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CANINE SPERMATOZOA: PURIFICATION, ASSESSMENT, AND PRESERVATION

By

TAMEKA CERISE PHILLIPS

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010

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© 2010 Tameka C. Phillips

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To my Lord and Savior Jesus Christ, because all that I am is because of you

Faith without works is dead. (James 2:20)

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ACKNOWLEDGMENTS

First and foremost, all praises goes to God who is the head of my life, for without

him I am nothing. I would like to express my appreciation to those who contributed in

any fashion for the completion of this dissertation. I am equally grateful for my Ph.D.

funding provided by the Florida Education Fund McKnight Doctoral Fellowship and the

College of Veterinary Medicine Office of Research and Graduate Studies.

To my PhD advisor, Dr. John Verstegen, thank for the decision to stay at the UF

as my advisor. To my committee, thank you for the support, guidance, edited materials,

and the ability to spend time in your labs and clinics. To Dr. Joseph DiPietro and Dr.

Louis Archbald, thank you for guiding me back to Florida to pursue my Ph.D. To my

wonderful family and friends, there is not enough space to speak the volumes of

acknowledgements that I have for you. So, I say thank you for all the food, laughter,

love, prayers, support, and correction. Finally, to my research dogs: Bashful, Borat,

Deogee, Freckles, and Jimbo this would not have been possible without each of you.

Thank you all. Kimmie we made it!

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...................................................................................................... 4

LIST OF TABLES ................................................................................................................ 7

LIST OF FIGURES .............................................................................................................. 8

LIST OF ABBREVIATIONS ................................................................................................ 9

ABSTRACT........................................................................................................................ 10

CHAPTER

1 INTRODUCTION ........................................................................................................ 12

Dissertation Objectives ............................................................................................... 13

2 LITERATURE REVIEW .............................................................................................. 16

Purification .................................................................................................................. 16 Assessment ................................................................................................................ 19

Microscopy ........................................................................................................... 19 Evaluation of Sperm Motility ................................................................................ 20 Evaluation of Sperm Morphology ........................................................................ 23 Evaluation of Sperm Functional Test and Membrane Integrity .......................... 25 Evaluation of Sperm at Molecular Level.............................................................. 28 Evaluation of Sperm Membrane Proteins (Antigens or Receptors) ................... 30

Preservation ................................................................................................................ 32

3 EFFICACY OF FOUR DIFFERENT DENSITY GRADIENT SEPARATION MEDIA TO REMOVE RED BLOOD CELLS AND NON VIABLE SPERMATOZOA FROM CANINE SEMEN ............................................................... 38

Introduction ................................................................................................................. 38 Materials and Methods ............................................................................................... 39

Materials ............................................................................................................... 39 Animals ................................................................................................................. 40 Experiments ......................................................................................................... 40 Statistical Analysis ............................................................................................... 42

Results ........................................................................................................................ 42 Discussion ................................................................................................................... 43

4 IDENTIFICATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN THE CANINE ..................................................................................... 50

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Introduction ................................................................................................................. 50 Materials and Methods ............................................................................................... 54

Materials ............................................................................................................... 54 Animals and Sperm Preparation.......................................................................... 55 Experiments ......................................................................................................... 56 Statistical Analysis ............................................................................................... 57

Results ........................................................................................................................ 57 Discussion ................................................................................................................... 60

5 CHARACTERIZATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN CANINE BY WESTERN BLOT ANALYSIS ...................................... 70

Introduction ................................................................................................................. 70 Materials and Methods ............................................................................................... 71

Materials ............................................................................................................... 71 Animals and Sperm Preparation.......................................................................... 72 Experiments ......................................................................................................... 73

Results ........................................................................................................................ 75 Discussion ................................................................................................................... 76

6 EFFECTS OF CANINE SEMEN PRESERVATION ON SPERM PROTEIN EXPRESSION............................................................................................................. 84

Introduction ................................................................................................................. 84 Materials and Methods ............................................................................................... 87

Materials ............................................................................................................... 87 Animals ................................................................................................................. 88 Sperm Preparation and Preservation .................................................................. 89 Experiments ......................................................................................................... 90

Effect of chilled extenders on motility and acrosomal status ....................... 90 Effect of chilling and different extenders on sperm protein expression ....... 90 Effect of acrosome reaction induction on sperm protein expression ........... 91

Statistical Analysis ............................................................................................... 92 Results ........................................................................................................................ 92

Effect of chilled extenders on motility and acrosomal status .............................. 93 Effect of chilling and different extenders on sperm protein expression ............. 94 Effect of acrosome reaction induction on sperm protein expression ................. 94

Discussion ................................................................................................................... 95

7 GENERAL DISCUSSION ......................................................................................... 107

LIST OF REFERENCES ................................................................................................. 123

BIOGRAPHICAL SKETCH.............................................................................................. 139

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LIST OF TABLES

Table page 5-1 Molecular weight standard densitogram data. ...................................................... 81

5-2 SPAG6 at 1:200 dilution densitogram data. .......................................................... 82

5-3 SP17 at 1:100 dilution densitogram data. ............................................................. 82

5-4 CATSPER2 at 1:2 dilution densitogram data. ....................................................... 83

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LIST OF FIGURES

Figure page 3-1 DGC media separation of sperm and RBCs with a 45% and 90% Percoll

gradient.. ................................................................................................................. 47

3-2 Sperm separation media parameters for the collected fractions. ......................... 48

3-3 Characteristics of fractions (%) with optimal recovery for the different DGC: ...... 49

4-1 Diagram of a sperm cell. ........................................................................................ 65

4-1 SPAG6 immunocytochemistry. .............................................................................. 66

4-2 SPAG6 confocal immunofluorescence. ................................................................. 67

4-3 Canine SP17 with fresh negative and positive controls.. ...................................... 68

4-4 SP17 localization on fresh canine spermatozoa.. ................................................. 68

4-5 SP56 localization on fresh canine spermatozoa. .................................................. 69

4-6 CATSPER2 localization on canine spermatozoa. ................................................. 69

5-1 Canine sperm protein western blot. ....................................................................... 81

5-2 Molecular weight standard densitogram................................................................ 81

5-3 SPAG6 at 1:200 dilution densitogram. .................................................................. 82

5-5 CATSPER2 at 1:2 dilution densitogram. ............................................................... 83

6-1 Overall motility (%) of sperm preserved at 4°C overtime in different chilled semen extenders. ................................................................................................. 101

6-2 Mean (%) of acrosome-intact sperm overtime at 4°C in different chilled semen extenders. ................................................................................................. 102

6-3 SPAG6 chilled spermatozoa Day 3 of preservation at 4°C. ............................... 103

6-4 SP17 chilled spermatozoa Day 10 and Day 20 of preservation at 4°C.............. 104

6-5 Mean (%) of acrosome-intact sperm before and after acrosome reaction induction. .............................................................................................................. 105

6-6 Effect of acrosome reaction induction on sperm proteins................................... 106

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LIST OF ABBREVIATIONS

BSA Bovine Serum Albumin

CASA Computer Assisted Sperm Analysis

CATSPER2 Cation Channel, Sperm Associated 2

DGC Density Gradient Centrifugation

ICC Immunocytochemistry

IVF In vitro Fertilization

PBS Phosphate Buffer Saline

RBCs Red Blood Cells

SP17 Sperm Protein 17

SP56 Sperm Fertilization Protein 56

SPAG6 Sperm- Associated Antigen 6

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment

of theRequirements for the Degree of Doctor of Philosophy

CANINE SPEMATOZOA: PURIFICATION, ASSESSMENT, AND PRESERVATION

By

Tameka Cerise Phillips

May 2010

Chair: John Verstegen Major: Veterinary Medical Sciences

A series of experiments were conducted to determine optimum methods to purify,

assess canine spermatozoa, and detect the presence of sperm-associated proteins and

the effect of preservation on these proteins.

Density gradient centrifugation media (DGC) were compared (Isolate, Percoll,

PureCeption, and PureSperm) for separation of viable, motile sperm from nonmotile or

dead sperm and red blood cells (RBCs). DGC media were effective tools for separation

of RBCs from motile sperm. PureCeption more efficiently allowed for purification of

motile sperm than Isolate and Percoll (P<0.01) in the optimal fraction.

Using immunocytochemistry, fertility associated sperm proteins: SPAG6, SP17,

SP56, and CATSPER2, were identified. SPAG6 (61.55 kDa) was strongly localized at

the acrosomal region with none to weak localization at the flagellum. SP17 (15.45,

28.75, and 53.75 kDa) was strongly localized at the acrosome and principal piece with

weak to moderate expression at the midpiece. SP56 was moderately localized at the

acrosome with moderate to strong localization at the flagellum, and was not confirmed

using western blot. CATSPER2 (48-49 and 63 kDa) was weakly to moderately localized

at acrosome with moderate to strong localization at flagellum.

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Chilled semen preservation (4ºC) using semen extenders [Camelot, Synbiotics,

CaniPRO Chill 5 and Chill 10] had decreased overall motility (%) overtime with a

difference between the extenders when motility was assessed. No significant

differences overtime of the acrosome reaction, with an average of 40% of sperm being

acrosome reacted by day 20. Chilled sperm protein expression, no labeling was present

by day 1 for CATSPER2, weak or no labeling from day 3 for SPAG6, but SP17

remained labeled up to day 20. When acrosome reaction was artificially induced with

SP-TALP, acrosome-intact sperm decreased ~40% when compared to controls. SPAG6

and CATSPER2 had post acrosomal region staining with minimal staining of the

flagellum. SP17 had no labeling present.

In conclusion, PureCeption more efficiency separated motile sperm from

contaminants than Isolate, Percoll or PureSperm. The differential expressions of the

sperm proteins in fresh, chilled preserved or acrosome-induced sperm suggest a

possible role in canine spermatozoal function and their variable expressions may

correlate with different functional status of the spermatozoa.

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CHAPTER 1 INTRODUCTION

An owner comes into the clinic with a seven year old male dog. The owner

complains that recently bitches have not had litters after mating with this stud dog and

the owner suspects that the male dog is infertile. A breeding soundness evaluation

(Johnston et al. 2001) which includes a complete physical examination, semen

collection and evaluation (sperm concentration, progressive motility, morphology, color,

and volume) determines that the male dog suffers from asthenozoospermia, reduced

sperm motility without any other major signs. The presence of blood is also observed in

the ejaculate.

The owner is informed that the cause of the reduction in sperm motility is

unknown, as no evidence of disease or infection was noticed. The blood that was

observed in the ejaculate was linked to a little non significant trauma of the penis. The

owner is told to rest the dog and follow up with rechecks at intervals which take into

account the length of the canine spermatogenesis cycle that is over 2 months (Foote et

al. 1972).

As it has been ruled out that the different bitches mated to the stud dog were not

responsible for the observed infertility, the male factor for infertility needed to be

determined. There are many possible reasons why a male dog is suddenly termed

‘infertile’. Incomplete ejaculates, obstruction of the male reproductive tract, hormonal

abnormities, environmental toxins, trauma, or prostatic disease can be some of the

reasons reported(Harrop 1966; Larsen 1977). Also, as dogs get older there is an

association between decreased semen production and quality and the development of

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prostatic disease that has been shown to lead to infertility (England and Allen 1992;

Rijsselaere et al. 2004). A proper semen evaluation is thus needed.

However, it is generally admitted that the classical laboratory and clinical

assessment tools (Graham 2001; Henkel and Schill 2003) currently available can only

guess the potential fertility value of a semen sample; and that infertility can still be

observed in otherwise apparently normal samples. For these reasons, new and more

detailed approaches are required. A combination of fertility tests including classical

evaluation, semen purification, sperm protein assessment, effects of semen

preservation, in vitro and/or in vivo fertilization, and functional tests of acrosome

integrity is needed in order to gain a fuller understanding of the fertilizing ability of a

semen sample.

Dissertation Objectives

Currently, there is no single in vitro method providing an accurate fertility

prognosis. This is why for many years numerous studies have been conducted to find

fertility tests trustworthy enough to determine the fertilization ability of sperm (Mahi and

Yanagimachi 1978; Kawakami et al. 1993; Farstad 2000a). Nowadays, it is generally

recognized that a combination of tests, including classical semen evaluation for motility,

concentration, and morphology, in addition to sperm function tests including acrosome

reaction induction, will increase the accuracy of the prognosis concerning the potential

fertility of an evaluated semen sample. This dissertation describes a combination of

techniques for semen purification, sperm protein assessment, effects of semen

preservation on spermatozoa, and the functional test of acrosome status after acrosome

reaction induction as predictors of canine spermatozoa fertility. In order to accomplish

these goals the following objectives were defined and tested.

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The objective of the first experiment was to compare commercially available

density gradient centrifugation media [Isolate, Percoll, PureCeption, PureSperm] in their

ability to optimally separate viable, motile spermatozoa from nonviable (nonmotile

and/or dead) spermatozoa and red blood cells (RBC) contamination. The hypothesis

tested was that there would be differences between the four DGC media in terms of

their efficiency on canine spermatozoa, as determined by the ability to separate motile

sperm from non motile and RBC for optimal sperm recovery.

The objective of the second experiment was to identify and characterize using

immunocytochemistry the presence of sperm proteins SPAG6, SP17, SP56, and

CATSPER2 proteins in canine spermatozoa. The hypothesis was that sperm proteins

SPAG6, SP17, SP56, and CATSPER2 are localized and expressed in similar patterns

in canine sperm cells as in other species and may play a role in sperm function.

The objective of the third experiment was to confirm the presence of sperm

proteins SPAG6, SP17, SP56, and CATSPER2 by western blot analysis on canine

sperm, this way validating the immunocytochemistry results presented in the previous

chapter. The hypothesis to be tested was: western blot analysis will confirm by

immunolabeling in the dog the presence of sperm proteins with molecular weights

corresponding to the previously described SPAG6, SP17, SP56, and CATSPER2

proteins in other species spermatozoa.

The objectives of the last set of experiments were: a) to evaluate the changes in

selected sperm protein expression during chill preservation over a period of time, b) to

establish a possible relationship between sperm protein expression and different semen

parameters including motility, capacitation, and acrosome reaction. The hypotheses to

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be tested were: a) semen preservation overtime affect protein expression, b) differences

may be observed between the different evaluated extenders, c) acrosome induced

functional changes may be detected in relation to the preservation processes, d)

acrosome reaction induction is associated with changes in the expression of the

different semen proteins evaluated, e) overtime changes in motility may be related to

changes associated with acrosome reaction and modification in the expression of the

different proteins.

Male fertility is complex. In the dog, semen assessment has been limited in

comparison to other species such as mice and humans (Fulton et al. 1998; Farstad

2000a). Even in these other species, in vitro clinical and laboratory semen evaluations

can only partially predict the outcome of fertility: a successful pregnancy- the birth of

offspring. The achievement of our objectives will produce sound evidence in support for

in vitro canine semen assessment, which will improve chances of a successful

pregnancy and attaining a litter.

The overall hypothesis of this study was that the incorporation of semen

purification protocols, sperm protein assessment, and the effects of semen preservation

or acrosome reaction induction techniques to canine semen will improve the ability to

determine fertilizing potential of a semen sample.

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CHAPTER 2 LITERATURE REVIEW

Purification

Purification of semen prior to artificial insemination and semen preservation

freezing is vital to the success of canine fertility. There are many techniques for semen

purification, which can be classified in several families: the motility based separation

techniques (swim-up and migration sedimentation (Henkel and Schill 2003), filtration

based techniques (gel, wool, or glass bead filtrations (Henkel and Schill 2003), and the

density based (Percoll) techniques (Parrish et al. 1995). More recently, more complex

techniques have also been presented, one of which is centrifugal counter-current

distribution analysis (CCCD) an aqueous two-part partition system. This technique has

been proven to show sperm heterogeneity in semen samples (Rodriguez-Martinez

2003). Transmembrane migration has also been presented. It is a migration/filtration

system where the spermatozoa have straight channels to swim through the membrane.

Unfortunately, the membrane has a very low ratio of the total cross-sectional area of the

pores to the overall membrane area and the yield is extremely low (Henkel and Schill

2003). Many of these techniques are expensive, difficult to apply in clinical conditions

and most often still at the development phase. For these reason, we will only focus on

the technique of direct potential clinical application.

Swim-up is an aqueous two-phase sperm separation system that reveals sperm

heterogeneity in semen samples. The swim-up technique requires a short amount of

time (incubation 1 hour) and is indirectly correlated with fertility as it allows for the

separation of the ‘swimming’ spermatozoa from dead spermatozoa or inactive

contaminants. However, it is difficult to initially achieve high levels of repeatability and

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the recovery rate being low; it is difficult to recover adequate numbers of spermatozoa

for artificial insemination (Parrish and Foote 1987; Parrish et al. 1995; Chen et al.

1995b; Henkel and Schill 2003).

Migration-sedimentation is a very gentle sperm separation system that works as a

swim-up method combined with a sedimentation step. Sperm separated using

migration-sedimentation represent usually a very clean fraction of highly motile

spermatozoa. Nonetheless, special glass or plastic tubes are required and tubes are

expensive and sensitive (Henkel and Schill 2003). This technique has not yet been

applied or validated in dogs.

Glass Beads is a column separation technique of motile spermatozoa across glass

bead columns. Glass beads have a high yield selection of motile human spermatozoa.

However, with glass beads there is the risk of a possible spill over of beads into the

insemination medium (Henkel and Schill 2003). This technique has not yet been applied

or validated in dogs.

Glass wool filtration is a variation of the previous technique in which motile

spermatozoa are separated from nonmotile spermatozoa by means of densely packed

glass wool fibers. Glass wool filtration is simple to perform, a good yield with recovery of

spermatozoa with good motility, and contaminants are eliminated to a large extent.

However, the technique is expensive, the filtrate is not as clean as it is with other sperm

separation methods, and remnants of debris are still present (Henkel and Schill 2003).

Sephadex beads can also be used in a separation column also sold commercial

as SpermPrep (http://www.zdlinc.biz/) a sperm separation kit to purify good quality

sperm from poorly motile or dead sperm and contaminants. The Sephadex based

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semen separation technique (SpermPrep) is similar to the density gradient separation

methods using Percoll. However, it has significantly lower recovery and separation

abilities for sperm count and morphology (Ruiz-Romero et al. 1995; Henkel and Schill

2003) than density gradient separation methods.

The density gradient centrifugation method is well known in the literature for

separation of viable, motile spermatozoa from contaminants in several species i.e.: bull

(Parrish et al. 1995; Samardzija et al. 2006), dog (Hishinuma and Sekine 2004), human

(Miller et al. 1996; Centola et al. 1998; Hammadeh et al. 2001a). There are different

types of substrate available. The most commercially known substrate is Percoll® which

is composed of a silica-based colloidal/silica particles coated with polyvinylpyrrolidone

(PVP). Isolate®, PureCeption® and PureSperm® are silane-coated silica-based

substrates that are also currently available and often used.

Percoll is continuous or discontinuous density centrifugation media. Separation

with Percoll is based upon differences in densities (live and dead sperm cell have

different densities). Percoll is simple, repeatable, and faster, antibiotics are not needed

and has six-fold greater recovery rate of motile spermatozoa than swim-up procedure

as shown in human (Chen et al. 1995b) and in bovine (Parrish et al. 1995). However,

Percoll is less effective for the recovery rate of progressively motile spermatozoa than

swim-up in canine (Hishinuma and Sekine 2004). It allows for the separation of motile

from dead spermatozoa without high differentiation between motile cells. It has also

been suggested that contaminated cells often will be trapped at the wrong layer (Parrish

and Foote 1987; Parrish et al. 1995; Chen et al. 1995b; Miller et al. 1996; Hishinuma

and Sekine 2004).

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Isolate, PureCeption, and PureSperm are more recently developed density

centrifugation media that have primarily been used in human studies (Perez et al. 1997;

Hammadeh et al. 2001b; Hirabayashi et al. 2006). These silane-coated silica-based

substrates have become more widely used since the discontinuation of Percoll from

clinical human usage (Scott and Smith 1997). In some comparative studies (Perez et al.

1997; Politch et al. 2004; Mousset-Simeon et al. 2004), these substrates are listed as

very similar to Percoll in their ability to separate live from dead spermatozoa. However,

there are conflicting results as to whether PureSperm and PureCeption are as effective

as Percoll for spermatozoa separation (Centola et al. 1998; Chen and Bongso 1999;

Samardzija et al. 2006) i.e. for HIV-1 remove from motile spermatozoa (Politch et al.

2004; Kuji et al. 2008). Isolate, PureCeption, and PureSperm have not been evaluated

in dogs.

Assessment

Regardless, of whether a semen sample is purified or not the semen sample

should be analyzed for several factors such as motility, morphology, concentration,

acrosomal status, and membrane integrity. There are numerous techniques for

assessment of semen quality. In order to limit confusion, these techniques will be

placed into categories.

Microscopy

Brightfield microscopy was developed in 1590 by Z and H Janssen and improved

by Antonie van Leeuwenhoek in the late 1600’s. Microscopy allows for the direct

evaluation of sperm concentration and motility on non fixed, non stained samples.

However, viability, except on fixed stained samples, can poorly be analyzed. The use of

fluorescence microscopy, using a fluorescent dye which emits a specific color when

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excited, allows for the identification of either specific cells (dead or alive) or specific

location on the cell (Becker et al. 2003)

Transmission electron microscopy (TEM) and scanning electron microscopy

(SEM) allow for the observation at the subcellular level of details not visualized with light

microscopy. Transmission electron microscopy functions by transmitting elections

through the specimen and producing two-dimensional images. Scanning electron

microscopy functions by electron beams scanning the surface of the specimen and

producing three-dimensional images. Both TEM and SEM require expensive equipment

and specialized training (Rijsselaere et al. 2005; Pesch et al. 2006).

Evaluation of Sperm Motility

In vivo fertilization relies on the ability of the propelling force of the flagellum to

push the spermatozoa towards the oviduct to fertilize the ovulated oocyte. A disruption

of flagella movement can be detrimental to fertility. For this reason, assessing the

motility of the semen sample is a main component for a breeding soundness

examination. In most normal dog semen samples, motile spermatozoa are more than

70%. It is beneficial if the spermatozoa are not only motile but linear, progressively

motile (forward movement), so that the sperm can be transported to the oviduct for

capacitation and fertilization to occur. Capacitation causes a change in the pattern of

spermatozoa movement which becomes hyperactivated. Hyperactivated sperm have

increased flagella movement with decrease linear, forward progressive movement, and

increased curvilinearity. Capacitation and hyperactivation of sperm cells are

prerequisites for the acrosome reaction to occur, which is essential penetration of the

zona pellucida and fertilization (Ho and Suarez 2001b; Suarez 2008).

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Studies have shown (Gunzel-Apel et al. 1993; Takagi et al. 2001; Iguer-Ouada

and Verstegen 2001a) that information regarding sperm motion characteristics is

advantageous for assessment of male fertility. An example, the utilization of these

motility parameters have demonstrated that canine semen freezing extender induced

changes of sperm motion characteristics bear a resemblance to hyperactive motion

dynamics of spermatozoa associated with capacitation (Nizanski et al. 2009).

Furthermore, the combination of various motility parameters and other sperm function

tests were strongly correlated and produced predictive values for bovine fertility

(Januskauskas et al. 2001; Rodriguez-Martinez 2003).

Assessment of sperm motility has been subjectively performed for many years

with individual visual observation using light microscopy. These methods, though cheap

in cost, are variable in results due to individual interpretation and investigator level of

experience. Therefore, with the advent of semen analyzers and the standardization of

sperm parameters, objective analysis was developed. Objective analysis eliminated

most of the biases that were present with subjective analysis. In addition, with objective

analysis, multiple factors can be assessed simultaneously such as: motility differential

analysis of motion based on velocity (progressive, local, immotile, hyperactive) and path

(linear, curvilinear), concentration or morphology (Verstegen et al. 2002). Motility

parameters such as the following are generated by CASA systems: the percentage of

motile spermatozoa; velocity average pathway (VAP); the average velocity of the

smoothed cell path in µrn /sec; the velocity straight line (VSL); the average velocity

measured in a straight line from the beginning to the end of track in µrn/sec; the

curvilinear velocity (VCL); the average velocity measured over the actual point-to-point

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track followed by the cell in µrn/sec; the amplitude lateral head (ALH); amplitude of

lateral head displacement in lam; the beat cross frequency (BCF); frequency of sperm

head crossing the sperm average path in Hertz; the straightness (STR); the average

value of the ratio VSL/VAP in percentage (straightness estimates the proximity of the

cell path to a straight line, with 100% corresponding to the optimal straightness); and

the linearity (LIN); the average value of the ratio VSL/VCL in percentage (linearity

estimates the proximity of the cell track to a straight line).

The Computer Assisted Semen Analyzer (CASA) system, allow for large amounts

of spermatozoa to be analyzed in a short amount of time. A CASA can be used on

fresh, chilled, frozen-thawed, and capacitated sperm. However, appropriate technical

training to use the equipment is required. The CASA systems most often use phase

contrast microscopy for image analysis. However, fluorescence is also sometime used

particularly to evaluate viability of samples overtime. Phase contrast microscopy is used

for the examination of unstained specimens, especially living cells. Phase contrast

microscopy has the major advantage of quickly providing better discrimination of moving

cells from the background contaminants. Disadvantages to CASA usage are its

expensive equipment including phase contrast rings and objectives, variable results

depending on user experience, and potential variability in parameter settings in different

laboratories (Iguer-Ouada and Verstegen 2001a; Iguer-Ouada and Verstegen 2001b;

Iguer-Ouada and Verstegen 2001c; Verstegen et al. 2002; Martinez 2004; Verstegen et

al. 2005).The correlation between CASA results and fertility has also been questioned

(Rota et al. 1999a; Martinez 2004).

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If CASA systems appear optimal, intermediate systems, less expensive, easier to

use but providing less objective information are also available. The Spermacue provides

objective automated numeration of sperm cells. Similarly, the Sperm Quality Analyzer

(SQA) is a simple, less expensive system that can be used to assess fresh or

cryopreserved sperm. It functions by detection of the variations in optical density

included by sperm concentration and movement. The sperm motility index (SMI)

generated by the SQA is an accurate estimation of sperm concentration and motility.

The value is repeatable enough for canine sperm analysis (Iguer-Ouada and Verstegen

2001c; Rijsselaere et al. 2002b) and is of clinical interest even if not providing as

thorough and informative results as the CASA systems.

Evaluation of Sperm Morphology

Another main component for a breeding soundness examination is assessment of

sperm morphology. Morphological evaluation is important because a high number of

abnormal sperm can be associated with infertility. In most normal dogs, morphological

abnormal spermatozoa are less than 20%. Sperm with primary and secondary defects

such as: double head, proximal or distal droplets, coiled or kinked tails are to be less

than 20%. Morphology assessment, with the appropriate stain also allows for the

determination of acrosomal status. If sperm are already acrosome reacted, they may

not function correctly, due to the fact that proteins and enzymes that are a part of the

acrosome are no longer present and thus can not perform their specific function.

Staining: To identify sperm cell structure and viability, numerous stains have been

routinely used in clinic. Eosin-nigrosin staining is commonly used to discriminate “live”

from “dead” spermatozoa (sometime called the “live-dead stain”). It is simple and quick

to use, assessing membrane integrity (live sperm do not allow the stain to penetrate the

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cytoplasm while the modified membrane in dead sperm will not prevent the stain to

penetrate). Eosin-nigrosin stain has been shown to be hypotonic and possibly cause

sperm defects and incomplete staining of spermatozoa (Rijsselaere et al. 2002a;

Tsutsui et al. 2003; Rijsselaere et al. 2005).

Diff-Quik, a modification of the Wright Giemsa stain, is a basic commonly used in

practice stain that differentiates cells and cellular components based on eosinphilic

(acidic) or basophilic properties. This stain is easy to use and provides fast results. In a

comparative study (Root Kustritz et al. 1998), Diff-Quik was shown to significantly stain

a larger percentage of morphological normal spermatozoa when compared to eosin-

nigosin stains.

Spermac stain has a two-fold function. It differentiates normal from abnormal

sperm morphology and determines the acrosomal status of spermatozoa. The stain

provides quick results, easy to use, and can be used on fresh, chilled, or frozen semen

(Oettle 1986; Bencharif et al. 2010).

Coomassie Blue stain is an effective and inexpensive acrosomal staining method

that provides fast results. The acrosome is defined as intact (uniform dark-blue staining

overlaying the entire acrosome region), damaged (patchy staining over the acrosome

region), or non intact (total absence of staining or staining only in the equatorial

segment) (Brum et al. 2006).

Immunostaining is a different more specialized and specific type of staining where

the reaction can either induce a colorimetric (immunocytochemistry and

immunohistochemistry), a fluorescent (IF) or a luminescent reaction

(chemiluminescence). Immunocytochemistry (ICC) allows for the specific detection of

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the expression of molecules (antigens) at the cellular and/or tissue level depending on

the specific interaction of an antibody with the antigen of interest.

The immune reaction, specific to the antibody used, is detected either by an

enzyme mediated reaction associated with the local precipitation of a stain (i.e. 3, 3’-

diaminobenzidine tetrahydrochloride (DAB) and 5-bromo-4-chloro-3’-indolylphosphate

p-toluidine salt (BCIP) or the production of a chemiluminescent or fluorescent signal

such as the green one produced by fluorescein isothiocynate (FITC). There are several

advantages to using fluorescent staining: it has less interference due to

cryopreservation media; it can be used in solution to analyze fluorescent, living stained

sperm in culture or by means of flow cytometry. The immunostaining approach not only

is specific but allows for the detection of several parameters at the same time

(membrane integrity and acrosomal status); and stains can be combined.

Flow cytometry also called cell sorting, when used with semen analysis has the

advantages of allowing simultaneous evaluation of different and numerous

characteristics (i.e. viability, acrosomal integrity, DNA fragmentation and integrity or

mitochondrial function (Graham 2001; Evenson and Wixon 2006). However, to date, its

clinical applications are limited by cost, time, and the number of cells that can be

analyzed in a minimal amount of time (Brewis et al. 2001; Graham 2001; Petrunkina et

al. 2004).

Evaluation of Sperm Functional Test and Membrane Integrity

Assessment of sperm motility and morphology alone are not strong predictors of

fertilization ability (Amann 1989). The incorporation in the panel of evaluation of a sperm

functional test should provide an improved and more detailed evaluation of sperm

characteristics. Acrosomal status, membrane integrity, and fertilization ability can be

26

tested using functional tests (Amann 1989; Farstad 2000b; Rijsselaere et al. 2005). The

ultimate goal would be to establish a correlation between subjective and objective tests

(functional tests) to increase the probability that in vitro evaluation will result in favorable

fertility prognosis.

Outside the previously described basic stains to evaluate acrosomal status, more

complex and expensive technologies have been described. The Chlortetracycline Assay

(CTC) is used with a fluorescent antibody probe for detection of the acrosomal status

and advancement of spermatozoa capacitation. In canine, there are three fluorescent

patterns: uncapacitated and intact acrosome is represented with F-pattern, capacitated

and intact acrosome is represented with B-pattern, and capacitated and acrosome

reacted is represented with AR-pattern (Rota et al. 1999b; Iguer-Ouada and Verstegen

2001b; Rodriguez-Martinez 2003).

Fluorescent SYBR14-PI (PI- propidium iodide, which binds to DNA in membrane

defective cells) is a sensitive method for separation of live, dead, and dying state

spermatozoa (Kawakami et al. 2004; Rijsselaere et al. 2005; Samardzija et al. 2006).

Triple staining (SNARF-1/YO-PRO-1/ethidium homodimer) permits viability and

acrosomal integrity to be evaluated at the same time in fresh or frozen-thawed semen

utilizing fluorescent microscopy or flow cytometry without removal of cryopreservation

extender (Tsutsui et al. 2003; Pena et al. 2006).Epifluorescence microscopy (CFDA/PI

fluorophores) is used for the recognition of ruptured or damaged acrosomal membranes

and can be used with flow cytometry. Epifluorescence with CFDA-carboxyfluorescein

and PI-propidium iodide fluorophores are not adequate for evaluation of the existence of

27

acrosomal defects (Graham 2001; Rijsselaere et al. 2005). Moreover, availability of

equipment limits clinical application.

Fluoresceinated lectins combined with propidium iodide are regularly used to

detect the acrosome reaction in fresh or frozen-thawed dog sperm diluted in various

extenders. Fluoresceinated lectins, Pisum sativum agglutinin (PSA) and peanut

agglutinin (PNA) are harmful due to the fact that they are extremely toxic when added to

sperm incubating in capacitating environments (Graham 2001; Kawakami et al. 2004;

Rijsselaere et al. 2005). Moreover, availability of equipment limits clinical application.

Functional tests permit in vitro experimentation to test the potential fertilization

ability of sperm cells. Hypo-osmotic swelling test (HOST) evaluates the permeability of

the sperm membrane. The HOST is advantageous due to the fact that there is a high

correlation between assessment of membrane integrity and the ability of sperm to

penetrate the oocyte (Kumi-Diaka 1993; Hishinuma and Sekine 2003; Samardzija et al.

2006; Bencharif et al. 2010).

If in vivo fertilization is the definitive studies’ answer for the fertilization ability of

spermatozoa, the cost and the laborious nature of in vivo studies strongly and

negatively influence the use of such in vivo technologies to assess semen quality

through fertilization studies. To alleviate the difficulties in performing in vivo studies,

additional in vitro works have been described. In Vitro Fertilization (IVF) is a technique

that involves in vitro spermatozoa and oocyte interaction, which results in fertilization

outside of the body. The procedure is well established and commonly used in many

species: bull (Parrish et al. 1995; Samardzija et al. 2006), mice (Songsasen and Leibo

1997), human (Bungum et al. 2008). Unfortunately, this is not the case in the canine,

28

due to the low percentage of oocytes fertilized (low percentage passing from oocyte

maturation to morula and blastocyst stage), difficulties with in vitro development, and is

largely dependency on laboratory expertise (Verstegen et al. 2005; Rodrigues and

Rodrigues 2006; Songsasen and Wildt 2007).

Intracytoplasmic sperm injection (ICSI) involves the injection of one spermatozoon

directly into the ooplasm of the oocyte. This procedure is very useful for cases where a

small number of sperm is available (Evenson et al. 2002; Henkel and Schill 2003;

Rijsselaere et al. 2005). Intracytoplasmic sperm injection was performed using chilled

canine semen for treatment of canine infertility (Fulton et al. 1998). They reported that

the results were not as successful as in other species treated with by ICSI and that

further studies are warranted. Currently, this technique is not yet available in canine.

Zona pellucida binding assay functions to determine the ability of spermatozoa to

bind to and penetrate intact zona pellucida. The zona pellucida binding assay is able to

detect sperm damage at the molecular level. Unfortunately, canine salt-stored oocytes

and frozen-thawed oocytes do not do as well as fresh oocytes (Strom et al. 2000b); and

canine spermatozoa chilled for 4 days have the tendency to reduce zona-binding (Strom

et al. 2000a). Hemi-zona assay is performed by bisecting an oocyte by means of

microdissection. The two halves of the zona are then incubated with a semen sample.

Both the hemi-zona assay and the zona pellucida assays are time-consuming and

difficult procedures to perform that not yet readily and easily available in canine

(Mayenco-Aguirre and Perez Cortes 1998; Ivanova et al. 1999).

Evaluation of Sperm at Molecular Level

Upon visual observation, sperm may have normal motility and morphology. The

sperm may successfully pass functional tests. However, the male is still deemed

29

infertile. Indeed, molecular DNA defects may be present but not detected by classical

approaches. The next step is then to assess the sperm at a molecular level. At the

molecular level, DNA, RNA, protein, and/or subcellular structures such as chromatin

can be evaluated for defects. The detection of sperm molecular defects may be the

determinate for fertile or infertile status. Assessment of sperm at a molecular level, also

allows for identification of genes and proteins of interest.

Many modern techniques have been described to evaluate DNA integrity. In situ

Nick Translation assay allows for assessment of DNA integrity by quantifying the

incorporation of biotinylated dUTP at single-stranded DNA breaks in a reaction

catalyzed by the template-dependent enzyme DNA polymerase I. Flow cytometry can

also be used to evaluate DNA integrity. Single-cell electrophoresis assay (COMET) is a

technique that is used to assess DNA integrity. The COMET assay detects damage at

the level of single and double strand breaks. Terminal transferase dUTP nick end

labeling (TUNEL) assay is a common method used to assess DNA integrity by

quantifying the incorporation of dUTP at single and double stranded DNA breaks in a

reaction catalyzed by the template-independent enzyme, terminal transferase, TdT

(Evenson et al. 2002; Rodriguez-Martinez 2003). These techniques have not been

evaluated and validated in canine.

Sperm chromatin structure assay (SCSA) evaluates the vulnerability of chromatin

to denaturation when sperm are incubated under denaturing conditions. The sperm

chromatin structure assay is an objective assay that uses flow cytometry to detect

abnormalities. This assay, which was first described by (Evenson et al. 1980), can be

used on fresh as well as frozen-thawed semen. The assay also has the advantage of

30

repeatability and of having data supporting it as a strong predictor of semen quality

(Evenson et al. 1980; Evenson et al. 2002; Nunez-Martinez et al. 2005). Justified data

have been presented in dogs (Nunez-Martinez et al. 2005; Shahiduzzaman and Linde-

Forsberg 2007) .

Among the newer techniques developed, Fluorescent in situ hybridization (FISH)

is an interesting and sensitive technique with a promising future. It permits the analysis

of sperm by using chromosome-specific probes labeled with fluorochromes. Fluorescent

in situ hybridization is used to map out chromosomes that contain microsatellite markers

and to identify specific genes. FISH like ICC is specific and when an optimal probe is

available providing reliable results. However, FISH can not yet been applied on live

sperm cells and is missing sensibility due to the low amount of DNA available into the

sperm cells (Breen et al. 1999; Olivier et al. 1999; Breen et al. 2001; Gianaroli et al.

2005).

Evaluation of Sperm Membrane Proteins (Antigens or Receptors)

At the level of the spermatozoa, there are many proteins that can provide vital

information as it relates to fertility. These proteins can be receptors localized in the

membrane (proteins and peptides receptors) or within the cytoplasm (steroid receptors)

and their activation may be related to specific important functional events. They can

also be antigens localized on the membrane or within the cytoplasm and be important

for the overall spermatozoa activity and metabolism.

Sperm proteins localized on the flagellum may be associated with motility. Sperm

proteins localized at the acrosomal region may be associated with capacitation and/or

acrosome reaction. Knockout mice that are infertile or have reduced fertility as a result

31

of gene or protein disruption, might give clues as which sperm proteins are required for

fertilization (Ren et al. 2001; Sapiro et al. 2002).

Heparin-binding proteins are largely located on the surface of ejaculated

spermatozoa and less on the plasma membrane of epididymal spermatozoa. Heparin is

described as the most potent enhancer of capacitation of bovine and rabbit

spermatozoa. Thus, heparin-binding proteins function to increase affinity for heparin and

enhance capacitation (Miller et al. 1990; Braundmeier and Miller 2001). Fertilization

associated antigen (FAA) is a heparin-binding protein that binds to spermatozoa

(Braundmeier and Miller 2001; Gatti et al. 2004) and is secreted by the seminal vesicles

and the prostate gland. A study has been published (de Souza et al. 2006), stating that

there are heparin-binding proteins in canine seminal plasma. The report does not

mention however, which fraction the seminal plasma was taken from; as the only

seminal fluid in the canine would come from the prostate gland, its only sexually

accessory gland.

Estrogen receptor alpha (ERα), a truncated form of estrogen receptor alpha, and

estrogen receptor beta (ERβ) are present in human ejaculated spermatozoa (Aquila et

al. 2004; Lambard et al. 2004; Carreau et al. 2006). Estrogen receptor beta (ERβ) is

present in rodent ejaculated spermatozoa (Saunders et al. 1998; Carreau et al. 2006).

In the dog, ERβ is expressed in the round spermatids, spermatogonia, and

spermatocytes, but is not expressed in the spermatozoa (Nie et al. 2002).

A major olfactory receptor transcript (DTMT) and its corresponding protein have

been identified on canine spermatids and the midpiece of mature spermatozoa

(Vanderhaeghen et al. 1993). Due to the pattern of expression, it is hypothesized that

32

the function of the olfactory receptor and protein is associated with sperm maturation,

motility, or chemotaxis (Vanderhaeghen et al. 1997). Odorant receptors hOR17-4 in

human and mOR23 in mouse mediate calcium signals in mature spermatozoa.

However, the specific physiological function of hOR17-4 and mOR23 is unknown (Spehr

et al. 2006).

The presence of sperm-associated antigen 6 (SPAG6) has been linked with

sperm flagella motility and structural integrity of the axoneme central apparatus of

mature sperm in mice and humans (Neilson et al. 1999; Sapiro et al. 2000; Horowitz et

al. 2005). Sperm protein 17 (SP17), initially was reported as a rabbit sperm protein that

was highly expressed in spermatozoa and had a primary function of zona pellucida

binding (Richardson et al. 1994). Sperm fertilization protein 56 (SP56), is a sperm-

oocyte recognition sperm protein that has an affinity for ZP3 an oocyte glycoprotein

associated with sperm-oocyte interaction (Cheng et al. 1994). Cation channel, sperm

associated 2 (CATSPER2), is a voltage-gated protein that is present in spermatozoa

(Quill et al. 2001). Nothing is known of SPAG6, SP17, SP56, or CATSPER2

spermatozoa expression in dogs.

Preservation

The first reportedly successful canine artificial insemination (AI) using fresh semen

was done by an Italian physiologist and priest, Abbe Lazzaro Spallanzani in 1784. Dr.

AE Harrop, using heat-treated pasteurized milk performed the first successful canine

chilled semen insemination in 1954 (Harrop 1954). In 1969, Seager reportedly

performed the first successful canine insemination with frozen-thawed semen (Seager

and Fletcher 1972; Platz Jr. 1990; Farstad 2000a).

33

Preservation of semen involves providing an optimal in vitro environment for

preserving sperm viability. Semen preservation consists of fresh, chilled, and frozen-

thawed semen. Short-term preservation (days) includes fresh or chilled semen. Long-

term preservation (months and years) includes frozen semen. Normally, canine semen

is shipped chilled or frozen in canine semen extender. Semen, in chilled extender, is

chilled to 4-5°C and placed in a shipping box with ice packaging for transport. Frozen

semen, in freezing semen extender, is shipped in a liquid nitrogen container at a

temperature around -196°C (Linde-Forsberg 1991).

Since the first successful artificial insemination in canine species using chilled

semen in 1954, several studies have been conducted in order to evaluate the effects of

temperature over freezing point on semen conservation; the range of 4–5 °C was

determined as the best temperature conditions for sustaining semen motility by reducing

gamete metabolism (Province et al. 1984; Bouchard et al. 1990; Rota et al. 1995). This

temperature was demonstrated initially to allow semen conservation for up to 4–5 d, as

opposed to a few hours when semen was conserved either at room temperature or at

35 °C. Different cooling techniques and semen extenders were proposed during the last

30 years (Foote and Leonard 1964; Province et al. 1984; Bouchard et al. 1990; Brown

1992; Goodman and Cain 1993; Rota et al. 1995).

There are several essential components for semen extender: it must be isotonic

with the semen of interest, a good buffer to avoid significant pH changes overtime,

minimize cold shock and damage during chilling, provide appropriate nutrients, allowing

sperm to survive, prevent microbial growth, and be relatively low in cost (Wales and

White 1958; Foote 1964a; Linde-Forsberg 1995). Buffers such as Tris, sodium citrate,

34

and sodium phosphate are commonly used (Rota et al. 2001; Iguer-Ouada and

Verstegen 2001b; Verstegen et al. 2005). The main nutrients required for sperm

metabolism are glucose and/or fructose (Ponglowhapan et al. 2004). During

preservation when semen is chilled or frozen the semen extender must provide

protection against cold shock and damage. Egg yolk or low density lipoproteins (LDL)

are used to provide energy and prevent cold shock which effects cell membrane

integrity (Foote and Leonard 1964; Songsasen et al. 2002; Okano et al. 2004). Last,

antibiotics such as penicillin and streptomycin, or gentamycin will help prevent microbial

growth.

In a canine study (Rota et al. 1995), demonstrated that Tris egg yolk extender

appears to be better than other extenders, such as, egg yolk milk or egg yolk cream in

preservation of dog semen at 4°C. In another canine study (Iguer-Ouada and Verstegen

2001b), comparing different commercial and laboratory made extenders; it was

demonstrated that a Tris-glucose egg yolk based extender had the highest quality

motility and percentages of motile sperm. All extenders showed a significant

improvement in motility parameters with the addition of egg yolk. Among commercial

extenders that were examined, Biladyl had the best motility parameters when egg yolk

was added. While the Tris-glucose egg yolk extender, a laboratory created extender

had the best overall motility parameters and allowed for the longest viability after

extension (more than 80% of the initial motility up to 14 days after collection). Egg yolk

had an obvious effect for protecting the membranes and preventing acrosome reaction.

Glucose and fructose provide nourishment for metabolism and support for motility

in sperm. There is still significant controversy in the canine as to which of the two

35

sugars has the highest activity. If Iguer-Ouada and Verstegen (Iguer-Ouada and

Verstegen 2001b) did consider glucose as having the most activity, it was described

later that egg yolk Tris extender supplemented with fructose at a concentration of 70mM

was superior for long-term preservation of chilled canine semen (Ponglowhapan et al.

2004). On the other hand, Tris-glucose egg yolk extender was found, after several

changes of extender, to extend chilled semen motility up to 3 weeks with preservation of

good quality sperm for at least 15 days. When the extender was changed, sperm cells

that were thought to be nonmotile or of poor motility were reactivated. To verify

fertilization ability of this prolonged, chilled extended semen, 20 bitches were

inseminated with 7-11 day chilled semen; 12 became pregnant proving that motility can

be preserved while chilling semen in the dog and that as semen motility can be

reactivated, motility without further evaluation cannot always be a reliable predictor of

fertility (Verstegen et al. 2005). The Iguer-Ouada and Verstegen study (Iguer-Ouada

and Verstegen 2001b) was recently confirmed (Shahiduzzaman and Linde-Forsberg

2007) to maintain preservation of good quality semen up to 14 days at 5°C.

Other sugars added to a Tris-citric acid extender had variable effects. Galatose,

lactose, trehalose, maltose, and sucrose reduced damaged acrosome percentages in

equilibrated sperm. Monosacchrides (fructose and xylose) improved motility along with

post-thaw viability and intact acrosome percentages. Trehalose, xylose, and fructose

significant increased total active sperm rates in frozen-thawed semen samples (Yildiz et

al. 2000).

The inclusion of Equex STM paste (including sodium dodecyl sulfate (SDS)

demonstrated to prevent protein agglutination and coagulation) to semen extenders has

36

been confirmed to be beneficial for semen preservation (Rota et al. 1997; Pena and

Linde-Forsberg 2000; Petrunkina et al. 2005; Ponglowhapan and Chatdarong 2008). In

a study involving Equex (Petrunkina et al. 2005), before freezing, canine semen was

diluted with or without Equex STM paste. The extender containing Equex STM paste

had protective effect on isotonic cell volume, on regulatory function under hypertonic

conditions, and on the proportion of live acrosome reacted cells. The use of egg yolk

Tris-citrate-glucose extender with the addition of 0.5% Equex STM paste significantly

increased the proportion of spermatozoa having an intact plasmalemma after thawing

compared to without Equex STM paste (Rota et al. 1997). Furthermore, there is

increased longevity of the thawed spermatozoa which prolongs the maintenance of both

motility and plasma membrane integrity.

During freezing, a cryoprotectant such as glycerol, dimethyl sulfoxide (DMSO),

methanol, or methyl glycol will protect against damage due to intracellular ice formation.

Nevertheless, egg yolk has been used for many years as a membrane protectant and

energy substrate for the prevention of cold shock during semen chilling and freezing.

However, with the insurgence of bioterrorism and avian bird flu the usage of egg yolk in

semen extenders for international shipping has decreased. Pace and Graham, reported

that the low density fraction or low density lipoproteins (LDL) was the component of egg

yolk that in the absence of glycerol protects motility of sperm cells during the freezing

process (Pace and Graham 1974). They also suggested the presence of unknown

substances in purified egg yolk [without LDL] that diminish sperm motility. Low density

lipoproteins which adheres to cell membranes during chilling, freezing, and thawing

were shown to improve bull sperm motility when compared to an egg yolk extender

37

(Moussa et al. 2002). The specific protective effect of LDL on canine semen has never

been analyzed. Nevertheless, it has been demonstrated that canine semen frozen with

LDL instead of egg yolk had better post-thaw characteristics (Bencharif et al. 2008;

Bencharif et al. 2010)

38

CHAPTER 3 EFFICACY OF FOUR DIFFERENT DENSITY GRADIENT SEPARATION MEDIA TO REMOVE RED BLOOD CELLS AND NON VIABLE SPERMATOZOA FROM CANINE

SEMEN

Introduction

Many factors can affect seminal and prostatic fluid and consequently have an

impact on male fertility. Blood, urine, or other products (bacteria, debris, and pus) have

been demonstrated (England and Allen 1992; Chen et al. 1995a; Johnston et al. 2001)

to contaminate semen and prevent its use for artificial insemination (AI) and/or semen

processing including freezing. It is well known that in dogs, blood of different origins is

negatively affecting semen preservation (Rijsselaere et al. 2004). Moreover, the aging

dog is prone to prostatic disease. Prostatic disease commonly contributes to cellular

contamination (blood, bacteria, and neutrophils) that can compromise semen quality

and reduce its suitability for AI and preservation procedures. Therefore, at the time of AI

or prior to application of assisted reproductive technology (ART), it is critical to remove

contaminants prior to further processing.

Removal of liquid contaminants, such as urine, can easily be achieved by

centrifugation and multiple washes with semen extender. However, cellular

contaminants are more of a concern as they cannot easily be spun-down and excluded

from the viable, motile sperm cells. The density gradient centrifugation (DGC) method

has been demonstrated in many species (Miller et al. 1996; Centola et al. 1998;

Samardzija et al. 2006) including dogs as being not only simple and relatively

inexpensive, but also resulting in favorable sperm cell recovery. However, the first

published studies demonstrated a poor efficiency in separation of cellular contaminants.

39

Percoll is a commonly used DGC media for sperm separation. Unfortunately,

Percoll was removed from human clinical usage in 1996 and has been reported to have

deleterious effect on sperm membranes (Strehler et al. 1998). However, in many non

human clinics and laboratories Percoll is still used as the DGC media of choice. The

objective of the study was to compare commercially available prepared density gradient

centrifugation media (Isolate, Percoll, PureCeption, PureSperm) in their ability to

optimally separate viable, motile spermatozoa from nonviable (non-motile and/or dead)

spermatozoa and contamination of red blood cells (RBC). The hypothesis to be tested

was that there would be differences between the four DGC media in terms of their

efficiency on canine spermatozoa, as determined by the ability to separate motile from

nonmotile sperm and RBC for optimal sperm recovery.

Materials and Methods

Materials

SpermVision SAR was purchased from Minitube of America (Verona, WI).

CaniPRO Chill 5, canine semen extender was purchased from Minitube of America

(Verona, WI). Isolate, Percoll, and PureCeption DGC media were purchased from Irvine

Scientific (Santa Ana, CA), Pharmacia (Uppsala, Sweden), and SAGE In-Vitro

Fertilization, Inc. (Trumbull, CT), respectively. PureSperm in 40% and 80% ready-to-

use-solutions were purchased from Nidacon International AB (Molndal, Sweden). The

sperm separation media gradients (Isolate, Percoll, and PureSperm) were prepared by

combining the undiluted sperm separation media with CaniPRO Chill 5 semen extender

to reach desired working dilutions. For all media, the pH was adjusted to be within the

range of 6.8 to 7.2 and the osmolarity adjusted to be within the range of 280 to 325

40

mOsm. For RBC and spermatozoa staining Diff-Quik was purchased from Webster

Veterinary Supply (Sterling, MA).

Animals

Four healthy male dogs of unknown fertility, ranging between 2 to 5 years old were

used in this study. Semen was collected a maximum of three times each week with

significant periods of rest (more than a week) occurring between collection cycles as

collections were performed continuously during the year but only as needed.

To reduce variability in the different studies concerning semen, semen was pooled

from the four dogs. Using a pool of semen allowed for a reduction of variability due to

animals and allowed for a reduction of the number of animals needed for every study.

The pools of semen submitted to the different treatments will indeed vary only

depending on the effect of the treatment studied and not depending on the animals. The

number of dogs is then justified by the need to have access to sample volumes large

enough to allow the submission of the pools of semen to the different treatments

(extenders, sperm protein, etc.). Five animals have been demonstrated to be optimum

for this purpose during CASA validations and semen extenders studies (Verstegen et al.

2005). However, one of our animals had to be removed from the study just after the

start of this research for health reasons. Animal care and research was conducted

under the approved protocol (# E434) by the University of Florida Institutional Animal

Use and Care Committee.

Experiments

Semen was collected by digital manipulation (Foote 1964b; Linde-Forsberg 1991)

and blood was sampled by jugular venipuncture by means of a vacutainer containing K2

EDTA from BD (Franklin Lakes, NJ). The sperm-rich fractions of semen from the four

41

dogs were pooled to reduce individual variability between trials as previously described

by Silva & Verstegen (Silva and Verstegen 1995). The pooled semen was objectively

analyzed with a computer assisted semen analyzer (CASA), SpermVision 1.0 Minitube

of America (Verona, WI) for concentration, motility and patterns of motility. The semen

was then washed with CaniPRO Chill 5 canine semen extender at 700 x g for 15

minutes at room temperature. After centrifugation, the supernatant was removed and

the semen was re-extended if needed with CaniPRO Chill 5 canine semen extender to

reach a final volume of 4 ml and a sperm concentration around 100 x106/ml. When

needed, whole blood was then added to the washed sperm at concentrations of 1 to 4%

v/v to mimic a natural contamination. Then a volume of 1 ml of the blood/sperm

admixture was gently pipetted over 4 ml of a double layered-column (the higher density

mixture was layered on the bottom) (Fig. 3-1) of the following working gradients: 50/90%

Isolate, 45/90% Percoll, 40/80% PureCeption, and 40/80% PureSperm as

recommended by the manufacturers. The columns were then centrifuged at 400 x g for

20 min at room temperature and the different phases of the column gradients were

removed layer by layer into 1 ml fractions (A, B, C, D, E,) and when present an

additional layer or a pellet (F). The different fractions were finally analyzed using the

Spermvision SAR system for sperm concentration and motility parameters. Slides were

also prepared for the evaluation of the RBC/spermatozoa ratio as well as the sperm cell

morphology in the different fractions.

The smears were stained using Diff-Quik (Root Kustritz et al. 1998) and examined

under light microscopy (200-400x). A hundred spermatozoa were counted in three fields

of each slide and an average was obtained. To evaluate morphology, (normal, double

42

tail, distal droplet, coiled tail, kinked tail, abnormal head shape, and detached head)

smears of each collected fraction were stained and examined under light microscopy

under oil immersion (1000x). A hundred spermatozoa were counted for each slide with

a laboratory counter Fisher Scientific (Pittsburg, PA). A minimum of 3 slides were

analyzed per fraction. The study was repeated three times.

Statistical Analysis

One-way analysis of variance (ANOVA) was used to compare spermatozoa

parameters and efficiency of separation of contaminants of the different sperm

separation media. Two by two comparisons were performed when P was significant

(P<0.05) using the Bonferroni multiple comparisons test. Two-way ANOVA was used to

compare the different sperm separation media and fractions for morphology. Data were

expressed as mean ± SD. Differences were considered to be significant when P<0.05

Results

All media except Isolate presented a pellet which generally included the maximum

number of motile spermatozoa and the lowest concentration of dead or immotile

spermatozoa and RBC. The overall percent recovery of total and motile spermatozoa,

the percent motility of spermatozoa in the specific fractions and RBC/ spermatozoa ratio

(RBC/S) per fraction for each separation media are given in (Figs. 3 2A-D). All DGC

media allowed for separation of RBC from sperm cells and the highest ratio RBC/S for

each DGC media were: Isolate fraction A (29.4 ± 29.7), Percoll fraction A (28.2 ± 20.8),

PureCeption fractions A and B (37.0 ± 22.8, 39.6 ± 24.3, respectively), and PureSperm

fractions A and B (25.2 ± 5.9, 23.0 ± 3.9, respectively).

For all DGC media, the fractions with the highest RBC/S were significantly

different than all the remaining fractions (P<0.05). The optimal fraction (highest sperm

43

cell recovery, motile sperm cell recovery and overall motility respectively) in each of the

media were: Isolate fraction D (33.9 ± 29.4%; 40.99 ± 27.9%; 71.2 ± 21.8%), Percoll

fraction D (36.4 ± 14.5%; 39.3 ± 15.8%; 88.6 ± 2.3%), PureCeption fraction F (78.8 ±

28.3%; 88.0 ± 17.4%; 70.2 ± 11.1%), and PureSperm fraction F (73.1 ± 21.0%; 75.4 ±

24.6%; 80.6 ± 17.1%). The fraction F for PureCeption and the fractions E and F for

PureSperm were significantly different than all the other fractions (P<0.05). The optimal

fraction of PureCeption (Fig. 3-3) had a significantly higher recovery of total and motile

sperm cells compared to the optimal one for Percoll and Isolate (P<0.0163).

PureCeption and PureSperm fractions F were not significantly different; however,

the maximum number of total and motile spermatozoa was concentrated in only one

fraction (F) in PureCeption while for PureSperm they were observed in either fraction E

or F as demonstrated by the high standard deviations observed (39 and 21, 44 and 24

for total and motile sperm cell recoveries, respectively). DGC separation does not

improve morphology (data not shown).

Discussion

In the canine, it has been shown that the presence of increased concentrations of

blood in semen stored for a prolonged period of time has detrimental effects on sperm

motility parameters, membrane integrity, and acrosome status. The detrimental effects

are partially due to the high concentration of hemoglobin originating from RBC

hemolysis (England and Allen 1992; Rijsselaere et al. 2004). Using bovine sperm, it was

found that with an increase in blood and serum contamination, there is a decrease of in

vitro fertilizing capacity (Verberckmoes et al. 2004). These studies provide the

relevance for the removal of blood from canine semen.

44

In the present study, four density gradient centrifugation media were compared

taking into account not only the highest recovery of total and motile spermatozoa, but

also separation of red blood cells from spermatozoa and morphology preservation. The

ideal media should separate the ejaculate into different fractions allowing for the highest

recovery of total and motile spermatozoa and the lowest contamination with other cells

including RBC, immotile or dead spermatozoa. On a practical point of view to ease

separation, the location of spermatozoa would be best in the bottom of the column,

away from other cellular contaminants such as blood, which ideally would be located in

upper phases.

Among the evaluated media, Percoll is the most extensively used. However, it was

removed from human clinical usage because of a risk of endotoxin contamination (Scott

and Smith 1997). It has also been reported that Percoll has a deleterious effect on

sperm membranes (Strehler et al. 1998). In a recent study, in cats it was reported that

Percoll centrifugation was associated with a low final yield of sperm cells (Filliers et al.

2008). Since Percoll has been shown to provide poor results and negative effects on

sperm, there is a further need for comparison of other DGC media.

In the present study, Percoll centrifugation resulted in poor separation. To get a

good overall recovery we would have had to pool the fractions C to E making the

process more complicated. Although Isolate resulted in recovery of motile spermatozoa,

it had percentages of recovery which were significantly lower than PureSperm and

PureCeption. PureSperm had better percent recovery of total and motile spermatozoa

than Isolate and Percoll. However, the separation of motile spermatozoa was not

optimal as it was split with high variations between 2 fractions (E and F). Furthermore,

45

the percentages of recovery were lower than the one observed when PureCeption was

used (P<0.05 for fraction E). PureCeption resulted in the highest percent recovery of

total and motile spermatozoa concentrated in only one fraction (fraction F), which also

was characterized by a very low RBC/S ratio corresponding to our initial objective.

The centrifugation settings selected for this study were within the range that had

been shown to result in the least amount of damage to spermatozoa (Parrish et al.

1995; Hishinuma and Sekine 2003; Politch et al. 2004; Samardzija et al. 2006). Our

centrifugation speed being at the upper limit, made increasing the speed not an option

without inducing significant sperm cell damage while decreasing the speed has been

shown to reduce the separation efficacy. If the use of centrifugation has not been shown

to be optimal for the sperm cell (Aitken and Clarkson 1988; Alvarez et al. 1993;

Mortimer 1994), the only other alternative presently available (swim-up) has also been

associated with a poor recovery in the canine. Furthermore, several studies have

illustrated, that using Percoll at the present centrifugation speed and time, centrifugation

provided better recovery and is not associated with more sperm damage than swim-up

(Tanphaichitr et al. 1988; Moohan and Lindsay 1995; Chen et al. 1995b).

PureCeption usage has up to now only been reported in human studies (Politch et

al. 2004; Kuji et al. 2008) for separation of human immunodeficiency virus type 1 (HIV-

1) from spermatozoa or for sperm separation. Isolate and PureSperm have been used

in human (Politch et al. 2004; Mousset-Simeon et al. 2004) and animal studies

(Hernandez-Lopez et al. 2005; Underwood et al. 2009).

In conclusion, we have demonstrated in this study a significant difference in the

percent recovery of total and motile sperm between PureCeption and Percoll, and

46

difference in their ability to separation sperm between different DGC media and their

ability to separation sperm from RBC. On a practical and clinical point of view,

PureCeption more efficiently allowed separation of the motile sperm cells in different

fractions with the highest recovery concentrated in the bottom of the column and better

recovery than Isolate, Percoll, and PureSperm.

47

Figure 3-1. DGC media separation of sperm and RBCs with a 45% and 90% Percoll gradient. One ml of sperm mixed with 1-4% whole blood for contamination was added to the layered aliquots of 45% and 90% phases. Centrifugation was for 20 minutes and 100 x 106 sperm cells/ml were layered. One ml fractions (A-F) were pipetted from the column and number of motile sperm, nonmotile sperm, and RBCs were determined per fraction. Diagram created by Tameka C. Phillips.

48

Figure 3-2. Sperm separation media parameters for the collected fractions. A) Isolate, B) Percoll, C) PureCeption, D) PureSperm

49

Figure 3-3. Characteristics of fractions (%) with optimal recovery for the different DGC: Isolate D, Percoll D, PureCeption F, and PureSperm F.

50

CHAPTER 4 IDENTIFICATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2 IN

THE CANINE

Introduction

For many species, when semen characteristics are poor, procedures such as in

vitro fertilization and intracytoplasmic sperm injection provide successful outcomes

(Hammadeh et al. 2001a; Henkel and Schill 2003). In the dog, these in vitro approaches

are up to now disappointing and the results poor (Fulton et al. 1998; Songsasen and

Wildt 2007; de Avila Rodrigues et al. 2007): fertilization results are low, the embryos do

not develop, the oocytes availability (in number, maturation and quality) or accessibility

(high lipid content and dark aspect) are mediocre. To date, only in vivo approaches

remain available. Achieving optimal pregnancy rates requires good breeding

management, a fertile male and fertile female. The semen should be optimally

concentrated, devoid of contaminants, and of optimal motility and morphology.

However, even when these parameters are present, there are still numerous cases

where pregnancy is not obtained. It is believed that more than 70% of the infertile cycles

are related to female causes while the male seems to be responsible for the other 30%.

In the previous chapter, we saw that semen contamination could be an issue and that

we presently have approaches to purify contaminated semen, thereby improving the

quality of the ejaculate (Hishinuma and Sekine 2004; Mousset-Simeon et al. 2004). Low

concentration, abnormal motility, or abnormal morphology can be responsible for the

infertility. However, infertility can still be observed in classically evaluated normal

samples. For these reasons, there is a real need for other complementary approaches

to evaluate semen characteristics and function. Recently, several sperm proteins have

51

been identified some of which are directly related to sperm function and motility

(Bookbinder et al. 1995; Sapiro et al. 2000; Wen et al. 2001; Quill et al. 2001).

The Basic Local Alignment Search Tool (BLAST) for bioinformatics (Altschul et al.

1990) provided the technology to perform a search to scan the canine genome for

genes associated with fertility previously described in mice or other species. At the

initiation of the present work, a Protein-Protein BLAST (blastp) was completed to

determine in the dog if the equivalent sequences of proteins associated with sperm

function and identified in mice. With confirmed similarities between the two species of

specific sequences for sperm proteins, the expectation was that these proteins would be

conserved and potentially identified in canine sperm. Four proteins were identified:

SPAG6, SP17, SP56, and CATSPER2 with SPAG6: 11 blast hits, SP17: 2 blast hits,

SP56: 6 blast hits, and CATSPER2: 1 blast hit (www.ncbi.nlm.nih.gov/BLAST/). For

these reasons we considered that there was a high probability to be able to identify the

proteins also in the dog and developed the second part of our work. It was expected

that SPAG6 and SP56 with 11 and 6 blast hits, respectively, would have a high

probability to be identified in canine sperm cells. SP17 and CATSPER2 with 2 and 1

blast hits, respectively, were more questionable but worth investigating as they have

never been studied in the dog.

The presence of sperm associated antigen 6 (SPAG6), a sperm protein, which is

localized on the flagellum in mice and human, has not been investigated in canine. The

presence of SPAG6 has been linked with sperm flagella motility and structural integrity

of the axoneme central apparatus of mature sperm in mice and humans (Neilson et al.

1999; Sapiro et al. 2000). Specifically, SPAG6 is expressed on the principal piece of

52

epididymal sperm, with weaker expression located on the midpiece and head regions

(Sapiro et al. 2000). Mature SPAG6 knock out mice are infertile and produce sperm with

low concentration, abnormal motility and structural defects (fragmentation of midpiece,

shortened flagella, unstable central apparatus, missing axonemes, or decapitations)

(Sapiro et al. 2002). These mice have hydrocephalus, and 50% die within 8 weeks of

life. The presence of SPAG6 as observed in human and mice appear to be highly

conserved among mammals (Neilson et al. 1999; Sapiro et al. 2000; Horowitz et al.

2005). However, this sperm protein has yet to be identified in canine sperm.

Sperm protein 17 (SP17) was initially described as a rabbit sperm autoantigen

protein (RSA) that was highly expressed in spermatozoa and had a primary function of

zona pellucida binding (Richardson et al. 1994). Additional studies stated that SP17,

though strongly expressed in the testes, was also present in somatic tissues. It also

functions in somatic and germ cell migration and/or cell adhesion, and is vital for

heparin binding (Wen et al. 2001; Frayne and Hall 2002).

SP17 appears to be a highly conserved mammalian protein and has been

observed in mouse sperm where it is expressed on the principal piece, weakly on the

midpiece, and over the acrosomal region of the head (Kong et al. 1995). On human

sperm, SP17 appears to be located on the principal piece, midpiece, and in scattered

patches throughout the head region (Lea et al. 2004). While another group reported that

SP17 was detected throughout the principal piece, but not intermediate, head,

acrosome vesicle in human ejaculated sperm, and that spermatogonia, Sertoli and

Leydig cells were immunonegative for SP17 (Grizzi et al. 2003). SP17 has yet to be

identified in canine sperm.

53

Sperm fertilization protein 56 (SP56), is a sperm-oocyte recognition protein that

has an affinity for ZP3 an oocyte glycoprotein associated with sperm-oocyte interaction

(Cheng et al. 1994). Initially, SP56 was reported in mice as localized on the peripheral

sperm membrane and on the outer surface of the sperm head (Cheng et al. 1994). In

later reports, SP56 was demonstrated as a part of the acrosomal matrix and not a

component of the plasma membrane (Kim et al. 2001; Buffone et al. 2008). However, in

mice with an abnormal spermatozoon head shape mutation (Azh) that produced 100%

abnormal sperm, SP56 was localized on the flagellum as well as the acrosome (Moreno

et al. 2006). SP56 is presently accepted as an intra-acrosomal component, part of the

acrosomal matrix (Kim et al. 2001) expressed primarily in the acrosomal vesicle (Cohen

and Wassarman 2001). In the rat and hamster, SP56 is expressed on the sperm head

(He et al. 2003). SP56 has not been detected in human sperm (Bookbinder et al. 1995),

nor has it been identified in canine sperm.

Cation channel, sperm associated 2 protein (CATSPER2), is a voltage-gated

protein identified in mice spermatozoa (Quill et al. 2001). Catsper2 knockout mice are

infertile. Disruption of the CATSPER2 gene through gene mutation does not significantly

affect sperm production or overall motility; however, It was demonstrated in these

knockout mice, an inability to achieve hyperactive sperm motility at the time of

activation, which is vital for in vivo fertilization (Quill et al. 2003). Mouse CATSPER2 is

located on the plasma membrane of the principal piece of the sperm tail (Quill et al.

2001; Ren et al. 2001; Xia et al. 2007) while human CATSPER2 protein is located on

the flagellum (Avidan et al. 2003). CATSPER2 has yet to be identified in canine sperm.

54

These proteins have critical roles in sperm motility and in the process of

fertilization. The objective of this study was to use immunocytochemistry to identify and

characterize the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2

proteins in canine spermatozoa. The hypothesis was that SPAG6, SP17, SP56, and

CATSPER2 are localized and expressed in similar patterns in canine sperm cells as in

other species and may have a role in sperm motility, capacitation, and/or acrosome

reaction.

Materials and Methods

Materials

SPAG6 mouse monoclonal, SP17 rabbit polyclonal, SP56 mouse monoclonal and

CATSPER2 rabbit polyclonal antibodies were purchased from Abnova Corporation

(Taipei, Taiwan), Delta Biolabs (Gilroy, CA), GeneTex, Inc. (Irvine, CA), and Aviva

Systems Biology (San Diego, CA), respectively. Zamboni fixative was purchased from

Newcomer Supply (Middleton, WI). SpermVision SAR and Spermac stain were

purchased from Minitube of America (Verona, WI). Methanol (HPLC) was purchased

from Fisher Scientific (Fair Lawn, NJ). DakoCytomation LSAB + System-HRP Kit with

swine- anti mouse/rabbit/goat biotinylated secondary antibody were purchased from

DakoCytomation, Inc. (Carpinteria, CA). BX51 Olympus microscope was purchased

from Olympus Industrial America, Inc. (Orangeburg, NY). Alexa Fluor 647 IgG goat anti-

mouse antibody and Alexa Fluor 594 IgG goat anti-rabbit antibody were purchased from

Invitrogen (Eugene, OR). Leica TCS SP5 Laser Scanning Confocal microscope was

purchased from Leica Microsystems, Inc. (Bannockburn, IL).

55

Animals and Sperm Preparation

Semen was collected from four sexually mature dogs ranging from 2-6 years of

age according to IACUC authorization protocols # E434 and #200903106. Semen was

collected a maximum of three times each week with significant periods of rest (more

than a week) occurring between collection cycles as collections were performed

continuously during the year but only as needed. The sperm rich fraction of the

ejaculate was collected by digital manipulation in a calibrated plastic vial (Foote 1964b;

Linde-Forsberg 1991).

To reduce variability between trials concerning semen, the semen was pooled to

have access to sample volumes large enough to allow the submission of the pools of

semen to the different antibodies tested. Using a pool of semen allowed for a reduction

of variability due to animals and allowed for a reduction of the number of animals

needed for every study. After pooling, the semen was evaluated for concentration,

sperm motility using SpermVision SAR, and morphology using Spermac staining (Silva

and Verstegen 1995; Verstegen et al. 2002). Five animals have been demonstrated to

be optimum for this purpose during CASA validations and semen extenders studies

(Verstegen et al. 2005). However, one of our animals had to be removed for health

reasons from the program just after the start of this research.

Mature BALB/c male mice were euthanized by CO2 gas and cervical dislocation,

according to IACUC authorization protocol #200903106. Directly after euthanasia, the

cauda epididymides were excised and washed twice in PBS at room temperature. Each

cauda epididymis was punctured with a 23-guage needle and sperm were released

from the epididymis into the petri dish.

56

Experiments

Brightfield immunocytochemistry: Twenty µl of semen was deposited on a

Superfrost®/Plus microscope slide, a smear was prepared and allowed to air dry

overnight. The air dried slides were fixed with Zamboni fixative for one hour at 4 °C,

permalized with methanol for 10 minutes at -20 °C, and processed following Dako

standardized immunocytochemistry protocol (www.dako.com) using a primary mouse

monoclonal antibody against SPAG6 or SP56, or primary rabbit polyclonal antibody

against SP17 or CATSPER2. Primary antibody concentrations were optimized prior to

experimentation and were diluted as follows: SPAG6 at 1/200, SP17 at 1/200, SP56 at

1/100, and CATSPER2 at 1/50 with PBS, incubated overnight at 4° C, rinsed in distilled

water, washed 3 times in PBS, then incubated 1 hour at room temperature with a swine

anti-mouse/rabbit/goat biotinylated secondary antibody. Chromogenic DAB + Substrate

buffer were used to visualize the binding sites. In the negative controls (canine and

mice) the primary antibody was omitted. Mouse sperm was used for the positive control.

Visualizations were ascertained using a BX51 Olympus microscope at 200-600X

magnifications. This study was repeated five times.

Confocal immunofluorescence: One ml of sperm was incubated with one of the

following primary antibodies after dilution as follows: SPAG6 at 1/200, SP17 at 1/200,

SP56 at 1/100, and CATSPER2 at 1/50 with PBS. The diluted semen was then

incubated at room temperature for 30 minutes in the dark, centrifuged at 2000 x g for 5

minutes in 2 ml of 2% fetal bovine serum in PBS, the supernatant was removed,

incubated at room temperature for 30 minutes in the dark with a secondary Alexa Fluor

647 IgG goat anti-mouse antibody or Alexa Fluor 594 IgG goat anti-rabbit antibody, and

centrifuged at 2000 x g for 5 minutes in 2 ml of 2% fetal bovine serum in PBS, the

57

supernatant was removed. The negative controls were as before with the addition of a

tissue negative control consisting of immortalized cat T cells. Visualizations were

ascertained using a Leica TCS SP5 Laser Scanning Confocal microscope at 200-600X

magnifications. This study was repeated three times.

Assessment of immunostaining: Points of interest for localization are:

acrosome, equatorial band, post acrosomal region, midpiece, principal piece, or flagella

(both midpiece and principal piece (Fig, 4-1). Scoring for spermatozoa staining intensity

was classified: negative (no staining), weak (+), moderate (++), or strong (+++)

expression. Spermatozoa were reported negative when the stain did not differ from the

negative control.

Statistical Analysis

For each trial, three slides were labeled and 100 sperm cells were evaluated per

slide; thus µ=5 with 3 replicates. One-way ANOVA was used to compare sperm protein

expression of the individual sperm protein with the three other sperm proteins. Tukey-

Kramer multiple comparison test was performed when the P value was significant

(<0.05). Data were expressed as mean ± SD. Differences were considered significant

when P<0.05.

Results

The mean values for concentration, overall motility, and forward progression of

sperm samples were 596.14 ± 373.54 x 106 sperm/ml, 98.49 ± 1.1%, 94.19 ± 0.80%

respectively. For sperm morphology, the mean percentage of normal spermatozoa was

93 ± 5.70% sperm. Secondary sperm abnormalities were tail defects (5.8%). The mean

percentage of acrosome-intact spermatozoa was 95 ± 2.94%.

58

SPAG6 labeling was identified on 97.6 ± 2.88% spermatozoa counted per slide.

Brightfield and confocal allowed both for the detection of a similar pattern of expression

of SPAG6 immunostaining on fresh canine sperm. SPAG6 was mainly and strongly

expressed in the head of the spermatozoa (Fig. 4-2C) with a high incidence of pan-

acrosome labeling, but variation of the expression was also noticed with punctate and

equatorial (Fig. 4-3) SPAG6 labeling. In addition, SPAG6 had a variable (negative to

moderate) expression at the level of the midpiece section of the flagellum (Fig. 4-2C).

Midpiece expression was found in less than 20% of canine spermatozoa. The canine

SPAG6 negative control (Fig. 4-2A), mouse positive (Fig. 4-2D), and negative (Fig. 4-

2B) controls were as expected. No sperm were labeled in the canine or mouse sperm

negative controls. In agreement with previous reports, the mouse positive control had

stronger expression of the principal piece, with weaker expression of midpiece and

acrosome.

SP17 labeling was identified on 93.6 ± 4.0% spermatozoa counted per slide.

Brightfield and confocal allowed both for the detection of a similar pattern of expression

of SP17 immunostaining on fresh canine sperm. SP17 was strongly expressed at the

acrosome and principal piece, with weaker expression at the midpiece (Figs. 4-4 and 4-

5). Acrosome and principal piece expression were observed in more than 85% of canine

spermatozoa. The SP17 negative and positive (Fig. 4-4) controls were as expected. No

sperm were labeled in the canine or mouse sperm negative controls. In agreement with

previous reports, the mouse positive control had strong flagella and acrosomal labeling.

SP56 labeling was identified on 97.3 ± 2.5% spermatozoa counted per slide. SP56

was strongly expressed and localized mostly (>95%) at the principal piece. Weak to

59

moderate acrosomal expression (Fig. 4-6) was observed in more than 25% of the

sperm. Confocal results were negative for canine SP56 expression. The SP56 negative

and positive (Fig. 4-6) controls were as expected. No sperm were labeled in the canine

or mouse sperm negative controls. The mouse positive control had labeling at the level

of the acrosome as previously reported.

CATSPER2 labeling was identified on 75.0 ± 5% spermatozoa counted per slide.

CATSPER2 was expressed and localized at the flagella region. There was also weak

expression localized at the acrosomal region (Fig.4-7) and this was seen in more than

70% of the sperm. Confocal results were not consistent for canine CATSPER2

expression. No sperm were labeled in the canine or mouse sperm negative controls.

The mouse positive control had labeling at the level of the flagellum as previously

reported.

CATSPER2 labeling was identified on 75.0 ± 5% spermatozoa counted per slide.

CATSPER2 was expressed and localized at the flagella region. There was also weak

expression localized at the acrosomal region (Fig.4-7) and this was seen in more than

70% of the sperm. Confocal results were not consistent for canine CATSPER2

expression. No sperm were labeled in the canine or mouse sperm negative controls.

The mouse positive control had labeling at the level of the flagellum as previously

reported.

The percentages for the number of spermatozoa that were labeled of the four

proteins were compared. There was not a statistical difference in the labeling of SPAG6,

SP17, and SP56. However, CATSPER2 was significantly labeled in fewer spermatozoa

than the other three studied proteins (p<0.05).

60

Discussion

Brightfield immunocytochemistry and confocal fluorescent imaging allowed us, to

identify and localize the four tested proteins in canine sperm. To the best of our

knowledge, this is the first report of these proteins in canine sperm. Their pattern of

distribution and intensity of staining were clear. In the present study, immunolabeling

was observed on almost all sperm cells from neat fresh ejaculates from adult mature

male dogs for SPAG6, SP17 and SP56. The percentage of sperm cells labeled by these

antibodies (97.6 ± 2.88, 93.6 ± 4, 97.3 ± 2.5 for SPAG6, SP17 and SP56, respectively)

was not different from the percentage of acrosome normal sperm cells (95 ± 2.4).

CATSPER2 labeling (75 ±5.0) was the only one to be significantly lower than the other

labeled proteins and lower than the percentage of acrosome-intact spermatozoa

(P<0.05).

In the present study, it was shown that SPAG6 had strong expression localized

mainly at the acrosomal region of the sperm head. In addition, SPAG6 had negative to

moderate expression localized at the midpiece of the sperm tail. The strong expression

of SPAG6 localized to the canine acrosomal region of the sperm head has not been

shown in mice and humans (Neilson et al. 1999; Sapiro et al. 2000). In a mouse model,

SPAG6 was essentially localized to the principal piece of the flagellum and a weaker

signal in the sperm tail midpiece and head region (Sapiro et al. 2000).

Flagella localization of SPAG6 in the canine was less consistent than that seen in

mice and humans (Neilson et al. 1999; Sapiro et al. 2002). However, it may still indicate

a similar role in flagella movement and motility as found in other species. As illustrated

in mice and humans, it may be proposed that SPAG6 in the canine has a function in

sperm motility. However, the essential involvement of SPAG6 in fertility is still

61

controversial as SPAG6-deficent mice have several disruptions of the spermatozoa

particularly at the midpiece (Sapiro et al. 2002). However, in humans with a

heterozygous mutation that affects SPAG16L, which interacts with SPAG6, male

infertility is not observed (Zhang et al. 2007). The exact role of SPAG6 in the canine

needs to be investigated and warrants further studies.

In the canine, we have shown that SP17 is localized with strong and similar

intensity at the acrosomal region and the principal piece of the flagellum. These findings

are in agreement with those previously observed in the mouse and human (Kong et al.

1995; Grizzi et al. 2003). SP17 has yet to be clearly demonstrated to be involved in the

fertilization process. In fact, due to its localization within the tail region, it has been

postulated by (Frayne and Hall 2002) that the role of SP17 in oocyte binding through the

zona pellucida is unlikely to be its principal function. However, direct involvement in

sperm cell motility has not been demonstrated. SP17 seems to be nonspecific to sperm

cells as it is also expressed in tumors and normal somatic tissues (Wen et al. 2001;

Straughn, Jr. et al. 2004). In agreement with what has been shown in these species,

canine SP17 function can not yet be proposed except that it may be involved in both

fertilization and motility as seen with the other sperm proteins with flagella protein

expression.

In the present study, canine SP56 was consistently expressed predominantly at

the principal piece of the flagellum with some moderate acrosomal expression. The

observed primary localization of SP56 in the canine was different than that reported for

the mouse. In the normal mouse, SP56 is reported as essentially localized at the

acrosomal matrix (Kim et al. 2001; Buffone et al. 2008). However, it has been

62

demonstrated (Moreno et al. 2006) that Azh mutated mice have SP56 localized on the

flagellum as well as the acrosome. The reason for this discrepancy between normal and

mutated animals is currently unknown. When SP56 was first identified it was reported to

be localized on the peripheral sperm membrane (Cheng et al. 1994). This type of

localization could account for the strong expression found mostly at the principal piece

of the sperm tail in the current study.

CATSPER2 is part of a cation-channel family of four similar, non identical

(CATSPER1, CATSPER2, CATSPER3, CATSPER4) proteins. Catsper2 knockout mice

are void of Catsper1 protein expression and the inverse. Hence, both proteins are

required in order for the other to function (Carlson et al. 2005). The family of four

proteins is essential for male fertility and hyperactive sperm motility (Qi et al. 2007). In

the present study, canine CATSPER2 flagellar localization was shown to be in

agreement with that shown in other species (Ren et al. 2001; Xia et al. 2007). However,

there was also some weak to moderate expression of CATSPER2 at the level of the

acrosomal region. The localization at the level of the flagellum may indicate a role in

spermatozoal movement and calcium exchanges.

The flagella localization of the four proteins (SPAG6, SP17, SP56, CATSPER2),

comparable to that seen in mice and humans, may suggest a similar role in motility as

found in other species. For fertilization to occur, the sperm must be motile and have the

ability to capacitate and undergo an acrosome reaction (Kawakami et al. 1993; Brewis

et al. 2001; Witte et al. 2009). The changes to the acrosome will occur as part of sperm

activation. Capacitation is a stepwise process. During the progression through the

female reproductive tract, sperm change their pattern of motility from linear, forward

63

progression to a vigorous, non-progressive, curvilinear, hyperactivated movement. This

change in motility pattern is a result of capacitation (Mahi and Yanagimachi 1978). It is

understood that hyperactivated sperm assist with transport through oviductal secretions

and binding of sperm to the zona pellucida. Then the hyperactivated sperm will

penetrate the zona pellucida (Mahi and Yanagimachi 1978; Kawakami et al. 1993;

Suarez 2008). Only a capacitated sperm cell can fertilize an oocyte, and the inability of

spermatozoa to fertilize could be the result of failure in all motility and acrosome

associated changes. The localization and expression patterns of SPAG6, SP17, SP56,

and CATSPER2, to the acrosomal region of canine spermatozoa that were observed in

the present study suggest that these proteins have functions associated with

capacitation, acrosome reaction, and fertilization in the canine.

Lastly, as reported (Ho and Suarez 2001b), it was demonstrated that an increase

in cAMP and calcium stimulate sperm motility and hyperactivation. This is illustrated in

CatSper knockout mice that have defects in the cAMP-induced increase in calcium, and

where defective hyperactivated motility is observed (Carlson et al. 2003). The

confirmation of CATSPER2 protein presence in canine spermatozoa will warrant

investigation for its function. Similarly, it would be interesting to evaluate changes in the

expression of these proteins with acrosome reaction, capacitation and hyperactivation in

the dog like in other species.

In conclusion, we have demonstrated in this study the presence of sperm proteins

SPAG6, SP17, SP56, and CATSPER2 in canine spermatozoa using

immunocytochemistry and confocal immunofluorescence. The objective to identify and

characterize the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 in

64

canine spermatozoa was achieved. The sperm proteins SPAG6, SP17, SP56, and

CATSPER2 were localized and expressed similar pattern, with minor differences, to that

found in other species.

65

Figure 4-1. Diagram of a sperm cell. The head includes the acrosome, equatorial band, and post acrosomal regions. The principal piece is the tail according to this diagram. The picture is taken from Dr. Danton O’Day website (www.erin.utoronto.edu).

66

Figure 4-2. SPAG6 immunocytochemistry. A) canine negative control, B) SPAG6 mouse negative control, C) canine SPAG6, D) mouse positive control. Magnification at 200-600x.

A B

C D

67

Figure 4-3. SPAG6 confocal immunofluorescence. A) negative tissue control

(immortalized cat T cells), B) canine SPAG6 at 200x, C) canine SPAG6 at 400x, D) canine SPAG6 at 600x. Top) fluorescence, Center) phase contrast, Bottom: fluorescence/phase contrast.

A B

Top

B A

Center

Bottom

C

D

68

Figure 4-4. Canine SP17 with fresh negative and positive controls. A) mouse sperm positive control, B) canine sperm negative control. Black arrow: normal SP17 expression. White arrow: acrosome reacted sperm. Magnification at 400x.

Figure 4-5. SP17 localization on fresh canine spermatozoa. Confocal immunofluorescence red labeling at acrosome, midpiece, and principal piece of the canine spermatozoa. Magnification at 400x.

69

Figure 4-6. SP56 localization on fresh canine spermatozoa. Canine SP56 expressed mainly at the principal piece, with some expression at the acrosome. The insert is a mouse positive control. Magnification at 600x.

Figure 4-7. CATSPER2 localization on canine spermatozoa. Canine CATSPER2 expressed at the flagellum, with some expression at the acrosome. Magnification at 600x.

70

CHAPTER 5 CHARACTERIZATION OF SPERM PROTEINS SPAG6, SP17, SP56, AND CATSPER2

IN CANINE BY WESTERN BLOT ANALYSIS

Introduction

While immunocytochemistry can identify the presence of immunoreactivity, it does

not demonstrate the presence of the protein itself since similar labeling can be observed

in case of nonspecific reaction. In order to validate and confirm the identity of the

observed labeling, the uses of protein electrophoresis and protein characterization are

absolutely required. For these reasons, identification and characterization of protein

(including molecular weight) using western blot analysis is warranted.

Western blot is a powerful protein characterization tool in which small amounts of

protein can be detected, protein molecular weight can be determined, and antibody-

antigen interaction can be visualized as seen with specific band development. Western

blotting, also known as immunoblotting of proteins, was first described as a method for

detection and identification of proteins transferred from a gel to a membrane

(nitrocellulose or PDVF, or other membranes) and then detected with a substrate

(Towbin et al. 1979). Prior to transfer and detection the protein sample of interest along

with molecular weight markers are separated using electrophoresis either native without

denaturation and separation based on protein mass: charge ratio or SDS-PAGE (with

denaturation and separation of subunits and neutralization of charges) based on

molecular mass only.

After electrophoretic separation and transfer of a membrane, the actual process of

western blotting involves several blocking steps to neutralize the nonspecific binding

sites, washing between steps with or without detergents to reduce nonspecific binding,

incubation with primary and then secondary antibodies of interest, and finally the

71

addition of substrate reagent for chromogenic visualization (Towbin et al. 1979; Tash et

al. 1988; Sabeur et al. 2002). The disadvantages of using western blot are extensive

optimization, presence or absence of bands is subject to interpretation, and nonspecific

interactions may result in variability of results.

In the present study, western blot analyses were performed for each of the four

proteins of interest on canine spermatozoa. According to the literature, SPAG6 is listed

as a 55.5 kDa molecular weight protein (Neilson et al. 1999; Zhang et al. 2005). SP17 is

less homogenous and the following molecular weights have been presented: 14-15

kDa, 17-19 kDa, 21-24 kDa, 26 kDa, 29 kDa (Richardson et al. 1994; Wen et al. 1999;

Lea et al. 2002; Grizzi et al. 2003). SP56 has the following recorded molecular weights:

31 kDa, 40 kDa, 42 kDa, 43 kDa, and 67 kDa (Cheng et al. 1994; He et al. 2003).

CATSPER2 according to the manufacturing company has the band sizes of 48kDa and

61 kDa. However, Carson’s group has for CATSPER2 a band size of ~72 kDa (Carlson

et al. 2005). The objective of this study was to confirm the presence of sperm proteins

SPAG6, SP17, SP56, and CATSPER2 by western blot analysis in the canine. The

hypothesis tested was that western blot analysis will confirm in the dog the presence of

sperm proteins with molecular weights corresponding to the previously described

SPAG6, SP17, SP56, and CATSPER2 proteins in other species spermatozoa.

Materials and Methods

Materials

SpermVision SAR was purchased from Minitube of America (Verona, WI). BX51

Olympus microscope was purchased from Olympus Industrial America, Inc.

(Orangeburg, NY). Tris, EDTA, and Triton X-100 were purchased from Fisher Scientific

(Fair Lawn, NJ). PMSF was purchased from MP Biomedical, LLC (Solon, Ohio). N-

72

ethylmaleimide was purchased from Acros Organics (Morris Plaines, NJ). Pierce 660

nm Protein Assay and the Fast Blot Developer were purchased from Thermo Scientific

(Rockford, IL). Spectra Max Plus 384 plate reader was purchased from Molecular

Devices (Sunnydale, CA). NuPage 10% Bis-Tris Precast Gels, NuPage MOPS SDS

Running Buffer (20X), Sample Reducing Agent (10X), NuPage LDS Sample Buffer (4X),

NuPage Antioxidant, SeeBlue Plus 2 Prestained Standard (1X), iBlot® Transfer Stack,

Mini (Nitrocellulose), iBlot® Gel Transfer Device were purchased from (Invitrogen,

Carlsbad, CA). PowerPac Basic was purchased by Bio-Rad (Hercules, CA). Carnation

instant nonfat dried milk was purchased from a local store. Alkaline phosphatase -goat

anti-mouse IgG Fc specific-, alkaline phosphatase goat anti-rabbit IgG whole molecule,

NBT/BCIP substrate tablet, and Tween-20 were purchased from Sigma-Aldrich (St.

Louis, MO). PBS was purchased from Fisher Scientific (Fair Lawn, NJ). BSA was

purchased from Roche (Mannheim, Germany). SPAG6 mouse monoclonal, SP17 rabbit

polyclonal, SP56 mouse monoclonal and CATSPER2 rabbit polyclonal antibodies were

purchased from Abnova Corporation (Taipei, Taiwan), Delta Biolabs (Gilroy, CA),

GeneTex, Inc. (Irvine, CA), and Aviva Systems Biology (San Diego, CA), respectively.

SoftmaxPro version 5.3 (Molecular Devices, Sunnydale, CA) software was used to

determine total sperm protein concentration of the semen samples. A serial dilution BSA

standard was used as a reference. This study was repeated five times.

Animals and Sperm Preparation

Semen was collected from four sexually mature dogs ranging from 2-6 years of

age, according to IACUC authorization protocols # E434 and #200903106. The sperm

rich fraction of the ejaculate was collected by digital manipulation in a 15 ml calibrated

plastic vial (Foote 1964b; Linde-Forsberg 1991). To reduce variability between trials

73

concerning semen, the semen was pooled to have access to sample volumes large

enough to allow the submission of the pools of semen to the different antibodies tested.

Using a pool of semen allowed for a reduction of variability due to animals and allowed

for a reduction of the number of animals needed for every study. Five animals have

been demonstrated to be optimum for this purpose during CASA validations and semen

extenders studies (Verstegen et al. 2005). However, one of our animals had to be

removed for health reasons from the program just after the start of this research.

After pooling, the semen was evaluated for concentration, sperm motility using

SpermVision SAR, and morphology using Spermac staining (Silva and Verstegen 1995;

Verstegen et al. 2002). The fresh semen was then centrifuged twice at 700 x g for 10

minutes at room temperature in PBS and the supernatant was removed after each

wash. The sperm extract was serial diluted with 1% BSA from undiluted to 1:128

dilutions and analyzed for total protein concentration.

Mature BALB/c male mice were euthanized by CO2 gas and cervical dislocation,

according to IACUC authorization protocol #200903106. Directly after euthanasia, the

cauda epididymides were excised and washed twice in PBS at room temperature. Each

cauda epididymis was punctured with a 23-guage needle and sperm was released from

the epididymis into the petri dish. The sperm cells were then centrifuged twice at 700 x

g for 10 minutes at room temperature in PBS and the supernatant was removed after

each wash. The sperm extract was serial diluted with 1% BSA from undiluted to 1:128

dilutions and analyzed for total protein concentration

Experiments

During preliminary trials (data not shown), primary and secondary antibody

dilutions were optimized. Primary antibodies diluted in 1% BSA were used as follows:

74

SPAG6 at 1/100 and 1/200, SP17 at 1/50 and 1/100, SP56 at 1/100 and 1/200, and

CATSPER2 at ½ and 1/50. Secondary antibodies diluted in 1% BSA were used as

follows: alkaline phosphatase goat anti-mouse and goat anti-rabbit at 1:2000. Canine

and mouse sperm samples were analyzed on separate membranes to allow for

concurrent evaluation of sperm protein expressions.

Total protein concentration was determined with Pierce 660 nm protein assay.

Sperm samples were diluted with a NuPAGE sample reducing agent that contains

dithiothreitol, which denatures the protein, NuPAGE LDS sample buffer which

neutralizes the pH of the environment to minimize protein modifications, and PBS.

Samples were then heated at 70 ºC for 10 minutes to denature the proteins prior to

electrophoresis. The NuPage 10% Bis-Tris precast 2-lane gel cassette was prepared

along with the electrophoresis tank (MOPS SDS running buffer with antioxidant). In the

first lane, 200 µl of canine sperm sample (109 µg/per lane of total protein) and in the

second lane 7 µl of molecular weight standards (191, 97, 64, 51, 39, 28, 19, and 14 kDa

molecular weights used as recommended) were added to the gel cassette within the

electrophoresis tank. The voltage on the tank was set at 200V, constant for 40 minutes

at room temperature. After electrophoresis, the gel was removed from the cassette and

placed in an IBlot® Gel Transfer Device (Invitrogen, Carlsbad, CA) for 8 minutes. After

the transfer of proteins from the gel to the membrane, the membrane was blocked in 5%

non-fat dried milk in PBS for 1 hour at room temperature on a rocker. After blocking, the

membrane was briefly (5-10 seconds) rinsed twice in PBS-Tween (0.1%). The

membrane was then placed in a Fast Blot Developer (a 10-lane template that allows for

the detection of different antibodies or dilutions at the same time (Thermo Scientific,

75

Rockford, IL) and 1000 µl of each primary antibody were loaded into the 10-lane

template in the following order: lane 1-CatSper2 undiluted, lane 2-CatSper2 1:2, lane 3-

BSA only, lane-4 goat anti-rabbit secondary antibody only, lane 5-SP17 1:50, lane 6-

SP17 1:100, lane 7-goat anti-mouse secondary antibody only, lane 8-SPAG6 1:100,

lane 9-SPAG6 1:200, lane 10-BSA only. The primary antibodies were allowed to

incubate overnight at 4 ºC in a cold room on a rocker. The following day, the liquid was

discarded, the membrane rinsed in PBS-Tween (0.1%), aspirated again, and washed

three times for 5 minutes with PBS-Tween (0.1%) at room temperature. Secondary

antibodies diluted to 1:2000 in 1% BSA were added to the appropriate lanes and

incubated for 1 hour at room temperature on a rocker. Following completion of

secondary antibody incubation, the liquid was discarded, the membrane rinsed in PBS-

Tween (0.1%), aspirated again, and washed twice for 5 minutes with PBS-Tween

(0.1%). The membrane was then removed from the template and placed in a tray on a

rocker for a final rinse with PBS-Tween (0.1%). The wash buffer was removed and the

colormetric NBT-BCIP substrate tablet was then added to the tray for 10 minutes. If

color development did not occur after 10 minutes the substrate was discarded and new

NBT-BCIP tablet was added for an additional 10-20 minutes. To stop the reaction the

membrane was washed in distilled water and then allowed to air dry.

Results

The mean value for concentration, overall motility, and forward progression of

sperm samples were 596.14 ± 373.54 x 106 sperm/ml, 98.49 ± 1.1%, 94.19 ± 0.80%

respectively. For sperm morphology, the mean percentage of normal spermatozoa was

93 ± 5.70% sperm. The protein concentration of the sperm samples were canine (9.3

mg/ml) and mouse sperm (0.365 mg/ml). The canine negative controls and molecular

76

weight standards (Figs. 5-1 and 5-2; Table 5-1) were as expected. The molecular

weight standards lane had all associated bands accounted for and the calculated

standard curves were linear as expected. Based upon five trials, the coefficient of

variation for the measurement of molecular weight standards was estimated to be 10-

20%. The canine negative control lanes which contained one of the following: BSA only,

goat anti-mouse secondary antibody only, or goat anti-rabbit secondary antibody only,

had no visible bands present.

Under reduced conditions, (SDS-PAGE) western blot analysis of canine sperm

demonstrated SPAG6 was detected in a band at 61.57 kDa (Figs. 5-1 and 5-3; Table 5-

2). SP17 antibodies allowed us to localize different bands at 15.45 and 28.75 kDa (Figs.

5-1 and 5-4; Table 5-3). For SP56 there were no bands immunodetected in the canine

protein extracted sperm (data not shown). CATSPER2 antibodies allowed us to localize

different bands corresponding to molecular weights of 48.48, 49.18 and 63 kDa (Figs.

5.1 and 5-5; Table 5-4).

Mouse sperm was used as a positive control and results were as followed: SPAG6

immunoreactivity was detected in a band at 49.75 kDa. SP17 immunoreactivity was

detected in associated bands at 12.83 and 21.99 kDa. The SP56 immunoreactivity was

detected at 52-53 kDa. CATSPER2 immunoreactivity was detected in a band at 48.16

kDa.

Discussion

The results of this study confirmed the presence of SPAG6, SP17, and

CATSPER2 proteins and allowed us to define their molecular weight in canine

spermatozoa extracted samples. For sperm cell extraction, some authors (Sabeur et al.

2002; De Los et al. 2009), added protease inhibitors to their samples. Other authors do

77

not add protease inhibitors (Baxendale and Fraser 2003; de Souza et al. 2007), simply

washing the sperm sample after heating and SDS denaturation.

In the present study, sample preparation was initially done (data not shown) using

the addition of protease inhibitors (50 mM Tris, 20mM EDTA, 1mM PMSF, 5mM N-

ethylmaleimide) and solubilization using with 0.5-1%Triton X-100. Unfortunately, after

several attempts, western blot protein identification was unsuccessful. It was postulated

that the addition of the protease inhibitor cocktail was associated with protein

degradation preventing their detection by immunoblotting

Canine SPAG6 immunoreactivity was detected with a molecular weight of 61.57

kDa while in the control mouse samples; SPAG6 was detected in a band at 49.75 kDa.

Based on the published results, the expected molecular weight for mouse SPAG6 is

55.5 kDa (Neilson et al. 1999; Zhang et al. 2005). If we accept 10-20% variation in

molecular weight evaluation as estimated by western blot, the molecular weight

calculated here can be considered as correct. Canine SPAG6 is expressed on canine

spermatozoa and this was verified with the presence of a 61.57 kDa band on the

western blot. The difference between the 61.57 kDa molecular weight observed here for

the canine SPAG6 and the predicted 55.5 kDa band observed in mouse may again just

be related to the variability in molecular weight evaluation between western blot or may

be due to a different isoform of SPAG6 in the dog. To confirm the protein identity, an

amino acid composition will have to be performed. However, the existence of different

isoforms between species or within species has been postulated (personal

communication with Dr. Jerome Strauss) and needs to be evaluated in dogs.

78

Canine SP17 immunoreactivity was detected for proteins of 15 and 28-29 kDa.

These molecular weights are in agreement with the previously described values. The

presence of multiple bands for SP17 protein is a generalized observation has been

reported in several publications (Kong et al. 1995; Wen et al. 1999; Grizzi et al. 2003)

with variation in molecular weights as follows:14-15, 17-19, 21-24, 26, and 29 kDa.

However, in species were SP17 was observed, even if large variations in molecular

weights were noticed, two isoforms were always detected and were also detected in the

present study.In mouse SP17 was detected in associated bands at 12.83 and 21.99

kDa. In addition, some studies have also observed higher molecular weight bands

ranging from 54- 193 kDa. In rabbits, it has been reported that these higher molecular

weight bands may be are aggregates of rabbit sperm auto-antigen protein now known

as SP17 (or non-RSA) cross-reactive antigens (Richardson et al. 1994). Moreover,

recombinant mouse SP17 has higher bands present in western blots. Like for many

proteins, proteolytic product and proteolytic cleavage of pre-pro-proteins were the

explanation for some of the observations (Kong et al. 1995). It has also been mentioned

(Adoyo et al. 1997; Grizzi et al. 2003; Grizzi et al. 2004) that a detected 54 kDa band is

consistent with a multimeric form of SP17. In accordance with these findings canine

SP17 was also detected in a visible band located at the 53.27 kDa.

Sperm protein 56 was not detected in canine spermatozoa, while mouse SP56

was detected (data not shown) in a band at 52-53 kDa (Cheng et al. 1994; He et al.

2003). Canine CATSPER2 was detected at 48-49 and 63 kDa. Mouse CATSPER2 was

detected in a band at 48.16 kDa. The bands were in agreement with the predicted

molecular weight at 48 and 63 kDa as reported by the manufacturing company. Canine

79

and mouse CATSPER2 immunoreactivity of higher molecular weights were also

present, which could be due to the presence of pre or pre-pro proteins and their

cleavage products.

The present results confirm the presence of SPAG6, SP17 and CATSPER2

proteins in canine spermatozoa. The presence of SP56 was not confirmed as

immunodetection was not observed in western blot. As the sperm protein SP56 is

important for mammalian fertilization by functioning in the sperm ability to bind the zona

pellucida (Cheng et al. 1994; Cohen and Wassarman 2001) it was of interest to see if

this sperm protein would also be expressed and function in canine sperm. In chapter 3,

according to ICC results, SP56 was reported strongly expressed and localized at the

principal piece, with weak to moderate acrosomal expression. The SP56 expression

that was observed in ICC could be the result of denaturation of the sperm protein or

inaccessibility of isoforms. The fact that SP56 is present in mouse sperm and not canine

sperm indicates that further investigation is warranted to determine if the sperm protein

SP56 is present or not in canine sperm and to identify the immunoreactivity observed in

the present study.

The objective of this study was to confirm the presence of sperm proteins SPAG6,

SP17, SP56, and CATSPER2 by western blot analysis on canine sperm. The objective

to identify and characterize the presence of sperm proteins SPAG6, SP17, and

CATSPER2 in canine spermatozoa was achieved. Canine SPAG6 is expressed on

canine spermatozoa and this was verified with the presence of a 61.57 kDa band on the

western blot. Canine SP17 is expressed on canine spermatozoa and this was verified

with the presence of several SP17 associated proteins 15, 28-29, and 53 kDa. Canine

80

CATSPER2 is expressed on canine spermatozoa and was verified with the presence of

48-49 and 63 kDa bands which are in agreement with what has been reported by the

manufacturing company. In conclusion, the present study demonstrated the presence of

SPAG6, SP17, and CATSPER2 on canine spermatozoa using western blot analysis and

immunostaining.

81

Figure 5-1. Canine sperm protein western blot. Altered and original 1) CatSper2

undiluted, 2) CatSper2 1:2, 3) BSA only, 4) goat anti-rabbit secondary antibody only, 5) SP17 1:50, 6) SP17 1:100, 7) goat anti-mouse secondary antibody only, 8) SPAG6 1:100, 9) SPAG6 1:200, 10) molecular weight marker.

1

2

3 4 5 6

7

8

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Rf distance down track

0

10000

5000

15000

20000

Profile height

0 806403

Distance travelled (pixels)

1

20

5

100

500

Log molecular weight

A B Figure 5-2. Molecular weight standard densitogram. A) densitogram, B) standard curve

Table 5-1. Molecular weight standard densitogram data. Track 10 Number Mol. weight Height Raw vol. 1 191.00 7646.113 5290350.50 2 97.00 5139.493 12072106.00 3 64.00 9080.901 7600069.50 4 51.00 9199.746 13004836.00 5 39.00 9430.046 14444542.00 6 28.00 9136.664 17888004.00 7 19.00 5913.502 12070192.00 8 14.00 10629.005 11489455.00

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

A

B

82

1

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Rf distance down track

0

10000

5000

15000

20000

Profile height

Figure 5-3. SPAG6 at 1:200 dilution densitogram.

Table 5-2. SPAG6 at 1:200 dilution densitogram data. Track 9: SPAG6 Number Mol. weight Height Raw vol. 1(m) 61.57 4252.524 1351688.25

1 23

4

5

6

7

89

1011

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Rf distance down track

0

10000

5000

15000

20000

Profile height

Figure 5-4. SP17 at 1:100 dilution densitogram.

Table 5-3. SP17 at 1:100 dilution densitogram data. Track 6: SP17 Number Mol. weight Height Raw vol. 1 193.51 1166.964 528417.38 2(m) 163.35 1505.759 433985.44 3 59.24 2822.645 758897.25 4(m) 53.27 1466.241 380468.91 5 42.70 6552.807 2549770.50 6 39.28 16622.148 6909526.50 7 36.34 6148.989 3302094.00 8 35.08 8239.422 5784035.00 9 33.56 8251.163 6719155.00 10 28.75 3008.873 2853290.50 11 15.45 1847.011 1364073.88

83

1

2

3

4

56

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Rf distance down track

0

10000

5000

15000

20000

Profile height

Figure 5-5. CATSPER2 at 1:2 dilution densitogram.

Table 5-4. CATSPER2 at 1:2 dilution densitogram data. Track 2-: CATSPER 2 Number Mol. Weight Height Raw vol. 1(m) 289.81 7721.347 5181371.50 2 183.68 20769.453 15434651.00 3(m) 147.18 5741.048 3569350.00 4(m) 122.64 3517.732 1653897.50 5(m) 63.69 1856.448 573341.75 6 49.18 3065.417 5707022.00

84

CHAPTER 6 EFFECTS OF CANINE SEMEN PRESERVATION ON SPERM PROTEIN

EXPRESSION

Introduction

Protein detection by immunocytochemistry or western blotting does not

automatically imply that these proteins have any biological activity or function. However,

if functions in motility and/or fertilization are expected, any induced changes in their

motility and/or morphology should be associated with changes in their expression. If

involved in sperm motility, changes in their flagellar expression may be expected and

correlated with changes in motility. Similarly, if involved in fertilization, artificial induction

of acrosome reaction and hyperactivation should possibly be associated with changes

in their acrosome related expression.

Canine semen extenders protect spermatozoa by conserving motility and fertility

overtime by stabilizing the sperm membrane, providing energy substrates and

preventing deleterious effects of changes in pH and osmolarity (Foote and Leonard

1964; Province et al. 1984; Bouchard et al. 1990; Brown 1992; Goodman and Cain

1993; Rota et al. 1995). Cold shock is prevented in chilled semen by slowly cooling

(Bouchard et al. 1990; Ponglowhapan et al. 2004) in the presence of a defined buffer

and preserved at 4° C. There are commercially available and laboratory prepared

canine chilled semen extenders. Until recently, commercially and laboratory prepared

extenders allowed for the preservation of chilled semen at 4° C for only 4-5 days.

However, some of the more recently described and produced extenders allow for the

preservation of canine semen for more than 10-15 days. Using a laboratory prepared

extender (Verstegen et al. 2005), chilled semen was conserved up to 3 weeks.

85

Among the laboratory-prepared buffers, egg yolk Tris–fructose was tested as an

extender (Rota et al. 1995) and shown to possess the best properties to allow long-term

chilled semen preservation. Similarly, results from (Iguer-Ouada and Verstegen 2001b)

have confirmed the interest of the egg yolk Tris–based extenders (using glucose instead

of fructose) in terms of motility conservation assessed objectively using computer

assisted analyzer (CASA Hamilton-Thorn IVOS 10) (Iguer-Ouada and Verstegen 2001a;

Iguer-Ouada and Verstegen 2001b). The glucose-based extender was also evaluated

by the ability to limit the acrosome reaction, as determined using chlortetracycline

staining. In this extender, good semen quality, characterized by more than >70% motile

spermatozoa and less than <10% acrosome-reacted cells, were observed after

conservation for 10–13 days at 4 °C (Iguer-Ouada and Verstegen 2001b). At the end of

the storage period (day 17), no semen motility was observed. Overall, quality of stored

semen using glucose instead of fructose was improved, suggesting a major role of the

energy substrate glucose in maintaining canine semen motility. Studies in other species

horse (Love et al. 2002) or boar (Boe-Hansen et al. 2005) questioned the possibility to

preserve chilled semen for such a long period without reducing fertility. However, the

preserved fertility after long-term preservation in canine was demonstrated with

significant pregnancies obtained after insemination with chilled semen preserved for

more than 10 days (Verstegen et al. 2005).

In fresh canine sperm, SPAG6 has been demonstrated previously to have strong

expression localized mainly at the acrosomal region of the sperm head. In addition,

SPAG6 had negative to moderate expression localized at the midpiece of the sperm tail.

SP17 is localized with strong and similar intensity at the acrosomal region and the

86

principal piece of the flagellum. CATSPER2 had flagella localization and there was also

some weak to moderate expression of CATSPER2 at the level of the acrosomal region.

The localization of all three of these proteins either at the level of the head or at the

level of the flagella may be indicative of a role of these protein in either or both of the

fertilization and motility processes. Since the optimal chilling extenders were developed

for the preservation of semen (i.e. by stabilizing the membrane), there should be little to

no change in the plasma membrane and in acrosome integrity during chilled semen

preservation in these extenders.

Only at the appropriate time can capacitated sperm fertilize oocytes. It is then

logical to postulate that a sperm cell’s unable to undergo an acrosome reaction at the

optimal time or opposite, developing premature acrosome reaction can result in

fertilization failure. Acrosome reaction is associated with head permeabilization and

significant motility changes. In the dog acrosome reaction is associated with significant

changes in the motility pattern of the sperm cells and/or hyperactivation both needed for

oocyte penetration. If these events do not occur (cell damage) or do occur to early

(inappropriate early acrosome reaction and hyperactivation) fertilization will be impaired.

Disruption of plasma and acrosomal membranes induced by the preservation process

inhibit the ability of sperm cells to fertilize. These modifications can be artificially

induced and assessed by the in vitro acrosome reaction protocols and staining of the

sperm cells. Laboratory prepared capacitation media SP-TALP has been demonstrated

to induce an acrosome reaction. In a comparison study (Sirivaidyapong et al. 2000) of

sperm capacitation media it was concluded that SP-TALP induces a significantly higher

percentage of acrosome reacted sperm cells than other capacitation media tested.

87

Different commercially available chilling semen extenders were compared for their

semen preservation capabilities and their effects on the different membrane proteins

evaluated to analyze the changes in selected sperm protein expression during

preservation over a period of time and establish a possible relationship between this

expression and different semen parameters including motility, capacitation, and

acrosome reaction. The objectives of the experiments were: a) evaluate the changes in

selected sperm protein expression during preservation over a period of time, b)

establish a possible relationship between sperm protein expression and different semen

parameters including motility, capacitation, and acrosome reaction. The hypotheses to

be tested were: a) semen preservation over time affect protein expression, b)

differences may be observed between the different evaluated extenders, c) acrosome

induced functional changes may be detected in relation to the preservation processes,

d) acrosome reaction induction is associated with changes in the expression of the

different semen proteins evaluated , e) overtime changes in motility may be related to

changes associated to acrosome reaction and modification in the expression of the

different proteins.

Materials and Methods

Materials

SpermVision SAR, CaniPRO Chill 5, and CaniPRO Chill 10, and Spermac stain

were purchased from Minitube of America (Verona, WI). Camelot was purchased from

Camelot Farms (College Station, TX). Synbiotics was purchased from Synbiotics

Corporation (San Diego, CA). SP-TALP sperm capacitation media was laboratory

prepared according to published instructions (Sirivaidyapong et al. 2000). Briefly, the

ingredients were: 100 mMol sodium chloride, 3.1 mMol potassium chloride, 2.0 mMol

88

calcium chloride, 0.4 mMol magnesium chloride, 25 mMol sodium bicarbonate, 1 mMol

sodium pyruvate, 3.38 ml sodium lactate 60% syrup, 0.3 mMol sodium phosphate, 10

gm/L bovine serum albumin, 2.28 ml Hepes, and 0.25 mMol gentamycin S. At the start

of the experiment, all ingredients were dissolved in ultrapure water. Coomassie Blue

stain was purchased from Fisher Scientific (Fair Lawn, NJ) and laboratory prepared

according to published instructions (Larson and Miller 1999; Brum et al. 2006). SPAG6

mouse monoclonal, SP17 rabbit polyclonal, CATSPER2 rabbit polyclonal were

purchased from Abnova Corporation (Taipei, Taiwan), Delta Biolabs (Gilroy, CA), Aviva

Systems Biology (San Diego, CA). Zamboni fixative was purchased from Newcomer

Supply (Middleton, WI). DakoCytomation LSAB + System-HRP Kit with swine- anti

mouse/rabbit/goat biotinylated secondary antibody were purchased from

DakoCytomation, Inc. (Carpinteria, CA). BX51 Olympus microscope was purchased

from Olympus Industrial America, Inc. (Orangeburg, NY).

Animals

Semen was collected from four healthy dogs ranging from 2-6 years of age,

according to IACUC authorization protocols # E434 and #200903106. The sperm rich

fraction of the ejaculate was collected by digital manipulation in a calibrated plastic vial

(Foote 1964b; Linde-Forsberg 1991). To reduce variability between trials concerning

semen, the semen was pooled to have access to sample volumes large enough to allow

the submission of the pools of semen to the different antibodies tested. Using a pool of

semen allowed for a reduction of variability due to animals and allowed for a reduction

of the number of animals needed for every study. Five animals have been demonstrated

to be optimum for this purpose during CASA validations and semen extenders studies

89

(Verstegen et al. 2005). However, one of our animals had to be removed for health

reasons from the program just after the start of this research.

Mature BALB/c male mice were euthanized by CO2 gas and cervical dislocation,

according to IACUC authorization protocol #200903106. Directly after euthanasia, the

cauda epididymides were excised and washed twice in PBS at room temperature. Each

cauda epididymis was punctured with a 23-guage needle and sperm were released

from the epididymis into the petri dish.

Sperm Preparation and Preservation

After pooling, the semen was evaluated for concentration, sperm motility using

SpermVision SAR, and morphology using Spermac staining (Silva and Verstegen 1995;

Verstegen et al. 2002). The fresh semen was then centrifuged twice at 700 x g for 10

minutes at room temperature in PBS and the supernatant was removed after each

wash. The final concentration of the PBS extended semen was 300 x 106 sperm cell/ml.

To extend and process the semen samples, each extender was warmed to room

temperature before dilution of the samples. The different samples were extended

according to manufacture’s recommendations. Briefly, for the Camelot, and CaniPRO

Chill 5, and CaniPRO Chill 10 groups, 1 part of semen was diluted with 3 parts of

extender. For the Synbiotics group, 0.5 part of semen was diluted with 2 parts extender.

CaniPRO Chill 10 extender requires the addition of 20% egg yolk before being used to

extend the semen.

After extension, ten µl of each semen samples were used for assessment of

motility after dilution using the CASA analysis as previously described. Twenty µl of

each of the samples were also deposited on a Superfrost®/Plus microscope slide, a

smear was performed and allowed to air dry overnight before fixation with the Zamboni

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fixative. Another ten µl of sample was last deposited on a microscope slide, dried, and

stained using a Spermac stain to assess morphology and acrosomal status following

the manufactures recommendations. The rest of the extended chilled semen (1.5-2.5

ml) was placed in 15 ml conical tubes and stored in the refrigerator at 4ºC in alcohol-

filled containers up to day 20 post collection (evaluation was stopped when all sperm

were dead as demonstrated by CASA analysis). The alcohol-filled storage containers

prevented both cold shock during the chilling process and temperature variations

(Verstegen et al. 2005). Each CASA analysis included evaluation of sperm

concentration, motility, and forward progression parameters.

Experiments

Effect of chilled extenders on motility and acrosomal status

Sperm motility was assessed at different time points (Days 0 (before dilution with

the different extenders) and 1,3,5,7,10,13,15,18, and when needed day 20 after

dilution)]. The sperm were no longer assessed when sperm cells were no longer motile.

Acrosomal status was assessed at Days 0, 5, 10, 15, 20. This study was repeated three

times.

Effect of chilling and different extenders on sperm protein expression

At each time point listed above, a ten µl sperm sample from the different extenders

was overnight air dried on a Superfrost plus microscope slide and fixed with Zamboni

fixative for one hour at 4°C and processed following Dako standardized

immunocytochemistry protocol using a primary mouse monoclonal antibody against

SPAG6, or primary rabbit polyclonal antibody against SP17 or CATSPER2. Primary

antibodies were diluted as follows: SPAG6 at 1/200, SP17 at 1/200, and CATSPER2 at

1/50 with PBS, incubated overnight at 4° C, rinsed in distilled water and washed 3 times

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in PBS, then incubated 1 hour at room temperature with a swine anti-mouse/rabbit/goat

biotinylated secondary antibody. DAB + Substrate buffer was used to visualize the

binding sites. In the negative controls (canine and mice) the primary antibody was

omitted. Mouse sperm was used for the positive control. Visualizations were

ascertained using a BX51 Olympus microscope at 200-600X magnifications.

Effect of acrosome reaction induction on sperm protein expression

SP-TALP sperm capacitation media was laboratory prepared according to

published instructions (Sirivaidyapong et al. 2000). Briefly, the ingredients were: 100

mMol sodium chloride, 3.1 mMol potassium chloride, 2.0 mMol calcium chloride, 0.4

mMol magnesium chloride, 25 mMol sodium bicarbonate, 1 mMol sodium pyruvate,

3.38 ml sodium lactate 60% syrup, 0.3 mMol sodium phosphate, 10 gm/L bovine serum

albumin, 2.28 ml Hepes, and 0.25 mMol gentamycin S. All ingredients were dissolved in

ultrapure water when needed. The pH and osmolarity (7.26 and 311 nmol/L

respectively) of SP-TALP were recorded before the addition of the semen. The pooled

semen was divided into two groups: sperm with SP-TALP (1 part semen and 3 parts

SP-TALP) and sperm without SP-TALP but extended in PBS (1 part semen and 3 parts

SP-TALP). Prior to extension and incubation, semen was analyzed for motility and

evaluation of acrosome status (before incubation samples). Then, the extended

samples were covered in mineral oil and incubated for 5 hours at 37 °C in 5% C02

before being analyzed again as stated below (after incubation samples).

After the incubation period, 250 µl of each sample was fixed separately using 0.8

ml of 4% paraformaldehyde for 10 minutes at room temperature. The samples were

then centrifuged at 500 x g for 6 minutes, the supernatant was removed, and the pellet

was resuspended in 500 µl of ammonium acetate (0.1M) then centrifuged at 500 x g for

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5 minutes and the clear supernatant was removed. Finally, a 50 µl smear of sperm

suspension was placed on a microscope slide and allowed to air dry overnight. The

following day, the air dried slides were incubated at room temperature for 2 minutes in

freshly made Coomassie Blue stain, rinsed 3-4X in distilled water, and allowed to air dry

before analysis under brightfield microscopy.

Statistical Analysis

For the effect of extenders on motility and acrosomal status, three slides were

labeled and 100 sperm were evaluated per slide per group. Two-way ANOVA was used

to compare the different chilled extenders with percent motility and percent acrosomal

status overtime. Tukey-Kramer multiple comparison test was performed when the P

value was significant (<0.05). Data were expressed as mean ± SD. Differences were

considered significant when P<0.05. These studies were repeated three times.

For the acrosome reaction induction study, three slides were labeled and 100

sperm were evaluated per slide per group, thus µ=3 per group. One-way ANOVA was

used to compare the results. Tukey-Kramer multiple comparison test was performed

when the P value was significant (<0.05). Data were expressed as mean ± SD.

Differences were considered significant when P<0.05. These studies were repeated

three times.

Results

The mean values for concentration, overall motility, and forward progression of

sperm samples were 391 ± 80.9 x 106 sperm/ml, 94.12% ± 8.0, and 78.4% ± 5.4,

respectively. For sperm morphology, mean percentage of normal spermatozoa was 95

± 4.8% sperm. Data for normal sperm morphology was assessed prior to

experimentation.

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Effect of chilled extenders on motility and acrosomal status

There were no statistical differences between the CaniPRO 5, CaniPRO 10 and

the Camelot groups of chilled semen immediately after dilution. However, Synbiotics

extender induced a significant decline of sperm cell motility immediately after dilution.

One day later, all the chilled diluted samples were similar without statistical differences.

However, a trend for Synbiotics to be lower was already observed. Overtime, the

motility observed decrease progressively for all groups. However, significant differences

in the dynamics of the decrease were observed between groups. An overall motility of

50% was obtained for Synbiotics after 3 to 4 days, 7 days for the Camelot group while

only observed after 11 days for both the CaniPRO Chill 5 and CaniPRO Chill 10

extenders (P<0.05). The overall motility was lower than 30% for Synbiotics at day 5,

nearly all sperm cells being immotile at day 10, the same 30% were noticed at day 10

for Camelot with nearly all sperm cells being dead at day 15 while 30% of motility was

only observed at days14-15 with and nearly all sperm cells dead at days 18 for

CaniPRO Chill 5 and CaniPRO Chill 10 respectively. These differences were statistically

significant (P<0.05).

Overtime, the percentage of sperm undergoing the acrosome reaction was not

significantly different (P>0.5) between the different extenders (Fig. 6-2). Large variations

between samples and repetitions were noticed explaining these high standard

deviations. The fact that the number of living sperm cells was changing significantly

overtime for some extenders also dramatically influence the standard deviation. At 50%

motility (days 3-4, 7, and 11 for Synbiotics, Camelot, and CaniPRO Chill 5 or 10

respectively), 20 to 30% of the sperm cells were acrosome reacted. At the end of the

preservation period when motility was lower than 10%, the percentage of acrosome

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reacted sperm cells was between 30 and 50% with variations between groups however

without statistical differences.

Effect of chilling and different extenders on sperm protein expression

SPAG6 and CATSPER2 proteins were poorly preserved during the chilling

process. Indeed, if the labeling was considered as normal before dilution and incubation

at 4°C, none or only few cells were still identified after extension and incubation

whatever group considered for both proteins. For all extenders, SPAG6 labeling was

very weak or absent by day 3 and when observed, only the acrosome or equatorial

band were weakly marked for SPAG6 (Fig. 6-3). No or weak labeling were observed for

CATSPER2 at the same period.

For SP17, the typical labeling of the acrosome changed with time and acrosome

integrity. In acrosome-intact sperm cells, the acrosome is labeled, but this typical

labeling disappears in all acrosome modified or reacted sperm cells that become

detected with preservation. This observation was consistent with the four chilled semen

extenders (Fig. 6-4). The flagella labeling sometimes observed in control fresh non-

extended sperm cells was also observed in preserved sperm cells, but with an

increased intensity. The intensity of the labeling was negatively correlated with the

labeling of the acrosome. In the non acrosome reacted sperm cells, the principal piece

was weakly labeled while in the acrosome reacted sperm cells, the flagellum was more

clearly recognized by the antibody as shown by an increased labeling intensity.

Effect of acrosome reaction induction on sperm protein expression

Acrosome reaction induction with SP-TALP significantly (P<0.0001) caused a

decrease in the number of acrosome-intact sperm when compared to sperm not treated

with SP-TALP (Fig. 6-5) Less than 50% of acrosome-intact sperm cells were observed

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after 5 hour incubation and 20% of the sperm cells remained alive. Similarly, sperm

protein expression was dramatically changed with acrosome reaction induction (Fig. 6-

6).

For sperm proteins SPAG6 and CATSPER2 there was moderate expression localized

at the post acrosomal region and none to weak expression localized at the flagellum.

For sperm protein SP17, no labeling was observed on the sperm cells. The negative

controls had no labeling present. For each sperm protein, the mouse positive controls

were normal (data not shown).

Discussion

When sperm is not immediately used for artificial insemination, semen extenders

are needed and are important for sperm conservation. This present study compared

commercially available canine chilled semen extenders for their ability to maintain

sperm motility and acrosome integrity. Considering previously published studies on

canine chilled semen preservation, it was decided to follow some semen characteristics

(motility and acrosome reaction) until motility decreased below 10% for some extenders

up to a period of 20 days at 4ºC. Significant differences were observed between the

chilled semen. These differences were not statistically significant when semen were

compared at fixed days because of the large individual variations observed between

samples. Indeed, for an extender like Synbiotics, at day 5 in some samples the semen

was already dead while in some other samples at the same day 30 or 40% of the sperm

cells were still motile. However, a significant difference was noticed for the dynamic of

motility over time. If the samples were compared for percent of motility, we noticed that

the extenders can be classified with the Symbiotic one preserving motility over the

shorter period while the CaniPRO extenders allowed for a significantly extended

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duration of preservation. These results confirm those already published with similar

extenders: huge variation and differences in the overall ability of the extenders to

maintain motility. In the present study, the percentage of overall motility decreased

significantly overtime. On day 0, the extended sperm cells were motile in the range 78-

98% (with motility before extension being 94%) and all the samples were immotile after

11 days for Synbiotics, 15 days for Camelot and more than 18 days for the CaniPRO

extenders. The initial motility of 70% was preserved 3 days for the Synbiotics extender,

5 days for the Camelot and 10 days for the CaniPRO extenders. During the first 5 days,

no significant changes in the overall motility were noticed for the two CaniPRO

extenders. These two last observations are of significant interest in clinical conditions

where most often the preserved chilled semen is used within 3 to 5 days after collection.

Similarly 70% motility is most often considered as being the optimal for the success rate

after artificial insemination in the dog.

No significant differences were observed between the chilled extenders in regards

to acrosome integrity. The percentage of acrosome-intact spermatozoa was 100% on

day 0 and ranged from 45-70% on day 20. The absence of difference for this parameter

can be due to the limited number of samples used in this study. Though not significant,

Camelot appeared to have more damaged and acrosome reacted sperm compared to

the other semen extenders. Interestingly, no differences were noticed as far as

acrosome reaction is concerned when the two CaniPRO products were compared.

While the exact composition of the two extenders is not known and based on previous

studies demonstrating a beneficial effect of the egg yolk on semen preservation, we

were expecting not only a difference in term of motility preservation but also as far as

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acrosome reaction is concerned for the CaniPRO 10 that is added with 20% egg yolk.

The absence of difference can be due to the limited number of samples used in this

study or to possible differences in the composition or origin of the egg yolk used in this

study versus the previous published reports. It is, however, interesting to note that in the

best present extenders, acrosome reaction after long term preservation does not seem

to be a major issue.

On fresh sperm, SPAG6 was strongly expressed and localized mainly at the

acrosomal region of the sperm head. In addition, SPAG6 had negative to moderate

expression localized at the midpiece of the sperm tail. On chilled sperm and with all

extenders, SPAG6 had weak or absent expression by day 3. When present, the labeling

was located at the acrosome or equatorial band either similarly to what was observed in

fresh samples (acrosome) or with a new different pattern when the equatorial bands

were labeled. The presence of the labeling is not directly related to the acrosome

reaction as in nearly all samples at day 3 after collection, acrosome reaction is in

general of less than 30% and the labeling is gone in nearly all sperm cells. The fading of

the labeling for SPAG6 is probably not directly correlated to changes in motility as

during the first few days for all the extenders there no significant changes in motility.

A similar observation was made for CATSPER2. On fresh sperm, CATSPER2 was

expressed and localized at the flagellum and also had weak to moderate expression at

the level of the acrosomal region. On chilled sperm, CATSPER2 localization was

absent. In other species, the CATSPER family of proteins is known to play a role in

sperm motility. They are also proposed to have a role in sperm calcium regulation. The

absence (or weak) of labeling after 3 days of preservation without significant changes in

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both acrosome reaction and motility, either indicates that these proteins are not involved

in these mechanisms or that the proteins are involved in process associated with

prolonged motility conservation or cold shock protection. A last possibility is that the

extension with complex media including egg yolk made the availability of the protein to

the antibodies impossible.

On fresh sperm, we demonstrated earlier that SP17 is expressed and localized

with strong and similar intensity at the acrosomal region and the principal piece of the

flagellum. On chilled sperm and with all extenders, no significant changes in the

expression of SP17 were observed during the first week of preservation. Then it was

noticed that SP17 had weak to moderate expression by day 10 and weaker staining

present by day 20. The labeling that was present was located at the acrosome with

varying degrees of acrosomal damage and with variable expression at the level of the

principal piece. It was also observed that the principal piece was labeled more when the

acrosome was reacted than when the acrosome was intact, and vise versa. No labeling

of the head was noticed in acrosome reacted sperm cells. This observation was

confirmed, but the acrosome induction study was actively inducing the acrosome

reaction associated with a total inhibition of the expression of the protein. This

observation was of major interest clearly indicating that based on the changes in

absence of acrosome reaction induction, that SP17 may have a function in the

preservation of sperm cells, but not directly associated with the acrosome reaction.

Indeed, the dynamic change in the expression and pattern of expression of this protein

may be indicative of a functional role of SP17 which certainly should be further

investigated.

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The results demonstrated that chilling does cause a change in expression of

SP17. This was shown with the diminishing expression from day 10 to day 20. The

justification for the observation that the principal piece was labeled more when the

acrosome was reacted can best be explained by fact that a sperm cell that is acrosome

reacted no longer has plasma or acrosomal membranes in the head region of the

spermatozoa. Thus, expression of SP17 at the level of the acrosome should be absent.

An acrosome reacted sperm should not have disruption of the plasma membrane

covering the principal piece of the flagellum. The justification for the acrosome-intact

being expressed and the principal piece being no longer labeled can best be described

by the semen extender environment inducing the breakdown of the plasma membrane,

but not enough to cause an acrosome reaction; thereby preserving SP17 expression at

the level of the acrosome.

To verify that an acrosome reaction does cause changes in sperm protein

expression, an acrosome induction study was conducted. With the use of SP-TALP a

capacitation media, to first determine how many spermatozoa will acrosome react and

secondly to determine sperm protein expression following acrosome reaction induction.

It was believed that a significant change in acrosome reacted sperm cells will occur with

a decrease in protein expression. The study confirmed the belief that acrosome reaction

induction significantly decreased the amount of acrosome-intact sperm (60%) when

compared to acrosome-intact sperm not treated (98%) with SP-TALP capacitation

media. Sperm protein expression was dramatically changed with acrosome reaction

induction. For sperm proteins SPAG6 and CATSPER2 there was moderate expression

localized at the post acrosomal region and none to weak expression localized at the

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flagellum. For sperm protein SP17, no labeling was observed on the sperm cells. The

first observation was that SP17 was more so affected by semen preservation and

acrosome reaction induction when compared to the other sperm proteins. Under these

conditions, SP17 was either weakly expressed or absent. SPAG6 and CATSPER2 both

showed evidence that an acrosome reaction occurred by the presence of post

acrosomal region staining.

This is the first study to characterize the expression of SPAG6, SP17 and

CATSPER2 in canine spermatozoa. Furthermore, this study demonstrated that motility

overtime significantly decreased when sperm were preserved in chilled semen

extender. This study also showed that acrosome status overtime does not significantly

change regardless of the four canine semen extender used. Finally, that acrosome

reaction induction does significantly affect sperm protein expression and potential

sperm protein function.

In conclusion, we have demonstrated in this study differences in the ability of

different extenders to preserve motility. The present results, however, did not reveal

significant effects of the extenders and chill preservation on the induction of acrosome

reaction in the dog. Semen preservation was associated with significant changes in

sperm membrane protein expression without obvious correlation with changes in the

overall motility and acrosome status, except for SP17, which showed changes

correlated to modifications of the acrosome. The in vitro induced acrosome reaction

induction surprisingly was associated with changes in sperm cell labeling. These

observations seem to indicate possible changes in the protein expression patterns with

preservation and acrosome reaction and warrant further investigations.

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0

10

20

30

40

50

60

70

80

90

100

0 1 3 5 7 10 13 15 18 20

Days

% o

vera

ll m

otili

ty

Chill 5

Chill 10

Camelot

Synbiotics

Figure 6-1. Overall motility (%) of sperm preserved at 4°C overtime in different chilled semen extenders.

102

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20

Days

%

Chill 5

Chill 10

Camelot

Synbiotics

Figure 6-2. Mean (%) of acrosome-intact sperm overtime at 4°C in different chilled semen extenders.

103

Figure 6-3. SPAG6 chilled spermatozoa Day 3 of preservation at 4°C. A) Chill 5, B) Chill 10, C) Camelot, D) Synbiotics. Poor labeling is observed with non labeled sperm. Skinny black arrow: damaged, acrosome orange arrow: no labeling even if intact acrosome. Magnification at 600x.

10 µm

A B

C D

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Figure 6-4. SP17 chilled spermatozoa Day 10 and Day 20 of preservation at 4°C. A) Chill 5 D10, B) Chill 5 D20, C) Chill 10 D10, D) Chill 10 D20, E) Camelot D10, F) Camelot D20, G) Synbiotics D10, H) Synbiotics D20. Black arrow: acrosome-intact, dashed arrow: flagella labeled, skinny black arrow: acrosome damaged, orange arrow: no labeling. Magnification at 400x.

10

A B

C D

E F

G H

105

Figure 6-5. Mean (%) of acrosome-intact sperm before and after acrosome reaction induction.

106

Figure 6-6. Effect of acrosome reaction induction on sperm proteins. A, C, E, G) before acrosome reaction induction, B, D, F, H) after acrosome reaction induction. Magnifications at 400-600x.

Stain

SPAG6

SP 17

CATSPER2

A B

C D

E F

G H

107

CHAPTER 7 GENERAL DISCUSSION

The canine industry in the United States is a multimillion dollar business. Using a

valuable male dog that has a successful career for breeding can be a major investment

for the owner. Indeed, dog breeders want to breed their bitches to well known and

successful males with excellent fertility. Therefore, some breeding stud dogs are

expected to breed large numbers of bitches each year. In order to satisfy these

expectations, the testes must continuously produce a large number of good quality

spermatozoa and the ejaculates should be devoid of any factors possibly leading to a

reduced fertility. Any factor that affects semen quality and spermatozoa function may

have a great impact on a dog’s reproductive career and cause great financial and

emotional losses to the breeder.

There is a large body of evidence that semen and prostatic fluid qualities are

important factors involved in reproductive function and fertility (Foote 1964b; Strzezek

and Fraser 2009). With age, male dogs often develop prostatic disease which may lead

to the production of poor quality semen that often is contaminated with blood, urine, or

other products such as bacteria, debris, and pus (England and Allen 1992; Rijsselaere

et al. 2004; Sampson et al. 2007). Contaminated semen can not be used for artificial

insemination (AI) and semen freezing, the main reasons being risk of infectious

contamination of the female or poor freezability (England and Allen 1992; Chen et al.

1995a; Verberckmoes et al. 2004). Furthermore, various abnormal conditions of the

reproductive genital tract, including infection by herpes virus or Brucella canis bacteria,

balanoposthitis, prostatitis, abscesses, and hemospermia can affect the quality of the

ejaculate that can result in reduced fertility through direct effects on the semen or

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indirectly affecting female fertility. Moreover, in older dogs, the number of good quality

and highly motile sperm cells may decrease inversely proportionally to the increase

number of dead or abnormal cells.

For all these reasons, there is a need for the development of techniques which will

successfully allow the separation of the contaminants and/or poor quality sperm from

the progressively moving, highly viable cells. The discontinuous Percoll gradient

centrifugation technique has been developed with this objective to separate viable,

progressively motile sperm cells from dead/poor quality sperm cells in several species,

including bovine (Parrish et al. 1995), human (Chen et al. 1995b; Miller et al. 1996), and

more recently canine (Strom et al. 2000a; Hishinuma and Sekine 2004).

If new techniques are needed to improve semen quality by purification, sperm cells

need normal membrane integrity and functionality to be able to penetrate and fertilize

the oocytes. Recently several membrane proteins have been demonstrated to play a

significant role in sperm cell motility and function. Up to now, none of these sperm

membrane proteins have been analyzed in the dog.

Among the proteins of interest, the presence of sperm associated antigen 6

(SPAG6), a sperm protein, which is localized on the flagellum in mice and human

sperm, has not been investigated in canine. The presence of SPAG6 has been linked

with sperm flagellar motility and structural integrity of the axoneme central apparatus of

mature sperm in mice and humans (Neilson et al. 1999; Sapiro et al. 2000). Mature

SPAG6 knock out mice are infertile and produce sperm with abnormal motility and

structural defects (Sapiro et al. 2002). Knowing the localization and levels of SPAG6

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and other sperm proteins in the dog will allow for better identification of their potential

role in that species and evaluation of the effects of canine semen preservation.

For many years, in vitro assessment of sperm has been done in attempt to

accurately predict the fertility of the sample. This has not been without difficulty, as

fertilization is a complex process. There are many in vitro techniques to assess sperm,

including: evaluation of sperm motility, concentration, morphology, membrane integrity,

and acrosome reaction. Unfortunately, data have shown that these same parameters

alone are not always accurate predictors of fertility (Graham 2001; Henkel and Schill

2003; Rodriguez-Martinez 2003; Martinez 2004; Rijsselaere et al. 2005). According to

the literature a combination of these parameters in addition to others such as: oocytes

penetration, hemi-zona, and zona pellucida binding assays, will be the most useful and

accurate approach to evaluate the semen potential to be fertile (Henkel and Schill 2003;

Rijsselaere et al. 2005). However, even if these approaches are easily achievable in

many species, due to various reasons including ovulation occurring only once or twice

per year, oocyte maturation occurring after ovulation, or the dark lipid content in the

oocytes, there are many difficulties with conducting successful in vitro studies in the dog

(Rodrigues and Rodrigues 2006; Songsasen and Wildt 2007).

The American Kennel Club has over 150 registered breeds. These breeds are

placed throughout the nation and the world and in many cases to avoid displacement of

the dog; semen must be shipped for artificial insemination. The semen must be

extended and chilled or cryopreserved prior to shipping, in hopes that the sperm will be

viable and fertile upon arrival and insemination.

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Semen preservation offers many advantages to the canine industry, particularly in

conjunction with genetic evaluation and selection programs. However, the biggest

obstacle to using preserved semen is that cooling, conservation at 4° C, freezing

conservation at -196° C, and thawing generally damage sperm membrane structures

which lead to fewer viable and motile cells. Consequently, fertility following artificial

insemination with preserved semen might be decreased compared to that with fresh

semen. Due to increased demand for preservation, many laboratories have been

working to better understand the properties of preserved semen in order to find more

efficient methods for sperm storage (at either 4 or -196° C), recovery, viability, and

fertility. The most commonly described side effect of semen preservation is the dramatic

and sharp decrease in sperm viability, motility, and plasma membrane integrity

observed in some animals.

Prior to preservation of semen, in vitro assessment of the semen sample must be

performed. However, there are no guarantees that sperm that are alive and potential

fertile prior to chilling or cryopreservation will be viable and fertile at the time of

insemination. This is the reason why another in vitro assessment of the semen sample

must be performed prior to insemination. To get the optimal results after preservation

and before artificial insemination, the preservation techniques have to be optimized so

that an optimal semen extender will be chosen to provide a high quality environment for

sperm to remain fertile upon artificial insemination.

There are several steps to analyze a semen sample. Initially, after the semen has

been collected, the semen should be evaluated for sperm concentration, motility, and

morphology; then the semen may be purified. Purification may be important because it

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is necessary that non-contaminated, viable, and potentially fertile sperm are used

whether inseminating or freezing sperm. Before and after purification, the semen needs

to be assessed and finally processed for preservation.

In order to improve the predictability of a semen sample and gain a better

understanding of canine male fertility the following objectives and hypotheses were to

be tested at the beginning of this work: to compare commercially available density

gradient centrifugation media (Isolate, Percoll, PureCeption, PureSperm) in their ability

to optimally separate viable, motile spermatozoa from nonviable (nonmotile and/or

dead) spermatozoa and red blood cells (RBC) contamination. The hypothesis tested

was that there would be differences between the four DGC media in terms of their

efficiency on canine spermatozoa separation and purification, as determined by the

ability to separate motile sperm from nonmotile and RBC for optimal sperm recovery.

After collection of a semen sample, purification to remove contaminants (blood,

urine, pus) (England and Allen 1992; Chen et al. 1995a) should improve semen quality

and improve the overall pregnancy success rates. Density gradient centrifugation is a

valuable and practical tool for sperm separation. Based upon the principle that due to

centrifugal forces sperm are arranged with heads downward and flagella upward, that

motile sperm cells move while non motile sperm and contaminants stay static (Kuji et al.

2008). Density gradient centrifugation has been described in several species including

dogs (Parrish et al. 1995; Miller et al. 1996; Centola et al. 1998; Hishinuma and Sekine

2004), as a semen purification technique that can with little difficulty be done in a clinical

or laboratory setting. There are several density gradient centrifugation media available,

but the selected media Isolate, Percoll, PureCeption, and PureSperm have been

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predominantly used in animal and human studies (Politch et al. 2004; Mousset-Simeon

et al. 2004) for their ability to separate sperm from contaminants.

In this present study, significant differences were observed between the four

density gradient centrifugation media in their ability to separate viable and motile sperm

from non motile sperm and red blood cells. PureCeption and PureSperm abilities to

separate blood from sperm in the optimal fraction were significantly different than Isolate

and Percoll. Indeed, the optimal fraction of PureCeption had significantly higher

recovery of total and motile sperm cell compared to the optimal fraction for Isolate and

Percoll; though PureCeption and PureSperm optimal fractions did not significantly differ.

The maximum number of total and motile spermatozoa were located in one fraction in

PureCeption and located in two fractions in PureSperm. The practicality of density

gradient centrifugation is so that all the sperm cells of interest reside at the bottom of a

tube and the upper layers of fluid which contain contaminants can be decanted off

leaving the desired semen sample purified.

The direct clinical application for sperm separation using density gradient

centrifugation media is that it allows for efficient separation of motile sperm cells from

contaminants. The efficacy of viable sperm cell separation from non motile sperm cells

or contaminants is essential in the canine as it has been shown that detrimental effects

can occur when semen is preserved in the presence of blood (Rijsselaere et al. 2004).

Cushion centrifugation is a complementary process that has been reported recently in

equine (Volkmann 1987; Loomis 2006; Waite et al. 2008) and in porcine (Matas et al.

2007) to protect sperm cells during the centrifugation process. It has been associated

with less denaturation of sperm cells than during centrifugation only. The combination of

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our gradient centrifugation approach with the cushion centrifugation using iodoxynate

may be a further step that would be of interest in the dog allowing separation of sperm

cells without denaturation possibly with the centrifugation.

For practical use in a clinical setting, DGC is the method of choice. The ease of

the technique is obvious, does not require sophisticated equipment as all that is needed

is the DGC media and a centrifuge. The results are available within minutes and when

using DGC media the upper layers of fluid can be poured off leaving the viable, motile

sperm cells available for insemination or semen preservation. While Percoll is the most

often used DGC media of choice for many non human studies, Isolate, PureSperm, and

PureCeption are increasing in popularity with the beneficial characteristic that Isolate,

PureSperm, and PureCeption are all bioassay and endotoxin-tested.

In other species such as mice and humans (Sapiro et al. 2000; Ren et al. 2001; He

et al. 2003; Quill et al. 2003) sperm proteins have been reported to have a role in sperm

function. Our objective was to identify and characterize, using immunocytochemistry,

the presence of sperm proteins SPAG6, SP17, SP56, and CATSPER2 proteins in

canine spermatozoa. The hypothesis was that SPAG6, SP17, SP56, and CATSPER2

are localized and expressed in similar patterns in canine sperm cells as in other species

and may play a role in sperm function, such as sperm motility, capacitation and/or

acrosome reaction.

While immunocytochemistry is a protein characterization tool that localizes protein

expression it does not allow for confirmation of the presence of the proteins of interest.

Indeed, even if specific, the antibody may easily cross-react with similar sequences of

other proteins. Western blot analysis was used to confirm the presence of the sperm

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proteins of interest. The hypothesis tested was: western blot analysis will confirm in the

dog the presence of sperm proteins with molecular weights corresponding to the

previously described SPAG6, SP17, SP56, and CATSPER2 proteins in other species

spermatozoa (Cheng et al. 1994; Richardson et al. 1994; Neilson et al. 1999).

In the present study in canine spermatozoa, SPAG6 was demonstrated to be

strongly expressed and localized in the head region with high incidence of pan-

acrosomal labeling and variable punctate expression and equatorial localization.

SPAG6 was also variable (negative- moderate) in expression (less than 20%) labeling

at the midpiece of the flagellum. At the level of the midpiece, SP17 was demonstrated to

be strongly expressed (more than 85%) and localized at the acrosome and principle

piece, with weaker expression at the midpiece. SP56 was demonstrated to be strongly

expressed and localized predominately (more than 95%) at the principle piece, with

weak to moderate acrosomal expression (more than 25%). CATSPER2 was

demonstrated to be expressed and localized at the flagella region, with weak expression

(75%) localized at the acrosomal region.

According to what has been shown in mouse studies, SPAG6 is localized at the

principal piece with weaker expression at the midpiece and head region (Sapiro et al.

2000). Even though the level of expression is not the same, the localization is similar

and may still point to a role in sperm motility and/or capacitation and acrosome reaction

functions. Canine SPAG6 was detected in a band at 61.57 kDa. The predicted value for

SPAG6 was 55.5 kDa and based upon the coefficient of variation which was estimated

to be 10-20%, canine SPAG6 is within that range.

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The exact role of SP17 for fertilization is still unknown. It has been proposed that

based on flagella localization that the function of binding to the zona pellucida is not the

sperm protein’s main function (Frayne and Hall 2002). Furthermore, this protein is also

found in other tissues including somatic and tumor cells (Wen et al. 2001; Straughn, Jr.

et al. 2004). In the present study, SP17 immunoreactivity was detected as proteins of

different molecular weights: 15.45, 28.75, and 29.01 kDa which was in agreement with

predicted values. The presence of multiple bands for SP17 protein is a generalized

observation that has been reported in several publications (Kong et al. 1995; Wen et al.

1999; Grizzi et al. 2003) with variation in molecular weights as follows:14-15, 17-19, 21-

24, 26, and 29 kDa.

It was reported that (Lea et al. 2004) SP17 protein is found predominately in

spermatozoa, cilia, and human neoplastic cell lines. In the present study, SP17 was

present in the head and tail of spermatozoa. SP17 has been shown to bind A-kinase

anchoring protein 3 (AKAP3), but not to AKAP4. However, Akap4 knock out mice had a

reduction in the amount of SP17 expressed when compared to wild-type sperm (Lea et

al. 2004). Akap4 knockout mice have fibrous sheath that forms incompletely, that the

principal piece is shorter, and they are not progressive motile. From this information, it is

a reasonable assumption that SP17 has a function for sperm motility and due to the

SP17 association with anchoring proteins, that SP17 may have a role in sperm and

zona pellucida binding. As SP17 was detected with the same overall localization and

characteristics than SP17 in mice, we can probably suggest a similar role for SP17 in

the canine: involvement in sperm cell motility and oocyte binding.

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In the canine, SP56 was consistently expressed at the principal piece with

moderate expression at the acrosome. However, western blot analysis was unable to

detect sperm protein SP56. Our results in ICC for fresh canine sperm were different

than the one found in the mouse. In the mouse, SP56 was characterized as sperm

membrane protein (Cheng et al. 1994) and then later as an acrosomal matrix protein

(Kim et al. 2001). Yet, mice with an Azh mutation that produce 100% abnormal sperm

have SP56 localized on both acrosome and flagella (Moreno et al. 2006). In fact, the

studies concerning sperm protein SP56 have provided conflicting and controversial

results also in other studies. Different results have been reported for normal and

abnormal mice for SP56 expression and sperm function (Cheng et al. 1994; Kim et al.

2001; Moreno et al. 2006). Moreover, the sperm protein SP56 was reported

(Bookbinder et al. 1995) as a mouse sperm fertilization protein and detectable in mouse

and hamster sperm and not detectable in guinea pig or human sperm. Several years

later, another study (He et al. 2003), described SP56 localization in rat sperm.

Based on blast hits, it was expected that SP56 with 6 blast hits for similar mouse

and canine sequence to be identified in canine sperm cells. However, SP56 was not

detectable by western blot analysis in canine sperm cells. This lack of detection by

western blot analysis could be a possible result of denaturation of the protein. Due to

the conflicting published results in mice and contradictory results observed in the

canine, the sperm protein SP56 warrants further studies in all species to determine

localization, expression, and specific function.

CATSPER2 was demonstrated, in agreement with mouse sperm, to be localized to

the flagellum in canine spermatozoa. There was also a weak expression at level of the

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acrosome. The flagellar localization and the previous reports (Quill et al. 2003; Qi et al.

2007) of functionality with hyperactivated sperm imply that CATSPER2 has a role in

sperm motility and the calcium fluctuations which occur during capacitation and result in

sperm hyperactivation. CATSPER2 was detected in different bands at 48.48, 49.18 and

63 kDa. The predicted molecular weights are 48 and 61kDa.

Of the four sperm proteins selected for this dissertation, CATSPER2, with a single

blast hit, had the lowest probability to be detected in the dog. However, this study was

able not only to identify CATSPER2 in canine sperm cells by ICC, but also to confirm its

presence with the western blot analysis. CATSPER2 had weak expression at the level

of the acrosome. This presence at the level of the acrosome could be a result of cross-

reactivity of CATSPER2 with CATSPER3 or CATSPER4, which are present and

localized at the acrosome (Jin et al. 2005). It has been suggested that this family of four

proteins form a functional heterotetrameric channel in sperm (Quill et al 2003).

Semen preservation is a major part of canine reproduction management.

Increasingly, owners are shipping the semen to the female for artificial insemination

instead of allowing the dogs mate naturally. To improve the chances that the semen

sample is viable when it reaches its destination, the semen is either chilled or frozen

extended in canine semen extender for transport (Linde-Forsberg 1991). In our quest to

improve semen sample potential, our objectives were to evaluate the changes in

selected sperm protein expression during preservation over a period of time, and

establish a possible relationship between this expression and different semen

parameters including motility, capacitation, and acrosome reaction. The hypotheses

tested were: a) semen preservation over time affect protein expression, b) differences

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may be observed between the different evaluated extenders, c) acrosome induced

functional changes may be detected in relation to the preservation processes, d)

acrosome reaction induction is associated with changes in the expression of the

different semen proteins evaluated , e) overtime changes in motility may be related to

changes associated with acrosome reaction and modification in the expression of the

different proteins.

Preservation of semen involves providing an optimal in vitro environment for

preserving sperm viability (Linde-Forsberg 1991; Iguer-Ouada and Verstegen 2001b). It

is expected that semen extender will stabilize and protect the sperm cells from cold

shock and the plasma membrane from damage. In the current study, on day 0 no

statistical differences in motility were observed between CaniPRO Chill 5, CaniPRO

Chill 10, and Camelot groups directly after semen dilution. Also on day 0, the Synbiotics

group had a significant decline in motility directly after semen dilution. At day 1, all

diluted samples were not significantly different, though Synbiotics tended to be lower

than the other groups. Over a period of time, motility progressively decreased for all

groups. Synbiotics was below 50% motility on after days 3-4. Camelot was at 50%

motility on day 7. CaniPRO Chill 5 and CaniPRO Chill10 were below 50% motility after

days 12-13. Sperm classified as immotile or dead and were significant different for

Synbiotics day 10, Camelot day 15, and CaniPRO Chill 5 and CaniPRO Chill day 18.

There was not a significant difference between chilled semen extenders with

percentage of acrosome-intact sperm to transition to acrosome reacted sperm.

Semen preservation by chilling affected SPAG6 and CATSPER2 protein

expression. For all chilled extenders, SPAG6 expression was weak or absent by day 3

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and if weakly present only at acrosome or equatorial band. CATSPER2 had negative

expression at the same time period for all chilled extenders. For all chilled extenders,

SP17 expression varied with the time period and acrosome integrity. Meaning

acrosome-intact sperm had acrosomal expression of SP17 present. While, acrosome

damaged or acrosome reacted sperm, SP17 expression was diminished or absent. An

interesting occurrence was observed in normal acrosome-intact sperm, the principal

piece was weakly expressed. However, in acrosome reacted sperm the principal piece

was obviously expressed.

It has been reported (Zhang et al. 2005) that SPAG6 decoration of microtubules

increases their stability and renders them resistant to depolymerization by cold

(incubation at 4°C). Based on this observation, SPAG6 should be resistant and stable in

cold temperatures (4°C). However, the study did not mention how long they were

maintained at this temperature. In the canine, we found that cold temperature (4°C)

causes SPAG6 expression to be weak or absent by day 3. Furthermore, it was

mentioned (Zhang et al. 2007) that freezing and boiling of sperm result in loss of

SPAG6 also lost in mice with mutated sperm. Humans with heterozygous mutation are

fertile without sperm impairment and mice with heterozygous mutation have impaired

spermatogenesis (Zhang et al. 2007). Based on these observations, it appears that

changes in temperature result in loss of SPAG6 expression similar to that observed in

the present study in the canine where an obvious decrease or absence of expression of

SPAG6 during preservation at 4°C. This protein warrants further investigation.

For the functional test of acrosome status, SP-TALP a sperm capacitation media,

was significant in producing an acrosome reaction and caused a decrease (50%) in

120

number of acrosome-intact sperm when compared to non SP-TALP treated sperm. An

induced acrosome reaction substantially transformed sperm protein expression. SPAG6

and CATSPER2 were moderately expressed and localized to the post acrosomal

region. There was also negative to weak expression localized at the flagellum. SP17

labeling was absent from sperm cells.

In a functional test, (Kong et al. 1995) live, capacitated mouse sperm do not stain

with anti-SP17. However, live capacitated, ionophore-treated sperm do stain, indicating

that initially the polypeptide of SP17 is not available to bind to the surface of live sperm

but becomes available as the acrosome reaction begins. In the canine, SP-TALP

treatment resulted in an absence of SP17 staining. This result was in agreement with

the live, capacitation mouse sperm not expressing SP17. The Kong study, mentioned

that live, capacitated ionophore-treated mouse sperm did stain. This raises the following

question: were the canine sperm in our study capacitated? Based on light microscopy

observations after the acrosome reaction induction study, the few remaining active

sperm cells were traveling in a forward progressive movement, most were spinning

around in a pin wheel circular motion and the flagella had a high beat frequency typical

of hyperactivation.

Based on the above results, it appears that during sperm conservation in semen

extender at 4ºC, the sperm cells undergo physiological changes which result in the slow

progressive breakdown of plasma membranes overtime. The breakdown of plasma

membranes and the potential increase in acrosome damage could justifiable be the

explanation for changes of sperm protein expression observed. In addition, the slow

progression of the breakdown of the membranes could explain why overtime there was

121

not a significant decrease in acrosome-intact sperm. The slowness of the deterioration

may have been enough to cause some damage to the acrosome, but not adequate to

initiate an acrosome reaction.

On the other hand, induction of the acrosome reaction caused a rapid change in

the plasma membrane, which resulted in the deterioration of the plasma membrane and

the release of acrosomal enzymes. The sperm proteins that were released were no

longer present or were diminished in their availability. This was a justifiable reason for

the decrease or absence in sperm protein expression after acrosome reaction induction.

The supportive evidence was shown with post acrosomal region staining present for

SPAG6 and CATSPER2 proteins.

In conclusion, a complementary approach to the evaluation of canine semen and

potentially of semen fertility in the male dog was suggested based on the results of this

study. Based on the dissertation introductory analogy, the development of more

efficient, readily available, and quicker ways to assess fertilization potential of a semen

sample must be developed. Classical semen evaluation parameters alone are not

enough to predict fertility outcome. A different approach should be to evaluate sperm

characteristics and functions. Mouse research models, and especially knockout models

(Sapiro et al. 2002; Moreno et al. 2006), have been instrumental in characterizing sperm

functionality as it relates to fertilization ability. Research needs to be continued for the

development of additional knockout mouse models, as well as, sperm studies in other

species, that will help us further understand the roles that sperm proteins portray in the

fertilization process and to what degree the specific sperm protein functions.

122

Taken together, the results of this study demonstrated that the use of PureCeption

for canine semen purification is of interest to separate motile sperm cells from

contaminants including RBC. However, further work needs to be done to confirm these

results and its clinical implications. This is the first showing that sperm proteins SPAG6,

SP17, and CATSPER2 are localized in canine sperm and that they can potentially be

biochemical markers for canine fertility to predetermine if the sperm from an individual

canine will be a potentially good semen sample to undergo density gradient

centrifugation, functional tests, and semen preservation. With these techniques

combined, the chances of accurately predicting fertility of fresh and preserved sperm.

Above all these techniques must provide an accurate prediction of fertility, be objective,

repeatable, inexpensive, fast, and easy to do in a clinical or laboratory setting (Amann

1989; Rodriguez-Martinez 2003).

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BIOGRAPHICAL SKETCH

Tameka Cerise Phillips was born in Urbana, Illinois. She is the second of two

children that were born ten years apart. She has always had a fascination with animals

and the sciences. She was accepted into the University of Illinois at Urbana-Champaign

in 1994. She graduated with a bachelor’s degree in animal sciences in 1999. While an

undergraduate student, she developed a strong interest in reproductive physiology. In

1999, she was offered a master’s degree program on fellowship from the University of

Florida, College of Veterinary Medicine. In the spring of 2000, she began her master’s

degree program, in which she completed in 2001. She moved back to Illinois for a few

years before the decision was made for her to return to the University of Florida,

College of Veterinary Medicine to start a Ph.D. program. Upon the complete of Ph.D.

degree, Tameka plans to begin her career in science and government.