of progesterone
on the hypothalamic GnRH pulse generator (Clarke et al. 1987). Conversely, Guthrie and
Bolt (1990) and others have been unable to demonstrate an increase in LH

concentrations, frequencies or amplitudes in gilts undergoing natural luteolysis.

As the follicular phase progresses the amplitude of LH pulses varies between
species (Clarke 1989). In swine, the growth of ovarian follicles causes a progressive rise
in the plasma concentrations of estradiol that effect a short-term pituitary 'clamp' of LH
secretion during the late follicular phase of the cycle (Prunier et al. 1987; Clarke 1989).
Indeed, from studies in experimental models it is clear that estrogen can exert a short-
tenn negative feedback action at the pituitary level (Britt et al. 1991). Plasma LH levels
in ovariectomized pigs given a single injection of estrogen ﬁrst showed a negative
feedback response and then a positive feedback response. Estrogen apparently inhibits or
greatly reduces the release of GnRH for a period of approximately 54 to 60 h, and then
causes a release of GnRH that induces a surge of LH proportional to the amount of GnRH
released. The physiological signiﬁcance of such a 'clamp' would be to allow the

gonadotrophs to prepare cellular machinery for the subsequent positive feedback event.

FSH secretion and ovarian dynamics: In swine, changes in follicular ﬂuid
concentrations of steroids indicate two surges of follicle growth and increased
steroidogenesis (Guthrie and Cooper 1996), although follicle waves do not develop to the
same extent as in cattle probably due to ovarian progesterone suppression of LH

secretion. In the pig, the LH receptor appears at a relatively immature stage of follicular

II

development (~2 mm diameter; Liu et al. 1998, 2000), while in the cow LH receptors do
not appear until follicles reach 9 to 10 mm (Xu et al. 1995). Taken together, it may
explain why the cow has extended follicular waves with appearance of dominant follicles
during the luteal phase, whereas in the pig follicles do not grow further than 4 mm. The
ﬁrst surge of follicle activity consists of the replenishment of the follicle population
between days 2 and 8 of the estrous cycle, followed by an increased incidence of atresia
between days 5 and 7 of small and medium follicles. The second surge in follicle activity
consists of the selection and growth of ovulatory follicles, accompanied by atresia of
subordinate follicles during the follicular phase of the cycle (days 15-21). The pattern of
follicle growth during the mid to late luteal phase in pigs (7-15 d) has been characterized

as continual growth and atresia (Guthrie and Cooper 1996).

In cows, mares and ewes, the emergence of follicle waves is preceded by a
transient increase in plasma F SH secretion. In pigs, changes in follicle number and size
distribution during the estrous cycle are, in part, also associated with changes in
circulating concentrations of FSH (Guthrie et al. 1995). Increased atresia among small
and medium follicles coincides with a decrease in circulating levels of FSH starting 1.5 to
2 d after the initiation of the decline in circulating progesterone (Guthrie and Bolt 1990;
Knox et al. 2003). Secretion of FSH remains suppressed until the preovulatory LH surge.
Concentrations of FSH are transiently increased on days 2 and 3 after ovulation
(periovulatory FSH release), and at the same time the ovaries are replenished with small
(1 - 2 mm diameter) and medium (3 - 6 mm) follicles (35 to 40 per animal) (Guthrie

2005). Increased atresia on days 6 and 7 among the small and medium follicles coincides

12

with decreased circulating concentrations of F SH and may be a result of inhibin-induced

negative feedback on secretion of FSH (Guthrie et al. 1995).

Estrogen and progesterone secretion: Estradiol is secreted by follicles during
the entire estrous cycle, but concentrations remain low during the luteal phase due to the
inhibitory effect of progesterone at the hypothalamic level. Once luteolysis occurs and
preovulatory follicles are selected, circulating estradiol levels progressively increase until
triggering the preovulatory release of GnRH. The mechanism involved is that, during
follicular development LH binds to LH-speciﬁc membrane receptors located on the cells
of the theca intema of the developing follicle. The binding of LH to its receptors activates
a cascade of intracellular events ultimately resulting in conversion of cholesterol to
testosterone. Testosterone then diffuses out of the cells of the theca interna and enters the
granulosa] cells, which have receptors for FSH. When FSH binds to its receptor, it causes
the conversion of testosterone to estradiol via induced aromatase activity. During late
stages of development, preovulatory follicles obtain LH receptors in granulosa cells, so

allowing the LH surge to exert its full effect.

Concentrations of progesterone in circulation are low immediately after ovulation
and then begin to increase by day 3 or 4 of the estrous cycle. Luteal phase circulating
concentrations of progesterone are maximal by day 7 to 12 and then, with the regression
of corpora lutea (luteolysis; 14-15 d), concentrations of progesterone in circulation

decline rapidly and remain low throughout the follicular phase.

13

Follicular recruitment and selection

At recruitment, a cohort of follicles begins its ﬁnal growth phase while, at
selection, the preovulatory follicles are chosen and the remaining recruited follicles
become atretic. The relative roles of LH and F SH shift throughout the different stages.
During recruitment FSH plays a more important role than LH in antral follicle growth. As
the follicles enter the selection phase, inhibin and estradiol are produced (by the follicle)
and inhibit FSH secretion from the pituitary, while LH secretion increases. Finally, the
large follicles produce more and more estrogen that ﬁnally prompts the preovulatory
center to release a surge of LH.

Recruitment and selection are relatively short-term processes when compared to
the preantral stages. The time interval from when a follicle leaves the resting pool until
antrum formation is estimated to require 83 days in pigs while the time from antrum
formation until the follicle reaches the ovulatory stage takes only an additional 20 days
(Morbeck et al. 1992). From their estimate of antral follicle growth rate, Morbeck et al.
(1992) hypothesized that the follicles undergoing antrum formation at the beginning of
the cycle reach 3 mm in diameter on d 14 to 16 and constitute the population from which

the ovulatory follicles are recruited.

Recruitment: The term recruitment has been used to describe two points during
follicle development. The dormant primordial follicles are recruited into the growing
follicle pool in a continuous manner (initial recruitment), whereas increases in circulating
FSH during each estrous cycle recruit a cohort of antral follicles (cyclic recruitment)
(McGee and Hsueh 2000). During initial recruitment, intraovarian and/or other unknown

14

factors stimulate some primordial follicles to initiate growth, whereas the rest of the
follicles remain quiescent for months or years. After initial recruitment, follicles grow but
oocytes remain arrested in the prophase of meiosis. For those follicles not recruited, the

default pathway is to remain dormant.

Cyclic recruitment starts after puberty onset and results from cyclic increases in
circulating FSH forming cohorts of antral follicles, which begin ﬁnal growth after
entering the gonadotrophin-dependent stage of follicle growth. During cyclic recruitment,
only a limited number of follicles survive, and the default pathway is to undergo atresia.
Oocytes in these follicles have already completed their growth, acquired a zona pellucida,
and are competent to resume meiosis (McGee and Hsueh 2000). The number of recruited
follicles growing in the cohort appears to be highly variable between species. It ranges
from over 50 in pigs, to 5 to 10 in cattle and 1 to 4 in horses (Driancourt 2001). In pigs,
follicular recruitment occurs between days 14 and 16 of the estrous cycle (Clark et al.
1982; F oxcroft and Hunter 1985) or shortly after weaning in sows. On day 16,
approximately 40 to 50 follicles, 3 to 6 mm in diameter, are present in both ovaries and

represent the population of recruited follicles (Grant et al. 1989).

In cattle, the emergence of follicular waves is preceded by a transient increase in
FSH that stimulates the development of antral follicles. However, in pigs, a clear increase
in FSH concentrations coincident with the period of follicular recruitment has not been

detected (Flowers et al. 1989; Guthrie and Bolt 1990; Cardenas and Pope 2002).

Nevertheless, plasma FSH concentrations are consistently elevated at the time of

15

follicular recruitment and Guthrie and Bolt (1990) suggest that alterations in the F SHzLH

ratio may be critical in the recruitment process.

Selection: Only a small proportion of recruited follicles (about 30 to 40%) are
selected to complete ﬁnal maturation and ovulate. The remaining follicles and the
oocytes they contain will degenerate and disappear from the ovaries through atresia.
Therefore, the ﬁnal number of ovulatory follicles is determined by how many follicles are
recruited and by the ability of recruited follicles to continue to grow and avoid atresia
(Cardenas and Pope 2002). The intensity of selection (measured by the proportion of the
cohort follicles surviving) is highly variable among species. It can be very low in some
horses, which have a small cohort size, while it is very high for cattle (one selected
follicle out of 5 cohort follicles) and swine (12 selected follicles out of a cohort size of
50) (Driancourt 2001). Selection mechanisms seem to differ between cattle and pigs, due
to the different follicular receptors present at the time of selection (Liu et al. 1998, 2000;

Xu et al. 1995; Fig 1.5).

Lucy (2007) proposed two different models for follicular selection in cattle
(Missouri and Cornell, Fig. 1.6). Both models suggest that one follicle has a
developmental advantage over others at the time of follicular selection. This
developmental advantage may be conferred by individual aspects of the follicle at the
beginning of the follicular wave, including its vascular bed or the number or health of the

granulosa or theca cells.

16

FSH

 

 

 

 

LH
Selection: LH receptors
PIG
Recruitment H 1
. 1 J
15" 21d
FSH LH

Selection: FSH receptors
recruitment 1 1 l

Ill

1 l J
150 180 210

COW

Figure 1.5 Different selection mechanisms between cattle and pigs (based on data

from Lucy 2007 and Driancourt 2001).

According to the Missouri model (Xu et al. 1995), follicles that acquired LH
receptors before others could theoretically starve the other follicles of gonadotrophins by
selectively inhibiting F SH while not inhibiting LH. This model could explain selection in
pigs because the earlier development of the LH receptor in granulosa cells (Liu et al.
1998, 2000) may enable a cohort of follicles to respond to LH when blood FSH
concentrations are suppressed and so escape atresia. However, in cows the acquisition of
LH receptors by granulosa cells is not a key component of follicular selection, since it

appears to occur after, rather than before, selection has occurred (Fortune et al. 2001).

I7

-FSH -FSH

 

Missouri model Cornell model

Figure 1.6 Models for events during follicular selection. According to the Missouri
model, the initiating event is an increase in the LH receptor (LHR) in the
granulosa cell layer. According to the Cornell model, the initiating event
is the increase in pregnancy-associated plasma protein-A (PAPP-A). In
each case, there is an increase in estradiol (E) and inhibin (I) that feeds
back negatively on FSH secretion at the hypothalamo-pituitary axis. A =
androgen; FSHR = FSH receptor (From Lucy 2001).

According to the Cornell model (Fortune et al. 2004), an increase in FSH
enhances the IGF BP protease (pregnancy-associated plasma protein-A) that degrades
IGF BP within the follicular ﬂuid, leading to an increase in free IGF-I within the
follicular ﬂuid. The IGF-I is synergistic with FSH for estradiol synthesis within the
follicle, and therefore the dominant follicle grows faster and also produces more
estradiol. The increase in estradiol decreases F SH and starves the remaining follicles into
atresia. This model can explain selection in cows because the FSH receptor continues to

18

be expressed in the granulose cell layer throughout development (Xu et al. 1995). It is not
clear why granulosa cells develop LH receptors in cows. It was hypothesized that this
allows the granulosa cells to increase aromatase in response to both FSH and LH and that
this increases, or at least maintains, their capacity to produce more estradiol than
subordinate follicles. Another possibility is that LH receptors develop on granulosa cells
of dominant follicles to prepare them for further differentiation in response to the LH
surge, rather than to augment or maintain their estradiol-secreting capacity (Fortune et al.

2004)

Stimulating pubertal estrus

Weaned pig output per week is mostly dependant on breeding of a sufﬁcient
number of females each week (Dial et al. 1996). In order to have sufﬁcient females
available for breeding, a predictable supply of service-ready gilts (ie. gilts in estrus when
needed) is necessary. This is most easily achieved by having gilts show an early puberty
and then maintain regular cyclic activity. The only effective methods for stimulating an

earlier puberty are exposure to a boar and the injection of gonadotrophic hormones.

Boar exposure: Boar exposure is the most common practice for stimulating an
earlier puberty. Adequate estrus stimulation requires direct physical contact between the

boar and the gilts, while estrus detection may only need fence-line contact. To ensure

19

efﬁcacy of boar exposure, it is important to follow the rules of boar contact, as reviewed

previously (Kirkwood and Thacker 1992; Hughes 1997). These rules are:

1. Gilts must be old enough; the usual advice is at least 160 days of age although
recent evidence suggests that 180 days may be better. This is because the gilts need to be

mature enough to respond to the boar stimulus.

2. Boars must be old enough (ie. at least 10 months of age). A major part of the
boar stimulus is pheromone production from the submaxillary salivary gland, and this

gland is not fully developed until 9 or 10 months of age.

3. Gilts should be in physical contact with the boar for at least 15 min per day. As

stated above, fence-line contact is not as effective as full physical contact.
4. Twice daily exposure will improve the response.

5. Provide adequate space (eg. >l.5 m2 per gilt) since crowding may delay

puberty and will make estrus detection more difﬁcult.

6. House gilts at least 1 m away from boars to prevent gilts becoming refractory to

boar stimuli (a detection problem).
7. If gilts do not respond as expected, use a different stimulus boar.

If gilts are not bred at their ﬁrst estrus, boar exposure should continue (eg. at least

5 min every 2 to 4 d) in order to promote regular estrous cycles. In the absence of

20

continued boar exposure, many gilts exhibit irregular inter-estrus intervals making estrus

detection difﬁcult.

Exogenous hormones: If boar exposure does not appear to be effective, for
example due to a seasonal effect, then hormonal induction of estrus should be considered.
Commercially available gonadotrophin preparations contain equine chorionic
gonadotophin (eCG), which can be used alone or combined with human chorionic
gonadotrophin (hCG). A common combination is 400 IU eCG with 200 IU hCG
(PG600®; Intervet). The injection of PG600 is effective for the induction of fertile estrus
in prepubertal gilts but when administered to prepubertal gilts, up to 30% may not exhibit
behavioral estrus and about 30% of those exhibiting behavioral estrus may fail to cycle

regularly (Table 1.1).

Table 1.1. Estrus responses of gilts to PG600* injection

 

 

Percent estrous Percent ovulating Percent cycling Source
88 100 87 Paterson (1982)
-- 97 6O Paterson (1982)
70 99 -- Tilton et al. (1995)
78 -- 67 Kirkwood (I999)

 

*PG600®: combination of 4001U eCG + 200 IU hCG (Intervet Intenational)

21

The etiology of unpredictable responses is unknown, but possibly due to the hCG
component of PG600 inducing an immediate ovulation or luteinization of medium or
large ovarian follicles, and the subsequent progesterone production then suppressing

behavioral estrus.

Table 1.2 Long-term performance of gilts bred at a PG600*-induced or natural ﬁrst

 

 

estrus

Control PG600-bred PG600-skipped
Number of gilts bred 132 140 60
First farrowing rate 89.4 70.0 89.8
First litter size 9.9 9.6 10.7
Pl bred <7 days 68.3 65.4 67.3
Litters per sow 3.5 3.4 3.2
Pigs per sow 45.0 45.8 45.1

 

From Kirkwood et al. (2000)
*PG600®: combination of 4001U eCG + 200 IU hCG (Intervet)

Typical results from treating gilts with PG600 were documented by Kirkwood
(1999). Estrus detection rates during a 3-week period were 78% and 37.5% for PG600-
treated and untreated gilts, respectively. Further, for non-bred (skipped) and non-pregnant
PG600-treated gilts, a 2:1 ratio of regular to irregular returns was observed, conﬁrming

the 70% incidence of normal estrous cycles in hormone-stimulated gilts. The farrowing

22

rate of hormone-induced gilts was lower but there was no adverse effect on long-term
sow performance (Kirkwood et al. 2000; Table. 1.2). This suggests that if the use of
hormones is deemed necessary, it need not adversely affect long-term sow performance.
However, an improvement in estrus response of pre-pubertal gilts to PG600 is desirable.
However, in order to achieve this objective, it will be necessary to determine why some

gilts do not appear to respond to PG600 injection.

23

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27

CHAPTER 2
EFFECT OF ECG OR ECG PLUS HCG ON ESTRUS EXPRESSION AND
OVULATION IN PREPUBERTAL GILTS.

R Manjarinl, G Cassarz, DJ Sprecherl, RM Friendshipz, JC Dominguez3, and RN
Kirkwoodl

lDepartment of Large Animal Clinical Sciences, Michigan State University, East
Lansing, MI, USA, 2Department of Population Medicine, University of Guelph, Ontario,
Canada and 3 Department of Animal Reproduction and Obstetrics, University of Leon,
Leon, Spain (Reproduction in Domestic Animals, 2008. doi: 10.11 1 1/j.1439-
0531.2008.01089.x)

Abstract

To meet weekly breeding targets, it is occasionally necessary to inject exogenous
gonadotrophins to induce estrus in prepubertal gilts. However, the gilt estrus response to
equine chorionic gonadotrophin (eCG) either alone or in combination with human
chorionic gonadotrophin (hCG) can be unpredictable. The objective of the present study
was to examine possible reasons for this unpredictability. Prepubertal gilts (90 kg and
153 d of age, n=109) received an injection of either 600 IU eCG or a combination of 400
IU eCG and 200 IU hCG (PG600), or were not-injected (controls). The gilts were then
exposed to a mature boar for 15 min daily for 7 d for estrus detection. At the time of
injection, real-time ultrasound revealed that the gilt ovaries had primarily 1 to 2 mm
follicles. Blood samples were obtained at time of hormone injection (day 0) and at d 3, 7,
and 10 for assay of serum progesterone concentrations. The estrus responses by 7-d were
15.5%, 73.3%, and 0%, for eCG, PG600, and control gilts, respectively (P<0.001). The

estrus response improved (P<0.05) with increasing body weight. Based on circulating

28

progesterone levels, all estrous gilts ovulated except for 4 of the PG600 gilts. Failure to
express estrus in PG600 treated gilts was not associated with a premature rise in

progesterone.

Keywords: swine, gilts, gonadotrophins, estrus, ovulation

Introduction

The largest single component of herd non-productive days is often the gilt entry-
to-service interval. To minimize this interval, gilts should reach puberty as soon as
possible either after arrival on the farm or entry into the breeding herd. The ability to
meet the weekly breeding target requires a predictable supply of service-ready gilts (ie.
gilts in estrus when required), which is most easily achieved by having gilts show an
early puberty and then maintain predictable cyclic activity. When gilt availability is
limiting, a common method used to stimulate the onset of estrus is the injection of
gonadotrophins, such as the combination of 400 IU eCG and 200 IU hCG (PG600®,
Intervet). While this product is effective for the induction of a fertile estrus in prepuberal
gilts, in practice up to 30% of treated gilts do not show a behavioural estrus response
within 7 d (Kirkwood 1999). However, based on blood progesterone determinations at 10
d after treatment, most non-responding gilts do appear to have ovulated (Tilton et al.

1995).

Taking the above ﬁndings into consideration, we hypothesized that the 30% non-

estrous response is due to the hCG component of PG600, the hCG being a LH analogue

29

causing some gilts to either ovulate or suffer luteinization of medium or large follicles. In
either event, the premature production of progesterone would block behavioral estrus and
the gilts will be deemed to have failed to respond to treatment. We further suggest that if
our hypothesis were true, then injection of eCG alone (ie. no hCG component) would
result in an improved estrus response because used alone, this molecule would provide a

much lower LH activity (Combamous et al. 1984; Guthrie et al. 1990).

Materials and Methods

These studies were performed during October and November 2006 at Michigan
State University Swine Facility with the approval of the institutional Animal Care
Committee. To examine responses to different gonadotrophin preparations, 109
prepubertal Yorkshire x Landrace gilts (90 kg, 153 d) were employed. Gilts were housed
at 8 to 12 animals per pen and fed ad libitum a com-soybean meal ﬁnisher diet
formulated to provide 13.7 MJ ME/kg, 9.8% crude protein, 0.6% lysine. At selection,
gilts were assigned by weight and age to receive an intramuscular (1M) injection of 600
IU eCG (Pregnecol®; Bioniche Animal Health, Bellville, Ontario, n=45) or 400 IU eCG
plus 200 IU hCG (PG600®; Intervet, Millsboro, Delaware, n=45). A third group of gilts
served as uninjected controls (n=l9). All treatments were represented in each pen. The
dose of eCG (600 IU) was chosen on the basis of it being a label dose for sows at

weaning and that it is the ‘total’ gonadotrophin content of PG600.

30

From 2 (1 until 7 d after the time of hormone injection, all gilts were subject to
direct exposure to a mature boar for 15 min daily to facilitate detection of estrus. Estrus
was deﬁned as the expression of a rigid standing reﬂex in the presence of the boar. Fewer
control gilts were employed because we anticipated few, if any, would exhibit estrus
during the 7 d study period. To further characterize the estrus and ovulation responses to
hormone treatment, blood samples were obtained from all gilts via jugular venipuncture
at the time of hormone injection (0 d) and at 3, 7, and 10 (1. Serum samples were assayed
for progesterone (P4) concentrations in a single assay using a commercial kit (Diagnostic
Product Corp., Los Angeles, California). Assay sensitivity and intra-assay coefﬁcient of
variation were 0.1 ng per mL and 2.7%, respectively. An elevation in P4 to >1 ng per mL
on 3 d was considered indicative of premature ovulation (or luteinization), while low
levels on 3 d followed by elevations on 7 d and/or 10 d was taken to indicate a normal
ovulatory response. Immediately prior to hormone injection, the ﬁrst 25 gilts assigned to
each hormone treatment were subject to transrectal B-mode ultrasonography of their
ovaries using an Aloka SSD 500 (Aloka Inc, Wallingford, Connecticut) with a 7.5-MHz
linear array transducer as described by Knox and Althouse (1999). The diameters of the
largest 3 follicles were recorded. Data were analysed using the Number Cruncher
Statistical System (NJ Hintze, Kayeville, Utah). Treatment differences on the estrus
response rates were examined by Chi square and differences in age, weight, and follicle

diameter tested by GLM analysis of variance.

3]

Results

There were no differences between treatments for gilt age or weight at the start of
the study (Table 2.1). Similarly, there was no difference in ovarian follicular diameter
prior to hormone injection (Table 2.1). Most gilts had a maximum follicular size of 2 mm
although 3 mm follicles were measured in 7 of the eCG-treated and 5 of the PG600-
treated gilts, and 2 of the PG600-treated gilts had 4 mm follicles. The PG600-treated gilts

with the largest follicles ovulated normally.

All gilts had non-detectable P4 concentrations on 0 (I. As anticipated, no control
gilts exhibited estrus during the study period, and none had elevated serum P4
concentrations. Contrary to expectation, more (P<0.001) PG600-treated gilts than eCG-
treated gilts exhibited estrus by 7 d after injection (Table 2.1). All eCG-treated gilts
exhiting estrus had serum P4 concentrations indicative of normal ovulation. Elevated P4
concentrations were not detected in any anestrus eCG-treated gilt. Of the 33 (73%)
PG600-treated gilts exhibiting estrus, 4 failed to ovulate. Of the 12 anestrus PG600-
treated gilts, none had elevated P4 on d 3 after injection and only 1 had serum P4

concentrations indicating a normal ovulation.

There was an effect of body weight at treatment on the incidence of estrus, with
more of the gilts weighing >90 kg exhibiting estrus than their lighter counterparts
(P<0.05). However, because too few eCG-treated gilts exhibited estrus, the effect was
only signiﬁcant for PG600-treated gilts (Table 2.2). The two weight classes did not differ
in age (154.9i5.0 vs. 157.5i3.8 days for the lighter and heavier gilts, respectively,

P=O.8).

32

Table 2.1. Inﬂuence of eCG (equine chorionic gonadotrophin) or eCG plus hCG

(human chorionic gonadotrophin) on incidence of estrus by 7 d post-

treatment and ovulation as indicated by elevated progesterone (+P4) or

non-ovulation (-P4).

 

 

No. of gilts

Age, (1]

Weight, kgl

Follicle diameter, mmI
Gilts estrus by 7 d (%)2
Gilts estrus + P4 (%)
Gilts estrus — P4 (%)
Gilts anestrus + P4 (%)

Gilts anestrus — P4 (%)

eCG+hCG eCG Control
45 45 19

154.1i2.6 154.2i2.7 150.6i3.8

90.1i1.3 91 .4i1.3 88.5i2.0
2.43:0.1 2.3i0.1 --
73.3 15.6 0
64.4 15.6 --
8.9 O --
2.2 O --
24.4 84.4 --

 

' Means 1- SE

2 Effect of hormone preparation, P<0.001 by Chi square test

Table 2.2. Influence of gilt weight on the estrus response to eCG or eCG plus hCG

 

 

eCG+hCG eCG Control
No. of gilts 45 45 19
75-90 kg 13/22 (59.1%)a 2/23 (8.6%) 0
91-110 kg 20/23 (86.9%)b 5/22 (22.7%) 0

 

ab, Effect of weight, P<0.05 by ANOVA

33

Discussion

The results of this study conﬁrmed an approximate 70% estrus response to PG600
but did not support the hypothesis that injection of hCG concurrent with eCG results in
follicular luteinization or ovulation and premature elevation of circulating progesterone.
Indeed, the poor estrus response to eCG compared to the eCG/hCG combination suggests
an important role for the hCG in follicular development and subsequent estrus
expression. However, a follicular ovulatory or luteinizing effect may depend on the
follicular status of the ovaries at the time of stimulation (ie. presence or not of medium to

large follicles). In the present study, few gilts had follicles >2 mm in diameter.

Of the PG600-treated gilts exhibiting estrus, 4 of them did not have elevated
circulating progesterone levels. It is possible that these gilts underwent sufﬁcient
follicular development to cause circulating estrogen levels adequate to induce
behavioural estrus, but then failed to ovulate. A failure to ovulate may be due to the
PG600 activity being inadequate to complete follicular development to the point of
inducing the phasic LH release. However, in the absence of measurement of circulating
LH concentrations and ongoing follicular ultrasound examinations, these suggestions

remain speculative.

It is probable that the response to eCG treatment, with or without concurrent hCG
treatment, will depend on the gilt’s level of physiological maturity, which is supported by
the positive association between gilt weight and their estrus response in the present study,
although gilt age was not a signiﬁcant factor. Most of the gilts in the present study had

only small (1 to 2 mm) follicles on their ovaries, which should have been responsive to

34

FSH-like stimulation (Guthrie et al. 1990), although the possibility that they were too
immature to respond to gonadotrophic stimulation cannot be discounted. However, it was
shown previously that the presence of many or only a few large (>6 mm) follicles on the
ovary did not affect the estrus or ovulatory response of prepubertal gilts to eCG
treatment. This suggests that eCG can stimulate development of small follicles to the

point of ovulation (Bolamba et al. 1992).

It is possible that a period of FSH stimulation would advance follicular
development (Guthrie et al. 1988; Bolamba et al. 1996) such that they would then be
more responsive to eCG stimulation. Alternatively, it is possible that some minimum
level of LH-like stimulation is necessary to complete follicular development (Driancourt
et al. 1995). Previous studies examining estrus and/or ovulatory responses to eCG have
yielded variable results. Response rates of 70% to 100% have been noted when 725 to
1,000 IU eCG was injected (Guthrie 1977; Dial et al. 1984; Britt et al. 1986; Esbenshade
1987; Flowers et al. 1989; Bolamba 1992) although estrus rates of only 25% to 52% were
noted following injection of 363 to 600 IU (Britt et al. 1985; do Lago et al. 2005; Gama
et al. 2005). The difference in responses may be due to the degree of physiological
development and genetics of the gilts and/or the source of the eCG, although it is likely
that the greater LH-like activity associated with higher doses improved the likelihood of

ongoing follicular development.

We conclude from the present data that the gilt estrus responses to eCG will be
improved with concurrent use of hCG and that, depending on their degree of

physiological development, a failure of gilts to exhibit estrus in response to a

35

combination of eCG and hCG need not involve premature ovulation or luteinization of

follicles and an associated premature elevation in circulating progesterone.

Acknowledgements

Hormones were generously provided by Bioniche Animal Health, Bellville,
Ontario and Intervet, Milsboro, Delaware. Financial support for this study was provided

by Michigan State University.

36

References

Bolamba, D., Dubuc, A., Dufour, J.J., Sirard, M-A., 1996. Effects of gonadotropin
treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of
oocytes in prepubertal gilts. Theriogenology 46, 717-726.

Bolamba, D., Matton, P., Estrada, R., Dufour, J .J ., 1992. Effect of pregnant mare’s serum
gonadotropin treatment on follicular populations and ovulation rates in prepuberal
gilts with two morphologically different ovarian types. J. Anim. Sci. 70, 1916-1922.

Britt, J .H., Esbenshade, K.L., Heller, K., 1985. Responses of seasonally anestrus gilts and
weaned primiparous sows to treatment with pregnant mare’s serum gonadotropin and
altrenogest. Theriogenology 26, 697-707.

Combamous, Y., Guillou, F., Martinat, N., Cahoreau, C., 1984. [Origin of the FSH + LH
double activity of equine chorionic gonadotropin (eCG/PMSG)]. Ann. Endocrinol.
(Paris) 45, 261-268.

Dial, G.D., Dial, O.K., Wilkinson, R.S., Dziuk, P.J., 1984. Endocrine and ovulatory
responses of the gilt to exogenous gonadotropins and estradiol during sexual
maturation. Biol. Reprod. 30, 289-299.

do Lago, V., Vianna, W.L., Gama, R.D., de Campos Rosseto, A., Pinese, M.E., de
Sant’Anna Moretti, A., 2005. Second oestrus synchronization and precocious embryo
viability after puberty induction in gilts by the use of gonadotrophin treatment.
Reprod. Dom. Anim. 40, 141-144.

Driancourt, M.A., Locatelli, A., Prunier, A., 1995. Effects of gonadotrophin deprivation
on follicular growth in gilts. Reprod. Nutr. Dev. 35, 663-673.

Esbenshade, K.L., 1987. Ovarian response to pregnant mare serum gonadotropin and
porcine pituitary extract in gilts actively immunized against gonadotropin releasing
hormone. J. Anim. Sci. 65, 1768-1774.

Flowers, 3., Martin, M.J., Cantley, T.C., Day, B.N., 1989. The effect of pregnant mare
serum gonadotropin on follicle stimulating hormone and estradiol secretion in the
prepuberal gilt. Anim. Reprod. Sci. 21, 93-100.

Gama, R.O., Vianna, W.L., Pinese, M.E., de Campos Rosseto, A., de Sant’Anna Moretti
A., 2005. Different doses of porcine luteinizing hormone in precocious puberty
induction in gilts. Reprod. Dom. Anim. 40, 433-435.

Guthrie, H.D., 1977. Induction of ovulation and fertility in prepuberal gilts. J. Anim. Sci.
45, 1360-1367.

37

Guthrie, H.D., Bolt, D.J., Cooper, BS, 1990. Effects of gonadotropin treatment on
ovarian follicle growth and granulosa cell aromatase activity in prepuberal gilts. J.
Anim. Sci. 68, 3719-3726.

Guthrie, H.D., Bolt, D.J., Kiracofe, G.H., Miller, K.E., 1988. Ovarian response to
injections of charcoal-extracted porcine follicular ﬂuid and porcine follicle
stimulating hormone in gilts fed a progesterone agonist (altrenogest). Biol. Reprod.

38, 750-755.

Kirkwood, RN, 1999. Pharmacological intervention in swine reproduction. Swine
Health Prod. 7, 29-35.

Kirkwood, R.N., Soede, N.M., Dyck, G.W., Thacker, P.A., 1995. The effect of immuno-
neutralization of PMSG at a gonadotrophin-induced oestrus on the duration of
ovulation and reproductive performance of sows. Anim. Sci. 61, 321-324.

Knox, R.V., Althouse, GO, 1999. Visualising the reproductive tract of the female pig
using real-time ultrasonography. Swine Health Prod. 7, 207-215.

Tilton, S.L., Bates, R.O., Prather, RS, 1995. Evaluation of responses to hormonal
therapy in prepubertal gilts of different genetic lines. J. Anim. Sci. 73, 3062-3068.

38

CHAPTER 3
EFFECT OF PRIOR F SH TREATMENT ON THE ESTRUS AND OVULATION
RESPONSES TO ECG IN PREPUBERTAL GILTS

R. Manjarinl, J .C. Dominguezz, M.J. Castroz, B. Valladoz, S. Rodriguezz, D.J. Sprecher‘,
G. Cassar3, R.M. Friendship3, and RN. Kirkwood"

1Department of Large Animal Clinical Sciences, Michigan State University, East
Lansing, MI, USA, 2Department of Animal Reproduction and Obstetrics, University of
Leon, Leon, Spain and 3Department of Population Medicine, University of Guelph,
Ontario, Canada (Animal Reproduction Science, 2008; doi:
10. I 01 6/j. anireprosci. 2 008. 01 . 001 )

Abstract

The objective of this study was to determine the effect of pre-treatment of
prepubertal gilts with follicular stimulating hormone (FSH) on the estrus and ovulatory
responses to equine chorionic gonadotrophin (eCG) injection at two ages. A total of 149
prepubertal Hypor gilts were selected at 150 d (n=76) or 180 d (n=73) of age and
assigned to injection of 400 [U eCG plus 200 IU human chorionic gonadotrophin (hCG;
PG600®, Intervet International), 600 IU eCG alone (Folligon®, Intervet), pretreatment
with 72 mg FSH (Folltropin®; Bioniche Animal Health) administered as 6 x 12 mg
injections at 12 h intervals with 600 IU Folligon 12 h after last F SH injection, or non-
injected controls. To facilitate estrus detection, gilts were exposed to a mature boar for 15
min daily for 7 d. To determine ovulatory responses, blood samples were obtained on the
day of injection and 10 d later and assayed for progesterone content. Following treatment
at 150 d, I control gilt (5.3%) was deemed estrous but did not ovulate. Compared to
treatment with Folligon alone, PG600 injection tended (P=0.1) to increase the estrus

39

response (52.6% vs. 26.3%) and increased (P<0.01) the ovulatory response (89.5% vs.
47.4%). The estrus response in gilts pretreated with Folltropin was intermediate (42.1%)
but the ovulatory response (47.4%) was the same as for Folligon alone. Following
treatment at 180 d, 2 control gilts (10.5%) were deemed estrous and they also ovulated.
There was no difference between hormone-treated groups for estrus or ovulatory
responses, although the ovulatory response of PG600-treated gilts tended (P=0.l) to be
higher than for the Folligon-treated group (89.5% vs. 66.7%), with Folltropin-pretreated
gilts being intermediate (76.5%). These data demonstrate that the estrus and ovulatory
responses of gilts were higher for PG600 than for Folligon and that while responses to
PG600 were not affected by gilt age, for the combined Folligon groups, estrus response

(P<0.02) and ovulatory response (P<0.05) improved with increased gilt age.

Keywords: gilts, FSH, eCG, estrus, ovulation

Introduction

To minimize the gilt entry-to-service interval, or where availability of service-
ready gilts is limiting, gilts are often induced into estrus by injection of gonadotrophic
hormones. A common preparation used for estrus induction is a combination of 400 IU
equine chorionic gonadotrophin (eCG) and 200 IU of human chorionic gonadotrophin
(hCG; PG600®, Intervet International). Subsequent to injection of PG600, it is not
unusual to have only 70% of gilts show a behavioural estrus response within 7 days
(Kirkwood, 1999). It was hypothesized that the 30% failure to respond to PG600 was due

to its hCG component luteinizing or ovulating medium or large follicles, causing a

40

premature rise in circulating progesterone and blockade of behavioural estrus (Manjarin,
unpublished data). However, in this earlier work we did not observe any indication of
ovulation or follicular luteinization subsequent to PG600 injection into ISO-day old
prepubertal gilts. Interestingly, when we injected 600 IU eCG alone, it resulted in only a
15% estrus response in prepubertal gilts (Manjarin, unpublished data). The poor response
to this relatively low dose of eCG may have been due to the immaturity of ovarian
follicles (90% <3 mm), indicating a need for the concurrent biological activity of hCG in
order to adequately stimulate growth of small follicles. This is supported by the improved
estrus response observed with higher doses of eCG (Dial et al., 1984; Britt et al., 1985),
which will also have a higher LH-like activity (Dial et al. 1984).

Interestingly, while injection of eCG into prepubertal gilts caused atresia in the
small and medium follicle populations, injections of F SH promoted their growth (Guthrie
et al., 1990; Bolamba et al., 1996; Guthrie, 2005). Also, changes in follicle number and
size during prepubertal development were temporally associated with changes in
circulating F SH concentrations (Camous et al., 1985; Guthrie and Garret, 2000). Further,
suppression of FSH during the luteal phase of cyclic gilts decreased the small and
medium follicle populations (Knox and Zimmerman, 1993; Guthrie et al., 1987), which
were restored by FSH replacement (Guthrie et al., 1988). Taking all of the above into
account, we hypothesize that administration of FSH to prepubertal gilts will increase the
populations of medium sized ovarian follicles, which will enhance the gilt's ovarian

response to eCG injection.

41

Materials and Methods

These studies were performed on a 1,000-sow farrow-to-wean facility near Leon,
Spain during July and August 2006. A total of 149 Hypor gilts were employed to
examine responses to different gonadotrophin preparations. Gilts were housed at 6 to 8
animals per pen and fed ad libitum a standard ﬁnisher ration. Gilts were assigned by age
(150 d or 180 d) to receive an intramuscular (1M) injection of 400 IU eCG plus 200 IU
hCG (PG600®, Intervet International, Boxmeer, NL; n=19 and 19, at 150 and 180 (1,
respectively), 600 IU eCG (Folligon®, Intervet; n=19 and 18, respectively), or pre-
treatment with 72 mg F SH (Folltropin®; Bioniche Animal Health, Bellville, Ontario,
n=19 and 17, respectively) followed by 600 IU eCG. The F SH was administered as 6 x
12 mg injections at 12 h interval with the eCG being administered 12 h after ﬁnal F SH
injection. A fourth group of gilts served as uninjected controls (n=19 and 19,

respectively).

From 2 d to 7 d after hormone injection, gilts were subject to direct exposure to a
mature boar for 15 min daily to facilitate detection of estrus. To determine the ovulation
responses to hormone treatment, blood samples were obtained from all gilts via jugular
venipuncture at the time of hormone injection and 10 d later. Serum was harvested and
frozen (-20°C) until assayed for progesterone concentrations. Serum samples were
assayed for progesterone (P4) concentrations in a single assay using a commercial ELISA
kit (Immulite®, Siemens Medical Solutions Diagnostics, Tarrytown, NY). Assay
sensitivity and intra- assay coefﬁcient of variation were 0.2 ng/ml and 8.1%, respectively.
All gilts employed in this study had non-detectable P4 concentrations on the day of

injection. An elevation of progesterone concentrations on day 10 to a minimum of 4
42

ng/ml was deemed to indicate an ovulatory response to treatment. The effects of
treatment and gilt age on the proportional estrus and ovulation responses were examined
by Chi square. For the determination of age effects, the eCG and FSH followed by eCG

groups were combined.

Results

As shown in Table 3.1, a single 150 (I control gilt (5.3%) was detected as being
estrous but she did not ovulate. When compared to the gilts treated with eCG alone,
PG600 injection tended (P=0.l) to increase the estrus response (52.6% vs. 26.3%) and
increased (P<0.01) the ovulatory response (89.5% vs. 47.4%). The estrus response in
eCG-treated gilts that were pretreated with FSH was intermediate (42.1%) but their
ovulatory response (47.4%) was the same as for eCG alone. Following treatment at 180
d, 2 control gilts (10.5%) were deemed estrous and they also ovulated. There was no
difference between any horrnone-treated group for estrus or ovulatory responses,
although the ovulatory response of PG600-treated gilts tended (P=0.1) to be higher than
for the gilts receiving eCG alone (89.5% vs. 66.7%), with the FSH-pretreated gilts being
intermediate (76.5%). There was no effect of age on estrus or ovulatory responses of
PG600-treated gilts but the estrus and ovulatory responses to eCG were higher in older

gilts (P<0.05 for both).

43

Table 3.1. Inﬂuence of gilt age (150 vs. 180 d) on the estrus and ovulation responses
to eCG plus hCG combination, eCG alone, FSH pre-treatment then eCG,

or no treatment

 

 

eCG+hCG eCG FSH+eCG Control
150 days
No. ofgilts l9 I9 19 19
Gilts estrus by 7 d 10 (52.6%) 5 (26.3%) 8 (42.1%) 1 (5.3%)
Gilts ovulating 17 (89.5%) 9 (47.4%) 9 (47.4%) 0
180 d
No. of gilts 19 18 17 19
Gilts estrus by 7 d' 13 (68.4%) 12 (66.7%) 10 (58.8%) 2 (10.5%)
Gilts ovulating 17 (89.5%) 12 (66.7%) 13 (76.5%) 2 (10.5%)

 

lOne estrous eCG+hCG treated gilt failed to ovulate

Discussion

The primary objective of the present study was to test the hypothesis that gilts
pre-treated with FSH would have higher estrus and ovulation responses to eCG injection,
presumably due to enhanced follicular development. The hypothesis was not supported
since we observed no difference in proportions of gilts ovulating between the two eCG-
treated groups. Although we did not assess ovarian follicular dynamics in this study, our
FSH treatment protocol was similar to others where an effect on follicle growth was

evident (Guthrie et al., 1990; Bolamba et al., 1996). This suggests that even if F SH was

44

able to stimulate follicular grth to the medium follicle size, these follicles were not
more responsive to eCG at either 150 or 180 days of age. It is probable that the ovarian
response to eCG stimulation will depend on the gilt’s level of physiological maturity,
which will include the entire hypothalamo-pituitary-ovarian axis, not just at the ovarian
level. This is supported by a positive association between gilt weight and their estrus
response noted in our earlier study (Majarin, unpublished data) and the effect of age in

the present study.

The absence of a gilt age effect in the ovarian response to PG600 indicates that
during the prepubertal phase, concurrent LH activity is necessary for continued follicular
development. With PG600, this LH activity is supplied by the hCG component of the
product. The results of the present study, where no effect of F SH pretreatment on gilt
estrus or ovulatory responses to eCG were evident also supports, albeit indirectly, the
suggestion of an indispensable role for LH activity in follicular development. Indeed,
development of follicles beyond 4 mm in diameter was not FSH dependent but seemed to
be controlled primarily by LH pulses (Driancourt et al., 1995) and is coupled with a
change in FSH and LH follicular receptors (Nakano et al., 1983; Liu et al., 1998; Liu et
al., 2000). The improved estrus induction response of prepubertal gilts to higher doses of
eCG (Dial et al., 1984; Britt et al., 1985) also argues for an indispensable role for LH
activity in development of follicles to the preovulatory stage. The eCG molecule does
have some LH activity (Combamous et al. 1984; Guthrie et al. 1990) and at higher doses
it is possible that the LH activity of eCG becomes sufﬁcient to allow development of
large follicles. Indeed, it has been shown that LH (Huff and Esbenshade, 1992; Guthrie et

al., 1990) and hCG (Bolamba et al., 1991; Driancourt et. al., 1992) can stimulate
45 '

follicular development, estrus and ovulation in prepubertal gilts if given in sufﬁcient
quantities, whereas treatment with FSH alone did not have any effect on the number of
large follicles (Guthrie et al., 1988, 1990). In contrast, however, Bolamba and Sirard
(2000) did note growth of large follicles when FSH was administered for three days
instead of two (93-187 ug/kg BW 24 h interval), although this could be attributable to LH
contamination of the FSH used. Additional evidence in support of the role of LH in

follicular development was provided in the review of Evans and O’Doherty (2001).

In conclusion, the lower ovulatory response to a relatively low dose of eCG
compared to the combination of eCG and hCG, likely reﬂects an inability to stimulate
growth of follicles, particularly at younger gilt ages. The absence of an effect of FSH pre-
treatment on the response to eCG indicates that either our protocol did not affect
follicular growth or that there is an indispensable role for LH activity in ﬁnal follicular

development.

Acknowledgements

We gratefully acknowledge Bioniche Animal Health for the provision of
Folltropin, UVESA for allowing access to the gilts, Evaristo Aguado for his expert
assistance throughout the study, and Laboratorio de Tecnicas Instrumentals, University of

Leon, for provision of laboratory facilities.

46

References

Bolamba, D., Matton, P., Sirard, M.A., Estrada, R., Dufour, J.J., 1991. Ovarian
morphological conditions and the effect of injection of human chorionic gonadotropin
on ovulation rates in prepuberal gilts with two morphologically different ovarian
types. J. Anim. Sci. 69, 3774-3779.

Bolamba, D., Dubuc, A., Dufour, J.J., Sirard, M.A., 1996. Effects of gonadotropin
treatment on ovarian follicle growth, oocyte quality and in vitro fertilization of
oocytes in prepubertal gilts. Theriogenology 46, 717-726.

Bolamba, D., Sirard, M.A., 2000. Ovulation and follicular growth in gonadotropin-treated
gilts followed by in vitro fertilization and devlopment of their oocytes.
Theriogenology 53, 1421-1437.

Britt, J .H., Esbenshade, K.L., Heller, K., 1985. Responses of seasonally anestrus gilts and
weaned primiparous sows to treatment with pregnant mare’s serum gonadotropin and
altrenogest. Theriogenology 26, 697-707.

Camous, S., Prunier, A., Pelletier, J., 1985. Plasma prolactin, LH, FSH and estrogen
excretion patterns in gilts during sexual development. J. Anim. Sci. 60, 1308-1317.

Combamous, Y., Guillou, F., Martinat, N., Cahoreau, C., 1984. [Origin of the FSH + LH
double activity of equine chorionic gonadotropin (eCG/PMSG)]. Ann. Endocrinol.
(Paris) 45, 261-268.

Dial, G.D., Dial, O.K., Wilkinson, R.S., Dziuk, P.J., 1984. Endocrine and ovulatory
responses of the gilt to exogenous gonadotropins and estradiol during sexual
maturation. Biol. Reprod. 30, 289-299.

Driancourt, M.A., Locatelli, A., Prunier, A., 1995. Effects of gonadotrophin deprivation
on follicular growth in gilts. Reprod. Nutr. Dev. 35, 663-673.

Driancourt, M.A., Prunier, A., Bidanel, J.P., Martinat-Botte, F ., 1992. HCG induced
oestrus and ovulation rate and FSH concentrations in prepuberal gilts from lines
differing by their adult ovulation rate. Anim. Reprod. Sci. 29, 297-305.

Evans, A.C.O., O’Doherty, J.V., 2001. Endocrine changes and management factors
affecting puberty in gilts. Livest. Prod. Sci. 68, 1-12.

Guthrie, H.D., 2005. The follicular phase in pigs: Follicle populations, circulating
hormones, follicle factors and oocytes. J. Anim Sci. 83, E79-E89.

Guthrie, H.D., Bolt, D.J., Cooper, BS, 1990. Effects of gonadotropin treatment on
ovarian follicle growth and granulosa] cell aromatase activity in prepuberal gilts. J.
Anim. Sci. 68, 3719-3726.

47

Guthrie, H.D., Bolt, D.J., Kiracofe, G.H., Miller, K.F., 1987. Effect of charcoal-extracted
porcine follicular ﬂuid and 17 B-estradiol on follicular growth and plasma
gonadotropins in gilts fed a progesterone agonist, altrenogest. J. Anim. Sci. 64, 816-
826.

Guthrie, H.D., Bolt, D.J., Kiracofe, G.H., Miller, K.F., 1988. Ovarian response to
injections of charcoal-extracted porcine follicular ﬂuid and porcine follicle
stimulating hormone in gilts fed a progesterone agonist (altrenogest). Biol. Reprod.
38, 750-755.

Guthrie, H.D., Garrett, W.M., 2000. Factors regulating apoptosis during folliculogenesis
in pigs. J. Anim. Sci. 77, 1-15.

Huff, B.G., Esbenshade, K.L., 1992. Induction of follicular development and estrus in
prepuberal gilts with gonadotropins. Anim. Reprod. Sci. 27, 183-194.

Kirkwood, RN, 1999. Pharmacological intervention in swine reproduction. Swine
Health Prod. 7, 29-35.

Knox, R.V., Zimmerman, D.R., 1993. Effect of administration of porcine follicular ﬂuid
to gilts during the luteal phase of the estrous cycle on plasma gonadotropins,
follicular development and ovulation rate. J. Anim. Sci. 71, 1546-1551.

Liu, J., Aronow, B.J., Witte, D.P., Pope, W.F., La Barbera, A.R., 1998. Cyclic and
maturation-dependent regulation of follicle-stimulating hormone receptor and
luteinizing hormone receptor messenger ribonucleic acid expression in the porcine
ovary. Biol. Reprod. 58, 648 - 658.

Liu, J., Koenigsfeld, A.T., Cantley, T.C., Boyd, C.K., Kobayashi, Y., Lucy, M.C., 2000.
Growth and the initiation of steroidogenesis in porcine follicles are associated with
unique patterns of gene expression for individual components of the ovarian insulin-
like growth factor system. Biol. Reprod. 63, 942-952.

Nakano, R., Sasaki, K., Shima, K., Kitayama, S., 1983. Follicle-stimulating hormone and
luteinizing hormone receptors on porcine granulosa cells during follicular maturation:
an autoradiographic study. Exp. Clin. Endocrinol. 81, 17-23.

48

CHAPTER 4
EFFECT OF HCG TREATMENT ON THE ESTROUS AND OVULATION
RESPONSES TO FSH IN PREPUBERTAL GILTS

R Manjarin', JC Dominguezz, MJ Castroz, DJ Sprecher', G Cassar3, RM Friendship3, and
RN Kirkwoodl

lDepartment of Large Animal Clinical Sciences, Michigan State University, East
Lansing, MI, USA, 2Department of Animal Reproduction and Obstetrics, University of

Leon, Leon, Spain and 3 Department of Population Medicine, University of Guelph,
Ontario, Canada (Reproduction in Domestic Animals, 2 008; doi: 10.1 1 1 1/j.1439-
0531.2008.01103.x)

Abstract

To ensure sufﬁcient numbers of pregnant females, particularly at hotter times of
the year, hormonal induction of gilt estrus may be necessary. However, the gilt estrus and
ovulation responses to gonadotrophin treatment have often proven unpredictable. The
objective of the present study was to examine possible reasons for this unpredictability.
Prepubertal gilts (approximately 150 d of age, n=63) were assigned to one of three
treatments: injection of 300 IU hCG (n=15); pretreatment with 100 mg F SH in
polyvinylpyrrolidinone administered as 2 x 50 mg injections 24 h apart, followed by 600
IU eCG at 24 h after the second F SH injection (n=23); or FSH pretreatment as above
followed by 300 IU hCG at 24 h after the second FSH injection (n=25). To facilitate
estrus detection, gilts were exposed to a mature boar for 15 min daily for 7 (I. Blood
samples were obtained on the day of eCG or hCG injection and again 10 d later and gilt
ovulation responses determined on the basis of elevated progesterone concentrations. The
estrus responses by 7 d were 6.7%, 17.5%, and 64.0%, for gilts treated with hCG,

49

FSH+eCG, and FSH+hCG, respectively (P<0.001). The estrous gilt receiving hCG alone
and one estrous F SH+hCG gilt did not ovulate, all other estrous gilts ovulated. A further
2 anestrus FSH+eCG-treated gilts ovulated. These data suggest that FSH pre-treatment
facilitated the development of ovarian follicles to the point where they became responsive

to hCG, but had little effect on the response to eCG.

Keywords: swine, gilts, gonadotrophins, estrus, ovulation

Introduction

The output of the breeding herd is weaner pigs, and it has been suggested that the
factor most affecting the predictability of weaner pig output is the meeting of breeding
targets (Dial et al. 1996). The ability to meet the weekly breeding target requires a
predictable supply of service-ready gilts, which may necessitate the hormonal induction
of estrus to ensure the availability of a sufﬁcient number of breeding females.
Gonadotrophic preparations used to stimulate estrus in gilts are equine chorionic
gonadotrophin (eCG) and combinations of eCG with human chorionic gonadotrophin
(hCG). We have previously conﬁrmed that, compared to treatment with 600 IU eCG
alone, estrus and ovulation responses of gilts were improved when injected with 400 IU
eCG combined with 200 IU hCG (PG600®) (Manjarin et al. 2008a). However, responses
to PG600 remained unpredictable, with 73% exhibiting estrus, but only 64% both
exhibiting estrus and ovulating. We now speculate that the failure of overtly estrous gilts
to ovulate may be due to the 200 IU hCG in PG600 providing an inadequate duration of

LH-like activity to complete follicular development. Interestingly, the serum half-life of

50

hCG in humans was longer after subcutaneous than after intramuscular injection (Saal et
al. 1991) and on one occasion an improved estrus response of gilts to PG600 was noted
after subcutaneous injection compared to intramuscular injection (Knox et al. 2000). This
suggests a longer duration of gonadotrophic biological activity may be necessary for
some gilts to both express estrus and ovulate. However, prolonging the duration of
gonadotrophin activity by simply increasing the initial dose of PG600 did not prove
useful. The administration of 1.5 or 2.0 times the label dose of PG600 to gilts did
increase the number of corpora lutea but also increased the numbers of gilts with

follicular cysts as well as the numbers of cysts per gilt (Breen et al. 2006).

Several earlier studies have shown that LH (Huff and Esbenshade, 1992) and hCG
(Guthrie et al. 1990; Bolamba et al. 1991; Driancourt et al. 1992), in addition to their
ovulatory and steroidogenic actions, can also stimulate follicular development. However,
gilts with different ovarian status (number and size of follicles) at the time of hCG
injection had different rates of ovulation (Bolamba et al. 1991), suggesting that hCG is
unable to initiate growth of small follicles. In contrast, FSH seems to play a key role
during initial follicle growth (Guthrie and Bolt, 1990; Knox et al. 2003). Taking all
together, we now hypothesize that pretreatment of gilts for 2 days with FSH may advance
follicular development to the point where the follicles will respond to hCG injection with

onset of estrus and ovulation.

51

 

 

Material and methods

These studies were performed during October to December 2007 at a commercial
1,000-sow farrow-to-wean facility near Leon, Spain. Animals were cared for humanely in
accordance with institutional animal care guidelines. To examine responses to different
gonadotrophin preparations, 63 prepubertal Hypor gilts (approximately 150 d of age)
were employed. Actual gilt ages were between 140 and 160 d but the farm recorded all
gilts within this range as being 150 d. Gilts were housed at 6 to 8 animals per pen and fed
ad libitum, a wheat/soybean meal ﬁnisher diet formulated to provide 14 MJ DE/kg and

0.8% lysine.

At selection, gilts were assigned to receive an intramuscular (IM) injection of 300
IU hCG (Veterin Corion®; Divasa, Spain) (n=15), or 100 mg FSH (Folltropin®;
Bioniche Animal Health, Bellville, Ontario) mixed with polyvinylpyrrolidinone K-30
(PVP; Plasdone C-30, average mol wt. 58,000, ISP Technologies Inc., Wayne, NJ, USA),
administered as 2 x 50 mg injections 24 h apart, followed by 600 IU eCG (Folligon®;
Intervet International, Boxmeer, NL) at 24 h after the second FSH injection (n=23), or
100 mg FSH as above, and then 300 IU hCG at 24 h after the second FSH injection
(n=25). The dose of eCG was chosen because of it’s known efﬁcacy in weaned sows and
it also reﬂects the total gonadotrophin content of PG600 (400 IU eCG plus 200 IU of
hCG), a product of known efﬁcacy in gilts. The dose of hCG was chosen to reﬂect the
LH-like activity of PG600, ie. 200 IU of hCG with the 400 IU eCG likely having 20 to
40% LH-like activity (Combamous et al. 1984; Guthrie et al. 1990). The objective of
administering FSH in PVP solution was to prolong the period of biological activity

(Jackson et al. 2006). The PVP solution (30%) was prepared by dissolving 15 mg of PVP
52

in 40 ml of distilled and deionized water and was stored at 4°C until use. For each

injection, 3.125 ml PVP solution was mixed with 0.875 ml (50 mg) of FSH.

To facilitate detection of estrus, gilts were subject to direct exposure to a mature
boar for 15 min daily from 1 d to 7 d after time of hormone injection. Gilts were deemed
to be estrous if they adopted a rigid stance in the presence of the boar. To determine the
ovulation responses to hormone treatment, blood samples were obtained from all gilts via
jugular venipuncture at the time of hCG or eCG injection (day 0) and 10 days later.
Serum was harvested and frozen (-20°C) until assayed for progesterone (P4)
concentrations. Serum samples were assayed for P4 in a single assay using a commercial
ELISA kit (Immulite®, Siemens Medical Solutions Diagnostics, Tarrytown, NY). Assay
sensitivity and intra-assay coefﬁcient of variation were 0.2 ng/ml and 8.1%, respectively.
All gilts employed in this study had non-detectable P4 concentrations on the day of
injection. An elevation of P4 concentrations on day 10 to a minimum of 5 ng/ml was
deemed to indicate an ovulation response to treatment (Althouse and Hixon 1999).
Treatment differences for estrus and ovulation response rates were examined by Chi

square.

Results

As shown in Table 4.1, of the 25 gilts receiving FSH plus hCG, 16 (64%)

exhibited estrus by 7 days and all but one of these estrous gilts had serum P4

53

concentrations indicative of ovulation. None of the 9 anestrus F SH plus hCG-treated gilts
ovulated. One gilt receiving hCG alone exhibited estrus, but neither she nor any other gilt
in this group ovulated. Of the 23 FSH plus eCG-treated gilts, 4 (17.4%) exhibited estrus
but only 3 of these ovulated. Of the 19 FSH plus eCG-treated gilts that remained

anestrous, 3 had elevated serum P4 concentrations indicating ovulation.

Table 4.1. Effect of injecting hCG, or FSH followed by eCG, or F SH followed by
hCG, into 150-day gilts on the incidence of estrus by 7 days and ovulation

as indicated by elevated progesterone (+P4), or non-ovulation (-P4).

 

 

hCG‘ FSH + eCG2 FSH + hCG3
No. of gilts 15 23 25
Gilts estrus by 7 d (%)4 1 (6.66%) 4 (17.4) 16 (64.0)
Gilts ovulating (%) 0 6 (26.1) 15 (60.0)

 

1300 IU hCG
2100 mg FSH in PVP as 2 injections 24 h apart then 600 IU eCG 24h after last FSH
3100 mg FSH in PVP as 2 injections 24 h apart then 300 IU hCG 24h after last FSH

4 Effect of hormone preparation, P<0.001 by Chi square test

Discussion

The results of this study support our hypothesis that pre-treating gilts with F SH
will enhance the estrus and ovulation responses to injection of hCG. Further, although we

did not monitor ovarian follicular dynamics, we suggest that the better responses were

54

due to an F SH-associated advancement of follicle maturity, presumably to the 4 mm
stage that becomes responsive to LH-like activity. That prior F SH activity was necessary
for expression of the hCG effect on estrus and ovulation responses is also indicated by

the lack of effect of hCG when not pre-treated with FSH.

The present results also support our previous ﬁndings (Manjarin et al. 2008a,b)
and literature evidence (do Lago et al. 2005; Gama et a1. 2005) that, in the absence of
additional LH-like activity, 600 IU eCG, is relatively non-efﬁcacious for induction of
estrus and ovulation in prepubertal gilts. The eCG molecule does have some LH-like
activity (Combamous et al. 1984; Guthrie et al. 1990) but apparently not enough to drive
follicular development when only 600 IU is administered. Estrus responses to eCG are
increased when higher doses are administered (eg. Bolamba et al. 1992; Dial et al. 1984),
presumably due to the associated increase in LH-like activity. In a previous study, we
noted no effect of FSH pre-treatment on the efﬁcacy of eCG for estrus induction,
although in that study only 72 mg of FSH was injected and it was administered as 6 serial
injections (Manjarin et al. 2008b). The present study employed PVP to extend the
biological activity of the 100 mg FSH but, again, FSH failed to affect the ovarian
response to eCG. The F SH pretreatment was effective for priming the ovary to respond to
hCG, which does suggest an effect on follicle development. However, based on the
present data, the magnitude of the effect was not sufﬁcient to allow the follicles to

respond to eCG stimulation.

55

Driancourt et al. (1995) observed in gilts that follicle development to about the 4
mm stage was dependant only on FSH while growth beyond this diameter appeared to be
controlled by LH pulses. Indeed, granulosa cells of immature follicles contained
exclusively FSH receptors, but these declined by the 4 mm stage and were absent in
larger follicles (Liu et al. 1998; 2000). Interestingly, treatment in vitro with FSH
promoted LHr mRNA expression (LaBarbera, 1981), which likely explains how the FSH

pre-treatment was able to prime the ovary to respond to the hCG injection.

In conclusion, pretreatment with FSH increased the ovulation response to hCG
compared to hCG alone, supporting an indispensable role for F SH during early follicle
maturation but a transition to LH dependence. A better understanding of this relationship
may facilitate the development of improved protocols for hormonal induction of estrus

and ovulation.

Acknowledgements
We gratefully acknowledge Bioniche Animal Health for the provision of

Folltropin, UVESA for allowing access to the gilts, Evaristo Aguado for his expert
assistance throughout the study, Labatorio SYVA S.A., Leon, Spain for provision of
PVP, and Laboratorio de Tecnicas Instrumentals, University of Leon, for provision of

laboratory facilities.

56

References

Althouse, G.C., Hixon, J.B., 1999. Use of commercially available ELISAs to help
determine estrous status in female swine. Swine Health Prod. 7, 65-68.

Bolamba, D., Matton, P., Sirard, M.A., Estrada, R., Dufour, J.J., 1991. Ovarian
morphological conditions and the effect of injection of human chorionic gonadotropin

on ovulation rates in prepuberal gilts with two morphologically different ovarian
types. J. Anim. Sci. 69, 3774-3779.

Bolamba, D., Matton, P., Estrada, R., Dufour, J .J., 1992. Effect of pregnant mare’s serum
gonadotropin treatment on follicular populations and ovulation rates in prepuberal
gilts with two morphologically different ovarian types. J. Anim. Sci. 70, 1916-1922.

Breen, S.M., Rodriguez-Zas, S.L., Knox, R.V., 2006. Effect of altering dose of PG600 on
reproductive performance responses in prepubertal gilts and weaned sows. Anim.
Reprod. Sci. 95, 316—323.

Combamous, Y., Guillou, F., Martinat, N., Cahoreau, C., 1984. [Origin of the FSH + LH
double activity of equine chorionic gonadotropin (eCG/PMSG)]. Ann. Endocrinol.
(Paris) 45, 261-268.

Dial, G.D., Dial, O.K., Wilkinson, R.S., Dziuk, P.J., 1984. Endocrine and ovulatory
responses of the gilt to exogenous gonadotropins and estradiol during sexual
maturation. Biol. Reprod. 30, 289-299.

Dial, G.D., Duangkaew, C., King, V., Rademacher, C., 1996. The inﬂuence of the gilt
pool on weaned pig output. Proc. AD Leman Conf. 23, 39-41.

do Lago, V., Vianna, W.L., Gama, R.D., de Campos Rosseto, A., Pinese, M.E., de
Sant’Anna Moretti, A., 2005. Second oestrus synchronization and precocious embryo
viability after puberty induction in gilts by the use of gonadotrophin treatment.
Reprod. Dom. Anim. 40, 141-144.

Driancourt, M.A., Locatelli, A., Prunier, A., 1995. Effects of gonadotrophin deprivation
on follicular growth in gilts. Reprod. Nutr. Dev. 35, 663-673.

Driancourt, M.A., Prunier, A., Bidanel, J.P., Martinat-Botte, F ., 1992. HCG induced
oestrus and ovulation rate and FSH concentrations in prepuberal gilts from lines
differing by their adult ovulation rate. Anim. Reprod. Sci. 29, 297-305.

Gama, R.D., Vianna, W.L., Pinese, M.E., de Campos Rosseto, A., de Sant’Anna Moretti
A., 2005. Different doses of porcine luteinizing hormone in precocious puberty
induction in gilts. Reprod. Dom. Anim. 40, 433-435.

57

Guthrie, H.D., Bolt. D.J., Cooper, BS, 1990. Effect of gonadotropin treatment on
ovarian follicle growth and granulosa cell aromatase activity in prepuberal gilts. J.
Anim. Sci. 68, 3719-3726.

Guthrie, H.D., Bolt, D.J., 1990. Changes in plasma follicle-stimulating hormone,
luteinizing hormone, estrogen and progesterone during growth of ovulatory follicles
in the pig. Dom. Anim. Endocrinol. 7, 83-91.

Huff, B.G., Esbenshade, K.L., 1992. Induction of follicular development and estrous in
prepuberal gilts with gonadotropins. Anim. Reprod. Sci. 27, 183-194.

Jackson, A.L., Breen, S.M., Rodriguez-Zas, S.L., Knox, R.V., 2006. Evaluation of
methodology for administration of porcine FSH for use in estrus induction and for
increasing ovulation rate in prepuberal gilts. Theriogenology 66, 1042-1047.

Knox, R.V., Tudor, K.W., Rodriguez-Zas, S.L., Robb, J .A., 2000. Effect of subcutaneous
administration of PG600 on estrual and ovulatory responses of prepuberal gilts. J
Anim Sci 78, 1732-1737.

Knox, R.V., Vatzias, G., Naber, C.H., Zimmerman, D.R., 2003. Plamas gonadotropins
and ovarian hormones during the estrous cycle in high compared to low ovulation rate
gilts. J. Anim. Sci. 81, 249-260.

LaBarbera, A.R., Ryan, R.J., 1981. Porcine granulose cells in suspension culture. I.
Follicle-stimulating hormone induction of human chorionic gonadotropin binding
sites on cells from small follicles. Endocrinology 108, 1561-1570.

Liu, J., Aronow, B.J., Witte, D.P., Pope, W.F., La Barbera, A.R., 1998. Cyclic and
maturation-dependent regulation of follicle-stimulating hormone receptor and

luteinizing hormone receptor messenger ribonucleic acid expression in the porcine
ovary. Biol. Reprod. 58, 648-658.

Liu, J., Koenigsfeld, A.T., Cantley, T.C., Boyd, C.K., Kobayashi, Y., Lucy, M.C., 2000.
Growth and the initiation of steroidogenesis in porcine follicles are associated with
unique patterns of gene expression for individual components of the ovarian insulin-
like growth factor system. Biol. Reprod. 63, 942-952.

Manjarin, R., Cassar, G., Sprecher, D.J., Friendship, R.M., Dominguez, J.C., Kirkwood,
R.N., 2008a. Effect of eCG or eCG plus hCG on estrus expression and ovulation in
prepubertal gilts. Reprod. Dom. Anim.

Manjarin, R., Dominguez, J .C., Castro, M.J., Vallado, B., Rodriguez, D.J., Sprecher, D.J.,
Cassar, G., Friendship, R.M., Kirkwood, R.N., 2008b. Effect of prior FSH treatment
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Saal, W., Glowania, H.J., Hengst, W., Happ, J., 1991. Pharrnacodynamics and
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gonadotropin. Fertil. Steril. 56, 225-229.

59

CHAPTER 5
SUMMARY AND CONCLUSIONS

Summary

Three studies were performed to examine the reasons for unpredictability of
estrus and ovulatory response to equine chorionic gonadotrophin (eCG) either alone or in
combination with human chorionic gonadotrophin (hCG) in prepubertal gilts. Estrus
responses were determined on the basis of expression of a standing heat reaction in the
presence of a boar while ovulation responses were assessed on the basis of elevations of

circulating progesterone concentrations 7 to 10 days after treatment.

In the ﬁrst study, prepubertal gilts (90 kg and 153 d) received an injection of
either 600 IU eCG or a combination of 400 IU eCG and 200 IU hCG (PG600), or were
non-injected controls, and were then exposed to a mature boar for 15 min daily for 7 days
for estrus detection. Also at the time of injection, gilt's ovaries were subjected to real-
time ultrasound examination. Blood samples were obtained at time of hormone injection
((1 O and at d 3, 7, and 10) for assay for serum progesterone. Both the estrus and ovulation
responses were better following PG600 injection than following eCG injection, with
responses improving with increasing gilt weight. There was no evident association

between reproductive responses and size of ovarian follicles.

In the second study, prepubertal gilts were selected at 150 d or 180 d of age and
assigned to injection of PG600, 600 IU eCG alone, pretreatment with 72 mg FSH

administered as 6 injections at 12 h intervals with 600 IU eCG injected 12 h after last
60

FSH injection, or to be non-injected controls. Gilts were exposed to a mature boar for 15
min daily for 7 d and blood samples obtained on the day of injection and 10 d later and
assayed for progesterone content. Again, responses to PG600 were superior to those
following eCG injection with no evident effect of FSH pre-treatment. Responses were

improved for older gilt's age.

In the third study, prepuberal gilts (150 d of age) were assigned to treatments
involving injection of 300 IU hCG alone, pretreatment with 100 mg FSH administered as
2 injections 24 h apart, followed by 300 IU hCG at 24 h after the second F SH injection or
FSH pretreatment as above followed by 600 IU eCG at 24 h after the second FSH
injection. Gilts were exposed to a mature boar for 15 min daily for 7 d and blood samples
obtained on the day of eCG or hCG injection and again 10 d later. Estrus and ovulation
responses were minimal to hCG treatment alone but were improved by F SH pretreatment
prior to eCG injection. When FSH pretreatment was followed by hCG, estrus and

ovulation responses rivaled those following PG600 injection.

Conclusions

The results of experiment 1 indicate that when ovaries contain only small (<3
mm) follicles, the failure of 30% of gilts to exhibit estrus in response to a combination of
eCG and hCG does not involve premature ovulation or luteinization of follicles caused by
the hCG component. Data from experiment 2 demonstrate that the lower ovulatory

response to a relatively low dose (600 IU) of eCG compared to the combination of eCG

61

and hCG likely reﬂects an inability to stimulate follicle growth, particularly at younger
gilt ages. However, the absence of an effect of F SH pre-treatment on the response to
eCG, compared to eCG alone, indicates that the failure to stimulate growth is likely not
due to an inability to initiate development of small follicles. Interestingly, pre-treatment
with FSH increased ovulation responses to hCG compared to hCG alone, suggesting that
FSH may play a crucial role during ﬁrst steps of follicular maturation while the greater
ovulation response obtained to hCG compared to eCG after F SH pretreatment suggests an

indispensable role for LH-like activity once follicles have reached certain development.

62

 

 

 

 

 

 

 
 

 

 

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