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thesis entitled

Decapacitation of Boar Spermatozoa

presented by
Melissa L. Vadnais

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DECAPACITATION OF BOAR SPERMATOZOA

By

Melissa L. Vadnais

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

2004

ABSTRACT

DECAPACITATION OF BOAR SPERMATOZOA

By

Melissa L. Vadnais

This thesis describes three studies that were completed to demonstrate the
decapacitation ability of boar seminal plasma (SP) when added to incubated, cooled, and
frozen-thawed (F T) boar spermatozoa. In the first study, chlortetracycline (CTC) staining
analysis was used to investigate the effects of 20% (v/v) SP addition at different time
points on the temporal pattern of capacitation in spermatozoa incubated at 39 C or cooled
to 5 C and then rewarmed to 39 C. Seminal plasma was able to prevent and reverse
capacitation in spermatozoa incubated in a capacitation-supporting environment as well
as capacitation induced by cooling the spermatozoa to 5 C. In the second study, the
addition of either 10 or 20% (v/v) SP to frozen boar spermatozoa at thawing were
investigated using CTC analysis. The addition of either 10 or 20% SP to FT spermatozoa
resulted in the reversal of capacitation within 30 min of its addition. In the third study, the
decapacitating effects of 10% (v/v) SP addition to FT spermatozoa were examined in
different media and at different incubation temperatures. It was demonstrated that the
effects of SP were dependent on the temperature of the spermatozoa suspension as well
as the media the spermatozoa were suspended in. The results from these three studies
demonstrated that capacitation in boar spermatozoa is regulated by some constituent(s) of

boar SP.

ACKNOWLEDGMENTS

This project was made possible by the help, support, and encouragement of many people.
I am especially grateful to all of those who have provided me with education, training,
and guidance. In particular, I thank my advisor Dr. Roy Kirkwood and committee

members Dr. David J. Sprecher and Dr. Karen Chou.

I would also like to thank Dr. Gary Althouse for his continued guidance and mentoring
through both my undergraduate and graduate education. His influence, support, and

encouragement have been extremely influential in where I am and where I am going.

I also acknowledge Dr. John B. Kaneene and his team of graduate students and staff.
Their support, wisdom, and optimism have been great strengths to me during my time

here at the Michigan State College of Veterinary Medicine.
Finally, I would like to thank both Julie Lanivich and Faith Peterson for their patience

and kindness. Without their help I would have been lost! I also thank the staff at the

University’s Swine Farm.

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES .......................................................................................................... vii
LIST OF ABBREVIATIONS ............................................................................................ ix
OVERVIEW ........................................................................................................................ 1
Chapter 1
Review: Capacitation of spermatozoa ................................................................................. 2
Abstract .................................................................................................................... 2
Introduction .............................................................................................................. 2
Spermatozoa Maturation .......................................................................................... 3
Capacitation ............................................................................................................. 4
Fertilization and Acrosome Reaction ....................................................................... 7
Decapacitation Factors ............................................................................................. 8
Conclusion ............................................................................................................... 9
References .......................................................................................................................... 10
Chapter 2
Effect of cooling and the addition of seminal plasma on the capacitation status of fresh
boar spermatozoa as determined using chlortetracycline staining ..................................... 15
Abstract .................................................................................................................. 15
Introduction ............................................................................................................ 15
Materials and Methods ........................................................................................... 17
Medium ...................................................................................................... l7
Semen collection and assessment .............................................................. 18
Processing for non-cooling treatments ....................................................... 18
Processing for cooling treatments .............................................................. 19
Chlortetracycline staining analysis ............................................................ l9
Bis-Benzimide Hoechst 33258 staining analysis ....................................... 20
Treatments .................................................................................................. 21
Statistical Analysis ..................................................................................... 22
Results .................................................................................................................... 23
Viability assessment ................................................................................... 23
Non-cooling treatments .............................................................................. 23
Cooling treatments ..................................................................................... 24
Discussion .............................................................................................................. 25
References .......................................................................................................................... 29
Chapter 3
The effect of the addition of seminal plasma and the duration of incubation time on the
post-thaw capacitation status of frozen-thawed boar spermatozoa .................................... 4O

Abstract .................................................................................................................. 40

Introduction ............................................................................................................ 40

Materials and Methods ........................................................................................... 42

Medium ...................................................................................................... 42

Spermatozoa processing for treatments ..................................................... 43

Treatments .................................................................................................. 43

Chlortetracycline staining analysis ............................................................ 44

Statistical analysis ...................................................................................... 45

Results .................................................................................................................... 45

Discussion .............................................................................................................. 47

References .......................................................................................................................... 49
Chapter 4

The effects of egg yolk freezing extender, incubation temperature, and the addition of
seminal plasma on cryocapacitation of frozen-thawed boar spermatozoa as determined by

chlortetracycline analysis ................................................................................................... 56
Abstract .................................................................................................................. 56
Introduction ............................................................................................................ 56
Materials and Methods ........................................................................................... 58

Media ......................................................................................................... 58
Treatments .................................................................................................. 58
Chlortetracycline staining analysis ............................................................ 60
Bis-Benzimide Hoechst 33258 staining analysis ....................................... 61
Statistical analysis ...................................................................................... 62
Results .................................................................................................................... 62
Motility and Viability ................................................................................ 63
Discussion .............................................................................................................. 63

References .......................................................................................................................... 68

Chapter 5

Summary and Conclusions ................................................................................................ 73
Summary ................................................................................................................ 73
Conclusions ............................................................................................................ 74

Appendices ......................................................................................................................... 76
Appendix I ............................................................................................................. 77
Appendix II ............................................................................................................ 80
Appendix III ........................................................................................................... 82
Appendix IV ........................................................................................................... 85

Table 4.1.

Table 4.2.

LIST OF TABLES

Effect of seminal plasma on capacitation status of frozen-thawed
spermatozoa after a 30 min incubation period in commercial semen
extender at 17 or 39 C. ............................................................................... 71

Effect of seminal plasma on capacitation status of frozen-thawed
spermatozoa after a 30 min incubation period in phosphate buffered saline
(Treatments 5 through 8) or commercial semen extender (Treatments 9
and 10) at 17 or 39C .................................................................................. 72

vi

Figure 2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 2.6.

Figure 2.7.

Figure 2.8.

Figure 3.1.

LIST OF FIGURES

In Figures 2.1 through 2.8, time points ‘Pre’ and ‘Post’ represent readings
prior to and after the 100-min pre-incubation or cooling, respectively.

Effect of all treatments on the viability of spermatozoa. Each point
represents the mean :1: SE of all determinations. ........................................ 32

In Figures 2.2 through 2.8, A represent Fresh spermatozoa, I represent
Capacitated spermatozoa (B), and 0 represent Acrosome Reacted
spermatozoa. Each time point represents the average of 5 counts 1: SE.

Pre-incubation and incubation in li99 containing 0% SP. ................... 33

Pre-incubation with 0% SP and then incubation in li99 containing 20%
SP. .............................................................................................................. 34

Pre-incubation and initial incubation in li99 containing 0% SP with
20% SP added at Hr2. ................................................................................ 35

Cooling/warming and incubation of spermatozoa in li99 containing
0% SP ......................................................................................................... 36

Cooling in li99 containing 0% SP with warming and incubation in
li99 containing 20% SP. ...................................................................... 37

Cooling in li 99 containing 20% SP with warming and incubation in
li99 containing 0% SP. ........................................................................ 38

Cooling/warming in li99 containing 0% SP with 20% SP added at Hr2
of incubation .............................................................................................. 39

In Figures 3.1 through 3.4, A represent Fresh spermatozoa, I represent
Capacitated spermatozoa (B), and 0 represent _A_crosome Reacted
spermatozoa. Each point represents the average of 6 counts i SE.

Frozen spermatozoa thawed in li99 containing 0% SP. ...................... 52

vii

Figure 3.2. Frozen spermatozoa thawed in li99 containing 10% SP. .................... 53

Figure 3.3. Frozen spermatozoa thawed in li99 containing 20% SP. .................... 54

Figure 3.4. Frozen spermatozoa thawed in li99 containing 10% SP with readings
taken at 15 min intervals over 1 h. ............................................................. 55

viii

LIST OF ABBREVIATIONS

AR ........................................................................................................... Acrosome Reacted
B ................................................... .................................................................. Capacitated
CTC ............................................................................................................ Chlortetracycline
F ......................................................................................................... Fresh (uncapaCitated)
FT ................................................................................................................... F rozen-thawed
GAG ....................................................................................................... Glycosaminoglycan
HA ....................................................................................................................... Hyaluronan
HDL .............................................................................................. Hi gh-density Lipoprotein
mM199 .............................................................................................. Modified Medium 199
MSU ............................................................................................ Michigan State University
PBS ............................................................................................. Phosphate Buffered Saline
PS ............................................................................................................ Phosphatidylserine
SP ................................................................................................................. Seminal Plasma
ZP .................................................................................................................. Zona Pellucida

OVERVIEW
Chapters 1, 2, 3, and 4 in this thesis are written in a format suitable for publication
independently. Thus, those chapters each have an abstract, introduction, methods, and

discussion. A list of literature cited concludes each chapter.

Chapter 1 is a literature review of capacitation across species. It includes a discussion on
spermatozoa maturation, fertilizing ability, and decapacitation. Chapter 2 describes our
initial study investigating the effects of SP addition on capacitation in vitro. Chapter 3 is
a study examining the effects of SP on cryocapacitation caused by the freezing and
subsequent thawing of spermatozoa. Chapter 4 describes a study to determine if the
effects of SP are dependent on the incubation temperature and/or presence of egg yolk in
the spermatozoa suspension. The thesis concludes with Chapter 5, which is an overall

summary and synthesis of the findings of this research.

CHAPTER 1

Review: Capacitation of spermatozoa
Abstract
Spermatozoa are required to undergo the processes of capacitation as well as acrosome
reaction before they obtain fertilizing ability. The molecular changes of capacitation are
still not fiilly understood. However, it is accepted that capacitation is a sequential process
involving numerous physiological changes including membrane destabilization, alteration
of membrane potential, and protein phosphorylation. There are no known morphological
changes to the spermatozoa during capacitation. The purpose of this review is to
summarize the data available on the biochemical aspects of capacitation both in vivo and

in vitro.

Introduction

Early work with in vitro fertilization led to the postulate that spermatozoa must reside
within the female reproductive tract for a period of time before they acquire the ability to
fertilize an oocyte. It was later demonstrated that spermatozoa introduced into rabbit
oviducts shortly after ovulation were unable to fertilize the oocytes, despite rabbit ova
remaining fertilizable for up to 8 h after ovulation; however, when spermatozoa were
inseminated into the oviduct several hours prior to ovulation a high percentage of
fertilization occurred [1, 2]. Similarly, fertilization occurred 4 h after rat spermatozoa
were deposited into the postovulatory ovarian capsule [3]. This amount of time required
before the spermatozoa were capable of fertilizing an oocyte was termed capacitation and

was discovered independently by Austin [3] and Chang [4] over fifty years ago.

The evidence for the need for capacitation in various species is based on this temporal
pattern between mating or insemination and penetration of the zona pellucida (ZP).
Copulation after the time of ovulation in rats demonstrated that spermatozoa were present
in the oviduct after 1 hour, but it wasn’t until 2-3 hours afier copulation that spermatozoa
penetrated the oocyte [5]. Also, ejaculated spermatozoa incubated in a golden hamster
uterus for several hours fertilized a higher proportion of oocytes 4-5 hours afier surgical

insemination then did spermatozoa from the epididymis [6].

Capacitation of a freshly ejaculated spermatozoa population appears to take several hours
in most mammalian species. For example, in the rabbit, a period of 5-6 hours is required
for capacitation [4]. Conversely, within a spermatozoa population, individual cells
respond to capacitating conditions at widely different rates [7]. Capacitation within an
individual spermatozoon can take place over a short period of time [8]. The length of the
time needed for capacitation suggests that capacitation is a sequence of events. Therefore,
capacitation is a specific, initiated, and controllable process rather than a natural

continuation of maturation [9].

Spermatozoa Maturation
Spermatogenesis is the biological process of maturation of germ cells into spermatozoa
within the seminiferous tubules of the testis over a period of time. This process involves

cellular proliferation by repeated mitotic divisions, duplication of chromosomes, genetic

recombination through cross-over, reduction-division by meiotic division to produce

haploid spermatids, and terminal differentiation of the spermatids into spermatozoa [10].

Maturation of the spermatozoa is not completed in the seminiferous tubules but continues
as the spermatozoa are transported through the epididymis. Protein addition and lipid
membrane alteration occurs as the spermatozoa transit through the caput, corpus, and
cauda epididymides and at ejaculation [ 1 1-14]. An example of this is the secretion of the
25 kDa protein, anti-agglutinin, by the epididymal duct of the corpus epididymides and

the subsequent binding to the acrosome of spermatozoa [15].

Capacitation (Appendix I)

Capacitation has been described as a biochemical and reversible process which enables
the spermatozoa to penetrate the ZP of the ovum [16]. There is much evidence that
explain the molecular modifications of the spermatozoa membrane during capacitation.
In vivo, capacitation occurs in the isthmus of the oviduct after the spermatozoa contact

the oviductal epithelial cells [17-20].

Capacitation in vitro can occur during incubation at ambient temperature in the presence
of bicarbonate, calcium, and serum albumin in a balanced salt solution in which the pH is
controlled at 7.8 [11, 21, 22]. Incubation of spermatozoa in media devoid of serum
albumin, calcium, or bicarbonate components prevents spermatozoa from undergoing

capacitation, undergoing a ZP induced acrosome reaction, and fertilizing metaphase II

arrested oocytes in vitro [23]. In each of these cases, tyrosine phosphorylation of the

capacitation-associated proteins did not occur.

Several studies have shown that bicarbonate plays a key role in the ability of spermatozoa
to undergo capacitation and fertilization both in vivo as well as in vitro [21, 23-25]. Low
levels of bicarbonate are maintained in the epididymis, but bicarbonate levels are high in
the female reproductive tract where capacitation occurs [21]. This effect of bicarbonate
may be one of the initial events in the capacitation process by altering the plasma
membrane lipid architecture. This was demonstrated by Harrison et al. [25] using the
fluorescent lipophilic molecule merocyanine. Merocyanine is membrane-impenneable
and binds to the outer area of the plasma membrane of intact cells in relation to the
degree of lipid disorder. Bicarbonate causes an increase in membrane lipid disorder
therefore enhancing the ability of spermatozoa to bind merocyanine. The merocyanine-

detected change took place within 5 min of spermatozoa exposure to bicarbonate [25].

Bicarbonate activates adenylate cyclase to produce an increase in the intracellular CAMP
concentration, which in turn stimulates a protein kinase A to phosphorylate unknown
protein components of the spermatozoa membrane [24, 26, 27]. The relationship between
these proteins and the plasma membrane lipid architecture is not fully understood;
however, they are related to a continual cycle of phosphorylation and dephosphorylation
[7]. Recent work by Tardif et al. [28] demonstrated that a 32 kDa tyrosine kinase-like
protein is auto—phosphorylated in the capacitating boar spermatozoa. Moreover, the

capacitation-associated phosphorylation of this protein appears to be calcium dependent.

The bicarbonate induction of merocyanine binding is temperature dependent and
reversible, occurring at 38 C but not at 25 C [25]. The reversibility of the bicarbonate
effects may also be related to the continual cycle of membrane protein phosphorylation

and dephosphorylation.

Capacitation involves a reversible range of membrane-related events resulting in a
decreased membrane stability and increased permeability. These events include the
removal of protein ‘decapacitation’ factors acquired in the epididymis or at ejaculation, a
decrease in the cholesterol: phospholipids ratio [29], an increase in membrane lipid
fluidity, activation of phospholipases, and a readiness to undergo the acrosome reaction
[30, 31]. In boar spermatozoa, capacitation induces changes in lipid membrane structure
via a cAMP-dependent protein phosphorylation pathway. These membrane changes
include phosphatidylserine (PS) inversion, the relocation of cholesterol to the apical head,
and a decrease in lipid packing [26]. The lipid scrambling allows for cholesterol
relocation. Lipid scrambling, mainly PS inversion, is a recognized stage of the apoptotic
death pathway [32]. However, there is no evidence that PS inversion is related to the
apoptotic death pathway [26]. Serum albumin has been considered to act as an acceptor
of cholesterol from the cell membrane resulting in the decrease in cholesterol:

phospholipids ratio which contributes to the increase in membrane fluidity [29].

The increased membrane fluidity allows for a shift in membrane potential. The
membrane potential of the spermatozoa becomes more negative as potassium ions leave

the spermatozoa. This change in membrane potential may allow calcium channels to be

opened permitting calcium ions to enter the spermatozoa. Calcium and bicarbonate ions
may be critical in the activation of cAMP production leading to protein phosphorylation

[23, 33].

Fertilization and Acrosome Reaction

Shortly after binding to the ova’s ZP, the spermatozoa undergo a cellular exocytosis
referred to as the acrosome reaction. The acrosome is a large, Golgi-derived, lysosome-
like organelle that overlies the nucleus in the apical region of the spermatozoa head [34].
It is made up of an inner membrane that overlies the nucleus and an outer membrane that
underlies the plasma membrane. Binding of the spermatozoa to the ZP3 protein of the

ova initiates the acrosome reaction [35-37].

The acrosome reaction involves multiple firsions between the outer acrosomal membrane
and plasma membrane at the anterior region of the spermatozoa head, extensive
formation of hybrid membrane vesicles, and exposure of the inner acrosomal membrane
and acrosomal contents [30]. Only spermatozoa that have completed the acrosome

reaction can penetrate the ZP and fuse with the ova plasma membrane.

Acrosome reacted spermatozoa remain bound to the ZP through their association with
ZP2 [3 8, 39]. Bound spermatozoa must penetrate the ZP and pass through the
perivitelline space to reach and fuse with the ova’s plasma membrane. Penetration of the

ZP is a result of both spermatozoa motility and enzymatic hydrolysis [30, 40]. An

acrosomal serine protease called acrosin catalyzes this enzymatic hydrolysis [41]. When

the spermatozoa body is adsorbed into the ova, syngamy ensues.

Decapacitation Factors

Ej aculated spermatozoa are more difficult to capacitate then epididymal spermatozoa.
Coating of the spermatozoa surface with seminal plasma proteins that inhibit processes
normally associated with capacitation may be responsible for these findings [23]. Seminal
plasma proteins have been postulated to act as decapacitation factors that prevent a

premature acrosome reaction [42-44]. Their removal may initiate capacitation.

Bovine seminal plasma proteins (BSP-All-AZ, BSP-A3, BSP—30-kDa) bind to choline
phospholipids on the spermatozoa membrane at ejaculation. Subsequently these proteins
interact with high-density lipoproteins (HDL) and heprin-like glycosarninoglycans
(GAG) in the female reproductive tract resulting in capacitation [45]. In the boar, the
heparin-binding protein pBl has been characterized and identified to bind to lipids
containing the phosphorylcholine group on the spermatozoa membrane at ejaculation
[28]. Recently, an increase in capacitated spermatozoa were demonstrated after frozen-
thawed spermatozoa were incubated with hyaluronan (HA), a GAG [46]. This suggested
capacitation-causing actions of HA. Heparin [47, 48] and HDL [45] have also been
shown to stimulate capacitation. Glycosaminoglycans have been localized in the pre- and
post-ovulatory oviducts of swine reproductive tracts [49] and may play a role in the
removal of seminal plasma proteins on the spermatozoa membrane resulting in

capacitation both in vitro and in the female reproductive tract.

Conclusion

Spermatozoa’s ability to fertilize an oocyte is dependent on their ability to successfully
undergo capacitation and the acrosome reaction. The correct environment is required for
this to occur whether the spermatozoa are in the oviduct of the female reproductive tract
or an in vitro system. Capacitated spermatozoa have a short period of time to meet and

fertilize an oocyte. If an oocyte is not present the spermatozoa die shortly thereafter.

A large number of spermatozoa are deposited into the female reproductive tract at
artificial insemination. Cryopreservation results in a higher proportion of spermatozoa
being effectively capacitated upon thawing [50, 51]. As a result, the spermatozoa have a
shorter period of time for oocyte fertilization. In the swine industry, the use of frozen-
thawed spermatozoa results in lower farrowing rates and smaller litter sizes [52, 53] in

part due to the spermatozoa being effectively capacitated upon thawing.

A complete understanding of the process of capacitation would not only benefit the
scientific community but also the food animal production industry. The ability to reverse
the capacitation-like effects of cryopreservation would allow for the widespread
application and acceptance of frozen-thawed spermatozoa for artificial insemination,
specifically within the swine industry. As a result, the biosecurity of the international

food animal industry would be greatly enhanced.

References

1.

10.

ll.

12.

13.

Adams, C., Chang M. The effect of delayed mating on fertilisation in the rabbit. J
Exper Zoo, 1962. 151: 155-8.

Chang, M. Fertilisability of rabbit ova and the effects of temperature in vitro on
their subsequent fertilisation and activation in vivo. J Exper Zoo, 1952. 121: 351-
81.

Austin, C. Observations on the penetration of the sperm into the mammalian egg.
Aust J Sci Res Ser B, 1951. 4: 581-9.

Chang, M. Fertilising capacity of spermatozoa deposited into the Fallopian Tubes.
Nature, Lond, 1951. 168: 697.

Austin, C. The 'capacitation' of the mammalian sperm. Nature, 1952. 170: 326.

Hunter, R. Capacitation in the golden hamster, with special reference to the
influence of the uterine environment. J Reprod F ertil, 1969. 20: 223-37.

Harrison, R.A. Sperm plasma membrane characteristics and boar semen fertility. J
Reprod Fertil Suppl, 1997. 52: 195-211.

Roldan, E., Vazquez, J. Bicarbonate/C02 induces rapid activation of
phospholipase A2 and renders boar spermatozoa capable of undergoing acrosomal
exocytosis in response to progesterone. F EBS Letters, 1996. 396: 227-32.

Bedford, J. Report of a workshop, maturation of the fertilizing ability of

mammalian Spermatozoa in the male and female genital tract. Biol Reprod, 1974.
11: 346-59.

Hess, R. Spermatogenesis, Overview, in Encyclopedia of Reproduction. 1999,
Academic Press.

Watson, R, Green, C.E. Cooling and Capacitation of Boar Spermatozoa: What do
They Have in Common? In: Boar Semen Preservation IV, 2000. 53-60.

Bwanga, C.O. Cryopreservation of boar semen I: a literature review. Acta Vet
Scand, 1991. 32: 431-53.

Rana, A., Majumder, G.C., Misra, S., Ghosh, A. Lipid changes of goat sperm

plasma membrane during epididymal maturation. Biochem Biophys Acta, 1991.
1061: 185-96.

10

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

Nikolopoulou, M., Soucek, D., Vary, J. Changes in the lipid content of boar
sperm plasma membranes during epididymal maturation. Biochem Biophys Acta,
1985. 815: 486-98.

Harayarna, H., Magargee, S.F., Kunze, E., Shidara, O., Iwamoto, E., Arikawa, S.,
Miyake, M., Kato, S., Hammerstedt, R.H. Changes in epididymal protein anti-
agglutinin on ejaculated boar spermatozoa during capacitation in vitro. Reprod
Fertil Dev, 1999. 11: 193-9. '

Chang, M. A detrimental effect of seminal plasma on the fertilizing capacity of
sperm. Nature, Lond, 1957. 179: 258-9.

Pollard, J ., Plante, C., King, W.A., Hansen, P.J., Betteridge, K.J., Suarez, S.S.
Fertilizing capacity of bovine sperm may be maintained by binding of oviductal
epithelial cells. Biol Reprod, 1991.44: 102-7.

Ericsson, R. Technology, physiology and morphology of spermatozoa
capacitation. J Reprod Fertil Suppl, 1967. 2: 65-74.

Soupart, P. Studies on the hormonal control of rabbit sperm capacitation. J
Reprod Fertil Suppl, 1967. 2: 49-64.

F azeli, A., Duncan, A.E., Watson, P.F., Holt, W.V. Spenn-oviduct interaction:
Induction of capacitation and preferential binding of uncapacitated spermatozoa
to oviductal epithelial cells in porcine species. Biol Reprod, 1999. 60: 879-86.

Harrison, R. Capacitation mechanisms, and the role of capacitation as seen in
eutherian mammals. Reprod F ertil Dev, 1996. 8: 581-94.

Visconti, P., Kopf, G. Regulation of protein phosphorylation during sperm
capacitation. Biol Reprod, 1998. 59: 1-6.

Visconti, P.E., Bailey, J .L., Moore, G.D., Pan, D., Olds-Clark, P., Kopf, G.S.
Capacitation of mouse spermatozoa 1. Correlation between the capacitation state
and protein tyrosine phosphorylation. Development, 1995. 121: 1129-37.

Okamura, N., Tajima, Y., Soejirna, A., Masuda, H., Sugita, Y. Sodium
bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa
through direct activation of adenylate cyclase. J Biol Chem, 1985. 260: 9699-
705.

Harrison, R.A., Ashworth, P., Miller, N.G.A. Bicarbonate/C02, an effector of

capacitation, induced a rapid and reversible change in the lipid architecture of
boar sperm plasma membranes. Molec Reprod Dev, 1996. 45: 378-91.

11

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

Gadela, B.M. Harrison, R.A. Capacitation Induces Cyclic Adenosine 3',5'-
Monophosphate-Dependent, but Apoptosis-Unrelated, Exposure of
Animophospholipids at the Apical Head Plasma Membrane of Boar Sperm Cells.
Biol Reprod, 2002. 67: 340-50.

Visconti, P.E., Galantino-Homer, H., Moore, G.D., Bailey, J .L., Ning, X., Fomes,
M., Kopf, G.S. The molecular basis of sperm capacitation. J Androl, 1998. 19:
242-8.

Tardif, S., Dude, C., Chevalier, S., Bailey, J.L. Capacitation is associated with
tyrosine phosphorylation and tyrosine kinase-like activity of pig sperm proteins.
Biol Reprod, 2001. 65: 784-92.

Cross, N.L. Role of cholesterol in sperm capacitation. Biol Reprod, 1998. 59: 7-
11.

Yanagimachi, R. Mammalian Fertilization, in "The Physiology of Reproduction."
J .N. E Knobil, Editor. 1994, Raven Press: New York. 189-317.

Topfer-Petersen, E., Friess, A.E., Stoffel, M., Schill, W.B. Boar sperm
membranes antigens. I. Topography of a mobile glycoprotein of the sperm cell
membrane. Histochemistry, 1990. 93: 485-90.

Fadok, V.A., Bratton, D.L., Rose, D.M., Pearson, A., Ezekewitz, R.A., Henson,
P.M. A receptor for phosphatidylserine-specific clearance of apoptotic cells.
Nature, 2000. 405: 85-90.

Amoult, C., Kazam, I.G., Visconti, P.E., Kopf, G.S., Villaz, M., Florman, H.M.
Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3
and by membrane hyperpolarization during capacitation. Proceedings of the
National Academy of Sciences of the United States of America, 1999. 96: 6757-
62.

Abou-Haila, A., Tulsiani, D. Mammalian sperm acrosome reaction: formation,
contents, and function. Arch Biochem Biophys, 2000. 379: 173-82.

Florman, BL, Amoult, C., Kazam, I.G., Li, C., O’Toole, C.M.B. An intimate
biochemistry: egg-regulated acrosome reactions of mammalian sperm. Adv Dev
Biochem, 1999. 5: 147-86.

Bleil, J ., Wassarman, W. Sperm-egg interactions in the mouse: sequence of events
and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev
Biol, 1983. 95: 317-24.

12

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

O'Toole, C., Amoult, C., Darszon, A., Steinhardt, R.A., Florman, H.M. Ca2+
entry through store-operated channels in mouse sperm is initiated by egg ZP3 and
drives the acrosome reaction. Molec Biol Cell, 2000. 11: 1571-84.

Bleil, J ., Greve, T., Wassarman, P. Identification of a secondary sperm receptor in
the mouse egg zona pellucida: Role in maintenance of binding of acrosome-
reacted sperm to eggs. Dev Biol, 1988. 128: 376-85.

Mortillo, S., Wassarman, P. Differential binding of gold-labeled zona pellucida
glycoprotwins mZP2 and mZP3 to mouse sperm membrane compartments.
Development, 1991. 113: 141-51.

Bedford, J. Mammalian fertilization misread? Sperm penetration of the eutherian
zona pellucida is unlikely to be a lytic event. Biol Reprod, 1998. 59: 1275-87.

Eddy, E., O'Brien, D. In: The Physiology of Reproduction, E. Knobil and J. Neill,
Editors. 1994, Raven: New York. 29-77.

Dukelow, W., Chernoff, H., Williams, W. Properties of decapacitation factor and
presence in various species. J Reprod Fertil, 1967. 14: 393-9.

Fraser, L., Harrison, R.A., Herod, J. Characterization of a decapacitation factor
associated with epididymal mouse spermatozoa. J Reprod Fertil, 1990. 89: 135-
48.

Oliphant, G., Reynolds, A., Thomas, T. Sperm surface components involved in
the control of the acrosome reaction. Am J Anat, 1985. 174: 269-83.

Therien, I., Soubeyrand, S., Manjunath, P. Major proteins of bovine seminal
plasma modulate sperm capacitation by high-density lipoprotein. Biol Reprod,
1997. 57: 1080-8.

Suzuki, K., Asano, A., Eriksson, B., Niwa, K., Nagai, T., Rodriguez-Martinez, H.
Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal

plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan. Int J
Androl, 2002. 25: 84-93.

Therien, I., Bleau, G., Manj unath, P., Soubeyrand, S. Phosphatidylcholine-
binding proteins of bovine seminal plasma modulate capacitation of spermatozoa
by heparin. Biol Reprod, 1995. 52: 1372-9.

Kim, N.H., Day, B.N., Lim, J .G., Lee, H.T., Chung, K.S. Effects of oviductal

fluid and heparin on fertility and characteristics of porcine spermatozoa. Zygote,
1997. 5: 61-5.

13

49.

50.

51.

52.

53.

Tienthai, P., Kjellen, L., Pertofi, H., Suzuki, K., Rodriguez-Martinez, H.
Localization and quantitation of hyaluronan and sulfated glycosarninoglycans in
the tissues and intraluminal fluid of the pig oviduct. Reprod Fertil Dev, 2000. 12:
173-82. ~

Bailey, J ., Bilodeau, J ., Cormier, N. Semen cryopreservation in domestic animals:
a damaging and capacitating phenomenon. J Androl, 2000. 21: 1-7.

Maxwell, W., Johnson, L. Chlortetracycline analysis of boar spermatozoa afier
incubation, flow cytometric sorting, cooling, or cryopreservation. Molec Reprod
Dev, 1997. 46: 408-18.

Reed, H. Current use of frozen boar semen-future need of frozen boar semen. In:
Deep freezing of boar semen, J. LA and L. K, Editors. 1985, Swedish University
of Agricultural Science: Uppsala. 225-37.

Almlid, T., Hofmo, P. A brief review of frozen semen application under
Norwegian AI service conditions. Reprod Dom Anim, 1996. 31: 169-73.

14

CHAPTER 2

Effect of cooling and the addition of seminal plasma on the capacitation status of fresh

boar spermatozoa as determined using chlortetracycline staining

Abstract

Insemination of swine with frozen-thawed spermatozoa results in lower fertility, in part
due to spermatozoa having undergone a capacitation-like reaction. The present study
employed chlortetracycline staining analysis to investigate the effect of adding 20% (v/v)
boar seminal plasma (SP) to boar spermatozoa on the temporal progress of capacitation
and the acrosome reaction when spermatozoa were cooled to 5 C or incubated at 39 C.
Seminal plasma reversed capacitation induced by incubation at 39 C in a capacitation-
supporting medium from 59.7% to 36.6% capacitated spermatozoa (P<0.001). Similarly,
the addition of SP to boar spermatozoa cooled to 5 C resulted in both the prevention of
the capacitation-like reaction and the reversal of an established capacitation-like reaction
from 63.3% to 34.2% capacitated (P<0.001). These observations indicated that the
addition of boar SP can both prevent spermatozoa from undergoing capacitation as well

as reverse capacitation in spermatozoa that have already undergone the process.

Introduction

Artificial insemination of swine with frozen-thawed (FT) spermatozoa results in
farrowing rates and litter sizes 20 to 30% below those observed following insemination
of fresh spermatozoa [1]. This is believed to be a result of spermatozoa acquiring

cryoinjury, including a capacitation-like reaction (cryocapacitation), during the freezing

15

and thawing process. Capacitated spermatozoa do not associate with the oviductal
epithelium and so do not enter the functional spermatozoa reservoir [2, 3]. Although
capacitated FT spermatozoa are capable of fertilizing oocytes, their life span in the
female reproductive tract is considerably shorter than that of freshly ejaculated
spermatozoa [4, 5]. Therefore, the use of FT spermatozoa for swine artificial

insemination requires that an oocyte be almost immediately available for fertilization.

Capacitation is a sequential process involving numerous physiological changes including
membrane destabilization [6], an influx of calcium ions [7], and protein phosphorylation
[8]. Cryocapacitation is a similar, capacitation-like, process that displays a different
pattern and number of tyrosine-phosphorylated proteins [9]. Membrane destabilization
consists of the removal of protein decapacitation factors, acquired during epididymal
transport or after mixing with seminal plasma (SP) during ejaculation, as well as
cholesterol from the spermatozoa membrane [10]. In the boar, the epididymal protein
anti-agglutinin that is bound to spermatozoa acrosomes during maturation is released at
an early stage of capacitation; this release was attenuated in spermatozoa incubated in 5

or 10% (v/v) SP [11].

Green and Watson [9] demonstrated that the capacitation-like reaction was induced by
the cooling of boar spermatozoa to 5 C and was observed once the spermatozoa were re-
warmed to 39 C. Further, the addition of 20% (v/v) boar SP to cooled spermatozoa
suspensions resulted in a decreased percent of spermatozoa displaying the capacitation-

like reaction [12]. The addition of boar SP also reduced the percent of capacitated

16

spermatozoa when incubated in a capacitation-supporting media at 39 C [11].
Interestingly, it has been shown that the cold shock-induced membrane damage in ram
spermatozoa was reversed following incubation with a solution containing proteins
extracted from ram SP [13]. It is evident that SP has a role in the regulation of
capacitation. The objective of the present study was to examine the effect of

supplemental SP on the cooling-induced capacitation-like changes in boar spermatozoa.

Materials and Methods
All chemicals were of analytical grade. Unless otherwise stated, reagents were purchased
from Sigma Chemicals (St. Louis, MO) and solutions prepared with sterile deionized

water.

A pool of SP from four boars was accumulated for these studies. The sperm-rich fractions
of the ejaculates were centrifirged at 1000 x g for 10 min, and the pellet discarded. The
SP was stored in 15 mL conical tubes at —80 C until required. Prior to use, a tube was

thawed overnight in a humidified incubator at 39 C and 5% C02 in air.

Medium

The capacitation-supporting medium (li99) consisted of 87% (v/v) Medium 199
Hanks (Gibco Invitrogen Corporation, Grand Island, New York), 12% (v/v) heat-
activated fetal calf serum, 1% (v/v) penicillin-streptomycin, 2.3% (wt/vol) bovine serum

albumin, 3.05 mM D-glucose, 2.91 mM calcium lactate, and 0.91 mM sodium pyruvate,

17

with pH adjusted to 7.8. The li 99 was equilibrated overnight in a hunridified

incubator at 39 C and 5% C02 in air prior to use.

Semen collection and assessment

Ej aculates from three Yorkshire boars between 12 and 20 months of age were used for
this study. All boars were housed at the Swine Research Facility at Michigan State
University where their fertility had been proven. The sperm-rich fraction was collected
by a manual method into a thermally protected vessel and transported to the laboratory in
a 37 C water bath. Upon arrival to the laboratory, spermatozoa concentration was
evaluated using a Bright-Line Hemacytometer (Hausser Scientific, Horsham, PA). The

spermatozoa concentration in each treatment suspension was approximately 250 x 106

spermatozoa per mL.

Processing for non-cooling treatments (Appendix II)

A 5mL aliquot of each ejaculate was centrifuged at 23 C for 10 min at 600 x g. The
spermatozoa pellet was then resuspended in 5mL of li99 at 39 C and incubated at 39
C in a humidified incubator in 5% CO2 for a 100 min initial incubation. At the end of the
100 nrin initial incubation, each suspension was centrifuged for 10 min at 600 x g at 23
C. The pellet was then resuspended in 5mL of li99 at 39 C and immediately placed in
a humidified incubator at 39 C and 5% C02 in air for 10 min. This period of 10 min is
designated as the warming period. After the warming period, suspensions remained in the

incubator for an 8 h incubation period. The 100 min and 10 min periods were chosen to

18

allow for temporal comparisons with the cooling treatments (see below) and are based on

the protocol of Green and Watson [9].

Processing for cooling treatments (Appendix II)

A 5mL aliquot of the ejaculate was centrifuged at 23 C for 10 min at 600 x g, and the
spermatozoa pellet resuspended in 5mL of li99 at 39 C. The spermatozoa suspension
was then cooled to 5 C over 100 min in a Digital Temperature Control Electronic
Cooler/warmer (TR Enterprises Inc, Aberdeen, NC). At the end of the 100 min cooling
period, each suspension was centrifuged for 10 min at 600 x g in a refrigerated centrifuge
at 5 C. The pellet was then resuspended in 5mL of li99 at 5 C. The spermatozoa
suspensions were immediately placed in a humidified incubator at 39 C and 5% C02 in
air for a 10 min warming period; after which, they remained in the incubator for an 8 h

incubation period.

Chlortetracycline (CTC) staining analysis (Appendix II)

CTC staining analysis [14] was used to identify the progress of capacitation and
acrosome reaction of boar spermatozoa. For CTC staining, 20 ul of 500 uM CTC in a
chilled buffer of 20 mM Tris, 130 mM sodium chloride, and 5 mM L-cysteine prepared
the day of each treatment was added to a warm (37 C) microscope slide followed by 20
ul of spermatozoa suspension and nrixed. After 10 sec, spermatozoa were fixed with 10
ul of 12.5% glutaraldehyde in Tris buffer (pH 7.8) and mixed again. A cover slip was
applied, and the slide was compressed between bibulous papers to remove excess fluid

and maximize the number of spermatozoa lying flat on the slide prior to observation.

19

Prepared slides were placed in a black box until read within 2 h of preparation.
Spermatozoa were illuminated with a 100 W mercury bulb and examined under a Nikon
Optiphot microscope equipped with a 380-425 nm excitation filter, a 520 nm barrier

filter, and a 510 nm dichroic mirror (Nikon Co., Tokyo, Japan).

Slides were prepared for CTC staining analysis directly before (designated as ‘Pre’) and
afier (designated as ‘Post’) the 100 min initial incubation or cooling period; directly afier
the 10 nrin warming period (Hour 0); and at 1, 2, 4, 6, 7, and 8 h afier Hour 0. Two slides
were prepared for each sample, and 100 spermatozoa were counted on each slide. The
average of both slides was determined. Four fluorescent patterns were used to
demonstrate the progress of capacitation and the acrosome reaction. Fresh (F)
spermatozoa (uncapacitated) were characterized by a bright anterior region with a bright
acrosome cap, a relatively faint posterior region, and a dark half circle at the equatorial
region. Those with equally bright anterior and equatorial regions and a less bright
posterior region were also scored as F. Capacitated (B) spermatozoa were consistent with
a faint anterior region, a bright equatorial region, and a faint posterior region. Acrosome
reacted (AR) spermatozoa were characterized by a very faint anterior region, a slightly

bright equatorial region, and a faint posterior region.

Bis-Benzimide Hoechst No. 33258 staining analysis
For all treatments, Hoechst 33258 was used to determine effect of treatment on viability
of the spermatozoa. A 5 ul sample of chilled stock solution (200 uM Hoechst 33258) was

added to 100 pl of spermatozoa suspension and incubated for 15 min at 39 C and 5% C02

20

in the dark. Subsequently, 20 pl of stained spermatozoa suspension was added to a warm
(37 C) microscope slide and fixed with 5 pl 12.5% glutaraldehyde. A cover slip was
applied and the slide was compressed between bibulous papers. Prepared slides were
placed in a black box until read within 2 h of preparation. Spermatozoa were illuminated
with a 100 W mercury bulb and examined under a Nikon Optiphot microscope equipped

with a 365/ 10 nm excitation filter, a 400 nm barrier filter, and a 400 nm dichroic mirror

(Nikon Co., Tokyo, Japan).

Slides were prepared for staining analysis at Pre; Post; Hour 0; and at 2, 4, 6, and 8 h
after Hour 0. Two slides were prepared for each sample, and 100 spermatozoa were
counted on each slide. The average of both slides was determined. Spermatozoa

exhibiting bright blue fluorescence over the entire head were considered nonviable cells.

Treatments (Appendix III)

Seven treatments were performed with each of the three Yorkshire boars. All treatments
were replicated 3 times with boar Y27-10 and performed once each with boars Y43-11
and Y64-11 totaling 5 replicates per treatment. Where indicated, SP was added at 20%
v/v. This inclusion level was chosen based on previously demonstrated efficacy [11, 12].
Times for SP addition were chosen to examine its effect on the capacitation-like reaction
prior to or following cooling and its efficacy in reversing an established spermatozoa

capacitation reaction.

21

Three non-cooling treatments (Treatments 1, 2, and 3) were completed where the
spermatozoa suspensions were incubated at 39 C for an initial 100 min followed by
continued incubation for 8 h: Treatment 1 no SP was added; Treatment 2 SP was added
immediately prior to the 10 min warming period; Treatment 3 SP was added at Hour 2 of

the 8 h incubation.

Four treatments (Treatments 4, 5, 6, and 7) were completed where the spermatozoa
suspensions were cooled to 5 C over 100 min followed by warming and incubation:
Treatment 4 no SP was added; Treatment 5 SP was added immediately prior to the 10
rrrin warming period; Treatment 6 SP was added immediately before the cooling period,
and the pellet was re-suspended in li99 containing no SP after the cooling period;

Treatment 7 SP was added at Hour 2 of the 8 h incubation.

Statistical Analysis

Data were analyzed using Statistical Analysis Systems software (SAS Institute Inc., Cary,
NC). Percentage data for each response category (live, fresh, capacitated, and acrosome-
reacted) were transformed based on the arcsin of the square roots of the proportions prior
to analysis. Repeated measures AN OVA was conducted to determine treatment
differences at each time point separately for each of the response categories. Least-
squares means for each treatment and time were back-transformed for the purposes of

reporting point estimates and preparing figures.

22

Results

Viability assessment

The percent of viable spermatozoa decreased from 73% at time Pre to 60% at time Post.
Thereafter, the percent viable sperm remained relatively constant throughout the
experimental period with no significant difference between treatments (P>0.05; Figure

2.1).

Non-cooling treatments
In Treatment 1, SP was not included. The percent of F spermatozoa steadily decreased
from 59.6% to 10.9% over a total of 10 h of incubation. Concurrently, B spermatozoa

rose fi'om 39% to 58.8%, and AR increased from 1.4% to 30.3% (Figure 2.2).

In Treatment 2, SP was added at the end of the initial incubation period. The percent F
spermatozoa decreased from 58.1% to 46.6% while the percent B spermatozoa increased
fiom 39.4% to 50.3% by the end of the initial incubation period. The CTC analysis at
Hour 0 (57.3% F, 40.8% B) demonstrated that SP reversed capacitation within 10 min of
introduction (Figure 2.3). After reversal, the percent of F and B spermatozoa remained
relatively constant throughout the 8 h incubation period. The percent of spermatozoa that
had undergone an acrosome reaction increased fiom 2.5% to 11% during the

experimental period.

The changes in capacitation status observed in Treatment 3 were similar to those noted in

Treatment 1 until SP was added at Hour 2. At Hour 2, the percent F and B spermatozoa

23

were 32.4% and 62.3%, respectively, but were reversed to 36% and 59.6%, respectively,
at Hour 4 (Figure 2.4). The percent F and B remained relatively constant during the
remaining incubation period. The percent of spermatozoa that had undergone the

acrosome reaction increased throughout the treatment period from 1.4% to 9.75%.

Cooling treatments

In Treatment 4, no SP was included. The percent F and B spermatozoa remained
relatively unchanged during the 100 min cooling period. However, a sharp decrease in F
spermatozoa from 58.7% to 35.1% and an increase in B spermatozoa from 36.9% to
59.2% occurred upon warnring (Figure 2.5). Fresh spermatozoa continued to decline to

l 1.2% while the percent B spermatozoa increased slowly to 63.7% at Hour 8. Acrosome
reacted spermatozoa increased steadily throughout the treatment period from 1.8% at Pre

to 25.1% at Hour 8.

In Treatment 5, SP was added after the 100 min cooling period (Figure 2.6). The percent
F and B spermatozoa were initially 57.9% and 39%, respectively, and remained relatively
constant throughout the treatment period changing only to 47.8% and 39.8%,
respectively, by the end of the 8 h incubation. During the treatment period acrosome

reacted spermatozoa increased from 3.1% to 12.4%.

In Treatment 6, SP was included in the spermatozoa suspension during cooling but
removed for the incubation period. This resulted in the percent F and B spermatozoa

remaining relatively constant throughout the experimental period (Figure 2.7). Percent

24

acrosome reacted spermatozoa increased from 1.4% to 10.9% during the experimental

period.

In Treatment 7, changes in percent F and B spermatozoa were similar to those observed
in Treatment 4 until the addition of SP at Hour 2. The percent F and B spermatozoa were
29.5% and 64.4%, respectively, at Hour 2 but were reversed to 59.5% and 37.8%,
respectively, at Hour 4 (Figure 2.8). Acrosome reacted spermatozoa increased from

0.75% to 6.0% during the experimental period.

Discussion

Our findings confirmed that SP reduced the percent capacitated boar spermatozoa when
incubated in a capacitation-supporting media at 39 C [11,15] and extend this observation
to demonstrate that capacitation was reversed within 10 min of introduction of SP. The

addition of SP also reversed capacitation that had been well established after several

hours of incubation.

The present data support previous studies [9, 12, 16] in that rate controlled cooling of
boar spermatozoa to 5 C resulted in a capacitation-like reaction. This effect was not
evident until the spermatozoa were re-warmed to ambient temperature suggesting a
possible involvement of enzymes. The addition of 20% (v/v) SP to the spermatozoa
suspension before cooling or after cooling prevented the cooling-induced capacitation-

like reaction for a period of at least 8 h. The addition of SP after the warming period or

25

after 2 h of incubation resulted in the reversal of the cooling-induced capacitation-like

reaction.

The data presented are limited to a SP inclusion level of 20% v/v. However, additional
treatments that examined the effect of inclusion of 10 or 40% (v/v) SP have indicated
almost identical responses (data not shown) and also support previous studies [17, 18].
Therefore, the effect of SP appears to be independent of the time of addition, percent
inclusion (10%, 20%, or 40%), or the type of capacitation reaction (time-dependant or

cooling-induced).

Seminal plasma, produced by the accessory glands of the male reproductive system, has
several reported effects on spermatozoa in vitro: modulating capacitation in epididymal
bull spermatozoa [19, 20], reversing capacitation in fresh ejaculated boar spermatozoa
[11, 15], and improvement of motility of spermatozoa (boar: [21, 22]; ram, bull, boar:
[16]). Further, spermatozoa maintained in their own SP prior to its removal for extension
intended for cryopreservation demonstrated increased resistance to cold shock [23, 24].
The composition of SP includes inorganic compounds, amino acids, peptides, as well as
low and high molecular-weight proteins [25]. For artificial insemination, SP is highly

diluted with an isotonic extension media.

Serrrinal plasma proteins have been postulated to act as decapacitation factors that prevent

a premature acrosome reaction [26], and their removal may initiate capacitation. Bovine

SP proteins (BSP-A1/-A2, BSP-A3, BSP-30-kDa) bind to choline phospholipids on the

26

spermatozoa membrane at ejaculation subsequently interacting with high-density
lipoproteins (HDL) and heparin-like glycosaminoglycans (GAG) in the female
reproductive tract promoting capacitation [27]. Bovine spermatozoa that have been
exposed to SP possess more binding sites for heparin than spermatozoa obtained from the
caudal epididymis [20]. In boar, the heparin-binding protein p31 has been characterized
and identified to bind to lipids containing the phosphorylcholine group on the
spermatozoa membrane at ejaculation [28]. Recently, when FT spermatozoa were
incubated with the GAG hyaluronan (HA) an increase in the percent of B spermatozoa
was demonstrated [15]. This suggested a capacitation-promoting action of HA. Heparin
[29, 30] and HDL [27] have also been shown to promote capacitation.
Glycosaminoglycans have been localized in pre- and post-ovulatory oviducts of swine
reproductive tracts [31] and may play a role in the removal of SP proteins on the
spermatozoa membrane resulting in capacitation both in vitro and in vivo. As the present
study has demonstrated, by adding back a percentage of SP to the spermatozoa,
capacitation and the capacitation-like reaction were either prevented or reversed. This
prevention and reversal suggests that SP maintained proteins on the spermatozoa
membrane or replaced SP proteins previously lost to the membrane. Further
investigations are needed to identify the component(s) (i.e. possible proteins) of the SP

that result in these outcomes.

An important implication of the ability to reverse capacitation is the possibility to

significantly extend the fertile life of cryopreserved spermatozoa. An increased fimctional

spermatozoa reservoir within the oviduct as a result of the addition of SP to extended FT

27

spermatozoa would improve sow fertility when this technology is employed. The ability
to successfully use FT spermatozoa would allow the swine industry to gain the maximum
potential in terms of efficiency of spermatozoa use. More importantly, use of FT
spermatozoa would allow greater protection of herd health by permitting spermatozoa to

be quarantined prior to use in the breeding herd.

28

References

1.

10.

11.

12.

Johnson, L.A., Weitze, K.F., Fiser, P., Maxwell, W.M.C. Erratum to "Storage of
boar semen": [Anim. Reprod. Sci. 2000;62:143-72]. Anim Reprod Sci, 2000. 64:
133-4.

F azeli, A., Duncan, A.E., Watson, P.F., Holt, W.V. Sperm-oviduct interaction:
Induction of capacitation and preferential binding of uncapacitated spermatozoa
to oviductal epithelial cells in porcine species. Biol Reprod, 1999. 60: 879-86.

Tienthai, P., Johannisson, A., Rodriguez-Martinez, H. Sperm capacitation in the
porcine oviduct. Anim Reprod Sci, 2004. 80: 131-46.

Holt, W., Medrano, A. Assessment of boar sperm function in relation to fi'eezing
and storage. J Reprod Fertil Suppl, 1997. 52: 213-22.

Bailey, J ., Bilodeau, J ., Connier, N. Semen cryopreservation in domestic animals:
a damaging and capacitating phenomenon. J Androl, 2000. 21: 1-7.

Harrison, R. Capacitation mechanisms, and the role of capacitation as seen in
eutherian mammals. Reprod Fertil Dev, 1996. 8: 581-94.

Yanagimachi, R. Mammalian Fertilization. In: Knobil E, Neill JD (eds): The
Physiology of Reproduction. New York: Raven Press, 1994; 189-317.

Visconti, P., Kopf, G. Regulation of protein phosphorylation during sperm
capacitation. Biol Reprod, 1998. 59: 1-6.

Green, C., Watson, P. Comparison of the capacitation-like state of cooled boar
spermatozoa with true capacitation. Reproduction, 2001. 122: 889-98.

Watson, P., Green, C.E. Cooling and capacitation of boar spermatozoa: What do
they have in common? In: Boar Semen Preservation IV, 2000. 53-60.

Harayama, H., Magaree, S.F., Kunze, E., Shidara, 0., Iwamoto, E., Arikawa, S.,
Miyake, M., Kato, S., Hammerstedt, R.H. Changes in epididymal protein anti-
agglutinin on ejaculated boar spermatozoa during capacitation in vitro. Reprod
Fertil Dev, 1999. 11: 193-9.

Kaneto, M., Harayama, H., Miyake, M., Kato, S. Capacitation-like alterations in
cooled boar spermatozoa: assessment by the chlortetracycline staining assay and
irnmunodetection of tyrosine-phosphorylated sperm proteins. Anim Reprod Sci,
2002. 73: 197-209.

29

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Barrios, B., Perez Pe, R., Gallego, M., Tato, A., Osada, J., Muino Blanco, T.,
Cebrian Perez, J .A. Seminal plasma proteins revert the cold-shock damage on ram
sperm membrane. Biol Reprod, 2000. 63: 1531-7.

Lin, C. Regulation of sperm capacitation. Doctoral Thesis. Michigan State
University, East Lansing, Michigan 1996.

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

Maxwell, W., Johnson, L. Chlortetracycline analysis of boar spermatozoa after
incubation, flow cytometric sorting, cooling, or cryopreservation. Molec Reprod
Dev, 1997. 46: 408-18.

Maxwell, W.M.C., Long, C.R., Johnson, L.A., Dobrinski, J .R., Welch, OR. The
relationship between membrane status and fertility of boar spermatozoa after
cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil
Dev, 1998. 10: 433-40.

Maxwell, W.M.C., Johnson, L.A. Physiology of spermatozoa at high dilution
rates: The influence of seminal plasma. Theriogenology, 1999. 52: 1353-62.

Lee, C., Handrow, R.R., Lenz, R.W., Ax, R.L. Interactions of seminal plasma and
glycosaminoglycans on acrosome reactions in bovine spermatozoa in vitro.
Garnete Res, 1985. 12: 345-55.

Miller, D., Winer, M., Ax, R. Heparin-binding proteins from seminal plasma bind
to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod, 1990.
42: 899-915.

Okamura, N., Tajima, Y., Soejirna, A., Masuda, H., Sugita, Y. Sodium
bicarbonate in seminal plasma stimulates the motility of mammalian spermatozoa
through direct activation of adenylate cyclase. J Biol Chem, 1985. 260: 9699-
705.

Rodriguez-Martinez, H., Ekstedt, E., Einarsson, S. Acidification of epididymal
fluid in the boar. Int J Androl, 1990. 13: 238-43.

Tamuli, M.K., Watson, P.F. Cold resistance of live boar spermatozoa during
incubation after ejaculation. Vet Rec, 1994. 135: 160-2.

Catt, S.L., O’Brien, J .K., Maxwell, W.M.C., Evans, G. Assessment of ram and

boar spermatozoa during cell-sorting by flow cytometry. Reprod Dom Anim,
1997. 32: 251-8.

30

25.

26.

27.

28.

29.

30.

31.

Pursel, V., Johnson, L., Schulman, L. Effect of dilution, seminal plasma and
incubation period on cold shock susceptibility of boar spermatozoa. J Anim Sci,
1973. 37: 528-31.

Oliphant, G., Reynolds, A., Thomas, T. Sperm surface components involved in
the control of the acrosome reaction. Am J Anat, 1985. 174: 269-83.

Therien, I., Soubeyrand, S., Manjunath, P. Major proteins of bovine seminal
plasma modulate sperm capacitation by high-density lipoprotein. Biol Reprod,
1997. 57 : 1080-88.

Calvete, J ., Raida, M., Gentzel, M., Urbanke, C., Sanz, L., Topfer Petersen, E.
Isolation and characterization of heparin- and phosphorylcholine-binding proteins
of boar and stallion seminal plasma. Primary structure of porcine pBl. F EBS
Letters, 1997. 407: 201-6.

Therien, 1., Bleau, G., Manjunath, P., Soubeyrand, S. Phosphatidylcholine-
binding proteins of bovine seminal plasma modulate capacitation of spermatozoa
by heparin. Biol Reprod, 1995. 52: 1372-9.

Kim, N., Day, B.N., Lim, J .G., Lee, H.T., Chung, K.S. Effects of oviductal fluid
and heparin on fertility and characteristics of porcine spermatozoa. Zygote, 1997.
5: 61-5.

Tienthai, P., Kjellen, L., Pertoft, H., Suzuki, K., Rodriguez-Martinez, H.
Localization and quantitation of hyaluronan and sulfated glycosaminoglycans in
the tissues and intraluminal fluid of the pig oviduct. Reprod F ertil Dev, 2000. 12:
173-82.

31

 

 

 

. l
‘ Figure 2.1 i
r 80 l
l
E 60 ~ ‘
c2:
:3
.5
°\. 40
Q l
i i 5
l 20 i l
i i E
1 l l
l l l
5 o l‘ "’7‘ _‘"_i _—— ‘ _ T “ _‘ ‘_ l‘ __ _‘ _‘7_— _— ‘T‘ '“k _ _—l — ‘ j)
g Pre Post Hr0 Hr2 Hr4 Hr6 Hr8 l
l Time (Hours) l
l

l__+,_ __.__+ ..__L E.‘____,__ __, -____, .__, ‘_ WE __ __ __* _ ‘, ___ ._E

 

In Figures 2.1 through 2.8, time points ‘Pre’ and ‘Post’ represent readings prior to and
after the lOO-min pre-incubation or cooling, respectively.

Figure 2.1: Effect of all treatments on viability of spermatozoa. Each point represents the
mean :1: SE of all determinations.

32

 

 

 

""166 I ” ’ ‘ ’ " I

Frgure 2.2 I

I I

so i .

I |

~43 I

‘3 60* . I

E .

|

g i... I

,3- 40 -

g I
20 4 j 4:

O l . 7 I» l 7* ’— T. T if 7 T '7? * T“ 7 ~ Ft I T“ " W-T _. 7 -‘ T ’ 'I _ W" 'h I

Pre Post HrO Hr1 Hr2 Hr4 Hr6 Hr7 Hr8 I

Timed-lotus) I

 

In Figures 2.2 through 2.8, A represent _Eresh spermatozoa, I represent Capacitated
spermatozoa (1}), and 0 represent Acrosome Reacted spermatozoa Each point represents

the average of 5 counts t SE.

Figure 2.2: Pre-incubation and incubation in li99 containing 0% SP.

33

 

80

60*

40-

% Sperm Population

20“

 

 

 

I Pre

L _ A ,_,, A ___ _ _ A __.

Figure 2.3: Pre-incubation with 0% SP and then incubation in li99 containing 20%

SP.

 

 

r J

Time (Hours)

 

34

Hr1 Hr2 Hr4 Hr6 Hr7

 

0 I — ——-.——-r~—-

Post Hr 0

 

Hr8

 

 

,AA __A _A_ A .A I

 

 

 

 

100 I *—
Figure 2.4
80 . I
I
I i
5’ I
'5 60-
E
e <
0
,3- 40-
,\°
20-
I o-I- MA
I Pre Post Hr0 Hr1 Hr2 Hr4 Hr6 Hr7 Hr8
I Time(Hours)

 

Figure 2.4: Pre-incubation and initial incubation in li99 containing 0% SP with 20%
SP added at Hr 2.

35

 

 

___.

% Sperm Population

80
6O 4
40 i

20 'I

 

0 I

Figure 2.5

_A ”_l"

o
” Thfiw _fil"‘ "WT“ T ”W

Pre Post HrO Hr1 Hr2 Hr4 Hr6 Hr7 Hr8

Time (Hours)

:—-e "

“H" ‘7 w” ‘T ’T—__ T '_H T

 

l

 

 

Figure 2.5: Cooling/warming and incubation of spermatozoa in li99 containing 0%

SP.

36

 

 

A A, I
Figure 2.6 I
I 80f I
I
a I
E 60
2. I
a
E
‘3' 40‘ W
,\°
I
20I

 

 

Pre Post HrO Hr1 Hr2 Hr4 Hr6 Hr7 Hr8
Time (Hours)

AM
I

l
' T’ __“_T ‘M‘T‘ __ l l l f - _TMT"—_ “_T__ ___—I
J

 

 

Figure 2.6: Cooling in li99 containing 0% SP with warming and incubation in
li99 containing 20% SP.

37

 

 

 

“ so I

3 I
a 60 A Mr: I

a.
o I
°~ I
E I
‘2‘ 4O 4 W I
°‘° I
20 - I
O I 7‘ T‘TTHT fi—“GT‘M‘” 7*" ihflT—m ’fi'h' t" "T ”hf-7 [*7 *7 _Tm’” — l "T “7“” 7

Pre Post HrO Hr1 Hr2 Hr4 Hr6 Hr7 Hr8
Time (Hours)

 

 

I

 

Figure 2.7: Cooling in li99 containing 20% SP with warming and incubation in
mM 199 containing 0% SP.

38

I ' ‘166 I '
Figure 2.8 I
I 80 I

60

40 ~

% Sperm Population

 

 

 

 

 

 

Figure 2.8: Cooling/warming in li99 containing 0% SP with 20% SP added at Hr 2 of
incubation.

39

CHAPTER 3

The effect of the addition of seminal plasma and the duration of incubation on the post-

thaw capacitation status of frozen-thawed boar spermatozoa

Abstract

The present study investigated the effects of adding either 0%, 10%, or 20% (v/v) boar
seminal plasma (SP) at thawing to fiozen-thawed boar spermatozoa on the status of
capacitation. Chlortetracycline staining analysis was used to determine the capacitation
and acrosome reaction status of the spermatozoa. One hour after the addition of 10 or
20% (v/v) SP the percent of capacitated spermatozoa decreased from 59.7% to 30.3% and
from 59.5% to 26.8%, respectively, (P<0.001). The effects of SP on the reversal of
capacitation were determined to be complete by 30 min after SP addition to the
spermatozoa. These results indicated that SP is able to reverse cryocapacitation of frozen

boar spermatozoa within short period of time.

Introduction

The routine use of frozen-thawed (FT) spermatozoa by the swine industry would offer
such benefits as enhanced biosecurity with the safe introduction of new genetics into a
herd, decreased risk for the dissemination of pathogens capable of transmission via
semen, and the preservation of superior genetics. Transmission of viruses such as porcine
reproductive and respiratory syndrome virus can occur through boar semen [1]. The use
of FT spermatozoa would allow for the identification and regular use of non-infected

boars.

40

Unfortunately, the use of FT spermatozoa results in lower farrowing rates and smaller
litter sizes compared to the use of fresh-extended spermatozoa [2, 3]. This effect relates,
in part, to cryocapacitation at thawing. The term cryocapacitation describes the
capacitation-like process that occurs during cryopreservation and is expressed at the
subsequent thawing [4-6]. Capacitated spermatozoa show a decreased ability to bind to
the oviductal epithelial cells both in vitro as well as in the female reproductive tract [7, 8]
resulting in a decreased number of spermatozoa in the sperm reservoir available to
fertilize the ova. Presumably, cryocapacitated spermatozoa demonstrate a similar

inability to bind to the oviductal epithelial cells.

Capacitation is a sequential process that may involve the removal of protein
decapacitation factors from the spermatozoa membrane that were acquired in the
epididymus or at ejaculation [9]. It has been shown that the cold shock-induced
membrane damage in ram spermatozoa was reversed following incubation with a solution
containing proteins extracted fi'om ram seminal plasma (SP) [10]. In the boar, the
epididymal protein anti-agglutinin that is bound to spermatozoa acrosomes during
maturation is released at an early stage of capacitation, but this loss was attenuated in
spermatozoa incubated in 5 or 10% (v/v) SP [11]. The addition of boar SP also inhibited
the capacitation of spermatozoa when incubated in a capacitation-supporting media at 39

C[11,12].

41

Our previous work has demonstrated that the addition of 20% SP prevents fresh
spermatozoa from undergoing capacitation in a capacitation-supporting environment.
Spermatozoa that had already undergone the capacitation-like process experienced a
reversal. The objective of the present study was to examine the effect of 10 and 20%

(v/v) SP on cryocapacitation of FT boar spermatozoa.

Materials and Methods
All chemicals were of analytical grade. Unless otherwise stated, reagents were purchased
from Sigma Chemicals (St. Louis, MO) and solutions prepared with sterile deionized

water.

A pool of SP from four boars was accumulated for these studies. The spenn-rich fractions
of the ejaculates were centrifuged at 1000xg for 10 min, and the pellet discarded. The SP
was stored in 15mL conical tubes at —80 C until required. Prior to use, a tube was thawed

overnight in a humidified incubator at 39 C and 5% C02 in air.

Medium

To assure the proper environment for capacitation a capacitating-supporting medium
(li99) was used. The medium contained three ingredients that support capacitation in
vitro: bicarbonate, bovine serum albumin, and calcium [13, 14]. The li99 consisted of
87% (v/v) Medium 199 Hanks (Gibco Invitrogen Corporation, Grand Island, New York),
12% (v/v) heat-activated fetal calf serum, 1% (v/v) penicillin-streptomycin, 2.3%

(wt/vol) bovine serum albumin, 3.05 mM D-glucose, 2.91 mM calcium lactate, and 0.91

42

mM sodium pyruvate, with pH adjusted to 7.8. The li99 was equilibrated overnight in

a humidified incubator at 39 C and 5% C02 in air prior to use.

Spermatozoa processing for treatments

Frozen semen from three Yorkshire boars was purchased from lntemational Boar Semen
(Eldora, IA) in 5mL maxi-straws. Straws were held at room temperature for 7 see before
being submerged horizontally into a 42 C water bath for 43 sec. Immediately after
thawing, the straws were emptied into 15mL conical tubes and centrifuged for 10 min at
600xg. The supernatant was discarded, and 5mL of li99 at 39 C was added. The
spernnatozoa suspension was then centrifuged for 5 min at 600xg. Again the supernatant
was discarded, and the spermatozoa pellet was resuspended in 5mL of li99 with or
without SP at 39 C. A bright-line microscopic assessment (400x) of progressive motility
was performed directly after thawing and repeated after the final centrifugation. A
percent motile was assigned within 10%. The spermatozoa suspension remained in a

humidified incubator in 5% C02 in air for the remainder of the treatment period.

Treatments

Four treatments were performed with each of the three boars. In Treatments 1, 2, and 3
the spermatozoa suspensions were incubated at 39 C for 8 h after thawing. In Treatment
4 the spermatozoa suspension was incubated at 39 C for l h after thawing. No SP was
added in Treatment 1. Treatments 2 and 4 included 10% (v/v) SP added after the final

centrifugation. Treatment 3 included 20% (v/v) SP added after the final centrifugation.

43

Chlortetracycline (CTC) staining analysis (Appendix H)

The CTC staining assay is based on the transfer of neutral, uncomplexed CTC across the
spermatozoa membrane resulting in the fluorescent patterns characteristic of the various
transitional phases [15] that spermatozoa display (flesh, Icapacitated, acrosome reaction).
F rozen-thawed spermatozoa display an increase in the CTC fluorescence pattern

corresponding to the capacitated state [16-18].

For CTC staining [19], 20 pl of 500 pM CTC in a chilled buffer of 20 mM Tris, 130 mM
sodium chloride, and 5 mM L-cysteine prepared the day of each treatment was added to a
warm (37 C) microscope slide followed by 20 pl of spermatozoa suspension and mixed.
After 10 sec, spermatozoa were fixed with 10 pl of 12.5% glutaraldehyde in Tris buffer
(pH 7.8) and mixed again. A cover slip was applied, and the slide was compressed
between bibulous papers to remove excess fluid and maximize the number of
spermatozoa lying flat on the slide prior to observation. Prepared slides were placed in a
black box until read within 1 h of preparation. Spermatozoa were illuminated with a 100
W mercury bulb and examined under a Nikon Optiphot microscope equipped with a 380-

425 nm excitation filter, a 520 nm barrier filter, and a 510 nm dichroic mirror (Nikon Co.,

Tokyo, Japan).

For Treatments 1, 2, and 3 slides were prepared for CTC staining analysis directly after
the final centrifugation and resuspension (Hour 0); and at 1, 2, 4, 6, 7, and 8 h after Hour
0. For Treatment 4 slides were prepared at Hour 0, 15 min 30 min, 45 min, and at Hour 1.

Two slides were prepared for each sample, and 100 spermatozoa were counted on each

slide. The average of both slides was determined. Fluorescent patterns were used to
demonstrate the progress of capacitation and the acrosome reaction. Fresh (F)
spermatozoa (uncapacitated) were characterized by a bright anterior region with a bright
acrosome cap, a relatively faint posterior region, and a dark half circle at the equatorial
region. Those with equally bright anterior and equatorial regions and a less bright
posterior region were also scored as F. Capacitated (B) spermatozoa were consistent with
a faint anterior region, a bright equatorial region, and a faint posterior region. Acrosome
reacted (AR) spermatozoa were characterized by a very faint anterior region, a slightly

bright equatorial region, and a faint posterior region.

Statistical Analysis

Data were analyzed using Statistical Analysis Systems software (SAS Institute Inc., Cary,
NC). Repeated measures AN OVA was conducted to determine treatment differences at
each time point separately for each of the response categories (flesh, capacitated,
acrosome reacted) and for percent motile. Least-squares means for each treatment and

time were back-transformed for the purposes of reporting point estimates and preparing

figures.

Results
The average percent of progressive motile spermatozoa was 60% at post-thaw and 55%
after the final centrifugation. No significant differences (P>0.05) were observed between

treatment groups or times.

45

In Treatment 1, where no SP was included, the percent F spermatozoa remained
relatively unchanged throughout the 8 h incubation period only decreasing flom 31.2% to
30.5%. The percent B spermatozoa decreased flom 59.7% to 48.3% while the percent AR

increased from 9.2% to 21.2% (Figure 3.1).

In Treatment 2, where 10% SP was included, there was an increase in the percent of F
spermatozoa flom 31.7% at Hour 0 to 61.2% at Hour 1. Conversely, there was a decrease
in the percent of B pattern spermatozoa flom 59.7% at Hour 0 to 30.3% at Hour 1 (Figure
3.2). After reversal, the percent F and B spermatozoa remained relatively constant
throughout the remainder of the incubation period. The percent AR spermatozoa

increased flom 8.7% at Hour 0 to 13% at Hour 8.

In Treatment 3, where 20% SP was included, changes in percent F, B, and AR
spermatozoa were similar to those observed in Treatment 2 (Figure 3.3). The percent F
spermatozoa were 31.7% and 63.3% at Hours 0 and 1, respectively. The percent B
spermatozoa decreased flom 59.5% at Hour 0 to 26.8% at Hour 1. The percent AR

spermatozoa increased flom 8.8% to 12.7% during 8 h of incubation.

In Treatment 4, where 10% SP was included, the percent F spermatozoa increased flom
29%, to 55%, to 64% while the percent B spermatozoa decreased flom 64.5%, to 39.5%,
to 30%, at 0 min, 15 nrin, and 30 rrrin respectively. Percentages remained relatively

unchanged over the next 30 min of incubation (Figure 3.4). The percent spermatozoa that

46

had undergone the acrosome reaction remained relatively unchanged over the 1 h

incubation, increasing from 6.5% to 7.5%.

Discussion

Our findings confirmed that the majority of FT spermatozoa are cryocapacitated upon
thawing [5, 20, 21]. The present data also supports our previous studies with cooled, flesh
spermatozoa [12] in that 20% (v/v) SP was capable of reversing the capacitation-like
process. We now extend those observations to include the observation that 10% (v/v) SP
was capable of reversing cryocapacitation and that the effect of SP was evident within 15
rrrin of its addition to the spermatozoa and complete by 30 min. We also determined that
centrifugation and resuspension of the FT spermatozoa did not significantly alter their

motility.

The entire sequence of events involved in capacitation is not fully understood; however,
several major molecular changes have been linked to capacitation. In boar spermatozoa,
bicarbonate and exposure to C02 cause a reversible increase in plasma membrane lipid
disorder resulting in an increased concentration of cholesterol at the apical ridge [22].
This increased apical concentration of cholesterol may be an intermediate capacitation
state that allows for albumin-mediated cholesterol extraction [23]. Further membrane
destabilization occurs with the loss of proteins and carbohydrates flom the plasma
membrane. The membrane potential shifts with the increased membrane fluidity causing

an influx of calcium ions that drive the CAMP-protein phosphorylation [9].

47

The observations in the late 1950’s that SP caused a reversible inhibition of fertilizing
ability in rabbit spermatozoa [24] and ultracentrifugation of rabbit SP sedirnented a
decapacitation factor [25] have lead to the concept that capacitation involves the removal
of coating macromolecules flom the spermatozoa plasma membrane. The loss of proteins
and carbohydrates from the plasma membrane during capacitation may be the loss of the
decapacitation factors resulting in a reduced ability to bind to the oviductal epithelial
cells. In bull, cryopreservation resulted in a 70 to 80% decrease in sperrn-bound bovine
seminal plasma (BSP) proteins [26]. These proteins are responsible for controlling

cholesterol efflux and are mediators in spermatozoa-oviduct binding [27].

The results of this study predict that the addition of SP to FT boar spermatozoa would
prevent and/or reverse cryocapacitation, thereby, potentially improving fertility after
artificial insemination. Greater utilization of FT spermatozoa would allow the swine
industry to maximize the efficiency of genetically superior boars. Swine farms could
introduce superior genetics, disease-flee, without having to quarantine new boars.

Through increased biosecurity the industry-wide risks to swine health would decrease.

48

References

1.

10.

ll.

12.

Hennings, C., Holler, L., Benfield, D., Nelson, E. Detection and duration of
porcine reproductive and respiratory syndrome virus in semen, serum, peripheral
blood mononuclear cells, and tissues flom Yorkshire, Hampshire, and Landrace
boars. J Vet Diagn Invest, 2001. 13: 133-42.

Almlid, T., Hofrno, P. A brief review of flozen semen application under
Norwegian AI service conditions. Reprod Dom Anim, 1996. 31: 169-73.

Reed, H. Current use of flozen boar semen-future need of frozen boar semen, in
Deep freezing of boar semen, J. LA and L. K, Editors. 1985, Swedish University
of Agricultural Science: Uppsala. 225-37.

Curry, M.R., Kleinhans, F .W., Watson, P.F. Measurement of the Water
Permeability of the Membranes of Boar, Ram, and Rabbit Spermatozoa Using
Concentration-Dependent Self-Quenching of an Entrapped Fluorophore*1.
Cryobiology, 2000. 41: 167-73.

Bailey, J ., Bilodeau, J ., Connier, N. Semen cryopreservation in domestic animals:
a damaging and capacitating phenomenon. J Androl, 2000. 21: 1-7.

Watson, RE The causes of reduced fertility with cryopreserved semen. Anim
Reprod Sci, 2000. 60-61: 481-92.

Medeiros, C., Forell, F ., Oliveira, A.T.D., Rodrigues, J .L. Current status of sperm
cryopreservation: why isn't it better? Theriogenology, 2002. 57: 327-44.

Tienthai, P., Johannisson, A., Rodriguez-Martinez, H. Sperm capacitation in the
porcine oviduct. Anim Reprod Sci, 2004. 80: 131—46.

Watson, P., Green, C.E. Cooling and Capacitation of Boar Spermatozoa: What do
They Have in Common? In: Boar Semen Preservation IV, 2000. 53-60.

Barrios, Perez Pe, R., Gallego, M., Tato, A., Osada, J ., Muino Blanco, T.,
Cebrian Perez, J .A. Seminal plasma proteins revert the cold-shock damage on ram
sperm membrane. Biol Reprod, 2000. 63: 1531-7.

Harayama, H., Magargee, S.F., Kunze, E., Shidara, 0., Iwamoto, E., Arikawa, S.,
Miyake, M., Kato, S., Hammerstedt, R.H. Changes in epididymal protein anti-
agglutirrin on ejaculated boar spermatozoa during capacitation in vitro. Reprod
Fertil Dev, 1999. 11: 193-9.

Vadnais, M.L., Kirkwood, R., Sprecher, D.J., Tempelman, D., Chou, K. Effect of
cooling and seminal plasma on the capacitation status of boar sperm as

49

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

determined by chlortetracycline staining. Submitted to Anim Reprod Sci, June
2004.

Harrison, R. Capacitation mechanisms, and the role of capacitation as seen in
eutherian mammals. Reprod Fertil Dev, 1996. 8: 581-94.

Visconti, P., Kopf, G. Regulation of protein phosphorylation during sperm
capacitation. Biol Reprod, 1998. 59: 1-6.

Ward, C., Storey, B. Determination of the time course of capacitation in mouse
spermatozoa using a chlortetracycline fluorescence assay. Dev Biol, 1984. 104:
287-96.

Connier, N., Sirard, M.A., Bailey, J .L. Premature capacitation of bovine
spermatozoa is initiated by cryopreservation. J Androl, 1997. 18: 461-8.

Gillan, L., Evans, G., Maxwell, W. Capacitation status and fertility of flesh and
flozen-thawed ram spermatozoa Reprod Fertil Dev, 1997. 9: 481-7.

Perez, L.J., Valcarcel, A., de las Heras, M.A., Moses, D., Baldassarre, H.
Evidence that flozen/thawed ram spermatozoa show accelerated capacitation in
vitro as assessed by chlortetracycline assay. Theriogenology, 1996. 46: 131-40.

Lin, C. Regulation of sperm capacitation. Doctoral Thesis. Michigan State
University, East Lansing, Michigan 1996.

Maxwell, W., Johnson, L. Chlortetracycline analysis of boar spermatozoa after
incubation, flow cytometric sorting, cooling, or cryopreservation. Molec Reprod
Dev, 1997. 46: 408-18.

Watson, P.F. Cooling of boar spermatozoa and fertilizing capacity. Reprod Dom
Anim, 1996.31: 135-40.

Harrison, R.A., Ashworth, P., Miller, N. Bicarbonate/C02, an effector of
capacitation, induced a rapid and reversible change in the lipid architecture of
boar sperm plasma membranes. Molec Reprod Dev, 1996. 45: 37 8-91 .

F lesch F.M., Brouwers, J ., Nievelstein, P., Verkleij, A.J., van Golde, L.M.G.,
Gadella, B.M., Bicarbonate stimulated phospholipid scrambling induces

cholesterol redistribution and enables cholesterol depletion in the sperm plasma
membrane. J Cell Biol, 2001. 114: 3543-55.

Chang, M. A detrimental effect of seminal plasma on the fertilizing capacity of
sperm. Nature, Lond, 1957. 179: 258-9.

50

25.

26.

27.

Bedford, J ., Chang, M. Removal of decapacitation factor flom seminal plasma by
high speed centrifugation. Am J Phys, 1962. 202: 179-81.

Nauc, V., Manjunath, P. Radioirnmunoassays for bull seminal plasma proteins
(BSP-All-A2, BSP-30 Kilodaltons), and their quantification in seminal plasma
and sperm. Biol Reprod, 2000. 63: 1058-66.

Therien, 1., Soubeyrand, S., Manjunath, P. Major proteins of bovine seminal
plasma modulate sperm capacitation by high-density lipoprotein. Biol Reprod,
1997. 57: 1080-8.

51

% Sperm Population

 

 

 

 

 

 

 

 

In Figures 3.1 through 3.4, A represent Fresh spermatozoa, I represent Capacitated (B)
spermatozoa, and 0 represent Acrosome Reacted spermatozoa Each point represents the
average of 6 counts i SE.

Figure 3.1: Frozen spermatozoa thawed in li99 containing 0% SP.

52

 

 

 

 

 

 

 

 

100 *
anure 3.2
E L I T I I
a: 60- I T T ‘1
9°.
E
5- 4o
o\° { ‘H
20 ~
. a ALA ._ -—-r; :t/n
; Hr0 Hr1 Hr2 Hr4 Hr6 Hr7 Hr8
L Time(Hours)

 

Figure 3.2: Frozen spermatozoa thawed in li99 containing 10% SP.

53

I 100 ~

 

 

 

 

 

Figure 3.3

I I
g T M I

T I I

g 60 —‘ ‘f f I I

a. I

g I

g 40 -« I

,- '

i I
I 20- I + N/l I
I . -————-~-— _A W I

 

Figure 3.3: Frozen spermatozoa thawed in li99 containing 20% SP.

54

I 100

 

 

 

 

 

 

 

Figure 3.4
80
l J
2. 6° " I 1
:3
E
g 40
.\° {\I
I 20
I ‘5 V + 4cm ’9
I OI - - w- _, A--- A-
I HrO Hr0.25 Hr0.50 Hr0.75 Hr1
I Time(Hour)

I A A A AA A A

Figure 3.4: Frozen spermatozoa thawed in li99 containing 10% SP with readings

taken at 15 min intervals over 1 h.

55

CHAPTER 4

The effects of egg yolk fleezing extender, incubation temperature, and the addition of
seminal plasma on cryocapacitation of flozen-thawed boar spermatozoa as determined by
chlortetracycline analysis

Abstract

The addition of seminal plasma (SP) to flozen-thawed (FT) spermatozoa separated from
the egg yolk-based freezing extender decreased the percent of capacitated spermatozoa
within 30 min of its addition. The present study examined the effect of 10% (v/v) boar SP
addition to FT boar spermatozoa extended in either X-cell thawing extender with or
without the egg yolk-based fleezing extender or in phosphate buffered saline (PBS) at
either 17 C or 39 C. No effects of SP resulted from addition to spermatozoa when the egg
yolk-based fleezing extender was included or when the temperature was maintained at 17
C (P>0.001). However, the percent of capacitated spermatozoa were 36.5% and 37.3%
(P<0.001) when spermatozoa were incubated at 39 C with 10% (v/v) SP for 30 min in
either PBS or X-cell thawing extender without the egg yolk-based fleezing extender,
respectively. The effects of SP addition to the spermatozoa are dependent on both

temperature and the type of extender used to support fleezing.

Introduction

Regardless of the nearly 30 years of commercial availability of flozen-thawed (FT) boar
spermatozoa, it has not received widespread acceptance for commercial breeding in the
swine industry. This is in part due to the higher semen costs, lower farrowing rates, and

smaller litter sizes associated with the use of FT spermatozoa [1, 2]. The decrease in

56

fertility with FT spermatozoa is believed to be a result of the spermatozoa being
effectively capacitated upon thawing [3 -5]. This reaction has been termed
cryocapacitation. Capacitated spermatozoa do not enter the spermatozoa reservoir
therefore having a shortened period of time to meet and fertilize an oocyte [6, 7]. If there

is not an oocyte present, the spermatozoa die shortly after capacitation

Capacitation is a multi-step process that involves the removal of epididymal and seminal
plasma (SP) proteins flom the spermatozoa membrane [8]. The boar epididymal protein
anti-agglutinin is bound to spernnatozoa acrosomes during maturation and is released at
an early stage of capacitation, but this loss was reduced in spermatozoa incubated in 5 or
10% (v/v) SP [9]. Similarly, the addition of 10 or 20% (v/v) SP to spermatozoa

capacitated in vitro at 39 C decreased the percent of capacitated spermatozoa [9, 10].

In our previous experiments, when 10% (v/v) SP was added to washed FT boar
spermatozoa that were incubated at 39 C, cryocapacitation was reversed within 30 min of
the SP inclusion. It is evident that SP plays an important role in the capacitation process.
It is unclear if the effects of SP addition are dependent on the incubation temperature or
the media it is added to. The objective of the present study was to examine the effects of
egg yolk fleezing extender, incubation temperature, and the addition of SP on the

cryocapacitation of FT boar spermatozoa.

57

Materials and Methods
All chemicals were of analytical grade. Unless otherwise stated, reagents were purchased
flom Sigma Chemicals (St. Louis, MO) and solutions prepared with sterile deionized

water.

A pool of SP flom four boars was accumulated for these studies. The sperrn-rich fiactions
of the ejaculates were centrifuged at 1000 x g for 10 min, and the pellet discarded. The
SP was stored in 15mL conical tubes at —80 C until required. Prior to use, a tube was

thawed overnight in a humidified incubator at 39 C and 5% C02 in air.

Media

The Phosphate Buffered Saline (PBS) solution contained 160 mM sodium chloride, 8
mM NaHP04 (anhydrous), and 2 mM Nal-IP04°H20 with pH adjusted to 7.4. The PBS
was equilibrated overnight in a humidified incubator at 39 C and 5% C02 in air prior to

use. The commercial semen extender employed was X-cell (IMV, Maple Grove, MN).

Treatments (Appendix IV)

Frozen semen from three Yorkshire boars was purchased from lntemational Boar Semen
(Eldora, IA) in 5mL maxi-straws. For thawing, straws were held at room temperature for
7 see before being submerged horizontally into a 42 C water bath for 43 sec. Temperature
was maintained in either a humidified incubator at 39 C and 5% C02 in air or in a Digital
Temperature Control Electronic Cooler/warmer (TR Enterprises Inc, Aberdeen, NC) at

17 C for a 30 min incubation period prior to CTC analysis. Where indicated, 10% (v/v)

58

SP was added to the spermatozoa suspension. A microscopic assessment of progressive
motility was performed immediately after thawing, the final centrifugation, and the 30

min incubation period. A percent motile was assigned within 10%.

Experiment 1a: For this experiment, thawed semen was extended directly in X-cell and so
included the egg yolk-based fleezing extender in the extended semen. Treatments were a
2x2 factorial arrangement with main effects being temperature during the holding period
(17 or 39 C) and presence of SP (0 or 10%). After the 30 min holding period a 5mL
aliquot was extracted and centrifuged at 600xg for 10min The pellet was resuspended in
5 mL X-cell at either 17 or 39 C to remove the egg yolk and immediately used for CTC

analysis.

Experiment 1b: This experiment was a repeat of experiment 1a, except it was performed
in PBS without the egg yolk-based fleezing extender included in the frnal spermatozoa
suspension. Immediately after thawing, semen straws were emptied into 15 mL conical
tubes and centrifuged for 10 min at 600xg. The supernatant was discarded, and 5mL of
PBS at 17 or 39 C was added. The spermatozoa suspension was then centrifuged for 5
min at 600 x g. Again, the supernatant was discarded and the spermatozoa pellet
resuspended in 5mL of PBS with or without SP. All procedures were performed at either

17 or 39 C.

Experiment 1c: This experiment was performed using X-cell but without the egg yolk-

based fleezing extender included in the final spermatozoa suspension. Immediately after

59

thawing, the straws were emptied into 15 mL conical tubes and centrifuged for 10 min at
600 x g. The supernatant was discarded, and 5mL of IBS thawing extender at 39 C was

added. The spermatozoa suspension was then centrifuged for 5 min at 600 x g. Again the
supernatant was discarded, and the spermatozoa pellet was resuspended in 5mL of X-cell

extender with or without SP, all at 39 C.

Chlortetracycline (CTC) staining analysis (Appendix H)

CTC staining analysis [11] was used to identify the progress of capacitation and
acrosome reaction of boar spermatozoa For CTC staining, 20 pl of 500 pM CTC in a
chilled buffer of 20 mM Tris, 130 mM sodium chloride, and 5 mM L-cysteine prepared
on the day of each treatment was added to a warm (37 C) microscope slide followed by
20 pl of spermatozoa suspension and mixed. After 10 sec, spermatozoa were fixed with
10 pl of 12.5% glutaraldehyde in Tris buffer (pH 7.8) and mixed again. A cover slip was
applied, and the slide was compressed between bibulous papers to remove excess fluid
and maximize the number of spermatozoa lying flat on the slide prior to observation.
Prepared slides, read within 1 h of preparation, were placed in a black box prior to
reading. Spermatozoa were illuminated with a 100 W mercury bulb and examined under
a Nikon Optiphot microscope equipped with a 380-425 nm excitation filter, a 520 nm

barrier filter, and a 510 nm dichroic rrrirror (Nikon Co., Tokyo, Japan).

Slides were prepared for CTC staining analysis directly after the 30 min incubation

period. Two slides were prepared for each sample, and 100 spermatozoa were counted on

each slide. The average of both slides was determined. Four fluorescent patterns were

60

used to demonstrate the progress of flesh, capacitated, and acrosome reacted
spermatozoa Fresh (F) spermatozoa (uncapacitated) were characterized by either a bright
anterior region with a bright acrosome cap, a relatively faint posterior region, and a dark
half circle at the equatorial region or by equally bright anterior and equatorial regions and
a less bright posterior region. Capacitated (B) spernnatozoa were consistent with a faint
anterior region, a bright equatorial region, and a faint posterior region. Acrosome reacted
(AR) spermatozoa were characterized by a very faint anterior region, a slightly bright

equatorial region, and a faint posterior region.

Bis-Benzimide Hoechst No. 33258 staining analysis

Hoechst 33258 was used for all 10 treatments to determine effect of treatment on viability
of the spermatozoa A 5 pl sample of chilled stock solution (200 pM Hoechst 33258) was
added to 100 pl of spermatozoa suspension and incubated for 15 min at 39 C and 5% C02
in the dark. Subsequently, 20 pl of stained spermatozoa suspension was added to a warm
(37 C) microscope slide and fixed with 5 pl 12.5% glutaraldehyde. A cover slip was
applied and the slide was compressed between bibulous papers. Prepared slides were
placed in a black box until read within 1 h of preparation. Spermatozoa were illuminated
with a 100 W mercury bulb and examined under a Nikon Optiphot microscope equipped
with a 365/10 nm excitation filter, a 400 nm barrier filter, and a 400 nm dichroic mirror

(Nikon Co., Tokyo, Japan).

Slides were prepared at the same time points as for CTC staining analysis. Two slides

were prepared for each sample, and 100 spermatozoa were counted on each slide. The

61

average of both slides was determined. Spermatozoa exhibiting bright blue fluorescence

over the entire head were considered nonviable cells.

Statistical Analysis

Data were analyzed using Statistical Analysis Systems software (SAS Institute Inc., Cary,
NC). Two-way AN OVA was conducted to infer upon treatment differences separately for
each of the response categories (flesh, capacitated, and acrosome-reacted). Incubation
temperature and the media used for spermatozoa suspension were used as the variables in

the tvvo-way ANOVA.

Results

The effect of serrrinal plasma and holding temperature on capacitation status of
spermatozoa in X-cell commercial semen extender are shown in Table 1. As can be seen,
when the egg yolk-based fleezing extender was included in the final spermatozoa
suspension, there was not effect of seminal plasma on capacitation status at either

temperature.

When the egg yolk-based fleezing extender was removed prior to extension in PBS
(treatments 5 to 8), seminal plasma reversed capacitation (P<0.001) only in spermatozoa
suspensions held at 39 C (Table 2). Similarly, when extended in X-cell without the
freezing extender and held at 39 C (treatments 9 and 10), seminal plasma reversed

capacitation (P<0.001, Table 2).

62

Motility and Viability
The average percent of viable spermatozoa was 52% with no significant difference (P
>0.05) across treatment groups. Similarly, the average percent motile was 55% with no

significance (P>0.05) across treatment groups or time of evaluation.

Discussion

The present data support previous studies [3, 12, 13] in that cryopreservation of boar
spermatozoa results in cryocapacitation. These results also agree with our previous work
in that addition of 10% SP to FT spermatozoa at 39 C without the egg yolk-based
fleezing extender results in a decrease in the percent of capacitated spermatozoa These
results indicate that the effects of SP are dependent on the temperature of the
spermatozoa suspension as well as the presence or absence of egg yolk in the fleezing

extender.

When SP was added to spermatozoa at 17 C regardless of the extender used, no decrease
in the percent of capacitated spermatozoa was noticed. Similarly, when SP was added to
spermatozoa suspended in the egg yolk-based fleezing extender regardless of the
temperature no decrease in the percent of capacitated spermatozoa was noticed. The
addition of 10% (v/v) SP resulted in a reversal of capacitation only when added to
spermatozoa suspended in either X-cell thawing extender or PBS at 39 C without the egg

yolk-based fleezing extender.

63

Based on our work [10] and work by others [9, 14] a SP inclusion level of 10% (v/v) is
recommended to achieve the expected reversal of cryocapacitation. Our previous studies
[10] indicated that the effect of SP appears to be independent of the time of addition and
percent of inclusion (10, 20, 40%). It was concluded that the de-capacitating effect of SP
was complete within 30 min of its addition. This was used to determine that the length of
each treatrnent’s incubation period would be 30 min for this study. Incubation
temperature and the presence of egg yolk in the fleezing extender can interfere with the
ability of SP to reverse cryocapacitation in FT boar spermatozoa. Further trials need to be

performed to establish these effects in vivo.

Temperature change induces membrane stress and lipid phase change in spermatozoa [15,
16]. The major phase change occurs in the vicinity of 5-15 C [17]. A capacitation-like
reaction was induced by cooling boar spermatozoa to 5 C, and this reaction was displayed
upon re-warming the spermatozoa to 39 C [18]. Membrane destabilization associated
with such cooling increases the fluidity of the plasma membrane [19] but does not affect
the fertilizing capacity of the cells. These membranes may be more permeable allowing
flee calcium ions to enter the cell and stimulate calcium-dependent events associated with

capacitation [13].

The extent of cold shock to bull spermatozoa is decreased when the spermatozoa are
flozen with a protective compound such as egg yolk [20]. The component(s) in egg yolk
and the mechanisms by which they alter the membrane are not fully understood. It is

known that egg yolk contains lipids that interacts with the spermatozoa membrane [21-

24]. Presumably, the phospholipids and low density lipoproteins of egg yolk attach to the
membrane preventing lipid disorganization. The same level of protection to boar
spermatozoa is not achieved with egg yolk alone [20]; however, its protection can be
improved by added Orvus Es Paste (OES) to the fleezing extender [25]. Orvus Es Paste
only offers protection against cold shock when included with egg yolk. This suggests that
CBS acts to alter the constituents in egg yolk to improve its protection against cold shock
in boar spermatozoa Interestingly, boar spermatozoa incubated in their own SP became
resistant to cold shock over 16 h after ejaculation [26]. Similarly, it has been shown that
the cold shock-induced membrane damage in ram spermatozoa was reversed following
incubation with a solution containing proteins extracted flom ram SP [27]. This study
demonstrated that the presumed binding of egg yolk constituents to the spermatozoa
membrane obstructs the presumed binding of SP constituents to the lipid membrane;

therefore, preventing the reversal of cryocapacitation

The extent of damage that results flom temperature change depends on a combination of
elements: cholesterol/phospholipids ratio, content of non-bilayer-preferring lipids, degree
of hydrocarbon chain saturation, and protein/phospholipids ratio in the membrane [28]. In
boar spermatozoa, capacitation induces changes in lipid membrane structure via a cAMP-
dependent protein phosphorylation pathway. These membrane changes include
phosphatidylserine (PS) inversion, relocation of cholesterol to the apical head, and a
decrease in lipid packing [29]. The lipid scrambling allows for cholesterol relocation.

Lipid scrambling, mainly PS inversion, is a recognized stage of the apoptotic death

65

pathway [30]. However, there is no in evidence the PS inversion in temperature induced

capacitation is related to the apoptotic death pathway [29].

Seminal plasma is produced by the accessory sex glands and is composed of inorganic
compounds, amino acids, peptides, as well as both low and high weight-molecular
proteins [31]. The existence of decapacitation factors in SP has been known since
Chang’s work in the 1950’s [32]. Heparin-binding proteins in bovine SP bind to cauda
epididymal spermatozoa and regulate capacitation by heparin [33]. In boar, the heparin-
binding protein pBl has been characterized and identified to bind to lipids containing the
phosphorylcholine group on the spermatozoa membrane at ejaculation [34]. Recently,
when spermatozoa were incubated with the glycosaminoglycan (GAG) hyaluronan (HA)
an increase in the percent of capacitated spermatozoa was demonstrated [14]. This
indicated an effect of GAG’s such as HA and heparin on capacitation mediated through
SP proteins bound to the spermatozoa membrane. In addition, when SP was introduced
into the uterus before the spermatozoa an increase in fertilization rate and number of
accessory spermatozoa in the zona pellucida was obtained [35]. This beneficial affect
could be attributed to the decapacitation of spermatozoa therefore increasing the number

in the spermatozoa reservoir.

Further investigation is needed to fully identify all constituents of SP and their
mechanisms of action on capacitation both in vitro and in vivo. However, it is evident
that some constituent(s) of SP acts on the process of capacitation through pathways that

are affected by temperature and the cryoprotectants used during fleezing. The ability to

66

allow widespread use of PT spermatozoa in the swine industry would eventually result in
the preservation of good genetics, lower production costs, and an increase in herd
biosecurity. It would allow safe international trade of good genetics to improve herd
production worldwide. This study demonstrates concerns that need to be considered when
designing in vivo experiments that test the ability of post-thaw SP addition to the

improvement of swine fertility with FT spermatozoa.

67

References

1.

10.

ll.

12.

Almlid, T., Hofino, P. A brief review of flozen semen application under
Norwegian AI service conditions. Reprod Dom Anim, 1996. 31: 169-73.

Reed, H. Current use of frozen boar semen-future need of flozen boar semen, in
Deep freezing of boar semen, J. LA and L. K, Editors. 1985, Swedish University
of Agricultural Science: Uppsala 225-37.

Bailey, J ., Bilodeau, J ., Connier, N. Semen cryopreservation in domestic animals:
a damaging and capacitating phenomenon. J Androl, 2000. 21: 1-7.

Curry, M.R., Kleinhans, F.H., Watson, P.F. Measurement of the Water
Permeability of the Membranes of Boar, Ram, and Rabbit Spermatozoa Using
Concentration-Dependent Self-Quenching of an Entrapped F luorophore" l .
Cryobiology, 2000. 41: 167-73.

Watson, RE The causes of reduced fertility with cryopreserved semen. Anim
Reprod Sci, 2000. 60-61: 481-92.

Fazeli, A., Duncan, A., Watson, P.F., Holt, W.V. Sperm-oviduct interaction:

Induction of capacitation and preferential binding of uncapacitated spermatozoa
to oviductal epithelial cells in porcine species. Biol Reprod, 1999. 60: 879-86.

Tienthai, P., Johannisson, A., Rodriguez-Martinez, H. Sperm capacitation in the
porcine oviduct. Anim Reprod Sci, 2004. 80: 131-46.

Yanagimachi, R. Mammalian Fertilization, in "The Physiology of Reproduction."
J. N. E Knobil, Editor. 1994, Raven Press: New York. 189-317.

Harayama, H., Magargee, S.F., Kunze, E., Shidara, 0., Iwamoto, E., Arikawa, S.,
Mihake, M., Kato, S., Hammerstedt, R.H. Changes in epididymal protein anti-
agglutinin on ejaculated boar spermatozoa during capacitation in vitro. Reprod
Fertil Dev, 1999. 11: 193-9.

Vadnais, M.L., Kirkwood, R., Sprecher, D.J., Tempelman, D., Chou, K. Effect of
cooling and seminal plasma on the capacitation status of boar sperm as
determined by chlortetracycline staining. Submitted to Anim Reprod Sci, June
2004.

Lin, C. Regulation of sperm capacitation. Doctoral Thesis. Michigan State
University, East Lansing, Michigan 1996.

Maxwell, W., Johnson, L. Chlortetracycline analysis of boar spermatozoa after

incubation, flow cytometric sorting, cooling, or cryopreservation. Molec Reprod
Dev, 1997. 46: 408-18.

68

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Watson, P.F. Cooling of boar spermatozoa and fertilizing capacity. Reprod Dom
Anim, 1996. 31: 135-40.

Suzuki, K., Asano, A., Eriksson, B., Niwa, K., Nagai, T., Rodriguez-Martinez, H.
Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal

plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan. Int J
Androl, 2002. 25: 84-93.

De Leeuw, F .E., Chen, H.C., Colenbrander, B., Verkleij, A.J., Cold-induced
ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology,
1990. 27: 171-83.

Holt, W.V., North, R.D. Partially irreversible cold-induced lipid phase transitions
in mammalian sperm plasma membrane domains: fleeze-flacture study. J Exper
Zoo, 1984. 230: 473-83.

Drobnis, E.Z., Crowe, E.Z., Berger, T., Anchordoguy, T.J., Overstreet J .W.,
Crowe, J .H. Cold shock damage is due to lipid phase transitions in cell

membranes: a demonstration using sperm as a model. J Exper Zoo, 1993. 265:
432-7.

Green, C., Watson, P. Comparison of the capacitation-like state of cooled boar
spermatozoa with true capacitation. Reproduction, 2001 . 122: 889-98.

Robertson, L., Watson, P. Calcium transport in diluted or cooled ram semen. J
Reprod Fertil, 1986. 77: 177-85.

Benson, R.W., Pickett, B.W., Komarek, R.J., Lucas, J .J . Effect of incubation and
cold shock on motility of boar spermatozoa and their relationship to lipid content.
J Anim Sci, 1967. 26: 1078-81.

Foulkes, J. The separation of lipoproteins flom egg yolk and their effects on the
motility and integrity of bovine spermatozoa J Reprod Fertil, 1977. 49: 277-84.

Watson, P.F. The interaction of egg yolk and ram spermatozoa studied with
fluorescent probe. J Reprod Fertil, 1975. 42: 105-11.

Pursel, V.G., Johnson, LA., Schulman, L.L. Interaction of extender composition
and incubation period on cold shock susceptibility of boar spermatozoa J Anim
Sci, 1972. 35: 580-4.

Gebauer, M., Pickett, B.W., Romarek, R. Motility of bovine spermatozoa
extended in 'defmed' diluents. J Dairy Science, 1970. 53: 817-23.

69

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Pursel, V.G., Schulman, L.L., Johnson, L.A. Effect of Orvus ES Paste on
acrosome morphology, motility and fertilizing capacity of flozen-thawed boar
sperm. J Anim Sci, 1978.47: 198-202.

Tamuli, M.K., Watson, P. Cold resistance of live boar spermatozoa during
incubation after ejaculation. Vet Rec, 1994. 135: 160-2.

Barrios, Perez Pe, R., Gallego, M., Tato, A., Osada, J ., Munino Blanco, T.,
Cebrian Perez, J .A. Seminal plasma proteins revert the cold-shock damage on ram
sperm membrane. Biol Reprod, 2000. 63: 1531-7.

Medeiros, C., F orell, F ., Oliverira, A., Rodrigues, J .L. Current status of sperm
cryopreservation: why isn't it better? Theriogenology, 2002. 57: 327-44.

Gadela, B.M., Harrison, H.A. Capacitation Induces Cyclic Adenosine 3',5'-
Monophosphate-Dependent, but Apoptosis-Unrelated, Exposure of
Animophospholipids at the Apical Head Plasma Membrane of Boar Sperm Cells.
Biol Reprod, 2002. 67: 340-50.

Fadok, V.A., Bratton, D.L., Rose, D.M., Pearson, A., Ezekewitz, R.A., Henson,
P.M. A receptor for phosphatidylserine-specific clearance of apoptotic cells.
Nature, 2000. 405: 85-90.

Pursel, V., Johnson, L., Schulman, L. Effect of dilution, seminal plasma and
incubation period on cold shock susceptibility of boar spermatozoa J Anim Sci,
1973. 37: 528-31.

Chang, M. A detrimental effect of seminal plasma on the fertilizing capacity of
sperm. Nature, Lond., 1957. 179: 258-9.

Miller, D., Winer, M., Ax, R. Heparin-binding proteins flom seminal plasma bind
to bovine spermatozoa and modulate capacitation by heparin. Biol Reprod, 1990.
42: 899-915.

Calvete, J ., Raida, M., Gentzel, M., Urbanke, C., Sanz L., Topfer Petersen, E.
Isolation and characterization of heparin- and phosphorylcholine-binding proteins
of boar and stallion seminal plasma Primary structure of porcine pBl . FEBS
Letters, 1997. 407: 201-6.

Willmen, T., Weitze, K.F., Habeck, 0., Waberski, D. Effect of seminal plasma
and different sperm numbers in the insenrinate on fertilization, accessory sperm
number, and ovulation time in the gilt. In Proc. 3rd Int. Conf. Pig Reprod. 1989.

Nottingham.

70

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Egg Yolk Incubation SP Fresh Capacitated Acrosome
Inclusion Temperature Inclusion Reacted
1 Yes 17C No 31.67 58.17 10.17
2 Yes 17 C Yes 37.83 50 12.17
3 Yes 39 C No 34 56.6 9.5
4 Yes 39 C Yes 33 56.3 10.67

 

 

Table 1: Effect of seminal plasma on capacitation status of flozen—thawed spermatozoa

after a 30 min incubation period in commercial semen extender at 17 or 39 C.

71

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Egg Yolk Incubation SP Fresh Capacitated Acrosome
Inclusion Temperature Inclusion Reacted

5 No 17 C No 32 57.17 10.83

6 No 17C Yes 37 51.17 11.83

7 No 39 C No 37.5 51.5 11

8* No 39 C Yes 55 36.5 8.5

9 No 17 C Yes 34.83 55.67 9.5
10* No 39 C Yes 54.17 37.3 8.5

*P-value<0.001

Table 2: Effect of seminal plasma on capacitation status of flozen-thawed spermatozoa
after a 30 min incubation period in phosphate buffered saline (Treatments 5 through 8) or
commercial semen extender (Treatments 9 and 10) at 17 or 39 C.

72

CHAPTER 5

Summary and Conclusions
Summary
The effects of boar SP addition to capacitated boar spermatozoa were examined. Fresh
spermatozoa flom 3 boars as well as FT spermatozoa from 6 boars were used for these
studies. The spermatozoa were capacitated in a capacitation-supporting environment, by
cooling the spermatozoa to 5 C over 100 min, or via cryopreservation. The status of
capacitation was examined using CTC staining analysis. The viability of the spermatozoa

was examined using bis-benzirrride stain.

Seminal plasma was added at 0, 10, or 20% (v/v) to the spermatozoa suspension
depending on the treatment used. The temporal pattern of capacitation was initially
examined. In treatments where flesh spermatozoa were used, capacitation was examined
over a 10 hr period of time. In initial treatments with FT spermatozoa the process of
capacitation and acrosome reaction were examined oven an 8 hr period. After the
decapacitation pattern was repeatedly demonstrated, the effects of SP on FT spermatozoa

were examined over an hour period.

Seminal plasma was added at different time points depending on the treatment used.
Primary studies with flesh spermatozoa in a capacitation-supporting environment
observed the effect of SP addition at the start of the treatment but removed after 100 min,
added at 100 min, or added after several hr of incubation. Primary studies with flesh

spermatozoa cooled to 5 C over 100 min to initiate capacitation observed the effect of SP

73

 

If

 

addition at the start of the treatment, added at the start of the treatment but removed after
cooling, or added after rewarming to 39 C and several hr of incubation. When FT

spermatozoa were used SP addition occurred immediately after thawing.

In experiments 1 and 2 (Chapters 3 and 4, respectively) an egg yolk-based fleezing
extender was not a factor with the use of flesh spermatozoa and was washed flom the FT
spermatozoa. Also, the temperatures in both experiments were controlled at 39 C.
Temperature as well as the presence and absence of egg yolk in the fleezing extender
were examined using FT spermatozoa in the third experiment (Chapter 4). The

temperature in experiment 3 was at either 17 or 39 C.

Conclusions

The addition of either 10 or 20% SP was capable of preventing and reversing capacitation
in spermatozoa capacitated in a capacitation-supporting environment or in spermatozoa
capacitated by slow cooling to 5 C. Similarly, 10 and 20% SP addition were capable of
reversing cryocapacitation in FT boar spermatozoa This reversal is apparent within 15

min of SP addition and is complete within 30 min

The effects of SP appear to be independent of the time or percent of addition. However, it
was demonstrated that the effects of SP are dependent on both the temperature of the
spermatozoa suspension at the time of addition as well as the presence of egg yolk-based

fleezing extender. Seminal plasma is capable of reversing cryocapacitation in

74

spermatozoa when it is added at ambient temperature (39 C) in the absence of the egg

yolk-based fleezing extender.

75

 

 

APPENDICES

76

 

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APPENDIX I

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79

APPENDIX II

80

Fluorescent patterns (Fresh, Capaciated, Acrosome Reacted) displayed by spermatozoa
after staining with CTC:

Fresh Fresh Capacitated Acrosome Reacted

   
    
 

Brnght Brrght Fannt Dark Anterior

.- ---------------------------------- - - «0
Dark Bright Bright F annt

.- ------------------------------------- o
Faint Faint Faint Dark

Posterior

 

 

 

 

81

 

APPENDIX III

82

Time line depicting spermatozoa preparation and SP addition in non-cooling
Treatments 2 and 3 (Treatment 1 0% SP was added):

 

I 10 min centrifugation

1

Suspend pellet
in 5 mL of

 

 

 

 

 

li99 at 39 C

 

 

it

A I

 

lst CTC
reading
(‘Pre’)

 

 

Incubate at 39 C
for 100 min

 

 

I 10 min centrifugation I

 

 

I -.

 

 

 

 

 

  
   

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

— — — ————— >
,SusPend Pellet I SP addition
m 5 mL 0f Treatment 2
li99 at 39 C
2nd CTC
’ reading
W (‘Post’)
[10 min warmng ”
period at 39 C
l 3rd CTC
l ’ reading
(‘Hf0’)

 

 

 

Continued 8 h
incubation at 39 C - - - r

 

 

 

ADD '-—_

 

 

 

83

Time line depicting spermatozoa preparation and SP addition in cooling
Treatments 5, 6, 7 (Treatment 4 0% SP was added):

 

I 10 min centrifugation J

 

 

   

 

 

 

--2 Ann I —————— ->

 

  
 

SP addition

Suspend pellet
Treatment 6

in5mLof

li99 at 39 C
I lst CTC

f 1 f ' reading
I Cool to 5 C over ( Pre )

  

 

 

 

 

 

 

 

 

100 min

I 10 min centrifugation I

 

 

 

 

 

——‘ REMOVE P-—"

 

 

 

 

 

II

 

 

 

   

 

     
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

--* Ann ——————— ->
SUSpend pellet SP addition
in 5 mL of Treatment 5
li99 at 5 C
2nd CTC
; reading
,, w (‘Post’)
10 min warming ,,
I period at 39 C 1 3rd CTC
I a reading
1 e (‘Hr0’)
Continued 8 h
incubation at 39 C l” — An" ______ ’

 

 

 

 

84

APPENDIX IV

85

Experimental Design for Experiment 3 (Chapter 4):

0% 1P in Egg Yolk-based Freezing ExtInder

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Temperature Temperature
at 17 C at 39 C
Treatment 1 Treatment 3
10% SPi in Egg Yolk-based Freezing Exjender
Temperature Temperature
at 17 C at 39 C
Treatment 2 Treatment 4
9% SP in PBS 10% SP in PB§
Temperature Temperature
at 17 C at 17 C
Treatment 5 Treatment 6
Temperature Temperature
at 39 C at 39 C
Treatment 7 Treatment 8
10% jP in Extender without Egg jolk
Temperature Temperature
at 17 C at 39 C
Treatment 9 Treatment 10

 

 

 

 

 

 

86

 

   

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