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This is to certify that the
thesis entitled

SOW FERTILITY TO INSEMINATION OF
CRYOPRESERVED SPERM

presented by

MIGUEL ABAD-VALCARCE

has been accepted towards fulﬁllment
of the requirements for the

MS. degree in Lag; Animal Clinical Sciences

 

 

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SOW FERTILITY TO INSEMINATION OF CRYOPRESERVED SPERM

By

Miguel Abad-Valcarce

A THESIS
Submitted to
Michigan State University

In partial fulﬁllment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Large Animal Clinical Sciences

2006

ABSTRACT

SOW FERTILITY TO INSEMINATION OF CRYOPRESERVED SPERM

By

Miguel Abad-Valcarce

This thesis describes studies to assess the effect of the insemination-ovulation interval
and the addition of seminal plasma (SP) on the establishment of the sperm reservoir and
subsequent sow fertility following artiﬁcial insemination (AI) of cryopreserved sperm.
In initial experiments, the numbers of sperm located in the reservoir after A1 of fresh
(FS) or frozen-thawed (FT) semen was studied in gilts. Ovulation was synchronized and
gilts inseminated with FS sperm, FT sperm, or FT sperm supplemented with 10% SP.
After slaughter, uterotubal junctions (UTJs) were recovered and ﬂushed to recover
sperm. The number of sperm cells located in the reservoir was consistently lower in
gilts inseminated with FT sperm and results were not affected by the addition of seminal
plasma. In subsequent experiments, multiparous sows were inseminated 12 h before
ovulation with either F S sperm, FT sperm, or FT sperm with 10% SP. Other sows were
inseminated with FT sperm at 2 h before ovulation. lnseminations were either cervical
(experiment 2.1) or intrauterine (experiment 2.2). Farrowing rates were lower in sows
receiving FT sperm regardless of timing of AI and supplemental SP. Litter size was
unaffected. The smaller sperm reservoir and low fertility rates achieved after A1 of FT
sperm suggests that the injuries produced by the cryopreservation to the sperm cells

involves more than just cryocapacitation.

DEDICATION

To the eternal care, to my parents and brother

ACKNOWLEDGMENTS

I am extremely grateful to many people that have contributed widely to the thesis

presented here. Risking forgetting many, I would like to mention some.

Firstly Dr Roy Kirkwood, advisor not only limited to that, because of his professional
and personal excellence, identifying the time to push me, and the time to push me more.
None of these studies would have existed without him.

Dr Dave Sprecher, who repeatedly taught me the way to face the adversity, regardless
the size in which it arrives. He did show me the true American character and values.

Dr Bob Friendship, who host me as one more of his privileged students. His guidance
and never-ending enthusiasm was my main support during the stay in Canada.

Dr Jose Cibelli, who kindly took interest in this work. His generous character and
exceptional leadership have been a permanent referent in my time spent at MSU.

I would also like to acknowledge Arturo, Christine, Jessika, Jose, Pablo and all the
graduate students that collaborated to this thesis with their time and comprehension.
Special thanks to Glen Cassar and Missy Vadnais for their help and friendship.

Finally, I would like to thank Faith Peterson and also the staff at MSU and Arkell Swine

Farms for their continuous support and availability.

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................... vii
LIST OF FIGURES .................................................................................................... viii
LIST OF ABBREVIATIONS ......................................................... ix
Chapter I
Literature Review: Reproduction and artificial insemination ....................................... l
Swine reproductive anatomy and Physiology ............................................................... I
History of artiﬁcial insemination ....................................................................... 5
Sow fertility to AI .............................................................................................. 7
Semen backﬂow .................................................................................... 8
Sperm reservoir ..................................................................................... 9
Insemination of low sperm numbers ................................................... 10
Timing of insemination relative to ovulation ...................................... ll
Sperm capacitation .......................................................................................... 15
Boar semen cryopreservation .......................................................................... I7
Freezing protocols ............................................................................... I8
Capacitation-Iike status ........................................................................ 20
Seminal plasma ................................................................................................ 21
Effect of seminal plasma on sperm capacitation and fertility ............. 22
References ....................................................................................................... 24
Chapter 2
Effect of sperm cryopreservation and supplementing semen doses with seminal plasma
on the establishment of a sperm reservoir in gilts ....................................................... 32
Contents ........................................................................................................... 32
Introduction ..................................................................................................... 33
Material and methods ...................................................................................... 35
Results ............................................................................................................. 38
Discussion ........................................................................................................ 39
Acknowledgements ......................................................................................... 41
References ....................................................................................................... 42
Chapter 3
Effect of insemination-ovulation interval and addition of seminal plasma on sow
fertility ......................................................................................................................... 44
Contents ........................................................................................................... 44
Introduction ..................................................................................................... 45
Material and methods ...................................................................................... 47
Results ............................................................................................................. 49
Discussion ........................................................................................................ 51
Acknowledgements ......................................................................................... 53
References ....................................................................................................... 54
Chapter 4
Summary and Conclusions .......................................................................................... 57

Summary .......................................................................................................... 57

Conclusions ..................................................................................................... 58
Appendices .................................................................................................................. 59
Appendix I ...................................................................................................... 59
Appendix 2 ...................................................................................................... 61

vi

LIST OF TABLES
Table 1.1. Characteristics of a typical boar ejaculate .................................................... 5

Table 1.2. Volume and number of sperm in the semen backﬂow during insemination
(M1), 0-0.5h aﬁer insemination (M2) and 0.5-2.5h after insemination (M3). Semen
doses were 80ml with 1, 3 or 6x109 sperm .................................................................... 8

Table 1.3. Best time to inseminate relative to the ﬁrst time detection of standing estrous
..................................................................................................................................... 12

Table 1.4. hCG based ovulation synchronization protocols ........................................ 14

Table 2.1. Effect of freezing and supplementation with seminal plasma (SP) on number
of sperm recovered form the oviduct and uterotubal junction (experiment I) ............ 38

Table 2.2. Effect of freezing and supplementation with seminal plasma (SP) on number
of sperm recovered from the oviduct and uterotubal junction (experiment 2) ............ 39

Table 3.1. F arrowing rates and total born litter sizes following artiﬁcial insemination of
fresh (F S) sperm or cryopreserved (FT) sperm with or without 10% supplemental
seminal plasma (SP) inseminated 2 or 12 hours before ovulation (experiment one) ..50

Table 3.2. Farrowing rates and total born litter sizes following artiﬁcial insemination of
fresh (FS) sperm or cryopreserved (FT) sperm with or without 10% supplemental
seminal plasma (SP) inseminated 2 or 12 hours before ovulation (experiment two).. 51

vii

LIST OF FIGURES

Figure 1.1. Reproductive anatomy of the sow ............................................................... 3

Figure 1.2. Fertilization rates from Al with fresh and frozen semen at different times of

insemination relative to ovulation ............................................................................... 13
Figure 1.3. Model of sperm capacitation ..................................................................... 16
Figure 3.1. Sow ovulation time (hours post pLH injection) ........................................ 49

viii

LIST OF ABBREVIATIONS

AC ............................................................................................................ adenyl cyclase
AI .................................................................................................. artiﬁcial insemination
BSA ........................................................................................... bovine serum albumine
CAMP ............................................................................ cyclic adenosin monophosphate
CTC ...................................................................................................... chlortetracycline
ECG .............................................................................. equine chorionic gonadotrophin
FS ....................................................................................................... fresh (sperm cells)
FT ....................................................................................... frozen-thawed (sperm cells)
GnRH ......................................................................... gonadotrophin releasing hormone
HCG .............................................................................. human chorionic gonadotrophin
IM .............................................................................................................. intramuscular
IU ......................................................................................................... international unit
LH .................................................................................................... Iuteinizing hormone
LSD ....................................................................................... least signiﬁcant difference
MSU ...................................................................................... Michigan State University
PGF ..................................................................................................... prostaglandin F2a
PKA ........................................................................................................ protein kinase a
PKC ........................................................................................................ protein kinase c
PMSG .................................................................... pregnant mare serum gonadotrophin
PTO ...................................................................................... predicted time of ovulation
PRRS .................................................... porcine respiratory and reproductive syndrome
PSP ................................................................................ porcine seminal plasma protein
ROS ........................................................................................... reactive oxygen species

ix

SEM ....................................................................................... standard error of the mean

SP ............................................................................................................ seminal plasma
SR ........................................................................................................... sperm reservoir
UTJ .................................................................................................... uterotubal junction
V/V ...................................................................... . ................................... volume/volume
ZP ............................................................................................................. zona pellucida

CHAPTER 1

Literature Review: Reproduction and Artiﬁcial Insemination

Swine Reproductive Anatomy and Physiology

The reproductive organs of the female pig (Sus scrofa) are shown in Figure 1.1. The
vulva is the most external and visible portion of the reproductive tract and responds to
the cyclic levels of estrogen by showing a red edematous status during the proestrous
period. The vulva leads to the vagina, on the ﬂoor of which the urethra opens. By
directing artiﬁcial insemination (AI) catheters dorsally during catheter entry, urethral
penetration is prevented. The cervix is located immediately cranial to the vagina, the
lumen of which contains multiple interdigitating folds to prevent bacteria and other
foreign material from entering the uterus. The cervix is the site of semen deposition
during natural breeding and also during conventional AI. In addition, during
pregnancy the cervix secretes highly viscous mucus under the inﬂuence of
progesterone to keep the products of conception isolated from the exposed vaginal
tract. The uterus of the sow consists of a small uterine body and two long
(approximately 1 m) uterine horns. The oviducts extend from the end of each uterine
horn to the ovary. The junction of the uterus and the oviduct is called the uterotubal
junction (UTJ). Aﬁer sperm traverse the UTJ they remain in the ﬁrst 1 to 2 cm of the
oviductal isthmus, which is the site of the functional sperm reservoir (SR). Sperm in
the SR are primarily non-capacitated and remain so under the inﬂuence of the local
microenvironment until near the time of ovulation. The primary function of the
ovaries is to produce the female gametes, but they also produce hormones (mainly

estrogen and progesterone, but also oxytocin, relaxin, inhibin and activin).

As described by Kirkwood (1999), the 21-day porcine estrous cycle is composed of
an approximately 16—day Iuteal phase and a 5-day follicular phase. During the luteal
phase the ovarian corpora lutea produce progesterone, which limits ovarian follicular
development to the medium follicle stage and so prevents the onset of estrus. At
about 12 tol4 days of the luteal phase endometrial production of prostaglandin F 2,,
(PGF) causes regression of corpora lutea and so terminates progesterone production.
This removal of the progesterone block allows resumption of appropriate secretory
patterns of the pituitary gonadotropins, luteinizing hormone (LH) and follicle
stimulating hormone (FSH) which permits the resumption of ovarian follicular
development. The developing ovarian follicles secrete estrogen, which ultimately
results in behavioral estrus. In weaned sows, a 4 or 5-day wean-to-estrus interval is
equivalent to the follicular phase of the estrous cycle. Approximately coincident with
the onset of estrus, the increasing circulating levels of estrogen drive a surge release
of LH, which causes a cascade of events within the follicle including a switch from
estrogen to progesterone production and culminating about 40 hours later with

ovulation.

The male reproductive system consists of the testis, epididymis, accessory sex glands,
the spermatic cord and the penis. The primary function of the testes is to produce the
male gametes (sperm), but they also produce hormones (androgens, estrogens, and
progesterone). The epididymides are tubular structures that lie along the testis and
they store sperm while they mature. The vas deferens connect each epididymis to the
urethra. The boar accessory glands include the seminal vesicles, the prostate and the
bulbourethral glands, and these glands secrete the protective non-cellular portion of

semen known as seminal plasma.

Testicular function is controlled by pulsatile release of GnRH from the hypothalamus,
which stimulates secretion of FSH and LH by the anterior pituitary. The LI-I
stimulates progesterone and testosterone production by the Leydig cells while FSH
acts on the Sertoli cells to aromatize androgen to estrogen and to support
spermatogenesis. Inhibin, also produced by the Sertoli cells, regulates FSH secretion

via a negative feedback loop.

Uterus body

Uterus horn

Ovary

Oviduct

 

Cervix

  
 

Urinary bladder Vagina

Vestibule

Urethra o enin ;

Figure 1.] Reproductive anatomy of the sow

http://w" wist‘ ‘ ' ' " " ""‘""‘" histhtml

The normal boar ejaculate is composed of several fractions, an initial gel fraction
followed by a small volume of clear ﬂuid from the prostate, then the sperm-rich
fraction, then more clear prostatic ﬂuid and ending with the majority of the gel
fraction. The gel fraction secreted by the bulbourethral glands has a very high
estrogen content. Most of the sperm are located in the sperm-rich fraction of the
ejaculate. Aﬁer copulation, the sperm start arriving in the oviduct within 5-30 min
(Rodriguez-Martinez et a1. 2005). Sperm transport from the uterine body to the UTJ is
achieved by coordinated uterine contractions, the result of oxytocin release as well as
endometrial PGF released in response to seminal estrogen content (Willemburg et al.

2004)

Currently, techniques for evaluating the quality of boar sperm are similar to those
used for bull sperm (Johnson 1998). Differences are found among breeds and seasons,
but generally boar ejaculate volumes range from 160 to 266 ml, with concentrations
of 374 to 548 x 10‘5 sperm per ml (Ciereszko et al. 2000). Sperm motility immediately
after collection ranged from 70-90% (Vyt et al. 2004). For swine Al, the minimum
volume of ﬂuid and number of motile sperm necessary at each insemination is
suggested to be 50 ml and 3 x 109 motile sperm, respectively. However, insemination
of 2 x 109 sperm has been used successfully (Watson and Behan 2002). Currently,

typical AI doses have 3 x 109 sperm in 80ml extender.

Table 1.1. Characteristics of a typical boar ejaculate

 

 

Criterion Amount
Total volume, ml 300
Strained volume, ml 210
Gelatinous volume, ml 90

Sperrnatozoa per ml of strained semen 200 x 106

Total spermatozoa per ejaculate 42 x 109
Motile sperm, % 70

Total motile sperm per ejaculate 29.4 x 109
Number of Al doses possible 10

 

Straw (http://www.msu.edu/°/o7Estraw/)

History of Artiﬁcial Insemination

Artiﬁcial insemination involves the collection of semen from the male, its extension
(dilution) to produce multiple semen doses and subsequent deposition into the female
reproductive tract. In 1779, the Italian Lazaro Spallanzani performed the ﬁrst AI in
Milan. Using dog sperm to breed a bitch on heat, he obtained a group of normal
puppies with the same morphological characteristics as their progenitors. This event,
conﬁrmed by Pietro Rossi in 1782, is considered as the beginning of artiﬁcial

insemination in domestic animals (Abad 2001).

As reviewed by Abad (2001), food animal Al was introduced by Smith in 1891 and
was initially a way to control infertility. However, in 1902 Sand raised the possibility
of enhancing domestic animal reproduction by means of AI. During the following 30

years the main advances in Al were made in Russia, where in 1922 Ivanov developed
5

the ﬁrst extender for semen and his student, Milanov, designed the ﬁrst artiﬁcial
vagina for use in sheep. Ivanov initiated A1 of swine in 1907, and the procedure was

then introduced into the United States, Japan and Western Europe.

The rapid adoption of artiﬁcial insemination in swine originated from a more
judicious use of semen and easier accessibility to producers of high-quality genetics.
The uptake of Al was rapid in Europe, especially during 1990-2000 (Gerrits et al.
2005), leading to a current AI rate of 90% in most West European countries. The
United States was slower to adopt AI (Weitze 2000), having a 15% use rate by 1994
and barely 50% by 1999 (Buhr 2001). Currently, it has been estimated that 70% of

sows and gilts in North America are bred by Al (Rodriguez-Martinez et al. 2005).

Many different extenders have been developed to preserve sperm cell viability after
collection. The ﬁrst Russian extenders for boar semen were based on glucose
solutions with sodium potassium tartrate or sodium sulfate and peptone, keeping the
concentration of electrolytes low (F oote 2002). The recommended storage
temperature for semen extended in this manner was from 7 to 12°C. After yolk-
phosphate, yolk-citrate, and milk-based extenders were developed for bull semen,
they were also used or modiﬁed for boar semen (Polge 1956). Ito et al. (1948) were
the ﬁrst group recommending storage temperatures of 15 to 20°C for cooled boar

56111611.

At temperatures between 15 and 17°C, boar sperm storage times of up to 7 days are
achievable (Johnson 1998; Levis 2000). However, the majority of producers use

semen within 3-days of collection. The advantages of AI include the widespread use

of superior genetics; increased control of breeding and more uniform pigs; better
control of semen quality; decreased risk of injury to boar, sows and personnel
handling them; the ability to use heavy boars on light females; elimination of isolation
and testing of newly introduced boars; decreased boar housing and feeding costs;
reduced risk of disease transmission via boar introductions; reduced breeding work
load. The disadvantages of AI include the increased level of management required to
implement an AI program and, if collecting boars on farm, the need to train boars for

collection and redesign of facilities for semen collection and processing.

In swine, about 99% of AI procedures are performed with fresh-extended (liquid)
semen (Johnson et al. 2000). Bull semen extenders and freezing procedures were
modiﬁed for use with boar semen (for review see Johnson 1998). Initially, the pellet
method of freezing developed in Japan (Nagase and Graham 1964) was the most
common method used. Today frozen-thawed (FT) boar semen packaged in straws is
available. The higher price and the reduced pregnancy rates and litter sizes obtained in
comparison to fresh semen limits the use of cryopreserved sperm to speciﬁc breeding

programs (see “Boar Semen Cryopreservation”, p 23).

Sow Fertility to AI

About 50 years ago several authors, including Wiggins et al. (1951), Polge (1956) and
Dziuk and Henshaw (1958), recommended the intracervical deposition of a large
volume of liquid (50-200ml) and high numbers of spermatozoa (5 to 6 x 109) in every
semen dose to obtain maximum levels of fertility to AI. Few modiﬁcations have been

made to this practice since, as the current AI procedure in swine involves the

deposition in the sow’s cervix of about 3 x 109 sperm cells in a total volume of 70-

100ml per dose (for review see Martinez et al. 2001 a).

Semen backﬂow: With intra cervical deposition, approximately 25-30 % of the
inseminated sperm cells and up to 70% of the volume are lost by retrograde ﬂow
(backﬂow; Steverink et al. 1998). Although backﬂow of semen during or shortly after
insemination has been a concern, recent evidence suggests its relationship to fertility
may be minimal. Steverink et al. (1998) inseminated 1 to 6 x 109 sperm in 80ml and
collected semen backﬂow occurring: during the insemination, for the ﬁrst 30 min
after insemination, and from 0.5 h to 2.5h after insemination. Semen backﬂow
occurred in 63% of the sows during AI and in 98% after AI (Table 1.2). However, a
reduction in fertility was only evident after relatively large backﬂows (>5% number
of sperm deposited) during insemination of semen doses with 1x109 sperm.
Presumably, under these conditions, the backﬂow reduced the numbers of sperm in

the SR.

Table 1.2. Volume and number of sperm in the semen backﬂow during insemination
(M1), 0-0.5h after insemination (M2) and 0.5-2.5h after insemination (M3). Semen
doses were 80ml with l, 3 or 6 x 109 sperm.

 

 

 

 

Backﬂow n %1 Volume % Sperm %

Mean : se Range Mean : se Range
MI 63.3 7:1.1 1-56 8:1.3 0.3—50
M2 98.2 31 i 1.7 3-76 14 i- 1.0 0.3 — 79
M3 97.5 36 i 2.6 1-94 9 i 0.8 0.3 - 30

 

From: Steverink et al. (1998)
' Percent of sows exhibiting backﬂow

The female reproductive tract is very effective at moving sperm out of seminal plasma
or extender and up into the oviduct. Consequently, backﬂow during the 30 min
following AI, which was observed after 98% of inseminations, contained less and less
sperm per ml ﬂuid lost. With this loss of progressively fewer sperm, the impact of
backﬂow on sow fertility also lessens. Indeed, if the backﬂow occurred between 0.5
and 2.5h after AI, no effects on fertility were observed, regardless of numbers of
sperm originally inseminated. When intra-cervical tampons were used to prevent
backﬂow, sperm numbers in the upper uterus and the oviduct were not increased

(Pursel 1982).

Sperm reservoir: For successful fertilization, sperm must be able to reach the
oocytes soon after ovulation. With conventional AI, sperm are deposited in the cervix
and are then transported through the uterine lumen by myometrial contractions. Sperm
reach the UTJ within about 5 min (Rodriguez-Martinez et al. 2005), where a
functional sperm reservoir is created (Hunter 1981). This reservoir serves to provide
the appropriate number of sperm at the appropriate time and physiological state,
therefore ensuring successful fertilization of maximum numbers of oocytes (Suarez

2002)

The oviducts play a key role modulating sperm capacitation (F azeli et al. 1999;
Petrunkina et al. 2001; Hunter 2002) due to the different ﬂuid compartments that can
be demonstrated between the isthmus and the ampulla. Non-capacitated sperm are
stored in the reservoir by speciﬁc carbohydrate-mediated binding to the surface of the

mucosal epithelium of the oviduct (Suarez et al. 1998). Low levels of bicarbonate in

the region of the oviductal sperm reservoir act to delay or prevent capacitation prior to

sperm release in response to ovulatory signals.

Insemination of low sperm numbers: Currently, swine AI involves the cervical
deposition of 3x109 sperm diluted to a total: volume of 80-90ml with extender.
However, backﬂow rapidly reduces the liquid portion of inseminates. Within
approximately 75 min after AI almost no seminal ﬂuid/extender can be recovered.
However, from personal experience, semen backﬂow is minimized with intra-uterine
insemination. Therefore, the bypassing of the cervix may increase viable sperm
numbers available to reach the sperm reservoir, permitting an original insemination of
lower sperm numbers. Indeed, it has been shown that for optimal fertility,
progressively fewer sperm are required the closer they are deposited to the uterotubal
junction. When deposited near the uterotubal junction using either surgical (Krueger
and Rath 2000) or endoscopic techniques (Vazquez et al. 2000; Martinez et al.
2001a), very low numbers of sperm (10 to 150 x 106) are required. Such deep
intrauterine A1 techniques would only be justiﬁed when dealing with sperm of
extremely high genetic value, or for sex pre-selected sperm. However, the effect of
site of sperm deposition has received less attention than other fertility-related
parameters, such as the addition of different components to seminal doses, the effect
of the seminal plasma on sperm transport, or the timing of insemination relative to

ovulation (Martinez et al. 2001b).

Recently, several commercial companies have developed AI catheters that are capable
of being passed via manual manipulation through the cervix of the sow to allow

deposition of the semen dose into the uterine body. Using this technique allows the

10

insemination dose to be reduced to 1 x 109 per insemination (Watson and Behan
2002). Field trials have conﬁrmed that farrowing rates statistically comparable to
standard AI can be maintained using intrauterine insemination, although there may be
a reduction in litter size (Watson and Behan 2002; Rozeboom et al. 2004). Further, it
is likely that the timing of insemination relative to ovulation will become

progressively more important as the number of sperm deposited is reduced.

Timing of insemination relative to ovulation: The time of insemination relative to
ovulation is important for sow fertility (Kemp and Soede 1996). Inseminations
performed outside a window of time from 24h before to 4h after ovulation resulted in
a reduced fertilization rate and a consequent reduction in farrowing rates and litter
sizes (Waberski et al. 1994a,b; Nissen et al. 1997). Currently, there are no practical
methods for the detection of ovulation in large groups of sows. Consequently, the
timing of insemination is based on detection of estrus, although the time from

detection of estrus to ovulation is known to vary considerably.

Group weaning serves as an effective tool for synchronizing estrus and approximately
95% of sows are likely to express estrus between 3 and 8 days after weaning (Knox
and Rodriguez-Zas 2001). The duration of estrus averaged 40 hours for gilts and 48
hours for sows (Steverink et al. 1997), but ranged widely (12-88 h) depending on
parity, season, weaning-to-estrus interval and farm management (Soede et al. 2000).
Ovulation has been shown to occur approximately 70% of the way through the estrous
period independent of duration of estrus, although the exact timing relative to

ovulation was variable (10 to 85 hours; Weitze et a1. 1994; Nissen et al. 1997).

Il

Although the onset of estrus is the parameter used in commercial practice to time

swine AI, it is evidently not a good predictor of the time of ovulation.

As a consequence of variable estrus to ovulation intervals, multiple inseminations are
performed in order to ensure sperm deposition at a time associated with optimal
fertility. However, since studies have shown that Al causes an inﬂammatory reaction
in the uterus that is detrimental to sow fertility (Rozeboom et al. 1998; Kaeoket et al.
2003), insemination too frequently is not recommended. Common commercial
practices of timing AI relative to heat detection are described in Table 1.3. Although
not recommended by these authors, my personal recommendation is that females

standing on day 3 of estrus should also be bred.

Table 1.3. Best time to inseminate relative to the ﬁrst detection of standing estrous

 

Best time to inseminate

 

Frequency of estrus detection Gilts Sows
Once daily 0 and 24 hours 0 and 24 hours
Twice daily (12 hours apart) 12 and 24 hours 24 and 36 hours

 

From Estienne et al. (2005).

Currently, optimal fertility is achieved when inseminations with fresh-extended semen
are performed during the 24 hours prior to ovulation, with the peak fertility obtained
from insemination about 12 hours before ovulation (Waberski et al. 1994a; Kemp and
Soede 1996). When employing frozen semen, fertility rates are considerably lower.

However, fertilization rates similar to those following insemination of fresh semen

12

have been reported when insemination of frozen sperm occurred in the 4 hours before

ovulation (Figure 1.2; Waberski et al. 1994b).

If we knew when ovulation was to occur, we could use a single Al, or possibly
increase the use of frozen semen. However, to obtain this degree of predictability of
ovulation, control of follicular development and ovulation is required, which is

achievable via the administration of exogenous hormones.

 

 

 

10 -
93. I Fresh
80‘ - Frozen
70‘ -
60< ‘
50 V

  

 

\\\\\\\\\\\\\\\\
III-I
WW2;
III-III-
W:

 

I; A

4;:

+4-8 h +8-12 h

 

0-4

y'—
'1’
on
:r'
I
‘P
A
=-
I
1‘
c
=-
:-

Figure 1.2. Fertilization rates from A1 with fresh and frozen semen at different times
of insemination relative to ovulation (0h). From Waberski et al. (1994b).

Several hormones, such as human chorionic gonadotrophin (hCG), gonadotrophin
releasing hormone (GnRH) or porcine luteinizing hormone (pLH) have been used to
control ovulation in sows (De Rensis et al. 2003; Cassar et al. 2004; 2005). Although
equally efficient for induction of ovulation, the difference between protocols is the
duration of the interval from injection to ovulation, and possibly the synchrony of the

13

ovulation in the responding animals. Classically hCG was believed to cause a very
synchronized ovulation at around 40-42h post injection (Table 1.4). However, recent
studies have revealed a relatively wide variation in the interval to ovulation (Soede et
al. 1998; Nissen et al. 2000). When ovulation was induced using GnRH or pLH, the
interval from injection to ovulation was shorter (36 to 38h) and the degree of
synchrony appeared greater (Cassar et al. 2005). Sow fertility to A1 at a controlled
estrus and ovulation was improved (Candini et al. 2001; De Rensis et al. 2005; Cassar
et al. 2004, 2005) even when single inseminations were employed (Cassar et a1.

2005)

Table 1.4. hCG based ovulation synchronization protocols.

 

 

Synchronization protocol Ovulation Source

750 IU hCG 68h after weaning £13) Soede et al. 1998

1275 50 01316;: g ng‘hag eerrweaning 40-42 Martinez et al. 2001a,b
750 IU hCG 96h after weaning 38-48 Nissen et al. 2000

750 IU hCG 76h after weaning 40 Nissen et al. 1995
PG600 + SOOIU hCG 72h later 42 Bertani et al. 1997

 

l4

Sperm Capacitation

Mammalian sperm are not able to fertilize eggs immediately aﬁer ejaculation. They
acquire fertilizing capacity after residing in the female tract for a certain period of
time during which several biological changes occur to render the sperm cells capable
of binding with the oocyte and fertilizing it (Visconti et al. 2002). The physiological
changes that sperm undergo are collectively called ‘capacitation’ (Yanagimachi
1994). Capacitated sperm cells show a number of properties including an inability to
bind oviductal epithelium in the sperm reservoir, penetration of the cumulus layers
surrounding the ova, and binding to the zona pellucida (ZP) that permits the acrosome
reaction (Fazeli et al. 1999; Rodriguez-Martinez et al. 2005). During capacitation,
several proteins derived from the cauda epididymidis and from seminal plasma are
removed from the sperm surface, which then becomes accessible to lipid-binding
components of oviductal luminal ﬂuids that extract cholesterol from the sperm plasma
membrane. In this state, membrane ﬂuidity is enhanced, causing lipid scrambling and
initiating further capacitation changes such as the uptake of extracellular Ca2+,
tyrosine phosphorylation (Tardif et al. 2001; 2003), and reorganization of the sperm
membrane (Topfer-Petersen et al. 2002). In the pig, bicarbonate appears to be the
effector molecule that initiates membrane disorder, which is considered one of the
earliest signs of capacitation (Harrison et al. 1996; Gadella and Harrison 2000).
Bicarbonate is virtually absent from epididymal ﬂuids [0.5 mM] whereas high
concentrations [20 mM] are detected in the oviduct, except in the region of the sperm
reservoir (Suarez et al. 1998). These modiﬁcations of membrane ﬂuidity precede
speciﬁc changes in Ca2+ movement and of motility patterns (e.g. hyperactivated
motility) that predispose to the acrosome reaction and penetration of the ZP

(Rodriguez-Martinez et al. 2005).

15

In vitro, capacitation has been accomplished using sperm cells incubated under a
variety of conditions in deﬁned media (Breitbart et al. 1997; Visconti and Kopf 1998;
Baldi et al. 2000; 2002). It has been suggested that during capacitation intracellular
pH and Ca2+ rise, adenylate cyclase is activated resulting in a rise in CAMP levels, and
speciﬁc proteins (including extracellular signal regulated cyclases) are then tyrosine
phosphorylated (Tardif et al. 2003; De Lamirande 1997). Although the molecular
basis of capacitation is still not well understood, the following model (Figure 1.3)

proposed by Baldi et al. (I 996) is currently among the most accepted:

Extracellular

BSA —. BSA-cholesterol (332+ HCO3 - ROS

 

 

 

l
Cholesterol efﬂux

Destabilization
E a A it 6 v
Ca2+ -> 4- mm-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

».‘A m ’
L , K‘ t. ('1 1‘ cAMp,l pKA
r 1 1 J a
Na ——1——F:—9 'o. (T)
E l 1 ﬂ 34—4 Tyrosine phosphorylation
“SP—if CI '-,
I l '.

 

 

 

 

 

 

<({ i J th.

Intracellular

Figure 1.3. Model of sperm capacitation. Cholesterol removal from the cell membrane
(accelerated by the presence of bovine serum albumin (BSA) in the medium)
increases the ﬂuidity to ions, including bicarbonate (HCO3’) and calcium (Ca2+). Both
ions activate adenyl cyclase (AC) inside the cell, raising the levels of cyclic AMP
(CAMP) and subsequently activating the protein kinase A (PKA). PKA and the
incorporated reactive oxygen species (ROS) increase the tyrosine phosphorylation,
activating the phospholipases (PLCyL), which destabilize the cell structure,
remodeling the sperm membrane phospholipids. A possible activation of the protein
kinase C (PKC) has also been reported (Baldi et al. 1996).

16

Boar Semen Cryopreservation

The ﬁrst attempts at freezing boar sperm were described by Polge (1956). After
repeated failures with cervical and deep intrauterine inseminations, Polge et al. (1970)
became the ﬁrst group obtaining progeny from frozen-thawed (FT) boar sperm by
surgical deposition of the FT sperm directly into the oviduct. One year later, using the
same cryopreservation protocol, three other groups (Crabo and Einarsson 1971;
Graham et al. 1971; Pursel and Johnson 1971) obtained similar success (for review
see Pelaez 2004). Following these initial studies, efforts were directed towards
improving the procedures as the potential beneﬁts of FT sperm to the swine industry
were recognized. These beneﬁts, reviewed by Eriksson (2000), include an ability to
store semen until completion of any health test, allowing the semen to be quarantined
before use in the herd. Also, speciﬁc genetic lines may be preserved and used in
future breeding programs. Similarly, improved genetics could be introduced from
worldwide sources, eliminating the current geographic limitations. Frozen semen
would allow planned matings, which are essential at the top of the breeding pyramid

for optimal progress in every gestation.

The utilization of FT boar semen is estimated to be less than 1% of all inseminations
worldwide (Johnson et al. 2000). The limitations for a wider use have remained
invariable for years, being associated with lower fertility (farrowing rates of 40 to
70% and litter sizes of 7 to 10) compared with those achieved with fresh semen
(farrowing rates of 75 to 90% and litter sizes of 9 to 12). A large variation exists
between individual boars in freezing success measured as both in-vitro sperm viability
and in-vivo fertility post-thaw (Holt 2000a; Eriksson 2000). This, added to the lack of

reliable laboratory tests for assessment of sperm quality becomes an even larger

17

problem when trying to relate in vitro viability with fertility outcomes in-vivo (Selles
2001). The time of AI relative to ovulation must be accurate, as the optimal fertility
“window” for FT boar sperm appears to be substantially shorter than that for fresh
semen (Waberski et al. 1994b). In addition, costs are higher when using FT semen,
due to the more expensive laboratory procedures and higher number of sperm cells

needed per insemination dose (5 x 109) compared with fresh semen (3 x 109).

Although FT semen has been commercially available since 1975 (Johnson and
Larksson 1985), it has been used primarily for export and transfer of speciﬁc genetics
within the domestic market. Some usage of frozen semen for market hog production
has been reported (Hofmo and Grevle 2000) although it is not widely used in the
commercial production of swine because it is not economical in comparison to the use

of fresh semen (Wagner and Thibier 2002).

Freezing protocols: The susceptibility of sperm to low temperatures was ﬁrst
described by Milanov in 1934 (see Parks 1997), who observed the irreversible loss of
motility of bull sperm after cooling quickly to temperatures near 0°C, an event that he
termed “cold shock”. Boar semen differs from the semen of other domestic species in
that it is produced in large volumes and the sperm cells are particularly sensitive to
cold shock. The severity of the shock depends on cooling rate and ﬁnal temperature
obtained, being maximal at high cooling rates and ﬁnal temperatures of 12 to 2°C and

minimum at cooling rates below 10°C/min and temperatures above 4°C (Pelaez 2004).

Success in the freezing of boar sperm depends on an understanding of how several

factors inﬂuence the capacity of sperm to survive freezing and thawing while

18

retaining their ability to fertilize. These factors are either internal, such as the innate
differences among boars and ejaculates, or external such as the composition of
extenders, type and concentration of the cryoprotective agent applied, rates of
extension and cooling or equilibration, and the method of freezing and thawing of the
semen (Johnson et a1. 2000). Only external factors can be modiﬁed to optimize

freezing protocols.

Semen cryopreservation involves dilution, cryoprotection, cooling, freezing, storage
and thawing, steps which may cause a detrimental effect on cell structure and function
(Hammerstedt et al. 1990). Immediately after collection, the semen is diluted in an
appropriate medium, often based on egg yolk or milk, at temperatures close to body
temperature (30 to 39°C; Bailey et al. 2003). The usual extension rate is 1:1 v/v
because high dilution rates reduce sperm function, likely due to the reduced contact
between sperm and components of seminal plasma (Garner et al. 2001), the so-called
“dilution effect” (Mann and Lutwak-Mann 1981; cited in Maxwell and Johnson

1999).

For most domestic species, cooling of sperm from body temperature to near freezing
represents a major stress to the sperm plasma membrane resulting in the
rearrangement and destabilization of membrane components and calcium inﬂux
(Bailey et al. 2003). The addition of a cryoprotective agent increases both the viability
and the motility of the sperm cells at thawing. Glycerol is the cryoprotectant of choice
for most mammalian sperm, and is usually added at rates below 3% aﬁer cooling of
sperm to 5°C (Holt 2000b). The efﬁcacy of glycerol is partially due to its osmotic

effect that dramatically increases the osmolality of the medium, retarding intracellular

19

ice formation (Hammerstedt et al. 1990). The osmotic effects of glycerol are marked,
but transitory, and a rapid change in cell volume does not appear to be detrimental to
sperm function (Liu and F oote 1998). Furthermore, glycerol acts directly on the sperm
plasma membrane, possibly reducing some of the phase transitions and increasing
membrane ﬂuidity during cooling (Noiles et al. 1995). The most reliable boar sperm
cryopreservation protocols, described in 1975, are freezing in straws (Westendorf et

a1. 1975) and pellets (Pursel and Johnson 1975), or modiﬁcations of these.

Capacitation-like status: The cooling of sperm to 4°C, a step inherent in standard
freezing protocols, causes molecular changes to the sperm that effectively mimic
capacitation (Bailey et al. 2003). This capacitation-like reaction, or “cryo-
capacitation”, does not occur in the entire population of sperm. Cryopreserved boar
sperm supplemented with heparin, an established capacitating agent for bull sperm
(Parrish et al. 1988), penetrated nearly twice as many oocytes as the heparin-free
cryopreserved sperm (Bailey et al. 2000). Further, these authors provided evidence
that both cooling and full cryopreservation induced precocious capacitation of bull
sperm. Similar ﬁndings have been reported for boar sperm (Gillan et al. 1997). The
manifestation of the capacitation-like effect in a larger proportion of the sperm
population after cooling between 22°C and 5°C indicates that membrane lipid phase
transitions occur between these temperatures in boar spermatozoa (Green and Watson

2001).

Capacitation is known to involve a destabilized sperm membrane (Harrison 1997),
and the same is true for cryocapacitation. In this state the sperm membrane is fragile

and susceptible to deterioration or to undergoing spontaneous acrosome reactions and

20

death if fertilization has not occurred. Therefore, the in-vivo fertile lifespan of FT
sperm is reduced and the fertility of the insemination dose is compromised, based on

the proportion of cryocapacitated sperm present (Bailey et al. 2000).

Seminal Plasma

Seminal plasma is the major portion of a boar’s ejaculate and plays an important role
in carrying the sperm through the female reproductive tract. Produced by the
accessory glands of the male reproductive system, seminal plasma nourishes the
sperm cells and also inﬂuences the capacitation and fertilization processes (Zhu

2000)

The composition of seminal plasma varies with species, the interval between
ejaculations, and the health of the animal (Maxwell and Johnson, 1999). Seminal
plasma is especially rich in proteins, each of which may play different roles at
different stages in the fertilization process. Sperrnadhesins are the predominant family
of boar seminal plasma proteins, accounting for over 90% of total proteins in seminal
plasma (Calvete et al. 1997). Several studies by this latter author have reported that
Porcine Seminal Plasma Protein I (PSP-I) and II (PSP-II) are the two major members
of the sperrnadhesin family, representing well over 50% of total proteins in seminal
plasma (Rodriguez-Martinez et al. 2005). They are usually found combined in
heterodimers that show a marked zona pellucida glycoprotein-binding activity,

suggesting an important role during gamete interaction (Zhu 2000).

Seminal plasma hormones are potentially associated with sperm function. The Leydi g

cells secrete large amounts of estrogens, which modulate protein synthesis and

21

secretion as well as possibly affecting the timing of ovulation (Claus 1990). However,
the role of seminal plasma in the timing of ovulation remains unclear, as its addition
pre-insemination advanced ovulation time in some studies (Waberski et al. 1997) but

not others (Soede et a1. 1998).

Effect of seminal plasma on sperm capacitation and fertility: Incubation of boar
sperm in the presence of seminal plasma induces resistance to cold shock compared
with sperm incubated in the absence of seminal plasma (Pursel et al. 1973). In
contrast, at freezing temperatures the presence of seminal plasma caused a detrimental
effect on post-thaw sperm motility (Memon et a1. 1985). Thus, boar sperm
cryopreservation protocols usually include an incubation phase of several hours at
room temperature in the presence of seminal plasma before centriﬁJgation for its
removal prior to cooling and freezing. In both fresh and frozen-thawed boar sperm,
chlortetracycline staining indicated that incubation in the presence of seminal plasma
can both prevent and reverse capacitation (Vadnais et al. 2005a,b). Although the
addition of seminal plasma to the thawing extender resulted in a decrease in the
percent of capacitated spermatozoa, the effects of seminal plasma addition on
cryocapacitation are dependent on the temperature of the sperm suspension as well as

the presence or absence of egg yolk from the thawing extender (Vadnais et al. 2005b).

Rath and Nieman (1997) showed that the in vitro fertilizing capacity of frozen-thawed
boar epididymal sperm was better than that of frozen-thawed ejaculated sperm,
reﬂecting the absence of decapacitation factors that would have been added to the
sperm in seminal plasma during ejaculation. Furthermore, the motility of frozen-

thawed ram sperm was better after the addition of seminal plasma, and there were

22

fewer capacitated and acrosome reacted cells than in controls without seminal plasma
(Maxwell et al. 1999). Although boar sperm cells are less susceptible than are rams’
to the dilution effect (Ashworth et al. 1994), the optimal level of seminal plasma in
the extender to improve sperm motility is currently considered to be 10% (Maxwell
and Johnson 1999). Lower (5%) and higher (20%) concentrations of seminal plasma
appeared to be detrimental to the survival and membrane integrity of boar sperm

(Maxwell et al. 1997).

Various authors have studied sperm fertility after the addition of seminal plasma to AI
doses, reporting inconsistent results (Maxwell and Johnson 1999; Bellin et al. 1998;
Graham 1994). The variation obtained among species and individuals could be
associated with the presence, absence or critical concentration of certain components,
probably proteins, in some seminal plasma’s but not in others (Maxwell and Johnson
1999). Indeed, in bulls, heparin-binding proteins are secreted in a testosterone dose-
dependent manner. Further, some seminal plasma proteins have been related to higher
conception rates in cattle (Bellin et al. 1998) and concentrations of these seminal

plasma proteins vary among males.

Boar differences were noted by Rozeboom et al. (2000) in the immunological
response of the uterus after seminal plasma addition during AI. Seminal plasma from
some boars may have more of the component(s) that stimulate the uterus to protect the
new embryos from the dam's immune system. These various studies all support the
idea that seminal plasma has valuable components, which might beneﬁt establishment
of pregnancy. However, it appears clear that both the components and their actions

must be much better understood before commercial application is possible.

23

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29

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30

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31

CHAPTER 2
Effect of sperm cryopreservation and supplementing semen doses with seminal

plasma on the establishment of a sperm reservoir in gilts

M Abad', DJ Sprecher', P Ross', RM Friendshipz, RN Kirkwood'
1Department of Large Animal Clinical Sciences, Michigan State University, East Lansing,

USA. 2Department of Population Medicine, University of Guelph, Canada

Contents

Frozen-thawed (FT) boar sperm have a reduced fertile life, due in part to a
capacitation-like status induced by cooling. Reversal of this cryocapacitation in-vitro
by exposure to boar seminal plasma (SP) has been demonstrated. The objective of
these studies was to determine the effect of SP on the ability of FT sperm to create an
oviductal sperm reservoir following artiﬁcial insemination (AI). In Experiment One,
35 prepubertal gilts were injected (IM) with 400 IU equine chorionic gonadotropin
(eCG) plus 200 IU human chorionic gonadotropin (hCG) to induce estrus. At
detection of estrus, gilts were inseminated with 3x109 live sperm, either fresh (FS;
n=13), FT (n=10), or FT supplemented with 10% v/v SP (n=12). Gilts were sacriﬁced
8 h later, their reproductive tracts recovered and the uterotubal junctions (UTJs)
ﬂushed to recover sperm. Fewer (P<0.01) sperm were recovered following FT
compared to F8 inseminations, and there was no evident effect of SP. In Experiment
Two, 30 prepubertal gilts received 1M injections of 1,000 IU eCG followed by 5 mg
porcine luteinizing hormone (pLH) 80 h later to control time of ovulation. Gilts were
inseminated with 3x109 live FS sperm (n=6), FT sperm (n=15) or FT sperm plus 10%
SP (n=9) at 12 h before ovulation and then sacriﬁced 8 h later. The UTJs were
dissected and ﬂushed for sperm recovery. Fewest (P<0.001) sperm were recovered

following FT insemination and there was no evident effect of SP. These data
32

demonstrate that the size of the sperm reservoir is markedly reduced in gilts
inseminated with FT sperm. However, the lack of effect of SP suggested that either it
did not reverse cryocapacitation or that such a reversal does not impact the in-vivo

ability to create a sperm reservoir.

Introduction

After deposition into the female reproductive tract, boar sperm are passively
transported by gravity and uterine contractions to the tubal end of the uterine horns.
Thereafter, the sperm are passively and actively (sperm motility) transported through
the uterotubal junction (UTJ) into the oviduct (Langendijk et al. 2002). Only a small
percentage of the inseminated cells get to the UTJ (Rodriguez-Martinez et al. 2005),
where due to the narrower lumen of the region (Fazeli et al. 1999), binding of non-
capacitated sperm cells to oviductal epithelial cells (Suarez 1998; Petrunkina et a1.
2001), and the local oviductal ﬂuid environment (Rodriguez-Martinez et al. 2005), a
ﬁrnctional sperm reservoir is formed in the distal l to 2 cm of the isthmus (Hunter
2002). The sperm remain in this region until immediately prior to ovulation, at which
time they are released in waves to redistribute towards the ampulla-isthmusjunction
for fertilization. Presumably, a sufﬁcient number of viable sperm cells are needed in

the sperm reservoir in the peri-ovulatory period to maximize fertility rates.

In pigs, artiﬁcial insemination (A1) with frozen-thawed semen results in reduced
farrowing rates and litter sizes (Johnson et a1. 2000) due to cryoinjury to sperm
including the induction of a capacitation-like status of the sperm cells, so-called
cryocapacitation (Bailey et al. 2003). Since capacitated cells are not stored in the

sperm reservoir, to be able to fertilize ova cryopreserved sperm must presumably

33

encounter oocytes soon after insemination. Therefore, greater accuracy in timing of
the insemination relative to time of ovulation is likely needed when using frozen-
thawed semen (Larsson 1976). Hormones such as equine chorionic gonadotropin
(eCG), gonadotropin-releasing hormone (GnRH), human chorionic gonadotropin
(hCG) or porcine luteinizing hormone (pLH) and their combination can be used
effectively to control the time of ovulation in pigs, which may be monitored by real

time Ultrasonography (Soede et al. 1994).

If the primary cryoinj ury associated with reduced fertility is cryocapacitation, then
reversal of cryocapacitation should enhance sperm fertility. It has recently been
demonstrated that based on chlortetracycline staining patterns, exposure to seminal
plasma will reverse cryocapacitation of boar sperm in-vitro (V adnais et al. 2005a,b).
Further, it has been documented that addition of seminal plasma to cryopreserved ram
semen improved subsequent ewe fertility (Maxwell et al. 1999). A beneﬁcial effect of
thawing cryopreserved boar sperm in seminal plasma on in-vivo sperm longevity
(Einarsson and Viring 1973) and gilt and sow fertility (Crabo and Einarsson 1971;
Larsson 1976) has also been documented. However, we are not aware of any recent
studies examining the effect of a relatively low (10%) content of seminal plasma in
thawing diluents on in-vivo fertility in pigs. The present experiments were designed to
test the hypothesis that cryopreserved boar sperm are less able to form a sperm
reservoir in the oviduct and that this relative inability would be reversed by prior

exposure to seminal plasma.

34

Materials and Methods
These studies were performed at Michigan State University Swine Facility and
University of Guelph (Arkell) Research Station and were approved by the institutional

Animal Care committees.

In Experiment One, 60 prepubertal Landrace x Yorkshire gilts (124.3 : 1.8 kg) were
housed in groups of approximately 10 and were allowed ad libitum access to a
standard ﬁnisher ration. Gilts received an IM injection of 400 IU equine chorionic
gonadotrophin (eCG) plus 200 IU human chorionic gonadotrophin (hCG) (PG600,
Intervet) to induce a synchronous onset of estrus. From Day 1 post-injection, gilts
were exposed to a mature boar for 15 min daily to facilitate detection of estrus. Only
those gilts exhibiting standing estrus on Day 5 were selected for the study (35/60).
Selected gilts were assigned by weight to receive a single insemination either of fresh
semen (FS; n=l3), frozen-thawed semen (FT; n=10), or FT semen supplemented with
10% v/v seminal plasma (SP; n=12). The seminal plasma was pre-mixed in the
thawing extender, thus allowing the thawed sperm immediate contact with seminal
plasma constituents. Commercial semen from 2 boars was used in this study and both
fresh and frozen doses contained 3 x 109 live sperm with boars equally represented
within treatment. All inseminations were cervical and were performed by the same

personnel using conventional foam-tip catheters in the presence of a mature boar.

In Experiment Two, 30 prepubertal Yorkshire gilts (127.5:2.4 kg) without previous
boar contact were housed as groups of approximately 10 and fed ad libitum a standard
dry sow ration. Following formation of groups (Day 0), ovulation was induced by IM

injection of 1,000 IU eCG (Pregnecol, Bioniche Animal Health, Bellville, Ontario,

35

Canada) followed by 5 mg pLH (Lutropin, Bioniche Animal Health) 80 h later.
Ovulation was predicted to occur 38 h after pLH injection. Gilts were allowed 15 min

exposure to a mature boar at 12 h intervals to facilitate detection of estrus.

At 26 h after pLH injection (12 h before the predicted time of ovulation) gilts were
assigned by weight to receive a single insemination of FS semen (n=6), FT semen
(n=15), or FT semen + 10% v/v seminal plasma (n=9). The FT semen group was
over-represented because of an expectation of lower sperm recovery. The same
personnel using foam-tip catheters in the presence of a mature boar performed all of

the inseminations.

The boars supplying semen and seminal plasma for Experiment Two were housed in
the Ponsonby Research Facility, University of Guelph. All boars were 8 to 12 months
of age and were routinely collected weekly using the gloved-hand method.
Immediately after collection, entire ﬁltered ejaculates were transported to the
laboratory at 37°C and analyzed for volume, motility and sperm concentration. The
same pool of 3 boars was used to obtain both fresh and FT semen doses. Sperm
cryopreservation employed the method of Pursel and Johnson (1975), as modiﬁed by
Buhr et al. (2001). The vitality of cryopreserved sperm at thawing was determined
using Eosin-Nigrosin staining. All doses (FS and FT) were extended in Beltsville

Thawing Solution to a total volume of 80 ml and 3 x 109 live sperm.

In Experiments One and Two, seminal plasma (SP) was obtained after centrifugation
(800 x g for 10 min at 25°C) of a pool of semen derived from 5 and 10 Yorkshire

boars, respectively, and was stored at —80°C until needed. Thawing of SP was done in

36

a 37°C water bath and semen doses that included the 10% v/v SP were incubated for
20-30 min at 39°C before insemination. This interval was chosen based on previous
data showing that reversal of cryocapacitation in vitro required at least 15 min

(Vadnais et al. 2005b).

At 8 h after insemination, gilts were either subjected to commercial slaughter
procedures (Experiment One) or euthanized by IV injection of 100 mg/kg
pentobarbital (Experiment Two), and their reproductive tracts recovered. Both UTJs,
deﬁned as the portion of the tract comprising the last 1 cm of the uterine horn and ﬁrst
3 cm of oviduct, were dissected and clamped. For ﬂushing of sperm from the UTJs, a
blunted 18 g 12 mm needle was introduced into the oviduct and the UTJ’s ﬁlled with
0.5 ml of Dulbecco’s PBS. A gentle massage was performed for 1 min to detach
sperm cells from the oviductal epithelium and the volume emptied into a 15 ml tube.
This was repeated once and thereafter, two more ﬂushings were performed by forcing
5 ml DPBS to pass through the UTJ lumen. An average total volume of 11 ml was

recovered for each UTJ.

The recovered ﬂuid was centrifuged at 800g for 10 min (25°C), and the supernatant
discarded. The pellet was resuspended to 0.5 ml and then frozen overnight (-20°C) to
destroy any maternal cells. After thawing by submersion in a 37°C water bath, the
tubes were gently agitated to re-suspend sperm and the number of cells counted using
a hematocytometer. The average of two readings was recorded for each oviduct and
the average from both oviducts was recorded. If sperm were evident on the slide but
absent from the designated counting area, the gilts were given a nominal total sperm

count of 500.

37

Statistical analysis

Data were analyzed using INFOSTAT/E v2.0 (University of Cordoba, Argentina).
Differences in mean numbers of recovered spermatozoa were tested using analysis of
variance with LSD Fisher approximation. Body weight was used as a blocker in all
analyses. Results are presented as meaniSEM and P<0.05 was considered statistically

signiﬁcant.

Results

In both experiments, examination of gilt ovaries postmortem revealed that none had
ovulated. The effect of semen type and supplemental SP in Experiment One are
shown in Table 2.1. The insemination of FT sperm resulted in a signiﬁcantly smaller

sperm reservoir (P<0.01). There was no effect of SP on numbers of sperm recovered.

The effect of semen type and of supplemental SF in Experiment Two are shown in
Table 2.2. The results were very similar to those of Experiment One, with the
insemination of FT sperm resulting in a signiﬁcantly smaller sperm reservoir

(P<0.001) and with sperm numbers unaffected by supplemental SP.

Table 2.1. Effect of freezing and supplementation with seminal plasma (SP) on
number of sperm recovered from the oviduct and uterotubal junction (Experiment 1)

 

 

 

 

 

 

AI group

Fresh Frozen F rozen+SP
N 13 10 12
Body weight (kg) 125.2i2.7 123.2i2.9 124.2139
Sperm recovered (x103)* 211.8:849 91:24 8.6122

 

 

 

 

* P<0.01

38

 

Table 2.2. Effect of freezing and supplementation with seminal plasma (SP) on
number of sperm recovered from the oviduct and uterotubal junction (Experiment 2)

 

 

 

 

 

 

 

 

 

 

AI group

Fresh Frozen Frozen+SP
N 6 15 9
Body weight (kg) 1 19.2i-5.3 - 126.1:33 l35.6i3.9
Sperm recovered (x103)* 141.6:229 19311.3 94:10
* P<0.001
Discussion

The absence of evidence of ovulation in the gilts was taken to indicate that we had
achieved our objective, i.e., induction of estrus followed by insemination and
establishment of sperm reservoirs in the preovulatory period. Regarding the
establishment of the sperm reservoir, the numbers of sperm recovered following the
fresh semen inseminations were of the same order of magnitude as that reported
previously (Langendijk et al. 2002; Willenburg et al. 2003). Therefore, the numbers of
sperm observed are likely sufﬁcient for normal fertility, although the minimum
number of sperm within the sperm reservoir at the time of ovulation necessary to
maximize oocyte fertilization rates remains unknown. We are not aware of any recent
studies examining establishment of a sperm reservoir subsequent to A1 with
cryopreserved semen. However, the present results clearly indicate that
cryopreservation markedly reduces the ability of sperm to associate with the oviductal
epithelium of the sperm reservoir. It is now accepted that only non-capacitated sperm
reside in the sperm reservoir (Fazeli et al. 1999; Tienthai et al. 2004). Therefore, the
capacitation-like reaction induced by cooling (Vadnais et al. 2005a,b) may explain the

inability of cryopreserved sperm to enter the sperm reservoir. If it is accepted that a

39

 

small sperm reservoir will compromise fertility, then this inability to form an
adequate sperm reservoir may partly explain fertility depressions associated with

insemination of cryopreserved sperm.

Interestingly, using chlortetracycline (CTC) staining, seminal plasma has been shown
to prevent and/or reverse capacitation in-vitro (Maxwell et al. 1999; Vadnais et al.
2005a,b). Others have demonstrated improved gilt and sow fertility when frozen
sperm were thawed in seminal plasma (Crabo and Einarsson 1971; Einarsson et al.
1973; Larsson and Einarsson 1975). Also, thawing in seminal plasma increased sperm
survival time in-vivo (Einarsson et al. 1973; Einarsson and Viring 1973). Clearly,
some component(s) of seminal plasma can affect the fertility of cryopreserved boar
sperm. However, in the present study the addition of 10% v/v seminal plasma to
cryopreserved semen at thawing had no effect on the establishment of the sperm
reservoir in-vivo. This may indicate that the effect of 10% seminal plasma observed
with CTC staining in vitro is not biologically meaningful. However, we have shown
by ﬂow cytometry of sperm stained with merocyanine and yo-pro that seminal plasma
did prevent further capacitation when cryopreserved sperm were incubated in a
capacitation-supporting medium (unpublished data). Therefore, if it is accepted that
seminal plasma will affect the capacitation status of cryopreserved sperm, the
implication is that the failure of cryopreserved sperm to form an effective sperm
reservoir involves aspects of cryoinjury in addition to cryocapacitation. In support of
this suggestion, it has been shown that approximately 60% of cryopreserved sperm
exhibit cryocapacitation on thawing, with about 40% appearing “normal”. If these
normal appearing sperm are indeed normal, a markedly larger number of sperm would

be anticipated in the sperm reservoir.

40

From our data, we conclude that cryopreserved boar sperm did not form an effective
sperm reservoir subsequent to insemination. Further, the apparent lack of effect of
seminal plasma on the establishment of a sperm reservoir suggests that aspects of
cryoinjury in addition to the cryocapacitation effect are involved. Whether a greater
seminal plasma inclusion level, as used by earlier workers, would alter the observed

effect remains unknown.

Acknowledgements

We gratefully acknowledge Ontario Pork, Michigan State University Foundation, and
the University of Guelph-Ontario Ministry of Agriculture and Food Animal Research
Program for ﬁnancial support of these studies. We are indebted to Dr. Jose Cibelli for
allowing access to his laboratory (Experiment One) and to Glen Cassar, Christine
Pelland and Arturo Ruiz for invaluable assistance in the performance of Experiment

Two.

41

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43

CHAPTER 3

Effect of insemination-ovulation interval and addition of seminal plasma on sow
fertility to insemination of cryopreserved sperm

M Abad', JC Garcia‘, DJ Sprecher', G Cassarz, RM Friendshipz, RN Kirkwood'

lDepartment of Large Animal Clinical Sciences, Michigan State University, East Lansing, USA.

2Department of Population Medicine, University of Guelph, Canada.

Contents

In swine, the use of frozen-thawed (FT) sperm for artiﬁcial insemination (Al) is
limited due to poor sow fertility, possibly associated with a post-thaw capacitation-
like status resulting in fewer fully viable sperm. Sow fertility to A1 with FT sperm
may improve with deeper deposition of sperm within the female tract, insemination
very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We
performed two experiments to examine these suggestions. In Experiment One, 122
multiparous Yorkshire sows received 600 IU eCG at weaning and 5mg pLH 80 h later
to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after
pLH injection. Thereafter, sows were assigned on the basis of parity to a single A1 of
FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with
10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO.
All semen doses were adjusted to 3x109 live cells and deposited into the cervix.
Experiment Two employed 99 multiparous sows and was a repeat of Experiment One

except that all FT inseminations were intrauterine.

In both experiments, farrowing rates were lower (P<0.01) following FT inseminations
with no effect of time of insemination or of supplemental SP. In Experiment One,

litter size was smaller following FT insemination, but no effect on litter size was
44

evident in Experiment Two. Supplemental SP had no effect on litter size in either
experiment. The lack of effect of either SP or timing of FT insemination on sow
fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more

than just cryocapacitation.

Introduction

Artiﬁcial insemination (AI) is the technique of choice in the breeding of domestic
animals (Verberckmoes et al. 2004). However, in contrast to AI in cattle, swine AI
involves almost exclusively liquid semen collected within the previous 3 to 7 days.
This use of semen within a short interval from collection limits the time available to
test for pathogenic contamination and, as such, increases the risk of disease
transmission. Indeed, liquid semen is known to be a vehicle for dissemination of
pathogens including the Porcine Reproductive and Respiratory Syndrome (PRRS)
virus and porcine circovirus. In addition to improved ﬂexibility in timing of matings
and access to worldwide genetics (reviewed by Eriksson, 2000), the cryopreservation
of boar sperm would allow sufﬁcient time to ensure absence of pathogens and so

improve herd biosecurity.

The limited use of frozen-thawed (FT) sperm in swine is due primarily to relatively
poor farrowing rates and litter sizes associated with FT inseminations (Johnson et al.
2000). This poor performance results from cryoinjury to sperm including the
induction of a capacitation-like status of the sperm cells, so-called cryocapacitation

(Bailey et al. 2003).

45

Since cryocapacitated sperm cells are not stored in the oviductal sperm reservoir
(Abad et al. 2006), their fertile lifespan will be very short. Therefore, for acceptable
fertilization rates cryopreserved sperm must presumably encounter ova soon after
insemination. Indeed, when inseminated very close to the time of ovulation (eg. 34 h),
fertility to FT inseminations was greatly improved (Roca et al. 2003; Bolarin et al.
2006), with fertilization rates being comparable to those from inseminations of fresh

semen (Waberski et al. 1994).

Potentially, a further way to improve fertility of FT sperm is to reverse
cryocapacitation. Using chlortetracycline staining, it has been demonstrated that
incubation of fresh or FT sperm in media supplemented with seminal plasma (SP) will
reverse (cryo)capacitation (Suzuki et a1. 2002; Vadnais et al. 2005a,b). Further,
supplementing semen doses with SP has been shown to improve in-vivo fertility of
ewes (Maxwell et a1. 1999) and mares (Alghamdi et al. 2005). A beneﬁcial effect of
thawing cryopreserved boar sperm in seminal plasma on in-vivo sperm longevity
(Einarsson and Viring 1973) and on gilt and sow fertility has also been documented
(e.g. Crabo and Einarsson 1971; Einarsson et al. 1973). However, to our knowledge,
sow fertility to insemination of FT sperm in a commercial extender supplemented
with SP has not been examined. The present experiments were undertaken to test the
hypothesis that supplementing FT sperm with SP will extend in-vivo sperm longevity
and so allow maintenance of sow fertility when insemination occurs 12 hours before

ovulation.

46

Material and methods
These studies were performed at the University of Guelph, Arkell Research Station
(Experiment One) and Michigan State University Swine Facility (Experiment Two).

The respective institutional Animal Care committees approved the studies.

Prior to conducting the insemination studies, a pilot study was performed to verify the
efﬁcacy of a protocol for hormonally controlling the time of ovulation (Cassar et al.
2005). At weaning, 37 multiparous sows received an injection (IM) of 600 IU equine
chorionic gonadotrophin (eCG; Pregnecol®, Bioniche Animal Health, Bellville,
Ontario, Canada) to stimulate ovarian follicular development. At 80 h after the eCG,
sows received an injection (IM) of 5 mg porcine luteinizing hormone (pLH;
Lutropin®, Bioniche) to induce ovulation. Sows were subjected to transrectal real-
time ultrasound examinations of the ovaries using an Aloka 500 with a 7.5 MHz
linear array transducer every 2 h from 34 h after pLH injection until ovulation was
complete. Ovulation time was deﬁned as the time midway between an observed
absence of ovulatory follicles (>6mm) and the previous examination (Weitze et al.

1939).

For Experiment One, 122 multiparous sows were assigned at weaning to estrus and
ovulation control as described above. The predicted time of ovulation (PTO) was 38h
after pLH injection. Immediately before breeding, the sows were assigned by parity to
insemination with: 1) fresh sperm 12h before PTO, 2) FT sperm 12h before PTO, 3)
FT sperm supplemented with 10% (v/v) SP 12h before PTO, 4) FT sperm 2h before
PTO. The SP was obtained after centrifugation (800 x g for 10 min at 25°C) of a pool

of semen derived from 10 Yorkshire boars, and was stored at —20°C until needed.

47

Thawing of SP was done in a 37°C water bath and the SP was added to extender prior
to thawing sperm to allow immediate contact between the sperm and SP. Both fresh
and FT sperm were obtained from the same pool of boars housed at a commercial
boar stud (Ontario Swine Improvement, Innerkip, ON). Sperm were cryopreserved by
the method of Pursel and Johnson (1975) as modiﬁed by Buhr et al. (2001), and
thawed by submersion for 5 sec in a 60°C water bath. The post-thaw vitality of sperm
was determined using Eosin-Nigrosin staining. All doses (fresh and FT) were
extended in Beltsville Thawing Solution to a total volume of 80 ml and 3 x 109 live
sperm and were deposited intracervically in the presence of a boar. Sows were

allowed to go to term and farrowing rates and subsequent litter sizes were recorded.

For Experiment Two, 99 multiparous sows were assigned at weaning to treatments as
described for Experiment One, except that all FT inseminations were intrauterine. The
SP was obtained after centrifugation (800 x g for 10 min at 25°C) of a pool of semen
derived from 5 Yorkshire boars, and was stored at -20°C until needed. Thawing of SP
was done in a 37°C water bath and the SP was added to extender prior to thawing
sperm to allow immediate contact between the sperm and SP. The fresh and FT semen
was obtained commercially (International Boar Semen, IA) with boars equally
represented among treatments. Semen thawing was performed by exposure to room
temperature for 7 sec followed by submersion in 42°C water bath for 43 sec. The post-
thaw vitality of sperm was determined using Eosin-Nigrosin staining and doses (fresh
and FT) were extended in X-cell® extender (IMV, Maple Grove, MN) to a total
volume of 80 ml and 3 x 109 live sperm. Doses of FT were deposited using an

intrauterine catheter (IMV) while fresh semen was deposited intracervically, all in the

48

presence of a boar. Sows were allowed to go to term and farrowing rates and

subsequent litter sizes were recorded.

Statistical analysis

Data were analyzed using INFOSTAT/E v2.0 (University of Cordoba, Argentina).
Differences in farrowing rates and litter sizes (total born) were tested using analysis of
variance with LSD Fisher approximation, and with parity as a covariable. Results are

presented as meaniSEM and P <0.05 was considered signiﬁcant.

Results

The distribution of ovulation times in response to injections of eCG and pLH is shown
in Figure 3.1. All ovulations occurred between 35 and 42 hours, with 75.7% of the
animals ovulating between 37 and 39 hours. The time of ovulation (mean:SEM) was

38.4:03 hours after pLH injection.

Tlme post pLH (h)

 

50

42

30
Saw In

Figure 3.1. Sows ovulation time (hours post-pLH injection)

49

In Experiment One, a mean of 34.1% of the thawed sperm cells were observed to be
alive in randomly selected semen samples. The effects of semen type, supplemental
SP, and time of AI relative to ovulation are shown in Table 3.1. The insemination of
FT sperm resulted in signiﬁcantly reduced farrowing rates and smaller litter sizes
(p<0.01). There was no effect of supplemental SP on farrowing rate or litter size

(Table 3.1).

In Experiment Two, a mean of 65.4% of the thawed sperm cells were observed to be
alive in randomly selected semen samples. The effects of semen type, supplemental
SP, and time of AI relative to ovulation are shown in Table 3.2. The insemination of
FT sperm resulted in signiﬁcantly reduced farrowing rates (p<0.01) but semen type
did not affect litter size. There was no effect of supplemental SP on farrowing rate or

litter size (Table 3.2).

Table 3.1. Farrowing rates and total born litter sizes following artiﬁcial insemination
of fresh (FS) sperm or cryopreserved (FT) sperm with or without 10% supplemental
seminal plasma (SPmseminated 2 or 12 hours before ovulation (Experiment one).l

 

 

 

 

 

 

FS-12h FT-12h FT-12h+SP FT-2h
Number of sows 32 26 30 34
Parity2 32:03 33:03 37:04 30:02
Farrowing rate, % 68.8a 7.7b 10.0b 14.7b
Total born litter size2 12.1:0.8a 5.0:1.Ob 3.3:0.9b 8.0:1.8ab

 

 

 

 

 

I All inseminations were intracervical
2Means : SEM
ab, means followed by different letters differ, P<0.01

50

 

Table 3.2. F arrowing rates and total born litter sizes following artiﬁcial insemination
of fresh (FS) sperm or cryopreserved (FT) sperm with or without 10% supplemental
seminal plasma (SP) inseminated 2 or 12 hours before ovulation (Experiment two).l

 

 

 

 

 

 

FS-12h FT-12h FT-12h+SP FT-2h
Number of sows 25 24 24 26
Parity: 28:03 27:03 27:03 30:04
Farrowing rate, % 92.0a I 37.5b 41.7b 38.5b
Total born litter sizez 103:0.9 8.1:13 9.4:1.o 9.6:1.4

 

 

 

 

 

IF S sperm were inseminated intracervically but all FT sperm were inseminated
transcervically into the uterine body

2Means i SEM
ab, means followed by different letters differ, P<0.01

Discussion

Our data conﬁrms that of others (Cassar et al. 2005), in that the described ovulation
control protocol resulted in ovulation consistently at about 38 hours after the pLH
injection. Therefore, we are conﬁdent that the targeted times of insemination relative
to time of ovulation were accurate. This is further supported by the 92% farrowing
rate observed in Experiment Two following a single insemination of fresh semen on a

farm with a historical average of about 70% farrowing rate.

In the present studies, insemination of FT sperm was associated with reduced
farrowing rates regardless of site of sperm deposition or time of sperm deposition
relative to ovulation. Others have previously observed that fertilization rates
following FT inseminations were comparable to those following insemination of fresh
sperm, if the FT inseminations occurred with 4-hours of spontaneous ovulation
(Waberski et al. 1994). However, comparison with the present work is problematic

because sow fertility beyond determination of fertilization rates was not measured.
51

 

More recently, Wongtawan et al. (2006) observed that pregnancy rates in sows
following deep intrauterine insemination of non-extended FT sperm were improved
with closer timing of insemination relative to spontaneous ovulation. However, using
a model of spontaneous ovulation resulted in very few sows within any speciﬁed time
period relative to ovulation. Indeed, using a model of spontaneous ovulation, sow
fertility to FT insemination was less than that from fresh semen inseminations, but
were statistically comparable when FT inseminations occurred at 2 to 4 hours before
hormonally controlled ovulation (Roca et al. 2003). However, the latter authors
employed only 150 x 10° fresh sperm and a greater distinction between fresh and FT
insemination results may have been evident if a larger number of fresh sperm had
been used. Alternatively, we may have observed an effect of timing of insemination if
sperm deposition had been deeper. Although speculative, if this were the case it
would implicate a problem with transport of FT sperm into the oviduct, as suggested

by Holt (2000).

It has been suggested that the poor fertility associated with insemination of FT sperm
involves the cooling-associated induction of a capacitation-like state
(cryocapacitation), which would render the FT sperm incapable of forming an
enduring oviductal sperm reservoir. That FT sperm do not form an effective sperm
reservoir has been documented (Wongtawan et al. 2006; Abad et al. 2006).
Interestingly, although seminal plasma has been shown to reverse cryocapacitation,
such exposure did not affect the ability of FT sperm to form a sperm reservoir (Abad
et al. 2006). In addition to reversing cryocapacitation, seminal plasma has also been

shown to modulate uterine immune responses to sperm (Rozeboom et al. 1998) and to

52

be involved in the regulation of early embryo development (O’Leary et al. 2004).

However, there were no effects of seminal plasma on litter size.

The lack of effect of seminal plasma observed in our studies suggests that either the
supplemental seminal plasma did not affect the cryocapacitation, or more likely that
cryopreservation caused sperm injuries in addition to cryocapacitation that adversely
impacted fertility. Indeed, others have observed effects of cryopreservation on
integrity of sperm membranes and nuclear DNA (Rybar et al. 2004, Maldjian et al.
2005). Alternatively, it is possible that the speciﬁc seminal plasma used in these
studies was not effective. Others have shown that seminal plasma from different boars
can have different effects (Caballero et al. 2004). However, we used different pools of
seminal plasma in each experiment and neither indicated a positive effect. What
remains to be resolved is whether a higher concentration of seminal plasma would

yield a different response.

From the data presented, we conclude that sow fertility to insemination of
cryopreserved sperm is not improved by insemination close to the time of ovulation.
Further, there was no beneﬁt associated with supplementing semen doses with
seminal plasma, suggesting that the reduced fertility results from sperm cryoinjury in

addition to cryocapacitation.

Acknowledgments
We gratefully acknowledge Ontario Pork, Michigan State University Animal Industry
Coalition, and the University of Guelph-Ontario Ministry of Agriculture and Food

Animal Research Program for ﬁnancial support of these studies.

53

References

Abad M, Sprecher DJ, Ross P, Friendship RM, Kirkwood RN, 2006: Effect of sperm
cryopreservation and supplementing semen doses with seminal plasma on the
establishment of a sperm reservoir in gilts. Reprod Dom Anim (in press).

Alghamdi AS, Madill S, Foster DN, 2005: Seminal plasma improves fertility of
frozen equine semen. Anim Reprod Sci 89, 242-245.

Bailey J, Morrier A, Cormier N, 2003: Semen cryopreservation: Successes and
persistent problems in farm species. Can J Anim Sci 83, 393—401.

Bolarin A, Roca J, Rodriguez-Martinez H, Hernandez M, Vazquez JM, Martinez EA,
2006: Dissimilarities in sows’ ovarian status at the insemination time could explain
differences in fertility between farms when frozen-thawed semen is used.
Theriogenology 65, 669-680.

Buhr M, 2001: Emerging tools in artiﬁcial insemination. Proc London Swine
Conference 2001, pp 123-135.

Caballero I, Vazquez JM, Centurion F, Rodriguez-Martinez H, Parrilla I, Roca J,
Cuello C, Martinez EA, 2004: Comparative effects of autologous and homologous
seminal plasma on the viability of largely extended boar spermatozoa. Reprod Dom
Anim 39, 370-375.

Cassar G, Kirkwood RN, Poljak Z, Bennett-Steward K, Friendship RM, 2005: Effect
of single or double insemination on fertility of sows bred at an induced estrus and
ovulation. J Swine Health Prod 13, 254-258.

Crabo B, Einarsson S, 1971: Fertility of deep frozen boar spermatozoa. Acta vet scand
12, 125-127.

Einarsson S, Swensson T, Viring S, 1973: A ﬁeld trial on the fertility of deep-frozen
boar spermatozoa. Nord Vet —Med 25, 372-376.

Einarsson S, Viring S, 1973: Distribution of frozen-thawed spermatozoa in the
reproductive tract of gilts at different time intervals after insemination. J Reprod F ert
32, 117-120.

Eriksson B, 2000: Cryopreservation of boar semen. Studies on sperm viability in vitro
and fertility. PhD Thesis. Swedish University of Agriculture Sciences.

Holt WV, 2000. Fundamental aspects of sperm crybiology: The importance of species
and individual differences. Theriogenology 53:47-58.

Johnson LA, Witze KF, Fiser P, Maxwell WMC, 2000: Storage of boar semen. Anim
Reprod Sci 62, 143-172.

Maldjian A, Pizzi F, Gliozzi T, Cerolini S, Penny P, Noble R, 2005: Changes in
sperm quality and lipid composition during crypreservation of boar semen.
Theriogenology 63, 41 1-421.

54

Maxwell WMC, Evans G, Mortimer ST, Gillan L, Gellatly ES, McPhie CA, 1999:
Normal fertility in ewes alter cervical insemination with frozen-thawed spermatozoa
supplemented with seminal plasma. Reprod Fertil Dev 11, 123-126.

O’Leary S, Jasper MJ, Wames GM, Armstrong DT, Robertson SA, 2004: Seminal
plasma regulates endometrial cytokine expression, leukocyte recruitment and embryo
development in the pig. Reproduction 128, 237-247.

Pursel VG, Johnson LA, 1975: Freezing of boar spermatozoa: fertilizing capacity with
concentrated semen and a new thawing procedure. J Anim Sci 40, 99-102.

Roca J, Carvajal G, Lucas X, Vazquez JM, Martinez EA, 2003: Fertility of weaned
sows alter deep intrauterine insemination with a reduced number of frozen-thawed
spermatozoa. Theriogenology 60, 77-87.

Rozeboom KJ, Troedsson MHT, Molitor TW, Crabo BG, 1998: The effect of
spermatozoa and seminal plasma on leukocyte migration into the uterus of gilts. J
Anim Sci 77, 2201-2206.

Rybar R, F aldikova L, F aldyna M, Machatkova M, Rubes J, 2004: Bull and boar
sperm DNA integrity evaluated by sperm chromatin structure assay in the Czech
Republic. Vet Med 49, 1-8.

Suzuki K, Asano A, Eriksson B, Niwa K, Nagai T, Rodriguez-Martinez H, 2002:
Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal
plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan. Int j
andrology 25, 84-93.

Vadnais M, Kirkwood RN, Tempelman RJ, Sprecher DJ, Chou K, 2005a: Effect of
cooling and seminal plasma on the capacitation status of fresh boar sperm as
determined using chlortetracycline assay. Anim Reprod Sci 87, I21-32.

Vadnais ML, Kirkwood RN, Sprecher DJ, Chou K, 2005b: Effects of extender,
incubation temperature, and added seminal plasmas on capacitation of cryopreserved,
thawed boar sperm as determined by chlortetracycline staining. Anim Reprod Sci 90,
347-3 54.

Verbeckmoes S, Van Soom A, de Kruif A, 2004: Intra-uterine insemination in farm
animals and humans. Reprod Dom Anim 39, 195-204.

Waberski D, Weitze KF, Gleumes T, Schwartz M, Willmen T, Petzold R, 1994:
Effect of time of insemination relative to ovulation on fertility with liquid and frozen
boar semen. Theriogenology 42, 831-840.

Weitze KF, Habeck O, Willmen T, Rath D, 1989: Detection of ovulation in sow using
transcutaneous sonography. Zucht-hygiene 24, 40-42.

55

Wongtawan T, Saravia F, Wallgren M, Caballero I, Rodriguez-Martinez H, 2006.
Fertility after deep intra-uterine artiﬁcial insemination of concentrated low-volume
boar semen doses. Theriogenology 65, 773-787.

56

CHAPTER 4

Summary and Conclusions

Summary
The insemination-ovulation interval and the addition of seminal plasma on the
establishment of the sperm reservoir and the subsequent sow fertility following

artiﬁcial insemination (A1) of cryopreserved sperm were studied.

Firstly, gilts were injected 5mg of PG600 (n=35, experiment 1.1) or a combination of
eCG plus pLH (n=30, experiment 1.2) to induce ovulation. At breeding animals were
assigned by weight to receive single A1 of either FS sperm, FT sperm or FT sperm
plus 10% v/v seminal plasma. Semen was obtained from the same commercial boars
equally represented among treatments. Gilts were sacriﬁced 8h post AI and their
reproductive tracts recovered. All uterotubal junctions (UTJs) were dissected and
ﬂushed with PBS to recover sperm, which were counted using a hematocytometer.

The total number of sperm recovered per gilt was analyzed.

Secondly, multiparous crossbred sows were randomly selected to follow an ovulation
synchronization protocol (eCG at weaning plus pLH 80 hours later). This protocol
was veriﬁed previously by monitoring the ovulation of 37 animals by transrectal real
time ultrasonography. The predicted time of ovulation (PTO) was 38 hours post pLH
injection. Immediately before breeding, sows were assigned by parity to insemination
with either: 1) FS sperm 12h before PTO; 2) FT sperm 12h before PTO; 3) FT sperm
supplemented with 10% (v/v) SP 12h before PTO; 4) FT sperm 2h before PTO. The

FT sperm was deposited cervically in the Experiment 2.1 and intrauterine in

57

Experiment 2.2. Sows were allowed to go to term in both cases and farrowing rates

and litter sizes recorded.

Conclusions

The number of sperm located at the sperm reservoir was signiﬁcantly reduced after A]
with FT sperm in comparison with A1 with F S sperm. These data clearly indicated
that FT sperm are not capable of forming an adequate sperm reservoir. This inability
was assumed to be a result of a cooling-induced capacitation-like reaction
(cryocapacitation). However, exposure of FT sperm to seminal plasma had no effect
suggesting that the adverse fertility to A1 of FT sperm involves more than

cryocapacitation.

Neither the addition of seminal plasma to FT sperm not the timing of insemination of
FT sperm affected sow fertility. These data support the earlier conclusion that the
occurrence of cryoinjuries in addition to cryocapacitation either at membrane and/or
at nuclear levels, negatively affect fertility. Our studies indicate that the direction for
further studies should be to determine the speciﬁc damage the cryopreservation

process produces to the integrity of the sperm membrane and DNA.

58

APPENDIX 1

59

Sperm cryopreservation protocol

1- The rich sperm fraction of the boar ejaculate is regularly collected by the gloved-
hand method in a 37C prewarmed thermos.

2- Sperm motility is evaluated subjectively, the total volume by weight and the sperm
concentration by spectrophotometry (Spectronic 20D+) within 20 minutes from
collection.

3- Ejaculate is divided into fractions of 15ml and allowed to cool to room temperature
at the rate of 0.1C/min.

4- Samples are centrifuged at 800g for 10 min at 25C (Jouan CT 422) to remove
seminal plasma. The obtained sperm pellets are re-suspended in extender Beltsville
F 5-A to 1.2x109 sptz/ml and cool to SC over 3 hours.

5- Extender Beltsville F5-B is added to a ﬁnal concentration of 6x108 spz/ml and
0.5m] straws are ﬁlled and sealed (Filling Frigory, IMV).

6- Straws are frozen by contact with nitrogen vapor about 3cm above the liquid
nitrogen level for 20 min in an expandable polystyrene box, plunged into the nitrogen
tank and stored until use.

60

APPENDIX 2

61

Sperm Vitality - Eosin-Negrosin

Principles

A cell with an intact cell membrane does not take up the stain eosin Y, while a dead
cell (i.e. one with damaged cell membrane) takes up the red stain. Nigrosin is used as
a background stain to provide contrast for the unstained (white) live cells.

Procedure

- One drop (50ul) of undiluted, well-mixed liqueﬁed semen is mixed with one drop
(50u1) of eosin-nigrosin solution (eg in a porcelain spotting plate) and incubated
for 305ec.

- A drop of the solution (12-15ul per microscope slide) is placed on a clean
microscope slide, and a smear made. The drop is smeared by sliding a cover slip
in front of the drop. If two smears are made simultaneously by spreading between
two microscope slides without feathering, one drop of 20-30 ul is used.

- The smear is allowed to air dry and examined directly, or mounted permanently
the same day and examined after the mount has dried (eg overnight). Mounted
smears are stored at room temperature.

- At least 200 sperm are assessed at 1000x (or 1250x) magniﬁcation under 011
immersion with a high-resolution 100x objective (not phase contrast) with correct
adjustment of the bright ﬁeld optics. Sperm that are white (unstained) are
classiﬁed as ‘live’ and those that show any pink or red coloration are considered
as ‘dead’.

- WHO does not specify duplicate counting for vitality assessment.

Reagents

0.67g of Eosin Y and 0.9g sodium chloride were dissolve in 100ml distilled water
under gentle heating to afterwards add 10g Nigrosin. The solution was brought to
boil and allowed to cool to room temperature. Paper ﬁlter was used to ﬁlter the
solution before stored in a sealed glass bottle until needed, always at room
temperature.

 

ESHRE Monographs: Manual on Basic Semen Analysis p.12, 2002
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