cryopreservation of equine and porcine spermatozoa …

CRYOPRESERVATION OF EQUINE AND PORCINE SPERMATOZOA

WITH A UNIQUE FREEZING TECHNOLOGY (UFT)

by

HEATHER A. VARTORELLA, B.S., M.S.

A DISSERTATION

IN

ANIMAL SCIENCE

Submitted to Uie Graduate Faculty

of Texas Tech University in Partial Fulfilhnent of the Requirement

for the Degree of

DOCTOR OF PHILOSOPHY

Approved

Chairperson of the Committee

Accepted

Dean of the Graduate School

May, 2003

2003, Heather Ann Vartorella

ACKNOWLEDGEMENTS

From an academic standpoint, this dissertation represents an organized, written

collection of research and knowledge. In the real world though, this dissertation

represents a conglomeration of late nights, experiences, relationships, deadlines,

education, and emotions. By no means was this research project purely a feat of my own.

With that in mind, I would like to acknowledge a few people who added to the following

pages.

First off, I would like to acknowledge the people in my life that are close to my

heart. To my family in Michigan and my friends, in Texas and abroad, I am always

gratefiil for their continuous love and support. A special thanks to my future husband,

J.W. Goolsby, for helping me keep my sanity during my various academic challenges.

In regards to the actual production of this research project, there are several

people I need to mention. In appreciation of their collaboration with Texas Tech

University, I would like to recognize Brian Wood and Supachill USA, Inc., for

supporting this project. I also need to acknowledge all the staff at the TTUHSC

OB/GYN Laboratory, especially Susan Chavez and Roland Pirtle, thank-you for your

encouragement and help. 1 am also very grateftti for the support 1 received from Pete

Cotter, since two sets of hands are definitely quicker than just one. Also 1 need to thank

the management and staff at the TTU Ranch Horse Center and TTU Swine Unit for the

extra time taken to facilitate this project. To my committee. Dr. Kevin Pond, Dr. Sam

Jackson, Dr. Gail Comwall, and Dr. John Blanton; I would like to thank-you for your

time and supervision.

Finally, I need to convey my admiration and gratitude for Dr. Sam Prien, my

committee chair, mentor, and above all else, my friend. Without your guidance, my

graduate career would have not been possible.

i l l

TABLE OF CONTENTS

ACKNOWLEDGEMENTS Ji

ABSTRACT vh

LIST OF TABLES ix

LIST OF FIGURES x

CHAPTER

I. n^TRODUCTION 1

n. LITERATURE REVIEW 4

Introduction 4

Current Status of Equine Cryopreservation 5

Current Status of Porcine Cryopreservation 6

Cellular Effects of Cryopreservation 7

Diluents 9

Thermal Gradients 13

Pre-Freezing Protocols 15

Packaging Systems for Cryopreservation 18

Summary 20

111. EFFECTS OF SEMEN HANDLING ON POST-THAW MOTILITY

OUTCOMES OF CRYOPRESERVED EQUINE SPERMATOZOA 22

Abstract 22

Introduction 23

Materials and Methods 24

iv

Results 28

Discussion 28

IV. COMPARISONS OF A UNIQUE FREEZING TECHNOLOGY (UFT) TO TRADITIONAL LIQUID NITROGEN METHODOLOGY FOR CRYOPRESERVATION OF EQUINE SPERMATOZOA 31

Abstract 31

Infroduction 32


Results 37

Discussion 42

V. EFFECTS OF SEMEN HANDLING ON POST-THAW MOTILITY

OUTCOMES OF CRYOPRESERVED PORCINE SPERMATOZOA 45

Abstract 45

Introduction 46


Results 51

Discussion 52 VI. COMPARISONS OF A UNIQUE FREEZING TECHNOLOGY (UFT)

TO TRADITIONAL LIQUID NITROGEN METHODOLOGY FOR CRYOPRESERVATION OF PORCINE SPERMATOZOA 55

Abstract 55

Introduction 56


Results 60

Discussion 64

VII. LIPID PEROXIDATION EFFECTS FROM SEMEN HANDLING

DURUSIG CRYOPRESERVATION OF PORCINE SPERMATOZOA 66

Abstract 66

Infroduction 67


Results 73

Discussion 80 Vin. CONCLUSIONS 83

LITERATURE CITED 86

VI

ABSTRACT

The cryopreservation of sperm offers several benefits to various biological

sciences including clinical medicine, biotechnology and agriculture. This conservation

process requires a reduction in cellular metabolism while maintaining viability and

stractural stability through freezing and eventually thawing thus allowing for an

extension of viability past normal limitations. However, the application of sperm

cryopreservation has not been successfiil in all species. For example in the livestock

sector, advancements in semen technology have been made in the dairy industry, but not

as significantiy within the equine and swine areas.

Recently, a new Unique Freezing Technology (UFT) was developed to freeze

foodstuffs at a quick rate while maintaining freshness. This dissertation represents the

initial series of trials utilizing this new technology on cellular specimens not of meat

origin. From August 2001 to March 2003, numerous ejaculates from five different

Quarter Horse stallions and seven boars were collected. From these samples, over 1000

treatments were created to test various cryopreservation procedures and techniques

including extenders, freezing rates, thawing rates, male influence, and storage containers.

The design of this study was to improve post-thaw motility rates of stallion and boar

spermatozoa.

Several factors were found to affect the post-thaw motility outcome. In the

stallion trials significant factors included preparative handling, stallion influence and

freezing technique. The results of the boar trials also revealed the significant influences

vu

of the pre-freezing protocols, boar influence and freezing techniques. Further testing was

conducted on the boar sperm samples to evaluate the stability of the phospholipid

membrane post-thaw. Large variations were noted in the amounts of lipid free radicals

and lipid peroxidation from the fresh samples compared to those found from the post-

thaw samples. This idea was ftirther demonstrated by an analysis of the progressive

sfress accumulated during pre-freezing preparation.

The unique freezing technology could be potentially considered to be used for

spermatozoa cryopreservation for swine. However, ftirther studies need to be

implemented to standardize techniques and find alterative extenders.

Vlll

LIST OF TABLES

3.1 Comparison of Post-Thaw Motilities from Four Pre-Freezing Protocols 28

4.1 Post-Collection Evaluation Values of Stallion Semen 41

4.2 Comparison of Initial, Post-Thaw and Retum Motilities from Four StaUions 41

4.3 Egg Yolk and Glycerol Composition of Biladyl AB and Test Yolk Buffer Extenders 43

5.1 Comparison of Pre-Freezing Procedural Trials on Boar Spermatozoa 52

6.1 Post-Thaw Motility Outcomes from Cryopreserved Boar Semen 64

7.1 Averaged Lipid Peroxidation and Free Radical Amounts from Three Freezing Treatments in Experiment #1 74

7.2 Averaged Lipid Peroxidation Amounts from Fresh and Extended Boar Samples in Experiment #2 74

7.3 Averaged Lipid Peroxidation Amounts from Procedural Steps 76

7.4 Averaged Lipid Free Radical Amounts from Procedural Steps 78

7.5 Motility and Lipid Stability Trends from the Samples of Experiment #3 80

IX

LIST OF FIGURES

4.1 Comparison of Post-lliaw Motilities from Stallion Semen Frozen with Various Freezing Treatments 37

4.2 Comparison of Post-Thaw Motilities of Semen from Acceptable Breeding StaUions Frozen with Various Freezing Treatments 38

4.3 Comparison of Human and Bovine Freezing Extenders on the Post-Thaw Motility of Frozen Stallion Spermatozoa 39

4.4 Comparison of Freezing Techniques used with the Human Extender on

the Post-Thaw Motility of Frozen Stallion Spermatozoa in Experiment #2 40

6.1 Cooling Rates of the UFT 61

6.2 Post-Thaw Motility Percentages of Cryopreserved Boar Semen 62

6.3 Original Motility Percentage Retums of Cryopreserved Boar Semen 63

7.1 Procedural Lipid Peroxidation Trends 77

7.2 Procedural Lipid Free Radical Trends 79

CHAPTER I

INTRODUCTION

Mammalian mating requires a series of specific events with various sequences

and timing ratios to lead to a successfiil fertilization. In simple terms, fertilization is

commonly referred to as an interaction between an ova and a sperm. However, in the

complicated world we live in, it is not that simple, especially in the area of timing. In

response to infertility issues and genetic preservation where timing is the key, assisted

reproductive techniques (ARTs) have been developed and advanced. Though several

techniques have been utilized, including; in vitro fertilization (FVF) and intracytoplasmic

sperm injection ICSI, no other ART offers the availability for sperm conservation other

than cryopreservation. Cryopreservation of sperm requires a reduction in cellular

metabolism while maintaining viability and stractural stability through freezing and

eventually thawing. Depending on the mammalian species, sperm can live up to 120

hours (Hafez and Hafez, 2000) once ejaculated. Holding a sample in a frozen state until

needed allows the useftil lifespan of a sample to be extended up to 50 years (Yoshida,

2000). This process would allow genetic preservation in a variety of scenarios including:

intemational exchange, availability of laboratory evaluation, conservation of neiir-

extinction, transgenic or superior genetic lines, or supply in response to illness, death or

unavailability of the male. This conservation would benefit various biological sciences

such as clinical medicine, biotechnology and agriculture.

The use of assisted reproductive techniques (ARTs) has become increasingly

widespread in the livestock industry. The acceptability of artificial insemination, embryo

fransfer, and cryopreservation of sperm has lead to the establishment of breeding

organizations, semen banks, ART courses, and sire stations. This is especially tine in the

dairy cattle industry, where semen technological advancements have been the most

successful (Vishwanath and Shannon, 2000) and conception rates from frozen semen are

comparable to natural mating (Watson, 2000). However, the resulting quality of

cryopreservation in many species is not remotely similar to that of the initial specimen

collected. In the swine industry, the cryopreservation of bozir semen has repeatedly

produced low pregnancy rates and reduced litter size (Yoshida, 2000). The estimated

average farrowing rate from frozen sperm was 50% with seven piglets to a litter (Johnson

et al., 2000). The acceptability of this technique is newer to the equine industry,

especially within two of the major American breed organizations. Yet a survey of 1999

and 2000 revealed that commercial program conception rates from frozen semen

averaged was only 51.3% during first estras cycle attempts emd 74.7% for the season

(Loomis, 2001). The different success rates between species are not understood, even

though variations in cold shock sensitivity and the molecular composition differences

among sperm types have been confirmed (Parks and Lynch, 1992).

Since the first endeavors of freezing in 1776 by Spallanzani, the techniques

involving cryopreservation of sperm have improved. Although the expansion within the

past few decades has been progressively slow (Yoshida, 2000). Several factors involved

with the handling process during the pre-freeze, freezing and thawing phases have been

suggested as the cause. Nevertheless, the key to answer has not been found and

conceptive rates from spermatozoa following cryopreservation rates remain less than

optimal. Currently, it is estimated that 40-50% of a semen sample will be lost during

cryopreservation, even when following what are considered to be optimum protocols

(Watson, 2000). Thus, previous investigations of cryopreservation concerning the

reductions of fertility have focused on sperm viability and stractural integrity and suggest

significant room for improvements. The objective of the present study was to improve

post-thaw freezing rates while evaluating the effects of thermal gradients, packaging

systems, male specimen variation, membrane stability, and cryoprotectants on equine and

porcine spermatozoa.

CHAPTER II

LITERATURE REVIEW

Introduction

The preservation of sperm through cryopreservation offers a varied of advantages

to the biological sciences, yet the full potential has yet to be realized. Current estimations

project that 40-50% of a semen sample will be lost during cryopreservation even with

optimum protocols (Watson, 2000). Therefore, it is well understood that as the

temperature declines, there may be a reduction in the functionality, biochemical

comp>osition and stracture of the sperm cell. Though a various specie cell lines seem

more susceptible to cold shock, such as the pig, the mechanisms are not totally

understood (Holt, 2000; Johnson et al., 2000). It is assumed cryostress is due to the

variations of composition and the varying functions of homologous component parts

could be the cause for species-specific cryopreservation differences (Calvete et al., 1997).

Affecting factors can be separated into two groups, external and internal. The

external group refers to the alterations of stress due to preparative handling including

diluents, thermal gradient changes during freezing and thawing, cryoprotectants, and

holding periods. The internal factors of cryopreservation are involved with the

biochemistry and composition of the sperm cell itself; including protein stability,

membrane integrity, and oxidative damage. The following is a literature review to briefly

summarize and address these topics.

It should be noted that there is a lack of standard handling and freezing

techniques. Several of the reports reviewed use different practices and various markers

as a means for measuring success (Day, 2000; Love et al., 1988). Due to the absence of

standardized procedures, the findings of some published reports may not be presented as

completely as intended (Samper et al., 2002).

Current Status of Equine Cryopreservation

With the acceptance of cooled, shipped semen, artificial insemination (Al) has

become routine practice in most commercial equine operations (Sieme et al., 2002). The

utilization of frozen semen however has not progressed as quickly (Squires et al., 2002).

This is due in part to breed registry regulations and limitations. Though the European

based breed organizations such as the Warmblood and Trakehner have embraced this

technology, the American associations have recently amended their breeding rales. For

example in 2001, the American Quarter Horse Association and American Paint Horse

Association, two of the world's largest breed associations, permitted the artificial

insemination of frozen-thawed semen (Loomis, 2001). Yet with this new opportunity and

its benefits of genetic transport and decreased venereal disease risk, horse breeders have

not fully adopted this technique. Reports of lower pregnancies rates per cycle have been

commonly observed compared to natural mating or cooled semen (Samper et al., 2002).

Theses reported outcomes makes the utilization of this reproductive technique less

desirable to producers. It is estimated that only 24% of stallions are able to produce an

ejaculate suitable for cryopreservation (Linfor et al., 2002; Vidament, et al., 1997). In a

recent worid-wide survey of commercial breeding centers, Samper and colleagues (2002)

found pregnancy rates from frozen semen ranged from 12.5-60%, with an average of

45.9%. Loomis (2001) reported that pregnancy rate averages are 81.9% outside North

America and only 65.6% within with frozen semen.

Though offering long-term benefits, the possible financial losses from frozen

semen without a fertilization guarantee are a concern. Frozen semen also requfres a

stringent adherence to mare management and specialized personnel fraining. Samper et

al. (2002) noted breeding centers that bred more than 15 mares had a 10% higher

pregnancy rate than those that bred less. However, errors in semen processing,

equipment, and management have also been reported as a cause of reduced fertility rates

(Loomis, 2001). To improve fertility chances, increased inseminations have

demonstrated higher pregnancy rates. In spite of this, stallion owners usually limit frozen

availability since increased use reduces semen reserves (Squires et al., 2002). Regardless

of cause, the equine industry does not currently have a standardized cryopreservation

technique. This variability makes improvements, trends and comparisons hard to

investigate at this time (Samper et al., 2002). Furthermore in the United States, there is a

lack of a unified breeding certification program for facilities, technicians and sire registry

as seen in other countries (Loomis, 2001).

Current Status of Porcine Cryopreservation

In the swine industry, the use of artificial insemination (Al) of sows with

preserved semen has tripled within the past 15 years. It is estimated that more than 99%

of the inseminations performed worldwide are performed with extended liquid state

semen. Semen cryopreservation has been commercially available since 1975. In the last

15 years, the utilization of frozen semen in Europe has significantly increased (Johnson et

al., 2000). Yet with the increased utilization, the fertility performance of cryopreserved

samples is not efficient as emphasized by low litter rates (Yoshida, 2000). The estimated

average farrowing rate from frozen semen is 50% with seven piglets to a litter (Johnson

et. al., 2000).

Porcine semen has been recognized for its sensitivity to cold shock, though the

mechanisms are not totally understood (Holt, 2000; Johnson et al., 2000; Yoshida, 2000).

Even with successful motility levels, fertilization and embryonic development are not

guaranteed. As a litter bearing animal, the sow presents a unique challenge to the ART

through prolonged ovulations requiring sperm viability to encompass 40 hours. Though

fresh semen is able to maintain this longevity, frozen sperm by this point commonly

show a rapid decline (Watson, 2000). Thus, advances in the understanding of the porcine

physiology and development of reproductive techniques are paramount (Yoshida, 2000).

Cellular Effects of Cryopreservation

Mammalian spermatozoa are very temperature sensitive. This sensitivity, known

as cold shock, refers to the irreversible alterations commonly observed during

cryopreservation (Medeiros et al., 2002). Rapid thermal stress on spermatozoa can cause

abnormal swimming pattems, loss of motility, acrosomal and/or plasma membrane

damage, altered metabolism, and infracellular component loss (Amann and Graham,

1993). Once this damage has occurred, it cannot not be overcome since sperm lose the

ability to self repair, grow and biosynthesis after spermatogenesis (Yoshida, 2000). Plus,

this damage can occur in any region of the sperm either independently or collectively

(Zhu and Liu, 2000).

During the cooling process, the plasma membrane will enter a transition phase

changing from a selectively permeable fluid state to one with increased rigidity by

changing the arrangement of the membrane constituents. This will limit the lateral

movement of phospholipids and cause the proteins to become irreversibly clustered.

Functionality of protein-lipid complexes forming membrane receptors and ion pores may

be lost (Amann and Graham, 1993). As the fransition continues, unstable gel-fluid

portions form to create ion gaps. These conformational changes to the inner membrane

can lead to an increased permeability of exfracellular calcium (McLaughlin and Ford,

1994) possibly triggering calcium-dependent processes associated with capacitation

(Buhr et al., 1989; Green and Watson, 2001; Johnson et al., 2000; Watson, 2000). If tiie

membrane is leaking, ATP levels may decrease from the mid-piece signaling cryolysis

(Bosman et al., 1994). While the solutes and cryoprotectants increase outside the sperm,

phospholipid damage increases, protein-lipid complexes weaken and the sperm

dehydrates (Gao et al., 1993). If ice crystal formation occurs, the membrane will most

likely rapture. As the phase fransition completes, protein function may cease due to

denaturation (Amann and Graham, 1993). Thus, protein and DNA conformations may be

altered in the head portion.

Lipid peroxidation, which leads to lipid radical formation, is a normal mechanism

in cellular aerobic metabolism. Reactive oxygen species (ROS) including the superoxide

anion (O2") and hydrogen peroxide (H2O2) are involved in capacitation of the sperm

(Medeiros et al., 2002). However at toxic levels, peroxidation can have degradative

effects on the phospholipid membrane of spermatozoa. To briefly review, during the

oxidation of various metals including iron, an electron is transferred to oxygen (Huang et

al., 2001). This single electron on the oxygen produces a negative charge and unstable

molecule, free oxygen radical or anion. With addition of superoxide dismutase and

hydrogen molecules, the oxygen radicals can convert to peroxide. During the reduction

of iron, peroxide can fransform in to radical hydroxyls (OH). In the presence of

hydroxyl radicals, the stable polyunsaturated fatty acids (LH) of the membrane will lose

its hydrogen to become a lipid radical (L). In an aerobic environment, the lipid radicals

will begin to accelerate a cascade of lipid degradation. To reduce the accumulation of

peroxide, cells possesses catalase and gluthathione, to convert peroxide to water.

However, mammalian sperm have veuious, reduced levels of protective enzymes (Alvarez

and Storey, 1984; Bilodeau et al., 2000; Mennella and Jones, 1980).

With the possibility of any one of these events occurring, the ability of

cryopreserved sperm to sustain embryonic development is questionable (Watson, 2000).

Diluents

In an attempt to create the ideal environment for sperm viability, several studies

have been conducted to determine the effects of the diluents used in cryopreservation.

Diluents for cryopreservation are very similar to those used for storage at a liquid state.

The basic requirements for a cryopreservation solution must include an energy source,

cryoprotectant, osmolarity buffers, cold shock preventative substances, enzymes and

antibiotics (Vishwanath and Shannon, 2000).

When released from the male reproductive tract, sperm are submerged in a natural

buffering nutrient source known as seminal plasma. The presence of seminal fluid in the

female reproductive tract plays a role in the fertilization process both directly and

indirectly. As the sperm swims to its destination, the seminal plasma is an aid as a

nutrition source, buffering agent, and motility regulation while stimulating enhancement

of uterine contractions and the relaxation of the oviductal opening (Johnson et al., 2000).

Seminal plasma also down-regulates the uterine inflammation response to the presence of

sperm (Troedsson et al., 2002). Commonly seminal plasma is removed to increase

insemination rates and increase fertility of frozen-thawed sperm. However, some recent

studies have shown the possible positive influences of seminal fluid used as a diluent.

Using this concept, studies have demonstrated improved motility rates (Katila et al.,

2002; Pickett et al., 1975) and promotion of sperm viability normally inhibited by uterine

inflammation (Troedsson et al., 2002). Catalase activity, an enzymatic converter of

hydrogen peroxide, has been observed in bovine seminal fluid (Bilodeau et al., 2000,

LaPointe et al., 1998). It does need to be recognized that there are contradicting papers.

It has been suggested that the ferrous iron (Fe ^ ) within seminal plasma, is the driving

factor for malondialdehyde (MDA) production during incubation (Huang et al., 2001).

Verma and Kanwar (1999) confirmed increases in lipid peroxidation rates from samples

10

incubated with seminal plasma. Though Braemmer et al. (2002) found seminal plasma

had no effect on the post-thaw motilities of epididymal sperm. Love et al. (2002)

discovered detrimental motility and chromatin condensing effects.

At certain stages of the cryopreservation process, sperm cells may undergo

fluctuating environments due to the amounts of solutes inside or outside the cell. This

process is referred to as osmotic stress. During the freezing stage, the cells enter into a

hyperosmotic environment. As exfracellular ice crystals form, the solutes outside of the

cell increase in concenfration. In response, the sperm cell will dehydrate and shrink in

volume by losing infracellular water in attempt to equilibrate to the outer environment. A

hyperosmotic environment can also induce a reduction in motility while increasing

tyrosine phosphorylation, a step in the capacitation process (Linfor et al., 2002). During

the thawing stage, the sperm cell enters into a hypotonic envfronment where water will

passively enter into cell. This increase in volume can cause the cell to swell. Osmotic

sfress thus can cause irreversible damage, rendering the sperm cell incapable of

fertilization (Curry and Watson, 1994; Pommer et al., 2002).

More than one-half century ago, egg yolk was discovered to possess protective

attributes when added to spermatozoa during cryopreservation (Medeiros et al., 2002). It

has been proposed that the success of egg yolk extenders is due to the low density

lipoproteins that are found in two-thirds of the composition (Moussa et al., 2002).

Although egg yolk has been a successfiil addition to bull freezing extender (Vishwanath

and Shannon, 2000), the results have not been as successful for boars (Pursel et al, 1972).

Egg yolk extenders are still more commonly utilized, yet milk components have also

been developed into extenders (Vishwanath and Shannon, 2000).

To attempt to further reduce ice formation and osmotic situations, cryoprotectants

are usually added to freezing extenders. There are two types of cryoprotective agents,

permeable (i.e., glycerol, dimethyl sulfoxide, 1,2-propanediol) or non-permeable (i.e.

sucrose, sodium chloride, raffinose) (Liu and Foote, 1998; Sztein et al., 2001). In most

mammalian cryopreservation processes, glycerol is considered the standard

cryoprotectant (Silva et al., 2003; Watson, 2000). Glycerol is a permeating agent that

enters into the cell to replace the water that leaves during cooling, thus reducing the

osmotic sfress (Medeiros et al., 2002). The levels of glycerol added are dependant on the

cooling velocity. At each glycerol level, sperm can tolerate a variety of temperature

ranges without viability losses (Johnson et al., 2000). Glycerol levels are also dependant

on specie type. Glycerol is usually added at concentrations of 2.5 to 6% for stallions

(Samper and Morris, 1998; Vidament et al., 2002), 2.25 to 9% for bulls (Vishwanatii and

Shannon, 2000), 5 to 6% for goat bucks (Amoah and Gelaye, 1996), and 13-16% in

roosters (Gill et al., 1996).

However, permeating cryoprotective agents, such as glycerol, can be toxic to the

cell and cause membrane instability and motility loss (Medeiros et al., 2002). The toxic

effects are due to high glycerol concenfrations or too rapid of an addition and removal

from the cell (Katkov et al., 1998). Also, it has been observed that higher glycerol

concentrations cause an increase in acrosomal damage (Almid and Johnson, 1988).

Several articles have tested other cryoprotective agents in various species. A few of the

12

otiier cryoprotective agents evaluated include dimethyl sulfoxide (Amoah and Gelaye,

1996; Ball and Vo, 2001; Gilmore et al., 1998, 1995; Johnson et al., 2000; Sztein et al.,

2001), ethyl glycol (Ball and Vo, 2001; Gilmore et al., 1998, 1995; Henry et al., 2002),

and various amides (Blanco et al., 2000; Henry et al., 2002; Johnson et al., 2000;

Medeiros et al., 2002; Sztein et al., 2001; Vidament et al., 2002). Results vary

depending on the species, concentrations levels, and utilization of the agents in

combination or alone.

Thermal Gradients

The process of cryopreservation involves a range of temperatures during the

freezing and thawing process. A sperm cell is ejaculated close to body temperature,

cooled to below freezing, and returned to body temperature during thawing. The stress

upon the spermatozoa during these thermal insults can create irreversible damage to the

ftmctionality of the cell. In general terms, this damage is referred to as cold shock.

Though spermatozoa from any mammalian species can demonsfrate cold shock, the

thermal sensitivity or resistance varies between species (Medeiros et al., 2001). This is

due to the different of ultrastractural changes that occur and composition differences

between sperm (Curry and Watson, 1994; DeLeeuw et al., 1990; Parks and Lynch, 1992).

During the cooling process, several stractural and metabolic alterations occur

within the sperm cell at various temperatures. As the temperature nears 20-25°C, the

membrane fluidity will decrease in the samples from bulls, stallions, boars and roosters

(Parks and Lynch, 1992). This alteration is due to the stiffening of the hydrocarbon tails

13

of lipid within the bilipid layer to shift the membrane into a gel-like solid. Lowering the

temperature further from 22°C to 4°C, oxygen free radical amounts increase in bull sperm

(Chatterjee and Gagnon, 2001). This indicates an increase in lipid instability through

lipid peroxidation rates. Once the temperature falls to 4°C, there is also depression in

metabolic cellular activity (Medeiros et al., 2002) including the ftmctionality of sodium-

potassium (Na/K) ion pumps (Vishwanath and Shannon, 2000). Green and Watson

(2001) observed capacitation-like events in boar sperm cooled to 5*'C. This capacitation-

like event is related to flux of calcium, involved in hyperosmotic environments (Watson,

2000). At the freezing point, the latent heat radiated from the sperm cell during ice

formation must also be considered (Thurston et al., 2003). Past the freezing point, ice

formation occurs at about -10°C, depending on the cryoprotectant (Medefros et al., 2002).

Freezing rates must be fast enough to reduce infracellular ice formation, yet slow

enough to avoid osmotic alterations. As discussed in a previous section, glycerol is

commonly used as a cryoprotectant. However, there is a relation between the cooling

velocity and glycerol concentrations. The faster the cooling rate, the less glycerol needed

(Johnson et al., 2000).

Mammalian sperm are frozen at range of a rate of-15°C to -60°C/minute

(Watson, 2000). Various studies have reported a wide range of freezing rates. For

example, sperm viabilities were maintained in equine sperm at rates of-20°C to

-130°C/minute (Devireddy et al., 2002) and in swine sperm at -3°C to -80°C/minute

(Medrano et al., 2002). Recommended optimal freezing rates for each species are more

limited. Suggested rates include -5°C/minute in rams (Byme et al., 2000), -30°C/minute

14

in boars (Fiser et al., 1993; Johnson et al., 2000), and between -80°C and-120°C/minute in

bulls (Vishwanath and Shannon, 2000). Though the equine cryopreservation protocols

vary, most use liquid nitrogen vapor rate of-5°C/minute (Samper and Morris, 1998).

Stractural integrity due to thermal stress is not limited to the freezing rate but also

the thawing rate. Osmotic stresses have been recognized during the re-warming process

(Holt and North, 1994; Pommer et al., 2002; Watson, 2000). It has been suggested that

more damage occurs in hyposmotic environment than in a hyperosmotic environment.

These frends have been observed in the sperm of stallion (Ball and Vo, 2001; Pommer et

al., 2002), human male (Curry and Watson, 1994; Gao et al., 1993), boar (Gihnore et al.,

1996; Medrano et al., 2002; Zeng et al., 2001), mouse (Willoughby et al., 1996) and ram

(Curry and Watson, 1994; Holt and North, 1994). An exception though would be the bull

(DeJamettee et al., 2000). Regardless of these reports, the relation between freezing and

thawing rates must be taken in consideration (Henry et al., 1993). Post-thaw rates of

motility and acrosomal stability from fast freezing rates, 30°C/minute, were superior to

those from slow-freeze, l°C/minute, after a rapid thawing process (Fiser et al., 1993). In

general, faster rates of thawing are considered to be more beneficial. At slower rate of

thaw, the risk of ice reformation is more likely (Vishwanath and Shannon, 2000).

Pre-Freezing Protocols

Artificial insemination with the use of liquid-state semen has become common

procedure in several breeding facilities. Developments in this area of reproduction have

extended the life of semen from a few hours to several days. Yet, a few days is still

15

limiting in comparison to the potential genetic preservation gained through

cryopreservation. In attempt to refine cryopreservation protocols, these developments

have been integrated into the preparative handling techniques being utilized today.

Seminal plasma can only sustain sperm viability for a few hours after ejaculation

(Johnson et al., 2000). Boar semen incubated at 37°C in seminal plasma developed high

rates of peroxidative degradation (Comaschi et al., 1989). Though keeping a portion of

the seminal fluid amount may have beneficial properties, as discussed earlier, it is

common to remove the fluid through centrifugation. Samper and Morris (1998) found

that all horse breeders surveyed centrifuged samples after collection. Centrifugation

creates a more concenttated specimen while allowing for the limiting nutrient source to

be replaced. Removal of all seminal plasma also created a higher rate of chromatin

stability (Love et al., 2002) and higher post-thaw motilities (Martin et al., 1979) in

stallion samples. Nevertheless it has been suggested that centrifugation may cause undue

sfress to sperm (Gil et al., 1999). With dog semen, a higher amount of sperm was lost in

the discarded supemant at lower centrifiigal speeds. At faster centrifiigation speeds,

viability losses were higher (Rijsselaere et al., 2002). Neild and colleagues (2003) also

reported a capacitation-like phenomenon after centrifugation of stallion sperm. As a

compromise, Johnson and colleagues (2000) suggested the use of one of the most utilized

protocols for boar cryopreservation in a commercial setting, the Beltsville method. Initial

cooling involves incubating a sample in seminal fluid for 2 hours before centrifugation as

part of the protocol.

16

It has been suggested that post-thaw success may be achieved through a series of

osmotic steps (Curry and Watson, 1994; Gao et al., 1993). There is evidence of this idea

through the increases in cold shock resistance observed in sperm after incubation or

holding periods at cooler temperatures. Epididymal spermatozoa of the stallion held at

5°C for 24 hours demonstrated similar post-thaw motilities to those from samples

processed immediately (Braemmer et al., 2002). Similar trends in ejaculated sperm were

observed after 12 hours of incubation at 5°C (Crockett et al., 2001). Boar sperm

appeared to be more cold resistance after a 5 hour incubation period versus two or seven

hour periods (Pursel et al., 1972). More recentiy, Eriksson et al. (2001) demonstrated

higher retum rates with incubation periods of 10 and 20 hours for boar semen cooled to

17°C.

To create an optimal cryopreservation protocol, a cooling rate compatible with

osmotic environment must be considered (Watson, 2000). Glycerolized diluents have

reported to have varying success rates based on time to equilibration. Initially it was

thought that extended semen exposed to glycerol did not show any survival differences

post-thaw based on exposure time. However, a trial utilizing 0.5 mL straws demonsfrated

that an incubation period of 4 hours produced higher motilities and improved morphology

(Johnson et al., 2000). Temperature in which the glycerol is added also affects the post-

thaw outcomes. Vidament and colleagues (2000) found the addition of glycerol at 22°C

generated a higher retum rate than glycerol added at 4°C.

17

Packatzing Systems for Cryopreservation

In agriculture, frozen sperm offers an alterative solution to the labor and expense

of shipping live animals with the intent of spreading superior genetics. This advantage

not only allows for extension of travel distances and availability of semen supply, but

also helps ensure the health of a herd involved with breeding practices. As stated

previously, maximum fertility levels have not been reached for several species with

regard to cryopreservation. Aside from the challenges of optimum temperature gradients

and diluents, there is also a quest for the perfect containment system. Various types of

packaging systems have utilized in attempt to overcome the problems of identification,

contamination, thermal gradients and insemination amounts available associated with

some packaging units.

During the initial onset of cryopreservation use in the livestock industry, pelleting

semen on dry ice was a technique initially used in bulls (Martin et al., 1979; Merkt et al.,

1975). It has been discovered however that this methodology has several disadvantages.

Concems with identification, contamination and exchange of cellular material have been

recognized (Pickett et al., 1978). Straws with various volumes have also been utilized.

Though the 0.25 mL and 0.15 mL straws do not have a large volume capacity, there is a

higher rate of heat transfer allowing for a more uniformity in the freeze. From a

management standjwint, the 5 mL offers more desirable volume due to the required

eunount needed for an insemination dose.

Several studies have evaluated the efficiency of each package. Though the

seminal processing was not performed consistently, the results still show variation in

18

between species. Using bull (Johnson et al., 1995) and rooster (Duplaix and Sexton,

1983) semen, there was little difference in the post-thaw results between the 0.25 mL and

0.5 mL straws. However, Thomas and colleagues (1993) found higher rates of

progressive motile sperm post-thaw in canine semen with pellets when compared to

either of these sfraws. In lieu of the reduced capacity available from pellets and straws,

larger volume packages have also been investigated. Increased fertility rates were

reported in stallion semen was using 4 mL straws (Martin et al., 1979) and also in boars

semen using 5 mL straws (Cordova et al., 2002). Cordova and colleagues also observed a

fiigher rate of chromatin condensation with an increased stability in the larger straws.

Yet, aluminum tubes with larger volumes showed potential in stallion semen (Tischner,

1979) and later outperformed the enhanced straws with stallion sperm (Love et al., 1989)

and pelleting with canine semen (Ivanova-Kicheva et al., 1997). In the swine industry, 5

mL FlatPacks have shown potential in both the laboratory (Eriksson et al., 2001) and in

field trials (Eriksson et al., 2002). Several studies have evaluated the efficiency of each

package. Though the seminal processing was not performed consistentiy, the results still

show variation in between species. Using bull (Johnson et al., 1990) and rooster

(Duplaix and Sexton, 1983) semen, there was little difference in the post-thaw results

between the 0.25 mL and 0.5 mL straws. However, Thomas and colleagues (1993) found

higher rates of progressive motile sperm post-thaw in canine semen with pellets when

compared to either of these straws. In lieu of the reduced capacity available from pellets

and straws, larger volume packages have also been investigated.

The straw is considered to be the most accepted technique for cryopreservation of

many cryopreservation protocols. Species included in this group include bovine (Johnson

et al., 1995), canine (Pena and Linde-l'orsberg, 2000), and equine (Samper et al., 2002).

In the swine industry, the most utilized commercial packaging unit is the 5 mL Maxi-

straw (Cordova et al., 2002; Eriksson et al., 2000), even though all three packaging

variations are still used (Eriksson et al., 2002).

Summary

The potential benefits of cryopreservation of sperm could provide ways to

overcome the limits of time involving reproduction by increasing the long-term storage

and use of the genetic materials of the male. However, as presented in this section, the

cryopreservation process is multi-faceted with interactions of several factors to take in to

consideration, including the alterations of stractural integrity due to the effects of

preparative handling (Comaschi et al., 1989; Crockett et al.. 2001; Eriksson et al., 2001;

Love et al., 2002), extenders (Arrada et al., 2002; Ball and Vo, 2001; Wilhelm et al.,

1996) and thermal insults (Devireddy et al., 2002; Lagares et al., 2000; Linfor et al.,

2002; Pommer and Meyers, 2002). In several species, the optimal combination has not

yet been found. Two species included in this grouping are the boar and stallion. Both

have been shown to have significant problems with cold shock (Holt, 1999; Johnson et

al., 2000; Pursel et al., 1972; Yoshida, 2000) as well as sensitivity to cryoprotectants

(Henry et al., 2002; Johnson et al., 2000; Medeiros et al., 2002). The objective of tiie

present study was to determine if many of the problems described within this review

20

could be overcome in the boar and stallion using a variety of pre-freezing techniques in

conjunction with a new freezing technology (UFT).

21

CHAPTER 111

EFFECTS OF SEMEN HANDLING ON POST-THAW

MOTILITY OUTCOMES OF CRYOPRESERVED

EQUINIi SPERMATOZOA

Abstract

The equine industry current lacks a standard protocol for handling semen for

cryopreservation. Due to this variability, success of extenders, incubations periods,

cryoprotectant amount and thermal alterations are difficult to assess. The objectives of

this trial were to assess the post-thaw outcomes for various protocols. This study was

conducted in four trials. In trial #1, an extended post-collection sample was diluted with

cryoprotectant (0%-10%) and held at one of two incubation periods (10 minutes or 1

hour) before being frozen in a unique freezing technology (UFT) system. Trial #2

evaluated similar cryoprotectant concentrations with extended incubations periods (0, 2,4

or 6 hours) at 5* 0. In trial #3, glycerol levels were limited to 2%, 5%, or 10% with a two

hour 5°C incubation. These samples were divided ftirther for freezing in a unique

freezing technology (UFT) system, controlled freezing unit or liquid nifrogen mist. The

fourth trial, added the effects of centrifiigation, step-down cooling rate during various

incubations, and the addition of two freezing extenders. From the results, all of the post-

thaw motilities for trials #1, #2, and #3 were 0%. However, trial #4 produced post-thaw

motilities ranging from 2% to 43%, averaging 21%. Comparing the outcomes from each

trial, there was a significant difference in the results between the first three trials and #4

22

(P<0.001). Though there was some consistency between the initial extender and freezing

treatments utilized for this study, our results suggest that incubation periods with

decreasing temperatures, centrifugation and the addition of freezing extenders

demonstrated potential. Further research is needed to investigate the influences of these

specific factors.

Introduction

Artificial insemination of mares with cooled; shipped semen has become common

procedure in several breeding facilities (Sieme et al., 2002). The acceptance of this

artificial reproductive technique has grown since the 1980's with the elimination of breed

organization regulations and the development of an effective transport container fLoomis,

2001). The utilization of frozen semen however has not progressed as quickly (Squires et

al.. 2002). However, the acceptance of cryopreservation within the equine industry has

only occurred within the last few years (Loomis, 2001). As a consequence, there is a lack

of standardized techniques (Samper et al., 2002; Samper and Morris, 1998; Thurston et

al., 2003) unlike the more established programs involving boars (Johnson et al., 2000) or

bulls (Vishwanath and Shannon, 2000).

The objective of this study was to evaluate the effects of several different

preparative handling factors while implementing the UFT.

23

Materials and Methods

Post-Collection Processing

Several ejaculates from four Quarter Horse stallions were collected during the

2001 and 2002 horse breeding season. All of the stallions were collected at the Texas

Tech University Ranch Horse Center in New Deal, TX, by trained personnel. After each

collection, the ejaculate was initially evaluated for total volume, gel-free volume,

concentration and initial motility. The original motility of all samples needed to be equal

or greater than 60% to be considered acceptable for this project. A sample was extracted

and extended 2:1 with pre-warmed E-Z Mixin CST Extender (Animal Reproductive

Systems; Chino, CA) and packaged for transport to one of two off-site laboratories for

ftirther processing.

Freezing Technology

A new unique freezing technology (UFT) was utilized in this study. SupaChill®

(SupaChill, Inc.; Lubbock, TX) incorporates an organic freezing fluid with a heat index

similar to water; thus allowing for a high rate of heat dissipation. The freezing

technology of UFT has the unique ability to be set at temperature levels below 0°C

(freezing point) with out solidifying.

24

Procedural Methodology

Trial #1

Upon arrival at the off-site laboratory, two stallion samples were divided into six

5 mL amounts in pre-labeled tubes. Samples were then diluted with glycerol at

concentrations of 0%, 2%, 4%, 6%, 8% or 10%. Two aliquots of 1.5 mL were removed

from each tube and placed in 1.8 mL cryo-tubes (Nalge Nunc Intemational; Denmark)

witii an "A" or "B" label. The A tubes were held at room temperature (22°C) for ten

minutes before being plunged into the -25°C UFT. The B tubes were cooled in a 5°C

refrigerator for one hour before being frozen in the -25*'C UFT. After freezing, the 24

vials were stored in liquid nifrogen until post-thawing analysis could be completed.

Trial #2

Upon arrival at the off-site laboratory, a sample was divided into five 7 mL

amounts in pre-labeled tubes. Samples were then diluted with glycerol at concentrations

of 2%, 4%, 6%, 8% or 10%. One mL aliquots were removed from each tube and placed

in 1.8 mL cryo-tubes with a number label ranging from 0 to 6. These numbers

represented the number of hours in which the samples were held at 5°C before freezing.

Vials were frozen in the -25*'C UFT, after holding for one hour. After fi-eezing, the 35

vials were stored in liquid nitrogen until post-thawing analysis could be completed.

25

Trial #3

A third trial similar to the procedure of the second was also completed using two

stallion samples. However, only 2%, 5%, and 10% glycerol concentrations were used

with a two hour incubation in the 5°C. The freezing treatments were expanded to include

the UFT, a control freezing unit (Planar Unit, Planar Products Ltd.; United Kingdom) or

nittogen mist vapor. After freezing, the 18 vials were stored in liquid nifrogen until post-

thawing analysis could be completed.

Trial #4

Upon arrival at either laboratory, each sample was placed into a 17°C refrigeration

unit. TTie sample was held for one hour at this temperature after which a pre-cooled 17**C

wash buffer was added. The sample was held at this temperature for an additional 1 Vi

hour to equilibrate with the wash buffer. The sample then was centrifiiged to create a

sperm-composed pellet. The pellet was then dissipated in a pre-cooled 25% egg-yolk

extender at a 1:1 ratio with the original sample size. The sample was placed in a 5°C

refrigeration unit for 1 '/j hour before a secondary freezing extender was added in three

increments over 30 minutes. This 25% egg-yolk extender with 6% glycerol was also pre-

cooled to 5°C. Following the addition of the last increment, the sample was held at 5°C

for one hour before freezing. The sample was not broken into confrol and freatment

groups until immediately before freezing to ensure consistent handling. One ml aliquots

were extracted from the extended sample and placed into 1.8 ml labeled cryo-tubes for

freezing. Four freezing treatments were tested for this trial, three UFT and one confrol:

26

B: 4 minutes at-25°C

C: 4 minutes at -25°C with a proprietary filter #1

E: 4 minutes at -25°C with a proprietary filter #3

Control: 10 minutes in liquid nitrogen mist.

After freezing, the 24 vials were stored in a liquid nitrogen tank until the post-thaw

analysis could be completed.

Thawing Procedure

In Trials #1 and #2, a slow thaw protocol was utilized. The samples were

extracted from the nifrogen storage tank and placed in a 5°C refrigerator for 30 minutes.

This was followed by 5 minutes in a 37*'C water bath. For trials #3 and #4, a faster

thawing protocol was utilized. The samples were placed held in the air (22*'C) for 45

seconds before warming in a 37°C water bath for 5 minutes. All samples were

subjectively evaluated on a random and individual basis for motility.

Statistical Analysis

The post-thaw motilities from the four trials were compared using analysis of

variance (ANOVA). ANOVA was also used to compare the post-thaw outcomes from

the four freezing freatments (B, C, E, Control) within Trial #4. All statistics were

analyzed using the SPSS Version 8.0 Software (SPSS, Inc.; Chicago, IL).

27

Results

All samples from trials 1#, 2#, and #3 had 0% post-tiiaw motilities. Post-thaw

motilities from samples of trial #4 ranged from 2% to 43%. Consequentiy, a significant

difference was observed when comparing the post-thaw motilities of Trials #1, #2, and #3

to tiiose of Trial #4 (P<0.001). This is presented in Table 3.1.

Table 3.1 Comparison of Post-Thaw Motilities from Four Pre-Freezing Protocols

Motility

Trial n Avg Post-Thaw, % SD

#1 24 0%" 0.0

#2 35 0%" 0.0

#3 18 0%" 0.0

#4 24 21%** 9^4

Different superscripts represent significant differences in the post-thaw motihty percentages between the four trials as determined by P<0.001

Variations in between the four freezing treatments used in Trial #4 were also

investigated. The average post-thaw motilities for B (19%), C (18%). E (22%). and

Confrol (20%) groups were not significantly different.

Discussion

From our post-thaw comparison, the higher retums produced in Trial #4

emphasizes the need for a pre-freezing protocol with focus on osmotic equilibrium.

28

Though temperature shifts were utilized in the first three trials, trial #4 was the only

protocol with a two phase pre-freeze temperature shift, centrifiigation, and the addition of

an egg yolk freezing extenders.

Seminal plasma can only sustain sperm viability for a few hours after ejaculation

(Johnson et al., 2000). Higher rates of lipid peroxidation were observed in boar semen

incubated at 37°C in seminal plasma (Comaschi et al., 1989). This coincides with the

findings of Trials #1, #2, and #3, where seminal fluid was maintained in the sample

during the cooling process. Removal of the seminal plasma by centrifiigation created a

higher rate of chromatin stability (Love et al., 2002) and higher post-thaw motilities

(Martin et al., 1979) in stallion samples. The beneficial effects of removing the supemant

were observed in Trial #4.

It has been suggested that successfiil post-thaw results may be achieved through a

series of osmotic steps (Curry and Watson, 1994; Gao et al., 1993). Evidence of this idea

has been observed through the increases in cold shock resistance observed in sperm after

incubation or holding {jeriods at cooler temperatures. Species in which this included was

the stallion (Braemmer et al., 2002; Crockett et al., 2001) and boar sperm (Eriksson et al.

2001; Pursel et al., 1972). In several commercial boar settings, protocols involves

cooling a sample in seminal fluid for 2 hours before centrifiigation and cooling further

before freezing (Johnson et al., 2000).

Glycerol is usually added at concenttations of 2.5 to 6% for stallions (Samper and

Morris, 1998; Vidament et al., 2002). All four trials had glycerol concenfrations that fell

into this range. However glycerolized diluents have reported to have varying success

29

rates based on time to equilibration. Initially it was thought that extended semen exposed

to glycerol did not show any survival differences post-thaw based on exposure time.

However, a trial utilizing 0.5 mL straws demonstrated that an incubation period of 4

hours produced higher motilities and improved morphology (Johnson et al., 2000).

Temperature in which the glycerol is added also affects the post-thaw outcomes.

Vidament and colleagues (2000) found the addition of glycerol at 22°C generated a

higher retum rate than glycerol added at 4°C. Though trials #2 and #3 had longer

incubation periods at 5°C, the lack of an additional extender may lead to a metabolism

deficiency. Also egg yolk extenders possess protective attributes when added to

spermatozoa during cryopreservation (Medeiros et al., 2002).

In conclusion, refinement to preparative handling techniques of stallion sperm is

required for the development of an optimal protocol. Our results suggest that incubation

periods with decreasing temperatures, centrifiigation and the addition of freezing

extenders demonstrated potential. Further research is needed to investigate the influences

from these factors and the repeatability of this protocol with other stallions

30

CHAPTER IV

COMPARISONS OF A UNIQUE FREEZflvlG

TECHNOLOGY (UFT) TO TRADITIONAL

LIQUID NITROGEN METHODOLOGY FOR

CRYOPRESERVATION OF EQUINE SPERMATOZOA

Abstract

Several breed organizations in the equine industry have recently accepted the use

of frozen semen. However, cryopreserved stallion semen is commonly associated with

poor post-thaw semen quality and decreased pregnancy rates as compared to those

produced during normal mating or with cooled semen techniques. Therefore our

objective was to evaluate a new unique freezing technique (UFT) with the intent of

improving fertility outcomes. A series of experiments tested the UFT compared to

traditional liquid nitrogen methodology in combination with influence of the extenders

and stallions used. In Experiment #1, post-thaw motility results of UFT variations were

compared to those from liquid nitrogen methods. The averaged post-thaw motility

percentages of the four UFT treatments were similar when compared to the traditional

liquid nitrogen control group (P=0.845). In experiment #2, two egg-yolk based freezing

extenders, Biladyl AB® (Minitub GmbH; Germany) intended for bovine samples and

Freezing Medium Test Yolk Buffer® extender (Irving Scientific; San Jose, CA) used

human samples, were compared. A significant difference in the average post-thaw

motilities was found in between the Biladyl AB® (17%) as compared to the Test Yolk

31

Medium Buffer® extender (25%) (P<0.002). In the final experiment, we compared

variability between stallions using the UFT with the intensions of creating a more

consistent outcome. Post-thaw motilities and percent of original motility retum between

the four stallions were significantiy different (P<0.001).

In conclusion, the UFT may be potentially used as an alterative freezing method

to replacement current liquid nitrogen methodology. However, further investigation is

needed to refine techniques.

Infroduction

Until recently, freezing of equine spermatozoa was limited due to poor post-thaw

results (Devireddy et al., 2002; Samper et al., 2002) and breed regulations. Though the

reproductive capacity of equine frozen semen is still low, the acceptance of this

technology is slowly growing due to the elimination of other breed registry barriers

(Loomis, 2001).

Traditional freezing methodology of equine semen involved placing a sample in

liquid nitrogen vapor for 10 to 20 minutes before plunging. Though still used today, the

post-thaw results are less than optimal. Within the last few years, a high speed freezing

system has emerged for freezing foodstuffs with an increased ability of preserving

freshness (SupaChill®, SupaChill USA; Lubbock, TX). This Unique Freezing

Technique (UFT) contains an organic fluid with a heat capacity similar to water with a

freezing rate of-6.1°C/minute. This technique has been applied to various cell samples

and tissue biopsies with successful thawing results in preliminary trials. Our objective

32

was to compare the Ul i to traditional nitrogen methods while investigating various

cryopreservation factors.

Two cryopreservation factors were addressed in this study while studying the

UFT. More than one-half century ago, egg yolk was discovered to possess protective

attributes when added to spermatozoa during cryopreservation (Medeiros et al., 2002).

At least two egg-yolk cryoprotectant have demonstrated successfiil results in two other

species; Biladyl AB® (Minitub GmbH; Germany), a cryoprotectant is commonly used

for bovine semen cryopreservation, and freezing Medium Test Yolk Buffer® (Irving

Scientific; San Jose, Califomia), a cryoprotectant used on human samples. As no

standard cryoprotectant system exists in equine, the intention of this study was to assess

the post-thaw motilities of stallion semen frozen with these two extenders while studying

tiie UFT.

Several reports have discussed the variability seen in the cooling and freezing of

equine semen. Although several factors including health, season and age have been

known to affect semen quality, lower pregnancy rates from various stallions have been

related to the tolerance of semen sample (Samper et al., 2002). Our evaluation of the use

of the UFT in cryopreservation of stallion spermatozoa focused on the performance of

post-thaw motility, and comparing consistency between stallions.

33


Semen Collection

Several ejaculates from four Quarter Horses stallions were collected for this trial

during the 2002 breeding season. All stallions were collected off a breeding phantom

using a Missouri artificial vagina at the Texas Tech University New Deal Farm, TX.

After each collection, the ejaculate was initially evaluated for total volume, gel-free

volume, concentration and initial motility. The original motility of all samples needed to

be at least 60% to be considered acceptable for this project. A 30 ml sample was

exfracted and extended 2:1 with Equine E-Z Mixin CST Extender (Animal Reproductive

Systems; Chino, CA) and packaged for transport to one of two off-site laboratories for

ftirther processing.

Semen Processing

Up)on arrival to either laboratory, each sample was placed into a 17°C

environment. After holding for one hour at this temperature, a wash buffer pre-cooled to

the same temperature was added. The sample was maintained at this temperature for an

additional 1 '/2 hour, after which the sample was centrifuged. The supernatant fluid was

extracted and discarded. The remaining pellet was dissipated in 30 mL of a pre-cooled

freezing egg-yolk based extender (17°C). The sample was placed in 5°C refrigeration

unit for 1 '/z hours before 30 mL of the second pre-cooled freezing extender with 6%

glycerol was added. The second extender was added in increments over 30 minutes.

Following the addition of the final increment, the sample was held at 5°C for one hour

34

before freezing. The sample was not broken into control and treatment groups until

immediately before freezing to ensure consistent handling.

Unique Freezing Technology


(SupaChill USA; Lubbock, TX) incorporates an organic freezing fluid with a heat index

similar to water; thus allowing for a high rate of heat dissipation. The freezing fluid of

UFT has the unique ability to be set at temperature levels below O C (freezing point) with

out solidifying. Preliminary trials freezing foodstuffs and various cell lines with UFT

showed improved post-thaw retums versus other fraditional freezing methodology.

Freezing Methodology

One mL increments were extracted from the extended sample and placed into 1.8

mL cryo-tubes (Nalge Nunc Intemational; Denmark) for freezing. Preliminary trials

demonstrated higher post-thaw outcome with the cryo-tube than with the fraditional 0.5

ml straw. Cryo-tubes were grouped and labeled in sets of three for freezing. Preliminary

trials on boar semen determined the UFT freezing freatments selected. Freezing and

labeling for the UFT were as follows:


F: 5 minutes at -10°C, tiien 4 minutes at -25°C

G: 5 minutes at -10°C then 4 minutes at -25°C with a proprietary filter #1

35

J: 5 minutes at -10°C witii a proprietary filter #1 then 4 minutes at -25°C with a

proprietary filter #1

Control: 10 minutes in the liquid nitrogen mist.

Confrol vials were frozen using traditional liquid nitrogen methodology. Once initial

freezing was complete, all vials were stored in a nitrogen tank for at least 48 hours before

being evaluated for post-thaw values.

Thawing Process

Randomly selected samples were thawed individually. After extraction from the

liquid nifrogen tank, samples were warmed for 45 sec at room temperature (22°C) before

being placed in a 37°C water bath for 5 minutes. Samples were assessed manually by the

same observer for motility values.


All data was analyzed using the SPSS Version 8.0 Software (SPSS, Inc.; Chicago,

IL). In Experiment #1, the statistical significance of the post-thaw motility values

between the UFT treatment groups and control group was analyzed using analysis of

variance (ANOVA). In experiments #2 and #3, ANOVA was used to statistical

determine the differences between extenders and stallions. In the case that the

comparison was significantly different, such as in Experiment #3 the stallion comparison,

the stallions were separated using Fischer's Least Square Difference (LSD) procedure.

36

In each experiment, the effects of the specific UFT techniques used were also evaluated

by ANOVA.

Results

For experiment #1, 105 samples from seven different ejaculates were analyzed

diluted with various extender and wash buffer types. Preliminary results compared only

the effects of freezing on post-thaw motilities. Referring to Figure 4.1, the average

motilities for individual UFT treatments were significantly lower than the control

(P<0.001). Using LSD procedure, the significant differences between individual

treatments were separated (P<0.003).

CO

o Q-

40

35

30

25

20

15

10

5

27% "

13%" 1 5 % ' 16%'

^H

- m-'~ rrr. _, ^H

^1

— 1

UFT B UFT F UFT G

Freezing Technique

UFTJ ConLN

P<0.001

Figure 4.1 Comparison of Post-Thaw Motilities from Stallion Semen Frozen with Various Freezing Treatments

Different letter superscripts represent the significant differences in the average post-thaw motility percentages between the four freezing treatments as determined by P<0.003.

37

During the analysis of data presented in Figure 4.1, it was noted that one stallion's

sample returned no motility in any of the UFT treatment groups. To reduce the variation

that may be caused by stallion influence and extenders used, a second statistical analysis

was performed on the post-thaw motility data. In the second evaluation of this data,

samples from three different stallion collections were reanalyzed. These three samples

were processed with the same type of freezing extender. Freezing Medium Test Yolk

Buffer® extender (Irving Scientific; San Jose, CA). Referring to Figure 4.2, four of the

UFT techniques produced comparable results to the traditional nitrogen methodology

(P=0.845).

UFTB UFTF UFT G UFTJ

Freezing Technique

ConLN

P=0.845

Figure 4.2 Comparison of Post-Thaw Motilities of Semen from Acceptable Breeding Stallions Frozen with Various Freezing Treatments

38

Our results in Experiment #2, confirmed the influence of freezing extender as

observed in Experiment #1. To compare extenders, sixty samples were evaluated in this

experiment to compare the two extenders on four ejaculates from two Quarter Horses

stallions. From our study, the post-thaw motilities from Freezing Medium-Test Yolk

Buffer extenders (26%) were statistically different than those produced with the Biladyl

AB® (17%) as presented in Figure 4.3 (P=0.001).

35

30

25 •*- .»

a 0 20 4

1 15 H t); 10 o

O H

5

26%^

17%"

1

Freezing M edium TYB, Human Bilady i AB®, Bovine

Extender Type p < 0.002

Figure 4.3 Comparison of Human and Bovine Freezing Extenders on the Post-Thaw Motility of Frozen Stallion Spermatozoa.

Different letter superscripts represent the significant differences in the average post-thaw motility percentages between the two extenders as determined by P<0.002.

A second analysis to compare the influence of the freezing techniques within the

human extender experiment was completed. As presented in Figure 4.4, the UFT values

were not significantly different than the control

39

UFT F UFT G UFT J Freezing Technique

ConLN

P = 0.180

Figure 4.4 Comparison of Freezing Techniques used with the Human Extender on the Post-Thaw Motility of Frozen Stallion Spermatozoa in Experiment #2.

In the final experiment, four ejaculates from four different Quarter Horses

stallions were collected on separate days during the 2002 breeding season. Fertile

stallion ejaculation minimum requirements include 50 ml volume, 50% motile and 8 x

lO' sperm cells, were met and exceeded by the ejaculates used for this trial (Evans et al.,

1990). Post-collection values are presented in Table 4.1

40

Table 4.1 Post-Collection Evaluation Values of Stallion Semen

Gel-Free Motility, Concentration, Total Sperm

Stallion Volume, ml % sperm xlO^/ml Count, x lO '

Stallion B 110 ml 62% 145 9.89

Stallion 11 80 ml 63% 163 8.22

Stallion W 90 ml 77% 126 8.73

Stallion S 65 ml 73% 336 16J

Average motility values for post-collection, post-thaw, and motility percent

returned from original for each stallion are present below in Table 4.2. The averaged

post-thaw motilities were significant different between stallions (P<0.001) as were the

original motility retum percentages (post-thaw motility/post-coUection motility)

(P<0.001). Significant differences were separated using the LSD procedure for both the

post-thaw and original retum motility percentages.

Table 4.2 Comparison of Initial, Post-Thaw and Retum Motilities from Four Stallions

Stallion

Stallion B

Stallion H

Stallion W

Stallion S

Initial,

68

63

77

73

% Motility

Post-Thaw,

3a

12"

2,c

32"

% Retum, %

4W

19"

28^

43^

Different letter superscripts (a, b, c, d) represent significant motility percentage differences as determined byPO.OOl.

Different letter superscripts (w, x, y, z) represent significant original motility retum percentage differences as determined by P<0.001.

41

Variability caused by the UFT freezing treatments was also investigated. The

post-thaw motility averages of the UFT treatments, B (16%), F (19%), G(l 8%), and J

(15%), were found not to be significantly different (P=0.832). The average original

motility retum percentages for B (23%), F (27%), G (25%), and J (20%) were also not

found to be statistically different (P=0.782).

Discussion

The data has shown that the UFT has demonstrated post-thaw motility potential

comparable to that produced by liquid nitrogen methodology. In preliminary studies,

UFT freezing rate was determined to be -4.3°C/minute, which is vary similar to that of

liquid nitrogen.

It has also been suggested that an optimal freezing procedure would include

osmotic changes or temperature decreases in a series of steps (Curry and Watson, 1994;

Gao et al., 1993). The UFT has the unique ability to cool samples to set temperatures

below the point of freezing (0°C). This point is emphasized by the most successfiil UFT

treatment of this trial, G, in Experiments #1 and #2. In this freezing freatment, the

sample was lowered in steps to -10°C, -25°C, and then -196°C in the liquid nifrogen.

During the initial freeze, the sample enters a hyperosmotic environment where the

extracellular fluid freezes before the intracellular water at -10°C (Medrano et al., 2002).

Limiting the freezing rate to -5°C/minute through this critical temperature range before

increasing the freezing rate, has previously proven successful in rams (Byme et al.,

2000).

42

It should also be recognized that the fluid used within the UFT has a heat

dissipation rate similar to that of water. Thus the issue of non-uniform freezing involving

larger volume samples would not be a concern.

From our results, the Freezing Medium Test Yolk Buffer® extender appeared to

be more effective. This extender may have a cryoprotectant concenfration may be more

appropriate for the rate of freezing used (Table 4.3). Glycerol is usually added at

concenfrations of 2.5 to 6% to stallion samples (Samper and Morris, 1998; Vidament et

al., 2002). The bovine extender not only had a higher glycerol amount, but also

contained more active antibiotics such as Gentamycin, Spectinomycin and Linomycin.

Table 4.3 Egg Yolk and Glycerol Composition of Biladyl AB and Test Yolk Buffer Extenders

Biladyl AB®

20%

7%

Component

Egg Yolk

Glycerol

Test Yolk Buffer®

20%

6%

Reference: Insert instructions from Biladyl® (Minitub GmbH; Germany) and Freezing Medium-Test Yolk Buffer Freezing extender (Irving Scientific; San Jose, Califomia)

Male individual viuiation has been noted in several other mammalian species such

as tiie mouse (Holt, 1999), bull (Iritani, 1988; Vidament et al.. 2000), and ram (Byrne et

al., 2000) with regard to cryopreservation success. Therefore, some breeding stallions

may produce acceptable samples for natural or artificial cooled breeding standards; but

not all stallions produce samples acceptable for freezing. It is estimated that only 24% of

43

all stallions are to produce an ejaculate suitable for cryopreservation (Linfor et al., 2002;

Vidament et al., 1997). Our results also reflected an inconsistency of seminal retum

between the four different stallions using our new freezing technique. However, two of

the stallions had post-thaw motilities considered to at acceptable levels suggested from a

survey of commercial equine breeding stations (Samper and Morris, 1998). Thus, there is

potential for this new technique.

In summary, the UFT produced comparable results to those from the traditional

liquid nifrogen methodology. Due to the potential of this technology, ftirther

investigations involving stallion semen are merited.

44

CHAPTER V

EFl ECTS OF SEMEN HANDLING ON POST-THAW

MOTILITY OUTCOMES OF CRYOPRESERVED

PORCUME SPERMATOZOA

Abstract

Cryopreservation of boar semen within the swine industry has commonly been

correlated to lower piglet numbers per litter produced. However, swine reproduction

offers various challenges compared to other livestock animals including large ejaculate

volumes, litter bearing sows, and temperature sensitive semen. Due to this variability of

success, the aim of the present study was to assess the post-thaw outcomes of various

preparative protocols. This study was conducted in four trials. In trial #1, an extended

post-collection sample was diluted with cryoprotectant (0%-10%) and held at one of two

incubation periods (30 minutes or 5 hour) before being frozen in a unique freezing

technology (UFT). Trial #2 evaluated similar cryoprotectant concenfrations with

extended incubations periods (0, 2, 4, 6, 8 hours) at 5°C before UFT freezing. In trial #3,

extended samples are incubated at either 22°C or 17°C before centrifiigation and addition

of glycerolized media prior to UFT or nitrogen mist freezing. In the last trial, trial #4, the

effects of centrifiigation, step-down cooling rates during various incubations, and the

addition of two freezing extenders were examined. All of the post-thaw motilities for

trials #1 and #2 were 0%. Though trial #3 produced a few samples with significant

motility, the average was also 0%. However, there was a significant difference between

45

each trial with #4 (P<0.00l). Though there was some consistency between the initial

extender and freezing treatments utilized for this study, the variation observed between

the four trials emphasizes the need for a cooling incubation periods and centrifugation as

part of a freezing protocol for boar semen.

Introduction

Semen cryopreservation for boar semen has been commercially available since

1975. Yet, the fertility performance of cryopreserved samples is not considered efficient

as demonsfrated by lower average farrowing rates of seven piglets to a litter (Johnson et.

al., 2000). Swine reproduction, though, is unique compared to other livestock sjjecies.

Boars produce large ejaculate amounts with temperature sensitive sperm (Pursel et al.,

1972). Sows, as a litter bearing animal, create a fertilizing challenge through prolonged

ovulations (Johnson et al., 2000). Thus, insemination amounts must also be large (Holt,

2000).

Currently, two cryopreservation protocols, the Westendorf and Beltsville

methods, are being utilized in commercial boar settings. As part of the Westendorf

method, semen is diluted and then frozen in straws. As for the Beltsville method, a

sample is cooled for 2 hours prior to centrifugation and then cooled ftirther for 3 hours

before pelleting (Johnson et al., 2000). Even though the swine industry has established

protocols, as recognized with bulls (Vishwanath and Shannon. 2000), the procedures

need refinement to produce satisfactory results. Therefore, the objective of this study

46

was to evaluate the effects of several different preparative handling factors while

implementing a new UFT.


Post-Collection Processing

Several ejaculates from three boars were collected during the fall of 2001. All the

boars were collected at the Texas Tech University Farm at New Deal, TX, by frained

personnel. After each collection, the ejaculate was evaluated for total gel-free volume,

semen concenfration and initial motility. The original motility of all samples utilized had

60% or greater and meet concentration requirements for established breeding protocols.

A sample was extracted, extended 2:1 with pre-warmed X Cell Extender (IVM, Maple

Grove, MN), and packaged for transport to one of two off-site laboratories for further

processing.

Freezing Technology


(SupaChill, Inc.. Lubbock, TX) incorporates an organic freezing fluid with a heat index


UFT has the unique ability to be set at temperature levels below 0°C (freezing point) with

out solidifying. This technology was utilized in all four of the trials within this study.

47

Trial #1

Upon arrival at the off-site laboratory, a sample was divided into six 5 mL


of 0%, 2%, 4%, 6%, 8% or 10%. Two aliquots of 1.5 mL were removed from each ttibe

and placed in 1.8 mL cryo-tubes (Nalge Nunc Intemational, Denmark) with an "A" or

"B" label. The A tubes were held at cooled in a 5°C refrigerator for thirty minutes while

the B tubes were held at 5°C for one hour. Upon completion of this incubation, the

samples were plunged into the -25°C UFT and maintained in this environment for one

hour.

Trial #2

Upon arrival at the off-site laboratory, a sample was divided into four 6 mL


of 4%, 6%, 8% or 10%. One mL aliquots were removed from each tube and placed in 1.8

mL cryo-tubes with a number label ranging from 0 to 10. These numbers represented the

number of hours in which the samples were held at 5°C before freezing. Vials were

frozen and held in the -25°C UFT for one hour.

Trial #3

At the off-site laboratory, two samples were individually divided into two equal

parts. One half was placed in a 17°C refrigeration unit, the other held at room

temperature (22°C). Samples remained in this environment for one hour. After

48

centrifuging the sample and removing the supemant, a 6% glycerol media was added to

each sample. Cryo-tubes and sfraws with 0.5 mL volume were filled with samples from

both incubation groups. Upon freezing, the samples were divided again. One half of

each group was frozen in the liquid nifrogen mist for 30 minutes. The other half was

frozen in the UFT at -25°C for two minutes.

Trial #4

Upon arrival at either laboratory, each sample was divided and placed into a 17°C

refrigeration unit or held at room temperature (22°C). The sample was held for one hour

at this temperature after which a pre-cooled 17*'C wash buffer (IVM, Maple Grove, MN)

was added. The sample was held at this temperature for an additional 1.5 hour to

equilibrate with the wash buffer. The sample then was centrifiiged to create a sperm-

composed pellet. The pellet was then dissipated in a pre-cooled 25% egg-yolk extender

(Boarchipos A, IVM, Maple Grove, MN) at a 1:1 ratio with the origmal sample size. The

sample was placed in a 5° C refrigeration unit for 1.5 hour before a secondary freezing

extender was added in three increments over 30 minutes. This 25% egg-yolk extender

with 6% glycerol (Boarcipheos B, IVM, Maple Grove, MN) was also pre-cooled to 5° C.

Following the addition of the last increment, the sample was held at 5° C for one hour

before freezing. The sample was not broken into confrol and treatment groups until

immediately before freezing to ensure consistent handling.

49

One ml aliquots were exfracted from the extended sample and placed into 1.8 ml

labeled cryo-tubes for freezing. Only UFT freezing treatments were tested for this trial:

A: 10 minutes at-10°C

B: 4 minutes at -25° C

C: 4 minutes at -25° C with a proprietary filter #1

D: 4 minutes at -25° C with a proprietary filter #2

E: 4 minutes at -25° C with a proprietary filter #3

F: 5 minutes at -10° C, then 4 ntinutes at -25° C

G: 5 minutes at -10° C, then 4 minutes at -25° C with a proprietary filter #1

H: 5 minutes at -10° C, then 4 minutes at -25° C with a proprietary filter #2

1: 5 minutes at -10° C, then 4 minutes at -25° C with a proprietary filter #3

J; 5 minutes at -10° C with a proprietary filter #1, then 4 minutes at -25° C with a


K: 5 minutes at -10° C with a proprietary filter #2, then 4 minutes at -25° C with a


L: 5 minutes at -10° C with a proprietary filter #3, then 4 minutes at -25° C with a

proprietary filter #3.

Thawing Procedure

All samples were held in a liquid nitrogen tank for at least 48 hours before

assessment. In Trials #1 and #2, the thawing protocol included 2 minutes at ambient

temperature (22°C) before placement into a 37°C water bath for 2 minutes. For trial #3,

50

five different thawing procedures were utilized. The procedures involved warming at

room temperature for 10 or 45 seconds before incubation in the 37°C water bath for 5 or

10 minutes. The samples from Trial #4 were thawed using three thawing rates. Either

warming for 45 seconds at 22°C before 5 minutes in a 37°C water bath, 45 seconds at

22°C before 5 minutes in a 37°C water bath and 15 seconds in a 55°C water bath, or 15

seconds in a 55°C water bath before 5 minutes in a 37°C water bath.


On the data from the four trials was analyzed using analysis of variance

(ANOVA). All statistics were analyzed using the SPSS Version 8.0 Software (SPSS,

Inc., Chicago, IL).

Results

Post-thaw motilities from trials #1 and #2 were 0%. Post thaw motilities from

samples of trial #3 ranged from 0% to 15%. However the majority were 0% (8 motile

samples: 140 total samples). Trial #4 produced the highest post-thaw motilities,

averaging 24%. Consequently, a significant difference was observed between the

treatments (P<0.001). This is emphasized in Table 5.1.

51

Table 5.1 Comparison of Pre-Freezing Procedural Trials on Boar Spermatozoa

Motility

Trial Avg Post-Thaw, % SD Range

#1 0%" 0.0 0

#2 0%" 0.0 0

#3 0%' 0.0 0-15

#4 24%" n j 0-45

Different superscripts represent significant motility percentage differences as determined by P<0.001

Discussion

The higher motilities reported in trial #4 reflect the importance of incubation

cooling periods and centrifugation of the semen sample as part of the boar

cryopreservation protocol. Trial #4 had a similar procedure to the Beltsville method,

used in commercial; breeding facilities (Johnson et al., 2000). Trials #1 and #2, lacked

centrifugation, as does the Westendorf method. However, these procedures did not prove

to be successful.

Seminal plasma cannot sustain sperm viability beyond a few hours after

ejaculation (Johnson et al., 2000). In boars, higher rates of lipid peroxidation were

observed in semen when incubated at 37°C within this native plasma (Comaschi et al.,

1989). These findings concur with the poor performance of trials #1 and #2, where

seminal fluid was maintained in the sample during the cooling process. In stallion semen

samples, removal of the seminal plasma by centrifugation created a higher rate of

chromatin stability (Love et al., 2002) and higher post-thaw motilities (Martin et al..

52

1979). This beneficial effect of centrifugation was observed in trial #3, but more

significantly in trial #4.

Boar spermatozoa are very temperature sensitive (Watson, 2000). The Beltsville

Method involves cooling a sample in seminal fluid for 2 hours before centrifiigation and

cooling further before freezing (Johnson et al., 2000). It has been suggested that

successful post-thaw results may be achieved through a series of osmotic steps (Curry

and Watson, 1994; Gao et al., 1993) such as thermal reductions. Evidence of this trend

has been observed in sperm after holding periods at cooler temperatures. Improvements

in cold shock resistance post-incubation have been reported in seminal samples from

stiillions (Brummer et al., 2002; Crockett et al., 2001) and boars (Eriksson et al. 2001;

Pursel etal., 1972).

Due to the sensitivity of boar sperm, the recommended glycerol concenfration is

only 3% (Johnson et al., 2002). Of the four trials tested in this study, all four trials had

glycerol concenfrations that were similar to this percentage. In reviewing the total

sample composition, trial #4 had a 3% glycerol concentration. However, not only is the

amount of glycerol important, so is the way in which it is added. Incubation timing

involving glycerol is necessary to reduce osmotic stress from the cryoprotectant, itself. It

was suggested initially it was thought that extended semen exposed to glycerol did not

show any survival differences post-thaw based on exposure time. Higher motilities and

improved morphology were observed after incubating samples for 4 hours (Johnson et al.,

2000). The temperature in which the glycerol is added also affects the post-thaw

outcomes. Vidament and colleagues (2000) found the addition of glycerol at 22°C

53

generated a higher return rate than glycerol added at 4°C. Though trials #1 and #2 had

longer incubation periods at 5°C. the lack of an additional extender may lead to a

metabolism deficiency. Also egg yolk extenders possess protective attributes when

added to spermatozoa during cryopreservation (Medeiros et al., 2002).

In conclusion, refinement to preparative handling techniques of boar spermatozoa

is recommended for the development of an optimal protocol. It should be noted that some

variation within the results may be due to varying thawing procedures. However, our

results suggest that incubation periods with decreasing temperatures, centrifugation and

the addition of freezing extenders demonstrated post-thaw potential.

54

CHAPTER VI

COMPARISONS OF A UNIQUE FREEZING

TIXIJNOLOGY (UFT) TO TRADITIONAL

LIQUID NITROGEN METHODOLOGY FOR

CRYOPRESERVATION OF PORCINE SPERMATOZOA

Abstract

Due to the poor results from current cryopreservation methodology in several

species, especially the boar, new techniques need to be investigated. Therefore our

objective was to investigate newly developed freezing techniques using a new Unique

Freezing Technology (UFT) with the intent of improving current fertility outcomes. Four

ejaculates from four different boars were used to compare the results of a UFT to those

from traditional liquid nitrogen methods on the cryopreservation of boar semen. Though

treatment and control groups were handled similarly during pre- and post-freezing

procedures, the samples were divided into freatment groups immediately before freezing.

The effects of the freezing freatments demonstrated significantly different post-thaw

motilities (P<0.02). As expected there was a significant difference between freezing

treatments as evaluated through original motility percent retums (P<0.05). When

assessing the individual UFT techniques, all of six UFT techniques had significantly

higher post-thaw motilities (P<0.05) and original motility percent retums (P<0.05)

compared to either the control vial or straw groups. The preliminary data suggests that

the UFT could improve current cryopreservation fertility results of boar semen.

55

Introduction

The use of artificial reproductive techniques in the swine industry has increased

greatly within the past twenty years. It is estimated that 45-50% of all sows in the United

States are bred by artificial insemination, and of those inseminations 99% are extended

for same day use or cooled for 1-5 days (Johnson et al., 2000). However, the capacity of

cryopreservation is very limited due to porcine sperm's high vulnerability to cold shock

(Buhr et al.. 1989; Holt, 1999).

Freezing rates must be fast enough to reduce intracellular ice formation, yet slow

enough to avoid osmotic alterations (Watson, 2000). Currentiy, most boar samples are

frozen as pellets on dry-ice or in maxi-straws in nitrogen vapor (Johnson et al., 2000).

The procedures are very similar to those used thirty years ago (Larsson, 1978). This

produces a dramatic temperature change from the holding temperature of 5°C degrees to

that of liquid nitrogen, -196°C. Medrano and colleagues (2002) demonstrated higher

rates of membrane damage at a faster freeze rate of 24°C/minute versus 3°C-12°C/minute

while cooling to -5**C. Thurston et al. (2003) showed higher post-thaw viabilities and

motilities in boar sperm which were cooled at 6°C/minute to -5°C. then froze at higher

rate of-40°C/minute to -80°C.

Within the last few years, a new freezing system has developed for freezing

foodstuffs with an increased ability to dissipate heat. This unique freezing technology

(UFT) has a relatively high dissipation rate which appears to vitrify water and preserve

freshness (SupaChill®, SupaChill USA; Lubbock, TX). Due to the unique freezing

capacity, the temperature can be adjusted at range of freezing levels below 0°C.

56

Preliminary trials on various cell and tissue types have shown very promising results.

Our objective was to compare the effects of freezing swine spermatozoa using this UFT

and traditional nitrogen methods in attempt to create a more optimal standardized

cryopreservation technique.


Freezing Technology


(SupaChill USA; Lubbock, TX) incorporates an organic freezing fluid with a heat index


UFT has the unique ability to be set at temperature levels below 0°C (freezing point) with

out solidifying. To evaluate the rate of freezing of the UFT used, samples of water and

the two egg-yolk freezing extenders used in this study were placed in 1.8 mL cryo-tubes

(Nalge Nunc Intemational; Denmark) and froze in the -25°C UFT unit. Temperatures

inside each vial were monitored using Thermocouple Thermometer (Cole-Parker

Instrument Company; IL). Data was collected every 10 seconds for 9 minutes.

Post-collection Processing

Four ejaculates from four boars were collected during four separate occasions.

All the boars were collected at the Texas Tech University Farm at New Deal, Texas by

trained personnel. After each collection, the ejaculate was evaluated for total volume,

gel-free volume, concentration and initial motility. To meet the standards of the

57

experiments the original motility of all samples needed to be equal or greater than 60%

and the concentrations had to be consistent with farm breeding practices. A sample was

extt^cted and extended 2:1 with a tris-egg yolk extender (X Cell Extender, FVM; Maple

Grove. MN) and packaged for transport to one of two off-site laboratories for ftirther

processing.

Cryopreservation Processing

Upon arrival at either laboratory, each sample was placed into a 17°C refrigeration

unit. The sample was held for one hour at this temperature after which a pre-cooled 17*'C

wash buffer was added. The sample was held at this temperature for an additional 1.5

hour to equilibrate with the wash buffer. The sample then was centrifiiged to create a

sperm-composed pellet. TTie pellet was then dissipated in a pre-cooled 25% egg-yolk

extender, Boarchipos A (IVM; Maple Grove, MN (Ext #1)) at a 1:1 ratio with the original

sample size. The sample was placed in a 5°C refrigeration unit for 1.5 hour before a

secondary freezing extender, Boarchipos B (FVM; Maple Grove, MN (Ext #2)), was

added in three increments over 30 minutes. This 25% egg-yolk extender with 6%

glycerol was also pre-cooled to 5°C. Following the addition of the last increment, the

sample was held at 5°C for one hour before freezing. The sample was not broken into

confrol and treatment groups until immediately before freezing to ensure consistent

handling.

58

Freezing Methodology

Preliminary trials produced significant improvements in post-thaw outcome when

comparing the 1.8 ml cryo-tubes (Nalge Nunc Intemational; Denmark) to the 0.5 ml

straw in the UFT. Therefore, one ml aliquots were extracted from the extended sample

and placed into cryo-tubes for freezing in the UFT and control C groups. Additional

samples were also exfracted and placed in 0.5 mL straws for control S group. Eight

freezing treatments were tested for this trial, six UFT and two controls. Preliminary trials

demonstrated higher results with the selected six UFT treatments:


C: 4 minutes at -25°C with a proprietary filter #1

E: 4 minutes at -25°C with a proprietary filter #3

F: 5 minutes at -10°C. tiien 4 minutes at -25°C

G: 5 minutes at -10°C then 4 minutes at -25°C with a proprietary filter #1

J: 5 minutes at -10°C with a proprietary filter #1 then 4 minutes at -25°C with a


Con C (Cryo-tubes): 10 minutes in the liquid nitrogen mist

Con S (Straws): 10 minutes in the liquid nifrogen mist.

After initial freezing was complete, all cryo-tubes and straws were stored in liquid

nitrogen.

59

Thawing Methodology

The thawing method used is based on previous trials performed in our laboratory.

Samples were stored at -196 °C for at least 48 hours before being evaluated for post-thaw

values. Each sample was thawed on an individual, random basis by a trained observer.

The thawing protocol involved a 45 second duration at room temperature, then 5 minutes

in a 37°C water bath. Each vial was manually assessed immediately following thaw.


All data from the UFT thermal gradient evaluation was analyzed using a linear

regression model. Based on a best fit linear estimate, the average slope was considered to

represent the rate of temperature change.

Tbe analyses of the motilities, both post-thaw and original retum percentages,

from the eight freezing treatments were analyzed by ANOVA using the general linear

model (GLM) of tiie Statistical Program of the Social Sciences (SPSS Version 8.0; SPSS,

Inc.; Chicago, IL). Post-thaw motility averages and percentages of original motility

retumed averages of the individual freezing techniques were separated through the Post

Hoc fimction of Fisher's Least Squared Differences (LSD).

Results

UFT Thermal Gradient

Data from the three medias froze are represented in Figure 6.1. The average

temperature change was -6.1°C/minute.

60

• Water

-Ext# l

• Ext #2 E

B H

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55

Time (in 10 sec increments)

Figure 6.1 Cooling Rates of the UFT

Freezing Treatment Comparison

These preliminary results reflect the effects of freezing techniques on the post-

thaw motilities from each treatment group. Referring to Figure 6.2, the significant

differences between post-thaw motilities of freezing technique treatments is shown.

There was a significant difference between freezing techniques (P<0.02). Using the LSD

procedure, averages post-thaw motilities of the individual freezing techniques were

separated.

61

UFTB UFTC UFTE UFTF UFT G UFTJ Con C Con S

Freezing Technique p < o 02

Figure 6.2 Post-Thaw Motility Percentages of Cryopreserved Boar Semen.

Different letter superscripts represent significant difference between average post-thaw motilities as determined by P<0.05

A similar trend in improvement was observed in the original motility percentage

retums, referring to Figure 6.3. Again, there was significant differences between the

freezing techniques (P<0.05). Individual average original motility percentage retums for

each freezing technique were separated using the LSD.

62

50

45

40

35

u 30

I 20 w) 15 O

10

23%>' 23%>'

29%"

19%'

UFTB UFTC UFTE UFTF UFFG UFTJ Con V Con S

Freezing Techniques P < 0.05

Figure 6.3 Original Motility Percentage Retums of Cryopreserved Boar Semen.

Different letter superscripts represent significant difference between average original motility percent retums as determined by P<0.05.

This improvement in average post-thaw motilities by the UFT is amplified in

Table 6.1. Though all six UFT techniques showed improved post-thaw outcomes,

treatments E, F, G, and J showed higher average post-thaw motilities over the control vial

group and control straws group (P<0.05). Treatments E, F, G and J were also

significantly higher than the both control groups for the original motility percentage

rehims (P<0.05).

63

Table 6.1 Post-Thaw Motility Outcomes from Cryopreserved Boar Semen.

Treatment

B (n=8)

C (n=8)

E (n=8)

F(n=8)

G (n=8)

J (n=8)

Con Vials (n= =6)

Con Straws (n=6)

Motility Percentages

Post-Thaw

18%"

18%"

22%"

29%^

22%''

27%'

15%'

12%"

Retum

23%''

23%y

29%"

37%"

29%"

34%"

19%'

15%'

Different letter superscripts (a,b,c,d) represent significant difference between average post-thaw motilities as determined by P<0.05 Different letter superscripts (x,y,z) represent significant difference between average original motility percent retums as determined by P<0.05.

Discussion

With the low success of cryopreservation in the swine industry, new techniques must

be investigated. Commonly, damage is observed through reduced pregnancy and motility

rates produced in part to the freezing rate of liquid nifrogen vapor at 20°C/minute. Our

preliminary data suggests a slower pre-freeze phase below the freezing point before

acceleration to liquid nitrogen temperatures. Our data is in agreement with two other

reports demonstrating a step-down cooling below freezing from 5°C (Medrano, 2002;

64

Thurston et al., 2002). By nearing the point of intracellular ice formation at a slower rate

of 4°C/minute. the water is cooled to a stable transition point. Then the freezing rate is

accelerated by the liquid nitrogen mist, thus reducing ice formation.

Therefore, the UFT could potentially be considered as an altemative to replace the

current traditional liquid nifrogen methods. These improvement trends on post-thaw

results using the UFT have been also reported in similar UFT trials involving other

species and cell lines. However, ftirther studies need to be conducted to refine a standard

technique. Additional testing is needed to evaluate the effects of UFT on boar sperm

viability and structural integrity as well as pregnancy rates.

65

CHAPTER VII

LIPID PEROXIDATION EFFECTS FROM SEMEN

HANDLING DURING CRYOPRESERVATION OF

PORCUSIE SPERMATOZOA

Absfract

The potential advantages of sperm cryopreservation have not been fiiUy

accomplished due to the limiting detrimental effects on sperm stmcture and composition.

Previous studies have suggested that cells suffer lipid peroxidation damage during the

cryopresevation process, specifically suggesting the damage results from mechanical

stress during the preparatory and freezing processes. In this present study, sperm samples

were analyzed for lipid stability throughout sample processing through evaluations for

lipid peroxidation and lipid free radical amounts. Our analysis was completed in three

experiments. In Experiment 1, lipid stability levels were gained from five separate boar

ejaculates frozen using three different freezing methods to compare cryopreservation

techniques. In Experiment 2, lipid p)eroxidation amounts for fresh post-ejaculate and

albumin extended boar samples were compared. Experiment 3 involved evaluations of

the semen processing to evaluate sample and seminal fluid alterations. Samples tested

from the freezing protocol included fresh, extended, addition of a wash buffer, cooling to

]7°C, centrifugation, addition of two egg-yolk extenders, cooling to 5°C and post-thaw

values. Though there was no difference between the three freezing freatments, significant

differences were noted between the fresh and extended samples (P<0.001). These

66

findings were exemplified by the step by step analysis of the processing and freezing

protocol. The lipid peroxidation amounts accumulated after each the procedural step

(P<0.001). Significant differences were also observed in the lipid radical levels

(P<0.001). The largest gains of both lipid parameters developed after the addition of an

egg-yolk freezing extender. The results of the pre-freezing protocol, alterations in lipid

stability do not appear to be due to thermal or mechanical sfress. The results suggest

ftirther studies in alterative extenders need to be explored.

Introduction

In lieu of the different success rates of the varying freezing methodologies, this

study was performed to investigate the possible source of damage on a molecular level.

More specifically, we evaluated the lipid stability of the phospholipid membrane during

the freezing and pre-freezing process.

Several articles have been published based on lipid peroxidation and oxygen free

radicals in human and murine samples. Negative correlations have between associated

between increased levels of malondialdehyde (MDA) reduced motility (Engel et al.,

1999; Huang et al., 2001; Verma and Kanwar, 1999) and poor fertilization capability

(Zabludovsky et al., 1999) in human sperm. Though conclusions are made from trials

based on other species, species differences in composition have demonstrated the

presence of varying protective enzymatic mechanisms against peroxidation (Alvarez and

Storey, 1984; Medeiros et al., 2002; Mennella and Jones, 1980).

67

Very few articles have been published in relation to boar semen. Comaschi and

colleagues (1989) suggested the lipid radicals were more detrimental than the radical

hydroxyls. MDA levels have been identified in samples incubated at 37*'C (Comaschi et

al., 1989), cooled liquid storage (Cerolini et al., 2000) and post-thaw (Cerolini et al.,

2001).

Though peroxidation levels have been analyzed in previous reports specifically

studying the post-collection and post-thawed samples, the evaluation of the possible rate

during pre-freezing procedures has not been investigated. The objective was to

investigate the rate of lipid peroxidation of the porcine spermatozoa and amount of lipid

peroxidation and lipid free radicals during the cryopreservation process.


Semen Preparation

Semen specimens were collected artificially from boars at the Texas Tech

University New Deal Farm by trained staff. During the post-collection evaluation,

standard measurements including total gel-free volume, concentration, and motility were

obtained. To fulfill standard breeding practices, all samples used possessed a minimum

motility rate of 60% post-collection and met the breeding requirements of the swine urtit.

From the filtered ejaculate, a 30 mL sample was retrieved and extended with 60 mL of a

pre-heated X-Cell extender (IVM; Maple Grove, MN). The sample was then packaged

for transport to one of two possible laboratories.

68

Experiment #1

Upon arrival, the extended sample was placed in a refrigeration unit (17°C) for

one hour. At the end of the hour, 210 mL of a wash buffer maintained at 17°C was added

before continuing the storage in refrigeration unit for another 1.5 hours. The samples

were tiien centrifuge for 20 minutes at 1900-2000 rpm in a 15°C setting. The supemant

fluid was removed and discarded. The remaining pellets were reconstituted in 30 mL of a

25% egg-yolk extender, Boarciphos A (IVM; Maple Grove, MN), also maintained at

17°C. The sample was allowed to equilibrate for 1.5 hours in a secondary refiigeration

unit set at 5°C. With 10 minute intervals between, a second 25% egg-yolk extender with

6% glycerol, Boarciphos B (FVM; Maple Grove, MN), cooled to 5°C, was added three

times in 10 mL increments. After all of the 30 mL were added, the sample was

maintained at 5°C for one hour. One ml aliquots of the extended sample were placed in

1.8 mL labeled cryo-vials (Nalge Nunc Intemational; Denmark) before being

cryopreserved. Through labeling, specimens were separated into three freezing treatment

groups:

Control - liquid nifrogen mist for 10 minutes

F: 5 minutes at -10°C followed by 4 minutes at -25°C

J: 5 minutes at -10°C followed by 4 minutes at -25°C within a proprietary fluid

filter in each unit.

Freezing treatments "F" and "J" were two optimal freezing freatments of the UFT

(SupaChill®, SupaChill USA; Lubbock, TX) justified in previous trials within our

69

laboratory. Samples were plunged into liquid nitrogen and stored until testing began for

lipid peroxidation rates and lipid free radical amounts.

Experiment #2

For Experiment #2, we used fresh post-ejaculate boar samples and extended post-

ejaculate samples for lipid stability comparisons of five samples from five boars. The

above protocol discussed in semen processing was utilized and completed until arrival at

the off-site lab. One mL aliquots were exfracted from all ten samples and placed in 1.8

mL cryo-tubes (Nalge Nunc Intemational; Denmark). Immediately afterwards, each vial

was plunged in liquid nitrogen for freezing and storage.

Experiment #3

The third phase of this trial utilized the same protocol discussed above in

Experiment #1 on three semen samples from separate boars. However, the proportions of

original semen sample and the various extenders used were increased. This ensured

sufficient amounts for three 1 mL aliquots of semen samples and fluid to be extracted

during each major step of the protocol without influencing final ratios. The semen

samples represent the whole fractions available at that step. Fluid samples were created

by centrifuging addition samples during each step specified below. The procedural steps

represented include:

1. Ejaculation samples post-transport,

2. Extended samples post-transport.

70

3. Extended samples cooled to 17°C for 1 hour,

4. Samples equilibrated with a wash buffer at 17°C for 1 Vi hours,

5. Samples after centrifiigation for 20 minutes at 2500 rpm,

6. Samples equilibrated with the first freezing extenders at 5°C for 1 '/z hours,

7. Samples equilibrated with the second freezing extenders at 5°C for 1 Vi hours,

8. Post-thaw values of optimal freezing treatment.

Due to the density of the semen pellet after centrifugation and concems of altering final

concentrations, a semen sample was not exfracted for this step. While thawing the

samples, fluid specimens were not removed for analysis. After exfraction during each

step, the aliquots were plunged and stored in liquid nifrogen.

Quantification of Lipid Peroxidation

After thawing at room temperature and then being vortexed, a 200 pL sample was

extracted from each aliquot and utilized for this test. The semen samples were mixed

witii 200 pL of SDS solution, 1.5 mL of 20% AA solution (at 3.5 pH), 1.5 mL TBAR

solution (1.6 g 2-thiobaritric acid with 200 mL of deionized water) and 600 pL of

deionized water. After vortexing each sample, the samples were incubated in a 100**C

water bath for 2 hours. The samples were then cooled for 10 minutes at room

temperature before centrifuged at 2000 rpm for 10 min. A 0.5 mL sample of fluid was

again extracted from each tube and mixed with 2 mL of butanol. After a second

centrifugation at 2500 rpm for 10 minutes, each fluid sample was transferred into a glass

71

absorbance box one at a time for analysis at 532 nm wavelength in a spectrophometer

(Coleman 575, Perkin-Elmer; Oak Brook, IL).

Quantification of Free Lipid Radicals

A 250 pL sample was extracted from each specimen and placed into a pre-labeled

12 X 75 mm glass tube. After diluting each sample in 2 mL of a 2:1 chloroform:heptane

mixture, each tube was thoroughly vortexed. The samples were then centrifiiged for 10

minutes at 2500 rpm. Fluid in the amount of 700 |jL was extracted from each specimen

and transfer to a new set of glass tubes. Samples were dried under a flow of normal afr at

ambient temperatures for 15-20 minutes. The dried extracts were reconstituted in 2 mL

of heptane before performing a spectrophotometer (Coleman 575, Perkin-Elmer; Oak

Brook, IL) analysis at a 233 nm wavelength. The lipid radical confrol, requfred for

proper calculations, was processed the same as a sample except the confrol contained 250

pL of water instead of a sample.


The analysis of the lipid peroxidation and lipid free radical amounts for the three

freezing freatments was analyzed using analysis of variance (ANOVA). Boar and

freezing treatment were both used as part of the statistical model. The means were

separated through Fisher's Least Squared Differences (LSD).

72

ANOVA was also used to compare the results of the fresh and extended values for

lipid peroxidation. Though the results were significant, the sample size is too small to

complete a LSD analysis.

On the procedural analysis in Experiment #3, the lipid peroxidation and lipid free

radical amounts were also analyzed using the ANOVA model. Since these values

represent accumulations of lipid instability over time, a linear regression model function

was used to analyze each parameter.

All statistical analyses were performed using the SPSS Version 8.0 Software

(SPSS, Inc.; Chicago, IL).

Results

Experiment # 1

When comparing the three freezing treatments using five separate semen samples,

the differences between the treatments were not significant for either lipid evaluations.

However, there was an interaction between the boar and lipid peroxidation amounts

(P<0.005). These differences, shown in Table 7.1, concur with the variability of semen

quality in-between boars.

73

Table 7.1 Averaged Lipid Peroxidation and Free Radical Amounts from Three Freezing Treatments in Experiment #1

Boar

Yellow (n=3)

Pink (n=3)

Green (n=3)

White (n=3)

Orange (n=3)

Lipid Peroxidation (nM/mL)

62.3^

58.6^

52.2^

58.2^

50.8^

SEM

2.79

2.19

2.19

2.36

2.76

Radicals (A233/mL)

5.7

5.5

5.9

5.7

5.8

SEM

0.28

0.24

0.10

0.18

0.25

Different letter superscripts indicate significant differences between the lipid peroxidation of five boars (P<0.005)

Experiment #2

When comparing the amounts of membrane lipid stability between fresh, post-

ejaculation samples and extended post-ejaculation samples there was a significant

difference. Lipid peroxidation rates were much higher for the fresh samples as compared

to tiie extended samples (P<0.001; Table 7.2).

Table 7.2 Averaged Lipid Peroxidation Amounts from Fresh and Extended Boar Samples in Experiment #2

Lipid Peroxidation Sample n (nM/mL) SEM

Fresh 5 14.3' 2.24

Extended 5 1.6" 0.36

Different letter superscripts indicate significant differences between the lipid peroxidation levels of the sample types(P<0.001)

74

Experiment #3

Referring to Table 7.3, lipid peroxidation levels accumulated during the handling

procedure in both specimen types evaluated. The levels of peroxidation were

significantly different within the semen samples (P<0.001) and fluid samples (P<0.001).

Using the LSD procedure, significant differences were observed at various fransition

points of the procedure including steps 1 to 2, steps 4 to 6, and steps 7 to 8 in the sample

(P<0.05) and fluid (P<0.001).

Significant changes were found during the duration of the cryopreservation

protocol. Amounts of MDA were correlated between the semen and fluid levels for both

the lipid peroxidation (r=0.96). These increases in peroxidation rates are emphasized in

Figure 7.1.

75

Table 7.3 Averaged Lipid Peroxidation Amounts from Procedural Steps


Step n Sample SEM n Fluid SEM

1: Post-collection

2: Extended

3: Cool to 17"

4: Wash Buffer

5: Centrifugation

6: Freezing Extender at 5°C

3

3

3

3

0

3

7: Second Freezing Extender 3

8: Frozen-Thaw Samples 3

10.2'

22.6"

23.9"

23.4"

n/a

66.0'

62.2'

52.3"

6.97

4.90

3.14

2.52

-

1.83

4.42

6.74

3

3

3

3

3

3

3

0

5.2^

21.4"

23.3"

22.6"

23.5"

41.6^

50.4"

n/a

0.12

1.77

2.49

0.67

0.94

2.33

3.30

-

Different letter superscripts (a,b,c,d) indicate significant differences between lipid peroxidation levels of the procedural steps in the samples (P<0.05). Different letter superscripts (w,x,y,z) indicate significant differences between lipid peroxidation levels of the procedural steps in the fluid extracted (P<0.001).

76

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77

Referring to Table 7.4, lipid free radical levels also showed significant variations

during the procedure for the samples (P<0.001) and fluids (P<0.001) evaluated. The

amounts found from the samples and fluid extracted were again correlated (r=0.985).

Using the LSD procedure, a significant increase was found after the addition of the egg-

yolk freezing extender in the sample (P<0.001) and fluid (P<0.001). The lipid free

radical accumulation is presented in Figure 7.2. In lieu of the accumulation trends of

lipid instability, the reduction of motility is correlated with higher levels of both lipid

peroxidation (r= -0.913, P<0.001) and free radicals (r= -0.940, P<0.001) as demonstrated

in Table 7.5.

Table 7.4 Averaged Lipid Free Radical Amounts from Procedural Steps

Step


Sample SEM n Fluid SEM

1: Post-collection

2: Extended

3:Cooltol7*'C

4: Wash Buffer

5: Centrifugation

6: Freezing Extender at 5°C

7: Second Freezing Extender

8: Frozen-Thaw Samples

3

3

3

3

0

3

3

3

0.1"

0.1"

0.1"

0.0"

n/a

3.4"

3.2"

3.7"

0.13

0.03

0.02

0.0

-

0.81

0.78

0.20

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3

3

3

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3

3

0

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0.0''

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4.3^

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0.02

0.01

0.0

0.0

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0.74

1.75

-

Different letter superscripts (a,b) indicate significant differences between lipid fi^e radical levels of the procedural steps in the samples (P<O.OOI). Different letter superscripts (y,z) indicate significant differences between lipid free radical levels of the procedural steps in the fluid extracted (P<0.001).

78

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79

Table 7.5 Motility and Lipid Stability Trends from the Samples of Experiment #3

Sample

Post-collection

JTrt

FTrt

Control

n

3

3

3

3

Lipid Peroxidation Motility (nM/mL)

75%'

23%^

22%^

14%^

6.70"

53.2"

57.2"

58.3"

Radicals (A233/mL)

0.14^

3.66"

3.79"

3.72"

Different number superscripts indicate significant differences between motility percents (P<0.001). Different letter superscripts (a,b) indicate significant differences between lipid peroxidation levels (P<0.001). Different letter superscripts (y, z) indicate significant differences between lipid free radical amounts (P<0.001). Negative correlations were found between motility percentages and lipid peroxidation (r= -0.913) amounts and between motility percentages and lipid free radical (r= -0.940) amounts.

Discussion

High lipid peroxidation levels have been associated in several articles with

reduced sperm ftmctionality (Engel et al., 1999; Huang et al., 2001; Verma and Kanwar,

1999; Zabludovsky et al., 1999). Similar results were found in the present study as well

(refer to Table 7.5). However it has been suggested in other articles that the cause has of

this membrane degradation was due to the mechanical stresses of cryopreservation. Our

data suggests that the changes observed are due to the pre-freezing procedures, especially

the addition of egg-yolk extenders at a cooled state (refer to Tables 7.3 and 7.4).

Centrifugation is a common step to concenfrate the specimen in the

cryopreservation preparation process. Yet it has been suggested that centrifugation may

cause undue stress (Gil et al., 1999). With dog semen, lower centrifugal speeds resulted

in a higher amount of sperm lost in the supemate while viability losses were higher at

80

increased centrifugational velocities. Yet, membrane integrity was maintained at the

various speeds (Rijsselaere et al., 2002). Our evaluation of the seminal fluid extracted

before and after centrifugation, show very little difference in lipid stability (refer to Table

7.3 and 7.4). Neild and colleagues (2003) reported a capacitation-like phenomenon after

centrifugation of stallion sperm. Thus, permeability alterations may be due to other

sources than lipid peroxidation.

Protein degradation due to peroxidation has been observed (Medeiros et al.,

2002). Baumber et al. (2002) correlated high peroxidation levels in post-thawed semen

with DNA fragmentation. This may actually be the cause for the reduced fertilization

capability recognized in sperm after cryopreservation (Dobrinski et al., 1995).

As observed in bovine semen, our results showed higher levels of peroxidation in

frozen-thawed semen versus fresh or cooled (refer to Tables 7.3 and 7.4). However, due

to the trends at various points in the procedural analysis, our results do not demonstrate

the stress in relation to cooling and thawing as suggested by Chatterjee and Gagnon

(2001). Instead the largest accumulation of both lipid peroxides and lipid radicals was

observed during the addition of the freezing egg yolk extender (refer to Tables 7.3 and

7.4). Egg yolk contains phosphatidylcholine, similar to that already present in the

membrane of sperm. Though studies have demonstrated the protective effects of egg

yolk (Vishwanath and Shannon, 2000), our results project a negative side. Misik and

colleagues (1994) demonstrated the amplification in MDA levels in egg yolk held at 22°C

compared to 50*'C. The introduction of a diluent with ongoing lipid peroxidation or the

addition of a product, specifically lipid peroxides (LOOH), will enhance lipid

81

peroxidation (Tang et al., 2000). Thus, altemative extenders need to be investigated.

Braun and colleagues (1995) observed an increase in the membrane mtegrity and motility

of frozen stallion semen with the exclusion of egg-yolk. Dacaranhe and Terao (2001)

observed reduced iron-peroxidation levels with the presence of phosphatidylserine. A

trial on frozen stallion semen has already verified an improvement in motility with the

addition of phosphatidylserine (Wilhem et al., 1996).

The addition of antioxidants, such as Vitamin E, may need to be incorporated into

breeding schemes to reduce lipid peroxidation rates. Increases in semen quality have

been observed in turkey (Donoghue and Donoghue, 1997), boar (Brzeezinska-

Slebodzinska et al., 1995; Cerolini et al., 2000) and horse samples (Baumber et al., 2002)

with antioxidant supplementation. Dietary antioxidant supplementation has also

increased semen quality in boars (Brzeezinska-Slebodzinska et al., 1995) and roosters

(Neuman etal., 2002).

82

CHAPTER Vlll

CONCLUSIONS

Numerous groups have searched for an optimal freezing protocol for semen

cryopreservation. Their studies have combination of factors, including: cooling rates,

extenders, and packaging systems. Conversely, these comparisons are performed on a

limited basis with small animal samples. Though the final solution will include

interactions between cooling rates and cryoprotectant amounts and osmotic pressures,

consideration must be taken for what we do and do not understand. With the large

amounts of variation currently in both the literature and commercially practiced

cryopreservation protocols, this assessment is difficult to ascertain (Day, 2000; Samper,

2002; Samper and Morris).

Cryopreservation procedures are currently riddled with long standing tradition.

For example, pelleting initial began in the cattle industry over thirty years ago (Larsson,

1978) and has been replaced by liquid nitrogen and freezing straw storage (Vishwanath

and Shannon, 2000). Even though contamination and identification problems have

become a concern (Pickett et al., 1978), this technique is still used today in the swdne

industry (Johnson et al., 2000). In an attempt to improve boar freezing techniques, we

tested a unique freezing technology (UFT). Our investigation revealed a higher post-

thaw motility yields from UFT than those produced by traditional nitrogen freezing

methods on boar sperm.

83

Membrane integrity problems associated to cryopreservation have been attributed

to mechanical stresses such as rapid centrifugational velocities (Gil et al., 1999; Neild et

al.. 2003; Rijsselaere et al., 2002) or rapid freezing rates (Curry and Watson, 1994;

Pommer et al.. 2002). At extreme rates, we admit these areas can cause significant

damage. However, our lipid peroxidation and lipid radical data instead suggests that egg

yolk extender may actually cause the weakening of the membrane while using acceptable

levels of mechanical stress. Yet, egg yolk is the mostly commonly used a freezing

extender for mammalian sperm (Medeiros et al., 2002; Vishwanath and Shaimon, 2000).

Attention also needs to be given to preparative handling techniques. There have

been recommendations for a series of osmotic steps (Curry and Watson, 1994; Gao et al.,

1993) such as thermal reductions to be included in a protocol. Our step-down technique

utilized incubation periods in cooling environments demonstrated significant

improvements on both stallion and boar samples. This is in agreement with other studies

involving stallions (Bruemmer et al., 2002; Crockett et al., 2001) and boars (Eriksson et

al. 2001; Pursel et al., 1972). In spite of these findings, Johnson and colleagues (2000)

reported that one of the two most used protocols in commercial boar settings utilizes only

the addition of a diluent before freezing.

Tliough certain factors are specie specific, our trials applied techniques normally

used for species other than on those that were tested. Both the stallion and boar have

been shown to have significant problems with cold shock (Holt, 1999; Johnson et al.,

2000; Pursel et al., 1972; Yoshida, 2000). Yet, we attempted to utilize techniques used on

less thermally sensitive species, such as the human and bull (Vishwanath and Shannon,

84

2000; Watson, 2000). Our results actually showed superior trends using both medias and

packaging systems. Regardless of all attempts, certain challenges may not be overcome

such as infertility. The variability reported in stallions (Katila et al., 2002; Medeiros et

al., 2002; Pattie and Dowsett, 1982) also was apparent in our results.

Several alterations in our studies to current methodology demonstrated higher

trends or significant improvements. The utilization of these outdated techniques has

produced lower pregnancy rates. Though these techniques are not ideal, the blame for

poor fertility results are often placed upon the handlers (Holt,, 2000; Loomis, 2001).

However, without a set of standards to work from, the variation in results will remain to

be high (Day, 2000; Samper, 2002; Samper and Morris, 1998).

The potential preservation offered by semen cryopreservation has not been ftilly

grasped thus far. Currently, cryopreservation is commonly associated with poor fertility

performance, especially in boars (Thurston et al., 2003; Yoshida, 2000) and stallions

(Loomis, 2001; Medeiros et al., 2002). However, our results reflect the potential for

improvement. Additional studies will be necessary to establish the ftill potential of the

UFT in the cryopreservation of semen and to determine if this technology can be

combined with newer preparatory techniques to improve semen quality in species which

have previously been reported as poor candidates for cryopreservation.

85

LITERATURE CITED

Alvarez, J.G. and B.T Storey. 1984. Lipid peroxidation and the reactions of superoxide and hydrogen peroxide in mouse spermatozoa. Biol Repro 30: 833-841.

Amanda, R.P. ad Graham. Spermatozoal Function. 1993. In Equine Reproduction. Edited by A.O. McKinnon and J.L. Voss. Philadelphia: Lea and Febiger. pp. 715-740.

Amoah, E.A. and S. Gelaye. 1996. Biotechnological advances in goat reproduction. J Ani Sci. 75: 578-585.

Arruda, R.P., B.A. Ball, C.G. Gravance, A.R. Garcia and I.K.M Liu. 2002. Effects of extenders and cryoprotectants on stallion sperm head morphometry. Therio 58: 253-256.

Ball, B.A. and A. Vo. 2001. Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J Andro. Nov-Dec 22(6): 1061-1069.

Baumber, J., B.A. Ball, J.J. Linfor and S.A. Meyers. 2002. Reactive oxygen species and cryopreservation promote deoxyribonucleic acid (DNA) damage in equine sperm. Therio 58: 301-302.

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