THESIS 

CRYOPRESERVATION OF COOLED SEMEN FOR EQUINE 

INTRACYTOPLASMIC SPERM INJECTION 

Submitted by 

Bradford W. Daigneault 

Department of Biomedical Sciences 

In partial fulfillment of the requirements 

For the Degree of Master of Science 

Colorado State University 

Fort Collins, Colorado 

Spring 2010 

: 

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SF291 
.D355 
201 0 

COLORADO ST ATE UNIVERSITY 

December 16th, 2009 

WE HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER OUR 

SUPERVISION BY BRADFORD WILLIAM DAIGNEAULT ENTITLED 

CRYOPRESERVATION OF COOLED SEMEN FOR EQUINE 

INTRACYTOPLASMIC SPERM INJECTION BE ACCEPTED AS FULFILLING IN 

PART REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE. 

Committee on Graduate Work 

Graham 

~:?:;;?: 
David J Denniston 

( 

Advisor Elaine M Carnevale 

ting Department Head Thomas R Hansen 

.COLORADO STATE _{=1NIVER ITY LIBRARIES 
11 .. 



ABSTRACT OF THESIS 

CRYOPRESERVATION OF COOLED SEMEN FOR EQUINE 

INTRACTYOPLASMIC SPERM INJECTION 

The development of intracytoplasmic sperm injection (ICSI) for use in the horse 

has resulted in unique needs for semen. Modifications of ways to handle sperm for ICSI-

related procedures were investigated through a series of three experiments. In 

Experiment 1, semen was cooled for 24 h and then frozen to provide a viable population 

of sperm for ISCI. The objectives of this study were to compare the efficacy of: (1) two 

extenders for cooling semen prior to freezing and (2) four extenders for freezing cooled 

semen. Stallion semen was extended in two commercial cooling extenders (CST and 

INRA96) and cooled for 24 h before freezing in four extenders. Freezing extenders were 

a modified- French (FR8), Lactose-EDT A (LAC 10), glycerol (G), and glycerol egg yolk 

(GEY). Extenders were added directly to cooled semen and diluted to a final 

concentration of 20 x 106 sperm/mL before freezing. After thawing, sperm were 

evaluated at 0, 45 and 75 min. Cooling extender did not affect sperm motility after 

freezing. Total motility and progressive motility was similar (p>0.05) for all freezing 

extenders at O and 45 min, but sperm frozen in glycerol egg yolk (GEY) yielded the 

highest total and progressively motile sperm at 75 min. Differences in motility over time 

l1l 



were not detected except for sperm frozen in glycerol (G) in which total and progressive 

motility declined from Oto 75 min. For all extenders, some motile sperm were obtained 

from semen cooled for 24 h and then frozen. 

In Experiment 2, five media were evaluated for holding sperm to determine which 

best maintained the motility of stallion sperm over time. The experiment was designed to 

determine appropriate media in which to handle sperm for ICSI selection procedures. 

The media compared were: 1) GIVF, 2) FDCM, 3) TALP, 4) Emcare (EM) and 5) Mare 

Mojo (MM). The only media designed for holding sperm was T ALP; the remaining 

media were formulated as holding media for oocytes and embryos. Motility of sperm 

was analyzed every hour for 5 h. No differences in total or progressive motility were 

detected among groups until 5 h. At 5 h, sperm held in TALP had higher (p<0.05) total 

and progressive motility than all other groups. Sperm can be held in any of the five 

media evaluated and remain motile for ICSI. However, T ALP resulted in the best sperm 

motility over time. 

Experiment 3 was performed to evaluate a hyaluronic acid (HA) binding assay for 

the selection of sperm for ICSI. Sperm binding to hyaluronic acid has been characterized 

for many species. Sperm that bind to HA have been shown to exhibit more normal 

chromosomal structures and improve fertilization. The objective of Experiment 3 was to 

determine if fresh and frozen stallion sperm bind to HA when introduced to 

premanufactured hyaluronic acid binding kits (HBA, Biocoat Inc., Horsham, PA). Fresh 

semen pooled from two stallions exhibited approximately 15% of sperm bound to HA. 

Four frozen samples from Experiment 1 were thawed and pooled. Frozen sperm did not 

bind to the assays. For fresh and frozen samples, sperm displayed some hyperactivation, 

lV 



uncharacteristic circling, and tails bent at the mid-piece. Sperm were observed to 

agglutinate in congregations of fifty or more sperm. Hyaluronic acid seemed to have an 

effect on stallion sperm that has not been described for other species. Further 

investigation of HA binding for selection of sperm for ICSI is warranted. 

V 

Bradford W. Daigneault 
Department of Biomedical Sciences 

Colorado State University 
Fort Collins, Colorado 80523 

Spring 2010 



ACKNOWLEDGMENTS 

The work that I have accomplished herein would not have been possible without 

the advising and friendship of the many people that I have encountered over two years at 

this laboratory. My gratitude is extended toward my advisor Dr. Elaine Carnevale and 

my committee members Dr. Jason Bruemmer, Dr. James K. Graham, and Dr. David J. 

Denniston. I wish to thank all those people who have helped me through my work, 

including those who may not have played a direct role in research, but rather supported 

me through friendship and advice. 

This journey would not have been a success without prior preparation from years 

past. I wish to formally thank coaches, friends, immediate and extended family, as well 

as employers who gave me the chance to succeed through their generous and unselfish 

gifts that helped me to grow as a person. 

Finally, I owe all of my accomplishments to my wife Sarah, who has persevered 

with me for over six years through the good and the bad as I worked to further my 

education. I have only succeeded through her selfless support and undying love. 

Vl 



TABLE OF CONTENTS 

Page 
CHAPTER I Review of Literature 

Introduction 

A. Cooled Semen 2 
B. Cooling Extenders 3 
C. Cold Shock for Cooled and Frozen Semen 5 
D. Cooling Rates 6 
E . Freezing Extenders 7 
F. Cooling Rates for Freezing Sperm 9 
G. Seminal Plasma 12 
H. Processing Sperm for ICSI 14 
I. Fertilization Capability of Frozen Sperm for ICSI 17 

I 

J. HspA2 & Hyaluronic Acid Binding Glycoprotein 19 
K. Use ofHyalurnoic Acid for ICSI 22 
L. Methods of Sperm Evaluation 26 
M. Conclusions and Experiments 28 

CHAPTER II Cryopreservation of cooled semen for equine 
intracytoplasmic sperm injection 31 

Introduction 31 
Methods and Materials 33 
Results 36 
Discussion 36 

CHAPTER III Effect of five holding media on motility of 
stallion sperm 45 

Introduction 45 
Methods and Materials 46 
Results 47 
Discussion 48 

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CHAPTER IV Evaluation of a hyaluronic acid sperm binding assay 
for equine intracytoplasmic sperm injection 53 

Introduction 53 
Methods and Materials 55 
Results 57 
Discussion 58 

CHAPTER V Summary 60 

CHAPTER VI References 62 

CHAPTER VII Appendices 69 

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FIGURES AND TABLES 

Figure Number Page 

1. Diagram of experimental design for Experiment 1 40 

2. Time points for evaluation of controls and frozen straws 41 

3. Mean total motility of semen cooled for 24 h, frozen in 
four extenders and warmed for evaluation of motility at 
0, 45 and 75 min 42 

4. Mean progressive motility of semen cooled for 24 h, 
frozen in four extenders and warmed for evaluation of 
motility at 0, 45 and 75 min 43 

5. Means ± SEM of total and progressive motility over 
time for sperm frozen in glycerol (G) and evaluated at 
0, 45 and 75 min post thaw 44 

6. Mean total motility for sperm held in five media for 5 h 50 

7. Mean progressive motility for sperm held in five media for 5 h 51 

8. Percentage of total motile sperm extended to a concentration 
of 50 x 106 total sperm/mL and held in five media for 5 h 52 

9. Percentage of progressively motile sperm extended to a 
concentration of 50 x 106 total sperm/mL and held in five media for 5 h 52 

lX 



TABLE OF APPENDICES 

Appendix Number Page 

I. Recipes for freezing diluents for Experiment 1 and raw data 69 

II. Recipe for T ALP diluent for Experiment 2 and raw data 83 

III. Proposal and protocol for application of a sephadex/glass wool 
column to select frozen sperm for ICSI 89 

IV. Protocol to select frozen sperm at a low concentration for ICSI 
using a modified swim-up technique 91 

X 



Introduction 

CHAPTER I 

REVIEW OF LITERATURE 

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technology 

that has gained a great amount of success in recent years when applied to the mare. The 

use of ICSI in the horse has resulted in oocyte cleavage rates of 60% and blastocyst 

development of up to 30% (Squires et al. 2003). When compared to other assisted 

reproductive procedures, ICSI provides the most conservative use of sperm (Carnevale 

2008). The frequency of using frozen sperm for ICSI is as high as 85% (Carnevale 

2008). Therefore, improving upon the efficiency of using frozen-thawed sperm for ICSI 

is an important objective. A means of processing shipped, cooled semen for freezing 

would provide a more cost-effective method for obtaining sperm for clinical programs. 

Methods to process and cryopreserve shipped, cooled semen for use in an equine 

ICSI program have yet to be established. The processing and packaging of cooled semen 

for freezing at low concentrations could provide for extended use of limited amounts of 

semen. Packaging semen by means of individually segregated portions will allow for 

utilization of smaller amounts of semen without liquid nitrogen exposure to the unused 

portion. Methods developed to accomplish these goals will provide a more efficient 

means of utilizing equine semen for ICSI. 

1 



Determining which freezing extender will provide the highest post-thaw motility 

for individual stallions is difficult unless test freezes are performed. Although 

performing a test freeze is beneficial for freezing semen from stallions with unknown 

freezing potential, a test freeze may be unrealistic when considering semen that has been 

cooled and shipped unless performed at a very low concentration. 

Cooled Semen 

Improved methods for processing cooled semen to ensure maximal survival and 

motility are necessary in the equine industry. Semen used for insemination at a location 

distant to the stallion is often shipped in a passive cooling device that cools semen to 5°C. 

Semen can be held for a period of 24 to 48 h while maintaining acceptable motility for 

most stallions (Jasko et al. 1992). Cooled semen for insemination is commonly shipped 

at concentrations of 50 x 106sperm/mL (Crockett et al. 2001 ). 

One effect of cooling semen to 5°C is a change in sperm membranes from a 

liquid-crystalline state to a gel state (Squires et. al 1999). Byproducts of metabolism such 

as CO2 and lactic acid can cause permanent damage to sperm if they remain at room 

temperature. The occurrence of peroxidation of lipid membranes, or oxidation of lipids 

in the cell membrane, can lead to membrane damage and reduced fertilization capability 

after a period of 12 to 24 h (Squires et al. 1999). Every 10°C reduction in temperature of 

sperm results in a 50% decrease in metabolic activity. Consequently, semen held at 5°C 

metabolize at about 10% of what they would when stored at 38°C, the normal body 

temperature of a horse (Squires et al. 1999). Due to a decrease in metabolic activity, the 

lifespan of sperm can be lengthened. Decrease in metabolism allows storage of sperm for 

2 



a prolonged period of time when cooled at a controlled rate in a medium that has 

membrane-protective properties (Squires et al. 1999). 

The viability of cooled stallion sperm for future use is stallion dependent (Aurich 

2008). Not all stallion semen can be cooled and preserved to provide acceptable 

parameters for insemination (Brinsko et al. 2000). Quality of semen, composition of 

seminal plasma and integrity of plasma membranes are contributing factors influencing 

the ability to maintain cooled semen (Aurich 2005). The sensitivity of individual 

stallions' semen to cooling may render the use of cooled semen from individual stallions 

unrealistic for insemination regardless of cooling extender (Aurich 2008). However, 

enough sperm cooled from these stallions may remain viable for ICSI. 

Cooling Extenders 

Cooling extenders provide nutrients, a balance of minerals, and ensure proper 

cellular osmotic pressure. Extenders help to neutralize byproducts of metabolism that 

can be toxic, buffer temperature changes, and help to stabilize enzyme systems as well as 

membrane integrity (Squires et al. 1999). The addition of a cooling extender to semen 

can also be a useful means of introducing antibiotics. 

The most common semen extenders include milk, milk by-products, and buffers 

to help regulate pH and osmolarity. In 1975, Kenney et al. reported the successful use of 

dried skim milk extender for stallion sperm. Subsequent additives to stallion sperm have 

attempted to investigate the efficacy of additives to cooling extenders for improving 

fertility and survivability. The addition of glutamine to INRA82 cooling extender 

significantly increased motility compared to samples without glutamine ( 49% versus 

3 



46%) but did not alter fertility (Vidament et al. 2001). Bedford et al. (1994) noted a 

detrimental effect on progressively motile sperm when egg yolk was added to semen 

containing seminal plasma before cooling. Upon removal of seminal plasma, the addition 

of egg yolk or glycerol added to a nonfat dried skim milk-glucose (NDSMG) extender 

did not depress motility of sperm but did negatively affect fertility. Although many 

additives have been applied to cooling extenders, there has yet to be defined a superior 

cooling extender that works for all stallions. 

Milk or milk-based extenders continue to be commonly used for diluting and 

storing stallion semen. Milk contains more than 100,000 complex molecules. These 

molecules have been speculated to be both beneficial and deleterious to sperm (Battelier 

et al. 2001 ). Milk can be fractionized by microfiltration, ultrafiltration, and freeze-drying 

which allows for purification of fractions. The equine cooling extender INRA96 was 

developed based on this science (Battelier et al. 2001 ). 

INRA96 is supplemented with native phosphocaseinate (NPCC) and formulated 

based on Hanks ' salts, a collective group of salts with bicarbonate ions that helps to 

buffer pH (Battelier et al. 2001). Native phosphocaseinate is a milk fraction composed of 

micellar caesins and has been found to be protective to stallion sperm along with P-

lactoglobuline, a major soluble protein. The protein, a-lactoglobuline, has been identified 

as harmful to sperm and has been removed from some sperm diluents (Battelier et al. 

1997). Native phosphocaseinate was reported to be the most efficient fraction at 15°C in 

aerobic conditions when compared to milk-based extenders held at 4°C for maintaining 

sperm motility (Battelier et al. 1997). These findings ultimately led to the development 

of INRA96, often referred to as a chemically-defined extender. Sperm held at l 5°C in 

4 



INRA96 extender in aerobic conditions resulted in higher motility than milk-based 

extender. However, sperm held at 4°C in anaerobic conditions with a milk-based 

extender was superior to INRA96 at the same conditions when evaluated for motility 

after 4 days of storage (Battelier et al. 1997). Although sperm can be cooled and 

maintained for several days, motility of sperm could be improved upon with a shorter 

cooling time such as 24 h. 

Cold Shock for Cooled and Frozen Semen 

Methods to prevent cold shock, or cooling stress, reduce cellular damage and 

ensure post-thaw survivability during the cooling process. Cooling semen at a rapid rate 

can cause membranes to rupture, resulting in permanent cellular damage that can be 

observed after cooling and thawing (Squires et al. 1999). Abnormal sperm motility 

patterns can include circling, backward motility, and loss of motility. Acrosomal and 

plasma membrane damage as well as the absence of cellular components can also be seen 

with sperm subjected to cold shock (Squires et al. 1999). Lipids and lipophilic molecules 

are additives that help to protect the plasma membrane during the transition from the 

fluid phase to the solid phase during cooling. Ethylenediaminetetraacetic acid (EDT A) is 

utilized to bind calcium, magnesium, and other ions which likely function to stabilize ion 

diffusion across membranes during transition phases (Squires et. al 1999). Egg yolk is 

commonly used as a lipid additive in semen extenders to aid in the protection of the lipid 

membrane. Phospholipids, such as low-density lipoproteins present in egg yolk, aid in 

maintaining the sperm membrane during cooling. Lipoproteins in skim-milk extenders 

are commonly used to prevent cold shock and are thought to function similary to the 

5 



lipoproteins found in egg yolk (Squires et al. 1999). The phospholipids that are abundant 

in milk and egg yolk have been proposed to produce structural modifications to cell 

membranes that could help to prevent temperature-related damage (Varner et al. 1987). 

However, the amount of energy that would be necessary for lipid exchange is unlikely 

present (John Parrish; personal communication). Caseins, that are present in milk and 

have been purified for use in the cooling extender INRA96 (IMV Technologies, 

Normandy, France), have been shown to bind strongly with Ca++. It has been postulated 

that they prevent accumulation of intracellular Ca++ that can be toxic to sperm at high 

concentrations (Quinn and White 1967). 

Cooling Rates 

Different species have varying responses to cooling rates for sperm. The response 

of sperm to cold shock affected by cooling rate may be attributed to differences in plasma 

membrane compositions (Varner et al. 1987). Plasma membranes characterized by high 

levels of cholesterol, or an increased ratio of saturated to unsaturated phospholipids, are 

better able to withstand cold shock (Varner et al. 1987). The use of passive cooling 

devices for storage and transport of semen has been studied in the stallion to address 

these concerns. 

Cooled stallion sperm survive best when stored at a temperature range of 4 to 6°C 

(Moran et al. 1992). Membrane damage of stallion sperm is often associated with 

improper cooling rates. During cooling, stallion sperm are susceptible to cold shock 

between temperatures of 24 to 4 °C (Moran et. al 1992). Stallion sperm can be cooled 

rapidly (-7°C/minute) to l 9°C without increased cold shock damage when compared to 

6 



slow cooling (Moran et al. 1992). Rapid cooling did not induce cold shock when sperm 

reached a temperature of S°C (Moran et al. 1992). The hypothesis for the above 

experiment was that slow cooling at a rate of -0.05°C/min would allow adequate time for 

membrane reconstruction, which has presumably taken place during this phase transition 

stage (Moran et al. 1992). Stallion sperm are most susceptible to cold shock induced by 

rapid cooling from 19 to S°C. Sperm should be cooled at a slow cooling rate during this 

time interval (Moran et al. 1992). 

Freezing Extenders 

Stallion sperm was first frozen in 1950 (Smith and Polge 1950). The first equine 

pregnancy from frozen sperm was reported in 1957 (Barker and Gandier 1957). 

Glycerol is the most common cryoprotectant used for freezing sperm in a number of 

species, including the horse. The concentration of glycerol observed in stallion freezing 

extenders ranges from 2.5 to 6% (Vidament et al. 2001). However, glycerol toxicity 

could be a reason for variations observed with individual stallion freezability (Alvarenga 

et al. 2005). Furthermore, glycerol has been shown to have contraceptive effects for 

some species. Although glycerol functions as a moderator of cellular osmolarity, it can 

also cause damage to sperm that has already been compromised during the freezing 

process. 

Protein denaturation, changes in actin interactions, and changes in cytoplasmic 

properties are a few byproducts of glycerol that can induce damage to cells (Alvarenga et 

al. 2005). Due in part to these findings and the need to increase the success of freezing 

stallion sperm, alternative cryoprotectants, such as amides, have been investigated and 

7 



utilized in a commercial industry (Alvarenga et al. 2005). These alternative 

cryoprotectants have a lower molecular weight and greater membrane permeability than 

glycerol, which help to minimize osmotic damage to sperm (Scherzer et al. 2009). 

Scherzer et al. (2009) designed a randomized 2 x 3 block experiment to compare 

differences in motility when using a skim-milk glucose extender (EZ Mixin Original 

Formula) or a milk-free extender (INRA96). These extenders were used in combination 

with 2, 3 or 4% glycerol for freezing. There was a difference between the two extenders 

in motility of sperm after cryopreservation, with INRA96 being superior. When glycerol 

was added at a concentration of 4%, the highest post-thaw motility was observed. The 

addition of 4% glycerol was significantly different than 2% glycerol. In conclusion, 

INRA96 when combined with 4% glycerol provided the highest average post-thaw 

motility (Scherzer et al. 2009). However, sperm for this experiment was cooled for 1 h to 

4°C and then centrifuged at 4°C to remove seminal plasma prior to freezing . This 

method is not common practice when cooling stallion sperm for commercial purposes. 

Motility may be improved by centrifuging semen at room temperature rather than after 

cooling. The alternative method has been shown to be deleterious to sperm (Backman et 

al. 2004). 

Although egg yolk has been added to extenders at concentrations ranging from 2 

to 20%, the optimal concentration has not been clearly defined for a number of freezing 

extenders (Vidament et al. 2001). An additional class of additives shown to have positive 

effects on improving post-thaw motility is the addition of amino acids. Glutamine is an 

example of an amino acid that improves frozen semen parameters across a variety of 

species including the stallion (Vidament et al. 2001 ). 

8 



Cooling Rates for Freezing Sperm 

The use of a cryoprotectant is essential to ensure survivability when freezing 

stallion sperm, as is the rate of temperature decline at which semen is cooled. In order to 

fully understand the effects of freezing on stallion sperm, measuring the biophysical 

response, water transport, and intracellular ice formation in sperm during freezing must 

be addressed (Devireddy et al. 2002). 

The addition of cryoprotectant helps to ensure that ice nucleation is formed in the 

extracellular space. A cryoprotectant, such as glycerol, creates an osmotic gradient 

across the plasma membrane to draw water out of intracellular solution that was initially 

isotonic. A fast or slow cooling rate can determine if intracellular water will move across 

the cell membrane to become part of the extracellular ice phase, or if it will remain inside 

the cell and freeze as ice crystals (Devireddy et al. 2002). 

There are differences in membrane permeability between cell types. Intracellular 

ice formation is dependent on different cooling rates between cells and media. Therefore, 

knowing the cell membrane ' s permeability to water in the presence of extracellular ice 

and cryoprotectants is essential (Devireddy et al. 2002). Generally, cells that undergo 

intracellular ice formation become osmotically inactive or lysed due to the loss of cell 

membrane integrity (Mazur 1984). The opposite occurs, but with the same effect, when 

too much water leaves the cell because of exposure to high solute concentrations 

(Lovelock 1953). Therefore, the cooling rate must be optimized to ensure cell survival. 

The best cooling rate to optimize the freeze/thaw results of any cellular system 

has been defined as the fastest cooling rate in a given medium that precludes the 

formation of damaging intracellular ice (Mazur et al. 1972). Stallion sperm viability 

9 



remains constant when cooled at a rate between 20°C/min and 130°C/min (Devireddy et 

al. 2002). If cooling is too rapid, cells freeze intracellularly because an insufficient 

amount of water has left the cell (Mazur 1984). In contrast, slow freezing can cause ice 

to form outside the cells, and the remaining unfrozen medium forms channels that shrink 

in size and consequently increase in solute concentration. The cells trapped inside these 

channels also begin to shrink due to an osmotic response elicited by the high 

concentration of solute in the gradient. Ultimately, these changes can cause harm to the 

cells (Mazur 1984). 

Semen cooled for transport is not cooled below 4°C; because between -5 °C and 

-l 5°C, ice begins to form in the external holding medium. The cell contents are not 

frozen, but they are supercooled due to the protective properties of the plasma membrane 

(Mazur 1984). Changes elicited in sperm depend on the velocity of cooling. When 

subjected to a sufficient slow cooling process, the cells can adequately remove enough 

water to maintain equilibrium between intracellular and extracellular water. The cell 

dehydrates and does not freeze intracellularly. However, if the cell is cooled too fast, it 

becomes increasingly supercooled, and the only way to obtain equilibrium is by freezing 

intracellulary because water is not able to leave the cell at a fast enough rate (Mazur 

1984). Consequently, similar results can be observed when thawing semen. Slowly 

frozen sperm cells are sensitive to rapid thawing due to osmotic shock. Solute loading 

into the cell during slow cooling may not have enough time to diffuse out if a cell is 

subjected to a rapid thaw, causing the cell to swell and ultimately lyse (Mazur 1984). 

A common misconception is that cells are most challenged when stored at low 

temperatures. An intermediate zone of approximately -l 5°C to -60°C has the most 

10 



potential lethality, because cells must pass through that zone twice (Mazur 1984). Sperm 

are commonly stored in liquid nitrogen at -196°C, because liquid water does not exist 

below -130°C. Only crystalline or glassy states exist below this temperature in which the 

viscosity is too high for diffusion. Sperm is protected indefinitely when stored in liquid 

nitrogen. At -196°C, an insufficient amount of thermal energy exists for chemical 

reactions. The only reactions that occur at such a temperature are photophysical 

reactions. These reactions are breaks in macromolecules, which are caused by ionizing 

radiation or cosmic rays. Such reactions could ultimately cause enough breaks in DNA 

to be detrimental to sperm after thawing (Mazur 1984). 

The properties of cell membranes play critical roles during the freezing process. 

As previously discussed, the structure of the surface membrane functions to protect the 

cell during the cooling process and allows it to supercool. This membrane most likely 

determines the ice-nucleation temperature for a particular cell. These two major 

components ultimately determine if cells will equilibrate with extracellular medium via 

dehydration or by intracellular freezing (Mazur 1984). Membranes on the surface of cells 

as well as the interior are main subjects to injury. Furthermore, membranes allow 

permeability to the cell allowing access to protective solutes during the freezing process. 

Depending on the cell type, most cryoprotectants and solutes are low in molecular 

weight, allowing permeabilization into the cell. If additives are not able to permeabilize, 

they have the potential to induce osmotic stress. Lastly, damage caused to intracellular 

organelles such as mitochondria and microtubules are detrimental to cell survivability 

(Mazur 1984). 

11 



Seminal Plasma 

Seminal plasma has both detrimental and beneficial effects on frozen stallion 

semen (Jasko et al. 1991 , Dawson et al. 1999). Several studies have been conducted to 

investigate the significance of removing seminal plasma from semen and the 

concentrations of seminal plasma that can be added back to sperm for freezing. Seminal 

plasma must be diluted to obtain maximum sperm motility and survivability after 

cooling. When seminal plasma is removed, egg yolk in an extender is beneficial when 

cooling for 24 h (Bedford et al. 1995). Similar results were obtained when egg yolk 

extenders were compared to skim milk for a 24 h cooling period (Jasko et al. 1992). 

Completely removing seminal plasma before cooling has been shown to be detrimental. 

A concentration of 5 to 10% seminal plasma in semen maintains sperm motility and 

acrosome integrity (Jasko et al. 1991 , Dawson et al. 1999). 

The speed, timing, and temperature of sperm during centrifugation have been 

investigated. When semen is cooled before freezing, optimal survival is achieved by 

centrifuging semen upon collection before cooling to 5°C at 200 x 106sperm/mL 

(Backman et al. 2004). Sperm processed in this manner without the addition of glycerol 

for 18 h before freezing provided the best results regarding post-thaw motility of 

previously cooled sperm (Backman et al. 2004). Also, sperm survived better when frozen 

at 200 rather than 400 x 106sperm/mL (Backman et al. 2004) . 

Semen should not be centrifuged after cooling (Backman et al. 2004). Warming 

semen after cooling and centrifuging before freezing has been shown to result in 

detrimental effects on motility of frozen sperm (Backman et al. 2004). Low-level forces 

12 



-

of centrifugation (370 to 829 x g) are necessary to avoid detrimental effects on equine 

sperm with the presence of 10% seminal plasma (Pickett et al. 1975). 

Freezing stallion sperm that has been previously cooled can be accomplished 

(Crockett et al. 2001 ). An appropriate extender for cooling, packaging time, removal of 

seminal plasma, concentration and length of time after collection are described (Crockett 

et al. 2001). Semen was first centrifuged at 300 x g for 8 min then cooled to 5°C. The 

sample was held for 24 h in a skim milk extender before cryopreservation. Post-thaw 

motilities were similar for skim milk-egg yolk-sugar extender and skim milk-egg yolk-

salt extender (Crockett et al. 2001). All extenders contained 10% glycerol. Progressive 

motilities averaged 10% and were considered unacceptable for insemination. The overall 

conclusions for this experiment were: semen could not be held at 5°C for 24 h prior to 

freezing; the addition of egg yolk aided post-thaw motility; and packaging cooled 

samples into 0.25-mL versus 0.5-mL straws did not change motility (Crockett et al. 

2001). Furthermore, freezing semen that was centrifuged before cooling resulted in 

improved motility when compared to centrifuging after cooling (Crockett et al. 2001). 

Similar results have been obtained by other researchers (Backman et al. 2004). Based on 

the above-described experiments, conventional methods exist for shipping cooled semen 

for the purpose of freezing. 

Optimal concentrations for freezing stallion sperm combined with the absence or 

presence of seminal plasma have been described (Crockett et al. 2001 ). The effects on 

sperm cooled to 5°C for 12 h at three different concentrations followed by freezing were 

examined. Egg yolk helped to maintain sperm motility after freezing and thawing and 

stallion sperm should be centrifuged to remove seminal plasma at 25°C. Sperm should 

13 

" 
II 

I 

1, 



be resuspended to 50 x 106sperm/mL or 250 x 106/mL for shipping up to 12h. If sperm 

are to be stored for more than 2.5 h before freezing, they must be centrifuged to remove 

seminal plasma at 25°C (Crockett et al. 2001). This information provides insight to 

diluting shipped, cooled semen into small aliquots to be frozen for later use in an ICSI 

program. 

Processing Sperm for ICSI 

Methods of processing sperm for ICSI have been explored with fresh, cooled, 

and frozen equine sperm. Sperm have been thawed and refrozen at low concentrations to 

help preserve limited amounts of sperm (Choi et al. 2005). Choi and colleagues thawed 

frozen sperm from a fertile stallion and diluted the sample 1: 100 (EZ Mixin ™ MFRS 

and LE; Animal Reproduction Systems, Chino CA). Sperm were refrozen in 0.5-mL 

straws at a final concentration of 2 million sperm/mL. Refrozen sperm diluted with both 

extenders provided motile sperm that could fertilize oocytes using intracytoplasmic 

sperm injection. Thawed and refrozen samples did not contain enough sperm for a swim-

up; therefore, the samples were washed, centrifuged, and resuspended in the remaining 

medium after aspiration of the supernatant. Motile sperm were then placed on top of 

Chatot-Ziomek-Bavister medium modified for sperm (Sp-CZB) containing 10% 

polyvinylpyrrolidone (PVP). This method allowed sperm that swam into the droplet to 

be visualized and selected for ICSI. The dilution of semen to 2 million sperm/mL 

allowed harvesting of sufficient numbers of motile, functional sperm for ICSI. The same 

results were obtained for both a fertile and subfertile stallion, albeit the subfertile stallion 

had sperm with 3% total motility after thawing (Choi et al. 2005). This data is 

14 



convincing evidence that sperm may be diluted to low concentrations and cryopreserved 

to provide adequate numbers of motile sperm for ICSI. 

Frozen sperm have been prepared for ICSI using swim-up procedures (Altermatt 

et al. 2009). The following method describes utilization of a minimal number of sperm 

frozen in a 0.5-mL straw by cutting a portion (~5mm) of the straw under liquid nitrogen. 

This technique preserves the remaining portion of the straw (Altermatt et al. 2009). The 

cut portion of the straw is introduced into a round-bottom tube containing 1 mL of the 

holding medium F-CDM (CDM (Olsen and Seidel 2000) supplemented with 0.5% fatty 

acid-free bovine serum albumin 2 mM caffeine and 2µg mL-1 heparin). The tube is placed 

into incubation at 38.5°C at a 45° angle for 20 min in 6% CO2 and air. A total of 500 µI 

of supernatant is aspirated from the top of the tube and placed into 2 mL of pre-

equilibrated F-CDM for centrifugation at 300 x g for 5 min at room temperature. 

Following centrifugation, 1 µl of sperm pellet is placed in 5 µl of G-MOPS (Vitro life AB, 

Goteborg, Sweden) containing 0.5% bovine serum albumin and 5% 

polyvinylpyrrolidone. Sperm is then selected for injection based on morphology and 

motility characteristics (Altermatt et al. 2009). 

The use of a Percoll gradient to select frozen sperm for ICSI has been successfully 

employed in the horse (Lazzari et al. 2002). The protocol includes thawing frozen sperm 

1 hr prior to injection followed by separation on a 45-90% Percoll gradient. The sperm 

pellet is diluted to a concentration of 4 million sperm/mL and then 1: 1 with a 12% PVP 

solution in synthetic oviductal fluid. The medium is supplemented with BSA, amino 

acids, heparin and penicillamine, hypotaurine, and epinephrine. One motile sperm is then 

selected in PVP medium after immobilization by Piezo pulses (Lazzari et al. 2002). 

15 



The description of methods to freeze semen for ICSI from taxonomic species 

related to the horse has potential application. Such information has been described for 

the rhesus monkey (Yeoman et al. 2005). Semen was collected via penile 

electroejaculation from nine rhesus monkeys and subjected to four freezing treatments. 

Prior to freezing, semen was allowed to liquefy for 10-15 min followed by two washes in 

HEPES-buffered TALP containing 0.3% BSA. The four treatments consisted of: 1) 

extension in a medium consisting of 6 % glycerol and 0.1 M sucrose followed by cooling 

to 4°C for 90 min and floating over liquid nitrogen (LN2), 2) dilution in a 30% egg yolk, 

20% skim milk, 0.06 M glucose medium and 3% glycerol followed by 60 min 

equilibration at 4°C and frozen in LN2, 3) same as treatment two with the addition of a 

post-thaw density gradient centrifugation using Percoll, 4) modified freezing protocol 

using dry ice before plunging into LN2 for storage. Reduced fertilization unrelated to 

post-thaw motility was observed when sperm was frozen in LN2 vapors; however, 

blastocyst development was not different among treatments (Yeoman et al. 2005). 

Freezing sperm in drops on dry ice resulted in similar fertility rates to fresh semen. The 

slower cooling rates of -60 to -85°C/min, compared to LN2 vapor of -150°C/min is 

attributed to the different success rates (Yeoman et al. 2005). A total of 23 pregnancies 

have been achieved to date using this method of cryopreservation (Yeoman et al. 2005). 

The acrosomal status of fresh sperm is thought to affect fertilization rates 

achieved by ICSI (Mansour et al. 2008). Increasing the incubation time of human sperm 

perhaps increases the rate of acrosome reaction, thus increasing fertilization rates 

(Mansour et al. 2008). Acrosome reacted sperm may have decreased sperm chromatin 

decondensation rates (Mansour et al. 2008). When acrosome reaction is induced, 

16 



electron microscopy shows the rate of acrosome reaction is time dependent (Mansour et 

al. 2008). Fertilization achieved by ICSI is most successful when sperm are incubated in 

tissue culture medium for 3 hours (Mansour et al. 2008). 

Fertilization Capability of Frozen Sperm for ICSI 

Changes that could affect the ability of a frozen-thawed sperm to fertilize an 

oocyte after ICSI are not well defined and are theoretical in nature. Exempting physical 

and cellular changes that have already been described, specific fertilization factors could 

be impacted during the freezing process. Certain sperm-borne, oocyte-activating factors 

that are generally insoluble may be compromised during freezing and thawing (Yeoman 

et al. 2005). 

Equine oocytes in metaphase II of meiotic arrest require a number of factors to 

initiate fertilization. These factors include a decrease in maintenance of maturation 

promotion factor (MPF) composed of a complex of p34cd2 kinase and cyclin B (Meng and 

Wolf 1997). The entry or injection of a sperm into the oocyte results in cyclin B 

degredation and a decrease in MPF activity allowing the oocyte to activate (Y eng and 

Wolf 1997). Sperm-induced changes in intracellular calcium concentrations function to 

initiate oocyte activation (Meng and Wolf 1997). Sperm-induced calcium changes in the 

oocyte could be initiated when sperm bind to the oocyte membrane receptor, resulting in 

G-protein and phospholipase C activation and the production of inositol 1,4,5-

triphosphate, a calcium-releasing agent (Meng and Wolf 1997). 

A second hypothesis that may be relative to ICSI with frozen sperm involves a 

soluble cytostolic factor, oscillin, which has been characterized in several mammalian 

17 

I! 

1, 



species (Meng and Wolf 1997). The protein, oscillin, has been shown to induce Ca2+ 

oscillations that could aid in oocyte activation (Meng and Wolf 1997). The loss of 

structural integrity to oscillin during freezing and thawing of sperm could implicate 

fertilization. 

Incomplete or premature chromosome decondensation of sperm contribute to 

possible failure of oocyte activation (Sousa and Tesarik 1994). Although ICSI 

circumvents oocyte activation via the sperm-oocyte binding process, micromanipulation 

of the sperm and the physical act of injection into the oocyte are thought to aid in the 

release of sperm factors contributing to oocyte activation and fertilization (Yeoman et al. 

2005). 

Freezing and thawing of sperm could reduce chromosomal compaction of sperm, 

possibly contributing to fertilization failure (Hammadeh et al. 1999). Fertilization can 

still be achieved after intentional manipulation of protamines reducing chromosomal 

compaction (Ahmadi and Ng 1999). Sperm exposed to varying levels of gamma 

radiation prior to insemination in a mare were verified for DNA-breakage via a TUNNEL 

assay. No significant differences were detected for fertilization rates between the control 

group and those exposed to varying degrees of radiation. However, blastocyst 

development was 50% in the control group and 20% for sperm exposed to gamma 

radiation at the lowest treatment level. Like correlations for implantation and fetal 

development were observed (Ahmadi and Ng 1999). The study demonstrated that DNA-

damaged sperm have the ability to fertilize oocytes, but embryonic development is highly 

related to the degree of sperm DNA damage. These findings attribute to the ability of the 

oocyte to repair DNA of sperm with less than 8 % damage (Ahmadi and Ng 1999). 

18 



HspA2 and Hyaluronic Acid Binding Glycoprotein 

The testis-expressed heat shock protein chaperone (HspA2) is a 70 kDa protein 

formerly known as creatine kinase M isoform. This protein can be used as a measure of 

sperm cellular maturity, function and fertility in humans. The testis-expressed heat shock 

protein is expressed in elongating spermatids and is associated with the formation of 

hyaluronic acid (HA) and zona pellucida binding sites as well as plasma membrane 

remodeling. When the HA receptor of mature human sperm are introduced to HA-coated 

slides and/or Petri dishes, microscopic observation reveals sperm that are bound to HA, 

thus providing for selection of mature sperm that can be selected for use in ICSI. HA-

selected sperm for ICSI exhibited a 4 to 6-fold reduction in the frequency of 

chromosomal disomy and diploidy compared to sperm not selected using this method 

(Huszar 2007). 

The testis-expressed heat shock protein exhibits a two-wave expression. The first 

wave is seen in spermatocytes as a meiotic component within the synaptonemal complex. 

The second wave is exhibited during terminal spermiogenesis in elongating spermatids 

(Huszar et al. , 2000). In human and stallion sperm, all CK and HspA2-related 

maturational events are completed in sperm located in the caput epididymis (Huszar et 

al., 1998b). Elongated spermatids that express a substantial amount of HspA2 exhibit 

fully formed binding sites for hyaluronic acid (Huszar 2007). These sperm are indicative 

of cells that have undergone a normal maturation process and are capable of fertilizing an 

oocyte. 

The presence of a cell surface hyaluronic acid binding glycoprotein (HABP) in 

sperm was identified for several species including the rat, mice, bull, and human 

19 



(Ranganathan et al. 1994). The protein was found to be present on the head, midpiece, 

and tail depending upon the species. These proteins function as receptors that bind to the 

surface of cells. The HA-binding protein described has been shown to specifically bind 

to hyaluronan (Ranganathan et al. 1994). The localization of HA-binding protein on the 

sperm surface was detected by indirect immunofluorescence using anti-HA-binding 

protein antibodies. The protein was found to be present on the head and tail of rat sperm 

and less prominent on the midpiece. Human sperm were found to have varying 

intensities of staining, differing from sperm to sperm from the same ejaculate, with 

relatively more intense staining on the midpiece of human as compared to the rat sperm. 

Bull sperm had similar staining patterns as rat sperm, described as head and tail staining 

(Ranganathan et al. 1994). 

Similarly, stallion sperm express a surface-associated hyaluronidase that can be 

found on the posterior head of ejaculated sperm (Meyers 2001). The protein is similar to 

proteins that have been characterized in other species and is known as PH-20 (Meyers 

2001). Glycerolphosphatidylinositol-linked hyaluronidase (PH-20) translocates during 

final epididymal maturation in the stallion (Meyers 2001 ). When the acrosome of stallion 

sperm is exocytosed, the inner acrosomal membrane fluoresces when hyaluronidase is 

stained. The author suggested that hyaluronidase plays a role in zona penetration by 

sperm (Meyers 2001). Although stallion sperm have not been shown to bind in vitro to 

hyaluronic acid assays, post-thaw motility is increased when hyaluronan is added to 

cooled semen before freezing (Bruemmer et al. 2009). The characterization and 

localization of HA receptors on stallion sperm coupled with the positive effects of 

hyaluronan warrant further investigation as possible methods of sperm selection for ICSI. 

20 



The testis-expressed heat shock protein has a high ATP content (Huszar et al. 

2000). Associated with HspA2 in sperm is B-type creatine kinase (CK) that is found in 

significantly higher content in sperm of subfertile men (Huszar et al. 1998a,b and Cayli et 

al. 2003a). High sperm CK activity was directly correlated with increased cytoplasmic 

protein and CK concentrations, which indicate the amount of cytoplasmic retention in 

immature sperm (Huszar and Vigue 1992). The finding suggested a defect in sperm 

development during the latter parts of spermiogenesis, at which time excessive cytoplasm 

is commonly extruded from elongating spermatids (Clermont 1963). The HspA2 ratio 

(formerly known as the CK-M ratio) reflects the proportion of mature to immature sperm 

within a semen sample. Furthermore, proportions of mature and immature sperm, 

regardless of normozoospermic or oligozoospermic producing men, have resulted in day-

to-day and man-to-man variations between semen samples (Huszar et al. 1988b). 

The content of HspA2 in sperm associated with zona-binding capabilities has 

helped to demonstrate the importance of mature sperm for fertilization. Sperm bound to 

the zona pellucida of halved unfertilized human oocytes did not retain cytoplasm. 

Immature sperm exhibiting retained cytoplasm and low expression ofHspA2 were 

deficient in the zona-binding site (Huszar et al. 1994). 

Due to the above findings, investigation of a plasma membrane remodeling step 

followed. Remodeling of the plasma membrane occurs only during spermiogenesis. The 

enzyme B 1,2-galactosyltrasferase is present only in the plasma membrane. The 

membrane density of B 1,2-galactosyltransferase and cytoplasmic concentrations of 

HsP A2 or CK were significantly related (Huszar et al. 1997). Further analysis concluded 

that the formation of the hyaluronic acid (HA) binding site also occurs at this time, 

21 



providing a stepping-stone for fertility testing and ICSI sperm selection in men (Huszar 

2007). 

The sperm-hyaluronan-binding assay (HBA) uses the hylauronidase (HA) 

receptor present on the intact acrosome of the sperm membrane surface to test for HA 

binding ability (Hong et al. 2006). When sperm are placed in 10-ul droplets into the well 

of pre-manufactured HBA kits (Biocoat Inc., Horsham, PA) and allowed to incubate for 

15 minutes, they can be visualized and counted for the total number of motile sperm 

bound to HA versus unbound motile sperm to gain a percentage. Sperm bound to HA 

can be detected due to the increase in tail cross-beat frequency as opposed to unbound 

sperm which remain swimming freely (Huszar et al. 2003). In addition to sperm 

selection, medium containing HA has been shown to increase sperm velocity, long-term 

motility, and viability of freshly ejaculated sperm as well as cryopreserved/thawed human 

sperm (Huszar et al. 1990b, Sbracia et al. 1997). 

Sperm that bind to hyaluronic acid are viable; non-viable sperm do not bind 

(Huszar et al. 2003). Sperm that bind to HA do not exhibit cytoplasmic retention, 

persistent histones, DNA fragmentation or capase 3, an apoptotic marker (Irvine et al. 

2000 and Sati et al. 2004). These findings are significant because immature sperm with 

DNA fragmentation are known to be a hindrance to fertilization (Huszar et al. 2003). 

Use of Hyaluronic Acid for ICSI 

Apart from motility, there is not a universally accepted method for distinguishing 

a fully functional sperm capable of fertilizing an oocyte from a less-capable sperm. 

However, HA binding score zones that have been established for human clinical 

22 



--

·---

laboratories have been applied to ICSI with some success. The binding scores have been 

shown to correlate to fertilization capability of sperm in the human (Huszar et al. 2002). 

Three binding zones indicate the percentage of sperm within a sample that bind to HA. A 

score of >80% is indicative of excellent binding. Intermediate binding is designated to 

samples binding 60 to 80% of sperm. Men with sperm in this category are advised that 

intrauterine insemination of their partner may be an option to enhance fertilization rates. 

Sperm exhibiting less than 60% binding to HA are considered to have diminished binding 

capabilities, and intrauterine insemination or ICSI is an option for couples wishing to 

reproduce (Huszar et al. 2002). 

The observance of chromosomal aneuplodies and diploidies in humans has been 

investigated in HA-bound sperm for potential use with ICSI (Jakab et al. 2005). In 

summary, HA- bound sperm were subjected to fluorescence in situ hybridization (FISH). 

Except for Y disomy, all other aneuploidies and diploidies declined 4 to 6 fold, resulting 

in HA-selected sperm being within the range of normozoospermic men. When sperm is 

selected by visual assessment, a direct correlation has been observed comparing the 5-

fold decline seen in X, Y and XY disomies to chromosomal abnormalities seen in ICSI-

derived children (Huszar 2007, Simpson and Lamb 2001 , Bonduelle et al. 2002). 

No adverse effects on fertilization or embryonic development have been observed 

in laboratories currently utilizing HA-selected sperm (Huszar 2007). HA-selected sperm 

used for ICSI in humans resulted in significantly higher pregnancy rates than visually 

selecting sperm (Warrilow et al. 2006). Furthermore, miscarriage rates were higher 

(p=0.0 13) in the visually selected group of embryos versus selecting sperm bound to HA 

(Warrilow et al. 2006). 

23 

It 



The use of HA-selected sperm has been reported in other species, including pigs 

(Park et al. 2005). More normal embryos were produced with HA selected sperm and 

ICSI versus visual selection of sperm for ICSI and IVF. Using HA-selected sperm 

reduced the number of early embryonic mortalities and chromosomal abnormalities (Park 

et al. 2005). Fluorescence in situ hyrbridization (FISH) analysis was used to analyze 

chromosome aneuploidy in sperm and embryos using chromosome 1 submetacentric 

probe. Although no significant differences were observed in blastocysts rates among 

embryos produced from IVF, ICSI, and ICSI-HABS (hyaluomic acid binding sperm), 

embryos derived from ICSI-HABS had a lower (p<0.05) chromosome abnormality rate 

(Park et al. 2005). Chromosomal abnormality in 4-cell porcine embryos derived from 

IVF, ICSI, and ICSI-HABS procedures were 43.0, 31.8, and 24.5%, respectively. The 

authors concluded that the selection of sperm with a hyaluronic acid binding assay may 

help to reduce early embryonic mortality in the pig. The conclusion is based on 

decreased incidence of chromosomal aneuploidy in HA-selected sperm and embryos 

produced by ICIS-HABS. Furthermore, ICSI-HABS is superior to conventional ICSI for 

higher production of porcine embryos with normal chromosomal complement (Park et al. 

2005). 

Nasr-Esfahani et al. (2008) compared the efficiency of routine sperm selection 

methods to HA-selection procedures for human sperm. The researchers' objectives were 

to observe fertilization rates, embryo development, implantation and pregnancy rates, and 

the relationship between HA-binding ability with sperm protamine deficiency. The 

percentage of sperm that bind to HA was statistically significant when compared to those 

with protamine deficiency, DNA fragmentation, and abnormal sperm morphology. 

24 



Although pregnancy and implantation rates were not shown to increase with HA 

procedures, oocytes injected with HA-selected sperm had significantly higher fertilization 

rates (Nasr-Esfahani et al. 2008). Sperm chromatin dispersion testing and chromomycin 

A3 staining were used to evaluate DNA fragmentation and protamine deficiency for 

human sperm (Nasr-Esfahani et al. 2008). In agreement with similar work conducted by 

Huszar et al. (2003), this study revealed that sperm samples with chromatin immaturity 

exhibited lower HA-binding ability. Sperm motility was not directly correlated with 

DNA fragmentation and abnormal morphology. 

Although HA selection improved fertilization rates, it did not have a significant effect 

on cleavage and embryo quality on day 2 and 3 after ICSI compared to routine sperm 

selection (Nasr-Esfahani et al. 2008). The authors suggest that embryo quality was not 

different between the HA and control group due to the possibility of paternal DNA 

anomalies occurring during post genomic activation and peri-implantation. 

Current methods of sperm selection for ICSI using motility and morphology versus 

HA-binding have been explored. Sperm hyaluronan-binding assay (HBA) scores are 

highly and significantly correlated with sperm motility and normal morphology (Hong et 

al. 2006). Commercial HBA kits (Biocoat Inc., Fort Washington, PA) were utilized on 

175 human patients participating in an IVF study. Objectives were to determine the 

relationship between HBA selected sperm and routine sperm analysis results and 

fertilization rates with conventional IVF (Hong et al. 2006). In contrast to previously 

mentioned studies, results from this experiment indicate that normal sperm morphology 

was more significant than HBA sperm in relation to fertilization rates, although HBA 

methods were significantly correlated with fertilization rate in conventional IVF (Hong et 

25 



al. 2006). The use of HBA' s was not shown to clearly identify patients with low and 

high fertilization rates. Furthermore, in contrast to other findings, the authors conclude 

that HBA is not superior to the selection of sperm through routine semen analysis as a 

predictor of fertility due to the lack of a distinct threshold value as a predictor of sperm-

fertilizing ability (Hong et al. 2006). 

Methods of Sperm Evaluation 

Sperm Chormatin Dispersion (SCD) is an assay for sperm DNA fragmentation. 

Sperm are subjected to an acid denaturing solution and then stained for fluorescence or 

brightfield microscopy to allow for detection of single-stranded DNA from DNA breaks 

(Fernandez et al. 2003). This assay demonstrates that sperm with fragmented DNA do 

not produce the normal halo of dispersed DNA loops. A halo is characteristic of normal 

sperm presenting non-fragmented DNA after acid denaturation and the removal of 

nuclear proteins (Fernandez et al. 2003). Several tests are available to assess sperm DNA 

fragmentation including the TUNNEL assay, the comet assay, chromomycin A3 testing, 

DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) test, and the 

sperm chromatin structure assay (SCSA) (Fernandez et al. 2003). 

Normal sperm with non-fragmented DNA can be monitored by fluorescence 

microscopy using specific DNA flurochromes to visualize halos of DNA dispersion. 

These halos can be evaluated after sperm are immersed in an agarose matrix and exposed 

to lysing solutions. The deproteinized nuclei, exibiting relaxed DNA loops or halos, are 

then referred to as "nucleoids." DNA damage can be assessed by looking for breaks that 

will facilitate the expansion of the halo. Sperm nuclei that have fragmented DNA can be 

26 



detected by the absence or minimal observance of the halo when treated with an acid 

solution before the addition of a lysis buffer using the SCD test (Fernandez et al. 2003). 

Acid or alkaline solution acts as a denaturing solution for somatic cells with fragmented 

DNA and allows for increased spreading of DNA fragments. The fragmentation is much 

more recognizable than when observed without a denaturing solution (Fernandez et al. 

2002). 

The SCSA is a flow cytometer assay that measures the susceptibility of sperm 

DNA to low pH-induced denaturation in situ (Larson et al. 2000). This test utilizes 

Triton X-100 which is a low pH solution used to permeate cell membranes allowing for 

access of arcidine orange to DNA. The solution acts to partially denature DNA in sperm 

that have abnormal chromatin structures. When passed through a flow cytometer, DNA 

fluorescing red indicates single-stranded (denatured) DNA that has associated with 

arcidine orange while green indicates normal double-stranded DNA. The results of DNA 

denaturation can be quantified by using an Ortho Diagnostic Cytoflurograf II (Larson et 

al. 2000). Results of one experiment utilizing SCSA for ICSI in humans revealed that no 

pregnancies occurred if>27% of sperm within a given sample showed DNA denaturation 

(Larson et al. 2000). SCSA is one of the most popular and widely used tests independent 

of sperm morphology that can be used as a predictor of pregnancy rates in human IVF 

clinics. 

27 



Conclusions 

Semen collected from stallions can be used for a variety of assisted reproductive 

techniques. Stallion sperm can be inseminated into a mare immediately after collection, 

cooled for use within 48 h, or cryopreserved for an indefinite period. Stallion sperm can 

also been cooled prior to freezing with some success. However, these techniques can be 

improved upon by conserving the volume and number of sperm needed to fit a specific 

application. Intracytoplasmic sperm injection is one such technique that requires a 

unique set of sperm parameters. ICSI differs from all other assisted reproductive 

techniques in that only a single sperm is required for injection into an oocyte. The 

cooling and cryopreservation of sperm can be modified to preserve low concentrations of 

sperm specifically designed for ICSI. Freezing sperm in smaller volumes and numbers 

than what is needed for insemination would allow for conservation of limited quantities 

of stallion sperm. The amount of time that semen can be cooled before freezing can be 

further evaluated to fit less strict parameters regarding overall motility and sperm 

numbers that are necessary for success with ICSI. 

The selection of sperm for ICSI involves techniques such as swim-up procedures 

and Percoll gradients with the objective to select a fully functional sperm capable of 

fertilization. Other than a visual assessment of motility, after initial selection procedures 

stallion sperm cannot be selected for fertilizing potential. Selecting sperm through an 

alternate method such as hyaluronic acid binding, would utilize a biochemical marker to 

help determine fertilizing potential of sperm. A method such as HA binding would help 

differentiate sperm that are more likely to lack chromosomal aberrations and improve 

fertilization, blastocyst, and pregnancy rates after ICSI. Advances such as those 

28 



described would ultimately aid to improve clinical ICSI programs. Therefore, a series of 

experiments were conducted help facilitate advances in the above described areas of 

interest: 

Experiment 1 

In Experiment 1 we determined if semen could be cooled for 24 h and then 

frozen to provide sufficient motility for ICSI selection. The objectives of Experiment 1 

were to compare: 1) two commonly used commercial diluents for cooling semen before 

freezing and 2) compare four freezing diluents added to semen that has been cooled for 

24 h prior to freezing. 

Experiment 2 

Maintaining the viability of sperm in a holding medium is essential for many 

assisted reproductive techniques. Sperm must survive the time required for processing 

and injection into an oocyte and any unforeseen circumstances that would increase 

processing time. A medium that does not require an altered environment, such as 

humidity and CO2, would be optimum for maintaining sperm over time. The objective of 

Experiment 2 was to determine which of five holding media best maintains the motility 

of stallion sperm over time. 

Experiment 3 

Experiment 3 tested the hypothesis that stallion sperm could bind to a 

commercially available HA-binding assay (HBA, Biocoat Inc., Horsham, PA). The 

29 



objective of this experiment was to determine if fresh and frozen stallion sperm bind to 

HA when introduced to pre-manufactured HBA kits. 

30 



CHAPTER II 

CRYOPRESERVATION OF COOLED SEMEN FOR EQUINE 

INTRACTOPLASMIC SPERM INJECTION 

Introduction 

Frozen sperm that have been previously cooled are of potential use for equine 

intracytoplasmic sperm injection (ICSI). Improving the efficiency of frozen sperm for 

ICSI is of importance when considering the frequency of use (85%) in recent years by 

one clinic (Carnevale 2008). Progressive motility is a parameter of successful freezing 

and is an indication of fertilizing ability. Although the motility of sperm that have been 

cooled and later frozen may be inadequate for insemination, there may be sufficient 

numbers of motile sperm for ICSI. 

Semen can be frozen after various cooling periods with acceptable post-thaw 

motility and fertility after insemination (Backman et al., 2004; Crockett et al., 2001). 

Results depended on the length of time sperm were cooled and the type of extender used. 

Cooling semen without glycerol at concentrations of 200 x 106sperm/mL before freezing 

provided numerically higher post-thaw motility rates (Backman et al. 2004). Semen 

cooled for 1 h at 4 °C and then centrifuged to remove seminal plasma survived best when 

4% glycerol was added to INRA96 prior to freezing. 

31 



Glycerol is the most common cryoprotectant utilized in stallion freezing 

extenders, but concentrations varying from 2.5 to 6% have proven to be effective 

(Vidament et al. 2001). The addition of 4% glycerol did not prove to be significantly 

different when compared to 3%, which is a common industry standard (Scherzer et al. 

2009). When combined, a positive effect is obtained with the addition of glycerol and 

egg yolk to cooling extenders for up to 24 h prior to freezing (Crockett et al. 2001 ). 

Similarly, egg yolk has been added to freezing extenders at different concentrations 

ranging from 2% (Palmer, 1984) to 20% (Martin et al. 1979). Although several 

commercial freezing extenders are available for the stallion, the optimal concentration of 

egg yolk that should be present within an extender is not clear. 

Stallion sperm can be frozen at several concentrations that preserve post-thaw 

motility equally well. Semen can be cooled and then frozen at lower than industry 

standard concentrations (50 x 106sperm/mL) and still provide similar post-thaw motility 

to industry standard concentrations of 250 x 106sperm/ml (Crockett et al. 2001 ). 

Although semen can be frozen at concentrations of 2 x 106sperm/mL for ICSI, this 

concentration does not provide enough sperm that can be used for swim-up selection as 

the concentration of sperm is too dilute (Choi et al. 2005). 

The overall goal of Experiment 1 was to develop a method for freezing stallion 

sperm that have been previously cooled for 24 h, while maintaining sufficient motility for 

ICSI selection. We tested the hypothesis that semen can be cooled for 24 hand then 

frozen to provide a viable population of sperm for ISCI. The objectives of this study 

were to compare: (1) two diluents for cooling semen prior to freezing and (2) four 

diluents for freezing cooled semen. 

32 



Materials and Methods 

A two-by-four factorial design was used to compare two cooling diluents and four 

freezing diluents at three time points after thawing semen (Figure 1 ). Eight stallions were 

chosen at random from approximately 18 stallions housed on the facility for research and 

commercial uses . Stallions were of light-horse breeds and ranged in age from 11 to 18 

yr. Prior to the beginning the experiment, five stallions were on an inconsistent 

collection schedule. These stallions were collected twice, approximately 1 h apart, to 

minimize extra-gonadal reserves of sperm. They were rested for 1 d prior to the 

collection for this experiment. Three stallions were being collected for a commercial 

breeding program, and semen for the project was collected in accordance with their 

normal collection schedule. 

Semen was collected using a CSU Model artificial vagina with an in-line gel filter 

(Animal Reproduction Systems; Chino, CA) and evaluated within 10 min of collection. 

The concentration was evaluated using a Densimeter (Model #543B, Animal 

Reproduction Systems; Chino, CA). Total and progressive motility was observed using a 

computer-automated sperm analysis system (CASA; Hamilton-Thom HT-IVOS Motility 

Analyzer, Hamilton-Thorn Research, Danvers, MA) by placing a 5.5-µl droplet ofraw 

semen, diluted to 50 x 106sperm/ml with CST (E-Z Mixin®- CST Animal Reproduction 

Systems, Chino, CA), onto a preheated (37°C) glass slide with a cover slip. Three 

random frames on each slide were obtained and averaged to count a minimum of 100 

sperm. System parameter settings were: 15 frames acquired at 30 frames/sec ; 

magnification factor 1.95; minimum contrast 75, minimum size 4 pixels; lower and upper 

33 



static size limits of 0.7 and 7.75, respectively; minimum velocity of the average path of 

motile sperm (V AP) was 20 µm/sec. 

Ejaculates were split and the semen was extended to 50 x 106 total sperm/mL in a 

total volume of23 mL using two cooling diluents: (1) INRA96 (IMV International 

Corporation, Minneapolis, MN) and (2) CST (E-Z Mixin®- CST Animal Reproduction 

Systems, Chino, CA). The samples were cooled in a passive cooling device (EST Equine 

Semen Transporter™, Baraka Enterprises Inc., Arkansas, USA) and held at 5°C for 24 h. 

After 24-h, 3 mL of sperm were removed from each sample to be used as the 

cooled control; the samples were further diluted to 20 x 106 total sperm/mL in their 

respective diluents and placed in an incubator at 37°C for 10 min prior to being evaluated 

with the CASA at 0, 45 and 75 min (Figure 2). 

The remaining sperm in INRA96 and CST diluents were diluted with their 

respective diluents to a concentration of 40 x 106 total sperm/mL before being assigned in 

3-mL aliquots to one of the following groups for freezing: (1) FR8, diluted with 0.75 mL 

of a modified French extender (Wilhelm et al. 1996) containing 0% glycerol then diluted 

1:1 (3.75 mL) with FR8 (the modified French extender containing 8% glycerol) for a 

final concentration of 4% glycerol and 20 x 106 total sperm/mL; (2) LAC 10, diluted with 

0.75 mL lactose EDTA extender (Cochran et al. 1984) containing 0% glycerol and then 

diluted 1: 1 with LAC 10 (lactose EDTA extender containing 10% glycerol); (3) G, diluted 

with 0.75 mL of CST and then 1:1 with 8% glycerol solution; and (4) GEY, diluted with 

0.75 mL of the respective cooling extender, then diluted 1: 1 with a solution containing 

8% glycerol and 8% egg yolk. The G solution was composed by diluting CST with 8% 

glycerol by volume. Solutions for GEY were composed with respective cooling 

34 



extenders with the addition of 8% glycerol and 8% egg yolk by volume (see Appendix I 

for composition of freezing extenders). 

Sperm were packaged into 0.5-mL polyvinylchloride straws (IMV International 

Corporation, Minneapolis, MN). Samples in FR8 were loaded into a cell programmable 

freezer (Kryo 10 Series III, Planer, Middlesex, UK) and cooled at a rate of -10°C/min 

from 20 to -l 5°C, at -l 5°C/min from -15 to -120 °C, then plunged into liquid nitrogen. 

Samples in LAC, G and GEY were floated 3 cm above liquid nitrogen for 10 min at 

approximately - l 60°C and then submerged. 

One straw from each treatment was thawed in a water bath at 37°C for 30 sec. A 

5.5-µl sample of thawed semen was analyzed immediately for motility using the CASA. 

Remaining sperm from samples were stored in an incubator at 37°C in 5-mL 

polypropylene tubes and evaluated at 45 and 75 min (Figures 1 and 2). 

Statistical Analyses 

All analyses were conducted using SAS 9.2 (SAS Institute Inc, Cary, NC); prior 

to analyses, data were arcsine transformed. In Experiment 1, total and progressive 

motility were compared between cooling diluents and among freezing treatments using 

ANOV A at 0, 45 and 75 min. No effect of cooling diluents (CST and INRA96) was 

detected; therefore, data from both diluents were combined for further analyses of 

freezing treatments. Freezing treatments were compared among groups at each time 

point by ANOV A, and if significant (p<0.05), differences were determined between 

treatments by least significant differences . The effect of time was evaluated for each 

freezing treatment by comparing the three time points using ANOV A. 

35 



Results 

Mean total and progressive motility were not different (p>0.05) for sperm cooled 

in CST versus INRA96 when evaluated at 0, 45 and 75 min. Total and progressive 

motilities among groups of frozen semen were not different (p>0.05) for cooling diluents; 

therefore, freezing treatments with different cooling diluents were combined. 

Mean total motility of sperm was not different among freezing groups after 

thawing for O and 45 min. However, by 75 min, differences were detected among 

freezing treatments (p=0.03), with motility for GEY higher (p<0.05) than LACIO and G, 

and motility for FR8 higher (p<0.05) than G (Figure 3). 

Mean progressive motility of sperm followed a similar pattern as total motility, 

with approximately half as many progressively motile sperm for GEY and FR8 as for 

LACIO and G at each time. No differences were observed at O and 45 min among 

groups, although groups tended (p<0.08) to differ at 45 min. A difference (p=0.01) was 

present among groups at 75 min, with motility for G being lower (p<0.05) than for FR8 

and GEY (Figure 4). 

Mean total and progressive motilities were not different for the three time points 

when analyzed across time for any group except G. Mean total and progressive motility 

for G were lower (p=0.02) for 75 versus O min after thawing (Figure 5). 

Discussion 

Frozen sperm are frequently used for equine ICSI (Carnevale, 2008). Sperm used 

for ICSI is often packaged at concentrations that are common for insemination. The need 

to have a large population of motile sperm for insemination is not relevant, as only one 

36 



sperm is required for injection into an oocyte. The concentration of frozen sperm stored 

for ICSI can be improved upon by freezing a minimal number of sperm. Any method of 

freezing sperm for ICSI must result in a viable population of sperm after thawing. 

Cooled semen has the potential to be frozen and later used for ICSI. Stallion 

semen can be cooled for various time lengths before freezing (Backman et al. , 2004; 

Crockett et al. , 2001). Previous groups have conducted experiments to determine if 

stallion sperm can survive prolonged cooling before freezing and still provide adequate 

sperm for future insemination (Backman et al. , 2004; Crockett et al. , 2001) . Our study 

differs from those described in that viable sperm with acceptable motility parameters for 

ICSI differs from that of what is required for insemination. Selection of sperm for ICSI 

requires swim-up and centrifugation; therefore, sperm were evaluated at 45 and 75 min 

after thawing. These time points were selected to simulate the time required for sperm 

preparation for ICSI (Altermatt et al. 2009). 

The removal of seminal plasma is not feasible for every facility that ships cooled 

semen. Seminal plasma has been shown to be detrimental when freezing stallion sperm 

(Jasko et al. 1991, Dawson et al. 1999, Crockett et al.2001). We have demonstrated that 

the removal of seminal plasma is not necessary for sperm being frozen for ICSI. This 

finding is significant because cryoprotectant can be directly added to cooled semen 

without the removal of seminal plasma and then frozen for ICSI. The methods described 

herein for freezing semen containing seminal plasma may be of application for facilities 

lacking the ability to remove seminal plasma before shipping. Although seminal plasma 

has the potential to be removed upon arrival, centrifugation would be an additional insult 

37 



to sperm survival. However, further investigation is needed to determine the effects on 

freezing sperm with seminal plasma and implications for ICSI. 

Differences in the composition of freezing diluents have likely contributed to the 

results described. Egg yolk is commonly used as a lipid additive in semen extenders to 

aid in the protection of the lipid membrane. Phospholipids, such as low-density 

lipoproteins present in egg yolk, aid in maintaining the sperm membrane during cooling 

(Squires et al. 1999). The highest motilities were observed with the addition of FR8 and 

GEY. These results are probably attributed to the egg yolk present in both diluents. 

When GEY was added to cooling diluents, the combination resulted in the GEY diluent 

being very similar in composition of ingredients to FR8. Our results are in agreement 

with other studies in which egg yolk has been shown to aid in preservation of motility 

(Crockett et al. 2001 ). Cooled sperm frozen with only the addition of glycerol resulted in 

the lowest motilities among groups. However, sperm frozen with only the addition of 

glycerol still resulted in motile sperm for ICSI. 

Freezing sperm at a concentration of 20 x 106sperm/mL has allowed for the 

cryopreservation of a low number of sperm. Low concentrations of frozen sperm are 

beneficial to preserving limited quantities of stallion sperm. Freezing sperm at this 

concentration in a 0.5-mL straw, results in a total population of approximately one tenth 

of industry standard concentrations. Conventional methods of selecting sperm for ICSI 

require cutting a portion of a straw with frozen sperm under liquid nitrogen (Altermatt et 

al. 2009). The total number of sperm obtained by freezing sperm in one straw at 20 x 

106sperm/mL is comparable to numbers of sperm obtained from a single cut portion of 

straw using current methods (Altermatt et al.2009). Therefore, freezing sperm at 20 x 

38 



106sperm/mL eliminates the need of liquid nitrogen for cutting a straw, eliminates 

contamination to an unused portion of straw, and is more efficient in a laboratory setting 

and conservative in quantity. 

The use of two different cooling diluents did not have any bearing on freezing 

ability of sperm or differences in controls after cooling. This finding is significant when 

considering that facilities shipping stallion semen may only have access to one commonly 

used cooling diluent at a given time. Sperm frozen at a slow cooling rate (FR8) did not 

perform better than sperm frozen at a faster rate (GEY). Motility rates after 24 h of 

cooling as well as after freezing may be improved if the length of cooling time is 

shortened. The ability of sperm to survive a 24 h cooling period before freezing is 

beneficial for shipments arriving overnight at a facility that lacks the personnel to freeze 

sperm immediately. The above-described methods for freezing semen that has been 

cooled for 24 h without the removal of seminal plasma demonstrate the ability to 

maintain sufficient numbers of motile sperm for ICSI. Clinical changes in mare 

management, such as a change in procedure from ooycyte transfer to ICSI, would 

potentially result in a standard insemination dose of semen that is no longer needed at 

such a quantity. The remaining sperm not used for ISCI could be frozen in this 

circumstance and others of unforeseen nature. Due to the observation of variability in 

stallion sperm surviving a cooling process, further investigation is warranted to 

characterize differences in seminal plasma between stallions. The ability to predict 

cooling and freezing potential for individual stallions based on characterization of 

seminal plasma would be of great benefit for a number of assisted reproductive 

techniques. 

39 



Figure 1. Diagram of experimental design for Experiment 1 

INRA96 

Ejaculate INRA96 

CST 

CST 

40 

Evaluate at 0, 45, 75 
min at 37°C 

FR8 

LACIO 

G 

GEY 

Evaluate at 0, 45, 75 
min at 37°C 

FR8 

LACIO 

G 

GEY 

Evaluate at 0, 
45, 75 min at 
37° C after 
thawing 

Evaluate at 0, 
45, 75 min at 
37° C after 
thawing 



Figure 2. Time points for evaluation of controls and frozen straws 

Control (warm to 37°C) 

Time after 24 h cool 0 min 45 min 75 min 

(thaw to 37°C) 
Straw 

Time after thaw 0 min 45 min 75 min 

41 



Figure 3. Mean total motility of semen cooled for 24 h, frozen in four extenders• and 
warmed for evaluation of motility at 0, 45 and 75 min (n = 16 samples). 

30 (5) 

25 -c 20 

.:; 15 0 
::li: 
iii ... 10 0 
I-

5 

0 
0 

(4) 

45 
Minutes 

(3) 

75 

b,c 

C 

-+- FR8 
- LAC10 
-t:r- G 
~GEY 

a,b,c Values with different superscripts at 75 min differed (p=.03) Values at O and 45 min 
were similar (p>0.05). The pooled SEM for each time point appears between 
parentheses. 

*FR8: Modified French, LACIO: Lactose-EDTA, G: glycerol, GEY: glycerol and egg 
yolk 

See Appendix I for table 

42 



Figure 4. Mean progressive motility of semen cooled for 24 h, frozen in four extenders* 
and warmed for evaluation of motility at 0, 45 and 75 min (n = 16 samples). 

12 - (2) (2) 
10 

a -+-FR8 .. 8 
0 a - LAC1 :!= 
Cl) 6 --lr- G > "iii ~ GEY Ill 4 Cl) ... a,b en 
0 2 ... 

Q. b 
0 

0 45 75 

Minutes 

a,b Values with different superscripts at 75 min differed (p=0.01) Values are similar 
(p>0.05) at O min. A trend towards significance is present at 45 min (p<0.08). The 
pooled SEM for each time point appears between parentheses. 

*FR8: Modified French, LAClO: Lactose-EDTA, G: glycerol, GEY: glycerol and egg 
yolk 

See Appendix I for table 

43 



Figure 5. Means± SEM of total and progressive motility over time for sperm frozen in 
glycerol (G) and evaluated at 0, 45 and 75 min post thaw (n = 16 samples). 

25 

20 

e.... 15 
>, 

;!: -+- Total rv1otility 
; 
0 10 :!!: 

- Progressive rv1otility 

b 
5 

b 
0 

0 45 75 
Minutes 

a,b Values with different superscripts for total or progressive motility differed over time 
(p<0.05). 

44 



Introduction 

CHAPTER III 

EFFECT OF FIVE HOLDING MEDIA ON MOTILITY OF 

STALLION SPERM 

Assisted reproduction for the equine requires the use of various holding media. 

Media are typically formulated for a specific purpose. Although many media specify a 

use, such as maintaining embryos or shipping oocytes, these media may be comparable in 

effectiveness to those designed specifically for holding sperm. Maintaining the viability 

of stallion sperm for a prolonged period of time is beneficial for many assisted 

reproductive techniques. Holding sperm in a medium that does not require altered 

environments, such as maintenance of humidity and CO2, would provide alternative 

sources of media that may be more compatible for use in research or a clinical equine 

program. 

Swim-up procedures are common practice when selecting sperm for 

intracytoplasmic sperm injection (ICSI);(Altermatt et al. 2009). Sperm must be able to 

survive and maintain motility over a period of time in order to be selected by swim-up. 

Swim-up techniques often involve centrifugation and washing of sperm which increases 

the length of time before sperm selection and injection into an oocyte (Altermatt et al. 

2009, Choi et al. 2005). The objective of Experiment 2 was to determine which of five 

holding media best maintains the motility of stallion sperm over time. 

45 



Materials and Methods 

Single ejaculates were collected from seven randomly chosen stallions out of a 

population of approximately 18 stallions of light-horse breeds housed on the facility. 

Stallions were between 4 and 15 years of age. After evaluation of initial concentration, 

semen was diluted in a skim-milk extender (E-Z Mixin®- CST Animal Reproduction 

Systems, Chino, CA) and placed into five media at a final concentration of 50 x 106 total 

sperm/mL. For 5 h, semen was analyzed for total and progressive motility at hourly 

intervals. 

Semen was collected and evaluated as described in Experiment 1. Five media 

were evaluated: 1) GIVF (Vitrolife; Sweden AB, Kungsbacka, Sweden), 2) FCDM 

[CDM (Olson and Seidel 2000) supplemented with 0.5% fatty acid-free bovine serum 

albumin 2 mM caffeine and 2µg rnL- 1 heparin], 3) TALP [Tyrodes Albumin Lactate 

Pyruvate; (see Appendix II)], 4) EM (EmCare™ Embryo Holding Solution; AgTech Inc., 

Manhattan, KS), and 5) MM (Mare Mojo Embryo Holding Medium; Jason Abraham, 

Canadian, TX). See Appendix II for TALP recipe. 

TALP, EM and MM were warmed to 37°C prior to the addition of sperm. GIVF 

and FCDM require CO2 for the maintenance of pH; therefore, they were equilibrated in 

an incubator at 38.5°C in 6.0% CO2 and air for approximately 2 h before addition of 

sperm. Sperm were added to 5-mL polystyrene round bottom tubes containing 1.5 mL of 

each of the five media to achieve a final concentration of 50 x 106 total sperm/mL. After 

the addition of sperm, GIVF and FCDM samples were held in an incubator at 38.5°C in a 

6.0% CO2 atmosphere. EmCare, TALP and MM were maintained at 37°C in an 

incubator without CO2 and capped to prevent evaporation. An aliquot of each sample 

46 



was removed at hourly intervals for a total of 5 h for evaluation of total and progressive 

sperm motility using the CASA. 

Statistical Analyses 

All analyses were conducted using SAS 9.2 (SAS Institute Inc, Cary, NC); prior 

to analyses, data were arcsine transformed. Total and progressive motility were 

compared among groups at each time point by ANOV A. When significant differences 

were detected, differences between groups were determined by least significant 

differences. Differences were considered significant at p<0.05. 

Results 

The mean ±SEM total motility for semen from the seven stallions at the time of 

collection was 85 ± 3%. Mean total motility was not different among groups from 1 

through 4 h, although means tended to differ at 3 hand 4 h (p = 0.12 and 0.09, 

respectively), with TALP having the highest numerical motility among all groups. Total 

motility was different (p = 0.03) among groups at 5 h, with sperm in TALP having a 

higher (p<0.05) total motility than sperm in other media (Figures 6 and 8). 

The mean ±SEM total progressive motility for semen from the seven stallions at 

the time of collection was 33 ± 5%. Mean progressive motility approached significance 

(p=0.055) at 1 h with progressive motility of sperm in TALP higher (p<0.05) than FCDM 

and GIVF. Differences among groups were not different at 2 h. At 5 h, groups differed 

(p=0.004), with TALP having higher (p<0.05) progressive motility than all other groups 

(Figures 7,and 9) . 

47 



Discussion 

Sperm subjected to five holding media were analyzed every hour for 5 h. These 

time points were selected to ensure the adequate time necessary for sperm selection and 

preparation before ICSI. Although sperm preparation does not usually require more than 

1 h, we evaluated the motility of sperm for 5 h to replicate a situation in which 

unforeseen circumstances would necessitate the maintenance of sperm in media for a 

prolonged time. 

Cell culture mandates the maintenance of pH. The incubation of five media 

required that GIVF and FCDM be maintained in an exogenous CO2 atmosphere. Both 

GIVF and FCDM are formulated with HEPES (N-2-Hydroxyethylpiperazine-N'-2-

Ethanesulfonic Acid) buffer. Media used for cell culture is often formulated with HEPES 

or sodium bicarbonate to maintain pH. Buffers are most effective when the pH is equal 

to the pKa (acidic strength) of that buffer. Therefore, when a higher pH is desired, 

HEPES if often added to a media to maintain a pH of 7 .2 to 7 .6 (Good et al. 1966). 

Although HEPES-buffered media such as T ALP can be maintained without CO2, 

extracellular CO2 is thought to play a novel, but not fully understood, role in cell 

proliferation (Chakrabarti R, 2001 ). Exogenous CO2 is used by one clinic for oocyte 

incubation and may be compatible for holding sperm (Altermatt et al. 2009). Apart from 

buffer, the incubation temperature of GIVF and FCDM was also different (38.5°C) than 

for the remaining three media (37°C). Discrepancy in temperatures is attributed to GIVF 

and FCDM being held in an incubator used for oocyte culture. Adjusting the temperature 

of incubation for holding sperm would not be practical for a clinical program. Although 

holding sperm in a cooler environment would have increased survival time, motility 

48 



patterns would have been depressed. Motility is the only indication of sperm fertilizing 

potential and is essential to repeatedly successful ICSI. 

The comparison of five media for maintaining sperm motility resulted in T ALP 

being superior over time. TALP was the only medium evaluated that is specifically 

designed for holding sperm. TALP was initially formulated in response to the sensitivity 

of hamster sperm to culture media (Bavister and Yanagimachi 1977). The composition 

of energy substrates within a solution (lactate, pyruvate, and glucose) were found to 

effect motility of sperm at different time points with varying concentrations. The 

ingredients in T ALP have since been modified for use with other species (Bavister and 

Yanagimachi 1977, Graham et al. 1986). Furthermore, T ALP is a clear medium and the 

use of a clear medium is essential for clear observation of sperm during a selection 

process. 

Although differences in media approached significance at 1 h, it is important to 

note that no significant differences were detected until analyses at 5 h. The preparation 

of sperm for ICSI may not require a full hour. Therefore, media that are not designed for 

holding sperm may be of acceptable use. Holding sperm in T ALP is beneficial, because 

TALP does not require an altered atmosphere. T ALP is relatively simple to make and 

can be frozen for later use. 

49 



Figure 6: Mean total motility for sperm held in five media* for 5 h (n = 7 stallions). 

90 • (7) (7) 
80 

(3) 
70 

:J 60 
j:: 

50 0 

..J 40 
<( 
I- 30 0 
I- 20 

10 

0 
0 1 2 

HOURS 

• Initial total motility after collection 
* p < 0.05 among groups 
** p < 0.09 among groups 

(7) (7) (7) 

3 4 5 

The pooled SEM for each hour appears between parentheses. 

* GIVF (Vitrolife; Sweden AB, Kungsbacka, Sweden) 

--+---- GIVF 

---- FCDM 
----...- TALP 
---EM 
---)1(-- - MM 

• INITIAL 

FCDM (Fertilization Chemically Definied Medium with BSA, Caffeine and Heparin) 
TALP (Tyrodes Albumin Lactate Pyruvate) 
EM (EmCare™ Embryo Holding Solution; AgTech Inc., Manhattan, KS) 
MM (Mare Mojo Embryo Holding Medium; Jason Abraham, Canadian, TX) 

50 



Figure 7: Mean progressive motility for sperm held in five media* for 5 h 
(n = 7 stallions). 

35 
• (5) 

(4) (4) (3) (3) (2) 
>- 30 I-
:::i • GIVF j:: 25 
0 • FCDM 
::1: 20 TALP w • > --- EM en 15 U) - --~·-MM w 
0::: 10 • INITIAL C) 
0 
0::: 5 a. 

0 
0 1 2 3 4 5 

HOUR 

• Initial progressive motility at collection 
* p = 0.004 among groups with T ALP differing (p<0.05) from all other media 
** p < 0.09 among groups 

The pooled SEM for each hour appears between parentheses. 

* GIVF (Vitrolife; Sweden AB, Kungsbacka, Sweden) 
FCDM (Fertilization Chemically Defined Medium with BSA, Caffeine and Heparin) 
T ALP (Tyrodes Albumin Lactate Pyruvate) 
EM (EmCare™ Embryo Holding Solution; AgTech Inc., Manhattan, KS) 
MM (Mare Mojo Embryo Holding Medium; Jason Abraham, Canadian, TX) 

51 



Figure 8. Percentage of total motile sperm extended to a concentration of 50 x 106 total 
sperm/mL and held in five media for 5 h 
(n = 7 stallions). 

Media GIVF FCDM TALP EMCARE MOJO 
lh 69ab±8 61ab±7 74a±5 51b±7 6lab±8 
2 h 63 ± 6 55 ± 8 62 ± 2 47 ± 8 49 ± 9 
3h 50ab±8 41b ±8 62a±8 40b±5 38b±7 
4 h 35ab ± 7 39ab ± 8 54a ± 9 28b ± 5 27b ± 5 
5h 24b±7 30b±9 54a±8 29b±6 28b±5 
a,b Values within individual rows with different superscripts are different (p<0.05). 

Figure 9. Percentage of progressively motile sperm extended to a concentration of 50 x 
106 total sperm/mL and held in five media for 5 h 
(n = 7 stallions). 

Media GIVF FCDM TALP EMCARE MOJO 
1 h 11 b ± 4 14 b ± 3 293 ± 5 1 7ab ± 4 1 gab ± 3 
2 h 12 ± 3 8 ± 3 14 ± 3 16 ± 4 15 ± 6 
3 h 1 ob ± 3 9b ± 2 21 a ± 6 9ab ± 2 9b ± 3 
4 h 1 oab± 2 gb ± 3 203 ± 5 gb ± 2 7b ± 2 
5 h 5b ± 1 5b ± 1 193 ± 4 6b ± 2 8b ± 2 
a,b Values within individual rows with different superscripts are different (p<0.05). 

52 



CHAPTERIV 

EVALUATION OF A HYALURONIC ACID SPERM BINDING ASSAY FOR 

EQUINE INTRACYTOPLASMIC SPERM INJECTION 

Introduction 

Intracytoplasmic sperm injection involves the selection of a single sperm chosen 

from a population of viable and non-viable sperm. Apart from motility, there is not a 

universally accepted method of selecting and distinguishing a fully functional sperm from 

one that may not be as likely to fertilize an oocyte. Current methods of selecting equine 

sperm include centrifugation and swim-up techniques (Altermatt et al. 2009). Others 

have described similar techniques that mandate swim up or Percoll gradients for selection 

of stallion sperm for ICSI (Choi et al. 2005, Lazarri et al. 2002). However, aside from 

visual assessment of motility, sperm cannot be selected for fertilization capability. 

Some human in vitro clinics implement a method of sperm selection that helps to 

distinguish a more fully functional and mature sperm from one that is not mature (Huszar 

2007). Hyaluronic acid binding assays (HBA) are used to distinguish sperm that are 

mature, and are likely to have a 4 to 6-fold decrease in chromosomal aberrations (Jakab et 

al. 2005). Similarly, others have shown that a 5-fold decrease in chromosomal 

abnormalities has been observed when sperm are selected with HA binding techniques 

53 



rather than visual assessment (Huszar 2007, Simpson and Lamb 2001 , Bonduelle et al. 

2002). 

The sperm-hyaluronan-binding assay uses the hylauronidase receptor present on 

the intact acrosome of the sperm membrane surface to test for hyaluronic acid (HA) 

binding ability (Hong et al. 2006). The presence of a cell surface hyaluronic acid binding 

glycoprotein (HABP) in sperm was identified for several species including the rat, 

mouse, bull and human (Ranganathan et al. 1994). Sperm obtain the HA receptor during 

spermiogeneis. This event occurs at the same time as plasma membrane remodeling 

(Huszar et al. 1997). Therefore, sperm that will bind to hyaluronidase are indicative of 

cells that have undergone a normal maturation process and are capable of fertilizing an 

oocyte. Sperm that are bound to HA can be distinguished by cessation of head 

movement, and an increase in tail cross-beat frequency, as opposed to unbound sperm 

which remain swimming freely (Huszar et al. 2003). 

Porcine sperm have also been shown to bind to HA and have been selected for 

ICSI using this technique (Park et al. 2005). The localization of HA-binding protein on 

the sperm surface has been detected by indirect immunofluorescence using anti-HA-

binding protein antibodies for many species (Ranganathan et al. 1994). Similarly, 

stallion sperm express a surface-associated hyaluronidase that can be found on the 

posterior acrosome of ejaculated sperm (Meyers 2001). Our hypothesis is that stallion 

sperm can bind to a commercially available HA-binding assay (HBA, Biocoat Inc. , 

Horsham, PA). The objective of this experiment is to determine if fresh and frozen 

stallion sperm bind to HA when introduced to pre-manufactured HBA kits (Biocoat Inc., 

Horsham, PA). 

54 



Methods and Materials 

Two preliminary studies were designed to test a hyaluronic-binding assay (HA) as 

a means to select stallion sperm for intracytoplasmic sperm injection in the mare. Boar 

sperm were used as a control to observe sperm binding to assays. Semen from two boars 

were pooled and subjected to a hyaluronic acid binding assay (HBA® Biocoat Inc., 

Horsham, PA) and visualized for binding of sperm. Shipped semen from two boars was 

cooled and extended at a distant facility. The boar semen arrived to our lab within 24 h 

of collection. Samples were pooled, analyzed for motility, and centrifuged at 600 x G for 

5 minutes. The supernatant was removed and the sperm pellet resuspended in T ALP 

(Tyrodes Albumin Lactate Pyruvate) and washed by centrifugation a second time at 600 

x G for 5 minutes. After removal of the supernatant, the pellet was resuspended, and the 

concentration of sperm obtained by spectrophotometer following dilution of sperm to 20 

x 166 total sperm/mL. A 10-µl sample of the extended semen was placed onto the center 

of premanufactured hyaluron coated slides with a coverslip. A separate sample was 

placed onto a glass slide with a coverslip and incubated simultaneously to serve as a 

control. Sperm were incubated for 10 min on the slide at room temperature (20 -25°C) 

for optimum binding to hyaluron. After 10 minutes, the slide was placed under 

microscope for visualization of binding. Binding was determined as cessation of head 

movement of the sperm and increased cross-beat frequency of the tail. The slide was 

then placed back into the humidified incubator and analyzed once more for binding 10 

minutes later. 

Upon confirmation that boar sperm would bind to the assay, two random stallions 

were collected; the semen pooled and analyzed for motility. Each sample was diluted to 

55 



200 x 106 sperm/mL in T ALP (Experiment 2). The pooled sample contained 4000 x 106 

total sperm. The sample was centrifuged at 1000 X G for 20 minutes. The supernatant 

was removed, and sperm were washed by centrifugation at 1000 x G for 20 minutes. The 

sperm pellet was resuspended in 5 mL of T ALP after removal of the supernatant. The 

concentration of sperm was obtained using a Nucleocounter (Nuclecounter™, New 

Brunswick Scientific; Edison, NJ). The sample was extended to 50 x 106sperm/mL, and 

a 10-µl droplet was placed onto the center of the premanufactured hyaluron coated slides 

with a coverslip. Slides were placed into a humidified chamber at room temperature and 

incubated for 10 minutes. Sperm were analyzed microscopically for visual confirmation 

of binding and then returned to incubation for 10 more minutes followed by a final 

binding evaluation. Controls were placed onto glass slides with coverslips as described 

for boar semen. 

Upon confirmation that fresh stallion semen bound to hyaluronic acid, four frozen 

samples of stallion semen from Experiment 1 were analyzed for binding to assays. 

Stallions were selected by eliminating the two stallions with the highest and lowest post-

thaw sperm motilities at 75 min, and then randomly selecting from the remaining six 

stallions. Straws frozen in GEY were selected for thawing by randomly choosing two 

samples that had been previously cooled in INRA 96 and two samples cooled in CST. 

Straws were placed in a 37°C water bath for 30 sec and pooled for a total volume of 2 mL 

containing 40 x 106 total sperm at a concentration of 20 x 106sperm/mL. T ALP held at 

37°C was added to the pooled sample bringing the total volume to 5 mL. Sperm were 

washed by centrifugation at 1000 x g for 10 min. Upon completion of centrifugation, no 

sperm pellet was observed, so a 100-µl sample was obtained from the bottom of the 

56 



centrifugation tube and visualized microscopically for confirmation of sperm. TALP was 

then added to the 100-µl sample to bring the total concentration to approximately 50 x 

106sperm/ml. A 10-µl droplet of sperm was then placed onto the hyaluron assay, and 

incubated as described above for a total of 20 min. Samples placed on glass slides with 

coverslips served as controls. 

Results 

Pooled boar semen that arrived cooled had an initial total and progressive motility 

of 35 and 20%, respectively. Sperm were introduced to the hyaluron assay and visualized 

for binding after a 10-min incubation, which did not result in any bound sperm. The 

assay was placed back in the humidified chamber for an additional 10 minutes . 

Approximately 15% of boar semen was observed bound to the slide after 20 min of 

incubation time. 

Fresh semen pooled from two stallions exhibited total and progressive motility of 

65 and 26%, respectively. The semen displayed approximately 5% bound sperm after 

incubation on hyaluron-coated assays for 20 min. Frozen sperm were pooled and had an 

initial post-thaw total and progressive motility of 69 and 33%, respectively. Frozen 

sperm that were washed by centrifugation, as described above, did not exhibit any 

binding during the total 20-min incubation period. Frozen stallion sperm were 

agglutinated in numerous congregations of up to 50 or more sperm. The sperm seemed to 

be attached to one another at the head with an increased affinity, when compared to 

controls on glass slides. Furthermore, uncharacteristic circling, tails bent at the midpiece, 

and some hyperactivation was observed. Stallion sperm seemed to be affected by the 

57 



hyaluron-coated slide, but did not behave in the same manner as described for other 

species. 

Discussion 

Reports indicating that stallion sperm may have the potential to bind to hyaluronic 

acid are vague and unclear at best (Huzar et al. 1998b ). However, stallion sperm have 

been shown to express a surface-associated hyaluronidase on the posterior acrosome 

(Meyers 2001). Binding characterization and localization of HA receptors on sperm have 

been described for other species (Ranganathan et al. 1994, Park et al. 2005). Boar sperm 

selected for ICSI using HA-binding techniques results in more normal embryos than 

visual selection of sperm for IVF and ICSI (Park et al. 2005). Chromosomal 

abnormalities in 4-cell porcine embryos are lower when sperm is selected using HA (Park 

et al. 2005). No adverse effects on fertilization or embryonic development have been 

observed in human laboratories currently utilizing HA-selected sperm (Huszar 2007). 

Sperm binding scores have been developed for use in human clinics and have been shown 

to be directly correlated to fertilization capability (Huszar et al. 2002). The success of 

sperm selected for ICSI using HA could have the potential to be replicated for the equine 

as demonstrated for other species. 

The objective for this study was to see if fresh and then frozen stallion sperm 

could bind to HA assays. Results from pooling fresh semen from two stallions indicated 

that a small percentage ( ~5%) of sperm was bound to HA after 20 min of incubation. 

Sperm that had been previously cooled and then frozen did not bind to the assay. 

Because frozen sperm used for this experiment had been subjected to a 24 h cool prior to 

freezing, this process may have had a negative impact on binding ability. Sperm that 

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have advanced levels of capacitation or a disrupted acrosome are not able to bind to HA 

(Huszar et al. 2003). However, when stallion sperm are in vitro capacitated an