2006 Cryopreservation and Intracytoplasmic Sperm Injection With Bovine Eith Egg Yolk Sigma E0625

CRYOPRESERVATION AND INTRACYTOPLASMIC SPERM INJECTION WITH BOVINE
EPIDIDYMAL SPERMATOZOA
A Dissertation
Submitted to the Graduate Faculty of the

Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Interdepartmental Program of

Animal and Dairy Sciences
by
Carlos A. Guerrero
B.S., Louisiana State University, 2002
August 2006
ACKNOWLEDGMENTS
I would like to start by thanking my major professor and advisor, Dr. Robert A. Godke,
for accepting me in his Reproductive Physiology Program. I want to thank him for the
encouragement, patience, guidance, friendship and financial support he has given me. Most
importantly, I want to thank him for the magnificent scientific training he gave me over the past 4
years. He made me the hard working person I am today. I really enjoyed working with him, and
thanks again for allowing me to learn such specialized skills in the field of reproduction.
The completion of this dissertation has definitely been a challenge and could not have
been accomplished without the assistance and support from many individuals. I want to thank
the members of my committee (Dr. Stanley Leibo, Dr. Jill Jenkins, Dr. John Lynn and Dr.
Kenneth Bondioli) for constantly providing advice and suggestions for my research.
Special thanks to Dr. Kenneth Bondioli for sharing with me his extensive experience in
the area of reproductive physiology. He constantly helped me overcome problems that I had in
my work. Thank you for helping me and other graduate students at the laboratory with many
protocols and procedures. I feel honored to have worked with a scientist of your prestige. Also,
thanks to Dr. Lynn for helping me out with his microscopy skills. He was always available and
willing to help me with my research. Special thanks to Dr. Jill Jenkins for teaching me flow
cytometry. Thanks for always being worried and interested about my research. You were always
there for me as a professor and as a friend.
I would like to thank the Director of the Embryo Biotechnology Laboratory, Mr. Richard
Denniston, for keeping running smoothly and up to date this state-of-the-art research facility. He
always gave me his support and friendship. The laboratory will miss you when you retire.
Thanks to all the fellow graduate students and student workers at the Laboratory, who
offered there help in all projects undertaken. Special thanks to Angelica Giraldo, for helping me
with research projects and revising my abstracts. Thanks for your friendship! Also, thanks to Dr.
Marina Sansinena, for teaching the art of micromanipulation and laboratory procedures. Thanks
to Jesse for being there for me all the time. Keep working hard and you will reach your goals.
Thanks to Casey Ballard and Jason Smith, for helping me with the embryo transfer of my ICSI
embryos.
I would like to thank the Theriogenology group (Dr. Bruce Eilts, Dr. Dale Paccamonti and
Dr. Sara Lyle) from the LSU School of Veterinary Medicine for their participation at our weekly
research meetings. Thanks to Bryan for providing me with biological tissues at the local abattoir,
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to Jerry for providing fresh chicken eggs for my cryopreservation projects and to Dr. David
Donze for allowing me to use his tissue sonicator.
Last but not least, I want to thank my Mother, Maria Islena Espinosa, and Father, Carlos
Leonardo Guerrero, for their infinite emotional and financial support. Without your unconditional
support, the pursuit of this degree would have been impossible. I love you very much and hope
that some day I can repay you for everything you have done for me. Also, my Brother, Jorge
Enrique Guerrero, for being such an excellent friend and brother. I am proud of you for
everything you have accomplished.
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS................................................................................................ii
LIST OF TABLES........................................................................................................vi
LIST OF FIGURES..................................................................................................... vii
ABBREVIATIONS USED IN THE DISSERTATION........... xii
ABSTRACT....xiii
CHAPTER I INTRODUCTION................................................................................1
CHAPTER II LITERATURE REVIEW......................................................................... 3

Functions of the Epididymis.................... 3
Maturation........... 3
Transport............ 3
Maintenance and Storage............4
Protection........... 4
Collection of Epididymal Sperm.......... 5
Incisions..............5
Mincing............... 5
Flushing.............. 5
PESA........... 6
MESA.............. 6
The Use of Epididymal Sperm for Assisted Reproductive Techniques................ 6
Artificial Insemination (AI)............ 6
In Vitro Fertilization (IVF).................9
Intracytoplasmic Sperm Injection (ICSI).............. 12
Intracytoplasmic Sperm Injection in Cattle................................... 16
Multicolor Flow Cytometry for Sperm Quality Evaluation.....................21
Viability.......... 22
Capacitation........................................................................................ 24
Acrosome Integrity.......... 25
Mitochondrial Function............... 26
DNA Integrity............... 28
CHAPTER III EFFECT OF STORAGE AND CRYOPROTECTANT ADDITION AND

REMOVAL AT 4C ON MOTILITY AND PLASMA MEMBRANE
INTEGRITY OF BOVINE CAUDAL EPIDIDYMAL SPERM................ 30
Introduction....... 30
Materials and Methods.... 32
Experimental Design... 32
Experimental Procedure..... 33
Statistical Analysis... 39
Results... 39
Discussion. 46
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CHAPTER IV THE USE OF ETHYLENE GLYCOL AND GLYCEROL FOR
THE CRYOPRESERVATION OF BOVINE CAUDAL EPIDIDYMAL
SPERM.....................................................51
Introduction........... 51
Materials and Methods..... .. 53
Experimental Design....... 53
Statistical Analysis.... .. 61
Results.... .. 61
Discussion...... ...70
CHAPTER V THE EFFECT OF ADDITION OF BOVINE SEMINAL PLASMA TO

BOVINE CAUDAL EPIDIDYMAL SPERM PRIOR TO
CRYOPRESERVATION..... 76
Introduction. .. 76
Materials and Methods.... 79
Experimental Design....... 79
Statistical Analysis.... .. 88
Results.... .. 88
Discussion....102
CHAPTER VI EFFECT OF DILUTION AND INCUBATION OF POST-THAW

BOVINE EPIDIDYMAL SPERM HARVESTED WITH SEMINAL
PLASMA PRIOR TO CRYOPRESERVATION.................................. 108
Introduction.. 108
Materials and Methods.. 110
Experimental Design. 110
Experimental Procedure... 111
Statistical Analysis. 115
Results. 115
Discussion... 138
CHAPTER VII THE USE OF CRYOPRESERVED BOVINE CAUDAL EPIDIDYMAL

SPERM FOR INTRACYTOPLASMIC SPERM INJECTION
(ICSI)...................................................................................... 141
Introduction......... 141
Materials and Methods.. 144
Experimental Design. 144
Experimental Procedure... 145
Statistical Analysis. 153
Results. 153
Discussion... 161
CHAPTER VIII SUMMARY AND CONCLUSIONS... 169
LITERATURE CITED..... 171
VITA...202
v
LIST OF TABLES
3.1 Mean overall motility values for individual males during a 5-day exposure
to different concentrations of CPAs at 4C................................. 43
3.2 Mean progressive motility values for individual males during a 5-day exposure
to different concentrations of CPAs at 4C.......................... 44
3.3 Mean plasma membrane integrity values for individual males during a 5-day
exposure to different concentrations of CPAs at 4C......................... 47
4.1 Testicular and epididymal values from bovine testes collected postmortem.... 62
5.1 Testicular and epididymal measurements from bovine testes collected

postmortem......... 90
5.2 Morphology values (mean%SEM) of bovine epididymal sperm prior to

and post-cryopreservation.... 92
6.1 Percent plasma membrane integrity (meanSEM) of post-thaw bovine

epididymal sperm before and after dilution from the freezing extender............. 116
6.2 Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

sperm before and after dilution from the freezing extender................................120
6.3 Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

sperm before and after dilution from the freezing extender..............124
6.4 Percent plasma membrane integrity (meanSEM) of post-thaw bovine

epididymal sperm during a 4-hour incubation period at 37C... 127

sperm during a 4-hour incubation period at 37C......................127

sperm during a 4-hour incubation period at 37C........133
6.7 Percent progressive motility (meanSEM) of post-thaw bovine epididymal

sperm during a 4-hour incubation period at 37C........133
7.1 Effect of activation treatments on pronuclear formation of epididymal sperm-

injected bovine oocytes... 156
7.2 Effect of activation treatments on the embryonic development of epididymal

sperm-injected bovine oocytes...158
7.3 Total cell number (meanSEM) of day-8 bovine blastocysts derived from
ICSI using different activation treatments........ 162
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LIST OF FIGURES
3.1 The process of scrotum dissection. A. Pair of bovine testes inside the
scrotum. B. Dissection of the scrotum to retrieve the testes..................... 34
3.2 The process of cauda epididymides dissection. A. Pair of bovine testes

outside the scrotum and tunica vaginalis. B. Three pairs of testes showing
the dissected cauda epididymides. Each cauda was dissected
from the testis by cutting the vas deferens and corpus epididymis....... 36
3.3 The process of epididymal sperm retrieval from a dissected

cauda epididymis. The cauda was placed on a 100 mm plastic dish for 10
minutes to allow sperm to swim out into the collection medium.........37
3.4 Effect of cryoprotectant toxicity during 5 days of storage at 4C on overall

motility of bovine epididymal sperm. GLY = glycerol and
EG = ethylene glycol............................................................................................ 41
3.5 Effect of cryoprotectant toxicity during 5 days of storage at 4C on progressive

motility of bovine epididymal sperm. GLY = glycerol and
EG = ethylene glycol............................................................................................ 42
3.6 Effect of cryoprotectant toxicity during 5 days of storage at 4C on plasma

membrane integrity of bovine epididymal sperm. GLY = glycerol and
EG = ethylene glycol.........................................................45
4.1 Representative plasma membrane and acrosome integrity triple stain assay.
Sperm that exhibit green fluorescence (SYBR 14) are considered membrane
intact (viable), those that exhibit red fluorescence (PI) were considered to have
damaged membranes (nonviable) and those exhibiting yellow fluorescence
(PE-PNA) were considered acrosome reacted...................................... 58
4.2 Flow cytometric out-gating of egg yolk particles based on scatter properties
after staining frozen-thawed epididymal sperm with SYBR 14, PI and
PE-PNA. Sperm were selected (R1) based on size, gating out small
particles.........................................................................................................59
4.3 PI (red) and SYBR 14 (green) fluorescence. Remaining egg yolk particles
having sperm-like scatter properties were gated out based on DNA
fluorescence. Events that had either positive PI and/or SYBR 14 (R1)
fluorescence were selected as DNA positive events. Particles exhibiting
no DNA fluorescence (lower left corner) were considered to be nonsperm
events........................................................................... 59
4.4 SYBR 14 (viable) and PI (nonviable) flow cytometric dot plot illustrating
frozen-thawed epididymal sperm after gating out all nonsperm events.
Acquisitions of 10,000 events were made from positive green and red
fluorescence...........................................................................................60
4.5 Flow cytometric graph illustrating PE-PNA (orange) and PI (red) fluorescence.
Events exhibiting high orange fluorescence were recorded as sperm with
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reacted acrosomes. Quadrants were set to identify viable acrosome intact
sperm (LL), viable acrosome reacted sperm (LR), nonviable acrosome intact
sperm (UL) and nonviable acrosome reacted sperm (UR)............. 60
4.6 Examples of two pairs of bull testes with abnormal morphology..........................63
4.7 Percent progressive motility (meanSEM) of prefreeze and post-thaw bovine

epididymal sperm cryopreserved with glycerol and ethylene glycol.....................64
4.8 Prefreeze and post-thaw percent progressive motility values (meanSEM) of

bovine epididymal sperm from individual bulls cryopreserved with glycerol or
ethylene glycol.......................... 65
4.9 Percent membrane integrity (meanSEM) of prefreeze and post-thaw bovine

epididymal sperm cryopreserved with glycerol and ethylene glycol................. 67
4.10 Prefreeze and post-thaw percent membrane integrity (meanSEM) of bovine

epididymal sperm from individual bulls cryopreserved with glycerol and
ethylene glycol........ 68
4.11 Post-thaw percent reacted acrosomes (meanSEM) derived from the viable
and nonviable populations of bovine epididymal sperm cryopreserved
with glycerol and ethylene glycol......................................69
4.12 Percent acrosome integrity values (meanSEM) of prefreeze and post-thaw

bovine epididymal sperm from individual bulls cryopreserved with glycerol and
ethylene glycol........ 71
5.1 Flow cytometric out-gating of egg yolk particles based on scatter properties
after staining frozen-thawed bovine epididymal sperm with SYBR 14 and
MitoTracker Red. Sperm were selected (R1) based on size, gating
out small particles.......................................... 85
5.2 MitoTracker Red (red) versus SYBR 14 (green) fluorescence. Remaining egg
yolk particles having sperm-like scatter properties were gated out based on
DNA fluorescence. Events that had SYBR 14 (R1) positive fluorescence
were gated out and used for analysis........ 85
5.3 Flow cytometric dot plot illustrating MitoTracker Red (red) and SYBR 14 (green)
fluorescence from sperm of a bull exhibiting high mitochondrial activity. Graph
displays an epididymal sperm sample (10,000 sperm) with 69% of its
population having active mitochondria. ....................... 87
5.4 Flow cytometric dot plot illustrating MitoTracker Red (red) and SYBR 14 (green)
fluorescence from sperm of a bull exhibiting low mitochondrial activity. Graph
displays an epididymal sperm sample (10,000 sperm) with only 17% of its
population having active mitochondria....... 87
5.5 Flow cytometric dot plot illustrating green versus red fluorescence of acridine
orange in the SCSA. Graph displays bovine sperm with no DNA damage as
green and sperm with denatured DNA (R1) fluorescing red... 89
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5.6 Flow cytometric histogram illustrating red fluorescence of acridine orange in
the SCSA. Sperm in the M1 region are exhibiting DNA damage
(% COMP)............................................................................................................ 89
5.7 Post-thaw percent overall motility (meanSEM) of bovine epididymal

sperm harvested with either extender (no seminal plasma) or seminal plasma
prior to cryopreservation........... 93
5.8 Post-thaw percent progressive motility (meanSEM) of bovine epididymal

prior to cryopreservation....... 94
5.9 Post-thaw percent membrane integrity (meanSEM) of bovine epididymal

prior to cryopreservation....96
5.10 Post-thaw percent reacted acrosomes (meanSEM) derived from the viable
and nonviable populations of bovine epididymal sperm harvested with either
extender (no seminal plasma) or seminal plasma prior to cryopreservation........ 97

prior to cryopreservation........... 98

prior to cryopreservation......100
5.13 Percent DNA damage (meanSEM) of post-thaw bovine epididymal

prior to cryopreservation..........................................................................101
6.1 Membrane integrity values (meanSEM) of post-thaw bovine epididymal

sperm before and after one step dilution for removal of glycerol. The bars
represent sperm from individual males harvested with the extender
(Treatment A) prior to cryopreservation.................................118

represent sperm from individual males harvested with seminal plasma
(Treatment B) prior to cryopreservation........... 119

(Treatment A) prior to cryopreservation...............121

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(Treatment B) prior to cryopreservation...............122

(Treatment A) prior to cryopreservation............125

(Treatment B) prior to cryopreservation....... 126

sperm before and after a 4-hour incubation period at 37C. The bars
(Treatment A) prior to cryopreservation...................128

(Treatment B) prior to cryopreservation............................................129
6.9 Acrosome integrity values (meanSEM) of post-thaw bovine epididymal sperm

before and after a 4-hour incubation period at 37C. The bars represent sperm
from individual males harvested with the extender (Treatment A) prior to
cryopreservation........................ 131
6.10 Acrosome integrity values (meanSEM) of post-thaw bovine epididymal sperm

before and after a 4-hour incubation period at 37C. The bars represent sperm
from individual males harvested with seminal plasma (Treatment B) prior to
cryopreservation..................... 132
6.11 Mitochondrial activity values (meanSEM) of post-thaw bovine epididymal

(Treatment A) prior to cryopreservation..........................134
6.12 Mitochondrial activity values (meanSEM) of post-thaw bovine epididymal

sperm before and after a 4-hour incubation period at 37C. The bars represent
sperm from individual males harvested with seminal plasma (Treatment B)
prior to cryopreservation.....................135
6.13 Progressive motility values (meanSEM) of post-thaw bovine epididymal

sperm from individual males harvested with the extender (Treatment A) prior
to cryopreservation.....................136
6.14 Progressive motility values (meanSEM) of post-thaw bovine epididymal

sperm from individual males harvested with seminal plasma (Treatment B)
prior to cryopreservation........................137
x
7.1 Bovine cumulus oocyte complexes (COCs) received from
a commercial supplier at 21 hours of maturation. A. COC at 20X magnification.
B. COCs at 10X magnification....... 146
7.2 Immobilization of a motile bovine epididymal sperm with the microinjection

pipette prior to sperm microinjection. Arrow for the tail of the sperm.......... 150
7.3 The piezo ICSI procedure (40X magnification). A. An oocyte with the polar
body in the 12 oclock position held by the negative pressure of the holding
pipette before sperm injection. B. The zona pellucida drilled by applying piezo
pulses. A small cylinder of the zona pellucida can be observed outside
the oocyte. The sperm is moved to the tip of the pipette before
penetrating the oolema. C. The pipette is mechanically moved forward to
stretch the oolema. A single piezo pulse is applied to break the oolema. D.
The sperm is pushed forward by applying positive pressure to the
injector. E. Excess medium is removed and the injection pipette is moved
out from the oocyte. F. A small amount of ooplasm at the place of injection
is removed to allow sealing of the oolema..................151
7.4 Cytolyzed bovine oocytes degenerated 4 to 5 hours after sperm injection. A.

Arrows indicates cytolyzed oocytes (20X magnification). B. Cytolyzed oocyte
(40X magnification).................154
7.5 Oocyte stained with aceto-orcein 18 hours after the ICSI procedure. Male and
female pronuclei are indicated by arrows............... 155
7.6 An example of a day-3 bovine embryo (8- to 16-cells) derived from epididymal
sperm-injected oocytes............................................................................... 159
7.7 Examples of a day-8 blastocysts derived from epididymal sperm-injected

oocytes. Blastocysts are hatching through the opening made by the
microinjection pipette.................................................................... 160
7.8 An example of a day-8 blastocyst stained with Hoechst 33342. A. Day-8

blastocyst at 20X magnification. B. An overlay of the stained cells of the same
day-8 blastocyst in panel A.................................................................................163
7.9 Ultrasound images of a 50-day frozen-thawed epididymal sperm

ICSI-derived fetus...............................................................................................164
xi
ABBREVIATIONS USED IN THE DISSERTATION
AI artificial insemination
ART assisted reproductive techniques
ATP adenosine triphosphate
BO Brackett Oliphant medium
BSA bovine serum albumin
BSP bovine seminal plasma proteins
CASA computer assisted sperm analysis system
CAM calcein acetoxy methyl ester
CFDA 6-carboxylfluorescein diacetate
CKIA computerized karyometric image analysis
CMFDA 6-carboxylmethylfluorescein diacetate
COC cumulus oocyte complex
EYT-GC egg yolk Tris-glucose citric acid monohydrate extender
FBS fetal bovine serum
FDA fluorescein diacetate
FITC fluorescein isothiocyanate
GFP green fluorescent protein
HCG human chorionic gonadotropin
ICI intra cervical insemination
ICSI intracytoplasmic sperm injection
IUAI intrauterine artificial insemination
IUI intrauterine insemination
IVF in vitro fertilization
IVM in vitro maturation
LN2 liquid nitrogen
MPF maturation promoting factor
MHV mouse hepatitis virus
PE phycoerythrin
POEC porcine oviductal epithelial cell monolayer
PI propidium iodide
PNA Arachis hypogaea agglutinin
PSA Pisum sativum agglutinin
PVP polyvinylpyrrolidone
R123 rhodamine 123
TCM tissue culture medium
TLH tyrodes lactate HEPES medium
xii
ABSTRACT
Recently, interest in the preservation of epididymal sperm as a potential source of

valuable genes for genome resource banks has escalated. The development of a successful
protocol to recover and cryopreserve sperm harvested from the epididymides would salvage
germplasm from genetically valuable males that are injured and can no longer mate or have
unexpectedly died and can be used as a model for the preservation of male gametes from
endangered species. In a series of experiments, epididymal sperm was successfully harvested,
cryopreserved and used for intracytoplasmic sperm injection. In Experiment I, ethylene glycol
was found to cause significantly (P<0.05) less osmotic damage to bovine sperm during a one
step addition and/or removal at 4C as compared with glycerol in all concentrations evaluated.
Furthermore, prolonged exposure (5 days at 4C) of ethylene glycol was found to be less toxic
than glycerol to sperm. In Experiment II, it was demonstrated that glycerol was more effective
than ethylene glycol in providing protection against freezing injury during the cryopreservation
process in the concentrations evaluated. In Experiment III, it was demonstrated that epididymal
sperm retrieval using seminal plasma is beneficial to enhance sperm overall and progressive
motility characteristics and to protect it from morphological abnormalities derived from the
freezing process. In Experiment IV, a one step dilution process for removal of glycerol from
cryopreserved epididymal sperm was found to significantly affect plasma membrane integrity
and mitochondrial function of sperm previously exposed to seminal plasma. However, seminal
plasma exposure did not have any significant detrimental effect on acrosome integrity.
Furthermore, it was demonstrated that the longevity and survivability in vitro during a 4-hour
incubation period at 37C of post-thaw epididymal sperm exposed to seminal plasma prior to
cryopreservation was not compromised when compared with the control extended sperm. In
Experiment V, we have demonstrated that fertilization, blastocyst and fetal development could
be achieved with cryopreserved bovine epididymal sperm by intracytoplasmic sperm injection
(ICSI). To our knowledge, this is the first report in the United States and second in the world to
use bovine epididymal sperm for ICSI. We achieved far markedly improved blastocyst rates
over those results recently reported in the first study originating in Japan.
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CHAPTER I
INTRODUCTION
The unexpected loss of breeding animals of high genetic value or zoological
interest for any reason, such as loss of libido, reproductive tract injury, or death, can be
costly in terms of the potential loss of genetically valuable germplasm. This loss can be
reduced by harvesting spermatozoa (more commonly referred to as sperm) from the
epididymis and using assisted reproductive techniques (ART) to continue propagating
these valuable genetics over an extended period of time.
Assisted reproductive techniques, such as artificial insemination (AI), in vitro
fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been used over the
years in an effort to bypass critical fertilization steps, bypass breeding problems,
increase animal production efficiencies, overcome infertility and to propagate valuable
genetics. Moreover, the use of ART procedures represent a plausible strategy to
circumvent sexual incompatibility, eliminate the risk associated with animal transport and
provide a method of using cryopreserved germplasm to infuse genes from wild animals
into captive breeding populations (Stachecki et al., 1994). The objective of this research
project was to develop a successful protocol for the cryopreservation of bovine
epididymal sperm. Furthermore, we wanted to use these valuable samples to produce
embryos and calves by developing an efficient ICSI protocol for cattle.
Reports on the use, cryopreservation, fertility and characteristics of bovine
epididymal sperm are limited. Even with all the research over the years to improve the
survivability of bovine sperm after cryopreservation, there is still a decline of ~50% in
sperm post-thaw viability due primarily to temperature and osmotic effects (Thomas et
al., 1998). Furthermore, due to intermale variation sperm from some bulls still can not be
cryopreserved with present bovine protocols.
ICSI can be defined as the process of mechanically inserting a whole sperm or
an isolated sperm head into the ooplasm of an oocyte. This becomes a powerful
technique when only a few sperm are available for insemination. Since ICSI requires far
fewer sperm to fertilize the same number of oocytes than does IVF, it is potentially
valuable for using semen samples more effectively (Horiuchi et al., 2002). The use of
postmortem epididymal sperm with ICSI will allow a more effective use of valuable
genetic material. This technique has become important in the area of human infertility
1
and in studies of the molecular mechanisms of fertilization. ICSI has become one of the
most successful techniques in humans for achieving fertilization and subsequent embryo
development in cases of male sterility (Palermo et al., 1992). In contrast, the use of ICSI
in farm animals to date is far from satisfactory. The embryo developmental rates of
sperm-injected oocytes are still markedly lower than after conventional IVF. The
blastocyst rates reported for ICSI using bovine sperm range from 1% to 20% (Goto et
al., 1990; Hamano et al., 1999; Horiuchi et al., 2002).
We must take into consideration that epididymal and ejaculated sperm are two
biochemically and functionally different cell types. Ejaculated sperm differ from
epididymal sperm in respiration rate (Hammerstedt et al., 1993), number of heparin-
binding sites (Nass et al., 1990), ability to capacitate (Miller et al., 1990), morphology
(Hewitt et al., 2001), types of proteins bound to the plasma membrane (Lee et al., 1985)
and they display different motion characteristics (Gooaverts et al., 2006). Thus, ART
protocols used for ejaculated sperm will likely need to be modified for epididymal sperm.
Once we develop a successful protocol to preserve bovine epididymal sperm
from postmortem animals and develop an efficient ICSI procedure to use these valuable
sperm more effectively, research can be conducted to modify these protocols to best fit
other species, including farm animal and endangered species.
2
CHAPTER II
LITERATURE REVIEW
FUNCTIONS OF THE EPIDIDYMIS

The epididymis is generally divided into three functional regions; caput (head),
corpus (body) and cauda (tail), where sperm undergo physiological and functional
maturation (Bedford, 1975). Sperm present in the testis or in the caput epididymis are
inmotile and immature, while those that reach the cauda are generally more motile and
mature and are thought to be capable of fertilization (Toshimori, 2003).
Maturation
Sperm maturation refers to the change in functional capacity that occurs in sperm
during their transit through the epididymis (Cooper, 1999). Modification of sperm to
achieve maturation requires enzymes and transfer proteins that are secreted from the
epididymal epithelium into the luminal fluid (Amann et al., 1993). Changes in sperm
associated with maturation fall into the following four areas, the relative importance of
which likely depends on the species (Amann et al., 1993).
Modification of the DNA-protein complex of the nucleus to minimize the
probability of environmental damage or premature degradation.
Modification of the plasma membrane, mitochondria, and fibrous and
microtubular components of the middle and principal pieces to enable energy
transduction for coordinated contractions of tail and motility.
Development of surface characteristics enabling prolonged survival within the
female reproductive tract.
Stabilization of the plasma and acrosome membranes and development of
multiple binding proteins to bind with ovum receptors.
Transport
Sperm transport is a consequence of frequent pendular contractions of smooth
muscle fibers in the wall of the duct within the head and body of the epididymis and low
frequency segmental contractions of periducal smooth muscle in the tail of the
epididymis (Jaakola and Talo, 1983; Turner, 1991). This spontaneous muscular activity
is augmented by adrenergic, cholinergic and purinergic nerve fibers along with blood-
borne angiotensins, vasopressin and oxytocin (Cooper, 1999). Although some sperm are
3
transported distally more rapidly than others, a typical bovine sperm is transported
through the caput and corpus of the epididymides in 2 to 4 days (Amann, 1972). Then,
sperm are transported through the cauda of the epididymides in 6 to 14 days or longer,
with this interval being influenced by frequency of ejaculation (Amann et al., 1993).
Maintenance and Storage
Storage and maintenance of fertile sperm is the primary role of the tail of the
epididymis. The number of sperm stored varies according to the reproductive pattern,
social and mating behavior of a given species (Amann, 1981). In all species known,
sperm are stored in a quiescent state before ejaculation. This gamete reserve permits
ejaculation of larger numbers of sperm than the daily testicular production (Copper,
1999). Once the sperm reserve is full, existing mature sperm are expelled through the
ductus deferens and the urethra either into the urine or via spontaneous ejaculation.
The two primary mechanisms for sperm storage (Copper, 1999) are:
1. Enforcing Metabolic Quiescence
The lowering of luminal sodium ion concentration (prevents proton efflux and a
rise in pH that triggers motility).
High concentration of sperm in billions/ml.
Secretion of viscous mucoprotein that restrict sperm movement.
Steady production of acids keeping intracellular sperm pH low.
2. Prevention of Premature Sperm Activation
Protection of sperm membranes by the secretion of decapacitation factor(s).
Sperm aggregation, including the pairing of sperm in New World marsupials and
rouleaux formation of New World eutherians (e.g., flying squirrel, guinea pig and
Naked Tail armadillo). The pairing and rouleaux formation involve stacking of
acrosome membranes for protecting this liable structure during transit (Bedford,
1979).
Protection
It is the responsibility of the epididymal epithelium to maintain stored sperm in a
viable state. This responsibility includes the rapid elimination of harmful metabolic by-
products, toxic substances, prevention of oxidative stress and protection from the
immune system (Hinton et al., 1995).
4
A series of enzymes produced by the epididymal epithelium, such as glutathione-
s-transferase, superoxide dismutase, glutathione peroxidase and catalase are important
for the elimination of reactive oxygen species and for the removal of toxicants (Cooper,
1999; Hinton et al., 1995). Protease inhibitors, such as cystatin-related epididymal-
specific protein prevents damage by lytic enzymes leaking from degenerating sperm
(Hinton et al., 1996). The blood-epididymis barrier is very important for the protection of
sperm against the immune system (Pollanean and Cooper, 1994). Sperm acquire many
different surface antigens during their development and immune system exposure of
these surface antigens often mounts an immune response (Pollanean and Cooper,
1994).
COLLECTION OF EPIDIDYMAL SPERM
There are five basic procedures for the collection of cauda epididymal sperm.
Three are used for harvesting sperm from deceased animals and two developed
specifically for live animals, and are often applied to humans.
Incisions
This method of collection is one of the fastest and easiest to perform. The cauda
is excised from the testis as aseptically as possible. Several longitudinal incisions are
made on the distal end of the cauda to expose the sperm to the outer environment.
Sperm are rinsed from the cauda with 3 ml of isotonic sperm wash media into a 100 mm
petri-dish. The cauda is placed on the dish for 5 minutes to allow the sperm to swim out
into the medium. This process has a good sperm recovery rate, however, there is
chance of contamination from blood during the process.
Mincing
The cauda epididymis is placed on a 100 mm petri-dish and minced for 10
minutes. Then 3 ml of sperm wash medium is added to the dish. The sample is filtered
and centrifuged twice. This method provides a high sperm recovery rate, however, there
is a high chance of contamination from blood and tissue. A longer process is required to
filter and remove pieces of tissue and debris from the sperm sample. Viability of sperm
can be affected due to centrifugation and exposure to blood components.
Flushing
The cauda is excised from the testis. A blunt needle (size depends on species) is
inserted into the vas deferens. A syringe is attached with 3 ml of sperm wash medium
5
and pressure is applied by depressing the plunger. Sperm is collected in a 15 ml test
tube. This method provides the highest recovery rate and the least contamination.
PESA
Percutaneous epididymal sperm aspiration (PESA) (Craft et al., 1995a) is a
relatively simple procedure involving removal of sperm directly from the epididymis by
percutaneous needle aspiration. The advantages to this technique are that it can be
performed without surgical scrotal exploration, it can be repeated easily, it is low cost
and it does not require an operating microscope or the expertise in microsurgery
(Patrizio, 2000).
MESA
Microsurgical epididymal sperm aspiration (MESA) (Patrizio et al., 1988) is the
microsurgical incision in a single epididymal tubule, very carefully prepared by
microsurgical exploration under an operating microscope. The aspiration is performed
using a special microsurgical capillary system. This procedure causes minimal trauma to
the epididymal tubular system (Schwarzer et al., 2003b). This approach provides a
higher recovery rate than PESA, however, it is more invasive and expensive (Patrizio,
2000).
THE USE OF EPIDIDYMAL SPERM FOR ASSISTED REPRODUCTIVE TECHNIQUES
Artificial Insemination (AI)
Barker and Gandier (1957) reported the birth of an equine foal using epididymal
sperm by AI. Epididymal sperm were frozen in milk diluent with 10% glycerol and stored
at -79C for 30 days. This was the first recorded birth resulting from the use of frozen-
thawed stallion epididymal sperm and AI. Decades later, Morris et al. (2002) reported 9
pregnancies from 20 mares using fresh epididymal sperm deposited directly into the
utero-tubal papilla, and they reported 9 pregnancies in 45 mares using the same
procedure with frozen-thawed equine epididymal sperm.
Barker (1954) reported the first pregnancy using frozen-thawed bull epididymal
sperm for AI. However, birth of the calf was not reported. Sperm were added to milk
diluent with 10% glycerol. The sample was placed in a refrigerator at 5C for an
equilibration period of 18 hours and further cooled to -79C in 1 ml ampoules. Amann
and Griel (1973) compared the fertility of bovine caudal epididymal sperm with
ejaculated sperm by AI. Insemination with 200 x 106 sperm into the uterine body of 106
6
cows resulted in 84% and 94% fertilized ova for caudal epididymal sperm and ejaculated
sperm, respectively. More recently, it was reported that bovine epididymal sperm stored
at 5C were viable for at least 60 hours when used for AI (Foote, 2000).
A 41-day pregnancy following AI with cryopreserved epididymal sperm harvested
from a gaur (Bos gaurus) bull that had been euthanized was reported (Hopkins et al.,
1988). Sperm were extracted 27 hours postmortem, diluted in an egg yolk-based
extender supplemented with 7% glycerol, placed at 12.5 cm above liquid nitrogen vapor
for 10 minutes and then plunged in liquid nitrogen for storage. Bartels et al. (2001)
maintained eland epididymal sperm at 4C for 5 hours postmortem before
cryopreservation. A live birth was then achieved following AI. More recently, Soler et al.
(2003a) reported an average fertility rate of 56% in Red deer using frozen-thawed
epididymal sperm subjected to different thawing procedures before AI.
In the rabbit, no difference was reported in the fertility after AI of sperm extracted
from the caudae epididymides when compared with its ejaculated counterpart after they
were incubated for 20 hours or for a longer interval in the female (Dott et al., 1966).
However, Cummins and Orgebin-Crist (1971) found that rabbit sperm from the caudae
epididymides fertilized 52% of oocytes when artificially inseminated at the same time as
human chorionic gonadotropin injection or 9 to 13 hours before ovulation in the
recipients, while ejaculated sperm fertilized 84% of oocytes under same experimental
conditions. Furthermore, Overstreet and Bedford (1976) reported 32 and 28 pregnancies
in does artificially inseminated with caudal epididymal and ejaculated sperm,
respectively, demonstrating that both were equally competent to support embryonic and
fetal development. Earlier, Adams (1975) produced offspring from 2 wild rabbits
(Oryctolagus cuniculus), which did not breed under laboratory conditions, using AI with
caudal epididymal sperm. These does were inseminated directly into the uterus
performing a laparotomy, however 17 other does inseminated into the vagina using
epididymal sperm produced no offspring.
Blandau and Rumery (1964) compared fertilization rates in the rat after AI using
caput and caudal epididymal sperm. Fertilization rates were 8% and 93% for the caput
and the cauda, respectively. Orihuela et al. (1999) studied sperm migration into and
through the oviduct following AI with epididymal sperm at different stages of the follicle
cycle in the rat. Nakatsukasa et al. (2001) reported birth of 41 normal offspring born from
7
9 female rats that were artificially inseminated with frozen-thawed epididymal sperm.
Sperm was frozen in a medium composed of 23% egg yolk, 8% lactose monohydrate,
10% Tris and 0.7% Equex Stem. This was the first successful production of rat offspring
using epididymal sperm cryopreserved at -196C. Recently, live young were produced
by AI with cryopreserved epididymal sperm extracted from closed colonies (Jcl:SD and
Jcl:Wistar), inbred (BN/Crj, F3441 DuCrj, LEW/Crj, Long-Evans and WKY/NCrj), mutant
(Zitter [WTC.ZI-zi] and Tremor [TRM]), transgenic (human A-transferase [A] and green
fluorescent protein [GFP]) strains of rats (Nakatsukasa et al., 2003).
Blash et al. (2000) reported the birth of one healthy kid from 20 inseminated does
by AI with frozen-thawed goat epididymal sperm. Epididymal sperm was obtained at
necropsy from goats and equilibrated with 20% egg yolk, 7% glycerol, 2.42% Tris, 1.38%
citric acid, 5.5 mg tylosin and 1% fructose. Samples were cooled to 5C for 4 hours,
placed into a -80C for 15 minutes and then plunged into liquid nitrogen.
In swine, live piglets were reported using AI and epididymal sperm harvested
from different regions of the epididymis (Holtz and Smidt, 1976). In this study, fertilization
rates improved significantly from the caput to the cauda regions.
Marks et al. (1994) reported the birth of a canine puppy after AI using frozen-
thawed epididymal sperm extracted postmortem to preserve the breeding line of a 9-
year old male boxer. Recently, Hori et al. (2004) compared the fertilizing ability of frozen-
thawed caudal epididymal and ejaculated sperm in beagle dogs. The post-thaw motility
was slightly lower for epididymal sperm, but the difference was not significant. However,
the pregnancy rate (16.7% and 90%) after AI was much lower for epididymal than for
ejaculated sperm, respectively.
Timing of AI in marsupials is critical because fertilization must occur before mucin
coats the oocyte during passage through the oviduct (Rodger and Bedford, 1982a).
Fertilization after intrauterine artificial insemination (IUAI) has been achieved using
epididymal sperm in the Brushtail possum (Trichosurus vulpecula) but no births were
reported (Molinia et al., 1998). Recently, the birth of two Tammar wallabies (Macropus
eugenii) were reported using IUAI and epididymal sperm (Paris et al., 2004). These
young represent the first macropodids born by AI and first marsupials conceived from
using epididymal sperm.
8
Morrel et al. (1997) used cooled epididymal sperm for AI in Marmoset monkeys
(Callithrix jacchus). Epididymal sperm was deposited in the cervix of 18 Marmoset
monkeys near the time of expected ovulation using 1, 2 or 3 inseminations. Six of 18
females conceived, resulting in the first reported births following AI in this species.
In humans, extraction of epididymal sperm and intrauterine insemination (IUI)
has been used to treat male infertility with obstructive azoospermia. In one report, from
66 IUI cycles, 3 pregnancies resulted giving birth to 3 healthy infants (Qiu et al., 2003).
Pregnancies have also been achieved in humans using intracervical inseminations (ICI)
using epididymal sperm (Cooper, 1990).
In Vitro Fertilization (IVF)
Goto et al. (1989) used frozen-thawed bovine epididymal sperm to examine
variation among bulls in fertilization and embryo development rates in vitro. Bovine
epididymides were transported from a local abattoir within 2 hours at 25C to the
laboratory. Sperm samples were frozen in egg yolk tris citrate extender supplemented
with 6.5% glycerol. The fertilization rates ranged from 55.2 to 64.3% and the day 7 to
day 8 blastocyst rates ranged from 9% to 12% among bulls, respectively. Subsequently,
Graff et al. (1996) reported 2 pregnancies obtained from IVF with noncapacitated
epididymal bovine sperm. Recently, Herbert and co-workers (2005) (unpublished data)
produced 5 live calves from IVF with bovine epididymal sperm harvested from
epididymides stored at 4C for 24 hours.
Herrick et al. (2004) used springbok (Antidorcas marsupialis) frozen-thawed
epididymal sperm for IVF to test its fertilizing ability. Epididymal sperm were extracted
following managed culls or hunts within 4 hours of death. From 132 in vitro matured
oocytes, only 4 had zona penetration. Winger et al. (1997) reported fertilization of
blesbok (Damaliscus dorcas phillipsi) oocytes using frozen-thawed epididymal sperm. In
African Buffalo (Syncerus caffer), epididymal sperm was harvested from testes stored at
4C for up to 24 hours, and used for IVF. Cleavage and morula rates were 50% and
65%, respectively (Shaw et al., 1995). Unfortunately, no live births have been reported
from these wild species.
Boar epididymal sperm show a higher resistance to cold shock than ejaculated
sperm, suggesting that they may be more suitable for cryopreservation (Losley and
Bogart, 1944). Jiang et al. (1991) reported the birth of live piglets after the transfer of 2-
9
to 4-cell embryos produced by in vitro matured porcine follicular oocytes and IVF using
frozen-thawed epididymal sperm. Rath and Niemann (1997) compared the use of
porcine frozen-thawed epididymal and ejaculated sperm for IVF obtained from identical
boars. Epididymal sperm were harvested immediately postmortem and cryopreserved in
an egg yolk lactose-based extender supplemented with 2% glycerol. The fertilization
(59.7% compared with 16%) and cleavage rates (21% compared to 5.3%) were higher
for frozen-thawed epididymal sperm than for frozen-thawed ejaculated sperm,
respectively. Subsequently, Kikuchi et al. (1998) studied the influence of prolonged
storage at 4C of boar epididymides on IVF. They reported lower oocyte penetration
rates for sperm collected on days 1, 2 and 3 from stored epididymides (12, 13 and 2%,
respectively) than control sperm (40%) collected from nonrefrigerated epididymides.
Recently, Romar et al. (2003) evaluated the effects of adding porcine oviduct epithelial
cell (POEC) monolayer during IVF. Frozen-thawed epididymal sperm were used to avoid
replicate variability. They reported an increase in oocyte penetrability in the presence of
POEC. However, a lower blastocyst rate was found with POEC incubation during IVF
than without POEC monolayer (21% and 14%, respectively).
Ogawa et al. (1972) reported the successful IVF of rabbit ova by epididymal
sperm in a synthetic medium without the need of follicular fluid, which in the past, was
normally required to induce capacitation in ejaculated sperm. They obtained a 59%
fertilization and a 23% blastocyst rate. Later, it was found that capacitation could be
induced in rabbit epididymal sperm by washing them twice (15 minute interval) in
isotonic or hypertonic defined medium (Brackett et al., 1978; Hosoi et al., 1981).
Niwa et al. (1985) studied the penetration rates of cat epididymal sperm for IVF.
They reported that 90% to 100% of the ova were penetrated between 0.5 and 5 hours
post-IVF. Female and male pronuclei were observed as early as 4 hours post-
insemination. Freistedt et al. (2001) used cat epididymal sperm and IVF to test the effect
of season and ovarian status on the efficiency to produce embryos. They reported that
although embryos could be produced year round, the efficiency was significantly affected
by season. Blastocyst rates produced from January to September and from October to
December were reported to be 28% and 18%, respectively.
In the field of transgenic production, the ability to maintain the genetic
contribution of the male after death is remarkably important. The first report of IVF using
10
goat epididymal sperm was in 1988 (Song and Iritani, 1988). They examined the effects
of length of pre-incubation and concentration during pre-incubation of fresh epididymal
sperm on IVF rates. The highest fertilization rates (57%) were obtained when sperm had
been pre-incubated at a concentration of 50 x 107 sperm/ml for 6 hours in a modified
Krebs Ringer bicarbonate solution. Blash et al. (2000) compared frozen-thawed
epididymal and ejaculated sperm for IVF. They reported 40% and 38% cleavage rates,
with 6% and 4% developing into blastocysts using frozen-thawed caprine epididymal and
ejaculated sperm, respectively.
Today, most of the basic research in mammalian genetics is undertaken in mice
and rats. The production of mice with transgenes and mutant genes is common, and an
abundance of valuable genomes are available for analysis (Szczygiel et al., 2002). Due
to the difficulty in collecting ejaculated sperm from mice and rats, most of the research
has been done using epididymal sperm. Toyoda and Chang (1974) used fresh rat
epididymal sperm for IVF. A total of 203 cleaved embryos where transferred to 14
pseudopregnant rats. A total of 43 live young were born.
Birth of live mouse pups have been reported using fresh (e.g., Miyamoto and
Chang, 1972; Songsasen et al., 1997) and frozen-thawed epididymal sperm (e.g.,
Songsasen et al., 1997; Sztein et al., 2000; Kawase et al., 2002). Lacham-Kaplan and
Trounson (1994) showed that the fertilizing ability of mouse epididymal sperm decreases
significantly from the caput to the cauda. Fertilization rates for caput, corpus and caudal
epididymal sperm were 10, 30 and 90%, respectively. Bongso and Trounson (1996) co-
cultured mouse epididymal sperm from the caput, corpus and cauda with epithelial cells
of their own region or more distal regions. They found that the presence of more distal
epithelial cells of the mouse epididymis increases the ability of caput and corpus, but not
cauda sperm, to fertilize zona-free oocytes. It was reported that epididymal sperm
collected from mouse cadavers kept at room temperature up to 24 hours can fertilize
oocytes in vitro (Christian et al., 1993; Songsasen et al., 1998). Sperm collected 15 and
24 hours after death resulted in fertilization rates of 25% and 20%, respectively. The
birth of 3 live female pups was reported after transfer of 11 blastocysts fertilized with
epididymal sperm harvested from cadavers after 24 hours of death. Subsequently,
Kishikawa et al. (1999) harvested epididymal sperm from mouse cadavers stored up to
15 days at 4C. Sperm motility decreased from 88% (day 0) down to 66% (day 5), 32%
11
(day 10) and 8% (day15). Day-5 epididymal sperm had a fertilization rate of 26%.
However, the sperm extracted from day 10 and day 15 had limited ability to fertilize
oocytes (2.8% and 2.7%, respectively). Tada et al. (1994) used epididymal sperm to
fertilize mouse vitrified oocytes. A 54% fetal development resulted after embryo transfer.
Sato and Ishikawa (2004) stored epididymal sperm at room temperature (22C) for 5
days in M2 medium and then used it for IVF. They reported a 7% fertilization rate with
12.5% of the transferred embryos resulting in live mouse pups. Scavizzi and Raspa
(2004) used epididymal sperm extracted from infected mice with mouse hepatitis virus
(MHV) to investigate its transmission by IVF. They reported that transmission was not
found when sperm from infected testes were used for IVF. These results suggested that
sperm do not transmit infection from actively infected animals and that IVF could be
considered a virus cleansing procedure.
In humans, epididymal sperm and IVF are used to treat patients who suffer from
azoospermia due to congenital absence of the vas deferens or secondary extended
obstruction of the spermic ducts (Bladou et al., 1991). Also, it is important to freeze
epididymal sperm to avoid additional epididymal surgery in the male. Pregnancies have
been obtained by IVF using epididymal sperm (e.g., Temple-Smith et al., 1985; Silber et
al., 1987, 1988; Silber, 1988; Bladou et al., 1991; Oates et al, 2004 and others). Bladou
et al. (1991) reported 5 pregnancies (35%) with embryos produced by IVF using caput
epididymal sperm. They found that caput epididymal sperm used for IVF gave a higher
rate of embryo degeneration (>50%) after embryo transfer than caudal epididymal
sperm. Patrizio et al. (1995) reported the birth of a healthy girl using frozen-thawed
epididymal sperm after conventional IVF. Belker et al. (2001) reported a live birth from
embryos produced by IVF using epididymal sperm retrieved from a moribund man.
Tzeng et al. (1996) reported the birth of a healthy girl by MESA and IVF.
Intracytoplasmic Sperm Injection (ICSI)
ICSI has become an important technique in the areas of infertility and for the
study of the molecular mechanisms of fertilization. This approach has become one of the
most successful techniques in humans for achieving fertilization and subsequent embryo
development in cases of male sterility (Palermo et al., 1992). ICSI has helped the
understanding of the early events of fertilization such as capacitation, the acrosome
12
reaction, the mechanisms of sperm oocyte interaction, sperm-induced oocyte activation,
pronucleus formation and the control of the first cell cycle (Keskintepe et al., 1997).
Caudal epididymal sperm for ICSI has been used extensively in the mouse (e.g.,
Markert, 1983; Iritani et al., 1988; Ahmadi et al., 1995; Kimura and Yanagimachi, 1995;
Ron-El et al., 1995; Suzuki and Yanagimachi, 1997; Moreira et al., 2003; Nagai et al.,
2003; Baart et al., 2004). Kishikawa et al. (1999) produced 6 mouse pups after ICSI with
immotile epididymal sperm harvested from cadavers stored at 4C for 20 days. Forty two
embryos were transferred and 6 implanted. When valuable male animals die
unexpectedly and facilities for sperm cryopreservation are not immediately accessible,
temporal storage of cadavers in a regular refrigerator followed by ICSI may help to
preserve the genome of individuals. Suzuki and Yanagimachi (1997) compared the use
of conventional ICSI with a novel piezo electrically driven pipette using mouse
epididymal sperm. They reported that although the efficiency of the piezo ICSI is much
more efficient, the inefficiency of the conventional ICSI could be improved by performing
the operation in a medium with high serum concentration. Kawase et al. (2005) studied
the effect of storing freeze-dried mouse epididymal sperm at either 4C or -80C on
embryo development after ICSI. They reported blastocyst rates of 21% and 62% for
freeze-dried epididymal sperm stored for 3 months, and 13% and 59% for sperm stored
for 6 months at 4C and -80C, respectively. Yanagimachi et al. (2004) reported the birth
of fertile mouse offspring after injecting epididymal sperm from an infertile male into
oocytes of normal females.
Re-establishment of mouse strains used for mutagenesis and transgenesis has
been hindered by difficulties in freezing sperm. The use of epididymal sperm and ICSI
enables the production of embryos for the restoration of mouse lines using low quality
sperm. Lacham-Kaplan et al. (2003) produced live offspring from inbred and hybrid
mouse strains using ICSI with epididymal sperm frozen without cryoprotectant. Li et al.
(2003) reported the birth of 26 live mutant mouse pups after ICSI using epididymal
sperm. They rescued 4 mutant lines that had become infertile and unresponsive to
conventional IVF by using ICSI. Moreira et al. (2004) reported the efficient generation of
transgenic mice by ICSI using epididymal sperm. They produced offspring carrying the
YAC transgene.
13
Hirabayashi et al. (2002) reported the birth of 18 transgenic rat pups carrying the
green fluorescent protein gene (GFP) from 168 transferred ICSI embryos using
epididymal sperm. Transgenic male rats carrying the GFP gene were used as the sperm
donors. Said et al. (2003) studied embryo development of rat oocytes after ICSI with
epididymal sperm heads. They reported 18% blastocyst rate and 6 pups were born after
embryo transfer.
Goto et al. (1990) used immobilized, killed epididymal sperm to fertilize IVM
bovine oocytes by ICSI. From 507 oocytes injected, 1.8% reached the blastocyst stage.
One healthy calf was born after embryo transfer. This is the first and only report of birth
of a calf using epididymal sperm and ICSI.
Ng et al. (2002) used Cynomolgus monkey (Macaca fascicularis) epididymal
sperm to produce embryos by ICSI. They reported 61% and 27% fertilization and
pregnancy rates, respectively. Four monkeys became pregnant resulting in the birth of
two healthy female offspring.
Richings et al. (2004) reported the use of ICSI with Tammar wallaby (Macropus
eugenii) epididymal sperm. Oocytes were assessed for the presence of pronuclei and
polar body extrusion and in vitro development was monitored for up to 4 days, resulting
in 3 of 4 follicular oocytes and 4 of 8 tubal oocytes cleaving. Embryo development halted
at the 4 to 8 cell stages. To date, this is the only known report of ICSI in marsupials.
Pushett et al. (2000) compared the developmental competence of cat embryos
developed by IVF and ICSI using frozen-thawed epididymal sperm. They reported higher
fertilization and blastocyst rates using ICSI from cryopreserved epididymal sperm than
by IVF. Bogliolo et al. (2001) studied the developmental competence of in vitro matured
(IVM) cat oocytes after ICSI with frozen-thawed epididymal sperm. A 7% blastocyst rate
was reported 7 days after sperm-injection.
Probst and Rath (2003) reported the birth of 13 male piglets from sex-sorted
frozen-thawed epididymal sperm and ICSI. Flow cytometrically sorted Y-chromosome
bearing sperm were injected into in vivo matured oocytes, activated with 1.2 pl of a 30
mM CaCl2 solution. Approximately 85 fertilized oocytes were transferred surgically into 4
recipient females.
Zheng et al. (2004) used fresh epididymal sperm collected from a fertile male
rabbit to compare different sperm injection methods into oocytes. They reported that the
14
highest blastocyst rate was achieved when the needle tip was pushed across half the
diameter of the oocyte, and when oolema breakage was achieved by repeated
aspiration and expulsion. Deng and Yang (2001) reported the birth of 7 live young from
the transfer of 113 ICSI embryos to 6 rabbit recipients. The fertilization and blastocyst
rates obtained were 78% and 39%, respectively.
Epididymal sperm for ICSI has been extensively used in humans for the last
decade to treat male caused infertility (e.g., Tournaye et al., 1994; Devroey et al., 1995;
Patrizio et al., 1995; Oates et al., 1996; Cha et al., 1997; Holden et al., 1997; Friedler et
al., 1998; Garrels et al., 1998; Nudell et al., 1998; Pasqualotto et al., 2002; DeVos et al.,
2003; Horak et al., 2003; Rhouma et al., 2003; Lin et al., 2004; Nicopoullos et al., 2004
and others). The ICSI procedure has revolutionized the treatment of male infertility
because ICSI is effective not only in cases of severe oligozoospermia but also in cases
of azoospermia, providing higher fertilization, pregnancy and implantation rates than that
of standard IVF (Schwarzer et al., 2003a). Live births have been reported in humans by
ICSI using frozen-thawed epididymal sperm recovered by PESA (Jin et al., 2004) and
MESA (Drouineaud et al., 2003). Nagy et al. (1995) compared the use of human
ejaculated, fresh and frozen-thawed epididymal and testicular sperm for ICSI.
Fertilization rates were reported to be 56% for fresh, 56% for frozen-thawed epididymal
and 48% for testicular sperm were used for the injection but these rates were
significantly lower than 70% for ejaculated sperm.
Shwarzer et al. (2003a) reported results from 1,079 ICSI cycles with aspirated
epididymal and testicular sperm. Epididymal sperm were used in 172 cycles and
testicular sperm or spermatids in 907 cycles. Retrieved sperm were used after
cryopreservation or immediately after aspiration. No significant difference was found
between epididymal (frozen-thawed or fresh) and testicular sperm. A 28% birth rate
resulted from these treatments. Ludwig and Katalinic (2003) analyzed 2,809 pregnancies
with 3,199 fetuses/children reported in Germany from ICSI using either testicular,
epididymal or ejaculated sperm. No statistical difference resulted from the origin of
sperm. Bonduelle et al. (2002) reported the birth of 2,840 ICSI children using either
ejaculated, epididymal or testicular sperm from 1983 to 1999 in Belgium. A year earlier,
Abdul-Jalil et al. (2001) had reported the first ongoing clinical twin pregnancy resulting
from ICSI using sperm retrieved by PESA into IVM oocytes. In recent years the recovery
15
of immature oocytes from unstimulated ovaries followed by IVM was found to be an
attractive alternative to conventional IVF in the treatment of female infertility.
Burrello et al. (2003) evaluated the impact of sperm aneuploidy on ICSI outcome.
Patients were divided into two groups based on sperm aneuploidy (<25% or >75%
aneuploidy). Significantly higher clinical pregnancy (75 versus 34%; P<0.001) and
implantation (34 versus 13%; P<0.001) rates were reported for the low and high
aneuploidy frequency groups, respectively. Recently, Ramos et al. (2004) reported the
use of computerized karyometric image analysis (CKIA) to select normal epididymal
sperm for ICSI from males with obstructive azoospermia. CKIA evaluates DNA
stainability, chromatin texture (nuclear condensation) and morphology. A two-fold
increase in selecting normal sperm for ICSI is achieved by this system.
INTRACYTOPLASMIC SPERM INJECTION IN CATTLE
The first report of ICSI in cattle (Younis et al., 1989) was performed 13 years
after the development of the basic ICSI technique by Uehara and Yahagimachi (1976).
In this report, sperm decondensation and cleavage development of sperm-injected
oocytes were evaluated using in vitro capacitated sperm and IVM oocytes. They
reported a 2% cleavage rate (2/101) when oocytes were not exogenously activated. In
addition, they stated that only 21% (21/101) of injected oocytes contained a sperm inside
the ooplasm at the time of evaluation. The addition of 1 M calcium ionophore (A23187)
for 5 minutes to artificially activate the oocytes increased sperm decondensation 3 hours
after sperm-njection from 0 to 37% (11/30) and cleavage rates 48 hours post-injection
from 2 to 36% (5/14). A year later, Goto et al. (1990) reported the production of the first
calf by ICSI using immobilized, killed cryopreserved bovine epididymal sperm. Oocytes
were chemically activated post-injection with 50 M calcium ionophore (A23187). This
was the first report to evaluate embryonic development of ICSI embryo up to the
blastocyst stage. Of 507 sperm-injected oocytes, only 9 (1.8%) reached the blastocyst
stage.
The high inefficiency of the ICSI procedure in cattle led researchers to evaluate
different chemical activation treatments and sperm pretreatments in order to increase
pronuclear formation and blastocyst rates. Li et al. (1999) investigated the effect of
activating sperm-injected oocytes with ethanol. In this study, sperm were decapitated by
sonication. They reported a 15.1% and 26.4% sperm decondensation and pronuclei
16
formation at 18 to 20 hours post-injection, respectively. The use of sperm heads for ICSI
will enable the use of flow cytometrically sorted sexed semen for gender preselection.
Hamano et al. (1999) reported the birth of 8 male and 2 female calves using flow
cytometrically sex sorted sperm heads. Oocytes were activated for 5 minutes in 7%
ethanol once before and twice after sperm injection (at 30 and 60 minutes). Cleavage
and blastocyst rates were 46.6% and 6.9%, respectively. They obtained a 20.8%
pregnancy rate (10/48) from the blastocysts nonsurgically transferred into recipient
females.
Sperm pretreatments were evaluated by Wei and Fukui (1999) in pursue for an
answer for the low fertilization rates of ICSI oocytes in cattle. They investigated the effect
of sperm type (dead, immotile or motile), sperm mechanical pretreatment (tail cutting or
scoring) and sperm chemical pretreatment (heparin, heparin and caffeine, calcium
ionophore (A23198) or dithiothreitol (DTT) on fertilization rates 20 hours after sperm
injection. No significant difference was found when dead (21.8%), immotile (20.8%) or
motile sperm (11.3%) without mechanical pretreatment were injected into oocytes.
However, the rate of pronuclear formation with motile sperm tended to be lower than
those with dead or immotile sperm. When motile sperm was used, tail scoring (36.4%)
yielded higher pronuclear formation than the control at 11.3%. However, no significant
difference was found between tail scoring (36.4%) and tail cutting (22.8%). All chemical
pretreatment agents used significantly increased the formation of the male pronucleus
20 hours post-injection. Pretreatment with calcium ionophore (A23187) (58.9%) or DTT
(61.3%) resulted in higher fertilization rates than those pretreated with heparin alone
(32.8%) or the control (23.2%). Fertilization rates were also higher with heparin and
caffeine treatment (46.4%) than in the control. In other studies DTT has also been
demonstrated to enhance bovine male pronuclear formation and blastocyst rates (Rho et
al., 1998a; Wei and Fukui, 2002; Galli et al., 2003; Ock et al., 2003).
The development of the piezo ICSI technique in the mouse by Kimura and
Yanagimachi (1995) led to a dramatic increase in fertilization and blastocyst rates when
compared with those of the conventional technique. With this novel technique, the zona
pellucida and oolema are penetrated by rapid vibrations produced at the pipette tip
caused by longitudinal wavelength produced by a piezo actuator. One of the main
advantages of the piezo technique is that it causes less deformation to the oocyte during
17
injection. The oolema is easily broken without suction of the ooplasm and produces less
ooplasm leakage increasing oocyte survival post sperm injection. Katayose et al. (1999)
were the first to apply this technique in cattle and reported a dramatic increase in
fertilization rates with piezo ICSI (55%) when compared with the conventional technique
(0.05%) when no exogenous chemical activation was used. The addition of 50 M
calcium ionophore (A23187) to activate the oocytes increased fertilization rates for the
conventional technique to 15%, while no beneficial effect was found in the piezo ICSI
group (58%).
In contrast, a year later Suttner et al. (2000) reported in cattle no significant
difference between the piezo and conventional ICSI techniques as assessed by
pronuclear formation, cleavage and blastocyst rates. They evaluated different chemical
agents to activate sperm injected oocytes. Ionomycin followed by 6-dimethylaminopurine
(DMAP) produced the highest quantity of blastocysts when compared with calcium
ionophore (A23187) and ionomycin followed by cycloheximide. This is in agreement with
others where the highest quantities of blastocysts were produced by using ionomycin
followed by DMAP (Ock et al., 2003). However, the use of ionomycin followed by DMAP
was found to produce up to 67% chromosomal abnormalities as assessed by polyploidy
and mixploidy (Ock et al., 2003). This high percentage of abnormal embryos was slightly
reduced by allowing a 3 hour delay in DMAP treatment (46% mixoploid and polyploid).
The occurrence of chromosomal abnormalities may be due to inhibition of extrusion of
the second polar body, with some nuclei presumably reentering S-phase of the cell cycle
without having passed through metaphase.
Horiuchi et al. (2002) produced 5 healthy calves from the transfer of 10
blastocysts activated with 7% ethanol 4 hours post-injection. They reported that ~60% of
the sperm-injected oocytes were activated by the spermatozoon as assessed by second
polar body extrusion 4 hours post-piezo ICSI. It was concluded that treating activated
oocytes 4 hours post-injection with 7% ethanol for 5 minutes, increased subsequent
embryo development. By treating activated oocytes with or without ethanol, they
obtained 20% and 11.9% blastocyst rates, respectively. Moreover, they demonstrated
that using killed sperm readily affects embryo development to the blastocyst stage when
compared with motile sperm immobilized prior to injection. Only 0.8% of activated
oocytes reached the blastocyst stage when killed sperm were used.
18
Fujinami et al. (2004) studied the effects of artificial activation with ethanol on
kinetics of maturation promoting factor (MPF) activity and development of bovine
oocytes following ICSI. They reported that MPF activity decreases immediately after
sperm injection but elevates temporarily at 6 hours post-injection. This temporary
elevation of MPF was inhibited when sperm-injected oocytes were treated with ethanol 4
hours post-injection. They obtained 14 and 4% blastocyst rates when sperm-injected
oocytes were treated with or without ethanol 4 hours after sperm injection, respectively.
Wei and Fukui (2002), using the piezo ICSI technique, were the first to report the
birth of 3 calves from ICSI derived embryos without any exogenous oocyte activation.
They felt that the poor outcome in bovine ICSI could be improved by making technical
improvements. The oocyte ooplasm was made clearer by centrifugation, the sperm tail
was cut leaving the midpiece intact and the amount of polyvinylpyrrolidone (PVP) was
reduced from 10% to 4% to ensure proper delivery of sperm into the oocytes. With these
adjustments, they reported 78.2, 71.8 and 22.7% fertilization, cleavage and blastocyst
rates, respectively.
Subsequently, Galli et al. (2003) also concluded that exogenous oocyte
activation did not improve the development of piezo ICSI embryos. They obtained 15, 6
and 17% blastocyst rates when sperm-injected oocytes were not treated or were treated
with ionomycin or ionomycin followed by cycloheximide, respectively. One healthy calf
was produced by the nonsurgical transfer of 11 nontreated piezo ICSI blastocysts into 6
recipient females.
Rho et al. (2004) reported the use of frozen-thawed bovine oocytes for ICSI.
Oocytes were cryopreserved by vitrification with copper electron microscope grid. They
reported that blastocyst rates of 9.8% and 16.3% were significantly different when using
frozen-thawed and fresh oocytes, respectively. They detected a significant difference
(P<0.05) between the treatment groups. Total cell counts on day 8 post-injection were
9919 and 12421 for frozen-thawed and fresh oocytes, respectively. It was concluded
that frozen-thawed bovine oocytes were suitable for ICSI to produce transferable
embryos.
Freeze drying sperm has been attempted as an alternative method to preserve
sperm in humans, (Katayose et al., 1992), hamsters (Hoshi et al., 1994), rabbits (Hoshi
et al., 1994), mice (Wakayama and Yanagimachi, 1998), cattle (Keskintepe et al., 2002),
19
swine (Kwon et al., 2004) and rats (Hirabayashi et al., 2005). Live offspring have been
reported in mice (Wakayama and Yanagimachi, 1998; Kusakabe et al., 2001; Kaneko et
al., 2003; Ward et al., 2003), rabbits (Liu et al., 2004) and rats (Hirabayashi et al., 2005)
after sperm injection of oocytes with freeze dried sperm.
Freeze drying is a procedure designed to achieve preservation by restricting the
active water in a biological system. In this process frozen material is dried through the
sublimation of ice (Keskintepe et al., 2002). In contrast, maintenance of cryopreserved
sperm is higher in cost due to the need of a constant supply of liquid nitrogen (Kawase
et al., 2005). Moreover, in some geographical areas, LN2 is very costly or in some cases,
not available to store sperm. Freeze drying allows the storage of sperm at room
temperature or in a household refrigerator and sperm can be reconstituted by the
addition of water when needed for ICSI (Wakayama and Yanagimachi, 1998). In cattle,
Keskintepe et al. (2002) reported cleavage and blastocyst rates for oocytes injected with
lyophilized sperm and activated with ionomycin followed by DMAP of 63.3% and 29.6%,
respectively. No significant difference was detected with lyophilized sperm when
compared with frozen-thawed sperm (72.4% and 34.2% cleavage and blastocyst rates,
respectively).
Recently, heat drying was tried as an alternate way to preserve sperm (Lee and
Niwa, 2006). The male gamete has been shown previously to have thermostability
(Yanagida et al., 1991). Hoshi et al. (1992) reported that Injecting rabbit sperm heated to
60C for 30 minutes into oocytes resulted in the development of embryos up to the 8 cell
stage. In addition, live offspring were produced in mice by injecting sperm that had been
heated at 56C for 30 minutes (Cozzi et al., 2001). The ability of sperm to withstand
heating, led investigators to evaluate heat drying as a method to preserve sperm. It is a
much simpler and less expensive way to preserve sperm than freeze drying and
conventional cryopreservation (Lee and Niwa, 2006). In cattle, Lee and Niwa (2006)
reported the development of ICSI blastocysts from sperm that had been heat dried in a
conventional oven at 50C for 8 hours and stored at 4C for up to 1 year. However, a
significant decline in blastocysts was found when heat-dried sperm was stored for longer
than 1 month before ICSI when compared with that of the control sperm.
The highest pregnancy and birth rates to date were reported for piezo ICSI
bovine embryos treated with 7% ethanol 4 hours after sperm injection (Oikawa et al.,
20
2005). This study compared activation protocols for blastocyst development. They
reported 8.0, 29.4 and 40.1% blastocyst rates for sperm-injected oocytes nontreated,
treated with 7% ethanol 4 hours post-injection or with ionomycin followed by DMAP,
respectively. Although the percentage of blastocysts was higher for ionomycin followed
by DMAP group. The subsequent pregnancy and birth rates were significantly higher for
the ethanol-treated group. One (12.5%) of the 11 recipients that received blastocysts
produced following the ionomycin-DMAP activation became pregnant. However,
blastocysts from the ethanol-treated group resulted in 10 of 17 (58.8%) pregnant
recipient females. The birth rate per transferred embryo was 47.4% and 9.2% for the
ethanol and ionomycin-DMAP-treated piezo ICSI oocytes, respectively. More recently,
Horiuchi (2006) reported that 24 calves were produced by the nonsurgical transfer of 61
ICSI-derived blastocysts activated with ethanol 4 hours after sperm injection.
With the exception of the birth of a few calves from sperm-injected oocytes
without the need of an exogenous activation stimulus (Wei and Fukui, 2002; Galli et al.,
2003), ICSI techniques have failed to produce expected rates of embryonic and fetal
development (Malcuit et al., 2006). Recently, Malcuit et al. (2006) attributed the ICSI
problem in bovine to a failure of the injected spermatozoon to initiate and maintain
prolonged [Ca2+] oscillations required for subsequent embryonic development. They
noted that less than 10% of sperm-injected oocytes displayed any [Ca2+] responses. It is
thought that some of the signaling mechanisms that lead to the activation of the
phosphoinositide pathway and generation of oscillations during natural fertilization are
not replicated by the ICSI procedure. The possibilities are that the release of the sperm
factor or the function and activation of the sperm factor after release may be
compromised after ICSI (Malcuit et al., 2006). It was recently demonstrated that injected
bovine sperm does not undergo comparable dissolution of the sperm perinuclear theca,
as occurs during IVF (Sutovsky et al., 1997; Sutovsky et al., 2003).
MULTICOLOR FLOW CYTOMETRY FOR SPERM QUALITY EVALUATION
Most laboratory assays used to assess the fertilizing capacity of a semen sample
before its use, have relied in the evaluation of motility, morphology and sperm
concentration. Unfortunately, these routinely used laboratory assays have not been very
successful in predicting the fertilizing capacity of sperm in a given sample (Gillian et al.,
2005). Correlations between sperm motility and fertility and/or sperm morphology and
21
fertility have been reported to range from 0.15 to 0.83 and 0.06 to 0.86, respectively
(Graham et al., 1980; Kjaestad et al., 1993; Stalhammar et al., 1994; Janaskuskas et al.,
2003). Sperm require a number of events to occur to achieve fertilization, such as, ATP
production for sperm movement, achieve hyperactivation, ability to capacitate, undergo
the acrosome reaction, a functional plasma membrane, have the ability to recognize and
bind the zona pellucida and possess a normal DNA complement (Gillian et al., 2005).
Multicolor flow cytometry is a powerful tool for evaluating the quality of sperm in a
given sample. This technology works by forcing individual sperm cells into a confined
stream of fluid that passes through the beam of a laser that will cause any fluorescent
stain associated with the sperm cell to fluoresce (Graham, 2001). Photomultiplier tubes
permit the determination of specific wavelengths emitted by individual compartments in
each sperm cell that have been previously stained with a fluorescent dye (Graham and
Moce, 2005). This permits the observation of physical characteristics including cell size,
shape and internal complexity (Gillian et al., 2005). The advantage of flow cytometry is
that thousands of cells can be evaluated in less than a minute, the amount of fluorescent
stain associated with cells is measured in an unbiased manner, it offers a high level of
accuracy and experimental repeatability, and can evaluate multiple sperm characteristics
at the same time (Graham, 2001; Gillian et al., 2005). Moreover, fixed or live sperm cells
can be used for evaluation and a large variety of fluorochromes are currently available to
measure most sperm characteristics (Graham and Moce, 2005). Using flow cytometric
assays, cell viability, capacitation status, acrosome integrity, mitochondrial function and
DNA integrity can be evaluated.
Viability
Cell viability is referred in most cases to whether or not the plasma membrane of
the spermatozoon is intact. One of the consequences of membrane disruption is the loss
of important intracellular components, such as metabolic enzymes and ATP (Graham
and Moce, 2005). In addition, plasma membrane integrity status is of extreme
importance due to its role in interacting with the female genital tract, the outer vestments
of the cumulus oocyte complex and fusion with the ooplasm (Rodriguez-Martinez, 2003).
Many different fluorochromes have been used to assess the integrity of the sperm
plasma membrane. This can be achieved by using fluorochromes that select viable or
nonviable sperm. Viable sperm can be measured using membrane permeable stains,
22
which are converted into a nonpermeant fluorescent compound by esterases in the
cytoplasm of live cells. The most used enzyme based compounds are fluorescein
diacetate (FDA) (Resli et al., 1983), 6-carboxylfluorescein diacetate (CFDA) (Garner et
al., 1986), 6-carboxylmethylfluorescein diacetate (CMFDA) (Thomas and Garner, 1994)
and calcein acetoxy methyl ester (CAM) (Kato et al., 2002). However, most recent
assessments of bovine sperm viability have used the membrane permeable DNA
fluorochrome, SYBR 14 (Garner et al., 1994). Viability assessments using nucleic acid
stains are considered less variable than enzyme-based stains. Enzyme-based stains
suffer from time dependency problems because they are based on enzyme substrate
conversion to a fluorescent product (Garner et al., 1995). Also, sperm DNA is considered
to be a more appropriate cellular target due to its stainability and staining uniformity
(Gillian et al., 2005).
The percentage of nonviable cells in a sperm sample can be measured using
membrane impermeable nucleic acid stains, which identify nonviable sperm by entering
cells with ruptured membranes. The most commonly used are Hoechst 33258 (Pintado
et al., 2000), ethidium homodimer-1 (Althouse and Hopkins, 1995), YO-Pro (Kavak et al.,
2003) and propidium iodide (PI) (Matyus et al., 1984). Each fluorochrome has different
excitation and emission ranges making it suitable for multicolor assays. Nowadays,
viability using a flow cytometer is routinely assessed by dual staining sperm with the
combination of a membrane permeable and membrane impermeable fluorochrome. The
two most common combinations are CFDA with PI or SYBR 14 and PI. CFDA is
converted to a green fluorescent molecule by esterases in the cytoplasm of viable
sperm, while PI is a red fluorescent molecule that labels the nucleus of sperm with
damaged membranes. SYBR 14 is also a green fluorescent molecule that labels the
nucleus of membrane intact sperm. By staining with any of these two combinations, two
populations of sperm can be identified. Events exhibiting brighter green fluorescence in
the FL1 channel are considered live sperm, while events exhibiting high red
fluorescence in the FL3 channel are considered dead sperm.
Before 1986, the supravital stains used to assess viability, such as eosin Y alone
or in combination with nigrosin or elanin blue, were not appropriate for analyzing
cryopreserved sperm because glycerol interfered with the stains (Mixner and Saroff,
1954). Sperm from bulls, boars, dogs, horses, mice and humans were evaluated to
23
examine the use of CFDA in combination with PI using flow cytometry (Garner et al.,
1986). They demonstrated that motile sperm retained the CFDA fluorochrome
throughout the cytoplasm of the cell and did not stain with PI. However, some immotile
sperm retained the CFDA fluorochrome in the cytoplasm. The authors claim that this
small population of sperm was composed of moribund sperm. Moreover, the percentage
of sperm that were positive for CFDA increased after a swim-up procedure. They found
a high correlation in the percentage of CFDA-positive sperm and progressive motility. In
addition, they reported that the major advantage of using this dual staining protocol was
that cryopreserved sperm containing glycerol could be analyzed.
Garner et al. (1994) were the first to use SYBR 14 in combination with PI to
evaluate viability in sperm. They validated the assay by evaluating mixtures of nonviable
and viable sperm. Viable bovine sperm were prepared by filtration through glass wool
and Sephadex to remove nonviable sperm. A sample from the viable population was
killed by repeated freezing and thawing without cryoprotectants. A mixture of 100:0,
75:25, 50:50, 25:75 and 0:100 of the filtered:killed samples were used to validate the
viability assay. The percentages of viable sperm for the ratios evaluated, as determined
by the green fluorescence of the SYBR 14 emitted in the FL1 channel, were: 85.1, 68.8,
39.8, 20.7, and 1.4%, respectively. Subsequently, Garner et al. (1995) evaluated the
viability assay in the mouse, boar, human, ram and rabbit. They reported that this flow
cytometric assay detected subtle differences between males in all the species evaluated.
To date, this staining combination has been shown to be a reliable way for determining
the proportions of viable and nonviable sperm in a given semen sample (Donoghue et
al., 1995; Maxwell et al., 1996; Garner et al., 1997; Maxwell and Johnson, 1997; Thomas
et al., 1998; Merkies et al., 2000; Garner et al., 2001; Love et al., 2003; Guerrero et al.,
2006; Saragusty et al., 2006). Viability stains can be used in combination with other
stains for evaluating multiple quality traits at the same time by using a flow cytometer
capable of detecting 3 or more colors.
Capacitation
For a sperm to undergo the acrosome reaction and achieve fertilization, the
sperm must first become capacitated. Capacitation is the process in which changes
occur within the sperm, such as cholesterol efflux from the plasma membrane (Langlais
and Roberts, 1985), increase in intracellular [Ca2+] (Parrish et al., 1999), bicarbonate,
24
protein phosphorylation and a decrease in intracellular pH. Cholesterol efflux and the
increase in disorder in lipid packaging leads to an increase in plasma membrane fluidity
(Gadella et al., 1999), which can be measured with the hydrophobic fluorochrome
Merocyanine 540 (M540). These changes lead to destabilization of the membrane,
which eventually leads to the acrosome reaction required to penetrate the outer
vestments of the oocyte. M540 is believed to detect a decreased packing order of
phospholipids in the outer leaflet of the plasma membrane lipid bilayer (Williamson et al.,
1983). Kavak et al. (2003) reported that the quantity of capacitated sperm increased
significantly after the incubation of bull sperm in a bicarbonate containing medium. It is
thought that membrane fluidity changes precede the increase in intracellular [Ca2+],
making M540 a better method for evaluating the early events of capacitation (Rathi et
al., 2001).
Acrosome Integrity
An intact acrosome is required for a sperm to penetrate the cumulus oophorus
cells and to attach and penetrate the zona pellucida of the oocyte at the time of
fertilization (Franklin et al., 1970). The acrosome reaction involves vesiculation of part of
the outer acrosome membrane and the overlaying plasma membrane (Bedford, 1968).
After vesiculation and release of the hydrolytic enzymes stored in the acrosome, some
sugar moieties, such as -mannose and -galactose remain exposed to the external
environment. There are a large number of plant lectins currently available that can be
used to detect reacted acrosomes by attaching to exposed sugar moieties in the reacted
acrosome compartment. A strong correlation between fertility and the percentage of
bovine sperm exhibiting intact acrosomes has been previously reported (Berndtson et
al., 1981).
Flow cytometry can be used to detect the percentage of sperm exhibiting a
reacted acrosome by using plant lectins labeled by a fluorescent probe (Gillian et al.,
2005). The most common plant lectins used to evaluate acrosome integrity are Pisum
sativum agglutinin (PSA) (Graham et al., 1990) and Arachis hypogaea agglutinin (PNA)
(Vasquez et al., 1990). PSA binds to -mannose and -galactose moieties in the
acrosome compartment, while PNA binds more specifically to -galactose moieties.
Since these lectins are not membrane permeable, only acrosome reacted or damaged
sperm will fluoresce (Farlin et al., 1992). The use of PSA to assess acrosome integrity
has been widely used (e.g., Cross and Meizel, 1989; Graham et al., 1990; Kinger et al.,
25
1995; Margalit et al., 1997; Pena et al., 1999; Pena et al., 2001), however, PNA is
preferred because it exhibits less nonspecific binding to other areas of the sperm and
has less affinity to egg yolk particles when working with cryopreserved sperm (Dalimata
and Graham, 1997; Thomas et al., 1997).
Plant lectins can be conjugated to many different commercially available
fluorescent probes in order to make them suitable for multicolor flow cytometric assays
(Graham, 2001). PI in combination with fluorescein isothiocyanate (FITC) PSA (Sukardi
et al., 1997) or FITC-PNA (Rathi et al., 2001) has been widely used to discriminate
between nonviable acrosome reacted and viable acrosome reacted sperm. Recently,
Nagy et al. (2003) developed a triple fluorochrome staining technique that involves the
well validated SYBR 14 and PI, with PNA conjugated to phycoerythrin (PE). PE
fluorescent moiety was used instead of FITC, because its fluorescence emission could
be measured independently from that of SYBR 14 or PI. This novel technique was
shown to assess viable acrosome intact, viable acrosome reacted, nonviable acrosome
intact and nonviable acrosome reacted sperm in a single assay.
Acrosome integrity can also be measured using the acidophilic probe
LysoTracker Green DND-26 (LysoTracker). This probe works by labeling acidic
organelles, such as the intact acrosome. Thomas et al. (1997) found a high correlation
between LysoTracker and the lectin PNA for determining acrosome integrity of bovine
sperm. In addition, they reported a high correlation between the percentages of sperm
stained positive for LysoTracker and sperm displaying normal acrosome ridges.
Mitochondrial Function
Motility is currently the most commonly used to evaluate sperm quality in
commercial bull stations. At best it is an indicator or an indirect measure of metabolic
activity and cell viability (Hallap et al., 2005). Mitochondria are the major organelles for
the production of adenosine triphosphate (ATP) required for sperm motility (Bartoov et
al., 1980). ATP production is very important for maintaining flagellar movement for a long
period of time. This is required for sperm to reach the site of fertilization (Windsor, 1997).
Changes in mitochondrial membrane potential has been proposed to be a good indicator
of sperm functional impairment (Pena et al., 2003). Pyruvate and lactate are produced
from glycolysis in the flagella and diffused to the midpiece where mitochondria
metabolize them to produce ATP via respiration (Mukai and Okuno, 2004). Flagellar
26
movement is the product of dynein ATPase activity that is localized along the entire
flagellum and depends on the supply of ATP (Mukai and Okuno, 2004).
There are ~100 mitochondria in the midpiece of each mammalian sperm, and
they can be visualized with various fluorochromes (Hallap et al., 2005). The evaluation of
mitochondrial function in a sperm sample should give us a more objective assessment of
the energetic status of the cells (Martinez-Pastor et al., 2004). Furthermore,
mitochondrial activity has been related to cell motility (Auger et al., 1989; Garner et al.,
1997; Thomas et al., 1998; Garner and Thomas, 1999; Garner et al., 2001; Martinez-
Pastor et al., 2004; Nunez et al., 2004; Hallap et al., 2005; Januskauskas et al., 2005),
viability (Garner et al., 1997; Januskauskas et al., 2005) and fertility (Kasai et al., 2002).
A number of commercially available fluorochromes have been used to assess
mitochondrial status of sperm. Rhodamine 123 (R123) is a cationic compound that
excites at 488 nm and emits at 515-575 nm (Evenson et al., 1982). R123 is transported
into actively respiring mitochondria and their accumulation in the mitochondria causes
them to fluoresce green (Graham, 2001). The mitochondrial inhibitors, monensin and
rotenone, were shown to decrease the uptake of R123 by sperm (Graham et al., 1990).
In humans, Auger et al. (1989) found a strong correlation between sperm motility and
R123 uptake during a 24-hour incubation period. Also, Graham et al. (1990) reported
that R123 was accumulated and retained most intensively in fully active bovine sperm
mitochondria. Mitochondria from nonviable sperm did not uptake R123. They
demonstrated that sperm treated with sodium fluoride (to inhibit glycolysis) displayed
positive R123 staining as a positive control. R123 is considered not to be very reliable
due to its low sensitivity, variability within samples and background staining (Garner et
al., 1997). In addition, its fluorescence is lost when mitochondria experience losses of
membrane potential, thus limiting its use for fixation. R123 also has been reported to
have a higher nonspecific affinity to other parts of the sperm than other mitochondrial
fluorochromes (Garner et al., 1997).
MitoTracker Probes offers a wide range of mitochondrion selective probes, such
as MitoTracker Orange CMTMRos, MitoTracker Red CMXRos and MitoTracker Deep
Red 633. These probes differ in spectral characteristics making them suitable for
multicolor flow cytometric analyses. These fluorochromes work by accumulating in active
mitochondria and are well retained during fixation. They contain a mildly thiol-reactive
27
chloromethyl moiety that is responsible for keeping the fluorochrome associated with the
mitochondria after fixation. Hallap et al. (2005) used MitoTracker Deep Red 633 in
combination with SYBR 14 to evaluate mitochondrial activity in bovine sperm. They
reported a high correlation with motility both after cryopreservation and the swim-up
procedure. Furthermore, no interference with glycerol or egg yolk from the freezing
extender was observed. Although functioning mitochondria are stained with these
fluorochromes, these stains do not permit one to differentiate among mitochondria that
exhibit different respiratory rates (Graham, 2001).
The lipophilic cationic fluorescent carbocyanine dye, JC-1, has been used to
evaluate mitochondrial membrane potential in sperm in the human (Kasai et al., 2002),
bull (Garner et al., 1997; Garner and Thomas, 1999), stallion (Grevance et al., 2000), rat
(Grevance et al., 2001), boar (Huo et al., 2002), Iberian Red deer (Martinez-Pastor et al.,
2002) and ram (Martinez-Pastor et al., 2004). This fluorochrome permits the distinction
of mitochondria exhibiting low or high membrane potential (Thomas et al., 1998). This
fluorochrome can reversibly change its emission from green to red with increasing
mitochondrial membrane potential (Garner and Thomas, 1999). In mitochondria with low
membrane potential, JC-1 forms monomers which emit in the green wavelength when
excited at 488 nm. In mitochondria exhibiting high membrane potential, JC-1 forms
aggregates emitting in the orange-red wavelength when excited at 488 nm (Pena et al.,
2003). In humans, in vitro fertilization rates were reported to be higher for sperm
exhibiting high mitochondrial membrane potential (Kasai et al., 2002). Furthermore, a
relationship was found between high membrane potential and fertility in humans
(Troiano et al., 1998). However, JC-1 appears to be not suitable for multi-parameter
assays due to its emission in both the green and red wavelengths. Moreover, Garner et
al. (1999) reported that it does not work effectively on frozen-thawed bovine sperm,
probably due to interactions with egg yolk particles from the freezing extender.
DNA Integrity
The nucleus of normal sperm has a highly condensed chromatin structure
(Yanagimachi and Noda, 1970). The major function of the male sperm cell is to transfer
genetic information to the future zygote. In mammals, the sperm nucleus is
transcriptionally inactive (Imschenetzky et al., 2003) and its chromatin is compacted at a
degree of condensation 6-fold higher compared with a somatic cell nucleus (Ward and
28
Coffey, 1991). This is achieved by the replacement of somatic histones by a series of
basic proteins and finally by protamines (Ward and Coffey, 1991). This structure of
protamine complexed DNA is likely important for protection of the genetic information
(Ward and Coffey, 1991). Various studies have demonstrated that chromatin structure is
related to the fertilizing ability of sperm, serves as a biomarker for exposure to toxic
chemicals and to detect disturbances during spermatogenesis (Evenson, 1986;
Ballachey et al., 1987; Dobrinski et al., 1994; Evenson and Jost, 1994). DNA
fragmentation can be caused during spermatogenesis by apoptosis caused by activation
of endonucleases or during ejaculation by reactive oxygen species (Evenson, 1999).
The DNA integrity of sperm can be determined by the sperm chromatin structure
assay (SCSA) (Evenson et al., 1980). The SCSA is a flow cytometric assay that
utilizes the metachromatic properties of the dye acridine orange (AO), staining single
stranded DNA red and double stranded DNA green (Evenson et al., 1980). It is based on
the principle that sperm with an abnormal chromatin structure have a greater
susceptibility to physical induction of partial DNA denaturation in situ (Darzynkiewicz et
al., 1975). The extent of DNA denaturation following acid treatment is measured by the
ratio of red to total fluorescence providing an index of normality to abnormality. The
percentage of sperm exhibiting denatured DNA has been related to high, moderate or
very low fertility rates when a semen sample has DNA damage between 0 to 15%, 16 to
29% and >30%, respectively (Evenson and Jost, 2000). The SCSA has been used in
the bull (Evenson et al., 1980; Ballachey et al., 1987; Bocheneck et al., 2001;
Januskauskas et al., 2003), rat (Evenson et al., 1983), mouse (Evenson et al., 1993;
Sailer et al., 1997), boar (Evenson et al., 1994; Boe-Hansen et al., 2005), Rhesus
monkey (Sivashanmugam and Rajalakshmi, 1997), stallion (Love and Kenney, 1998;
Love, 2005; Dias et al., 2006), human (Spano et al., 1999; Larson et al., 2000; Saleh et
al., 2002; Boe-Hansen et al., 2006; Evenson, 2006), rabbit (Gogol et al., 2002), cat
(Penfold et al., 2003), ram (Peris et al., 2004) and dog (Nunez-Martinez et al., 2005),
and has shown a good correlation with fertility in mice (Evenson et al., 1980), bulls
(Evenson et al., 1980; Ballachey et al., 1988; Karabinus et al., 1990; Sailer et al., 1996),
boars (Evenson et al., 1994), stallions (Love and Kenney, 1998) and humans (Evenson
et al., 1999; Spano et al., 2000; Evenson and Jost, 2000; Virro et al., 2004).
29
CHAPTER III
EFFECT OF STORAGE AND CRYOPROTECTANT ADDITION AND REMOVAL AT 4C

ON MOTILITY AND PLASMA MEMBRANE INTEGRITY OF BOVINE CAUDAL
EPIDIDYMAL SPERM
INTRODUCTION
Cryopreservation of sperm harvested from the epididymides would salvage
germplasm from genetically valuable males that are injured and can no longer mate or
have unexpectedly died. Over the past few decades, most research on sperm
cryopreservation has focused on comparative studies, such as changing cryoprotectant
(CPA) concentration, additives, cooling and warming rates and extender composition
(Holt, 2000). Although these studies have helped to improve sperm quality in a fixed set
of circumstances, they fail to identify important problems in sperm cryobiology, and are
of limited use in performing major advances in this area (Holt, 2000). In an effort to
develop a cryopreservation protocol for epididymal sperm, there needs to be a better
understanding of the underlying fundamental cryobiological properties of epididymal
sperm. Studies of membrane permeabilities, osmotic tolerance limits and on theoretical
approaches to predict minimally damaging cooling protocols have been successful in
allowing post-thaw sperm recovery (Gao et al., 1995; Guthrie et al., 2002).
Since the first use of glycerol for sperm cryopreservation by Polge et al. (1949),
the development of a more complete understanding of the mechanisms by which CPAs
protect cells during cooling and warming has been emphasized (Gilmore et al., 1997). It
is well known that the addition of CPAs is essential for sperm optimal survival following
the freezing process. However, CPAs can cause loss in sperm viability due to osmotic
damage or from true chemical toxicity. The addition of CPAs before freezing and their
removal after warming creates an anisotonic environment, resulting in potentially
damaging osmotically driven cell volume changes (Guthrie et al., 2002). During the
addition of permeating CPAs, sperm are exposed to a hypertonic environment, resulting
in cell shrinkage. During CPA removal, sperm are exposed to a hypotonic environment,
resulting in cell swelling. Such changes in solution osmolality can cause loss in the
functional integrity of sperm (Guthrie et al., 2002; Ball and Vo, 2001). It has become
evident that sperm from males of different species possess different sensitivities to
30
osmotic damage. Thus, it is of critical importance that the osmotic behavior of bovine
epididymal sperm be characterized to develop a successful cryopreservation protocol.
Tolerance to osmotic stress during the addition and removal of CPAs is highly
predictive of sperm survival after cryopreservation (Holt, 2000). This suggests that
sperm survival can be optimized by minimizing osmotic shock. There is limited
information regarding osmotic properties and cryopreservation of bovine caudal
epididymal sperm. Epididymal sperm and its ejaculated counterpart likely react
differently to cryoprotective agents due to differences in morphology and membrane
properties. Cytoplasmic droplets found on epididymal sperm are thought to cause an
increase in plasma membrane damage and cause bent tails during cryopreservation.
Theoretical predictions indicated that the optimal CPA would be one that could permeate
the cell in the shortest time causing the least amount of volume excursion during the
addition and removal of the CPA (Gilmore et al., 1995; Gao et al., 1995; Gilmore et al.,
1997). Thus, this problem might be overcome by using CPAs with higher membrane
permeabilities.
Considerable research efforts in rams (Curry and Watson, 1994), humans
(Gilmore et al., 1995; Gilmore et al., 1997), bulls (Liu and Foote, 1998; Guthrie et al.,
2002), boars (Gilmore et al., 1998), mice (Phelps et al., 1999) and stallions (Ball and Vo,
2001) have focused the attention to the osmotic stress attributed to the relative
permeabilities of different CPAs and water. However, no information is yet available
regarding addition and removal of CPAs in bovine epididymal sperm.
The use of ethylene glycol to cryopreserve different cell types has widely
increased after its first use with human erythrocytes by Lovelock (1954). Ethylene glycol
was found to have the highest membrane permeability of the most commonly used
CPAs (Gilmore et al., 1997; Gilmore et al., 1998). In swine, the rapid movement of
ethylene glycol through the plasma membrane was shown to cause less volume
excursions than glycerol (Gilmore et al., 1998). In the present study, we tested the
effects of addition and removal of two different concentrations of ethylene glycol and the
commonly used CPA, glycerol to select the cryoprotectant that offers the least osmotic
damage to epididymal sperm for future cryopreservation studies.
Another problem associated with permeating CPAs is the toxicity that they may
incur in sperm during the equilibration process prior to cryopreservation. Due to the
31
different protocols used in research laboratories, the length of sperm exposure to CPAs
varies widely. Sperm are usually in contact with permeating cryoprotectants for hours
before freezing. Prolonged exposure to cryoprotectants will likely shorten the life span of
sperm due to chemical toxicity. Field conditions often preclude the immediate availability
of liquid nitrogen, necessitating the development of alternative short term storage
methods. To our knowledge, no study has investigated the quality of bovine epididymal
sperm during cool storage (4C) for an extended length in a common Tris based
extender. In addition, the toxicity of prolonged exposure of different concentrations of
glycerol and ethylene glycol to bovine epididymal sperm during cool storage has not
been evaluated to date.
The objectives of this study were: (1) to assess the effect of addition and removal
of different concentrations of permeating cryoprotectants to epididymal sperm at 4C (2)
to test the toxicity caused by permeating cryoprotectants during prolonged exposure to
epididymal sperm during cool storage and (3) to investigate viability parameters of
epididymal sperm stored at 4C for 5 days.
MATERIALS AND METHODS
Experimental Design
Experiment 3.1
This experiment was conducted to determine the effect of the addition and the
removal of different concentrations of permeating CPAs on subsequent sperm quality.
Testes pairs were obtained from 10 mature bulls of various breed types at a local
abattoir and transported to the laboratory within 4 to 6 hours postmortem. Epididymal
sperm were harvested by multiple incisions from the caudae epididymides of each pair
of testes, then pooled and cooled in egg yolk Tris-glucose citric acid monohydrate
extender (EYT-GC) to 4C at ~0.1C/minute.
Pooled sperm from each bull were allocated to each of five treatment groups:
Control (no CPA), 7% glycerol (GLY), 14% GLY, 7% ethylene glycol (EG) and 14% EG.
Replicate samples (n=2) from each bull were diluted 1:1 in EYT-GC extender containing
twice the final desired concentration of CPA. After 10 minutes, each sample was diluted
directly into EYT-GC at 4C. Motility was assessed using a computer assisted semen
analysis system and sperm plasma membrane integrity was evaluated using fluorescent
microscopy following staining with SYBR 14 and propidium iodide.
32
Experiment 3.2
This experiment was conducted to determine the toxic effect of permeating CPAs
during 5 days of storage at 4C on sperm quality. Replicate samples (n=2) from
Experiment 3.1 were diluted 1:1 in EYT-GC extender containing twice the final desired
concentration of CPA and stored at 4C for 5 days. A 0.5 ml sample from each treatment
group was taken on days 0, 1, 3 and 5 and diluted directly into Brackett-Oliphant
medium to assess sperm quality. Motility was assessed using a computer assisted
semen analysis system and plasma membrane integrity was determined using
fluorescent microscopy.
Experimental Procedure
Testes Collection
Bovine testes were collected as pairs from mature mix breed bulls (n=10) at an
abattoir in Robert, Louisiana. Testes were transported to the Louisiana State University
Veterinary School individually packed in Ziploc plastic bags (Johnson & Son, Inc.,
Racine, WI) in a Styrofoam box at a temperature that ranged between 25C to 28C
within 4 to 6 hours postmortem. Immediately upon arrival to the laboratory, each pair of
testes was dissected away from its scrotum and tunica vaginalis (Figure 3.1). Testes
were positioned with the vas deferens facing ventrally and the corpus facing up to
distinguish the side of origin of each testis. Bulls with anatomically abnormal testes were
not used in this study.
Medium Preparation and Use
Egg yolk Tris-glucose citric acid monohydrate extender (EYT-GC) (250 mM Tris
(T-6066, Sigma-Aldrich Inc., St. Louis, MO), 69 mM glucose (6152, Sigma-Aldrich Inc.,
St. Louis, MO), 81 mM citric acid monohydrate (C-1909, Sigma-Aldrich, St. Louis, MO)
and 20% (v/v) dried egg yolk (E-0625, Sigma-Aldrich, St. Louis, MO) was prepared with
the addition of 50 g/ml of gentamicin (15750-060, Gibco, Grand Island, NY). Extender
was centrifuged at 20,000 x g in an ultra-fast centrifuge to remove large egg yolk
components. The supernatant was placed in a 50 ml plastic tube (BCT-P50RS, Biologix,
Shawnee Mision, KS) and warmed up to 37C in a water bath 1 hour prior to epididymal
sperm retrieval.
33
A
Figure 3.1. The process of scrotum dissection. A. Pair of bovine testes inside the
scrotum. B. Dissection of the scrotum to retrieve the testes.
34
Brackett-Oliphant medium (BO) was prepared as previously described (Brackett
and Oliphant, 1975). Briefly, BO-A stock solution contains 150.2 mM NaCl (S-5886,
Sigma-Aldrich, St. Louis, MO), 3.0 mM CaCl22H2O (C-7902, Sigma-Aldrich, St. Louis,
MO), 1.0 mM NaH2PO4H2O (S-9638, Sigma-Aldrich, St. Louis, MO), 0.6857 mM
MgCl26H2O (M-2393, Sigma-Aldrich, St. Louis, MO) and 0.1 ml of 0.5% phenol red (P-
0290, Sigma-Aldrich, St. Louis, MO) in a final volume of 500 ml of Milli-Q water. BO-B
stock solution contains 154.0 mM NaHCO3 (S-8875, Sigma-Aldrich, St. Louis, MO) and
0.04 ml of 0.5% phenol red in a final volume of 200 ml of Milli-Q water.
On the day of use, BO-AB medium was prepared by mixing 38 ml of BO-A stock
and 12 ml of BO-B stock with the addition of 1.2 mM pyruvic acid (P-4562, Sigma-
Aldrich, St. Louis, MO), 3 mg/ml of bovine serum albumin (Fraction V, A-4503, Sigma-
Aldrich, St. Louis, MO) and 50 /ml of gentamicin. Medium was warmed in an incubator
at 37C 1 hour prior to epididymal sperm collection.
Epididymal Sperm Retrieval
Each cauda was dissected from the testis by cutting the vas deferens and corpus
epididymis (Figure 3.2). Upon dissection, each cauda was rinsed with 0.9% saline to
remove any remaining materials before epididymal sperm extraction. Epididymal sperm
were harvested by making 5 to 6 incisions using a surgical blade (MDS-15110, Medline
Industries Inc., Mundelein, IL) in the cauda epididymides. The incisions were rinsed with
4 ml of BO medium into a 60 mm Falcon plastic petri dish (3037, Becton Dickinson
Labware, Franklin Lakes, NJ). The caudae were placed on the 60 mm plastic dish for 10
minutes to allow sperm to swim out into the medium (Figure 3.3). The sperm solution
was transferred into a 15 ml plastic tube (20171-024, VWR International Inc., Sugar
Land, TX). Specimens were centrifuged twice at 500 x g for 5 minutes to wash the
sperm. The pellet was resuspended in 8 ml of EYT-GC extender and placed for 15
minutes in a 37C water bath for sperm quality analyses.
Epididymal Sperm Analyses
Sperm analyses included plasma membrane integrity, overall motility and
progressive motility. A 100-l sample from the sperm solution was placed in a 1.7 ml
plastic micro-centrifuge tube (20170-355, VWR International Inc., Sugar Land, TX) and
diluted 1:10 sperm solution:EYT-GC for epididymal sperm quality analyses.
35
A
Figure 3.2. The process of cauda epididymides dissection. A. Pair of bovine testes
outside the scrotum and tunica vaginalis. B. Three pairs of testes showing the dissected
cauda epididymides. Each cauda was dissected from the testis by cutting the vas
deferens and corpus epididymis.
36
Figure 3.3. The process of epididymal sperm retrieval from a dissected cauda
epididymis. The cauda was placed on a 100 mm plastic dish for 10 minutes to allow
sperm to swim out into the collection medium.
37
Plasma membrane integrity was assessed using the LIVE/DEAD Sperm Viability
Kit (L-7011, Molecular Probes Inc., Eugene, OR). The kit consists of two nucleic acid
stains. SYBR 14 (1 mM solution in DMSO, Component A of the LIVE/DEAD Sperm
Viability Kit) is a permeable nucleic acid stain, which labels membrane intact cells green.
PI (2.4 mM solution in H2O, Component B of the LIVE/DEAD Sperm Viability Kit) is a
nonpermeable nucleic acid stain, which labels membrane ruptured sperm red. For
analysis, 250 l of the diluted sperm sample were placed in a 0.65 ml plastic micro-
centrifuge tube (20170-293, VWR International Inc., Sugar Land, TX). Staining was
conducted by the addition of 400 nM SYBR 14 and 24 M PI to the sperm solution. The
stained samples (n=10) were incubated for 10 minutes in the dark at 37C and mixed
gently before analyses.
For plasma membrane integrity, samples (n=10) were analyzed by counting 300
sperm with an inverted microscope (Nikon Diaphot, Tokyo, Japan) equipped with epi-
fluorescent illumination, a fluorescein isothiocyanate (FITC) filter set and a heated stage.
Sperm progressive motility was assessed by computer assisted sperm analysis
(CASA, Minitube of America, Verona, WI) equipped with a heated stage.
Epididymal Sperm Cooling
Sperm concentration was adjusted to 120 x 106 cells/ml using a hemacytometer
by the addition of more extender. The 15 ml plastic conical tube containing the
epididymal sperm sample was placed in a 250 ml Pyrex glass beaker (13912-207,
VWR International Inc., Sugar Land, TX) containing water. The beaker was placed in a
refrigerator for cooling to 4C for 4 hours.
The cooled sperm sample was thoroughly mixed using a plastic pipette and
placed equally (3 ml) into 5 previously cooled plastic tubes. Specimens were allocated
into 5 treatment groups: Control (no CPA), 7% GLY, 14% GLY, 7% EG and 14% EG.
Replicate samples (n=2) of cooled epididymal sperm were diluted 1:1 in EYT-GC
extender containing twice the final desired concentration of CPA. After being exposed for
10 minutes, a 0.5 ml sample from each treatment group was removed and diluted
directly into EYT-GC at 4C. Duplicate samples from each treatment group were stored
at 4C for 5 days. A 0.5 ml sample from each treatment group was removed on days 0,
1, 3 and 5 and then diluted directly into BO medium to assess CPA toxicity.
38
Subsequently, plasma membrane integrity and motility were analyzed as
described above to evaluate the effect of addition and/or removal and toxicity of the
different concentrations of CPAs.
Statistical Analysis
Variances in membrane integrity and overall and progressive motility were
statistically analyzed by ANOVA and the differences between bulls were calculated by a
Tukey Multiple Comparisons test. The sperm parameters are expressed as meanSEM
per treatment group. A P<0.05 value was considered significant in this study. Data were
analyzed using SigmaStat Statistical Software Version 2.0.
RESULTS
Experiment 3.1
Overall and Progressive Motility
The mean initial overall motility and progressive motility values before epididymal
sperm cooling were 75.11.3% and 58.92.4%, respectively. After cooling to 4C, these
mean values declined to 72.91.6% and 53.53.2%, respectively. No significant
detrimental effect (P>0.05) was detected during the cooling process of epididymal sperm
as assessed by both motility parameters. After CPA addition and removal, the mean
overall motility values for each treatment group were: 493.8, 234.5, 642.8, 48 4.4%
for 7% GLY, 14% GLY, 7% EG and 14% EG, respectively. The mean progressive
motility values for each treatment group were: 303.8, 103.9, 463.9 and 314.0% for
7% GLY, 14% GLY, 7% EG and 14% EG, respectively. No significant decline in sperm
overall and progressive motility was detected with the addition and removal of 7% EG.
However, a significant decline (P<0.05) in both motility parameters was noted in the 14%
EG group, 7% GLY group and 14% GLY group.
Membrane Integrity
No significant damage to the plasma membrane of sperm was detected during
the cooling process. Plasma membrane integrity values of epididymal sperm before and
after cooling to 4C were 83.21.1% and 78.12.5%, respectively. After CPA addition
and removal, the mean plasma membrane integrity values for each treatment group
were: 543.0, 293.0, 702.5 and 483.7% for 7% GLY, 14% GLY, 7% EG and 14% EG
treatment groups, respectively. No significant difference was detected in the 7% EG
group when compared with the control group. However, the plasma membrane was
39
damaged significantly in the 14% EG and 7% and 14% GLY groups when compared
with the control group.
Due to intermale variations, membrane integrity values ranged from 60 to 84%,
33 to 74%, 35 to 74% and 18 to 46% for 7% EG, 14% EG, 7% GLY and 14% GLY
treatment groups, respectively. There was a high variability in percentage of sperm with
intact membranes between males exposed to the same treatment groups. Some bulls
were apparently more sensitive to osmotic damage caused by the high concentration of
CPAs than others.
Experiment 3.2
The mean overall and progressive motility values during the 5 days of cool
storage are shown in Figure 3.4 and Figure 3.5, respectively. No significant decline in
overall and progressive motility was detected during the first 24 hours of storage for the
control and 7% EG-treated group. However, a significant decline (P<0.05) in overall and
progressive motility was found after CPA addition on day 0 for the 14% EG-treated
group and the 7% GLY- and 14% GLY-treated groups. The control group had a decline
in overall motility of 47.4% during the 5 days of storage, while the 7% EG, 14% EG, 7%
GLY and 14% GLY treatment groups had a decline of 42.3, 62.1, 63.9 and 74.8%,
respectively.
After 5 days of storage, progressive motility declined to 7.8, 12.0, 2.5, 2.2 and
0% in the control, 7% EG, 14% EG, 7% GLY and 14% GLY treatment groups,
respectively. Progressive motility was more severely affected than overall motility with
less than 20% progressively motile sperm in all CPA-containing treatment groups on day
3 of cool storage. The highest loss in progressive motility was observed in the 14% GLY-
treated group, which had a decline in progressive motility to less than 10% after the
addition of the CPA on day 0 and further declined to less than 3% after 24 hours of cool
storage.
Mean overall motility and progressive motility values for individual bulls during
the 5 day cool storage period are shown in Table 3.1 and Table 3.2, respectively. A
significant difference (P<0.05) was detected among males in both motility parameters
during the 5 day cool storage period. Epididymal sperm from 40% of the males were
40
100
Overall Motility (%)
80
Control
60 GLY 7%
GLY 14%
40 EG 7%
EG 14%
20
0
0 1 3 5
Days
Figure 3.4. Effect of cryoprotectant toxicity during 5 days of storage at 4C on overall

motility of bovine epididymal sperm. GLY = glycerol and EG = ethylene glycol.
41
100
Progressive Motility (%)
80
Control
60 GLY 7%
GLY 14%
40 EG 7%
EG 14%
20
0
0 1 3 5
Days
Figure 3.5. Effect of cryoprotectant toxicity during 5 days of storage at 4C on

progressive motility of bovine epididymal sperm. GLY = glycerol and EG = ethylene
glycol.
42
Table 3.1. Mean overall motility values for individual males during a 5-day exposure to different concentrations of CPAs at 4C
Overall motility
Trt Day 0 Day 1 Day 3 Day 5
Bull Cont GLY GLY EG EG Cont GLY GLY EG EG Cont GLY GLY EG EG GLY GLY EG EG
No. CONT 7% 14% 7% 14% CONT 7% 14% 7% 14% CONT 7% 14% 7% 14% CONT 7% 14% 7% 14%
1 73.0 35.0 20.0 50.0 66.0 60.0 25.0 5.0 35.0 40.0 58.0 25.0 8.0 38.0 29.0 48.0 20.0 3.0 34.0 29.0
2 66.0 49.0 38.0 57.0 50.0 57.0 14.0 6.0 38.0 50.0 60.0 12.0 3.0 34.0 36.0 50.0 10.0 0.0 35.0 34.0
3 66.0 30.0 10.0 63.0 33.0 51.0 13.0 4.0 36.0 23.0 15.0 0.0 0.0 25.0 4.0 0.0 0.0 0.0 5.0 0.0
4 69.0 45.0 9.0 50.0 17.0 65.0 22.0 6.0 52.0 35.0 20.0 5.0 0.0 29.0 10.0 5.0 0.0 0.0 12.0 5.0
5 75.0 64.0 46.0 73.0 60.0 70.0 37.0 30.0 70.0 55.0 60.0 19.0 5.0 32.0 14.0 20.0 12.0 0.0 17.0 0.0
6 80.0 55.0 20.0 77.0 54.0 77.0 44.0 16.0 66.0 40.0 44.0 30.0 9.0 40.0 24.0 15.0 5.0 0.0 35.0 19.0
7 70.0 60.0 15.0 67.0 48.0 60.0 30.0 5.0 56.0 17.0 60.0 52.0 5.0 56.0 27.0 54.0 34.0 0.0 57.0 12.0
8 75.0 38.0 10.0 65.0 50.0 75.0 46.0 5.0 62.0 20.0 35.0 43.0 0.0 38.0 6.0 20.0 8.0 0.0 46.0 10.0
9 80.0 65.0 44.0 70.0 55.0 75.0 50.0 5.0 56.0 45.0 51.0 17.0 0.0 48.0 23.0 31.0 5.0 0.0 33.0 10.0
10 75.0 50.0 15.0 65.0 49.0 70.0 49.0 16.0 67.0 46.0 50.0 28.0 5.0 58.0 30.0 34.0 18.0 0.0 54.0 11.0
CPAs = cryoprotectants; Trt = treatment; CONT = control; GLY = glycerol; EG = ethylene glycol.
43
Table 3.2. Mean progressive motility values for individual males during a 5-day exposure to different concentrations of CPAs at 4C
Progressive motility
Bull Cont GLY GLY EG EG Cont GLY GLY EG EG Cont GLY GLY EG EG Cont GLY GLY EG EG
1 40.0 18.0 10.0 27.0 36.0 30.0 5.0 0.0 10.0 20.0 27.0 5.0 0.0 11.0 10.0 20.0 3.0 0.0 10.0 10.0
2 45.0 34.0 20.0 39.0 35.0 35.0 2.0 0.0 20.0 35.0 33.0 0.0 0.0 13.0 15.0 18.0 0.0 0.0 11.0 12.0
3 40.0 19.0 3.0 39.0 20.0 30.0 6.0 0.0 22.0 10.0 3.0 0.0 0.0 10.0 0.0 0.0 0.0 0.0 0.0 0.0
4 45.0 20.0 0.0 26.0 5.0 39.0 7.0 0.0 21.0 11.0 5.0 0.0 0.0 12.0 0.0 0.0 0.0 0.0 0.0 0.0
5 60.0 45.0 33.0 50.0 45.0 58.0 22.0 25.0 55.0 43.0 40.0 5.0 0.0 5.0 5.0 0.0 0.0 0.0 5.0 3.0
6 63.0 33.0 5.0 60.0 30.0 65.0 25.0 0.0 49.0 25.0 15.0 5.0 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0
7 55.0 38.0 0.0 55.0 40.0 30.0 13.0 0.0 43.0 3.0 35.0 30.0 0.0 31.0 10.0 16.0 15.0 0.0 37.0 0.0
8 60.0 13.0 0.0 54.0 30.0 60.0 33.0 0.0 29.0 8.0 10.0 23.0 0.0 10.0 0.0 5.0 0.0 0.0 19.0 0.0
9 69.0 50.0 28.0 60.0 46.0 60.0 19.0 0.0 45.0 39.0 26.0 5.0 0.0 30.0 13.0 10.0 0.0 0.0 16.0 0.0
10 58.0 30.0 0.0 50.0 19.0 60.0 35.0 3.0 49.0 34.0 18.0 13.0 0.0 30.0 14.0 9.0 4.0 0.0 22.0 0.0
CPAs = cryoprotecants; Trt = treatment; Cont = control; GLY = glycerol; EG = ethylene glycol.
44
100
Membrane Integrity (%)
80
Control
60 GLY 7%
GLY 14%
40 EG 7%
EG 14%
20
0
0 1 3 5
Days
Figure 3.6. Effect of cryoprotectant toxicity during 5 days of storage at 4C on plasma

membrane integrity of bovine epididymal sperm. GLY = glycerol and EG = ethylene
glycol.
45
more vulnerable to the CPA than other males, as noted in a rapid depression of motility
parameters.
Membrane Integrity
The mean values for sperm exhibiting intact membranes during the 5 days of
cool storage are shown in Figure 3.6. No significant damage to the sperm plasma
membrane was detected during the first 24 hours of storage for the control and 7% EG-
treated group when compared with the 7% GLY, 14% GLY and 14% EG treatment
groups. However, a significant increase (P<0.05) in the percentage of sperm with
ruptured membranes was found immediately after the addition of CPA and further
increased after 24 hours of storage for the 7% GLY, 14% GLY and 14% EG treatment
groups. The highest reduction in membrane integrity was detected during the first 24
hours of storage for all 4 CPA treatment groups. The control group had a decline in
membrane integrity of 31.5% during the 5 days of storage, while the 7% EG, 14% EG,
7% GLY and 14% GLY treatment groups had a decline of 37.9, 63.1, 54.3 and 72.5%,
respectively.
The highest loss in membrane integrity was found in the 14% GLY-treated group.
In the 14% GLY treatment group, membrane integrity of epididymal sperm declined from
83.2% to 29.0% on day 0 and further declined to 10.9% on day 5 of cool storage.
Mean plasma membrane integrity values for individual bulls during the 5 day cool
storage period are shown in Table 3.3. A significant difference (P<0.05) was found
among 40% of the males used in this study. There was a high variability in the
percentage of sperm with intact membranes between males subjected to the same
treatment group for all treatments.
DISCUSSION
Glycerol is currently used as the cryoprotectant of choice to cryopreserve bull
sperm. The use of alternative permeating cryoprotectants with higher membrane
permeabilities might help reduce the osmotic damage caused to sperm during their
addition prior to freezing and removal after warming.
We found that the addition and removal of glycerol and ethylene glycol at 4C
resulted in a concentration dependent loss of motility and membrane integrity. Ethylene
glycol produced the least harmful effects when compared with glycerol in the
concentrations evaluated in this study. These results are in agreement with those
46
Table 3.3. Mean plasma membrane integrity values for individual males during a 5-day exposure to different concentrations of CPAs
at 4C
Plasma membrane integrity
Bull Cont GLY GLY EG EG Cont GLY GLY EG EG Cont GLY GLY EG EG Cont GLY GLY EG EG
1 79.0 58.0 36.0 70.0 74.0 68.0 40.0 31.0 60.0 49.0 63.0 42.0 18.0 55.0 38.0 64.0 34.0 17.0 47.0 34.0
2 77.0 58.0 42.0 65.0 56.0 69.0 28.0 20.0 53.0 45.0 65.0 32.0 15.0 51.0 35.0 54.0 30.0 13.0 49.0 30.0
3 64.0 54.0 23.0 65.0 46.0 58.0 27.0 12.0 51.0 25.0 42.0 9.0 7.0 40.0 21.0 30.0 8.0 5.0 31.0 15.0
4 79.0 51.0 26.0 64.0 33.0 63.0 34.0 16.0 52.0 34.0 59.0 30.0 15.0 49.0 20.0 55.0 28.0 13.0 47.0 20.0
5 80.0 56.0 30.0 78.0 41.0 60.0 34.0 27.0 65.0 30.0 69.0 25.0 9.0 38.0 15.0 50.0 36.0 7.0 40.0 11.0
6 80.0 50.0 23.0 79.0 43.0 71.0 36.0 16.0 72.0 30.0 74.0 36.0 20.0 51.0 23.0 58.0 38.0 15.0 44.0 27.0
7 72.0 49.0 18.0 65.0 39.0 65.0 40.0 10.0 60.0 19.0 59.0 50.0 12.0 56.0 23.0 57.0 35.0 9.0 55.0 13.0
8 70.0 35.0 19.0 60.0 40.0 72.0 50.0 15.0 64.0 20.0 44.0 47.0 13.0 50.0 15.0 46.0 28.0 10.0 49.0 16.0
9 90.0 74.0 46.0 84.0 53.0 84.0 48.0 8.0 65.0 30.0 65.0 30.0 7.0 53.0 21.0 49.0 20.0 7.0 39.0 13.0
10 90.0 50.0 27.0 74.0 54.0 84.0 47.0 24.0 64.0 42.0 60.0 40.0 14.0 59.0 31.0 54.0 32.0 11.0 52.0 22.0
CPAs = cryoprotectants; Trt = treatment; CONT = control; GLY = glycerol; EG = ethylene glycol.
47
reported for ejaculated bull and human sperm by Guthrie et al. (2002) and Gilmore et al.
(1997), respectively. Both groups found that the removal of ethylene glycol produced the
least harmful effects to sperm motility compared with that of dimethyl sulfoxide and
glycerol.
In our study, maximum survival as assessed by measurements of motility and
membrane integrity was achieved with spermatozoa exposed to 7% ethylene glycol.
Almost identical intermediate levels of survival were observed with sperm exposed to 7%
glycerol or 14% ethylene glycol. The lowest survival was found for sperm exposed to the
14% glycerol treatment.
There was a high variability in percentage of sperm with intact membranes
between males exposed to the same treatment groups. Some bulls were apparently
more sensitive to osmotic damage caused by the high concentration of CPAs than
others.
Ethylene glycol has been reported to permeate the plasma membrane of human
sperm four times as fast as glycerol (Gilmore et al., 1997). When a permeable
cryoprotectant is added to sperm, a hypertonic environment is created. The cell shrinks
as water leaves the cytoplasm into the extracellular environment and then swells as the
cryoprotectant enters the cell and water re-enters to maintain osmotic equilibrium.
During removal, the cell swells due to an influx of water and then returns to isosmotic
volume after the cryoprotectant and water leaves the cell.
In this study, the rapid movement of ethylene glycol through the plasma
membrane might have helped reduce shrinking and swelling of sperm reducing plasma
membrane damage and depression of motility to the sperm. The osmotic damage
caused by high concentration of cryoprotectants or cryoprotectants with low membrane
permeabilities can be reduced by step wise addition and dilution (Katkov et al., 1998).
Guthrie et al. (2002) reported that motility of ejaculated bull sperm was very
sensitive to osmotic changes. For bull sperm to maintain 90% motility of their isosmotic
value, sperm must be maintained within 92 to 103% of its original isosmotic volume.
Conversely, human sperm has a higher osmotic tolerance and can maintain motility
within 75 to 110% of its original isosmotic volume (Gao et al., 1995), while motility in the
boar is depressed outside 97 to 102% of its isosmotic volume (Gilmore et al., 1998).
Swelling of sperm was reported to be more detrimental to survival than shrinking
48
(Pommer et al., 2002). Therefore, most of the damage to sperm is likely during the
removal rather than during the addition of cryoprotectants. Due to the osmotic tolerance
differences between species and cell types, the osmotic tolerance limits of epididymal
sperm must be determined in an effort to reduce osmotic damage.
The protective effects of permeating cryoprotectants are so impressive that little
attention has been focused on the toxic effects of these chemical agents. Some cell
types are more vulnerable to damage caused by permeating cryoprotectants than
others. Hammerstedt and Graham (1992) reported that glycerol can affect physical
features in the cytoplasm, permeability and stability of the membrane bilayer,
metabolism and noncovalent attachment of proteins to the sperm surface. However, the
biochemical mechanisms of this type of toxicity are unknown.
To our knowledge, this is the first report to study the toxic effect of prolonged
incubation of epididymal sperm with different concentrations of permeable cryoprotective
agents. Ethylene glycol at low concentrations produced no evident toxic effect during the
5 days of cool storage, as assessed by sperm viability and both motility parameters.
However, when the CPA concentration was increased overall and progressive motility
was reduced and the percentage of membrane ruptured sperm was elevated. In the
present study, 7% and 14% glycerol was found to be more toxic than 7% and 14%
ethylene glycol concentrations. At the low concentration of glycerol (7%), overall and
progressive motility and membrane integrity were significantly reduced during storage.
At the high concentration of glycerol (14%), progressive motility was impaired after 24
hours of storage and membrane integrity was reduced to less than 20%. The instant
decline of motility and membrane integrity on day 0 was likely caused by osmotic
damage rather than true chemical toxicity. However, possible chemical toxicity by the
cryoprotectants can not be ruled out.
Toxicity of permeating cryoprotectants has been reported in mouse (Sztein et al.,
2001) and rooster sperm (Hammerstedt and Graham, 1992). Mouse sperm do not
tolerate the intracellular presence of permeating CPAs as well as sperm from other
species (Sztein et al., 2001). Nonpermeable CPAs, such as raffinose and trehalose are
well tolerated by mouse sperm (Storey et al., 1998). It is thought that permeable
cryoprotective agents affect mouse sperm due to true chemical toxicity rather than to
osmotic damage. Furthermore, glycerol has also been reported to increase membrane
49
disruptions in rooster sperm resulting in a reduction in fertility (Hammerstedt and
Graham, 1992).
In this study, motility and membrane integrity were the end points to assess
toxicity. The presence of permeable cryoprotectants might also affect other sperm
components lowering the functional integrity of the sperm, which were not assessed by
the assays used in this study. Poor fertility was reported in mice when sperm were
frozen using glycerol despite having good post-thaw sperm motility (Sztein et al., 1992).
A more rigorous study to try to understand the biochemical mechanism of toxicity caused
by permeating CPAs is needed in the future.
These results indicate that the use of ethylene glycol as a CPA may reduce
toxicity and osmotic damage to bovine epididymal sperm over that of glycerol. Future
research must evaluate its efficacy as a cryoprotectant for bovine epididymal sperm.
50
CHAPTER IV
THE USE OF ETHYLENE GLYCOL AND GLYCEROL FOR THE

CRYOPRESERVATION OF BOVINE CAUDAL EPIDIDYMAL SPERM
INTRODUCTION
The development of a protocol to cryopreserve sperm harvested from the
epididymides will enable the propagation of gametes from valuable animals that may die
unexpectedly. Recently, interest for the preservation of epididymal sperm as a potential
source of valuable genes for genome resource banks has escalated. In cattle, most
research has concentrated on improving freezing protocols for ejaculated bull sperm.
Reports on the use, characteristics, cryopreservation and fertility of bovine epididymal
sperm are limited. Even with all the work to improve the survivability of bovine sperm
after cryopreservation, there still remains ~50% decrease in sperm viability due to
temperature and osmotic effects (Thomas et al., 1998). Moreover, it is well known that
sperm from some bulls still can not be successfully cryopreserved with present day
protocols due to individual bull to bull variation. It has recently been emphasized that
there is marked diversity in the cryobiological response of sperm among different
mammalian species and among different individuals within a species (Agca and Crister,
2002).
Epididymal sperm has been cryopreserved in various species, including swine
(Rath and Niemann, 1997), dogs (Stilley et al., 1999), goats (Blash et al., 2000), mice
(Koshimoto et al., 2000), humans (Patrizio, 2000) and horses (Tiplady et al., 2002) with
variable results.
Research on the cryopreservation of bovine epididymal sperm is very limited.
Barker (1954) reported the first pregnancy using frozen-thawed bull epididymal sperm by
artificial insemination. Later, cryopreserved epididymal sperm was used for in vitro
fertilization (Goto et al., 1989) and intracytoplasmic sperm injection (Goto et al., 1990).
However, no data are available on the survivability and quality of epididymal sperm in
regards to post-thaw membrane integrity and acrosome integrity.
Most sperm are lethally damaged during the cooling and warming process of
cryopreservation in the absence of cryoprotective additives (CPAs) (Ashwood-Smith and
Farrant, 1980). The most widely used permeating CPAs since the 1950s have been
glycerol, dimethyl sulfoxide, ethylene glycol and propylene glycol. Permeating CPAs
51
function through colligative properties, which help maintain solute concentrations inside
and outside the cells at nondamaging levels (Gilmore et al., 1997). However, CPAs
produce a hyperosmolar environment to sperm cells inducing potentially damaging
volume changes. Sperm survival can be optimized by avoiding or minimizing osmotic
damage (Holt, 2000). Although functional differences exist between epididymal and
ejaculated sperm, it is proposed that epididymal sperm still require the presence of
similar components during the freezing process.
From early studies it is well known that the cryoprotective ability of any CPA
varies widely across different cell and tissue types (Karow, 1969). Therefore, an optimal
CPA for cryopreservation of bovine epididymal sperm needs to be determined.
Glycerol is routinely used at concentrations ranging form 5 to 8% to cryopreserve
bovine ejaculated sperm. Recently, the high membrane permeability of ethylene glycol
was shown to reduce the damage caused to the plasma membrane of bovine caudal
epididymal sperm during its addition and/or removal when compared with that of glycerol
(Guerrero et al., 2006). Gilmore et al. (1997) indicated that the optimal CPA would be
one that could permeate the cell in the shortest time causing the least amount of volume
excursions during its addition and removal. Ethylene glycol was chosen as a candidate
for the cryopreservation of bovine epididymal sperm in our study because of its high
membrane permeability.
Sperm are intricate cells that require a number of criteria to be met to achieve
fertilization. It is generally agreed that an intact acrosome and plasma membrane are
required for the sperm to transverse through the cumulus cells, zona pellucida and finally
fuse with the oolema of the oocyte.
The discovery of a variety of fluorochromes has made possible the analysis of
sperm at a functional, biochemical and ultrastructural level. Most fluorochromes have
been used by staining and examining cells with fluorescent microscopy. However,
microscopic analysis only measures a small number of sprem (200-300) within a
population, is time consuming and is considered subjective (Gillian et al., 2005). By
adapting these fluorochromes for flow cytometry, various sperm attributes can be
measured faster and on a larger scale (10,000-50,000).
The aim of all semen analysis procedures is to predict the potential for fertility in
a fast, objective and accurate way. The use of flow cytometry to assess sperm quality
52
has greatly improved the objectivity, accuracy and reproducibility of the quality
evaluation compared with traditional microscopy methods (Waterhouse et al., 2004).
Garner et al. (1994) developed a simple dual nucleic acid stain combination to
assess viability of frozen-thawed sperm in the presence of egg yolk particles. The two
stains, SYBR 14 and propidium iodide (PI) have the same cellular target making it less
variable than enzyme-based stains. Sperm DNA are believed to be a more appropriate
cellular target due to their stainability and staining uniformity (Garner et al., 1996). These
probes stain viable membrane intact sperm green, while nonviable membrane damaged
sperm stain red. After cell death, the PI rapidly overwhelms the fluorescence exhibited
by the SYBR 14 (Thomas et al., 1998).
Acrosome integrity has been measured using plant lectins conjugated to a
fluorescent probe. These lectins bind to sugar moieties exposed in reacted acrosome
membranes. Arachis hypogaea agglutinin (PNA) and Pisum sativum agglutinin (PSA)
are the most used plant lectins. However, PNA is believed to display less nonspecific
binding to other areas of the sperm and also to egg yolk particles, leading researchers to
favor PNA over PSA (Graham, 2001; Nagy et al., 2003).
Recently, Nagy et al. (2003) developed a triple fluorochrome staining technique
which involves the well validated SYBR 14 and PI, with PNA conjugated to phycoerythrin
(PE). The PE fluorescent moiety was used instead of fluorescein isothiocyanate (FITC),
because its fluorescence emission could be measured independently from that of SYBR
14 or PI. This novel technique was shown to assess viable acrosome intact, viable
acrosome reacted, nonviable acrosome intact and nonviable acrosome reacted sperm in
a single assay.
In the present study, this novel triple fluorochrome technique was duplicated for
the first time to determine the effect of ethylene glycol and glycerol on the cryopreser-
vation of bovine caudal epididymal sperm.
Experimental Design
Experiment 4.1
Paired testes were obtained from mature mixed breeds of beef and dairy bulls
(n=10) at a local abattoir and transported to the laboratory within 4 to 6 hours
postmortem. Epididymal sperm were harvested by multiple incisions from the caudae
53
epididymides of testes from each bull, pooled and then placed in egg yolk Tris-glucose
citric acid monohydrate extender (EYT-GC). Specimens were allocated into two
treatment groups: 7% ethylene glycol or 7% glycerol. Samples were diluted slowly over a
period of 30 min 1:1 in EYT-GC medium containing twice the final desired concentration
of cryoprotectant (CPA) and subsequently frozen over liquid nitrogen vapors. Plasma
membrane and acrosome integrity were evaluated by the same technician using
multicolor flow cytometry. Progressive motility was assessed subjectively with a light
microscope. The experiment was repeated three times with each replicate on a different
day.
Testes Collection
Bovine testes were collected as pairs from mature mixed breed bulls (n=10) at an
abattoir in Robert, Louisiana. Testes were individually packed in Ziploc plastic bags
(Johnson & Son Inc., Racine, WI) and transported to the Louisiana State University
Embryo Biotechnology Laboratory, St. Gabriel, Louisiana, in a Styrofoam box (at a
temperature that ranged between 25C to 28C) within 4 to 6 hours postmortem.
Immediately upon arrival to the laboratory, each pair of testes was dissected away from
its scrotum and tunica vaginalis. Testes were positioned on the table with the vas
deferens facing ventrally and the corpus facing up to distinguish the side of origin of
each testis. The weight of the testis was measured with a Pelouze balance scale
(Model SP5) before carefully dissecting the cauda epididymis.
Medium Preparation
Egg yolk Tris-glucose citric acid monohydrate extender (EYT-GC) (250 mM Tris
(T-6066, Sigma-Aldrich Inc., St. Louis, MO), 69 mM glucose (6152, Sigma-Aldrich Inc.,
St. Louis, MO), 81 mM citric acid monohydrate (C-1909, Sigma-Aldrich, St. Louis, MO)
and 20% (v/v) egg yolk) was prepared with the addition of 50 g/ml of gentamicin
(15750-060, Gibco, Grand Island, NY) and filtered through a 0.45 m Nalgene syringe
filter (190-2545, Nalge Company, Rochester, NY) to remove egg yolk materials. Egg
yolks were obtained from fresh chicken eggs at the Louisiana State University Poultry
research Farm. The extender was placed in a 37C water bath for at least 3 hours prior
to epididymal sperm retrieval.
54
Upon dissection, each cauda was weighed and rinsed with 0.9% saline before
epididymal sperm extraction. Epididymal sperm were harvested by making 5 to 6
incisions using a surgical blade (size 10, MDS-15110, Medline Industries Inc.,
Mundelein, IL) in the cauda epididymis. The incisions were rinsed with 4 ml of EYT-GC
into a 60 mm Falcon plastic petri dish (3037, Becton Dickinson Labware, Franklin
Lakes, NJ). The sperm solution was transferred into a 15 ml plastic tube (20171-024,
VWR International Inc., Sugar Land, TX) and placed for 15 minutes in a 37C water bath
for sperm quality analyses.
Epididymal Sperm Analyses
Sperm analyses included plasma membrane integrity, acrosome integrity and
progressive motility. A 100-l sample from the sperm solution was placed in a 1.7 ml
plastic micro-centrifuge tube (20170-355, VWR International Inc., Sugar Land, TX) and
diluted 1:10 sperm solution:EYT-GC for the epididymal sperm quality analyses.
Kit (L-7011, Molecular Probes Inc., Eugene, OR). The kit consists of two nucleic acid
Viability Kit) is a permeable nucleic acid stain, which labels membrane intact cells green.
nonpermeable nucleic acid stain, which labels membrane ruptured sperm red. For
analysis, 250 l of the diluted sperm sample were placed in a 0.65 ml plastic micro-
centrifuge tube (20170-293, VWR International Inc., Sugar Land, TX). Staining was
carried out by the addition of 400 nM SYBR 14 and 24 M PI to the sperm mixture. The
stained sample was incubated for 10 minutes in the dark at 37C and remixed gently
before analysis.
Acrosome integrity was assessed using the lectin peanut agglutinin conjugated to
FITC (FITC-PNA) (L-7381, Sigma-Aldrich Inc., St. Louis, MO) and the nucleic acid stain
PI. PNA labels acrosome reacted sperm green, while PI discriminates between viable
and nonviable sperm. For analysis, 250 l of the diluted sperm sample were placed in a
0.65 ml plastic micro-centrifuge tube. A final concentration of 5 g/ml of FITC-PNA and
55
24 M PI were added. The stained sample was incubated for 15 to 20 minutes in the
dark at 37C and mixed gently before analysis.
For plasma membrane and acrosome integrity, each sample was analyzed by
counting 300 sperm cells with an inverted microscope (Nikon Diaphot, Tokyo, Japan)
equipped with epi-fluorescence, a FITC filter set and a heated stage.
Sperm progressive motility was assessed subjectively by the same technician at
20X magnification using an inverted Nikon Diaphot microscope equipped with Hoffman
optics and a heated stage.
Sperm Cooling, Cryopreservation and Thawing
Sperm concentration was determined using a hemacytometer and adjusted to
100 x 106 cells/ml by the addition of more extender. The 15 ml plastic conical tube
containing the epididymal sperm sample was placed in a 250 ml Pyrex glass beaker
(13912-207, VWR International Inc., Sugar Land, TX) containing water. The beaker was
placed for 4 hours in a refrigerator for cooling the sperm samples to 4C.
Specimens from each bull were then allocated into two treatment groups: 7%
ethylene glycol (E-9129, Sigma-Aldrich Inc., St. Louis, MO) or 7% glycerol (G-5516,
Sigma-Aldrich Inc. St. Louis, MO). Samples were diluted slowly over a period of 30
minutes 1:1 in EYT-GC extender containing twice the final desired concentration of CPA
and subsequently loaded into previously cooled 0.5 ml straws (005569, Cassou straw,
IMV Technologies, Minneapolis, MN). Straws were placed in a custom built rack 2 cm
over liquid nitrogen (LN2) vapors for 10 minutes before being plunged in LN2. Between
15 to 20 of these 0.5 ml straws were frozen per bull.
Sperm samples were stored for 6 months prior to thawing. On the day of
analysis, 50 ml of Dulbeccos phosphate-buffered saline (D-PBS) (14287-080, Gibco,
Grand Island, NY) were placed in a 50 ml plastic tube (BCT-P50RS, Biologix, Shawnee
Mision, KS) and warmed in a 37C incubator 1 hour prior to sperm dilution. Three straws
per animal were thawed in a water bath at 37C for 40 seconds. The contents of each
straw (25 x 106 sperm) were placed in a 15 ml plastic tube and diluted 1:10 sperm
sample:D-PBS before motility and multicolor flow cytometric analyses.
Multicolor Flow Cytometry
Assessment of plasma membrane and acrosome integrity were carried out using
a novel triple stain protocol in the presence of egg yolk components (Nagy et al., 2003)
56
with minor modifications. The staining protocol consisted of SYBR 14, PI and the lectin
peanut agglutinin conjugated to PE (R-PE-PNA) (p-44, Biomeda Corporation, Foster
City, CA). A final concentration of 100 nM SYBR 14 solution, 4 g/ml of R-PE-PNA (1
mg/ml of stock solution in a buffer composed of 50 mM sodium phosphate and 0.05%
sodium azide, pH 7.0, and also containing 0.1 mM [Ca2+] and [Mn2+] ions) and 12 M PI
solution were added to 250 l of diluted sperm sample. The samples were thoroughly
mixed and incubated at 37C in the dark for 30 to 40 minutes and then remixed before
flow cytometric analysis.
Measurements were completed on a FACSCalibur flow cytometer (Beckton
Dickinson Immunocytometry Systems, San Jose, CA). Forward and side scatter values
were recorded on a linear scale, while fluorescent values were recorded on a logarithmic
scale. The three fluorochromes were excited at 488 nm with a 20 mW argon laser.
Plasma membrane ruptured cells were PI positive, and their red fluorescent signal was
detected on the FL3-channel using a 610 nm long pass filter. Membrane intact cells
were SYBR 14-positive, and its green fluorescent signal was detected on the FL1-
channel using a 530/30 nm band pass filter (Figure 4.1).
Acrosome reacted cells were PE-PNA positive, and its orange fluorescent signal
was detected at 585/42 nm on the FL2-channel (Figure 4.1). Three color compensations
were set according to the FACS training manual (Becton Dickinson Biosciences, San
Jose, CA). Nonsperm events (egg yolk components) were gated from the analyses as
judged on scatter properties when detected in the forward and side scatter detectors
(Figure 4.2). Moreover, events with similar scatter characteristics to sperm but with low
PI and SYBR 14 fluorescence were also gated out (Figure 4.3). Sperm acquisitions were
made using CellQuest software (Becton Dickinson, San Jose, CA). The flow cytometer
was kept at a low flow of less than 300 sperm/second. The recording of scatter and
fluorescent properties of all events stopped when 10,000 gated events were recorded.
Density and dot plots drawn for data analysis were generated by WinMDI 2.8
(free software by J. Trotter, available for downloading at http://www.facs.scripps.edu/
software.html). On SYBR 14 (FL1) and PI (FL3) dot plots, regions were drawn to
determine the percentage of viable and nonviable sperm (Figure 4.4). On PE-PNA (FL2)
and PI (FL3) dot plots, quadrants were set to measure the percentage of sperm
displaying intact plasma membranes and acrosomes (LL), intact plasma membranes
57
Figure 4.1. Representative plasma membrane and acrosome integrity triple stain assay.
Sperm that exhibit green fluorescence (SYBR 14) are considered membrane intact
(viable), those that exhibit red fluorescence (PI) were considered to have damaged
membranes (nonviable) and those exhibiting yellow fluorescence (PE-PNA) were
considered acrosome reacted.
58
Figure 4.2. Flow cytometric out-gating of egg yolk particles based on scatter properties
after staining frozen-thawed epididymal sperm with SYBR 14, PI and PE-PNA. Sperm
were selected (R1) based on size, gating out small particles.
Figure 4.3. PI (red) and SYBR 14 (green) fluorescence. Remaining egg yolk particles
having sperm-like scatter properties were gated out based on DNA fluorescence. Events
that had either positive PI and/or SYBR 14 (R1) fluorescence were selected as DNA
positive events. Particles exhibiting no DNA fluorescence (lower left corner) were
considered to be nonsperm events.
59
Figure 4.4. SYBR 14 (viable) and PI (nonviable) flow cytometric dot plot illustrating
frozen-thawed epididymal sperm after gating out all nonsperm events. Acquisitions of
10,000 events were made from positive green and red fluorescence.
Figure 4.5. Flow cytometric graph illustrating PE-PNA (orange) and PI (red)
fluorescence. Events exhibiting high orange fluorescence were recorded as sperm with
reacted acrosomes. Quadrants were set to identify viable acrosome intact sperm (LL),
viable acrosome reacted sperm (LR), nonviable acrosome intact sperm (UL) and
nonviable acrosome reacted sperm (UR).
60
and reacted acrosomes (LR), ruptured plasma membranes and intact acrosomes (UL)
and ruptured plasma membranes and reacted acrosomes (UR) (Figure 4.5).
Variances in testicular parameters, membrane integrity, acrosome integrity and
progressive motility were statistically analyzed by ANOVA and the differences between
bulls were evaluated by a Tukey Multiple Comparisons test. In addition, a t-test was
used to verify differences in sperm parameters before and after cryopreservation. The
sperm parameters are expressed as meanSEM per treatment group. A P<0.05 was
considered significant in this study. Data were analyzed using SigmaStat Statistical
Software Version 2.0.
RESULTS
Experiment 4.1
Testicular and Epididymal Weights
Testicular and caudae epididymal weights for individual bulls are shown in Table
4.1. Both weights from the right testis of bull number 10 were not included in the mean
values due to their abnormal weights (low). Testes and caudae epididymides weights
ranged from 241 to 490 g and 5 to 16 g for the bulls used in this study, respectively.
There was an intermale variability in both testicular parameters analyzed. No significant
difference (P>0.05) was detected between bulls for both testicular parameters.
A total of 32 pairs of testes were obtained from the abattoir. Only 10 bulls of the
32 obtained showed anatomically normal testis morphology, sperm production and
progressive motility greater than 60%. From the 22 bulls not used, 5 had no evidence of
sperm production, 7 had low sperm concentration and 10 had impaired motility and high
amount of morphological abnormalities. Of those 22 animals, 10 displayed abnormal
testicular morphology (Figure 4.6).
Progressive Motility
The mean initial progressive motility value after epididymal sperm retrieval was
662.1% for all the 10 bulls used in this study. Cryopreservation using both CPAs had a
significant detrimental effect (P<0.05) on sperm forward motility. The mean post-thaw
progressive motility values for samples frozen in 7% GLY and 7% EG were 313.3 and
50.8%, respectively (Figure 4.7). Progressive motility post-thaw for sperm frozen in 7%
61
Table 4.1. Testicular and epididymal values from bovine testes collected postmortem
Testicle weight (g) Cauda weight (g)
Bull No. Right Left Right Left

1 338 325 9 9
2 450 490 15 16
3 322 290 8 8
4 349 370 12 11
5 250 241 5 5
6 254 252 7 7
7 242 254 6 7
8 301 300 8 9
9 308 269 9 10
10* 92 470 0 11
Mean (SEM) 312.621.5 326.128.4 8.71.0 9.30.9
*Right testis not used in the study due to low weight and no sperm production. Means
values were not significantly different (P>0.05).
62
Figure 4.6. Examples of two pairs of bull testes with abnormal morphology.
63
100
80
60 Prefreeze
a Glycerol
40 b Ethylene Glycol
20
cc
0
Treatment
Figure 4.7. Percent progressive motility (meanSEM) of prefreeze and post-thaw bovine
epididymal sperm cryopreserved with glycerol and ethylene glycol. a,b,cMean values with
different letters are significantly different (P<0.05).
64
Prefreeze Glycerol Ethylene Glycol
100
80 a a
a a
a a a
a
a
60 a
b
b b
b
40 b b b b
b
20
c c
c
c
c c c c c c
c
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 4.8. Prefreeze and post-thaw percent progressive motility values (meanSEM) of
bovine epididymal sperm from individual bulls cryopreserved with glycerol or ethylene
glycol. a,b,cMeans with different letters within bull are significantly different (P<0.05).
65
GLY achieved a higher survivability than sperm frozen in 7% EG as assessed by post-
thaw progressive motility.
The mean progressive motility values for individual bulls prior to and post-
cryopreservation are shown in Figure 4.8. Sperm frozen in 7% GLY had a higher
progressive motility than sperm frozen in 7% EG after cryopreservation. However, sperm
from bull number 3 had little or no survivability post-thaw when compared with sperm
from the other bulls used using both CPAs in the study. Progressive motility declined
from 60 to 3.3% and from 60 to 0% when frozen in 7% GLY and 7% EG, respectively.
Post-thaw motility of sperm frozen in 7% GLY ranged from 25 to 40%, while sperm
frozen in 7% EG only ranged from 0 to 10%.
Membrane Integrity
The mean initial plasma membrane integrity value after epididymal sperm
retrieval was 831.5% for all the bulls used in this study. The mean percentage of sperm
with intact membranes after thawing was significantly (P<0.05) higher in samples frozen
in 7% GLY when compared with 7% EG (Figure 4.9). Moreover, there was a significant
decline in percent intact plasma membranes after cryopreservation using both CPAs.
The mean intact membrane values for individual bulls prior to and post
cryopreservation are shown in Figure 4.10. Sperm from bull number 4, 9 and 10 frozen
in 7% GLY had the highest number of viable sperm after thawing. These were
significantly different (P<0.05) in the incidence of live sperm after thawing from the
remaining bulls. Moreover, sperm from bull number 3 frozen in 7% GLY had the lowest
membrane integrity values of the 7% GLY-treated group. Conversely, only sperm from
bull number 1, 4 and 10 frozen in 7% EG had intact membrane values between 10 and
20%. These males had significantly higher sperm with intact membranes than those of
the remaining bulls in the 7% EG-treated group.
Acrosome Integrity
The overall mean prefreeze acrosome integrity value was 93.60.3% for all the
bulls used in this study. Overall, sperm frozen in 7% GLY and 7% EG treatment groups
had 81.22.0% and 73.33.4% intact acrosomes, respectively. No significant difference
was detected in acrosome integrity between the 7% GLY and 7% EG treatment groups.
However, there was a significant decline (P<0.05) in post-thaw acrosome integrity in the
66
100
a
80
60 Prefreeze
b Glycerol
40 Ethylene Glycol
20
c
0
Treatment
Figure 4.9. Percent membrane integrity (meanSEM) of prefreeze and post-thaw bovine
epididymal sperm cryopreserved with glycerol and ethylene glycol. a,b,cMeans with
different letters are significantly different (P<0.05).
67
100
a
a a a
a
a a
a
80 a a
b
b
60 b
b
b
40 b
b b b
c
c c
20
c
c c
c c
c c
c
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 4.10. Prefreeze and post-thaw percent membrane integrity (meanSEM) of

bovine epididymal sperm from individual bulls cryopreserved with glycerol and ethylene
glycol. a,b,cMean values with different letters are significantly different (P<0.05).
68
40
35
Reacted Acrosomes (%)
b
30
25
20 b
Viable
15 Nonviable
10
5 a
a
0
1
Glycerol 2 G.
Ethylene
Figure 4.11. Post-thaw percent reacted acrosomes (meanSEM) derived from the viable
and nonviable populations of bovine epididymal sperm cryopreserved with glycerol and
ethylene glycol. a,bMean values with different letters are significantly different (P<0.001).
69
7% GLY and 7% EG treatment groups when compared with the prefreeze acrosome
integrity value.
The overall mean number of live sperm with intact acrosomes was higher in the
7% GLY-treated group than in the 7% EG-treated group. There were 2.10.3% and
0.30.1% viable sperm with damaged acrosomes post-cryopreservation in the 7% GLY
and 7% EG treatment groups, respectively (Figure 4.11). In contrast, the number of
nonviable sperm with damaged acrosome membranes was lower in the 7% GLY-treated
group (16.51.5%) when compared with the 7% EG-treated group (26.02.1%). These
means were not statistically different (P>0.05). However, a difference (P<0.001) was
detected between the quantity of damaged acrosomes derived from membrane ruptured
than membrane intact sperm in both the 7% EG and 7% GLY treatment groups.
The mean acrosome integrity values for individual bulls before and after
cryopreservation are shown in Figure 4.12. Bull number 4, 5 and 10 in both treatment
groups did not have a significant decline in percent intact acrosomes after
cryopreservation as was the case for bull number 1, 2, 3, 6, 7, 8 and 9. Overall, there
was a high variability in acrosome integrity between bulls subjected to the same
treatments.
DISCUSSION
Reports comparing the quality of the sperm acrosome and the plasma membrane
of bovine caudal epididymal sperm cryopreserved from postmortem bulls are limited. In
this experiment, sperm progressive motility, acrosome and viability parameters of
epididymal sperm were evaluated before and after cryopreservation. Previous reports in
cats (Goodrowe and Hay, 1993), pigs (Kikuchi et al., 1998), mice (Songsasen et al.,
1998), cattle (Foote, 2000), horses (James et al., 2002), dogs (Yu and Leibo, 2002) and
sheep (Kaabi et al., 2003) and in exotic animals such as gaur (Hopkins et al., 1988),
rhinoceros (Lubbe et al., 2000), zebra (Lubbe et al., 2000), eland (Bissett and Bernard,
2005), Flying fox (De Jong et al., 2005), Common wombat (MacCallum and Johnston,
2005), Roe deer (Martinez-Pastor et al., 2005) and Red deer (Soler et al., 2005) have
focused on collection and/or cryopreservation of epididymal sperm from cooled testes
stored up to 7 days. In these studies most of the procedures use one epididymis to
evaluate sperm quality (control) and the sperm from the other testis are stored or frozen,
which increases the chance for error. Recently, Gooaverts et al. (2006) have indicated
70
100 a a
a a a a a a a
a
a a a a
b a
b
b b b
b
b
80 b
b
b
Acrosome Integrity (%)
c c
c
c c
60
40
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 4.12. Percent acrosome integrity values (meanSEM) of prefreeze and post-thaw
bovine epididymal sperm from individual bulls cryopreserved with glycerol and ethylene
glycol. a,b,cMean values with different letters within bulls are significantly different (P<0.05).
71
that sperm from both bovine caudae epididymides within the same male were not
comparable in most quality parameters.
In our experiment, it was decided to harvest sperm in the shortest time possible
(4 to 6 hours) without lowering the temperature of the testes to 4C. Various reports
have shown a lowered fertilizing ability in vitro of sperm that has was kept in testes
stored at low temperatures (Hay and Goodrowe, 1993; Kikuchi et al., 1998). Our aim
was to successfully develop a protocol to cryopreserve bovine epididymal sperm.
Therefore, we wanted to obtain sperm that were not exposed to the stress imposed by
low-temperature storage and possible degeneration caused by postmortem tissue
necrosis.
In the present study, progressive motility, acrosome integrity and plasma
membrane integrity of frozen-thawed bovine epididymal sperm cryopreserved with two
different CPAs were evaluated. Our results show that 66%, 94% and 83% of sperm
harvested from postmortem bulls have progressive motility, intact acrosome membranes
and intact plasma membranes, respectively. A detrimental effect of cryopreservation in
the survivability of epididymal sperm post-thaw was clearly evident from the use of both
glycerol and ethylene glycol as CPAs. Furthermore, sperm frozen in glycerol showed a
higher survivability post-thaw compared with that of ethylene glycol. These results differ
from those of various reports from other species using glycerol and ethylene glycol to
cryopreserve ejaculated sperm (Mahadevan and Trounson, 1983; Alvarenga et al.,
2000; Li et al., 2005; Rota et al., 2005; Sanitani et al., 2005). For example, Mahadevan
and Trounson (1983) reported a similar cryoprotective effect between 7.5% glycerol and
7.5% ethylene glycol for cryopreservation of human ejaculated sperm.
In contrast, Glander and Colditz (1981) reported a higher survivability of frozen-
thawed human ejaculated sperm as assessed by motility using glycerol compared with
that of ethylene glycol at three different concentrations (3.5%, 7% and 12%). Rota et al.
(2005) found a higher post-thaw survivability in canine ejaculated sperm cryopreserved
with ethylene glycol than with glycerol. Santiani et al. (2005) reported no difference
between glycerol and ethylene glycol for the cryopreservation of llama ejaculated sperm.
However, they reported plasma membrane integrity values as high as 20% post-thaw for
ethylene glycol. There range in values were similar to the results obtained in our study.
72
In sperm from Cynomolgus monkeys, ethylene glycol was reported to provide a
similar cryoprotective effect as glycerol (Li et al., 2005). Also, Alvarenga et al. (2000) and
Squires et al. (2004) found similar cryoprotective effects between glycerol and ethylene
glycol in post-thaw equine sperm. In contrast, Mantovani et al. (2002) noted a
significantly higher progressive motility in equine ejaculated sperm frozen with glycerol
than with ethylene glycol. They reported a detrimental effect of ethylene glycol on
progressive motility and membrane integrity at concentrations higher than 3%. In our
study, a 7% CPA concentration was used on the epididymal sperm, which could have
contributed to the decline in progressive motility, acrosome integrity and membrane
integrity post-thaw. It should be noted that bovine ejaculated sperm is routinely frozen in
CPA concentrations ranging from 5 to 8%, while in equine CPA concentrations used are
generally lower.
In previous studies we have verified that ethylene glycol at a 7% concentration
was not toxic to bovine epididymal sperm (Guerrero et al., 2006). This demonstrates that
ethylene glycol does not visibly affect bovine epididymal sperm during its addition
sequence prior to cryopreservation. It may be that ethylene glycol at lower concentration
(7%) does not provide protection against freezing injury.
In such species as mice, rats, pigs and horses ejaculated sperm are more
sensitive to CPAs than in other species (Sztein et al., 2001). In mice, higher post-thaw
survival of epididymal sperm has been obtained without the use of permeating CPAs
(Sztein et al., 2001). In contrast, bovine sperm require a higher concentration of
permeating CPA to successfully survive the freezing process. Therefore, the 7%
concentration of ethylene glycol used in our study may have been too low to provide
enough protection for bovine epididymal sperm. Furthermore, the same freezing rate
was used for both CPAs. It is possible that there is an interaction between CPA and
freezing rate. Ethylene glycol migh have a different optimum freezing rate than glycerol.
The success obtained with glycerol as a CPA to cryopreserve epididymal sperm
was shown to vary between bulls. Thus far, the understanding of the factors associated
with intermale variation in most species is very sparce.
We have demonstrated that the routine protocol to cryopreserve bovine
ejaculated sperm has to be modified to increase the survivability of post-thaw epididymal
sperm. It must be taken into account that ejaculated and epididymal sperm are
73
morphologically and functionally different (Hewitt et al., 2001). Hammerstedt (1993) has
shown that bull and ram epididymal sperm consume less ATP when compared with that
of ejaculated sperm. Recently, Goovaerts et al. (2006) demonstrated that bovine
epididymal sperm display a lower motility than ejaculated sperm from the same bull
when assessed by Computer Assisted Sperm Analysis System (CASA). Membrane
properties between epididymal and ejaculated sperm also differ, which may affect sperm
survival after cooling and freezing (Rath and Niemann, 1997). Moreover, the distal
cytoplasmic droplet found in epididymal sperm is thought to make them more vulnerable
to tail damage post-thaw (personal observation, unpublished). This morphological
difference may influence their susceptibility to osmotic damage during the
cryopreservation process. It has been shown that the fertilizing ability of canine
epididymal sperm is compromised after cryopreservation due to an increase in the
percent of sperm with abnormal morphology (Hori et al., 2004). However, no studies
have been reported to date to assess a direct comparison between the freezability of
epididymal and ejaculated sperm obtained from the same male.
We have successfully adapted the flow cytometric triple fluorochrome technique
for use with bovine epididymal sperm. This system has provided us with data showing
acrosome integrity from membrane damaged (nonviable) and membrane intact (viable)
sperm. Most dual stain techniques adapted to the flow cytometer to assess acrosome
integrity are compromised due to misreading sperm with egg yolk particulates. By using
the triple stain technique, egg yolk particles can successfully be removed from sperm
events by making gates based on DNA positive events. In the present study, acrosome
integrity readings were based only on events showing positive DNA staining.
Most sperm with reacted acrosomes were found to come from the nonviable
population than from the viable population in both CPA treatments (P<0.001). This
indicates that the plasma membrane is more sensitive to damage by the freezing
process than the acrosome compartments in epididymal sperm. Furthermore, the
plasma membrane was shown to be compromised prior to the acrosome. Most live
sperm were found to have an intact acrosome post-thaw. This is important for a sperm to
be able to penetrate the layer of cumulus cells and the zona pellucida surrounding an
oocyte during the fertilization process.
74
Factors that could have affected the success obtained with glycerol as a CPA
were that the testes were obtained from abattoir derived bulls. Bulls sent to be
slaughtered are sent there for numerous reasons, including low fertility. We confirmed
this by only using 10 bulls of 32 males that showed epididymal sperm motility
parameters higher than 60%.
It can be concluded that ethylene glycol at low concentrations does not provide
adequate protection against freezing injury during cryopreservation of bovine caudal
epididymal sperm. Moreover, the routine protocol used to cryopreserve bovine
ejaculated sperm must be improved to successfully cryopreserve sperm harvested from
postmortem animals. More research is needed to establish what concentration of CPA is
the optimal for the cryopreservation of bovine epididymal sperm.
75
CHAPTER V
THE EFFECT OF ADDITION OF BOVINE SEMINAL PLASMA TO BOVINE CAUDAL

EPIDIDYMAL SPERM PRIOR TO CRYOPRESERVATION
INTRODUCTION
Few studies on the cryopreservation of bovine epididymal sperm have been
reported. Recovery of epididymal sperm from postmortem animals will enable the
propagation of valuable gametes from genetically superior animals that may die or have
been injured. Development of a successful protocol to retrieve and cryopreserve bovine
epididymal sperm can be used as a model for the preservation of male gametes from
endangered bovid species.
In a recent study, Gooaverts et al. (2006) reported lower sperm motility
characteristics of bovine epididymal sperm when compared with ejaculated sperm. The
main difference between these two cell types is the presence or absence of accessory
sex gland fluids. Seminal plasma is a complex mixture of fluids from the testis,
epididymis and accessory sex glands and is used as a vehicle for sperm into the female
reproductive tract. Furthermore, it provides sperm with nutrients, a variety of biochemical
components for the regulation of sperm function post-ejaculation (Strzezek et al., 1992)
and factors that alter the sperm surface (Polakoski et al., 1982) and modulate the
fertilizing ability of sperm (Amann and Griel, 1974).
The details on how seminal plasma affects the function of ejaculated sperm
remain unclear and controversial. Some studies report beneficial effects (pigs: Pursel et
al., 1973; humans: Lindholmer, 1974; horses: Pickett et al., 1975; rabbits: Muller and
Kirchner, 1978 cattle: Dott et al., 1979; Bass et al., 1983; Acott and Hoskins, 1987;
Garner et al., 2001 and sheep: Graham, 1994; Barrios et al., 2000) while others report
detrimental effects (cattle: Shannon, 1965; pigs: Okagura and Sugita, 1983; horses:
Amann and Pickett, 1987 and humans: Han et al., 1990).
Studies examining the post-thaw influence of seminal plasma on the survivability
of sperm have been contradictory. One report stated that removal of seminal plasma
was necessary for sperm cryosurvival (Amann and Pickett, 1987). In contrast, other
reports indicate that seminal plasma is beneficial for sperm survivability post-thaw
(Aurich et al., 1996, Katila et al., 2002). Graham (1994) reported that the addition of
seminal plasma to washed bovine ejaculated or epididymal sperm had no effect on
76
motility post-thaw. However, post-thaw motility was higher for epididymal sperm when
treated with seminal plasma than for ejaculated sperm derived from the same male.
Removal of cytoplasmic droplets before cryopreservation may help reduce the
percentage of broken midpieces and circular swimming patterns normally found in post-
thaw epididymal sperm. It is probable that this morphological difference between
epididymal and ejaculated sperm renders epididymal sperm more susceptible to osmotic
damage during the cryopreservation process. It has been shown that the fertilizing ability
of canine epididymal sperm is compromised after cryopreservation due to an increase in
the percentage of sperm exhibiting abnormal morphology (Hori et al., 2004).
Selivanova et al. (1937) proposed that seminal plasma and its rate of dilution
were the primary cause of cytoplasmic droplet detachment. Moreover, Bialy and Smith
(1958) showed that seminal vesicle fluid reduces the quantity of distal droplets from
bovine caudal epididymal sperm. Subsequently, a phospholipid binding protein was
identified in the bovine ampulla and seminal vesicles, but not in epididymal fluid, which
was capable of liberating droplets from mature and immature sperm from various
species (Matousek and Kysilka, 1980). It is known that fewer sperm have droplets in the
ejaculates of boars, rams, bulls and bucks compared with epididymal sperm suggesting
that the droplets are removed near or prior to the time of ejaculation (Cooper, 2005).
Recently, Hori et al. (2005) reported a significant decrease in cytoplasmic
droplets in canine epididymal sperm recovered with prostatic fluid. They reported that
epididymal sperm motility, membrane integrity and pregnancy rates were higher after
thawing when sperm was recovered with prostatic fluid.
Extended exposure of bovine epididymal sperm to seminal plasma have been
reported to have detrimental effects on sperm viability (Way et al., 2000). In contrast, the
effects of epididymal sperm retrieval with seminal plasma followed by a short incubation
and its complete removal by density gradient centrifugation before cryopreservation has
not been carefully evaluated.
Flow cytometry is a valuable tool to evaluate frozen-thawed sperm since it is an
objective technique. This methodology allows accurate detection of small differences
between samples, with high repeatability and statistical reliability (Pena, 2000). Studies
on the use of flow cytometry with bovine epididymal sperm are uncommon. We have
previously adapted the triple fluorochrome staining technique (SYBR 14/PI/PE-PNA)
77
developed by Nagy et al. (2003) for the evaluation of frozen-thawed epididymal sperm.
This technique allows the accurate detection of sperm membrane integrity and
acrosome integrity in the presence of egg yolk particulates.
Sperm motility depends greatly on the energetic status of the cell. Since the
mitochondria of the sperm midpiece generate energy to support movements, changes in
mitochondrial membrane potential have been suggested as a good indicator of
functional sperm impairment (Pena et al., 2003). Furthermore, mitochondrial function
has been related to fertility in some species (human: Kasai et al., 2002; stallion: Kirk et
al., 2005).
Often variation in fertility found between individual males can not be adequately
explained by conventional methods of sperm quality evaluation. This led to speculations
that nonmorphological defects in sperm may be implicated in reduced fertility (Bedford et
al., 1973). Normal sperm development leads to a chromatin structure in which DNA is
fully resistant to denaturation (Gillian et al., 2005). Defects in the structure of chromatin
can severely reduce fertility, impair embryo development, increase spontaneous
abortions as well as birth defects (Evenson et al., 1980; Evenson and Jost, 2000;
Cordelli et al., 2005).
The DNA of sperm with an abnormal chromatin structure is susceptible to
denaturation and the extent of this abnormality can be detected using the metachromatic
properties of acridine orange in the sperm chromatin structure assay (SCSA) (Evenson
et al., 1980). This well validated method has had good correlation with male fertility in a
number of species, including mice (Evenson et al., 1980), cattle (Ballachey et al., 1987;
Karabinus et al., 1990; Januskauska et al., 2001; Januskauskas et al., 2003), pigs
(Evenson et al., 1994), horses (Love and Kenney, 1998) and humans (Hashimoto et al.,
2003; Boe-Hansen et al., 2006; Chohan et al., 2006; Erenpreiss et al., 2006).
Testing sperm for their susceptibility to DNA denaturation would increase our
ability to eliminate males with potentially low fertility. No information has been found on
chromatin stability from sperm of abattoir-derived bulls.
The objective of this study was to assess the effect of absence or presence of
seminal plasma during bovine caudal epididymal sperm retrieval as regard sperm
morphology, progressive motility, viability, mitochondrial function and chromatin stability
post cryopreservation. Furthermore, we will optimize the use of the SCSA and of
78
MitoTracker Red CMXRos in combination with SYBR 14 for the assessment of
chromatin stability and mitochondrial function of bovine epididymal sperm derived from
postmortem abattoir bulls.
Experimental Design
Experiment 5.1
Paired testes were obtained from mature bulls (n=10) at a local abattoir and
transported to the laboratory within 3 to 5 hours postmortem. Caudal epididymal sperm
were retrieved by multiple incisions from the caudae epididymides from each pair of
testes, sperm from each bull was pooled and placed in either egg yolk Tris-glucose citric
acid monohydrate extender (EYT-GC) (Treatment A) or bovine seminal plasma
(Treatment B). Epididymal sperm was incubated for 30 minutes before removal of
seminal plasma by centrifugation. Sperm from each treatment were then cryopreserved
in EYT-GC extender supplemented with 7% glycerol. Prefreeze membrane integrity and
acrosome integrity was evaluated by fluorescence microscopy. Post-thaw membrane
integrity, acrosome integrity, mitochondrial function and DNA integrity were evaluated
using multicolor flowcytometry. Overall and progressive motility were assessed
subjectively under a light microscope. Three replicate samples were evaluated per
animal each on different days.
Testes Collection
Testes were collected as pairs from mature mixed breed bulls at an abattoir in
Robert, Louisiana. The testes were then transported within 3 to 5 hours to the Louisiana
State University Embryo Biotechnology Laboratory individually packed in Ziploc plastic
bags (Johnson & Son Inc., Racine, WI) in a Styrofoam box with the temperature ranging
from 28C to 29C.. After arrival to the laboratory, each pair of testes was dissected
away from its scrotum and tunica vaginalis. Testes were positioned on the table with the
vas deferens facing down and the corpus facing up to distinguish the side of origin of
each testis. The weight of the testis was measured with a Pelouze balance scale
(Model SP5) before carefully dissecting free the cauda epididymis.
79
Medium Preparation
Egg yolk Tris-glucose citric acid monohydrate extender (Liu et al., 1998) (EYT-
GC) (250 mM Tris (T-6066, Sigma-Aldrich Inc., St. Louis, MO), 69 mM glucose (6152,
Sigma-Aldrich Inc., St. Louis, MO), 81 mM citric acid monohydrate (C-1909, Sigma-
Aldrich, St. Louis, MO) and 20% (v/v) egg yolk) was prepared with the addition of 50
g/ml of gentamicin (15750-060, Gibco, Grand Island, NY) and filtered through a 0.45
m Nalgene syringe filter (190-2545, Nalge Company, Rochester, NY) to remove large
egg yolk materials. Egg yolks were obtained from fresh eggs at the Louisiana State
University Poultry Research Farm. The extender was placed in a 37C water bath for 2
to 3 hours prior to harvesting the epididymal sperm.
Seminal Plasma Collection and Processing
Ejaculates for seminal plasma extraction were collected from mature beef bulls
(n=6) at a commercial bull stud (Genex Corporation, Baton Rouge, Louisiana).
Ejaculates were collected with an artificial vagina (AV) and placed in a 37C water bath
for motility analysis. Ejaculates were transported to the Embryo Biotechnology
Laboratory at 20 to 25C for seminal plasma collection and storage.
Collections with more than 50% progressively motile sperm were pooled and
used as part of this experiment. Samples were centrifuged at 2,000 x g in a general
purpose centrifuge (Model 5682 GP8R, Thermoforma, Marietta, OH) for 20 minutes. The
supernatant was placed into a new 15 ml plastic tube (20171-024, VWR International
Inc., Sugar Land, TX) and the sperm pellet was discarded. The centrifugation procedure
was repeated twice. The supernatant of each sample was filtered through a 0.8 m
Acrodisc syringe filter (PN 4618, Pall Corporation, East Hills, NY) for the complete
removal of any remaining sperm. Filtered seminal plasma from the six bulls was
thoroughly mixed together (pooled) and stored in 2 ml aliquots in a -20C freezer. On the
day of use, 3 vials of the seminal plasma were thawed in a 37C water bath ~30 minutes
prior to epididymal sperm extraction.
After dissection, each cauda epididymis was weighed with a balance scale and
washed from remaining materials with 0.9% saline before epididymal sperm retrieval.
Epididymal sperm were harvested by making 5 incisions using a surgical blade (size 10;
MDS-15110, Medline Industries Inc., Mundelein, IL) in the cauda epididymis. Half of the
80
sperm was scooped out from the incisions with a new surgical blade and placed in a 60
mm Falcon plastic petri dish (3037, Becton Dickinson Labware, Franklin Lakes, NJ)
containing 2 ml of EYT-GC extender (Treatment A). The other half was placed (with a
new blade) in another 60 mm plastic petri-dish containing 2 ml of seminal plasma
(Treatment B). Each sperm treatment sample was transferred into separate 4 ml plastic
tubes and placed in a 37C water bath for 30 minutes.
Assessment of Morphology
After the incubation period and after cryopreservation, a small droplet of each
sperm sample was mixed in ~20 l of eosin-nigrosin morphology stain (Lane
Manufacturing Inc, Denver, CO) previously prepared on a precleaned microscope slide
(3051, Gold Seal Products, Portsmouth, NH). Using a new slide, a smear was prepared
by spreading the sperm-stain mixture over the surface of the slide. Smears were allowed
to air dry for ~24 hours before evaluation. Smears were evaluated under light
microscopy using a 1,000X oil immersion objective (Nikon, Tokyo, Japan). A minimum of
200 sperm per treatment sample were evaluated to determine the percentages of lose
heads, abnormal heads, broken necks, bent tails, presence of distal and/or proximal
droplets. In this experiment, the presence of distal droplets was not recorded as sperm
abnormality at the time of evaluation.
Seminal Plasma Removal
Samples were placed over a two column sperm separation medium (99264,
ISolate, Irvine Scientific, Santa Ana, CA) (1 ml lower and 1 ml upper layer) in a 15 ml
plastic tube and centrifuged for 20 minutes at 300 x g to remove seminal plasma. After
centrifugation, the ISolate upper layer with the seminal plasma or extender was
removed. The lower layer containing the sperm suspension (1 ml) was resuspended in 8
ml of Tyrodes Lactate HEPES (TL-H) (04-616F, Cambrex Bio Science Walkersville Inc.,
Walkersville, MD) to wash sperm and remove silica particles. The sperm mixture was
then centrifuged at 350 x g for 5 minutes and resuspended in EYT-GC extender.
Epididymal Sperm Analyses Prior to Cryopreservation
Sperm samples were analyzed for plasma membrane integrity, acrosome
integrity, overall motility and progressive motility. A 100-l sample from the sperm
mixture was placed in a 1.7 ml plastic micro-centrifuge tube (20170-355, VWR
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International Inc, Sugar Land, TX) and diluted 1:10 sperm solution:EYT-GC for
epididymal sperm quality analysis.
Plasma Membrane Integrity
Kit (L-7011, Molecular Probes Inc., Eugene, OR)). The kit consists of two nucleic acid
Viability Kit) is a permeable nucleic acid stain that labels membrane intact sperm green.
nonpermeable nucleic acid stain which labels membrane ruptured sperm red. For
analysis, 250 l of the diluted sperm sample from each treatment group per bull were
placed in a 0.65 ml plastic micro-centrifuge tube (20170-293, VWR International Inc.,
Sugar Land, TX). Staining was completed by the addition of 100 nM SYBR 14 and 24
M PI to the sperm solution. The stained sample was incubated for 10 minutes in the
dark at 37C and mixed gently before analysis.
Acrosome Integrity
Acrosome integrity was assessed using the lectin peanut agglutinin conjugated to
FITC (FITC-PNA) (L-7381, Sigma-Aldrich Inc., St. Louis, MO) and the nucleic acid stain
PI. PNA labels acrosome reacted sperm green, while PI discriminates between viable
and nonviable sperm. For analysis, 250 l of the diluted sperm sample from each
treatment group per bull were placed in a 0.65 ml plastic micro-centrifuge tube. The final
concentration was 5 g/ml of FITC-PNA and 24 M PI. The stained sample was
incubated for 15 to 20 minutes in the dark at 37C and mixed before analysis.
For plasma membrane and acrosome integrity, each sample (n=10 bulls per
treatment) was analyzed by counting 300 sperm cells with an inverted microscope
(Nikon Diaphot, Tokyo Japan) equipped with epi-fluorescent illumination, a FITC filter set
and a heated stage.
Progressive motility
Sperm progressive motility was assessed subjectively before cooling on each
treatment for each of the 10 bulls by the same technician at 20X magnification using an
inverted microscope (Nikon Diaphot, Tokyo, Japan) equipped with Hoffman optics and a
heated stage.
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Sperm concentration was adjusted to 70 x 106 cells/ml using a hemacytometer by
the addition of more EYT-GC extender. The 15 ml plastic tubes containing the
epididymal sperm samples were placed in a 250 ml Pyrex glass beaker (13912-207,
VWR International Inc., Sugar Land, TX) containing water at room temperature. The
beaker was placed for 4 hours in a refrigerator for cooling the sperm samples to 4C.
Samples were diluted slowly over a period of 30 minutes 1:1 in EYT-GC medium
containing 14% glycerol and subsequently loaded into previously cooled 0.5 ml straws
(005569, Cassou straw, IMV Technologies, Minneapolis, MN). Straws were placed in a
custom built rack 2 cm over liquid nitrogen (LN2) vapors for 10 minutes before being
plunged in LN2. A total of 10 to 15 straws were frozen per bull.
Sperm samples were stored for 2 to 3 months prior to thawing. On day of
analysis, 50 ml of TL-H were placed in a 50 ml plastic tube (BCT-P50RS, Biologix,
Shawnee Mision, KS) and warmed in a 37C incubator 1 hours prior to sperm dilution.
Straws were thawed in a water bath at 37C for 40 seconds. The contents of each straw
were placed in a 15 ml plastic tube and diluted 1:5 sperm sample:TL-H before assessing
sperm morphology, sperm motility and multicolor flow cytometric analyses.
Flow cytometric measurements were carried out on a FACSCalibur flow
cytometer (Beckton Dickinson Immunocytometry Systems, San Jose, CA). Forward and
side scatter values were recorded on a linear scale, while fluorescent values were
recorded on a logarithmic scale for all assays, unless otherwise stated. All
fluorochromes were excited at 488 nm with a 20 mW argon laser. Density and dot plots
drawn for data analysis were generated by WinMDI 2.8 (software by J. Trotter, available
for downloading at http://www.facs. scripps.edu/software.html).
Assessment of Plasma Membrane and Acrosome Integrity Post-Cryopreservation
Assessment of plasma membrane and acrosome integrity were completed using
a triple stain protocol in the presence of egg yolk particles (Nagy et al., 2003) with minor
modifications. The staining protocol consisted of SYBR 14, PI and the lectin peanut
agglutinin conjugated to PE (R-PE-PNA) (p-44, Biomeda Corporation, Foster City, CA).
At a final concentration of 100 nM SYBR 14 solution, 4 g/ml of R-PE-PNA (1 mg/ml of
stock solution in a buffer composed of 50 mM sodium phosphate and 0.05% sodium
83
azide, [pH 7.0], and also containing 0.1 mM [Ca2+] and [Mn2+] ions) and 12 M PI
solution were added to 250 l of diluted sperm sample. Samples were thoroughly mixed
and incubated at 37C in the dark for 30 to 40 minutes and then gently remixed before
flow cytometric analysis.
Plasma membrane ruptured sperm were PI positive, and their red fluorescent
signal was detected on the FL3-channel using a 610 nm long pass filter. Membrane
intact sperm were SYBR 14 positive, and their green fluorescent signal was detected on
the FL1-channel using a 530/30 nm band pass filter. Acrosome reacted sperm were PE-
PNA positive, and its orange fluorescent signal was detected at 585/42 nm on the FL2-
channel. Three color compensations were set according to the FACS training manual
(Becton Dickinson Biosciences, San Jose, CA). Nonsperm events (egg yolk particles)
were gated out of the analyses, as judged on scatter properties detected in the forward
and side scatter detectors. Moreover, events with similar scatter characteristics to sperm
but with low PI and SYBR 14 fluorescence were also gated out in the procedure. Sperm
acquisitions were made using CellQuest software (Becton Dickinson, San Jose, CA,
USA). The flow cytometer was kept at a low flow of less than 300 sperm/second. The
recording of scatter and fluorescent properties of all events stopped when 10,000 gated
events were recorded.
On SYBR 14 (FL1) and PI (FL3) dot plots, regions were drawn to determine the
percentage of viable and non viable sperm cells. On PE-PNA (FL2) and PI (FL3) dot
plots, quadrants were set to measure the percentage of sperm showing plasma
membrane intact and acrosome intact sperm (LL), plasma membrane intact and
acrosome reacted sperm (LR), plasma membrane ruptured and acrosome intact sperm
(UL) and plasma membrane ruptured and acrosome reacted sperm (UR).
Assessment of Mitochondrial Function Post-Cryopreservation
Assessment of mitochondrial function was carried out using a novel dual stain
protocol in the presence of egg yolk particles developed in our laboratory. The staining
protocol consisted of SYBR 14 and MitoTracker Red CMXRos (MITO) (Molecular
Probes Inc., Eugene, OR, USA). A 1mM MITO stock solution was prepared in DMSO.
Then a 20 M working solution was prepared in TL-H before sample preparation. For
staining, 100 nM SYBR 14 and 100 nM MITO were added to 250 l of diluted sperm.
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Figure 5.1. Flow cytometric out-gating of egg yolk particles based on scatter properties
after staining frozen-thawed bovine epididymal sperm with SYBR 14 and MitoTracker
Red. Sperm were selected (R1) based on size, gating out small particles.
Figure 5.2. MitoTracker Red (red) versus SYBR 14 (green) fluorescence. Remaining egg
yolk particles having sperm-like scatter properties were gated out based on DNA
fluorescence. Events that had SYBR 14 (R1) positive fluorescence were gated out and
used for analysis.
85
Samples were thoroughly mixed and incubated at 37C in the dark for 30 minutes and
then remixed before flow cytometric analysis.
Detector FL-1 was used to detect SYBR 14 fluorescence (green). MITO
fluorescence was detected on detector FL-3. Data acquisition and compensations were
set, as described previously. The egg yolk particles were gated out based on scatter
properties (Figure 5.1) and on particles showing high green fluorescence (Figure 5.2).
Acquisitions were stopped after recording 10,000 SYBR 14 positive events.
On SYBR 14 (FL-1) and MITO (FL-3) dot plots, regions were drawn to determine
the percentage of sperm with active mitochondria and sperm with no mitochondrial
function. Sperm positive events showing a high red fluorescence were determined as
viable sperm with active mitochondrial membrane potential (Figure 5.3). Sperm positive
events showing a low red fluorescence were assessed as sperm with mitochondria that
had lost their transmembrane potential (Figure 5.4).
Assessment of DNA Integrity Post-Cryopreservation
DNA integrity was assessed using acridine orange (AO) (15855-0, Sigma-
Aldrich, St. Louis, MO) in the sperm chromatin structure assay (SCSA), as described by
Evenson (1980), with minor modifications. The utility of AO is based on its
metachromatic emission of fluorescence upon excitation. AO intercalated into double
stranded (dsDNA) nucleic acids fluoresces green. AO bound to single stranded (ssDNA)
nucleic acids emits a red fluorescence (Ballachey et al., 1987). When used with a flow
cytometer, the green and red fluorescence can be separated to indicate the amount of
ssDNA in the sperm sample. Evaluation of abnormal chromatin structure was assessed
as the increased susceptibility of sperm to acid-induced denaturation in situ.
Quantification was completed by measuring the percent of cells that shift from green to
red fluorescence. Cells outside the green population are determined as cells outside the
main population (COMP). COMP can be analyzed either by a flow cytometric dot plot or
histogram. The dot plot was used for analysis in this study.
Diluted sperm samples were further diluted 1:5 with TNE buffer (0.01 M tris
(hydroxymethyl) aminomethane (Tris) (15453-3, Sigma-Aldrich, St. Louis, MO), 0.15 M
NaCl (S5886, Sigma-Aldrich, St. Louis, MO) and 1 mM disodium ethylenediaminetetra
acetate [EDTA] [E-5134, Sigma-Aldrich, St. Louis, MO], pH 7.4) to a total of 0.2 ml.
Samples were maintained on ice at all times. Sperm were subjected to partial DNA
86
Figure 5.3. Flow cytometric dot plot illustrating MitoTracker Red (red) and SYBR 14
(green) fluorescence from sperm of a bull exhibiting high mitochondrial activity. Graph
displays an epididymal sperm sample (10,000 sperm) with 69% of its population having
active mitochondria.
Figure 5.4. Flow cytometric dot plot illustrating MitoTracker Red (red) and SYBR 14
(green) fluorescence from sperm of a bull exhibiting low mitochondrial activity. Graph
displays an epididymal sperm sample (10,000 sperm) with only 17% of its population
having active mitochondria.
87
denaturation in situ by mixing the 0.2 ml of diluted sperm sample with 0.4 ml of a low pH
detergent solution (0.15 M NaCl, 0.1% Triton X-100 [T-9284, Sigma-Aldrich, St. Louis,
MO] and 0.08 N HCl [SA48-1, Fisher Scientific, Fair Lawn, NJ], pH 1.4). Exactly 30
seconds later, 1.2 ml of AO staining solution (0.2 M NaH2PO4 [S-5011, Sigma-Aldrich,
St. Louis, MO], 1 mM disodium EDTA, 0.15 M NaCl, 0.1 M citric acid monohydrate
[C-1909, Sigma-Aldrich, St. Louis, MO], 6 g/ml of AO, pH 6.0) were added to the sperm
sample. The stained samples were analyzed 3 minutes later by flow cytometry.
Fluorescent values were recorded on a linear scale. Detector FL-1 was used to
detect the green fluorescence emitted by dsDNA. Detector FL-3 was used to detect the
red fluorescence emitted by ssDNA. The standard bull (CSS 7H5188, Genex
Cooperative Inc., Shawano, WI) used for in vitro fertilization at the Embryo
Biotechnology Laboratory was used to set up the machine and as a control. Data
acquisition and compensations were set, as described previously. Most egg yolk
particles were gated out by drawing a region on particles showing a low green and red
fluorescence. Acquisitions were stopped after recording 10,000 gated events.
On the red (FL-3) and green (FL-1) dot plot (Figure 5.5) and the (FL-3) histogram
(Figure 5.6) regions were drawn to determine the percentage of COMP. Sperm events
showing red fluorescence (% COMP) were determined as sperm with damaged DNA.
Variances in testicular parameters, morphology, membrane integrity, acrosome
integrity, mitochondrial function, % COMP and overall and progressive motility were
statistically analyzed by ANOVA and the differences between bulls were calculated by a
Tukey Multiple Comparisons test. Additionally, a t-test was used to verify differences
within each sperm parameter before and after cryopreservation. The sperm parameters
are expressed as meanSEM per treatment group. A P<0.05 was considered significant
in this study. Data were analyzed using SigmaStat Statistical Software Version 2.0.
RESULTS
Experiment 5.1
Testicular and Epididymal Weights
Testicular and caudae epididymal weights for individual bulls are shown in Table
5.1. Weights from the right testis of bull number 5 were not included in the mean values
due to the abnormally low weight. Testes weights ranged from 152 to 455 g and caudae
88
Figure 5.5. Flow cytometric dot plot illustrating green versus red fluorescence of acridine
orange in the SCSA. Graph displays bovine sperm with no DNA damage as green and
sperm with denatured DNA (R1) fluorescing red.
Figure 5.6. Flow cytometric histogram illustrating red fluorescence of acridine orange in
the SCSA. Bovine sperm in the M1 region are exhibiting DNA damage (% COMP).
89
Table 5.1. Testicular and epididymal measurements from bovine testes collected
postmortem
Testis weight (g) Cauda weight (g)
Bull No. Right Left Right Left

1 340 326 10 9
2 249 253 10 12
3 251 439 8 13
4 306 367 12 13
5* 90 455 0 9
6 317 364 6 8
7 368 379 15 18
8 218 243 6 5
9 152 154 3 3
10 184 145 8 7
MeanSEM 26524.3 31234.6 8.61.2 9.71.4
*Weight from the right testis and cauda of bull number 5 were not included in the total
*mean values due to the abnormally low weight. Means from testes and cauda weights
*were not significantly different (P>0.05).
90
epididymides weights ranged from 3 to 18 g. No significant difference (P>0.05) was
detected between bulls in both testicular parameters. There was a high intermale
variability in both testicular weights analyzed.
Morphology
Overall, epididymal sperm harvested with the addition of seminal plasma had
80.64.9% of sperm with normal morphology and was significantly higher (P<0.05) than
with without the addition of seminal plasma at 57.55.8%. Incubation in seminal plasma
significantly improved sperm quality, reducing the quantity of distal cytoplasmic droplets
and bent tails both prior to and post-cryopreservation (Table 5.2). However, the
incidence of other forms of sperm abnormalities were not increased or decreased by
treating sperm with bovine seminal plasma.
After cryopreservation, sperm previously exposed to seminal plasma had
76.23.3% of sperm with normal morphology compared with 39.93.7% in the
nonexposed treatment group. Cryopreservation significantly (P<0.05) increased the
percentage of sperm displaying bent tails and increased distal cytoplasmic droplet
detachment in both treatments.
Although the quantity of distal droplets was reduced after incubation in seminal
plasma, the extent of this removal was different among bulls. High distal cytoplasmic
droplet detachment was observed in 70% of the bulls after incubation in seminal plasma
while the remaining 30% showed no significant effect.
The mean initial overall motility and progressive motility values for all the bulls
used in this study after epididymal sperm retrieval were 75.51.7% and 68.02.1%,
respectively. Cryopreservation had an overall detrimental effect (P<0.05) on post-thaw
sperm motility for sperm harvested with or without seminal plasma treatment groups.
The mean overall post-thaw motility values for samples retrieved with or without seminal
plasma were 47.52.1% and 40.52.1%, respectively. In addition, the mean progressive
post-thaw motility values for samples retrieved with or without seminal plasma were
39.62.4% and 20.31.8%, respectively. Epididymal sperm retrieved with seminal
plasma achieved a significantly higher post-thaw overall and progressive motility than
the control (no seminal plasma).
91
Table 5.2. Morphology values (mean%SEM) of bovine epididymal sperm prior to and post-cryopreservation
Treatment Distal Proximal Bent Abnormal Detached Broken

group Normal droplets droplets tails heads heads necks
Control Pre 57.55.8a 65.82.0a 7.13.7a 21.95.4a 5.21.1a 7.22.2a 1.10.9a
Post 39.93.8b 52.73.7b 3.20.9b 47.34.3b 4.20.8a 5.11.6a 0.50.5a
.Seminal Pre 80.64.9c 45.91.3c 5.83.3a 3.70.6c 4.50.5a 4.41.3a 1.01.0a

.plasma
Post 76.23.3c 36.92.5d 3.01.2b 11.93.0d 3.70.7a 4.31.3a 0.70.5a
a,b.c
Means with different superscripts within columns are significantly different (P<0.05).
92
Prefreeze Extender Seminal Plasma
100
a
a a a
a
80 a
a a a
b a
Overall Motility (%)
b b
60 b
b
b
b
c
b c
b
c
c b
b
40 b
c
b
c
c
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 5.7. Post-thaw percent overall motility (meanSEM) of bovine epididymal sperm
harvested with either extender (no seminal plasma) or seminal plasma prior to
cryopreservation. a,b,cMeans with different letters within bull are significantly different
(P<0.05).
93
100
80
a a a a
a a
a
a a
60 b
b a
b b
b
b c
40 b
c
c c b
b
c
c
20 b
c
c b
c
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 5.8. Post-thaw percent progressive motility (meanSEM) of bovine epididymal

sperm harvested with either extender (no seminal plasma) or seminal plasma prior to
(P<0.05).
94
The mean overall and progressive motility values for individual bulls prior to and
post-cryopreservation are shown in Figures 5.7 and 5.8. Epididymal sperm collected
from animals harvested with seminal plasma had a higher (P<0.05) overall and
progressive motility than controls after thawing. However, bull number 6 and 7 showed
no difference (P>0.05) in overall motility between treatments. Furthermore, bull number
9 had a higher post-thaw overall motility in the control treatment than in sperm retrieved
with seminal plasma.
Membrane Integrity
The mean initial plasma membrane integrity value for the bulls used in this study
before cryopreservation was 81.22.1%. The mean percentages of post-thaw sperm
with intact membranes were 52.12.4% and 53.02.0% when retrieved with or without
seminal plasma, respectively. No significant difference was detected between
treatments. In contrast, there was a significant decline (P<0.05) in the percentage of
sperm with intact plasma membranes after cryopreservation in both sperm treatment
groups.
The mean intact membrane values for individual bulls prior to and post -
cryopreservation are shown in Figure 5.9. Bull number 7 had the highest number of
viable sperm after thawing in both treatments, while bull number 9 had the lowest
membrane integrity values prior to and post-cryopreservation of the bull group.
Acrosome Integrity
Overall, the mean prefreeze acrosome integrity value was 92.00.5%.
Epididymal sperm harvested with seminal plasma contained 75.03.1% intact
acrosomes, compared with 80.62.2% in the control group. No statistical difference was
detected between treatment groups. However, there was a significant decline (P<0.05)
in acrosome integrity after the cryopreservation process in sperm retrieved with or
without seminal plasma.
The mean numerical value of viable sperm with intact acrosomes was higher
(52.72.0%) when epididymal sperm were not exposed to seminal plasma but not
different than that for sperm exposed to seminal plasma (51.52.4%). Sperm exposed to
seminal plasma prior to cryopreservation had a mean of 0.60.1% viable sperm with
damaged acrosomes after thawing, while the control sperm had 0.30.1%. Furthermore,
the numerical value of post-thaw nonviable sperm with reacted acrosomes was higher
95
100
a
a a
a
a a
a
80
a
b a
a
b
b
b
b
b b
b
b
60
b
c
b c
b
c
c b
b
40 b
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 5.9. Post-thaw percent membrane integrity (meanSEM) of bovine epididymal

(P<0.05).
96
40
35
Reacted Acrosomes (%)
30
a
25
a Viable
20
Nonviable
15
10
5
b
b
0
1
Extender 2 Plasma
Seminal
Figure 5.10. Post-thaw percent reacted acrosomes (meanSEM) derived from the viable
and nonviable populations of bovine epididymal sperm harvested with extender (no
seminal plasma) or seminal plasma prior to cryopreservation. a,bMeans with different
letters within treatment are significantly different (P<0.05).
97
100
a a a a
a a b a a a a
b b b b
b b b b b
b b
80 b b
b b
c
c
60
c
40
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 5.11. Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

cryopreservation. a,b,cDifferent letters within bulls indicate significantly different (P<0.05).
98
(23.73.0%) in sperm exposed to seminal plasma but not different than that for sperm
not exposed to seminal plasma (18.11.8%) prior to cryopreservation (Figure 5.10). In
contrast, a higher quantity (P<0.001) of sperm with damaged acrosomes was found from
the nonviable than from the viable sperm population in sperm retrieved with or without
seminal plasma.
The mean acrosome integrity values for individual bulls before and after
cryopreservation are shown in Figure 5.11. Bull number 8, 9 and 10 had a significantly
higher (P<0.05) acrosome integrity when sperm were not exposed to seminal plasma
before freezing. However, bull number 1 had a higher acrosome integrity when exposed
to seminal plasma prior to cryopreservation. There was an intermale variation noted in
acrosome integrity post-thaw with males being exposed to the same treatments.
Mitochondrial Function
The overall mean percentages of post-thaw sperm with functioning mitochondria
were 49.32.0% and 47.91.7% when retrieved with or without seminal plasma prior to
cryopreservation, respectively. No significant (P>0.05) difference was detected between
treatments.
The overall mean values for sperm exhibiting functioning mitochondria for
individual bulls post-cryopreservation are shown in Figure 5.12. Bull number 1, 4 and 7
had a significantly higher percentage of sperm with active mitochondria after thawing
when sperm were exposed to seminal plasma prior to the freezing process compared
with the control extended sperm. However, bull number 9 had lower mitochondrial
function post-thaw when sperm were exposed to seminal plasma prior to
cryopreservation compared with the not exposed to seminal plasma treatment group.
DNA Integrity
The overall mean values of post-thaw sperm exhibiting chromatin damage were
1.60.3% and 2.50.5% when harvested with or without seminal plasma prior to
cryopreservation, respectively. The range of chromatin damage when sperm were
retrieved with or without seminal plasma were 0.9 to 3.5% and 0.8 to 7.8%, respectively.
The mean values of post-thaw sperm exhibiting chromatin damage for individual
bulls are shown in Figure 5.13. Bull number 1 had significantly (P<0.01) less DNA
damage when sperm were briefly exposed to seminal plasma prior to the
99
Extender Seminal Plasma
100
80
Mitochondrial Activity (%)
a a
a a a
60 a a a
b
a b a a
a
a b a a
40
b
b
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 5.12. Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

cryopreservation. a,bMeans with different letters within bull are significantly different
(P<0.05).
100
Extender Seminal Plasma
10
a
8
7
DNA Damage (%)
5
a
4 b
3 a a a
a a a
2
a a a a b a a
b b
a a
1
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 5.13. Percent DNA damage (meanSEM) of post-thaw bovine epididymal sperm
harvested with either extender (no seminal plasma) or seminal plasma prior to
cryopreservation. a,bMeans with different letters within males are significantly different
(P<0.05).
101
cryopreservation process compared to sperm not exposed to seminal plasma. There
was a variation noted between males exposed to the same treatment.
DISCUSSION
The number of studies conducted on the cryopreservation of bovine epididymal
sperm is very limited and, to the best of our knowledge, none has reported the effect of
epididymal sperm retrieval with seminal plasma followed by a short incubation and its
complete removal before cryopreservation.
In the work described herein we have demonstrated that the use of seminal
plasma to harvest bovine epididymal sperm is beneficial to enhance sperm quality post-
cryopreservation. Seminal plasma significantly improved overall and progressive motility
(P<0.05) characteristics when compared with the control (no seminal plasma) and
increased the quantity of sperm exhibiting normal morphology post-thaw. However,
seminal plasma exposure prior to cryopreservation had neither beneficial nor detrimental
effects on viability, mitochondrial function and DNA integrity after thawing.
Ejaculated sperm have been reported to differ from epididymal sperm in
respiration (Hammerstedt et al., 1993), heparin-binding sites (Nass et al., 1990), ability
to capacitate (Miller et al., 1990), morphology (Hewitt et al., 2001) and proteins bound to
the plasma membrane (Lee et al., 1985). Recently, bovine ejaculated sperm have been
shown to display different motion characteristics than bovine epididymal sperm when
assessed by computer-assisted sperm analysis (Gooaverts et al., 2006). Epididymal
sperm exhibits lower straight line and average path velocity and higher amplitude of the
head, which could hinder the penetration of the zona pellucida during fertilization
(Suarez et al., 1991).
In stallions, epididymal sperm have been shown to display progressive motility
similar to ejaculated sperm. However, the fertility of stallion epididymal sperm was much
lower than ejaculated sperm after cryopreservation (Tiplady et al., 2002). Also, canine
epididymal sperm have been reported to exhibit lower motility and pregnancy rates than
ejaculated sperm post-cryopreservation (Hori et al., 2004). Recently, the addition of
prostatic fluid to canine epididymal sperm prior to freezing enhanced motility, membrane
integrity and pregnancy rates after cryopreservation (Hori et al., 2005).
Various groups have reported seminal plasma factors that support or enhance
sperm motility and fertility (Baas et al., 1983; Okamura and Sugita, 1983; Killian et al.,
102
1993; Bellin et al., 1998; Love, 2005). However, seminal plasma is also known to contain
factors that inhibit motility (Iwamoto and Gagnon, 1988), inhibit capacitation (Chang,
1957) and negatively affect viability of sperm during extended incubation (Way et al.,
2000). These contradictory reports may be due to seminal plasma having different roles
in different species. Some species might only use it as a vehicle of transportation into
the female reproductive tract, while others might need it as a final maturation step prior
to fertilization.
In the present study, we found no detrimental effect in post-thaw viability when
epididymal sperm were harvested with seminal plasma. Most studies in the bull
regarding effects of seminal plasma on sperm have been made during long term
incubation and have not studied its possible cryoprotective effect. We avoided long term
incubation to minimize its possible detrimental effects. No difference in viability was
noted for epididymal sperm harvested and incubated for 30 minutes in seminal plasma
before cryopreservation compared with that of the control extender before
cryopreservation. In contrast, Way et al. (2000) found a 20% decrease in sperm viability
as soon as seminal plasma was added to bull epididymal sperm.
In the present study, seminal plasma enhanced both post-thaw overall and
progressive motility characteristics of epididymal sperm. This is in agreement with a
recent study reported were motility was greatly improved post-thaw when canine
epididymal sperm was harvested with prostatic fluid (Hori et al., 2005). This might have
been due to beneficial factors in seminal plasma as well as the reduction in cytoplasmic
droplets prior to cryopreser-vation. In the present study, the motion characteristics of
sperm incubated with seminal plasma both pre- and post-thaw was not assessed.
However, faster and straighter progressive motility was observed after sperm incubation
with seminal plasma.
Henault et al. (1995) demonstrated that incubation of epididymal sperm with
seminal plasma increased their ability to penetrate bovine oocytes in vitro. A possible
explanation may be that seminal plasma factors might have enhanced straight line
motility and reduced head amplitude increasing cumulus cells and zona pellucida
penetration rates. Also, seminal plasma might have caused modifications in the plasma
membrane that enhanced zona pellucida recognition and binding. This further verifies
103
that bovine seminal plasma might have uses other than transportation of sperm into the
female reproductive tract that are still unknown.
The overall mean number of epididymal sperm possessing droplets after
extraction was 65.8%. This value is in close agreement with 62.9% having droplets
reported by Branton and Salisburry (1947) but is lower than the 84% with droplets
reported later by Bialy and Smith (1957). This difference could be accounted to different
extenders used, dilution rates and possibly male to male variation. Furthermore, our
results are in agreement with previous studies (Selivanova et al., 1937; Bialy and Smith,
1958), that reported a decrease in cytoplasmic droplets after incubation of bovine
epididymal sperm in seminal plasma. Our results showed a significant decrease
(P<0.05) in cytoplasmic droplets when epididymal sperm was harvested and incubated
in seminal plasma at 37C for 30 minutes. The extent of this reduction in cytoplasmic
droplets was found in some bulls more than others. Part of the difference could be
accounted for by the inherent bull differences (Bialy and Smith, 1957).
In the present study, cryopreservation caused a significant elevation in the
percentage of epididymal sperm with abnormal morphology only when sperm had not
been exposed to seminal plasma prior to the freezing process. This could be due to the
reduction of cytoplasmic droplets when sperm was incubated in seminal plasma prior to
cryopreservation. However, 1 of 10 bulls showed no significant reduction in cytoplasmic
droplets after incubation with seminal plasma and had no elevation of post-thaw
morphological abnormalities. Thus, we propose that unknown factors in seminal plasma
are perhaps protecting sperm from osmotic or mechanical damage during
cryopreservation.
Bent tails were reported to increase in rooster sperm during the removal of
glycerol post-thaw (Westfall and Howarth, 1977). From previous observations at the
laboratory (unpublished observations), cytoplasmic droplets are thought to increase the
sensitivity of sperm to osmotic changes and to mechanical stress rendering them more
vulnerable to morphological damage at the tail and midpiece after thawing. These
observations were confirmed in this study, when the amount of bent tails increased
significantly after thawing in the treatment not exposed to seminal plasma. These results
are in agreement with those in a recent study using canine epididymal sperm, where an
104
elevation in morphological sperm abnormalities was noted post-cryopreservation (Hori et
al., 2004).
The lower progressive motility we observed in epididymal sperm not treated with
seminal plasma might have been due to the increase in bent tails post-thaw. However,
post-thaw epididymal sperm previously exposed to seminal plasma were observed to
possess faster velocity and straighter motility when compared with progressively motile
sperm from the control treatment. An increase in bent tails and circular swimming
patterns post-thaw have been observed in cattle and White Tail deer epididymal sperm
(personal observations, unpublished) cryopreserved with a routine freezing protocol
used for ejaculated sperm. This may be due to unknown biochemical components in
seminal plasma that permanently modify sperm enhancing motility characteristics.
A sperm motility factor found in bovine seminal plasma has been reported to
restore motility in nonmotile bovine sperm (Bass et al., 1983). In elephants, equine
seminal plasma was shown to initiate motility while almost no motility (<1%) was initiated
by the addition of extender (Schmitt et al., 2005). In humans, zinc and fructose levels in
seminal plasma were reported to be correlated with motility (Fuse et al., 1999; Patel et
al., 1988). Further investigations should include fractionation of bovine seminal plasma
to identify potential chemical components that enhance progressive motility.
Some of the published protocols developed to evaluate sperm function with a
flow cytometer appear to be compromised for an accurate analysis of frozen-thawed
sperm. This is likely due to interactions of the fluorochromes with egg yolk particles of
the freezing extender leading to misinterpretation of the results. The development of the
SYBR 14/PI/PE-PNA triple stain protocol by Nagy et al. (2003) allows the easy removal
of egg yolk particulates found in the freezing extender by gating using scatter properties
and by particles exhibiting DNA staining.
Although various fluorochromes are being used to assess mitochondrial function
in sperm, there are several problems connected with them for the evaluation of frozen-
thawed sperm. Some of the most commonly used fluorochromes involve Rhodamine
123 (R123), MitoTracker Green, JC-1 and recently MitoTracker Deep Red 633. R123 is
considered not reliable due to its low sensitivity, variability within samples and
background staining (Garner et al., 1997). In addition, its fluorescence is lost when
mitochondria experiences losses of membrane potential, thus limiting its use for fixation.
105
R123 also has been reported to have a higher nonspecific affinity to other parts of the
sperm than JC-1 and MtioTracker Green (Garner et al., 1997).
MitoTracker Green labels mitochondria regardless of membrane potential. Its
main use is to measure size and structure of mitochondria (Molecular Probes
Handbook). Therefore, it is not a reliable fluorochrome to evaluate mitochondrial activity
in sperm.
The usefulness of JC-1 is that this carbocyanine dye is capable of discriminating
between mitochondria exhibiting high membrane potential from those possessing
relatively low membrane potentials. However, by previous observations in our laboratory
and by those of Garner et al. (1999), it does not work properly on frozen-thawed sperm
due to interactions with egg yolk particles. MitoTracker Deep Red was shown to be
reliable to evaluate mitochondrial function in frozen-thawed sperm (Hallap et al., 2005).
However, a conventional flow cytometer equipped with a 488 nm argon laser can not be
used to excite this fluorochrome due to its high excitation wavelength.
We report for the first time the use of MitoTracker Red CMXRos in combination
with SYBR 14 to assess mitochondrial function in frozen-thawed epididymal sperm. This
combination allows us to gate nonsperm events depending on scatter properties and
DNA stainability.
Mitochondrial function in the present study was correlated to viability and overall
sperm motility, however, mitochondrial function was not correlated to progressive
motility. Previous studies have reported a high correlation between bovine ejaculated
sperm mitochondrial activity and motility (Januskauskas et al., 2005). However, our
results show that an increase in morphological abnormalities, such as bent tails, can
hinder sperm from moving in a progressive manner even though they exhibit functionally
active mitochondria.
Here we report the use of the well validated SCSA for the evaluation of bovine
epididymal sperm. To our knowledge, this is the first report to assess DNA damage from
abattoir-derived bulls. Although we discarded those bulls with low motility (<50%) and
high levels of abnormalities, this assay may also help discard bulls with spermatogenic
disturbances that can not be uncovered by conventional methods. Also, no detectable
detrimental effect on DNA was found in post-thaw bovine epididymal sperm previously
exposed to seminal plasma when compared with the control sperm (no seminal plasma).
106
We found that some bulls possess chromatin that is more sensitive to denaturation than
other bulls. It was noted that one bull possessed a significantly lower (P<0.01) percent of
denatured DNA when it was harvested with seminal plasma. During the 30-minute
incubation period, seminal plasma might have provided the necessary antioxidants to
help protect DNA from free radicals (Love, 2005). Furthermore, the sperm nucleus
absorbs zinc from seminal plasma during ejaculation (Bjorndahl et al., 1986). Zinc was
shown to enhance chromatin stability (Kvist et al., 1987). Sperm DNA from that one bull
might have been more susceptible to DNA damage when compared with the other males
used in this study. Furthermore, one can not preclude that unknown factors in the
seminal plasma might help protect or stabilize sperm nuclear chromatin.
The effect of seminal plasma from highly fertile bulls compared with low fertility
bulls should be carefully studied, since incubation of sperm from a low fertility bull with
seminal plasma from a highly fertile bull has been shown to increase the oocyte
penetration rates in vitro (Heneault et al., 1995). Killian et al. (1993) have shown that two
proteins (26 and 55 kDa) in seminal plasma predominate in higher fertility bulls, while
Bellin et al. (1994) found two proteins (16 kDa each) that predominate in lower fertility
bulls. The use of seminal plasma from highly fertile bulls might help enhance even more
the protective effect during cryopreservation. It should not be overlooked that seminal
plasma might help improve survivability of cryopreserved sperm from lower freezer
bulls.
We propose that sperm retrieval in the presence of seminal plasma is beneficial
to enhance bovine epididymal sperm motility and to protect it from morphological
abnormalities derived during the freeze-thaw process. Further research is needed to
evaluate the performance and longevity of epididymal sperm exposed to seminal plasma
during incubation post-thaw to address any possible long term effects.
107
CHAPTER VI
EFFECT OF DILUTION AND INCUBATION OF POST-THAW BOVINE EPIDIDYMAL

SPERM HARVESTED WITH SEMINAL PLASMA PRIOR TO CRYOPRESERVATION
INTRODUCTION
Cryopreservation of bovine epididymal sperm is still very inefficient.
Recently, the quality of post-thaw bovine epididymal sperm has been enhanced by a
short exposure to seminal plasma prior to cryopreservation (unpublished, personal
observations). Seminal plasma produces physiological changes to sperm preparing it for
capacitation (Miller et al., 1990) and fertilization (Bellin et al., 1998). Osmotic injury can
occur when cryopreserved epididymal sperm are diluted directly into an isotonic medium
after thawing. When sperm treated with glycerol are abruptly placed in an isotonic
environment, the intracellular environment is hyperosmotic relative to the extracellular
environment. The membrane permeability of glycerol is lower than that of water.
Therefore, there is a net influx of water resulting in an increase in cell volume (Ball and
Vo, 2001). This can cause cell lysis and damage to the plasma membrane due to the
fast water movement across the membrane (Curry and Watson, 1994). Osmotic
tolerance of sperm to hyperosmotic conditions differs between species. Post-
hyperosmotic stress is characterized by an irreversible loss of motility, membrane
integrity and mitochondrial function (Ball and Vo, 2001).
A one step addition and removal of glycerol to cooled bovine epididymal sperm
have been shown to be detrimental (Guerrero et al., 2006). Step-wise addition of
glycerol to ejaculated sperm prior to cryopreservation is done routinely to prevent
osmotic damage. However, glycerol is routinely removed from post-thaw ejaculated
sperm in a one step dilution process. Swelling of sperm was reported to be more
detrimental than shrinking, which indicates the importance to further evaluate the
removal than the addition of cryoprotectants (Pommer et al., 2002). Although multiple
studies have demonstrated the theoretical benefits of a stepwise dilution to remove
permeating cryoprotectants from post-thaw sperm (e.g., Gilmore et al., 1995; Gao et al.,
1995; Gilmore et al., 1998; Liu and Foote, 1998; Phelps et al., 1999; Ball and Vo, 2001;
Agca et al., 2005), studies examining this effect experimentally are rather uncommon
(Gilmore et al., 1997; Wessel and Ball, 2004). No studies have been found on the
sensitivity of post-thaw bovine epididymal sperm to a one step removal of glycerol.
108
Moreover, epididymal sperm previously exposed to seminal plasma prior to
cryopreservation may be more sensitive to osmotic changes due to physiological
modifications caused to the plasma membrane by biochemical components found in
seminal plasma (Harrison et al., 1992).
In the stallion, stepwise removal of glycerol from fresh ejaculated sperm prior to
freezing prevented the detrimental effects on membrane integrity and motility caused by
a rapid, one step removal (Wessel and Ball, 2004). However, they found no beneficial
effect from stepwise dilution for removing glycerol from cryopreserved sperm. In
humans, slow dilution for removal of glycerol from cryopreserved sperm resulted in a
higher recovery of motile cells than when glycerol was removed abruptly (Gilmore et al.,
1997). In the domestic cat, multi-step dilution with isotonic medium for the removal of
glycerol after cryopreservation of sperm minimized loss of sperm motility and membrane
disruption (Pukazhenthi et al., 2002). Sperm from various species are more susceptible
to osmotic damage caused by the hyperosmolar environment caused by glycerol,
therefore, stepwise dilution might help reduce its detrimental effects. In mice, no more
than 0.3 M glycerol is used in freezing extenders because of its toxicity and osmotic
damage caused at the 1.0 M concentration usually used in other species (Katkov et al.,
1998). However, stepwise dilution of glycerol minimized osmotic shock and allowed a
high percentage of mouse sperm to survive up to 0.8 M glycerol concentration (Katkov et
al., 1998).
In most mammals, fertilization requires that a sufficient number of sperm survive
in the female tract until ovulation. Cryopreservation exposes sperm to different stress
conditions resulting in a reduction in the proportion of sperm surviving for fertilization, as
well as altering functional capacity of those sperm that do survive (Watson, 2000). Even
the best cryopreservation protocols allow lethal and sublethal cryoinjury affecting the
efficiency of sperm to achieve fertilization (Holt, 2000). Assessing longevity of
cryopreserved sperm is of primary importance to know if the sperm would be able to last
long enough to achieve fertilization. Individual bulls that produce semen capable of
maintaining a competent population of sperm over time are generally the ones that
achieve acceptable fertilization rates, even with a lower number of sperm in the
inseminate (Den Daas, 1997). No studies have assessed the survivability and longevity
of bovine epididymal sperm subjected to 4 hours of stress in vitro. Moreover, it is not
109
known if epididymal sperm previously exposed to seminal plasma prior to
cryopreservation will have a longer or shorter life span.
The use of multicolor flow cytometry to assess membrane integrity, acrosome
integrity and mitochondrial function will allow the detection of subtle differences between
treatments. The overall objectives of this two part study were: (1) to assess the effects of
one step removal of glycerol from cryopreserved bovine epididymal sperm previously
exposed to seminal plasma and (2) to assess epididymal sperm longevity during a 4-
hour incubation period at 37C.
Experimental Design
Experiment 6.1
This experiment was conducted to evaluate the effect of a one step dilution for
removal of glycerol from epididymal sperm that had been previously harvested without
seminal plasma (Treatment A) or with seminal plasma (Treatment B). Paired testes were
obtained from mature bulls (n=10) at a local abattoir and transported to the laboratory
within 3 to 5 hours postmortem. Caudal epididymal sperm from each pair of testes were
harvested from the caudae epididymides, pooled and then split evenly into two
treatments. Each of the two treatment samples was incubated for 30 minutes in either
egg yolk tris-glucose citric acid monohydrate extender (EYT-GC) (Treatment A) or
bovine seminal plasma (Treatment B). The seminal plasma in Treatment B was then
removed by centrifugation. Treatments A and B were cryopreserved in EYT-GC
extender supplemented with 7% glycerol. Three treatment replicates per bull were
analyzed before and after a one step dilution (1:5) for removal of glycerol. Plasma
membrane, acrosome integrity and mitochondrial function were evaluated by the same
technician using multicolor flow cytometry.
Experiment 6.2
This experiment was designed to evaluate the survivability of post-thaw
epididymal sperm when exposed to a 4-hour in vitro stress test. Duplicate diluted post-
thaw epididymal sperm samples of both Treatments A and B from Experiment 6.1 were
incubated at 37C for 4-hours. Three treatment replicates per bull were evaluated at both
0 hours and 4 hours post-incubation. Progressive motility was assessed subjectively with
a light microscope. Three replicates were repeated on different days. Plasma
110
membrane, acrosome integrity and mitochondrial function were assessed by the same
technician using multicolor flowcytometry.
Testes Collection
Bovine testes were collected as pairs from 10 mature mixed breed bulls at an
abattoir in Robert, Louisiana. Testes were transported to the Louisiana State University
Embryo Biotechnology Laboratory individually packed in Ziploc plastic bags (Johnson &
Son Inc., Racine, WI) in a Styrofoam box (temperature ranging from 28 to 29C) within
3 to 5 hours postmortem. Upon arrival to the laboratory, testes were dissected away
from its scrotum and tunica vaginalis. Then, testes were positioned on a table with the
vas deferens facing ventrally and the corpus epididymis facing dorsally to distinguish the
side of origin of each testis.
Medium Preparation
Egg yolk Tris-glucose citric acid monohydrate extender (EYT-GC) (Liu et al.,
1998) (250 mM Tris (T-6066, Sigma-Aldrich Inc., St. Louis, MO), 69 mM glucose (6152,
Sigma-Aldrich Inc., St. Louis, MO), 81 mM citric acid monohydrate (C-1909, Sigma-
Aldrich, St. Louis, MO) and 20% (v/v) egg yolk) was prepared with the addition of 50
g/ml of gentamicin (15750-060, Gibco, Grand Island, NY). The extender was filtered
through a 0.45 m Nalgene syringe filter (190-2545, Nalge Company, Rochester, NY)
to remove large egg yolk particulates. Egg yolks from fresh chicken eggs were obtained
at the Louisiana State University Poultry Research Farm. The extender was placed in a
37C water bath 2 to 3 hours prior to epididymal sperm retrieval.
Seminal Plasma Collection and Processing
Ejaculates for seminal plasma extraction were collected from mature beef bulls
(n=6) at a commercial bull stud (Genex Corporation, Baton Rouge, LA). Ejaculates were
collected with an artificial vagina and placed in a 37C water bath for progressive motility
analysis. Samples with more than 50% progressively motile sperm were used for the
experiment. Ejaculates were transported to the Embryo Biotechnology Laboratory at 20
to 25C to harvest the seminal plasma.
Semen samples were centrifuged at 2,000 x g in a general purpose centrifuge
(Model 5682 GP8R, Thermoforma, Marietta, OH) for 20 minutes. The supernatant was
placed into a new 15 ml plastic tube (20171-024, VWR International Inc., Sugar Land,
111
TX) and the sperm pellet was discarded. This centrifugation procedure was repeated
twice on the seminal plasma. The supernatant of each sample was filtered through a 0.8
m Acrodisc syringe filter (PN 4618, Pall Corporation, East Hills, NY) for the complete
removal of any remaining sperm. Filtered seminal plasma from the 6 bulls was
thoroughly mixed together and stored in 2 ml aliquots in a -20C freezer. On the day of
use, 3 vials of seminal plasma were thawed in a 37C water bath 30 minutes prior
harvesting the epididymal sperm.
After dissection, caudae epididymides were rinsed with 0.9% saline to remove
any remaining materials before epididymal sperm retrieval. Epididymal sperm were
harvested by making 5 incisions using a surgical blade (size 10, MDS-15110, Medline
Industries Inc., Mundelein, IL) in each cauda epididymis. Half of the sperm was scooped
out from the incisions with a new surgical blade to a 60 mm Falcon plastic petri dish
(3037, Becton Dickinson Labware, Franklin Lakes, NJ) containing 2 ml of EYT-GC
extender (Treatment A). The remaining half of the sperm sample was placed with a new
blade in another 60 mm plastic dish containing 2 ml of seminal plasma (Treatment B).
Each sperm mixture was then transferred into separate 4 ml plastic tubes and placed in
a 37C water bath for 30 minutes.
Seminal Plasma Removal
Samples were placed over a two-column sperm separation medium (99264,
ISolate, Irvine Scientific, Santa Ana, CA) (1 ml lower layer and 1 ml upper layer) in a 15
ml plastic tube and centrifuged for 20 minutes at 300 x g to remove seminal plasma.
After centrifugation, the ISolate upper layer with the seminal plasma or egg yolk was
removed. The lower layer containing the sperm suspension (1 ml) was resuspended in 8
ml of Tyrodes-Lactate HEPES (TL-H) (04-616F, Cambrex Bio Science Inc., Walkersville,
MD) to wash sperm and remove silica particles. Sperm suspension was then centrifuged
at 350 x g for 5 minutes and resuspended in EYT-GC extender for both Treatments A
and B.
Sperm concentration was adjusted to 70 x 106 cells/ml using a hemacytometer by
the addition of more EYT-GC extender. The 15 ml plastic tubes containing the
epididymal sperm samples were placed in a 250 ml Pyrex glass beaker (13912-207,
112
VWR International Inc., Sugar Land, TX) containing water at room temperature. The
beaker was placed in a refrigerator for 4 hours to cool the sperm samples to 4C.
Samples were diluted slowly over a period of 30 minutes 1:1 in EYT-GC medium
containing 14% glycerol and subsequently loaded into previously cooled 0.5 ml straws
(005569, Cassou straw, IMV Technologies, Minneapolis, MN). The straws were placed
in a custom built rack 2 cm over liquid nitrogen (LN2) vapors for 10 minutes before being
plunged in LN2. A total of 10 to 15 straws were frozen per bull (35 x 106 cells/ml).
Sperm samples were stored for 2 to 3 months prior to thawing. On the day of
sperm analysis, 50 ml of TL-H supplemented with 3 mg/ml of bovine serum albumin
(Fraction V, A-7906, Sigma-Aldrich, St. Louis, MO) were placed in a 50 ml plastic tube
(BCT-P50RS, Biologix, Shawnee Mision, KS) and warmed in a 37C incubator 1 hour
prior to sperm dilution. Straws were thawed in a water bath at 37C for 40 seconds and
the contents of each straw were placed in a 15 ml plastic tube. Two 50 l droplets of
undiluted sperm were placed in each of two 5.5 ml plastic tubes. One tube was left
undiluted while the other was diluted 1:5 sperm sample:TL-H before multicolor flow
cytometric analyses. Duplicate diluted samples of each treatment were placed in an
incubator at 37C for 4 hours to expose thawed sperm to an in vitro stress test. After the
incubation period, sperm motility, membrane integrity, acrosome integrity and
mitochondrial function were assessed.
A FACSCalibur flow cytometer (Beckton Dickinson Immunocytometry Systems,
San Jose, CA) was used for sperm measurements. Forward and side scatter values
were recorded on a linear scale, while fluorescent values were recorded on a logarithmic
scale for all assays unless otherwise stated. All fluorochromes were excited at 488 nm
with a 20 mW argon laser. Density and dot plots drawn for data analysis were generated
by WinMDI 2.8 (software by J. Trotter, available for downloading at http://www.facs.
scripps.edu/software.html).
Assessment of Plasma Membrane and Acrosome Integrity
Assessment of plasma membrane and acrosome integrity were completed using
a triple stain protocol in the presence of egg yolk particulates (Nagy et al., 2003) with
minor modifications. The staining protocol consisted of SYBR 14, PI and the lectin
peanut agglutinin conjugated to PE (R-PE-PNA) (p-44, Biomeda Corp., Foster City, CA).
113
A final concentration of 100 nM SYBR 14, 4 g/ml of R-PE-PNA (1 mg/ml of stock
solution in a buffer composed of 50 mM sodium phosphate, and 0.05% sodium azide,
[pH 7.0] and also containing 0.1 mM [Ca2+] and [Mn2+] ions) and 12 M PI solution were
added to 250 l of diluted sperm suspension. Sperm samples were thoroughly mixed
and incubated at 37C in the dark for 30 to 40 minutes and then remixed gently by
pipetting before flow cytometric analysis.
Plasma membrane ruptured cells were PI positive, and their red fluorescent
signal was detected on the FL3-channel using a 610 nm long pass filter. Membrane
intact sperm were SYBR 14 positive, and its green fluorescent signal was detected on
the FL1-channel using a 530/30 nm band pass filter. Acrosome reacted sperm were PE-
PNA positive, and its orange fluorescent signal was detected at 585/42 nm on the FL2-
channel. Three color compensations were set according to the FACS training manual
(Becton Dickinson Biosciences, San Jose, CA). Nonsperm events (egg yolk particulates)
were gated out of analyses based on scatter properties detected in the forward and side
scatter detectors. Furthermore, events with similar scatter characteristics to sperm but
with low PI and SYBR 14 fluorescence were also gated out. Sperm acquisitions were
made using CellQuest software (Becton Dickinson, San Jose, CA). The flow cytometer
was kept at a low flow of less than 300 sperm/second. The recording of scatter and
fluorescent properties of all events stopped when 10,000 gated events were recorded.
On SYBR 14 (FL1) and PI (FL3) dot plots, regions were drawn to determine the
percentage of viable and non viable sperm. On PE-PNA (FL2) and PI (FL3) dot plots,
quadrants were set to measure the percentage of sperm showing plasma membrane
intact and acrosome intact sperm (LL), plasma membrane intact and acrosome reacted
sperm (LR), plasma membrane ruptured and acrosome intact sperm (UL), and plasma
membrane ruptured and acrosome reacted sperm (UR).
Assessment of Mitochondrial Function
Assessment of sperm mitochondrial function was conducted using a novel dual
stain protocol in the presence of egg yolk particles developed in our laboratory. The
staining protocol consisted of SYBR 14 and MitoTracker Red CMXRos (MITO)
(Molecular Probes Inc., Eugene, OR). A 1 mM MITO stock solution was prepared in
DMSO. Then a 20 M working solution was prepared in TL-H before sample
preparation. For staining, 100 nM SYBR 14 and 100 nM MITO were added to 250 l of
114
diluted sperm. Samples were thoroughly mixed and incubated at 37C in the dark for 30
minutes and then remixed gently before flow cytometric analysis.
Detector FL-1 was used to detect SYBR 14 fluorescence (green). MITO
fluorescence was detected on detector FL-3. Data acquisition and compensations were
set as described previously. The egg yolk particles were gated out based on scatter
properties and on particles showing high green fluorescence. Acquisitions were stopped
after recording 10,000 SYBR 14 positive events.
On SYBR 14 (FL-1) and MITO (FL-3) dot plots, regions were drawn to determine
the percentage of sperm with active mitochondria and sperm with no mitochondrial
function. Sperm positive events showing a high red fluorescence were determined as
viable sperm with active mitochondrial membrane potential. Sperm positive events
showing a low red fluorescence were assessed as sperm with mitochondria that had lost
their transmembrane potential.
Variances in progressive motility, membrane integrity, acrosome integrity and
mitochondrial function were statistically analyzed by ANOVA and the differences
between bulls were calculated by a Tukey Multiple Comparisons test. Additionally, a t-
test was used to verify differences in sperm parameters before and after
cryopreservation. The sperm parameters are expressed as meanSEM per treatment
group. A P<0.05 value was considered statistically significant in this study. All data were
analyzed using SigmaStat Statistical Software Version 2.0.
RESULTS
Experiments 6.1
Membrane Integrity
The mean overall percentage of sperm with intact membranes after thawing was
significantly (P<0.05) higher in undiluted samples when compared to the diluted samples
in the seminal plasma-exposed treatment (Treatment B) (Table 6.1). However, no
difference was detected before or after dilution for removal of glycerol in the control
extender group (Treatment A). Furthermore, the mean percent intact plasma
membranes in undiluted and diluted samples between both treatments (Treatments A
and B) were not significantly different.
115
Table 6.1. Percent plasma membrane integrity (meanSEM) of post-thaw bovine
epididymal sperm before and after dilution from the freezing extender
Plasma membrane integrity (%)

Treatment No. of
group bulls
Undiluted Diluted
Extender 10 57.92.0a 53.12.0a
Seminal Plasma 10 62.22.1a 51.52.2b
Extender = Treatment A; Seminal Plasma = Treatment B.

a,b
Means with different superscripts within rows are significantly different (P<0.05).
116
The mean intact membrane values for undiluted and diluted sperm samples from
individual bulls harvested without seminal plasma (Treatment A) or with seminal plasma
(Treatment B) or are shown in Figures 6.1 and 6.2, respectively. In both Treatments A
and B, bull number 2 appears to be the most affected by the one step dilution process,
while bull number 8 and 10 were the least affected by the one step dilution process.
Acrosome Integrity
The overall mean acrosome integrity values for undiluted and diluted post-thaw
sperm harvested with seminal plasma (Treatment B) or without seminal plasma
(Treatment A) prior to cryopreservation are shown in Table 6.2. The mean number of
viable sperm with intact acrosomes was not significantly different (P>0.05) before or
after dilution between both Treatments A and B. There were 0.20.3% and 0.60.1%
viable post-thaw sperm with damaged acrosomes before and after dilution, respectively,
for sperm harvested with seminal plasma (Treatment B). These values accounted for
0.11% and 0.31% for undiluted and diluted sperm post-cryopreservation in the control
group (Treatment A). The numerical value of nonviable sperm with damaged acrosomal
membranes was lower before and after dilution in the control group (Treatment A) than
in the seminal plasma group (Treatment B). These values accounted for 21.41.5%
before dilution and 24.91% after dilution for sperm exposed to seminal plasma
(Treatment B), as compared with 14.91% and 19.21% before and after dilution,
respectively, for sperm not exposed to seminal plasma (Treatment A). However, this
difference was not statistically significant (P>0.05). Furthermore, a difference (P<0.001)
was detected between the quantity of damaged acrosomes derived from nonviable than
from the viable sperm population in both Treatments A and B.
The mean acrosome integrity values for individual bulls before and after dilution
for sperm harvested without seminal plasma (Treatment A) or with seminal plasma
(Treatment B) are shown in Figures 6.3 and 6.4, respectively. Bull number 6 in the
seminal plasma group (Treatment B) and bull number 1 in the control group (Treatment
A) were the most affected by the dilution process. There was an intermale variation in
acrosome integrity between bulls subjected to the same treatments.
Mitochondrial Activity
The overall mean percentage of sperm with functioning mitochondria post-thaw
was significantly (P<0.05) higher in undiluted samples than in diluted samples in the
117
Undiluted Extender Diluted Extender
100
80 a
a a b
a a
a
a a
a b
60
b a a a
a
a
a a
40 a
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.1. Membrane integrity values (meanSEM) of post-thaw bovine epididymal

sperm before and after one step dilution for removal of glycerol. The bars represent
sperm from individual males harvested with the extender (Treatment A) prior to
cryopreservation. a,bMeans with different superscripts within males are significantly
different (P<0.05).
118
Undiluted Seminal Plasma Diluted Seminal Plasma
100
a
80
a
a b
a a
a
b a
a
60 a
a
a a
b a
b
b
40 a
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.

sperm from individual males harvested with seminal plasma (Treatment B) prior to
different (P<0.05).
119
Table 6.2. Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal
sperm before and after dilution from the freezing extender
Acrosome integrity (%)

Treatment No. of
group bulls
Undiluted Diluted
Extender 10 85.11.5 80.62.2
Seminal Plasma 10 78.63.2 75.03.1

Means were not significantly different (P>0.05).
120
100
a a
a a a a
a a a
a a a a
a a
a b
80 b
a
60
40
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.

different (P<0.05).
121
100
a
a
a
a b a
a a a
a a
80 b a a
b a
a
60 a
a
40
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.

different (P<0.05).
122
seminal plasma treatment group (Treatment B). However, no difference was detected
before or after dilution for removal of glycerol in the control treatment group (Treatment
A) (Table 6.3). In addition, there was no significant difference in the percentage of sperm
exhibiting active mitochondria in undiluted and diluted samples between both
Treatments A and B.
The mean values of sperm exhibiting functioning mitochondria for individual bulls
before and after dilution for sperm harvested without seminal plasma (Treatment A) or
with seminal plasma (Treatment B) are shown in Figures 6.5 and 6.6, respectively. Bull
number 1, 3, 4 and 7 had significantly higher post-thaw sperm displaying active
mitochondria in the undiluted sample when sperm were exposed to seminal plasma
(Treatment B) prior to the freezing process. However, bull number 9 had lower
mitochondrial function in the undiluted than in the diluted post-thaw sperm sample when
sperm were exposed to seminal plasma (Treatment B) prior to cryopreservation.
Experiment 6.2
Membrane Integrity
Incubation significantly (P<0.05) affected the post-thaw integrity of the plasma
membrane when epididymal sperm was harvested with seminal plasma (Treatment B) or
without seminal plasma (Treatment A) prior to cryopreservation (Table 6.4). However, no
difference in the percentage of sperm with intact membranes was detected between
Treatments A and B after 4 hours of incubation.
The mean membrane integrity values for sperm before and after being exposed
to the 4-hour in vitro stress test for epididymal sperm retrieved without seminal plasma
(Treatment A) or with seminal plasma (Treatment B) prior to cryopreservation are shown
in Figures 6.7 and 6.8, respectively. These values indicate that some bulls were more
sensitive to the 4-hour incubation period than the remaining bulls. Furthermore,
cryopreserved sperm from some males apparently deteriorate faster than others when
exposed to an in vitro stress test.
Acrosome Integrity
Acrosome integrity was significantly (P<0.05) affected by the 4-hour incubation
period in both Treatments A and B (Table 6.5). No difference was detected in acrosome
integrity at 0 hours post-cryopreservation between both Treatments A and B. However,
123
Table 6.3. Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal
sperm before and after dilution from the freezing extender
Mitochondrial activity (%)

Treatment No. of
group bulls
Undiluted Diluted
Extender 10 54.11.7a 47.11.7a

a,b
Means with different superscripts within rows and columns are significantly different
...(P<0.05).
124
100
80
a a
a a a
a
a a
60 a a
b a
a
a
a
a b a
a
40
a
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.

different (P<0.05).
125
100
a
80
a
a a
b a
b
a
a a
60 b a
b
a b a
b
a
40 a
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.

different (P<0.05).
126
Table 6.4. Percent plasma membrane integrity (meanSEM) of post-thaw bovine
epididymal sperm during a 4-hour incubation period at 37C
Plasma membrane integrity (%)

Treatment No. of
group bulls
0 hours 4 hours
Extender 10 52.72.0a 29.11.5b

a,b
Means with different superscripts within rows are significantly different (P<0.05).
Table 6.5. Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

sperm during a 4-hour incubation period at 37C
Acrosome integrity (%)

Treatment No. of
group bulls
0 hours 4 hours
Extender 10 80.62.2a 59.12.0b
Seminal Plasma 10 75.33.1a 49.01.9c

a,b,c
.....(P<0.05).
127
0 hours Extender 4 hours Extender
100
80
a
a
a a
a
60
a a
a
b a
b
40 a
b b
b b
b b
b
20
b
0
1 2 3 4 5 6 7 8 9 10
Bull No.

sperm before and after a 4-hour incubation period at 37C. The bars represent sperm
different (P<0.05).
128
0 hours Seminal Plasma 4 hours Seminal Plasma
100
80
a
a
a a
60
a a
a
a
a b
40
b b b
b b a
b
b
b
20 b
0
1 2 3 4 5 6 7 8 9 10
Bull No.

different (P<0.05).
129
there was significantly lower acrosome integrity for sperm exposed to seminal plasma
(Treatment B) when compared with the control group (Treatment A) after 4 hours of
incubation.
The overall mean acrosome integrity values for sperm at 0 and at 4 hours post-
incubation for sperm retrieved without seminal plasma (Treatment A) or with seminal
plasma (Treatment B) are shown in Figures 6.9 and 6.10, respectively. Cryopreserved
sperm from some males suffer acrosome damage faster than others when exposed to a
4-hour in vitro stress test.
Mitochondrial Activity
Exposing sperm to stress during incubation significantly (P<0.05) affected the
percentage of sperm exhibiting active mitochondria in both Treatments A and B (Table
6.6). However, no difference in the percentage of sperm with functioning mitochondria
was detected between both Treatments A and B at both 0 and 4 hours of incubation.The
mean values for sperm displaying functioning mitochondria for individual males at 0 and
4 hours post-incubation for sperm retrieved without seminal plasma (Treatment A) or
with seminal plasma (Treatment B) are shown in Figures 6.11 and 6.12, respectively.
Cryopreserved sperm from some males displayed a faster reduction in the percentage of
functioning mitochondria than others when exposed to a 4-hour in vitro stress test in
both Treatments A and B.
Progressive Motility
Sperm exposed to seminal plasma (Treatment B) displayed significantly higher
(P<0.05) post-thaw progressive motility than the nonexposed control group (Treatment
A), both before and after the incubation period (Table 6.7). The 4-hour incubation
significantly reduced progressive motility in both Treatments A and B. The mean values
for sperm displaying progressive motility for individual males at both 0 and 4 hours post-
incubation for sperm retrieved without seminal plasma (Treatment A) or with seminal
plasma (Treatment B) are shown in Figures 6.13 and 6.14, respectively. Some bulls
were more affected by incubation than others as observed in a decline in progressive
motility overtime. Sperm exposed to seminal plasma (Treatment B) maintained a higher
progressive motility overtime when compared with the control group (Treatment A).
130
100
a
a a
a a a
a a
80
a
a
a
60
b
b
b b
b b
40 b b b
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.9. Acrosome integrity values (meanSEM) of post-thaw bovine epididymal

different (P<0.05).
131
100
a a
a a
80 a
a
a a
a
a
a
60 b
a b b
b b b b
b
40
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.10. Acrosome integrity values (meanSEM) of post-thaw bovine epididymal

different (P<0.05).
132
Table 6.6. Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal
sperm during a 4-hour incubation period at 37C
Mitochondrial activity (%)

Treatment No. of
Group bulls
0 hours 4 hours
Extender 10 47.11.7a 28.81.7b

a,b
...(P<0.05).
Table 6.7. Percent progressive motility (meanSEM) of post-thaw epididymal sperm

during a 4-hour incubation period at 37C
Progressive motility (%)

Treatment No. of
Group bulls
0 hours 4 hours
Extender 10 20.31.8a 7.61.9c
Seminal Plasma 10 40.32.4b 21.02.5d

a,b,c,d
.(P<0.05).
133
100
80
a
a
60 a a
a
a
a a
a b
40 b b b
a b
b b b
b
20
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.11. Mitochondrial activity values (meanSEM) of post-thaw bovine epididymal

different (P<0.05).
134
100
80
a a
a
60 a
a
a a
a
a
40 b b
b
b
b b b a
b
20 b
b
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.12. Mitochondrial activity values (meanSEM) of post-thaw bovine epididymal

different (P<0.05).
135
100
80
60
a
40
a a a
a
a
20 b b
a
b b a
a b a
b
b
b b
b
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.13. Progressive motility values (meanSEM) of post-thaw bovine epididymal

different (P<0.05).
136
100
80
60 a
a
a a
a
a
40 a
b
b b b a
b a
b
20 a b
b b
0
1 2 3 4 5 6 7 8 9 10
Bull No.
Figure 6.14. Progressive motility values (meanSEM) of post-thaw bovine epididymal

different (P<0.05).
137
DISCUSSION
In the present study the one step dilution process for removal of glycerol from
cryopreserved epididymal sperm significantly affected plasma membrane integrity and
mitochondrial function of sperm previously exposed to seminal plasma. However,
exposure to seminal plasma did not produce any significant detrimental effect in
acrosome integrity when compared with the control treatment.
Seminal plasma has been shown to cause cholesterol efflux from the plasma
membrane to prepare sperm for capacitation, acrosome reaction and fertilization
(Osheroff et al., 1999; Iborra et al., 2000). The major protein fraction of bovine seminal
plasma is represented by a family of closely related proteins designated BSP proteins
(Manjunath and Sairam, 1987). These proteins represent ~65% of the total proteins in
seminal plasma (Bergeron et al., 2004). Also, these proteins have been shown to
interact with choline phospholipids in the plasma membrane and to potentiate sperm
capacitation induced by heparin (Therien et al., 1995) by stimulating cholesterol and
phospholipids efflux from the plasma membrane (Bergeron et al., 2004). We propose
that BSP proteins likely make the sperm more sensitive to osmotic changes during the
removal of glycerol when compared to sperm not exposed to seminal plasma during the
harvesting process.
During cryopreservation, sperm experience membrane perturbations and
membrane lipid and protein reorganization (Steponkus et al., 1983). High membrane
cholesterol levels inhibit crystallization of membrane hydrocarbons chains at low
temperatures (Rottem et al., 1973). This enhances membrane fluidity at low
temperatures and helps prevent damage to membranes as they undergo the phase
transition (Purdy and Graham, 2004). Treating sperm with cholesterol before
cryopreservation has resulted in the recovery of a higher proportion of membrane intact
and motile sperm in the bull (Purdy and Graham, 2004; Moce and Graham, 2006) and
the stallion (Moore et al., 2005). The short exposure of epididymal sperm to seminal
plasma prior to cryopreservation might have enhanced plasma membrane modifications
that rendered sperm more sensitive and vulnerable to dilution post-thaw. In the stallion,
rapid removal of glycerol was reported to cause a relative hyposmotic shock upon
dilution into isotonic medium resulting in loss of sperm motility, viability and
mitochondrial function (Ball and Vo, 2001). The rapid movement of water and glycerol
138
across the plasma membrane appears to account, in part, to the decline in membrane
integrity and mitochondrial function (Ball and Vo, 2001). Stepwise dilution with isotonic
media or multistep dilution with media of fixed molarities might help reduce the osmotic
damage caused by one step removal of glycerol.
In our second experiment, the long term effects of seminal plasma on post-thaw
epididymal sperm were assessed after a 4-hour incubation period at 37C. Incubation
significantly decreased progressive motility, membrane integrity, acrosome integrity and
mitochondrial function of post-thaw epididymal sperm harvested with or without seminal
plasma prior to cryopreservation. Membrane integrity and mitochondrial function did not
differ between the two treatments after 4 hours of incubation. However, acrosome
integrity was significantly lower at 4 hours of incubation when sperm were retrieved with
seminal plasma than without seminal plasma. In contrast, motility was higher at 4 hours
post-incubation for post-thaw epididymal sperm harvested with seminal plasma than
without seminal plasma.
In this study, we have demonstrated that the longevity of bovine epididymal
sperm harvested with seminal plasma prior to cryopreservation is not compromised
when compared with the extended sperm (control). To our knowledge this is the first
study to experimentally report that post-thaw bovine epididymal sperm are able to
survive a 4-hour stress test. In the stallion, no detrimental effect was noted on motility at
90 minutes post-thaw when 5% seminal plasma was added to the freezing extender
(Moore et al., 2005). However, it has been shown that increasing the content of equine
seminal plasma to 20% resulted in a significant decline in motility (Moore et al., 2005). In
our study, seminal plasma was completely removed 30 minutes after epididymal sperm
retrieval. This short incubation period could have helped reduce any long term
detrimental effects of seminal plasma.
Seminal plasma provides sperm with nutrients, a variety of biochemical
components for the regulation of sperm function (Strzezek et al., 1992) and factors that
alter the sperm surface (Polakoski et al., 1982). In addition, it has biochemical
components that permanently stimulate sperm enhancing motility characteristics. A
sperm motility factor found in bovine seminal plasma has been reported to restore
motility in nonmotile sperm (Bass et al., 1983). In our study, this sperm motility factor
might have increased sperm motility when compared with the treatment retrieved with
139
the extender. Seminal plasma has been shown to prepare sperm for capacitation and
the acrosome reaction. Thus, bovine epididymal sperm in our study might have been
capacitated faster than those in the control treatment during incubation. A higher
percentage of acrosome reacted sperm was found after 4 hours of incubation in the
seminal plasma exposed treatment. This might have been due to membrane
modifications caused by biochemical components in seminal plasma. Viable epididymal
sperm exhibiting damaged acrosomes after 4 hours were higher in the sperm seminal
plasma treatment than in the control treatment. However, these treatments were not
significantly different. It has been suggested that the reduced longevity and fertilizing
ability of cryopreserved sperm is due to accelerated capacitation (Perez et al., 1996).
In future experiments, membrane fluidity and calcium levels should be
investigated to assess the early stages of capacitation and determine if seminal plasma
is producing any significant effects. Further studies are needed to assess the effect of
harvesting epididymal sperm with seminal plasma from highly fertile bulls and with
seminal plasma collected by either artificial vagina or electroejaculation. Collection
techniques have been reported to affect the biochemical composition of bovine seminal
plasma (Bennett and Dott, 1967; Seidel and Foote, 1969). Furthermore, seminal plasma
might help improve survivability of cryopreserved sperm from lower freezer bulls.
140
CHAPTER VII
THE USE OF CRYOPRESERVED BOVINE CAUDAL EPIDIDYMAL SPERM FOR
INTRACYTOPLASMIC SPERM INJECTION (ICSI)
INTRODUCTION
ICSI is a procedure of mechanically inserting a whole sperm or an isolated sperm
head into the ooplasm of an oocyte. Since ICSI requires far fewer sperm to fertilize the
same number of oocytes than in vitro fertilization (IVF), it is potentially valuable
especially when a limited number are available (Keefer et al., 1990). The use of
postmortem epididymal sperm for ICSI will allow a more effective use of valuable
gametes if a breeding male unexpectedly dies.
In recent years, ICSI has been extensively used to treat male factor infertility in
humans, with thousands of babies born to date. Its importance in human infertility relies
on its ability to use semen samples with low sperm counts, low motile sperm and for
sperm with increased percentages of morphological abnormalities. In addition, sperm
can be used from patients with previous vasectomus by using testicular or epididymal
sperm. The ICSI procedure has revolutionized the treatment of male infertility providing
higher fertilization, pregnancy and implantation rates than standard IVF (Schwarzer et
al., 2003). ICSI bypasses the normal process of fertilization, where motile sperm are
needed to reach the oocyte, penetrate the cumulus cells, bind and penetrate the zona
pellucida and subsequently fuse with the oolema. This process requires the sperm to be
progressively motile, to be able to capacitate, undergo an acrosome reaction and
transform the plasma membrane for fertilization to be completed. Since ICSI bypasses
the initial steps of fertilization, nonfunctional or even dead sperm with a normal DNA
complement can be used to fertilize an ovum and produce healthy offspring (Goto et al.,
1990).
Since the first report of ICSI in hamsters (Uehara and Yanagimachi, 1976), this
technique has been used in various mammalian species. Live offspring have been
obtained using the ICSI procedure in the rabbit (Hosoi et al., 1988), cow (Goto et al.,
1990), human (Palermo et al., 1992), mouse (Kimura and Yanagimachi, 1995), sheep
(Catt et al., 1996), horse (Cochran et al., 1998), cat (Gomez et al., 2000), pig (Martin,
2000), rat (Hirabayashi et al., 2002), Cynomolgus monkey (Ng et al., 2002), goat (Wang
et al., 2003) and hamster (Horiuchi, 2006).
141
The efficiency of ICSI in cattle has been very limited because of the necessity for
additional oocyte activation before or after the sperm injection procedure (Suttner et al.,
2000). In contrast to mouse and human oocytes, bovine oocytes generally do not
activate by mechanical stimulus of the injection pipette (Goto et al., 1990). In mice,
~70% of sperm-injected oocytes reach the blastocyst stage (Kimura and Yanagimachi,
1995), while in cattle only between 1 to 20% develop into blastocysts (Goto et al., 1990;
Horiuchi et al., 2002). In contrast to humans, bovine sperm apparently need to be
pretreated by chemicals, immobilized before ICSI by freeze-thawing without
cryoprotectants or scoring the tail before injection to increase sperm head DNA
decondensation and pronuclear formation (Wei and Fukui, 1999). A detailed explanation
for these differences among various species has not been reported.
In mammals, the fertilizing sperm produces a series of long-lasting [Ca2+]
oscillations, which initiate shortly after sperm-oocyte fusion and can last up to 24 hours.
This continuous rise in [Ca2+] is required for normal oocyte activation and subsequent
embryo development. It is believed that a sperm factor is delivered to the ooplasm by
sperm at the time of fusion, which activates the phosphoinositide pathway (Swann,
1990). Recently, it was demonstrated that sperm injection into bovine oocytes produces
abnormal [Ca2+] responses and oocyte activation (Malcuit et al., 2006). Less than 10% of
the sperm-injected oocytes display any [Ca2+] responses. It is thought that the function or
activation of the sperm factor may be compromised after ICSI, thus leading to
incomplete [Ca2+] oscillations (Malcuit et al., 2006). This explains in part the need for
additional exogenous chemical activation of bovine sperm-injected oocytes.
Bovine oocytes for ICSI have been exogenously activated using electric
stimulation (Hwang et al., 2000), ethanol (Horiuchi et al., 2002; Fujinami et al., 2004;
Oikawa et al., 2005), calcium ionophore (A23187) (Goto et al., 1990; Keefer et al., 1990)
ionomycin (Chung et al., 2000; Keskintepe et al., 2002), ionomycin followed by DMAP
(Rho et al., 1998; Chung et al., 2000; Keskintepe et al., 2002; Ock et al., 2003; Li et al.,
2004; Oikawa et al., 2005) and ionomycin followed by cyclohexamide (Galli et al., 2003;
Rho et al., 2004), However, developmental rates of activated oocytes are still markedly
lower than after conventional IVF. Although there has been low ICSI efficiency in cattle,
there have been <35 calves reported after transfer of ICSI-derived blastocysts into
142
recipient females (Goto et al., 1990; Hamano et al., 1999; Horiuchi et al., 2002; Wei and
Fukui, 2002; Galli et al., 2003; Oikawa et al., 2005; Horiuchi, 2006).
Epididymal sperm have been used for ICSI in humans (Craft et al., 1995), mice
(Kimura and Yanagimachi, 1995), pigs (Kolbe and Holtz, 1999), cats (Pushett et al.,
2000), Cynomolgus monkeys (Ng et al., 2002), rats (Said et al., 2003), the Tammar
wallaby (Richings et al., 2004) and rabbits (Zheng et al., 2004). There has been only one
report of the use of bovine epididymal sperm for ICSI (Goto et al., 1990). In the latter
report, only 1.8% of 507 sperm-injected oocytes developed into blastocysts. One healthy
calf was born after transfer of 7 blastocysts into recipient females.
To our knowledge, pronuclear formation of epididymal sperm-injected oocytes
has not been evaluated in cattle. The low blastocyst development reported for
epididymal sperm-injected oocytes might have been due to low sperm head
decondensation of epididymal sperm. It is important to assess sperm head
decondensation and pronuclear formation of epididymal sperm before focusing on
blastocyst development. Also, due to chemical activation of sperm-injected oocytes in
present procedures, it can not be ruled out that some of the 7-day embryos produced to
date were of parthenogenetic origin.
Ejaculated and epididymal sperm may act differently when mechanically injected
into oocytes due to their biochemical and physiological differences. Ejaculated sperm
differ from epididymal sperm in respiration (Hammerstedt et al., 1993), motion
characteristics (Gooaverts et al., 2006), heparin-binding sites (Nass et al., 1990), ability
to capacitate (Miller et al., 1990), morphology (Hewitt et al., 2001) and proteins bound to
the plasma membrane (Lee et al., 1985). In humans, epididymal sperm are readily
capable of fertilizing oocytes by IVF, but the pregnancy rates are found to be significantly
lower than for ejaculated sperm (Hirsh et al., 1994).
The objectives of this study were: (1) to assess pronuclear formation of bovine
epididymal sperm-injected oocytes activated using two different protocols, (2) to assess
embryo development up to the blastocyst stage of epididymal sperm-injected oocytes
using two different chemical activation protocols and (3) to produce pregnancies from
the transfer of blastocysts derived from bovine epididymal sperm-injected oocytes.
143
Experimental Design
Experiment 7.1
This experiment was conducted to determine pronuclear formation in bovine
sperm-injected oocytes subjected to two different activation protocols using both
cryopreserved epididymal sperm and ejaculated sperm. Oocytes were obtained from a
commercial source and upon arrival oocytes exhibiting the presence of the first polar
body were selected and randomly allotted to one of six treatment groups. Treatments A
and B both consisted of the injection of a sperm head derived from cryopreserved
ejaculated sperm directly into the ooplasm, and served as controls for this study. In
Treatment A (n=4 replicates), sperm-injected oocytes (n=93) were chemically activated
in 7% ethanol in Tissue Culture Medium 199 (TCM) for 5 minutes. In Treatment B (n=3
replicates), sperm-injected oocytes (n=54) were chemically activated by exposure to 5
M ionomycin in CR1aa medium for 5 minutes followed by incubation in 10 g/ml
cycloheximide in CR1aa medium for 5 hours.
In Treatments C and D, a sperm head derived from cryopreserved epididymal
sperm was directly injected into the ooplasm of a matured bovine oocyte. The sperm-
injected oocytes (n=108) in Treatment C (n=4 replicates) were chemically activated 4
hours post-injection in 7% ethanol in TCM for 5 minutes. In Treatment D (n=3 replicates),
the sperm-injected oocytes (n=65) were chemically activated by exposure to 5 M
ionomycin in CR1aa medium for 5 minutes followed by incubation in 10 g/ml
Treatments E and F both consisted of sham-injected oocytes and served as
parthenogenetic controls for this study, where a similar volume of 10%
polyvinylpyrrolidone (PVP) in human tubal fluid medium was injected directly into the
ooplasm. Sham-injected oocytes (n=63) in Treatment E (n=4 replicates) were chemically
activated 4 hours post-injection in 7% ethanol in TCM for 5 minutes. In Treatment F (n=4
replicates), the sham-injected oocytes (n=59) were chemically activated by exposure to
5 M ionomycin in CR1aa medium for 5 minutes followed by incubation in 10 g/ml
The sperm-injected oocytes and sham-injected controls were then cultured in
CR1aa culture medium. Pronuclear formation was assessed 18 to 20 hours post-sperm
144
injection by fixing sperm-injected oocytes in acetic alcohol overnight. Fixed oocytes were
stained with 1% aceto-orcein, and the presence of male and female pronuclei were
examined with a bright field inverted microscope.
Experiment 7.2
This experiment was conducted to assess embryo development up to the
blastocyst stage of sperm-injected oocytes activated with two different activation
protocols using epididymal sperm. Oocytes were also obtained from a commercial
source and upon arrival oocytes exhibiting the presence of the first polar body were
selected and randomly alocatted to one of four treatment groups. Treatments A and B
consisted of injection of a single immobilized epididymal sperm into the ooplasm of a
matured oocyte. Treatments C and D (both controls) consisted of sham-injected oocytes,
where a similar volume of 10% polyvinylpyrrolidone (PVP) in human tubal fluid medium
was injected directly into the ooplasm.
Sperm-injected oocytes (n=118) in Treatment A (n=4 replicates) were chemically
activated 4 hours after sperm injection in 7% ethanol in TCM for 5 minutes. In Treatment
B (n=3 replicates), sperm-injected oocytes (n=75) were activated by exposure to 5 M
ionomycin in CR1aa medium for 5 minutes followed by 10 g/ml cycloheximide in CR1aa
medium for 5 hours.
Sham-injected oocytes (n=45) in Treatment C (n=4 replicates) were chemically
activated 4 hours post-injection in 7% ethanol in TCM for 5 minutes. In Treatment D (n=3
replicates), the sham-injected oocytes (n=35) were chemically activated by exposure to
5 M ionomycin in CR1aa medium for 5 minutes followed by incubation in 10 g/ml
Sperm-injected and sham-injected oocytes from all four treatment groups were
cultured in CR1aa medium from day 0 to day 3 post-injection and then in CR1aa
medium supplemented with 5% fetal bovine serum (FBS) from day 3 to day 8 of culture.
Cleavage and blastocyst formation rates were assessed on day 3 and day 8 of culture,
respectively.
Oocyte Preparation
Bovine oocytes were obtained from a commercial supplier (BoMed Inc., Madison,
WI and Concho Valley Genetics, San Angelo, TX) in groups of 50 and matured in a
145
A
Figure 7.1. Bovine cumulus oocyte complexes (COCs) received from a commercial
supplier at 21 hours of maturation. A. COC at 20X magnification. B. COCs at 10X
magnification.
146
portable incubator at 39C during overnight travel (Figure 7.1). Upon arrival to the
Embryo Biotechnology Laboratory, shipping vials were removed from the portable
incubator and stored in a 5% CO2 incubator in humidified air at 39C until use. After 21
hours of in vitro maturation, cumulus cells were removed by vortexing the cumulus
oocyte complexes for 4 minutes in Tyrodes lactate HEPES medium (TLH) (04-616F,
Cambrex Bio Science Walkersville Inc., Walkersville, MD) supplemented with 1 mg/ml of
hyaluronidase (H-3506, Sigma-Aldrich Inc., St. Louis, MO). After vortexing, oocytes were
washed through two Falcon 35 x 10 mm plastic petri dishes (353001, Becton Dickinson,
Franklin Lakes, NJ) of mTLH containing 10% (v/v) FBS (SH30070.02, Hyclone, Logan,
UT) and 50 g/ml of gentamicin (15750-060, Gibco, Grand Island, NY) to stop the
enzymatic action of the hyaluronidase.
Mature oocytes with an extruded first polar body were selected with a dissecting
microscope and placed in 500 l of mTLH in a 1.7 ml plastic micro-centrifuge tube
(20170-355, VWR International Inc., Sugar Land, TX). Oocytes were centrifuged at
6,000 x g for 5 minutes in a micro-centrifuge (AccuSpin, Fisher Scientific) to clear the
ooplasm to facilitate the microinjection procedure. Centrifuged oocytes were placed in
TCM 199 (11150-067, Gibco, Grand Island, NY) containing Earles salts, L-glutamine,
2,200 mg/l of sodium bicarbonate, 5% FBS and 50 g/ml of gentamicin until the time of
use.
Sperm Preparation
Both frozen-thawed ejaculated and epididymal bovine sperm were used for
Experiment 7.1. Ejaculated sperm were obtained from a Holstein bull (CSS 7H5188,
Genex Cooperative Inc., Shawano, WI) with proven in vitro fertilization ability.
Epididymal sperm were harvested from a abattoir-derived mature beef bull. No historical
information was available.
Straws (0.5 ml) of both ejaculated sperm and epididymal sperm were thawed in a
37C water bath for 40 seconds. Thawed sperm were carefully layered over 2 ml of both
lower and upper layer of ISolate sperm separation medium (99264, Irvine Scientific,
Santa Ana, CA) in a 15 ml plastic centrifuge tube (Corning, New York, NY). The tube
was then centrifuged at 300 x g for 20 minutes to obtain the motile fraction of sperm and
to remove egg yolk components to prevent sperm from sticking into the microinjection
pipette. The supernatant was discarded and the pellet and 500 l of the lower layer were
147
resuspended in 8 ml of mTLH medium supplemented with 3 mg/ml of bovine serum
albumin (Fraction V, A-7511, Sigma-Aldrich Inc., St. Louis, MO), 1 mg/ml of caffeine
sodium (C-4144, Sigma-Aldrich Inc., St. Louis, MO) and 50 ug/ml of gentamicin and
centrifuged twice at 350 x g for 5 minutes. After centrifugation, the supernatant was
removed and the remaining pellet was resuspended in 1 ml of mTLH and placed in a
water bath at 37C until time of use.
Sperm Head Preparation
For sperm head preparation, the 1 ml of sperm-TLH medium was placed on ice
and transported to Louisiana State University, Life Sciences Building for tail removal. A
500 l sample of the specimen was placed in a 1.7 ml micro-centrifuge tube and
sonicated at 20% power in a tissue sonicator for 1 second. The sample was kept on ice
to prevent the temperature from rising during sonication. With this procedure, 70 to 80%
of the sperm had their tails removed. Sperm heads were washed twice by centrifugation
at 350 x g for 5 minutes in mTLH and stored on ice until use.
Microinjection Apparatus
Holding pipettes were purchased from Cook (V-HPIP-1015, Cook, Australia).
They had a 1 mm taper with a 35 angle and a polished end with outer and inner
diameters of 120 m and 20 m, respectively. The holding pipette was connected to a
mineral oil-filled plastic tubing of a Narishige microinjector unit. The injection pipettes
were purchased from Humagen (PIEZO-8-25, Charlottesville, VA). They consisted of a 6
mm taper with a 25 angle and a blunt end with an inner diameter of 8 to 9 m. A small
volume of mercury (~1 l) was placed in the proximal end of the injection pipette. Then,
with an air-filled syringe the mercury was pushed forward around 0.5 cm to leave an air
column between the mercury and the proximal end of the pipette which was connected
to an oil-filled Eppendorf microinjector (Cell Tram, Eppendorf).
Sperm Injection Procedure
Sperm injection was performed in the lid of a Falcon 60 mm plastic petri dish
(353002, Becton Dickinson, Franklin Lakes, NJ) on a heated stage at 37C with 400X
magnification using an inverted Nikon microscope equipped with Hoffman optics and
Narishige micromanipulators. Each of four microdrops were aligned in the center of the
lid and covered with mineral oil (330779, Sigma-Aldrich Inc., St. Louis, MO). The first two
droplets (3 l) contained a 1:1 mixture of 10% (v/v) polyvinylpyrrolidone (PVP)
148
(reconstituted in isotonic HEPES buffered human tubal fluid medium) (99311, Irvine
Scientific, Santa Ana, CA) and mTLH to give a final PVP concentration of 5% (v/v).
A small volume of the sperm mixture was transferred into the two droplets by
using a pulled glass pipette. The third droplet was a 50 l oblong droplet of TLH
supplemented with 10% FBS for oocyte micromanipulation. The last droplet was 30 l in
volume and contained 10% (v/v) PVP for pipette washing after each injection procedure.
No more than 10 oocytes were placed in the TLH-FBS droplet at a time.
Immediately before sperm injection, a motile sperm was randomly selected from
one of the PVP-TLH droplets and immobilized by scoring its tail with the tip of the
injection pipette against the bottom of the dish (Figure 7.2). The sperm was aspirated
tail first into the injection pipette by applying gentle suction. When a sperm head was
used instead of whole sperm, 3 sperm heads were aspirated into the injection pipette at
the same time to decrease manipulation time of the oocytes.
In this study, ICSI was performed with a piezo unit (Piezo-Drill, Burleigh,
Germany) (Figure 7.3), as previously described (Kimura and Yanagimachi, 1995).
Briefly, a MII oocyte was picked up by the holding pipette by applying gentle suction and
the oocyte was rotated with the injection pipette until the polar body was located at either
the 6 oclock or the 12 oclock position. The injection pipette was then placed in close
contact with the oocyte at the 3 oclock position. The zona pellucida was drilled by
applying several piezo pulses while a light negative pressure was applied to it. When the
injection pipette had passed the zona pellucida, a cylindrical piece of the zona inside the
injection pipette was expelled into the medium outside the oocyte. After the injection
pipette was reinserted through the hole in the zona, the sperm or sperm head was
pushed slowly until it was positioned very close to the tip of the injection pipette before
penetrating the ooplasm. The pipette was advanced mechanically into the ooplasm until
it reached the center of the oocyte. A single piezo pulse was applied to break the
oolema. Then, the injection pipette was moved forward slightly and the sperm was
expelled slowly in the ooplasm with a minimum volume of medium. The pipette was
removed slowly while applying light negative pressure. The injection procedure lasted 3
to 5 minutes per oocyte. Sham injections were performed in the same manner, except
that no sperm was injected. After 8 to 10 oocytes were injected, they were washed twice
in mTCM and placed into their respective activation treatment groups.
149
Figure 7.2. Immobilization of a motile bovine epididymal sperm with the microinjection
pipette prior to sperm microinjection. Arrow for the tail of the sperm.
150
A B
C D
E F
Figure 7.3. The piezo ICSI procedure (40X magnification). A. An oocyte with the polar
body in the 12 oclock position held by the negative pressure of the holding pipette
before sperm injection. B. The zona pellucida drilled by applying piezo pulses. A small
cylinder of the zona pellucida can be observed outside the oocyte. The sperm is moved
to the tip of the pipette before penetrating the oolema. C. The pipette is mechanically
moved forward to stretch the oolema. A single piezo pulse is applied to break the
oolema. D. The sperm is pushed forward by applying positive pressure to the injector. E.
Excess medium is removed and the injection pipette is moved out from the oocyte. F. A
small amount of ooplasm at the place of injection is removed to allow sealing of the
oolema.
151
Oocyte Activation Treatments
Sperm-injected and sham-injected oocytes were subjected to two different
activation treatments. After injection procedure, oocytes in Treatments A, C and E were
cultured for 4 hours in TCM supplemented with 5% FBS and 50 g/ml of gentamicin.
Oocytes were then treated for 5 minutes in 7% (v/v) ethanol (060602, AAPER Alcohol
and Chemical, Shelbyville, KY) in TCM 199 containing 1 mg/ml PVP-40 (P-0930,
average molecular weight 40,000, Sigma-Aldrich Inc., St. Louis, MO). The remaining
sperm-injected oocytes in Treatments B, D and F were exposed to 5 M ionomycin (I-
9657, Sigma-Aldrich Inc., St. Louis, MO) in TCM 199 for 5 minutes followed by exposure
to 10 g/ml of cycloheximide (C-7698, Sigma-Aldrich Inc., St. Louis, MO) in CR1aa
medium supplemented with 3 mg/ml bovine serum albumin Fraction V (A-7511, Sigma-
Aldrich Inc., St. Louis, MO).
Embryo Culture
CR1aa medium was prepared as described previously (Rosenkrans and First,
1994). The CR1aa stock solution was prepared by adding 114.7 mM NaCl (S-5886,
Sigma-Aldrich Inc., St. Louis, MO), 1.6 mM KCl (P-5405, Sigma-Aldrich Inc., St. Louis,
MO), 0.39 mM pyruvic acid (P-4562, Sigma-Aldrich Inc., St. Louis, MO), 26.2 mM
NaHCO3 (S-8875, Sigma-Aldrich Inc., St. Louis, MO), 2.5 mM L (+) lactic acid (L-4388,
Sigma-Aldrich Inc., St. Louis, MO), 0.5195 mM glycine (G-8790, Sigma-Aldrich Inc., St.
Louis, MO), 0.5051 mM L-alanine (A-7469, Sigma-Aldrich Inc., St. Louis, MO) and 0.2 ml
of 0.5% phenol red per 100 ml of Milli-Q water. The stock solutions were kept unfiltered
at 4C for up to 2 months.
CR1aa culture medium for day 0 to day 3 of in vitro culture was prepared by
adding 1% (v/v) of Modified Eagle Medium (MEM) amino acid solution (10X, 11140050,
Gibco Laboratories, Grand Island, NY), 2% (v/v) of Basal Medium Eagle (BME) amino
acid solution (50X, B-6766, Sigma-Aldrich Inc., St. Louis, MO), 1.1 mM L-glutamine (G-
5763, Sigma-Aldrich Inc., St. Louis, MO), 3 mg/ml of BSA and 50 g/ml of gentamicin to
the CR1aa stock solution. CR1aa culture medium from day 3 to day 8 was the same as
from day 0 to day 3 of culture but with the addition of 5% FBS. The medium was
prepared the night before use and placed in a 5% CO2 incuabator at 39C to allow
proper temperature and CO2 equilibration.
152
Sperm-injected oocytes were cultured in groups of 10 in 50 l drops of CR1aa
medium at 39C in an atmosphere of 5% CO2, 7% O2 and 88% N2. Cleavage and
blastocyst rates were assessed on day 3 and day 8 of in vitro culture, respectively.
Assessment of Fertilization
Pronuclear formation was assessed 18 to 20 hours after ICSI. Oocytes were
mounted in groups of five between a precleaned Gold Seal microscope slide (3051,
Gold Seal Products, Portsmouth, NH) and a coverslip (size 18 x 18, 12-540A, Fisher
Scientific, Pittsburgh, PA) and fixed with methanol:acetic acid (3:1, v/v) for at least 24
hours. Fixed oocytes were stained with 1% aceto-orcein (O-7380, Sigma-Aldrich Inc., St.
Louis, MO) and unstained with acetic acid:glycerol:water (1:1:2 v/v) for easier evaluation
of the sperm. An oocyte with two polar bodies and two pronuclei were considered as
fertilized. In this study, an oocyte with two polar bodies, one pronucleus and a sperm
head were considered to be activated. Also, an oocyte displaying an enlarged sperm
head was considered as decondensing sperm head.
Blastocyst Staining
Day-8 blastocysts that were not transferred into recipient females were stained in
1 mg/ml of Hoechst 33342 (B-2261, Sigma-Aldrich Inc., St. Louis, MO) in TLH medium
for 15 minutes. After staining, blastocysts were washed twice in separate 75 l droplets
of TLH to remove excess stain. Embryos were placed between a slide and coverslip for
cell counts. Total number of cells was counted using an inverted microscope (Nikon
Diaphot, Tokyo, Japan) equipped with an ultraviolet light and a Hoechst filter.
Embryo developmental stages were analyzed across treatments by the Chi-
square test and Fishers Exact Probability test. Total cell counts for blastocysts are
expressed as mean valueSEM for each treatment group. Means were considered
significantly different at P<0.05 in this study. All data were analyzed using SigmaStat
Statistical Software Version 2.0.
RESULTS
Experiment 7.1
Pronuclei formation of sperm-injected oocytes was evaluated following aceto-
orcein staining. At 18 to 20 hours post-sperm injection, oocytes that had degenerated
153
A
Figure 7.4. Cytolyzed bovine oocytes degenerated 4 to 5 hours after sperm injection. A.
Arrows indicates cytolyzed oocytes (20X magnification). B. Cytolyzed oocyte (40X
magnification).
154
Figure 7.5. Oocyte stained with aceto-orcein 18 hours after the ICSI procedure. Male
and female pronuclei are indicated by arrows.
155
Table 7.1. Effect of activation treatments on pronuclear formation of epididymal sperm-
injected bovine oocytes
Treatment Sperm No. No. Total

groups source injected survived DSH 1 PN 2 PN activated
Ethanol Epi 108 97(90)a 8(8) 40(41)a 30(31)a 70(72)a

Ejac 93 86(93)a 5(6) 24(28)b 37(43)b 61(71)a
Sham 63 60(95)a - 27(45)a 1(2)c 28(47)b
Iono+Cyclo Epi 65 46(71)b 3(6) 15(33)b 14(30)a 29(63)a
Ejac 54 35(65)b 2(6) 11(31)b 13(37)a,b 24(69)a
Sham 59 43(73)b - 21(48)b 2(4)c 23(53)b
Iono+Cyclo = ionomycin treatment followed by cycloheximide, Epi = epididymal sperm,

Ejac = ejaculated sperm, DSH = decondensed sperm head, 1 PN = one pronucleus, 2
PN = two pronuclei.
Three to four replicates per treatment group. a,b,cValues with different superscripts within
columns are significantly different (P<0.05).
156
(Figure 7.4) were removed from the study. Only morphologically normal oocytes were
fixed for cytological assessments. The proportion of oocytes exhibiting male and female
pronuclei and two polar bodies (successfully fertilized) (Figure 7.5) was significantly
higher (P<0.05) when ejaculated sperm were used instead of epididymal sperm (43%
and 31%, respectively) (Table 7.1). However, significant difference was only found when
ethanol was used to exogenously activate the oocytes in place of ionomycin followed by
cycloheximide. Furthermore, no statistical difference was found in the percentage of
successfully fertilized oocytes when ionomycin followed by cycloheximide was used as
the activation protocol when both ejaculated and epididymal sperm were used.
The proportion of oocytes with one pronucleus was significantly higher (P<0.05)
when ethanol was used as activation protocol in the epididymal sperm-injected treatment
group compared with ionomycin followed by cycloheximide treatment. However, no
significant difference in the proportion of oocytes exhibiting one pronucleus was detected
when ejaculated sperm or sham injection were used. In more than 90% of sperm-
injected oocytes exhibiting one pronucleus a condensed sperm head was observed. In
sham-injected oocytes only 48% and 54% of oocytes formed a single pronucleus,
however, 2% and 5% of the oocytes formed two pronuclei when activated with ethanol
and ionomycin followed by cycloheximide, respectively. A significant difference (P<0.05)
was noted between the percentage of oocytes forming two pronuclei between the sham-
injected groups (Treatments E and F) and sperm-injected groups (Treatments A, B, C
and D) regardless of activation protocol used.
The proportion of cytolyzed oocytes was significantly greater (P<0.05) when
ionomycin followed by cycloheximide (Treatments B, D and F) was used compared with
ethanol activation (Treatments A, C and E). The percentage of cytolyzed oocytes was
not found to increase due to the source of sperm.
Experiment 7.2
Results of embryonic development from epididymal sperm-injected oocytes are
shown in Table 7.2. The percentage of sperm-injected oocytes reaching the blastocyst
stage (Figure 7.7) was significantly greater (P<0.05) when ethanol (14%) was used
compared with ionomycin followed by cycloheximide (4%) or the sham-injected control
(0%) groups. No difference in cleavage rates were found between the two different
157
Table 7.2. Effect of activation treatments on the embryonic development of epididymal sperm-injected bovine oocytes
Treatment Sperm No. No. Total Total

group injected injected survived 2-4 cell 4-8 cell 8-16 cell cleaved BLST
Ethanol + 118 108(92)a 19(18)a 33(31) 26(24)a 78(72)a 15(14)a
- 45 40(89)a 13(33)b 9(23) 0 22(55)b 0
Iono + Cyclo + 75 55(73)b 26(47)b 11(20) 4(7)b 41(74)a 2(4)b
- 35 26(74)b 6(38)b 3(19) 0 9(56)b 0
Iono + Cyclo = ionomycin followed by cycloheximide; BLST = blastocysts.

Three to four replicates per treatment group. a,b,cValues with different superscripts within columns are significantly different (P<0.05).
158
Figure 7.6. An example of a day-3 bovine embryo (8- to 16-cells) derived from
epididymal sperm-injected oocytes.
159
Figure 7.7. Examples of day-8 blastocysts derived from epididymal sperm-injected
oocytes. Blastocysts are hatching through the opening made by the microinjection
pipette.
160
activation treatment groups. However, a significantly higher percentage of oocytes
cleaved in the sperm-injected oocytes when compared with the sham-injected control,
regardless of activation treatment. A significantly higher percentage of epididymal
sperm-injected oocytes reached the 8- to 16-cell stages (Figure 7.6) when activated with
ethanol (28%) compared with ionomycin followed by cycloheximide (7%) at 72 hours of
embryo culture. No oocyte in the sham-injected control group developed further than the
4- to 8-cell stages in culture.
Furthermore, a significantly higher (P<0.05) proportion of ICSI oocytes were
observed at the 2- to 4-cell stages at 72 hours of culture when ionomycin followed by
cycloheximide treatment was used compared with ethanol treatment.
The mean total cell numbers of the stained blastocysts (Figure 7.8) that were not
transferred into recipient females are shown in Table 7.3. No difference was found
between treatments likely due to the low number of blastocyst in each group.
Experiment 7.3
Nine day-8 blastocysts were nonsurgically transferred into recipient beef females
on day 7 of the estrous cycle. Of the 9 blastocysts, 2 were excellent Grade 1 embryos
transferred to 2 recipients and 7 Grade 1 to Grade 2 embryos were transferred to 3
recipients. One ongoing 50-day pregnancy (with a heart beat) resulted from the transfer
of one of the 2 Grade 1 blastocysts transferred (Figure 7.9).
DISCUSSION
We have demonstrated that normal fertilization and blastocyst development
could be achieved with cryopreserved bovine epididymal sperm by piezo ICSI. Although
this is not the first report of ICSI in bovine using epididymal sperm, the blastocyst
success rate in this study is considerably higher at 14% than that reported previously by
Goto et al. (1990) at 1.8%. It should be noted that Goto et al. (1990) used epididymal
sperm that had been killed by repeated freeze-thawing without cryoprotectants.
Recently, Horiuchi et al. (2002) reported that embryonic development was drastically
reduced when killed sperm by multiple freeze-thawing was used compared with live
sperm that had been immobilized prior to the microinjection procedure. Also, our higher
success could be, in part, for the use of a piezo drill to penetrate the oolema to assure
the injection of sperm into the ooplasm.
161
Table 7.3. Total cell number (meanSEM) of day-8 bovine blastocysts derived from ICSI
using different activation treatments
Treatment No. of
group embryos Total no. of cells/embryo
Ethanol 5 964.2
Ionomycin + cyclohexamide 2 804.5
162
A
Figure 7.8. An example of a day-8 blastocyst stained with Hoechst 33342. A. Day-8
blastocyst at 20X magnification. B. An overlay of the stained cells of the same day-8
blastocyst in panel A.
163
Figure 7.9. Ultrasound images of a 50-day frozen-thawed epididymal sperm ICSI-derived
fetus.
164
Reports to date indicate that pronuclear formation and blastocyst rates following
ICSI with bovine ejaculated sperm range from 20 to 60% and 7 to 20%, respectively
(Keefer et al., 1990; Wei and Fukui, 1999; Hamano et al., 1999; Keskintepe et al., 2002;
Horiuchi et al., 2002; Galli et al., 2003; Ock et al., 2003; Rho et al., 2004; Li et al., 2004).
The marked difference in pronuclear formation and blastocyst rates between laboratories
may be because of different protocols used for pretreating sperm prior to microinjection,
including if whole sperm or sperm heads and if dead or live sperm are used, scoring or
cutting the tail before sperm injection and treating sperm with different chemicals to
accomplish disruption of plasma membrane and/or reduction of disulfide bonds. Also,
embryo manipulation, the use of conventional or piezo ICSI, expertise of the technician
and embryo culture play an important role in the success of the oocyte microinjection
procedure.
This is the first report that evaluated pronuclear formation of bovine ICSI oocytes
using epididymal sperm. A significantly higher fertilization rate was achieved, however,
when ejaculated rather than epididymal sperm was used in our procedure. This
difference in fertilization rates between ejaculated and epididymal sperm was only noted
depending on the activation protocol employed. No difference in fertilization rates was
found between activation treatments when the same sperm source (either ejaculated or
epididymal) was used. Furthermore, activation with ethanol produced higher fertilization
rates for ejaculated sperm than for epididymal sperm compared with ionomycin followed
by cycloheximide.
The lower pronuclear formation for epididymal sperm in this study may rely on
the difference in maturation status between ejaculated sperm and epididymal sperm.
Disruption of the plasma membrane to achieve nuclear decondensation and the release
of the sperm factor may be somehow compromised for epididymal sperm. The plasma
membrane is modified and prepared for fertilization by biochemical components in
seminal plasma at the time of ejaculation in the bull. Seminal plasma has been shown to
cause cholesterol efflux from the plasma membrane to prepare sperm for capacitation,
acrosome reaction and fertilization (Osheroff et al., 1999; Iborra et al., 2000).
Furthermore, it can not be ruled out that unknown factors in seminal plasma play an
important role in modifying sperm for the fertilization process.
165
Heneault et al. (1995) demonstrated that incubation of sperm from a low fertility
bull with seminal plasma from a highly fertile bull increased the oocyte fertilization rates
in vitro. Two proteins in seminal plasma of 26 and 55 kDa each have been shown to
predominate in higher fertility bulls (Killian et al., 1993). Thus, the role of seminal plasma
in preparing sperm for fertilization at the time of ejaculation can not be overlooked.
In addition, the differences in fertilization rates for IVF and ICSI due to a bull
effect using ejaculated sperm have to be considered. In a study using different bulls for
conventional IVF, fertilization rates have been reported to vary from 35 to 96% (Niwa
and Ohgoda, 1988). Recently, Wei and Fukui (1999) indicated that the ability of
microinjected sperm to decondense and form male pronuclei varies greatly between
bulls. In our study, epididymal sperm were harvested from testes obtained from abattoir
derived bulls with no previous reproductive history. This effect has to be considered
when comparing and analyzing the results reported herein.
Sperm pretreatment might help enhance sperm head decondensation and
pronuclear development of epididymal sperm-injected oocytes. The use of chemicals,
such as calcium ionophore, Triton-X 100 and dithiothreitol, can be used to increase the
rate of acrosome reaction, plasma membrane permeabilisation and sperm head
decondensation (Keefer et al., 1990; Malcuit et al., 2006; Wei and Fukui, 1999).
We found a significantly higher number of oocytes to degenerate 4 to 5 hours
after ICSI when they were activated with ionomycin followed by cycloheximide than with
the ethanol treatment. One difference between the two activation protocols was that the
oocytes in the ethanol group were kept for 4 hours in a medium containing 5% fetal
bovine serum before activation. Proteins, growth factors and unknown biochemical
components in serum might help with the wound healing of the oolema post-ICSI
(Suzuki et al., 1997). Suzuki et al. (1997) reported that a medium with high concentration
of serum was beneficial for the sealing process of the oolema after sperm injection in the
mouse. We are in agreement that serum-rich medium likely enhances the survival of the
oocyte after the injection procedure. This, in part, further demonstrates that the bovine
oocyte is very sensitive to the piercing of the microinjection pipette. We can not rule out
that the effect of the ICSI procedure is one of the reasons for the less than optimal
success in fertilization and embryo development rates of this technique in cattle.
166
The present study indicates that activation of bovine oocytes following ICSI by
ethanol was better in terms of blastocyst production than activation by ionomycin
followed by cycloheximide. The blastocyst rate of 14% obtained in our study using
epididymal sperm was in the range of 7 to 20% blastocysts reported over the years for
ejaculated sperm by other investigators (Hamano et al., 1999; Horiuchi et al., 2002; Galli
et al., 2003; Rho et al., 2004). We agree with previous investigations where ethanol was
reported to be the best activation treatment for bovine ICSI with ejaculated sperm when
applied 4 hours after sperm injection (Horiuchi et al., 2002; Fujinami et al., 2004; Oikawa
et al., 2005).
Recently, Horiuchi (2006) reported that more than 24 calves have been produced
by ethanol-activated ICSI oocytes using ejaculated sperm. In contrast, no calves have
been reported to date in ionomycin followed by cycloheximide-activated ICSI oocytes. It
has been reported that ~60% of the oocytes post-ICSI were activated by the sperm
when assessed by the extrusion of the second polar body 4 hours after sperm injection
(Horiuchi et al., 2002; Galli et al., 2003). This shows, in part, that a protein synthesis
inhibitor, such as cycloheximide, is not required during the first 4 to 5 hours after ICSI for
proper oocyte activation. Cycloheximide might rather affect embryo development or
enhance parthenogenesis of the oocytes not activated by the initial signal of the sperm.
The level of maturation promoting factor (MPF) activity was reported to decrease
upon sperm injection and temporarily increase after 4 to 6 hours post-ICSI (Fujinami et
al., 2004). Activation of bovine oocytes with ethanol 4 hours after sperm injection has
been shown to maintain the low level of MPF activity until 20 hours for the next cell
cycle, whereas, this elevation in MPF was not found in bovine oocytes without activation
(Fujinami et al., 2004). This rise in MPF may promote cleavage and subsequent embryo
development (Horiuchi, 2006). In addition, microtubules around the male and female
pronuclei were reported to be more defined and better developed when ethanol was
used to activate bovine oocytes 4 hours after ICSI compared with that of no activation
(43% vs. 73%) (Horiuchi, 2006). However, the exact mechanism by which ethanol
provides better subsequent embryo development remains largely unknown.
We can conclude that postmortem epididymal sperm can be collected from
genetically valuable males and used for the production of blastocysts using ICSI. At the
time of this writing 9 ICSI-derived bovine blastocysts were nonsurgically transferred into
167
5 recipient females resulting in a 50 day ongoing pregnancy. Further investigations
should evaluate the fertilizing ability of epididymal sperm harvested from testes that have
been stored for different time periods after death.
168
CHAPTER VIII
SUMMARY AND CONCLUSIONS

The primary objectives of these series of experiments were to develop a
successful protocol for cryopreservation and intracytoplasmic sperm injection (ICSI)
using bovine caudal epididymal sperm. Information on the cryopreservation and use for
ICSI of epididymal sperm is limited. The development of a protocol to recover and
cryopreserve sperm from postmortem animals will allow the propagation of valuable
gametes for future generations increasing the life span of an individual male.
Developing a successful ICSI protocol will allow the use of these rare and valuable
cryopreserved sperm samples for the production of future offspring.
In the first experiment, the effect of a one step addition and/or removal of
different concentrations of permeable cryoprotectants (CPAs) and their toxic effects
during cool storage were assessed. Results showed that ethylene glycol caused less
osmotic damage to sperm during addition and/or removal at 4C when compared with
glycerol in all concentrations evaluated. Maximum survival was achieved with sperm
exposed to 7% ethylene glycol. Furthermore, extended exposure (5 days at 4C) of
ethylene glycol was found to be less toxic than glycerol to epididymal sperm. We
conclude that ethylene glycol, used as a cryoprotectant, may help minimize toxicity and
osmotic damage to bovine epididymal sperm.
In the second experiment, the cryoprotective effect of glycerol and ethylene
glycol was evaluated. Results indicated that glycerol was far more effective in providing
protection against freezing injury during the cryopreservation process than ethylene
glycol.
In the third experiment, the effect of exposing epididymal sperm to seminal
plasma prior to cryopreservation was evaluated. It was demonstrated that epididymal
sperm retrieval using seminal plasma is clearly beneficial to enhance sperm overall and
progressive motility characteristics and to protect it from morphological abnormalities
derived from the freeze-thaw process.
In the fourth experiment, the effects of a one step removal of glycerol from
cryopreserved bovine epididymal sperm previously exposed to seminal plasma and its
longevity during a 4-hour incubation period at 37C were assessed. The results indicated
that a one step dilution process for removal of glycerol from cryopreserved epididymal
169
sperm affected the integrity of the sperm plasma membrane and the function of the
mitochondria of sperm previously exposed to seminal plasma. However, dilution did not
have any significant detrimental effect on the integrity of the acrosome. Furthermore, we
demonstrated that the longevity in vitro of post-thaw epididymal sperm exposed to
seminal plasma prior to cryopreservation showed no evidence of being compromised
when compared with epididymal sperm that was not exposed to seminal plasma.
In the final series of trials, an effort was made to evaluate fertilization and
blastocyst development rates from bovine epididymal sperm-injected oocytes. The
results demonstrated that acceptable fertilization and blastocyst development rates
could be achieved with cryopreserved bovine epididymal sperm by intracytoplasmic
sperm injection. Secondly, good quality ICSI-derived blastocysts were nonsurgically
transferred to evaluate if the protocols developed in our study would result in confirmed
pregnancies. One ongoing pregnancy at 50 days resulted from a day-8 Grade 1
blastocyst.
We can conclude that postmortem epididymal sperm can be collected from
genetically valuable males and used for the production of blastocysts using ICSI. This is
the first report to evaluate epididymal sperm quality using multicolor flow cytometry. In
addition, we have developed a successful protocol to cryopreserve bovine caudal
epididymal sperm. Furthermore, we have developed a successful ICSI protocol and
technique for generating embryos from sperm harvested from postmortem animals. The
protocols developed and designed herein can be adapted to best fit other species of
interests, including humans and endangered species.
170
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VITA
Carlos Andres Guerrero was born on June 23, 1980, in Cali, Valle, Colombia, to
Maria Islena Espinosa and Carlos Leonardo Guerrero. Carlos has an older brother,
Jorge Enrique Guerrero, who resides in Boston, Massachusetts, and works as a
physician. Carlos grew up in Cali, Colombia, were he attended the British school,
Colegio Colombo Britanico. Following graduation in 1998, Carlos came to the United
States to pursue a career in the area of animal science. Carlos enrolled at Louisiana
State University, in Baton Rouge, Louisiana, in the fall of 1998. During his junior year,
Carlos met Dr. Robert A. Godke, Boyd Professor of Reproductive Physiology, who
involved him in various reproductive physiology projects at the Embryo Biotechnology
Laboratory, St. Gabriel, Louisiana. After receiving his bachelor degree in animal science
in May 2002, Carlos started to pursue his doctoral degree under the guidance of Dr.
Robert A. Godke. He is a candidate for the Doctor of Philosophy degree in reproductive
physiology in the Department of Animal Sciences at Louisiana State University, Baton
Rouge, Louisiana.
202

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The Interdepartmental Program of

LIST OF FIGURES..................................................................................................... vii

ABBREVIATIONS USED IN THE DISSERTATION........... xii

CHAPTER II LITERATURE REVIEW......................................................................... 3

CHAPTER III EFFECT OF STORAGE AND CRYOPROTECTANT ADDITION AND

CHAPTER V THE EFFECT OF ADDITION OF BOVINE SEMINAL PLASMA TO

CHAPTER VI EFFECT OF DILUTION AND INCUBATION OF POST-THAW

CHAPTER VII THE USE OF CRYOPRESERVED BOVINE CAUDAL EPIDIDYMAL

CHAPTER VIII SUMMARY AND CONCLUSIONS... 169

LITERATURE CITED..... 171

5.1 Testicular and epididymal measurements from bovine testes collected

5.2 Morphology values (mean%SEM) of bovine epididymal sperm prior to

6.1 Percent plasma membrane integrity (meanSEM) of post-thaw bovine

6.2 Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

6.3 Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

6.4 Percent plasma membrane integrity (meanSEM) of post-thaw bovine

6.5 Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

6.6 Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

6.7 Percent progressive motility (meanSEM) of post-thaw bovine epididymal

7.1 Effect of activation treatments on pronuclear formation of epididymal sperm-

7.2 Effect of activation treatments on the embryonic development of epididymal

3.2 The process of cauda epididymides dissection. A. Pair of bovine testes

3.3 The process of epididymal sperm retrieval from a dissected

3.4 Effect of cryoprotectant toxicity during 5 days of storage at 4C on overall

3.5 Effect of cryoprotectant toxicity during 5 days of storage at 4C on progressive

3.6 Effect of cryoprotectant toxicity during 5 days of storage at 4C on plasma

4.6 Examples of two pairs of bull testes with abnormal morphology..........................63

4.7 Percent progressive motility (meanSEM) of prefreeze and post-thaw bovine

4.8 Prefreeze and post-thaw percent progressive motility values (meanSEM) of

4.9 Percent membrane integrity (meanSEM) of prefreeze and post-thaw bovine

4.10 Prefreeze and post-thaw percent membrane integrity (meanSEM) of bovine

4.12 Percent acrosome integrity values (meanSEM) of prefreeze and post-thaw

5.7 Post-thaw percent overall motility (meanSEM) of bovine epididymal

5.8 Post-thaw percent progressive motility (meanSEM) of bovine epididymal

5.9 Post-thaw percent membrane integrity (meanSEM) of bovine epididymal

5.11 Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

5.12 Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

5.13 Percent DNA damage (meanSEM) of post-thaw bovine epididymal

6.1 Membrane integrity values (meanSEM) of post-thaw bovine epididymal

6.2 Membrane integrity values (meanSEM) of post-thaw bovine epididymal

6.3 Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

6.4 Percent acrosome integrity (meanSEM) of post-thaw bovine epididymal

6.5 Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

6.6 Percent mitochondrial activity (meanSEM) of post-thaw bovine epididymal

6.7 Membrane integrity values (meanSEM) of post-thaw bovine epididymal

6.8 Membrane integrity values (meanSEM) of post-thaw bovine epididymal

6.9 Acrosome integrity values (meanSEM) of post-thaw bovine epididymal sperm

6.10 Acrosome integrity values (meanSEM) of post-thaw bovine epididymal sperm

6.11 Mitochondrial activity values (meanSEM) of post-thaw bovine epididymal

6.12 Mitochondrial activity values (meanSEM) of post-thaw bovine epididymal

6.13 Progressive motility values (meanSEM) of post-thaw bovine epididymal

6.14 Progressive motility values (meanSEM) of post-thaw bovine epididymal

7.2 Immobilization of a motile bovine epididymal sperm with the microinjection

7.4 Cytolyzed bovine oocytes degenerated 4 to 5 hours after sperm injection. A.

7.7 Examples of a day-8 blastocysts derived from epididymal sperm-injected

7.8 An example of a day-8 blastocyst stained with Hoechst 33342. A. Day-8

7.9 Ultrasound images of a 50-day frozen-thawed epididymal sperm

Recently, interest in the preservation of epididymal sperm as a potential source of

FUNCTIONS OF THE EPIDIDYMIS

EFFECT OF STORAGE AND CRYOPROTECTANT ADDITION AND REMOVAL AT 4C

Figure 3.4. Effect of cryoprotectant toxicity during 5 days of storage at 4C on overall