CRYOPRESERVATION OF EQUINE SEMEN WITH A MECHANICAL CONTROL RATE FREEZER
By
Sossi Marianne Iacovides, B.S.
A THESIS
IN ANIMAL SCIENCE
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE
Approved
Dr. Samuel Prien
Chairperson
Dr. Samuel Jackson
Dr. Leslie Thompson
Dr. Mark Sheridan
Dean of the Graduate School
December, 2014
Copyright 2014, Sossi Marianne Iacovides, B.S. Texas Tech University, Sossi M. Iacovides, December 2014
ACKNOWLEDGMENTS
‘It’s hard to soar with eagles, if you won’t jump off the cliff.’ At the beginning of my graduate school process, I had the choice to start somewhere fresh and new. While it may have been the right move for some, it was not for me; I knew that I wasn’t finished with here. The Department of Animal & Food
Sciences has become home, from the abundance of moral, ethical and personal support; it has been a blessing and privilege to spend the past 4 years here.
Dr. Glenn Blodgett, you believed in my research, and that means the world to me. Without the access to the 6666 Ranch, and your indispensible personnel, this project would have never gained flight. Mallory Canaday, the true
“Swimmie Specialist,” you were an indispensable resource for me and I have learned more from you then I could have ever imagined. The lab personnel: my countless questions never went unanswered. I hope that you’ll walk the hallways and hear my snaughles forever more. The minions, my fellow graduate students, from the countless nights of studying, to the late night research – I could not have hoped for a better group to have survived these last two years with! Dr.
Lindsey Penrose, where do I begin? You have been a foundation in my life since I began this journey. Your never-ending words of encouragement; the ideas we bounced back and forth; having been your chief minion in training…. You managed to keep this hot-blooded Greek girl off the edge! Can I finally have a
(small) crown, Turtle Queen?!
ii Texas Tech University, Sossi M. Iacovides, December 2014
Dr. Sam Jackson, starting with undergrad transfer orientation day, to sheep production and throughout my graduate career, you have always been available for help and encouragement. Thanks General. Dr. Leslie Thompson: I knew you would be a great asset after working with you for the IDEAL camps.
Your crucial guidance in my exploration of your Alma Mater in Florida was invaluable, and I cannot thank you enough.
The Captain, Dr. Sam Prien: I don’t think it’s possible to sum up what you mean to me in either a single word or even a few run on sentences. You have been so much more than just my mentor, confidant, and rock throughout my undergraduate and graduate careers. From presenting my research at different venues, to the times I have been at my wits end, I could not have made it this far without your endless understanding and support. I know my neon colors tend to get too bright and the shiny things too, but you put up with the true dark cutter and even managed to get a few songs out of it! You’re a phenomenal person, teacher and friend. Both you and your promise, “You will always have a home here,” will forever have a place in my mind and heart.
Girls, I don’t know how we all survived the past few years, but we definitely kept each other sane. The ranch, if it wasn’t for the push you gave me years ago, I never would have made it this far following livestock as my passion.
Mom, Mougs (and Chimmie) the late night phones calls; the crying over failed trials; the rejoicing when I finally finished. While I may sound like I’m speaking
Chinese, you know the blood, sweat and tears that I poured into this. Thanks for iii Texas Tech University, Sossi M. Iacovides, December 2014 putting up with my OCD, dark cutting ways and standing by my decisions to keep at it. A self-proclaimed perpetual student, without all of your help, I could not have lasted this long. But we did it! So the question is… Are y’all ready for the next 4 years?!
“You know what you gotta do when life gets you down? Just keep swimming, just keep swimming, just keep swimming, swimming, swimming!”
- Dory, Finding Nemo, Disney® (add a snaughle or two for good measure!)
iv Texas Tech University, Sossi M. Iacovides, December 2014
TABLE OF CONTENTS
ACKNOWLEDGMENTS ...... ii LIST OF TABLES ...... vi LIST OF FIGURES ...... vii ABSTRACT ...... viii
CHAPTERS I. LITERATURE REVIEW ...... 1 HISTORY OF ARTIFICIAL INSEMINATION ...... 1 HISTORY OF CRYOPRESERVATION ...... 4 MULTI SPECIES INDUSTRY TRANSFORMATION ...... 7 GENERAL CONCEPTS OF EQUINE SEMEN ...... 11 TYPES OF EXTENDERS ...... 19 CRYOPROTECTANT CLASSES ...... 25 FREEZING TECHNIQUES ...... 34 STALLION SPERM PHYSIOLOGY & CRYOPRESERVATION ...... 37 THAWING: ISSUES & IMPROVEMENTS TO POST-THAW QUALITY ...... 40 GENERAL DISCUSSION OF SPERM LOSS ...... 42 FUTURE OF SEMEN CRYOPRESERVATION ...... 48
II. CRYOPRESERVATION OF EQUINE SEMEN USING A MECHANICAL CONTROL RATE FREEZER ..... 50 INTRODUCTION ...... 50 MATERIALS & METHODS ...... 52 SEMEN COLLECTION & PREPARATION ...... 52 SEMEN FREEZING ...... 53 SEMEN THAWING ...... 54 MORPHOLOGY & ACROSOMES ...... 57 STATISTICAL ANALYSIS ...... 58 RESULTS ...... 58 DISCUSSION ...... 81
III. CONCLUSION ...... 83
LITERATURE CITED ...... 86 APPENDIX ...... 96
v Texas Tech University, Sossi M. Iacovides, December 2014
LIST OF TABLES
1. COMPARISON OF INTERMEDIATE SPEED PARAMETERS BY TREATMENT & TIME ...... 64 2. COMPARISON OF AVERAGE MOVEMENT CLASSIFICATIONS BY TREATMENT & TIME ...... 66 3. QUANTITATIVE COMPARISON OF TREATMENTS ...... 80 4. PERCENT RETENTION OF CONCENTRATION, MOTILITY & RAPID CELLS ...... 96 5. PERCENT RETENTION OF MORPHOLOGICAL VALUES ...... 97 6. PERCENT RETENTION OF ACROSOME VALUES ...... 97
vi Texas Tech University, Sossi M. Iacovides, December 2014
LIST OF FIGURES
1. COMPARISON OF CONCENTRATIONS BY TREATMENT & TIME ...... 60 2. COMPARISON OF MOTILITIES BY TREATMENT & TIME ...... 61 3. COMPARISON OF RAPID CELL PROGRESSION BY TREATMENT & TIME ...... 62 4. COMPARISON OF NORMAL MORPHOLOGY BY TREATMENT & TIME ...... 68 5. COMPARISON OF ABNORMAL HEAD MORPHOLOGY BY TREATMENT & TIME ...... 70 6. COMPARISON OF ABNORMAL MIDPIECE MORPHOLOGY BY TREATMENT & TIME ...... 71 7. COMPARISON OF OTHER ABNORMAL TAIL MORPHOLOGY BY TREATMENT & TIME ...... 73 8. COMPARISON OF CURLED ABNORMAL TAIL MORPHOLOGY BY TREATMENT & TIME ...... 74 9. COMPARISON OF INTACT ACROSOMES BY TREATMENT & TIME ...... 76 10. COMPARISON OF PARTIALLY ACROSOMES BY TREATMENT & TIME ...... 77 11. COMPARISON OF NON-INTACT ACROSOMES BY TREATMENT & TIME ...... 78
vii Texas Tech University, Sossi M. Iacovides, December 2014
ABSTRACT
With the evolving use of assisted reproductive technologies (ART) in the equine industry, there is a need for a cost effective, field method of preparing stallion semen for cryopreservation. Currently, semen is either suspended in the mist above liquid nitrogen or frozen in an electronic controlled rate freezer (ECRF).
Mist freezing (VM) has an uncontrollable temperature drop, increasing damage to cells. ECRF yields superior results, yet is cost prohibitive and complex to use.
The current study explored the use of a new mechanical controlled rate freezer
(MCRF) in a control environment. Samples for cryopreservation where obtained from 15 stallions, and frozen using MF, ECRF or MCRF. Once thawed, the samples were analyzed for standard semen parameters using a computer assisted semen analyzer at 0 and 3 hours post thaw, additionally morphology and acrosome slides were prepared. Initial post-thaw analysis demonstrates rapid cell movement in both MCRF and ECRF; but not MF. Further, morphology from MCRF and ECRF correlate with stallion standards. With current data, the
MCRF appears to produce higher quality samples than MF and comparable to
ECRF.
viii Texas Tech University, Sossi M. Iacovides, December 2014
CHAPTER I
LITERATURE REVIEW
History of Artificial Insemination
It has been widely reported that artificial insemination (AI) was first used in the twelfth century, but the accounts remain undocumented. In most reports,
"negative AI” was used by Arab horseman, who bred selective mares to their enemies’ stallions. The irony was the mares were reported to have been artificially impregnated with the sperm of weak and inferior stallions, which introduced an impure breed strain into the line [1], being the opposite of our goals today. In a second account, which more closely mimics the definition of AI used today, an Arab chief stole semen from the stallion of rival so he could breed his mares to improve his own bloodlines [2]. With no document available to support these reports, credit for the first attempted use of AI is most often given to Spallanzani who attempted the process in the dog four centuries later, and thus earned him a permanent place in AI history [1].
Sperm were first described by Leeuwenhoek and his assistant, Hamm, in
1678 [3]. Referring to their discovery as ‘animacules,’ they were unaware of the importance of the discovery they had made. Yet their simple description has led to techniques allowing the manipulation of genetics in cattle, dogs, goats, horses, poultry, rabbits, sheep, swine, and endangered species [3]. Their discovery led to the AI experiments of Spallanzani; who performed AI in a dog
1 Texas Tech University, Sossi M. Iacovides, December 2014 model which resulted in the birth of three pups 62 days later [3]. Spallanzani capitalized on a concept, which may have been around for hundreds of years; he manipulated genetics through arguably the first assisted reproductive technology (ART) procedure and changed the ideologies of the current world.
The discovery meant animals would no longer need to be ‘classically’ mated and led to a new wave of research. Yet it took over a century, until Heape in 1897 [4] and a variety of the other individuals from several countries, reported that AI had been used in isolated studies with rabbits, dogs, and horses [3].
Horses have always been a different and sometimes difficult species to work with, and such has been the case in the development of equine AI. With early domesticated horses being used for agriculture, and also military service, they were the first to be consistently worked with, for the betterment of their countries. In 1888, a French veterinarian, Repiquet, suggested AI to overcome subfertility in horses and cows, and later to breed more mares to one stallion, and to produce hybrid animals such as mules [5]. With WWI on the horizon, countries had already begun to explore his idea, and wanted to increase their equine military service forces at an expedited rate. Heape, described the aspiration of semen from bred mares, and used either a gelatin based capsule filled with semen or a syringe to transfer the semen to another mare’s uterus[5].
Later, G. Sand used porcine bladders as condoms to collect semen for breeding and birthed four foals from eight mares. Soon after, successful insemination of mares was reported across Europe from Austro-Hungarian Croatia, Hungary, 2 Texas Tech University, Sossi M. Iacovides, December 2014
and Russian Poland [5]. A Japanese scientist, Ishikawa, studied AI in Russia and
upon his return to Japan began an equine insemination program in 1912 [5]. By
the early 20th century, the AI was being practiced on a much larger scale, but
little research had been done to explain the mechanics of the process.
While a number of researchers around the world were experimenting
with AI, Russia had one of the active research programs. This was due in no small
part to Nicholas II, the Russian Czar, who was aware of his country’s growing
need for horses for the military service and agriculture and who recognized the
potential for AI. He supported the scientist Elia Ivanov, who constructed the first
controlled AI experiments and set a precedent for collecting horse semen [5].
With access to the Imperial Russian Studs, his research consisted of inserting silk
sponges into mares, allowed them to be mounted, removing the sponge,
collecting the semen with a press. He then used syringes to deposit the semen
transcervically with a rubber tube into other mares [5]. In his published work, he
systematically proved that he could use one stud to breed 500 mares, providing
the documentation needing to move AI from an experimental protocol to farm
procedure. Ivanov also discussed the idea of shipping semen to close locations
and in 1912 provided an instructional booklet showing photographic evidence of
foals on the ground to prove that there was no difference between the product
of a natural breeding and one of AI [5]. He is also credited to be the first to use AI
to produce interspecies hybrids in cattle, horses, poultry and zebroids by
inseminating horse mares with zebra semen [5]. 3
Texas Tech University, Sossi M. Iacovides, December 2014
Ivanov published his research in 1922, which included the development of some of the first semen extenders and training procedure for AI technicians.
His work was followed in 1938 by Milovanov who created artificial vaginas (AV) and other items still use practically today[3]. The development of AV’s represented a monumental advancement in AI, it provided a superior method for collecting semen and results in less overall loss [3]. Western breeders remained unaware of these latest industry advancements, until Japanese researchers Niwa and Nishikawa summarized the recent works into English in
1958, 1962, 1964 and 1972 [3]. In 1933, Walton published a book on AI, which included a procedure for shipping ram semen and two days later using it to successfully inseminate ewes [3]. Danish veterinarians Sørensen & Gylling-Holm established cervix manipulation with rectovaginal fixation, a practice still used today, which required fewer sperm for insemination, as semen was deposited deep within the cervix into the uterine body[3].
Another advancement was the packaging of semen in straws. This was first done by Sørensen, who used hollowed oat straws until he serendipitously saw some made of cellophane at his daughter’s birthday [3]. This evolved into the commercial straws designed by Cassou in 1964 and still used worldwide today [3].
History of Cryopreservation
Cryopreservation (CP) is the ability to store cells and maintain their integrity and viability at a sub-zero temperature until needed. Like some of 4 Texas Tech University, Sossi M. Iacovides, December 2014
the greatest discoveries, CP was the result of a fortunate lab error. In 1949,
Ernest John Christopher Polge and his colleagues were focused on trying to use
sugars as cryoprotectant’s (CPO) using what they thought was a stock fructose
solution [3]. They reported CP having been achieved with the fructose solution,
however after chemical analysis of the bottle was completed, they realized it
was all due to an error [3]. While history has provided varied accounts of the
story, it is conclusive that due to the mislabeling of a solution bottle in a
refrigerator, which led to fowl semen being frozen in a mixture of glycerol,
albumen, and water [6]. This was clearly different from the intended solution of
levulose, but allowing the fowl semen to be stored at -70oC, serendipitously
discovering the first CPO [6]. By adding the glycerol, accidentally discovered by
Polge et al. to a base media of yolk-citrate extender first described by Salisbury
et al. in 1941, the first CPO was formed [3]. Later work would describe a variety
of other CPO combinations, but the tris-buffered egg yolk-glycerol combination
remains as a standard for the protection of frozen and unfrozen sperm [3].
Prior to Sørensen’s discovery of the straw as a vessel for the freezing and
storage of semen, samples were frozen in glass ampules. This proved
problematic at the time as samples were frozen using dry ice and the ampules
would break during the freezing and thawing process [3]. However, with
Cassou’s modified straw, and the 1974 discovery by Pickett and Berndtson of an
efficient means for sealing plastic straws plus the AI gun for insemination [3], all
processes were in place to all widespread use of the technology. 5
Texas Tech University, Sossi M. Iacovides, December 2014
When significant biological changes were observed (including a significant loss of fertility) in semen frozen and stored via solid carbon dioxide
(−79°C), there was a shift towards liquid nitrogen −196°C, where sperm had be shown to survive with minimal biological changes [3]. A supporting factor pushing the shift to liquid nitrogen, was the lack of equipment necessary to maintain the dry ice, necessitating frequent resupply to maintain temperature.
Because of the inferior insulation within the tanks of the day, this same issue was initially seen with liquid nitrogen. However, a motivated investor, named J.
Rockefeller Prentice, who owned of American Breeders Service, convinced the
Linde Division of the American Cyanamid Company that there was a market for liquid nitrogen containers with improved insulation, which has led to today’s nitrogen tanks [3].
The basic concept of CP is to reduce or if possible, avoid intracellular freezing, ice crystal formation, and to overall minimalize damage to the cell from the environment during cooling or freezing [7]. There are two different classes of cryoprotective agents (CPA), both of which have their own beneficial and disadvantageous properties in the world of CP. Each facilitates different binding potentials of molecules from both the penetrating and macromolecules (non- pentrating) solutions of which they are made [7]. The use of a CPA is not only species dependent, but also can depend heavily on the chemical composition of the male’s semen. The underlying purpose of a CPA is to increase the cell survival rates under (harsh) imposed conditions during CP [7]. 6 Texas Tech University, Sossi M. Iacovides, December 2014
Current results indicate that penetrating and macromolecules agents accomplish this in different ways. Penetrating agents, such as dimethyl sulfoxide
(DMSO), allow the manipulation of the cellular environment. Dimethyl sulfoxide encourages the reduction of cell water content, so at sufficiently low temperatures, it can reduce the damaging effect of the concentrated solutes on the cells [7]. Non-penetrating agents, the most common of which is glycerol, osmotically “squeeze” water from the cells primarily during the initial phases of freezing, when these additives become concentrated in the extracellular regions, with temperatures ranging −10 and −20 °C [7]. However the successful use of glycerol is heavily species dependent.
Multi Species Industry Transformation
The gains of reproductive technology are widespread throughout the animal industry, and the successful CP of spermatozoa has enabled growth of livestock breeding industry wide. The momentum gained from research has greatly reduced costs and enabled ART not only to change breeding practices in animal, but to become a practical reality in the treatment of human infertility
[8]. Cryopreservation and cryobiology are multifaceted and a collaborative research effort between not only molecular biology and theriogenology, but also engineering and mathematics [8]. Due in part to the shape of sperm, the complex cytoskeletal structures, the cell’s molecular mechanism’s of activation and capacitation, there are a wide variety of applicable protocols from high tech laboratories to farm use [8]. Semen CP has helped to link all of these fields 7 Texas Tech University, Sossi M. Iacovides, December 2014 together for one common purpose: to further the superior genetics available for future generations.
As described earlier, the ability to freeze cattle semen, has turned out to be the specie that presents the fewest cryobiologic challenges, was discovered by accident, but by default became the starting point for semen preservation. As beef breed bull semen has been the least challenging to freeze efficiently, the general protocols for it have remained static for many years. Since CP is highly in demand with beef and dairy cattle, there has been an increase in the globalization of CP companies [8]. Yet, bovine semen post-thaw survival rates which use current methods range from 30%-40%, leaving much room for research to help improve CP technologies [8]. One notable modification was that of Purdy and Graham who investigated the effects of the additive cholesterol before CP and found that there was an increase in post-thaw motility in the cholesterol treated sperm when compared with control sperm [8].
However, the addition of cholesterol appeared to inhibited capacitation, which resulted in reduced fertility.
Sexed semen has added another dimension to the use of cryopreserved sperm within the cattle industry; especially with the dairy industry where cows represent potential income and bulls are generally an expense. However, it is well documented that the motility and concentration of semen from dairy bulls is considerably lower than that of beef cattle. Given that the number of sperm cells is significantly reduced when sexed, and with a lower recovery rate this 8 Texas Tech University, Sossi M. Iacovides, December 2014
endeavor has presented considerable challenges [8].
Global herd genetics have help to shape the demand on the preservation
of commercially important traits to the swine industry over the past ten years
[8]. Increased demand for swine CP has grown, but is impeded due to a reduced
recovery rate from porcine sperm being “cold–shock sensitive” [8, 9]. While
cryopreserved semen has been more than beneficial in the cattle industry,
currently there is a negative economic impact associated with frozen porcine
semen. A 10-50% decrease in farrowing rates, 1.5-3 piglet reduction in litter size,
and an increased AI dosage, are all due to poor recovery rates all seemingly stem
from cryopreserved semen [8]. Gilmore[10] reported reduced osmotic tolerance
levels in boar spermatozoa relative to sperm from other mammalian species, yet
Thurston identified 16 different molecular markers linked to cryosurvival that
potentially could be used for identifying inter-boar variation [8, 11]. Genetic
predetermination is a potential factor as they found Landrace boars had superior
quality sperm after freezing, but currently there is not a good genetic or
molecular predictor of cryosurvival [8].
As the use of CP is ever increasing across species, there are different
modifications of techniques and methods being applied. Similar to other species,
there has been an increased use of human sperm banks to treat infertility.
However, CP of human sperm is not thoroughly understood, and the standard
approach to it’s cryobiology has obstacles [8]. The World Health Organization
recently claimed on average about 50% of sperm cells are damaged by 9
Texas Tech University, Sossi M. Iacovides, December 2014 freezing and thawing; thus limiting the potential efficiency of human semen CP
[8].
Cryopreservation of men’s spermatozoa has been shown to cause an overall decrease in the progressive motility, and due to the differences seen in semen constituents between males no single CP procedure works for all[8]. With growing awareness of medical treatments that may contribute to subfertility,
(chemotherapy and radiation for cancer patients being at the top of the list), CP is currently the only long term storage option available for maintaining reproductive viability[8]. However, with recent improvements and increased access to available ART’s, even men with severe subfertility can benefit from conventional CP as procedures [8].
Ram semen has been cryopreserved by a variety of methods, pellet, ampule, and straw; but a major AI obstacle is establishment of a viable spermatozoa pool in the cervical canal of the ewe [12], due to multiple annular rings. Low fertilization results have been directly attributed to the failure of transport of sperm following insemination with frozen semen to the cervix and the low post-thaw viability [12]. The lack of rigidity found in the acrosomal membrane of frozen ram semen, leads to the less viable semen, which concurrently leads to lower quality semen being deposited as well as a higher number of insemination dosages necessary [12]. While ovine CP is feasible, with the appropriate cryoprotective agents, the physiological barriers encountered to date, have deterred the use of cryopreserved semen. However, 10 Texas Tech University, Sossi M. Iacovides, December 2014 recently studies have shown that rams producing smaller sperm heads show better cryoresistance to that of their larger counterparts[13], suggesting improvements can be made in ram CP techniques.
Aquaculture is a prime example of how, regardless of successful protocols being available, the use of cryopreserved sperm is narrow [8]. To date, the uses of reproductive cell cryobiology of any sort in aquaculture has been limited to Pacific oyster oocytes [14]. However, CP of semen in aquaculture would aid “sperm on demand” and increase the transport of sperm between farms for induced spawning[8]. Yet limited commercialization of the CP process in this industry has prevented this growth. With potentially huge genetic benefits for monogamous aquatic species (catfish) [8], it appears more research is need to improve techniques in aquaculture species. The combination of dramatically different physical and chemical conditions that aquatic species’ sperm are exposed to, in comparison mammalian sperm, must be a cornerstone in the research necessary to further new cryobiology discoveries [8]. However, caution must be used in dealing with the combination of wild and “farmed” animals in an aquaculture environment. As an example, the striped bass industry has grown astronomically, in part to the “sperm on demand” method, but there is the real potential to negatively impact the wild striped bass population as males have become selected for seed stock [8, 15].
General Concepts of Equine Semen
Stallions are a prime example of the species dependent nature of male 11 Texas Tech University, Sossi M. Iacovides, December 2014
reproduction. Generally, semen parameters in stallions are much different from
other mammalian species. This begins with the process of spermatogenesis (the
creation of spermatozoa from spermatogonium), which takes an average 60
days. This process results in sperm cells that generally have a different
morphology, lower motilities, and a more delicate acrosome cap than other
domesticated species. Further, semen production in the stallion is influenced by
the seasonality of the female. Looking at individual semen parameters, stallion’s
produce an ejaculate of 25mL to 60mL, much larger than that of others species.
While the morphological shape of an equine sperm cell follows the same general
shape: crescent shape acrosome cap, rounded head, neck, mid-piece, tail and
end piece, its head tends to be more symmetrical than other domesticated
species, making it more similar to human sperm. Further, unlike other
domesticated specie, the other semen parameters include: count, motility and
forward progression will vary widely between individual stallions, as animal are
selected more for performance traits or personal preferences rather than
reproductive potential. Finally the last parameter is the acrosomal cap, which
has three classifications: intact, partially intact and non-intact; with the
maintenance of the integrity of that membrane being vital to maintain it
fertilizing capacity.
In general, the evaluation of semen quality includes its functional ability
which is determined by several factors [16]. Numerous studies have correlated
morphology and motility of fresh spermatozoa with their fertilizing 12
Texas Tech University, Sossi M. Iacovides, December 2014
capability. However, there is data which demonstrates that the post-thaw
motility of cryopreserved stallion sperm alone is a poor predictor of sample
fertility; suggesting that post-thaw fertility of equine semen can be affected from
a variety of factors which may or may not impact sample motility [16]. Another
obstacle that stallion spermatozoa possess, is a coating of seminal plasma, which
is firmly attached and may be removed during capacitation [17].
Additionally, motility in fresh semen differs greatly from frozen semen.
With fresh semen, there comes an expectation of much higher percentages since
there is not superimposed cryodamage. Whereas with frozen the industry
accepts 30%, since there must be accountability for loss via cryoinjury.
Horses are generally classified as long day breeders, meaning they
physiologically anticipate mating season, which, in the North Hemisphere, begins
in February and can end as late as September. It is worth noting that stallions are
not considered to be seasonal breeders themselves, yet there are seasonal
differences in semen [16] and have clear differences in hormone concentrations.
Circannual rhythms are suggested to be a factor for difference seen in the
structure and functional integrity of the acrosome and plasma membrane
throughout the year. This is thought to be due to the hormonal changes within
the seminal plasma, specifically lower serum testosterone [18]; which cause a
fluctuation in the seminal plasma composition [16, 19] and an overall decrease
in testicular size [18]. However, studies agreeing that the membrane of sperm
cells are compromised during CP regardless of season [18], and that the 13
Texas Tech University, Sossi M. Iacovides, December 2014 removal of the seminal plasma is vital since it has proven almost impossible to freeze equine semen otherwise[17].
While the stallion might not be considered seasonal, a completed assay of sex hormone concentrations of animals in the northern hemisphere, conclusively demonstrated a clear pattern of seasonality; with a rise beginning in
February, plateauing in April and May, and plummeting in October through
January [19], then the cycle repeats. The hormonal differences can result in differences in morphology, acrosomal reactivity, as well as percentages of motility [16] in individual animals, but the effects are not universal. Extensive hormonal and morphological variation amongst stallions as well as within stallions [18] has been shown. For example, a recent study in Switzerland suggested that there is not a morphological difference between seasons [18].
Normal spermatozoa with appropriate morphological percentages imply there is more vitality and stability. However, another study demonstrated that frozen sperm undergo higher spontaneous acrosome reactions during winter [16].
Initial thoughts that breeding season was indicative of increased spermatozoa output and concentrations of gonadotropins, testosterone and estrogens [19], but recent findings have shown that only seminal parameters have an effect on motility. That knowledge has led to the supposition that testicular germinative functions are more affected by the endocrine system rather than seasons [19] which would correlate to the suboptimal conditions during the non-breeding season causing a change in sperm quality[16]. 14 Texas Tech University, Sossi M. Iacovides, December 2014
The year 1957 was monumental for CP of equine semen, as Canadians,
Barker and Gandier reported the first foaling using frozen epididymal
spermatozoa [17], demonstrating use of the technique was possible in the horse.
However, this early success has not led to widespread use of the technique as it
has in other industries, due to the unique nature of equine semen. Ultimately,
not only will the specificity of the species play a large role, but each individual’s
own body chemical composition may impact the process as well. Cryopreserved
equine semen faces differences in at least two areas: the physiological and
biochemical components of the spermatozoa themselves, and variations in the
anatomy and physiology of sperm transport in the female reproductive tracts
[20]. Currently, stallions generally do not fit the protocols of freezing programs
due to the unsatisfactory post-thaw sperm quality and fertility rates [21]. The
quantitative differences seen in the required number of spermatozoa necessary
for insemination between species is a largely important element when looking at
the potential fertility of cryopreserved semen [20], and if there are a larger
number spermatozoa required for insemination this means there can be less
tolerance of poor freezers [20] and poor survival rates. In the horse, the
accepted number of viable spermatozoa for insemination is more individual
dependent than in other species [20]. Studies have consistently shown CP sperm
lack in motility, viability and intact membranes when compared to that of fresh
semen [18]. Hence, in the horse, the development of successful freezing
procedures will involve more than the identification or application of novel 15
Texas Tech University, Sossi M. Iacovides, December 2014
CPO’s and additives [20].
Motility remains a major criterion used to determine the success or
failure of a new freezing procedure [17], but this is contradictory since motility
does not positively correlate with the sample’s future fertility. On the contrary,
the classical definition of a ‘successfully preserved sperm cell’ requires they have
the ability to fertilize an oocyte, and to produce a viable embryo via AI [17].
Using this definition, frozen/thawed sperm cells must be able to undergo
capacitation, activation of the enzymes within the acrosomal cap (while in the
female tract), which allows the sperm cell to penetrate through the zona
pellucida and fertilize the oocyte.
Cryopreserved stallion sperm exhibits a high degree of male-to-male
variability with respect to cell viability after thawing [21]. In order to adequately
classify the quality of frozen semen there must be an understanding of the
relative classification of a ‘good’ or ‘poor’ freezer. These concepts are based on
post-thaw motility characteristics, including percentages of progressively motile
sperm and velocity rate [21]. Tischner proved that only about 20% of the
stallions exhibit ‘good’ semen freezability with parameters being more than 40%
progressively motile sperm post-thaw [21, 22]. ‘Fair’ freezing stallions post-thaw
with 60% motility, and progressive motility range of 20–40% [21, 22]. ‘Poor’
quality semen has a < 20% survival whereby with a post-thaw progressive
motility rate of less than 20% [21, 22].
It is generally accepted that even under the best of conditions that 16
Texas Tech University, Sossi M. Iacovides, December 2014
40–50% of the sperm cell population will not survive CP even with optimized protocols [23]. In some species (including the horse) and specific individuals’ survival rates can be much lower, making them self-impeding to use of the CP process. Given the known limitations, Vidament [24] accepted stallions showing a post-thaw motility greater than 35% and sperm exhibiting ‘rapid velocity’ [21], while Loomis and Graham [25] accepted stallions with a post-thaw progressive motility greater than 30% [21]. Ultimately, these collaborative efforts have led to the commercially acceptable semen quality post-thaw of 30% progressively motile sperm, however even then there are many stallions that do not meet this standard. Previous studies have evaluated membrane structure characteristics, and suggested, in some cases, there may be genetic detriments, predisposing the cell to certain survival issues under CP stress [23]; supporting the concept of individuals being classified as ‘‘good or ‘‘bad” freezers. By developing an understanding the classes of ‘good’ and ‘bad’ freezing semen, patterns can be established allowing the modification of the components of CPA that permit the further improvement of this area of ART by suggesting which medias are more beneficial for each class and will produce optimal results.
Baird’s Tapir are evolutionarily related to equids and rhinoceros. Tapir’s semen osmolality has proven similar to that of the domestic horse, Prezwalskis horse, and the rhinoceros[26]. However, the average pH of the samples being lower than the three aforementioned species, the difference can be attributed to accessory gland contributions [26]. Due to the shared evolutionary 17 Texas Tech University, Sossi M. Iacovides, December 2014 relationship between these species, the industry knowledge that has been acquired for stallions may help to determine appropriate CP techniques and CPA that may be applicable for the other equid species. With stallions, it has been proven that the addition of cholesterol helps to increase the spermatozoa permeability to CPO thereby increasing the osmotic tolerance, and improving the sperm cryosurvival rates [26]. Given the semen osmolality similarities it has been suggested that cholesterol be included, to help facilitate better Tapir semen preservation [26].
One particular challenge for the Tapir, as with any non-domesticated specie, is the collection of the sample. While domesticated stallions are able to be collected via an artificial vagina, this is an unrealistic approach to nondomestic species, as Tapirs would be more at risk to injury [26].
Prezwalskis’ have not been cryobiologically studied, and therefore currently rely on research information gather from the domestic horse, especially concerning sperm cryosensitivity [27]. It is a well-established fact that less than 20% of domestic stallions produce sperm that are capable meaningful post-thaw survival, mainly due to the variations in individual CP capacities. This appears to be the case with the Prezwalskis as well. As in any species, there is the challenge of minimizing toxic CPO impacts are vital. However, there is evidence that amides will help to mitigate toxic impact of these compounds [27].
Current research has suggested that Prezwalskis spermatozoa are tolerant of cryoagents, cryodilutents, and the processing used in the domestic horse 18 Texas Tech University, Sossi M. Iacovides, December 2014 industry. These findings give hope for the potential of CP of a species that is facing extinction [27].
Types of Extenders
Extending semen is the first step in successful cryopreservation; enhancing the successful survival of sperm [28, 29] by increasing its storage time capacity [30]. With a wide variety of formulation of extenders available, varying to a large extent due to the protein source being used, the overall composition will play a significant role in cell survival [28]. The goal is to provide the best environment of support substances that will help to maintain the metabolic activity of the spermatozoa, serve as a buffer to pH changes, facilitate initial protection from cold shock damage [30] and maintain motility and fertilizing ability of semen for a time period of about 24 hours at 5°C [31]. Current protein bases can include, skim milk and/or egg yolk as the lipoprotein [32] source, and in more recent findings this relatively short list has grown to include egg yolk plasma [33] and soybean lecithin [32]. Other additives found in many extenders include: amides, antibiotics, fungicides, Hank’s salts (which maintain physiologic levels of calcium and magnesium levels in and surrounding the cells [34]), as well as a sugar source.
Sugars are a key component within the extender because of the lack of permeation within the sperm membrane. The sugar protects the cells against osmotic damage and lethal intracellular ice formation; especially when used in a combination with a penetrating CPO [35]. Seminal plasma contains a number 19 Texas Tech University, Sossi M. Iacovides, December 2014 of different sugar compounds (mainly fructose) that serve as natural energy source for the sperm. Studies have examined the use of a variety of sugar compounds at varying concentrations in CPA to limit the deleterious effects of sperm cryopreservation. Of the sugars currently being examined for their cryogenic properties, including: fructose and glucose, fructose has proven the least effective for preserving sperm quality [35]. Conversely, sperm frozen with extenders containing sorbitol exhibited better motility and viability at 10 min post-thaw; suggesting that sorbitol is more effective at protecting sperm against cryoinjury during cryopreservation [35]. Additionally, complex sugar combinations also show, in some cases, there are elevated rates of cryodamage between the supplemented glucose and the glycerol fractions [35].
Since the 1940 discovery of the cryoprotective properties of egg yolk
[36], it has become the most common extender option as well as protective agent used to aid spermatozoa in resisting cold shock [33, 37]. It has been successfully used to freeze and extend semen of many domestic or wild animals, including: cattle, gazelles, rabbits and horses [33]. However, without a full understanding of interactions between sperm and extenders, we are at a disadvantage to optimize extender use due to a lack of cryoprotective mechanism knowledge [36]. Egg yolk and egg yolk components [36] still have components which, although appear crucial to the process, have unknown mechanisms of action. An example would be the low-density lipoproteins (LDL) which are a major component of egg yolk and appear to be an active 20 Texas Tech University, Sossi M. Iacovides, December 2014
constituent of CPO, but little known about the nature of their interactions with
spermatozoa [36]. Recent research has suggested that egg yolk substrates
neutralize the sperm produced H2O2 during metabolism, which therefore
decreases the toxic effects of seminal plasma [32].
While effective in other species, egg yolk based media have proven
disadvantageous for stallions. Studies have suggested a number of issues. First,
fresh egg yolks are difficult to work with and given the history of the equine
industry it appears if the yolks are not powdered, most producers, are not willing
to put it into use [33]. Second, the non-processed animal origin of the material
represents a potential vector risk for bacterial contamination. This poses global
transport issues, as many countries are unwilling to import egg based products
[33]. Third, and from a more practical standpoint, the granular appearance and
density can interfere with microscope readings as well as assays. Finally, there is
variation amongst batches, since lipid composition of the yolks is directly
dependent on the hen’s diet[33].
While there are many varieties of milk-based extenders described in the
literature, skim-milk is the most commonly used. Additionally, as with the egg
yolk extenders, it is sold in a variety of forms, from a powdered mix, to a
premade solution, often requiring the necessity of warming prior to use. Skim-
milk based extenders have been shown to have positive effects on reducing the
antioxidative properties of semen, induced during the centrifugation process
[38]. 21
Texas Tech University, Sossi M. Iacovides, December 2014
Notably, the process of milk fractioning has allowed the preparation of purified or defined milk proteins [31]. Phosphocaseinate and β- lactoglobulin have been found to be the most effective in supporting long term sperm storage
[31] and short-term survival at cooler temperatures [39]. Currently, INRA96
(produced by IMV Technologies) is the most common defined milk protein extender, within the United States and Europe [39]. This premixed liquid formula is chemically defined, allowing standardization of manufacturing thereby eliminating variation in batch composition. With a custom formulation designed to incorporate only the beneficial components of the milk produces, studies suggest samples frozen in INRA96 maintain higher semen quality, and potentially better fertility as compared to biological skim milk extenders [39]. It is a combination of native phosphocaseinate, Hank’s salts, glucose, and lactose, supplemented with the purified milk-fraction, antibiotics and a fungicide [34].
Noted antibiotics include penicillin, gentamycin and amphotericin B, as they play a crucial role as they reduce contamination factors [34].
As with any biological substance, extenders exhibit significant variability due to the nature of their composition, and therefore are difficult to standardized between batches [31]. The use of additives can further increase this variation, and in some cases only certain components may be beneficial while others could be detrimental [31]. Therefore developing a chemically defined substance which will ultimately reduce the variablity currently observed between batches using skim milk and egg yolk bases has become a high 22 Texas Tech University, Sossi M. Iacovides, December 2014 priority for both researchers [32] and the CP industry [31]. The precise and defined composition that would be obtained from chemical extenders would also help to alleviate the deleterious effects and potentially enhance the beneficial protective properties of such compounds used in sperm storage [31].
Another supporting factor for the development of a chemically defined extender is while egg yolk and skim milk are both recognized as a good CPO, the nature of those same beneficial effects from both, have not yet been clarified in detail with the lifespan of the spermatozoa [31, 40]. Specifically egg yolk has been shown to stabilize the plasma membrane have been shown to bind with the plasma membrane of the sperm cell upon ejaculation, stimulating the cholesterol and a phospholipid efflux used in capacitation[40]. The CPO mechanisms of action for skim milk are still unknown, but studies has shown an increase in lipid peroxidation of spermatozoa membranes [40].
Given the nature of the semen sample itself, and the fact that most common extenders are comprised, at least in part, of components of animal origin, there is a consistent potential risk of contamination with bacteria in AI doses. Accordingly, manufacturers must include appropriate antibiotics as a precautionary measure [41].
Nowhere is this more true than in the collection of stallions; where the use of an enclosed AV, such as a Missouri, makes it virtually impossible to prevent bacterial contamination from external genitalia, as the surface and prepuce of the penis have a variety of non-pathogenic bacteria present [42]. 23 Texas Tech University, Sossi M. Iacovides, December 2014
To help mediate and prevent bacterial growth [42] within the semen during
storage, many manufacturers now provide a variety of antibiotic infused
extenders, in addition to the basic extender without antibiotics.
Per industry standards, the most commonly used antibiotics include
amikacin sulfate, gentamicin sulfate, streptomycin sulfate, sodium or potassium
penicillin, ticarcillin sodium and polymixin B sulfate [43]. While each has been
identified as covering a certain of spectrum of bacteria, the AI specialist on hand
should be able to recommend the most suitable extender and antibiotic
combination. It is worth noting, that none of these agents will completely
remove all bacteria present within the semen, more correctly, they serve to
retard the growth of new bacteria. Gentamicin and amikacin have proven to be
the best at preventing growth of aerobic bacteria in semen, while Polymixin B
has proven least affective[44]. While it is crucial to evaluate the effectiveness of
antibiotics in control bacterial growth during semen processing and storage, it is
also critical to know their effect on sperm motility [43]. With semen CP
becoming more prominent within the equine field, it has allowed researchers the
opportunity to address the issue of finding a suitable array of antibiotics that can
combat cold resistant bacteria, without causing more undue damage to the
motility of the sperm (Jasko et al., 1993). With little information available on cold
storage of semen with bacteria [41], the effects of potentially pathogenic
bacteria on frozen semen have yet to be analyzed [42]. Preliminary research has
implied antibiotics may be partly responsible for the detrimental effects 24
Texas Tech University, Sossi M. Iacovides, December 2014 seen in motility in frozen semen; either due to potential prolonged exposure or an interaction between cooling and the antibiotic [43].
Timectin® is a combination antibiotic formed by ticarcillin and clavulanic acid, and can be used in lieu of amikacin, which is not cost effective [44]. This combination allows for a broad spectrum of antibacterial activity and is known as an effective alternative to a variety of penicillin’s within extenders [44]. The addition of ticarcillin-clavulanic acid to INRA96 eliminated bacterial growth in stallion semen after extension and cooled storage without having a negative impact on sperm quality, i.e. motility features and sperm membrane integrity
[39].
Cryoprotectant Classes
Semen is highly individualistic, as no two stallions have the same chemical composition, and therefore each will freeze differently. Some individuals have been known to be hypersensitive to glycerol [45], whereas others may be able to tolerate it well. Following industry demands, a wide variety of CPO’s as well as many different commercially available CPA’s, have been developed. The exposure of equine semen to subzero temperatures demanded the discovery and further creation of CPO’s, which are vital to maintaining viability and survival of sperm cells while in storage. Even today, the exact mechanisms by which the CPA protects sperm during freezing and thawing, remains unclear. The current consensus is that CPO’s work by minimizing exposure to osmotic stress, stabilizing biomolecule and structure, 25 Texas Tech University, Sossi M. Iacovides, December 2014 and limit the effects of reactive oxidative species (ROS) [46]. The ideal CPA would not osmotically dehydrate the cell or induce cryoinjury and it would be non-toxic
[47].
The goal of a CPO should be to minimize intracellular freezing, minimize cell damage due to the freezing environment and promote cell survival upon thawing [7]. Larger amounts of CPA concentrations, have be shown to lead to more cellular damage; since cells exposed to those penetrating solutes undergo intense initial dehydration, then rehydration, resulting in a chance of gross cellular swelling to occur when the CPO is removed [48]. Ultimately these radical changes in volume and size can lead to damage and death of the sperm [48].
Previous studies suggest a CPO should have good water solubility properties, low molecular weights, and permeating capacities [21]. There are two classes of CPOs: penetrating and non-penetrating which when used together, increase the cells’ chance at survival while reducing the cellular water content to help prevent intracellular freezing [7].
Penetrating agents are micromolecules, which permeate through the plasma membrane of the sperm cell. Acting intracellularly, penetrating agents replace cellular water, as it pushed to the extracellular region, ultimately preventing internal ice crystal formation that could potentially rupture the membrane. Examples of penetrating agents would include DMSO, glycerol, methylformamide (MF) and dimethylformamide (DMF). Non-penetrating agents or macromolecules, capitalize on their increased concentrations within 26 Texas Tech University, Sossi M. Iacovides, December 2014 the extracellular regions during the first phase of freezing, generally -10 to -20°C, where they osmotically extract water from the cells [7]. Some non-penetrating agents worth noting include: egg yolk, sugars, liposomes, milk proteins and polymers that can form extensive hydrogen bonds with water.
Cryoprotectants are generally a combination of penetrating and non- penetrating agents each of which has a specific role in aiding the survival of the sperm cells during the freeze/thaw process. Further discussion of penetrating/ non-penetrating agents with CPA in relation to osmotic stress will follow in a later section.
The used of glycerol as a CPO for stallion semen was first described in
1950, by Smith and Polge. This formulation, remaining virtually unchanged, has be the mainstay of the freezing industry ever since [49]. It has been noted as the most effective CPA for lowering intracellular water freezing [50] while providing osmolality adjustments to the CPO via invasive thermal protection [51]. Research to understand the mechanisms of CPA led to the discovery of glycerol’s’ effectiveness in its ability to prevent various phase transitions while freezing via increased water permeability and fluidity of the sperm membranes [52].
However, while glycerol has allowed the CP of numerous species, it may not be ideal CPA. Results in cattle have shown a loss of fertility with aged sperm [51].
Further, while glycerol serves as the leading CPO for many species that was not the case for the equine species. While glycerol provides satisfactory protection for the roughly 20% of animals classified as “good freezers,” it has 27 Texas Tech University, Sossi M. Iacovides, December 2014
proven detrimental to remaining 80% due to its heavy viscosity and molecular
weight. Additionally, glycerol has been shown to be toxic to non-frozen sperm
and have contraceptive effects on mares [53].
Initial studies linked glycerol with the stabilization of semen membranes,
by its ability to cause a fluid to gel transition, however this finding led to the
expectation of higher CP survival rates [21]. However, recent research has
demonstrated glycerol induces cellular damage during the freezing process and,
in addition to cryoinjury [21], could be a largely contributing factor to poor post
that motility and fertility rates [49]. While the nature of semen glycerol toxicity
is not fully known, some data suggests its use may lead to protein denaturation,
directly altering the plasma membrane and the disrupting actin interactions [49].
Further, even though glycerol is a penetrating agent, it is extremely slow in
permeating the plasma membrane, which induces osmotic stress which may be
the ultimately cause of its toxicity. A number of studies have demonstrated that
the addition and removal of glycerol is an important factor responsible for the
reduction on post-thaw motility and viability of horse sperm. Equine
spermatozoa have been shown to have a limited osmotic tolerance. Glycerol has
been shown to induce more distinct osmotic stress with more severe alterations
on motion variables, cell viability and acrosomal integrity [49].
The issues with glycerol toxicity, have led to the research and testing of
countless other penetrating CPA’s, with the idea of finding one that will be less
toxic, while yielding comparable or better quality results [21]. This new era 28
Texas Tech University, Sossi M. Iacovides, December 2014
of CPO bases include combinations of penetrating CPA, for example glycerol and
dimethylformamide. Early results suggest these combinations have lower
molecular weights, increased water solubility and minimal toxicity [49], all of
which have proven to advantageous to the preservation of the delicate chemical
composition and plasma membrane structure of stallion semen.
Dimethyl sulfoxide is a sulfur containing, organic molecule, which easily
crosses cellular membranes. The fast penetrating capacity of DMSO helps to
decrease the amount of time necessary to displace water from the intracellular
fluid to the extracellular environment. Given the variability seen in stallion
sperm, a small amount of this strong compound is often used in conjunction with
glycerol or another CPA as an added speed component, and to help stabilize the
cell prior to freezing. However with some species DMSO is favored, and used in
much larger proportions than necessary for livestock. Species, whose semen
specifically perform better following freezing with DSMO may do so because
glycerol acts as a contraceptive for them. A few species that have benefited from
DMSO as a primary CPA include: mice, rabbits [54] and a variety of fish –
including: zebra fish [55], carp broodstock [56], seven-band groupers [57] and
mutton snapper[58]. Rabbit sperm appear to do best with a substantial
proportion of DSMO in relationship to glycerol for CP. Some research suggests
this could be do the lack of water channel protein Aquaporin 7 (AQP7), which
coincidentally serves as a glycerol transporter[54]. Dimethyl sulfoxide has also
been used for semen CP in some members of non-human primate family. 29
Texas Tech University, Sossi M. Iacovides, December 2014
Two species, both part of the macaque family: Cynomolgus monkey (Macaca
fascicularis [59] and Rhesus Monkey (Macaca mulatta; [60], have shown mixed
results. The Cynomolgus semen was successfully frozen when DMSO was in an
equal concentration to glycerol [59], whereas the Rhesus was unsuccessful after
using a stair step increasing trial of DSMO to glycerol [60].
Ashwood-Smith (1987) introduced the idea of creating a separate CPA
using two amide groups. The basic idea was that because amides have lower
molecular weight, DMF and MF have molecular weights of 73.09 and 59.07
g/mol respectively, (glycerol molecular weight of 92.09 g/mol), would more
easily and rapidly permeate the membrane, and most importantly leave the cell
more rapidly during sperm thawing, thus causing less osmotic stress [61].
Amides have proven to be a mostly beneficial CPA, having been shown to
decrease damaging results compared to those obtained when glycerol is solely
used as the CPO [61]. With stallions being sorted into different freezing classes,
poor freezers have required substantial work. Amides have increased the
freezing potential of this class while subsequently decreasing the overall
damaging results induced from glycerol [61]. While glycerol is still the main CPA
used for stallion sperm, the addition of amides, in part due to their lower
viscosity and molecular weight, may decrease sperm cell damage [28].
Dimethylformamide has been shown to enhance post-thaw motility,
preservation cellular membranes which may effectively enhancing semen
freezing potential[21]. With lower molecular weights, both DMF and MF 30
Texas Tech University, Sossi M. Iacovides, December 2014
are able to permeate stallion sperm, more efficiently than glycerol, which has
resulted in reduced swelling during equilibration in amide-containing diluents
and not as toxic as glycerol [61]. However, DMF and MF seem to be the only
amides with possible cryogenic effects. Studies have shown that other amides
have detrimental effects on semen not being cryoprotective.
Like most potential CPA, the use of a lipid based CPO has been
demonstrated to have both positive and negative effects on equine semen.
Previous studies have demonstrated CP can lead to loss of from the membrane
leading to peroxidation and continuing on to form reactive oxidative species
[62]. Lipid bases are multifaceted since they have been linked to both oxidation
of, as well as the protection of lipid bilayer infusions. The addition of a lipid
based CPO may destabilize the sperm membrane due to the formation of ROS
and recruitment of lipids from the membrane leading to lipid rearrangement
within the membrane itself causing additional oxidation to occur. Increased
peroxidation in turn might affect both motility and acrosomal activity. Sperm are
prone to cold shock damage due to osmotic stress and relative temperatures,
which in turn may lead to underlying damage to the integrity of membrane.
Further studies are need to determine the exact role lipids play in protecting
spermatozoa during freeze-thaw is unclear [62]. Therefore if lipids are to be
added as a cryoprotective agent to produce a more saturated CPO for semen
preservation, there are a few other issues that must be addressed. Numerous
studies have shown that ROS play a significant role in male infertility [63]. 31
Texas Tech University, Sossi M. Iacovides, December 2014
Further, cold shock damage has been directly linked to lipid phase transitions
that cause the sperm membrane to become leaky, thereby compromising
membrane integrity[62]. However, ROS been shown a double-edged sword.
While their detrimental effects are well documented, at low levels they are
involved in the normal physiological functions of sperm including capacitation,
acrosome reaction, and binding to the zona pellucida at physiological
concentrations [63-65].
As previously discussed, stallion sperm is highly individualistic, because of
this, research into additional CPA’s that would help to preserve frozen semen
has flourished. The discovery of MF and DMF as agents has been more than
beneficial to the industry and spurred investigation of other alternative agents.
Reductions of freezing and thawing damage, improving membrane integrity and
increasing progressive motility have been the goal of equine cryobiology
researchers over the last 30 years. During that time, reducing damage due to
freezing and thawing has been the focus of most investigating alternative CPA’s.
Recently, work with the addition of liposomes, which induce fusion to the sperm
plasma membranes, as well as the reversible binding of exogenous
phospholipids, have both shown to protect sperm from damage [66]. Cholesterol
and methyl-β cyclodetrin [67] have been shown to reduce membrane transition
temperatures, resulting in reduced cryoinjury, maintenance of the cellular
membranes and improve post-thaw motility.
While necessary to provide energy to the cells, some, but not all, of 32
Texas Tech University, Sossi M. Iacovides, December 2014
the sugars have proven to be effective non-penetrating CPA. The addition of
raffinose to extenders appears has no effect on post-thaw motility and viability
[53]. However, there are conflicting results when incorporating trehalose.
Squires et al [53] suggested it did not enhance post-thaw motility and viability;
yet Snoeck et al. [68] demonstrated an enhancement on post-thaw viability.
While the usage of amino acids with stallion sperm has not been studied
extensively, the few studies done today suggest they may be an important
addition to extender formulations. Koskinen et al. demonstrated the addition of
betaine, as a stallion CPA, which stimulated increased post-thaw motility [69].
Initial testing from Sanchez-Partida et al [70] working with frozen ram sperm
demonstrated low concentrations1 of proline, glycine and betaine could be used
to improve post-thaw motility as well. Trimeche et al [71] also showed that low
concentration=s of proline to be beneficial in enhancing the motility parameters
of stallion sperm. Glutamine has been helpful when combined with glycerol for
human sperm post-thaw motility and viability [72], and, at low concentrations, it
has worked effectively in stallion semen [71]. Conversely, high concentrations2 of
betaine, glutamine, histidine and proline were demonstrated to cause significant
dropped in sample motility [71].
As mentioned above, work has been done with other amides as well.
However, unlike the beneficial effects described for MF and DMF, acetamide,
and formamide [53] both appear to be toxic to stallion semen, and have poor
1 Low concentrations are defined at less than 120mM. 2 High concentration are defined at more than 160mM.33
Texas Tech University, Sossi M. Iacovides, December 2014
cryoprotective properties which make them unsuitable as a CPA [68].
Botu-crio® (Biotech Botucatu, Botucatu, São Paulo, Brazil) is relatively
new product with in the stallion CP world. Capitalizing on a market need,
preliminary studies and field trials suggest Botu-crio® provides far superior post-
thaw results compared to other available CPO; most likely due to the different to
the base medium in addition to CPA components, sugars and the buffer system.
This particular CPO includes a 1% concentration of glycerol in the presence of 4%
MF [73]. Using this CPA ratio, Botu-crio® it has shown to be non-detrimental to
the semen of good freezers and also appears to promote better post-thaw
viability. Additionally it contains a variety of sugars, plus both skimmed milk and
20% egg yolk, each with its own cryogenic properties [73]. Botu-crio® has been
designed to reverse the roles of the two main CPA, with glycerol, usually the
primary CPA, at lower concentrations than MF. Melo et al. [74] had previously
demonstrated that MF was a clearly superior amide when compared to DMF,
attributing most of success to the lower molecular weight, allowing faster
permeation and therefore resulting in less cellular osmotic damage, and
supporting its selection as the primary CPA in this product.
Freezing Techniques
While CPO’s are an essential part of semen CP the actual freezing process
also can determine the success or failure of the technique. Suitable procedures
with applicable freezing rates and parameters have been discovered for a
number of species, and research continues to provide the most successful 34
Texas Tech University, Sossi M. Iacovides, December 2014
results for improving CP of semen.
Since ejaculations are a heterozygous population of sperm that all will
respond individually to environmental conditions[75], different freezing
techniques must be examined. A technique that works well for one species may
be disadvantageous to another, meaning researchers and producers must be
willing to modify and adapt techniques in order to improve the final product.
There are currently a variety of techniques, machines, and protocols available
within the CP industry – none of which have been standardized.
During the freezing process, semen straws release latent heat of fusion
due to ice crystal formation, causing a sharp increase in temperature, showing
that while the straws are being frozen, the sample temperature does not
decrease in tandem with the decrease of the chamber temperature, remaining
static for 2-3 minutes before a further decrease is noticed[76]. While there are
suggested freezing rates for each species, there is always room for error, and
therefore always rooms for improvement. The electric programmable freezers
are widely used interspecies, and have proven to provide the best and most
reproducible results so far. However, these instruments are expensive, use a
large volume of liquid nitrogen and are fixed within a dedicated collection
facility.
Given an industry need for a more efficient and a more reliable means of
semen cryopreservation, researcher continues to look for alternative to meet
the need. One such system, the Styrofoam® box freezing method, is 35
Texas Tech University, Sossi M. Iacovides, December 2014
currently being debated. While it is cheap, easy to use, has low liquid nitrogen
requirements and is portable, studies suggest it produces highly varied results
[77]. The following section will review current available techniques for their
advantages and their limitations.
Vertical mist freezing is by far the simplest and most inexpensive
technique currently available. Normally, straws are simply loaded into canes,
which are then fastened into goblets, and hung interiorly from the lip of a liquid
nitrogen tank. The goblets then remain suspended in the mist for prescribed
amount of time; for stallions this usually means 30 minutes. After that, the
goblets are simply dropped into place down in the liquid nitrogen and stored
until future use. Since the goblets are cooled in the vapor phase of liquid
nitrogen, the initial ice crystallization will cause latent heat of fusion to be
released and the cooling continues at a non-equilibrated rate [75], freezing in an
uniform fashion.
As with most species, stallion semen frozen using this method
demonstrates a wide range of variability in their post-thaw survival. This may be
due to the fact straws are stored within different physical proximity while in the
mist, and therefore freeze at different rates.
The horizontal mist method was developed in an attempt to overcome
the variability seen in vertical mist freezing by standardizing the location of the
straws above the liquid nitrogen source. The container is first loaded with liquid
nitrogen on the bottom, and then semen filled straws are positioned to 36
Texas Tech University, Sossi M. Iacovides, December 2014
rest in a horizontal position at a proscribed height above the nitrogen pool
within the mist. This technique has been predominately used in cattle, with
positive results. However it has been shown in hogs[78] and stallions that when
the straws are placed above the liquid nitrogen and allowed to freeze, it was
disadvantageous to the sperm due to the lack of uniformity of the freeze
amongst the straws. While attempted in stallions, the general consensus has
been then inability to regulate the drop in temperature in a controlled fashion
results in cryoinjury to sperm, significant cell death and non-viable samples.
Programmable freezers are considered to be “slow freeze” as they have
been devised to decrease in temperature over a period of time, until the
optimum freezing temperature is achieved. This method, is currently considered
the “gold standard” within the industry as it achieves the most exact freezing
rate necessary for optimum post-thaw survival, by reducing the occurrence of
latent heat of fusion, by modifying the pulsing of nitrogen through the chamber
[76]. The controlled rate method of freezing allows for large quantities of straws
to be frozen uniformly with time and temperature [78]. Simultaneously achieving
consistent freezing rates from the constant flux of liquid nitrogen with is pumped
through the chamber at a controlled rate and evenly dispersed within the
chamber [78], provided straws with the optimum survival chance.
Stallion Sperm Physiology & Cryopreservation
Beginning formation within the seminiferous tubules of the testes, a
spermatozoon is made of nucleic acids, lipids and proteins. The two 37
Texas Tech University, Sossi M. Iacovides, December 2014
primary functions of the spermatozoa being to carry the male gamete to the
oocyte, as well as bind and fertilize the oocyte [79]. On first look, spermatozoa
appear to be very simple cells, composed simply of a head and flagella. While
seemingly simple, this is not accurate and they are actually intricate structures,
not only sophisticated and adaptable to that role of fertilization but also serving
as a highly organized intracellular matrix vital to the success of survival. The head
is comprised of a highly compacted nucleus [79] consisting of deoxyribonucleic
acid (DNA) and with the an acrosomal cap serving as a protective barrier. The
acrosomal cap not only serves as protection to the nucleus and DNA, but also
contains hydrolytic enzymes that allow the penetration for the zona pellucida for
fertilization to occur. In the event that the cap is damaged the proteolytic
enzymes will be released, the sperm will not be able to fertilize the oocyte [79].
While the flagella is broken into three portions: neck, midpiece and endpiece.
The midpiece generates the energy necessary for movement, and allows the cell
to become hypermotile once the cell has undergone capacitation within the
female tract.
Cryopreservation is the key to allowing the extension of genetic traits and
has become seen as a valuable commodity, which is applicable to many species.
The procedure itself as applicable in one beyond freezing and storage for
reproduction to a number of medical and veterinary applications. The use of CP
is rapidly progressing within the equine industry and significant research has
been undertaken to understand the different stresses that stallion 38
Texas Tech University, Sossi M. Iacovides, December 2014
spermatozoa experience during freezing and thawing, so that damage arising
from CP can be minimized [61]. This began with the creation of commercial
liquid cooled transported semen techniques within the last century. Breeders
quickly realized the benefits and the efficiency of transporting semen to mares,
rather than mares to stallions. However, liquid-cooled semen posed logistical
limitations on the industry such as time, distance and transportation delays.
Therefore breeders have looked towards frozen semen as a practical and
efficient method for transporting or storing stallion semen [25].
During the process of cryopreservation, stallion spermatozoa are exposed
to a variety of levels of osmotic stress due to osmotic pressures created prior to
the establishment of equilibrium with the CPA. When semen is cooled, cell
volumes change, resulting in an increased imbalance in water and solute
concentrations across the membrane, which result in cell damage to the sperm
[80]. Freezing protocols have been designed according to physical properties of
the various CPA, and have been designed to maximize the recovery rates in
correlation with the use of appropriate freezing and thawing rates, with the
ultimate goal being a maximization of viability [61].
Recently freeze-fracture techniques, freeze-substitution electron
microscopy, and ice formation measurement by differential scanning calorimetry
were used to demonstrate that there is no intracellular ice crystal formation in
human and stallion spermatozoa with the currently CP methodologies and
freezing rates. Intracellular water content has been classified a major 39
Texas Tech University, Sossi M. Iacovides, December 2014
source of damage from intracellular ice damage [8, 81]. Lovelock [82, 83]
deduced that CPA worked to lower the water within cells, while not having the
deleterious effect on the cell solute concentrations [8]. Given that finding, it can
be theorized that the major source of damage almost certainly comes from
osmotic stress, especially during thawing [61]. Further, the biophysical
parameters of cells change according to different stress influences they
encounter, and thus may cause damage to the membrane components [8]
resulting in reduced cell function or cell death.
Thawing: Issues & improvements to post-thaw quality
A major factor influencing the final post-thaw recovery rate of motile,
function sperm is the thaw process itself. A non-regulated thaw can damage the
cells, leading to decreased metabolic activity [84], which in-turn may lead to
further loss over an extended period of time. While directly associated to the
stallion’s semen ability to frozen (“good” vs. “poor” freezer), some research has
shown that selected CPOs, i.e. Botu-Crio® [84] are able to decrease the post-
thaw damage to sperm; especially when cooled in a refrigerator for 24 hours
prior. Salazar et al. [85] observed more cryoinjury when sperm cells which were
rapidly frozen and suggested stallion semen must go through a pre-freeze
cooling step in order to reduce the post-thaw damage. The cryoinjury caused a
disruption to the membrane, a separation factor within the lipid bilayers, and a
change to water transport rate. Gibbs et al. [86], used a 120 minute pre-freeze
cooling period, which resulted in far superior post-thaw results as well, 40
Texas Tech University, Sossi M. Iacovides, December 2014 leading to the suggestion that stallion sperm benefit from a equilibration period post addition of a CPO, allowing sufficient time for efficient permeation of the membrane and reduces cellular damage.
As shown above, CPO’s having lower molecular weights (DMF/MF), have been shown to more advantageous with post-thaw motility. The lower molecular weight of these CPA allows better cell permeability, therefore potentially reducing the overall stress induced on the cell. Studies have suggested that reducing the time of hypertonic or hypotonic influence on the sperm cells, is vital in their preservation; especially since this osmotic stress factor encourages the production of ROS, a severely DNA damaging issue [86]. Interestingly, while
Botu-Crio® has become known as an advantageous CPO, when used with tapirs 4 hours post–thaw motility numbers were higher comparatively to INRA96, but there was a sharp decrease in intact acrosomes immediately after thawing [26].
An alternate method that has been suggested is to add seminal plasma back into the post-thaw semen. While this method appears to be support the sperm cells, and be helpful in maintaining post-thaw viability, the task of re- adding seminal plasma might not be something that many breeders would be willing (or are equipped) to do. However, leaving a large amount of seminal plasma in the preparation prior to freezing has been suggested to reduce cryocapacitation [87]. Premature capacitation, which occurs during freezing, would render the cells unable to fertilize an egg, therefore stopping its occurrence would improve cell function. In addition to the inclusion of 41 Texas Tech University, Sossi M. Iacovides, December 2014 additives, certain processing steps have been shown to affect semen quality during cryopreservation. Melo et al. demonstrated that the use of a refrigerated centrifuge during processing degraded semen quality [74], yet this effect was not seen if cells were processed with a non-refrigerated unit.
General Discussion Sperm Loss
Mazurs’ [88] two factor hypothesis on freezing injury, helps to categorize and explain the freezing responses from different cell types. The osmotic behavior of cells is widely understood, respectively with each species and cell type, having unique boundaries. However, the increased variation between stallions compared to other species, make this less predictable. Recently, it has been shown that rapid cooling of human and stallion sperm infers a loss of viability, but more interestingly suggested that intracellular ice may not be the culprit[89]. Demonstrating how speeding up the cooling rates, Morris et al. [89] would reduce the ice crystallization damage by “solution effects.” They also suggested that those same higher cooling rates are found in glycerol solutions.
Given what we know about stallion sperm, this relationship appears counterintuitive, as the majority of glycerol based CPO’s have been shown to have a more deleterious effect on sperm cells. Morris also suggested that post- thaw semen quality might be just as dependent on semen concentration as well as any single CPA of the additive.
Finally, in addition to the biochemical and physiological issues above, both the methodologies used for collection as well as the mechanics preparing sperm 42 Texas Tech University, Sossi M. Iacovides, December 2014
for freezing may result in significant cell loss. While many techniques are more
than suitable for producing a fertile sample, the loss of semen is inevitable and
can be classified into the following: loss during receptacle transfer, loss in
centrifuge tubes, loss due to air exposure [90]. With cautionary techniques used,
there is still loss involved, most of which can be attributed to extended air
exposure. Stallion sperm can use both aerobic and anaerobic pathways [91]and
with fluxing temperature and air exposure , the concept of motility conservation
minus air exposure results in the decline of energy stores via glycolysis and
glycogen recruitment.
A basic understanding of the architecture of the sperm membrane is
crucial to understand how cryoinjury occurs to cells. Nowhere is this more true
and with more impact on fertility than damage to the acrosomal membrane.
Membrane composition and fluidity of the individual lipid bilayer is highly
dependent on the dietary intake [92]. The intercalation of CPO or other
compounds affect membrane fluidity, cause changes to the cytoplasmic
viscosity, ultimately affecting the cell’s metabolic capacity. Concurrently, when
cells are introduced to low temperatures that they would not normally
physiologically encounter, the membrane alters its mechanism of lipid packing,
which modifies enzymes within the membrane and the kinetic properties of the
cells. All of these factors lead to the imminent potential of cryoinjury, including:
cold shock, freezing damage or thawing damage[92].
Apoptosis can occur within all cells, resulting in programmed cell 43
Texas Tech University, Sossi M. Iacovides, December 2014
death. The physiological process of programmed cell death which affects single
cells and induces morphological and biochemical changes which lead to cell
death and acts as a homeostatic function within the body [93]. It has been
shown to occur within spermatogenesis as a homeostatic event, to help balance
the new and old cells. Since apoptosis is required to allow the normal
development of germ cells, spermatogenic apoptosis helps to maintain the
balance between germ and somatic cells, while also removing the defective germ
cells [93]. However if this process is interrupted it could lead to increased
quantities of ejaculates spermatozoa displaying apoptotic like changes and result
in decreased fertility [93]. With a two-pathway option for apoptotic initiation,
the intrinsic is due to pre-apototic signals that lead to the activation of caspases,
and extrinsically death receptor pathway receptors allow for ligand binding to
occur at the plasma membrane again leading to activation of capsases [93]. The
current two theory methodology for apoptosis include abortive apoptosis which
is the marking of defective germ cells during spermatogenesis, but instead of
apoptosis occurring, they are able to escape the testes. The second theory is
mature ejaculated spermatozoa are undergoing apoptosis or an apoptosis like
process; initially this was thought to have not occurred, but recent studies have
shown that ejaculated sperm are capable of triggering capsase activation [93].
Spermatozoa are exposed to a variety of physical and chemical stresses
during CP, changing the lipid composition of the plasma membrane, head size as
well as resulting in DNA damage [94] and increased plasma membrane lipid 44
Texas Tech University, Sossi M. Iacovides, December 2014
disorder[95] allowing the supposition that apoptotic like changes may be
induced in equine sperm CP [93].
A portion of the cell loss that occurs during CP is attributed to some cells
are already programmed to die are included in the freezing process and may
reduce the number of viable cells in an AI dosage. Moreover some of the more
subtle damage that is caused to sperm cells via CP may help to induce this
programmed cell death, and therefore lower viability numbers transferred
and/or reduced life span of those cells when in the female reproductive tract
[96]. The apoptotic phenomena [96] of cryopreserved stallions sperm is
attributed to oxidative stress, phase transitions of the plasma membranes,
cryocapacitation, as well as the premature activation of the pathway due to
subtle damages. Unfortunately, without extensive testing, there is no easy way
to determine a cell that has begun the apoptotic process. These defective cells
are programmed for removal, but unfortunately with only one pathway out, the
expulsion of dead cells occurs consistently within the ejaculate.
A cause of apoptosis normally overlooked during semen processing is the
presence of microbes in the semen sample [97]. Results from a more recent
study has shown that stallions ejaculate is more in line with that of humans due
to the bacteria which induce sperm apoptosis [98] and necrosis [99]; with the
microbial flora playing a critical role in the sublethal apoptotic damage that
stallion spermatozoa experience during CP and cooled storage [100].
A recent set of studies looked the activity of proteins, apoptosis and 45
Texas Tech University, Sossi M. Iacovides, December 2014
ROS, the proteins involved in the activation of apoptosis and the inductor protein involved in the activation of the mitochondrial pathway of apoptosis— have been found in fresh, frozen and thawed equine spermatozoa [101, 102].
Together these studies support the idea that ejaculated spermatozoa can trigger activation the nuclear matrix potentially leading to cleavage of the entire sperm
DNA into small fragments [103]. There is still controversy about the apoptotic markers that have been found in equine semen subpopulations and if this information is actually a significant, subsequent information collected post CP would need to be done for analysis for equine semen[80].
Further, depending on training of individuals involved, counts may include no sperm cells such as residual bodies. Residual bodies are made from cytoplasmic portions of elongated spermatids [96], and are subsequently shed with viable sperm cells into the seminiferous tubules, and therefore into the ejaculate.
Osmotic shock (OS) has long been associated with and a major factor in sperm damage during cryopreservation; and while this statement still holds true, newer research demonstrates it is just one potential problem. The influx of hypertonic concentrations while freezing, and the hypotonic concentrations when thawing have been shown to induce OS which has been shown to be detrimental to the integrity of sperm cells. Somatic cells have bene well documented to show that OS is responsible for apoptosis, cell cycle arrest, DNA damage, oxidative stress as well as a variety of other actions [104] 46 Texas Tech University, Sossi M. Iacovides, December 2014
This is especially true in stallions, as spermatozoa have a very limited
osmotic threshold [45] which ultimately results in uncontrollable shrinking and
swelling of the sperm head causing damage to the semen. Studies have shown
that stallion sperm damaged during flash freezing and morphologically abnormal
sperm generate greater amounts of ROS [61]. While the fluidity of the
plasmalemma is able to tolerate and adjust to these changes without penalty, OS
may lead to a loss in viability, poor motility, and/or a decrease in the
mitochondrial membrane potential [105].
Peroxidation of plasma membrane lipids (lipid peroxidation, LPO) has
been proposed to be a major factor involved in sublethal cryodamage of sperm
in many species, including horses [61]. Two pathways may result in the
formation of LPO’s; the enzymatic membrane system using NAD(P)H as a
substrate and the mitochondrial electron transport chain. The main source of
oxidative stress for spermatozoa is the mitochondria [106]. ROS production is
increased in sperm mitochondria due to freezing and thawing, while an osmotic
mechanism may increase mitochondrial membrane permeability, thus activating
apoptosis. In fact, oxidative stress is a well-documented inductor of apoptosis
[61].
The concept of relative osmotic stress to the hyper and hypotonic
environments has been shown to define the range, which may represent the
“osmotic tolerance limit.” Once these limits are surpassed, they cause
irreversible damage to the cell, preventing the spermatozoa from 47
Texas Tech University, Sossi M. Iacovides, December 2014 recovering its initial motility when returned to isosmolality environment.
Pommer et al. suggested this hypertonic limit for motility was reached at 450 mOsm/kg [107] while Garcia et al. demonstrated a hypertonic effect at 1500 mOsm/kg, which prevented stallion spermatozoa from recovering their initial volume once osmotic balance was restored [104].
Future of Semen Cryopreservation
As should be apparent from the forgoing material, CP of equine semen is desired by the industry as a means of long-term preservation and storage of superior genetics. While the vast majority of the research has focused on CPO’s, there remains a need for a simpler device that is able to provide the same quality results, which we are currently obtaining from the programmable freezers.
Equine CP, has be something which has been more than problematic for the industry, and just within the past ten years we have broken through to new methods which are allowing us to achieve the idea of a superior yet simple device. Programmable freezers using electricity have been the detriment to stallions, and the poor post thaw recovery rates generated from vertical mist have pushed the industry forward. The reduced quality of semen post-thaw is a clear response to the sub-optimal cryopreservation protocols that are in use, as the majority of cellular damage has been reported to occur between in the initial freezing stages [108]. Ideally an optimal freezing rate must be slow enough to prevent intracellular ice formation, but fast enough to avoid cryoinjury [108].
The necessity for a simpler device has been acknowledged and the equine 48 Texas Tech University, Sossi M. Iacovides, December 2014 reproductive industry has begun to explore alternative options. With sperm being susceptible to rapid cold shock injuries especially during the initial process and leading to membrane damage, the goal of this study was to slowly yet effectively control and decrease the temperature [109].
49 Texas Tech University, Sossi M. Iacovides, December 2014
CHAPTER II
CRYOPRESERVATION OF EQUINE SEMEN USING A MECHANICAL CONTROL RATE FREEZER
Introduction
As described earlier, there are only two current methods for freezing equine semen; mist freezing (MF) or an electronic controlled rate freezer (ECRF).
While mist freezing is both cheap and readily available to all facilities, the most efficient method is the ECRF; which requires electricity, and a large amount of liquid nitrogen. Because of these requirements, the ECRF is not easily moved.
Further it’s cost makes it prohibitively expensive for most smaller equine facilities, meaning to use the device they must ship either semen or horses to a centralized facility that can afford the equipment; risking either semen quality or the expense and possible injury to shipping animals. Therefore there is an industry need for a device with the ease and expense of MF but yields the results of the ECRF. For the past few years, this laboratory has been working on a mechanical controlled rate freezer (MCRF) unit. The device, which has been tested in cattle, appears to yield similar result to the ECRH[110].
The concept of the MCRF is currently under intellectual property review and details of the system cannot be discussed at the present time. However the concept is to use a non-electronic system to mimic the controlled temperatures seen in the ECRF. As freezing curves for horse semen in the ECRF are significantly different from those seen in cattle, the first step was to do a modification to 50 Texas Tech University, Sossi M. Iacovides, December 2014 the MCRF to adjust its nitrogen flow (thus its freezing rates) to that necessary for horse semen.
A series of adjustments were made to the MCRF designed to either increase or decrease the flow rate of liquid nitrogen through the system. The idea was to find a combination of nitrogen flow, which would decrease the temperature at a rate of approximately 4oC/10 sec. To test the modifications, straws loaded with a standard cryoprotectant, and temperature probes, and the probes were placed in four positions across the MCRF freezing chamber (right outside, right inside, left inside, left outside) to ensure consistency in the freezing rate. Nitrogen was then placed in the system (approximately 3 liters) and the freezing curves for each modification determined. The most crucial time points and temperature in cryopreservation of equine semen occur as the semen sample passes from 5oC to -40oC, during which semen pass through a crucial stage which can either result in membrane damage or successfully allow them to be exposed to further decreases in temperatures. Initial trials of the system approached the proper curves but never reached the exact curve desired as the sample frozen too fast or were slow to pass through the transition phase
(flashing from liquid to ice) and froze too slowly. The MCRF was then redesigned with modifications which allowed for mechanical seeding of ice crystals in the straws. Mechanical seeding uses a super cooled object to trigger ice crystal formation at one end of a straw[111] once it has reached a critical temperature in the freezing process (in the horse approximately -10oC). Once the seeding 51 Texas Tech University, Sossi M. Iacovides, December 2014 step was added, the freezing curve was within the desired range and further experimentation with semen samples could proceed.
Semen Collection & Preparation
For this trial, semen from 15 different stallions was obtained from the same facility using a phantom dummy and a Missouri AV, and prepared accordingly as per the facility standard. The collection procedures were part of the facility’s routine practice and did not require any additional specific handling for the research and were thus IACUC waived. Once collected, each sample underwent an on-site raw semen analysis using the computer assisted semen analyzer (CASA) (IVOS, Hamilton Thorn; Beverly, MA) and was then fresh extended at a 1:1 ratio with the INRA-96T (IMV Technologies) semen extender.
INRA-96T, commonly known as the “French extender,” contains purified milk micellular proteins, penicillin, gentamycin, amphotericin B, and Timectin®. This composition has been proven to be beneficial to the overall protection of sperm cells. Initial chilling ranging from 13-17oC began at the ranch with ice packs and transport Styrofoam® container and this range was maintained during transportation back to the central lab facility at Texas Tech University Health
Sciences Center, Lubbock, TX (an approximate 1 hr trip). Once back in the laboratory, each sample underwent a second semen analysis, using the CASA for base concentration and motility numbers, these were the base parameters we used for all future calculations.
Once the baseline analysis was completed, samples were prepared 52 Texas Tech University, Sossi M. Iacovides, December 2014
for freezing using the method for the cryoprotectant Botu-Crio® (State University
of São Paulo, Brazil) designed to result in a recommended post thaw
concentration of 200-400M/mL. In brief, samples were first centrifuged for 10
minutes, at 5000 revolutions/min in a static temperature of 5o to create a button
on the bottom of the 50mL polyproplylene conical tubes(BD Falcon) allowing
removal the excess extender and seminal plasma and increasing the
concentration per milliliter of volume prior to addition of the cryoprotectant.
Seminal fluid was then added back to the button to produce a sample
with a calculated to be twice that recommended by the cryoprotectant
manufacturer. As ideally, the experiments required 45 - .5 mL straws of semen
(103 straws were removed from the trial, due to unforeseen issues), 13 mL of
the processed semen was mixed with 13 mL of the cryoprotectant Botu-Crio®,
for a total volume of 26mL. Once the semen and Botu-Crio® were mixed, the
straw filler system from Animal Reproduction Systems (Animal Reproduction
Systems, Inc.; Chino CA) was used in conjunction with 5 straw adapter (ARS) to
load each set. After loading, the straws were heat sealed and refrigerated for 30
minutes prior to freezing.
Semen Freezing
The straws were then randomized into three groups according to the
type of freezing method, which they were to undergo: vertical mist (VM), ECRF
or the experimental MCRF. Straws were labelled as to treatment and then frozen
as follows: 53
Texas Tech University, Sossi M. Iacovides, December 2014
1. Straws frozen by VM were put into prechilled canes, and inserted into goblets, where they were suspended in mist of liquid nitrogen for 30 minutes and then plunged into the liquid nitrogen of the cryotank for storage.
2. Straws frozen in the ECRF: using the Planar system (TS Scientific, Perkasie, PA) was used, it had been preset to cool and freeze the semen at an appropriate speed and rate and predetermined by the industry as being the most beneficial.
The preset program dropped the temperature from 6 degrees to -196 by pulsing liquid nitrogen through its chambers, once the samples were completely frozen, they were transferred to a cryotank for storage.
3. Straws frozen in the inexpensive MCRF were placed on a holding rack in the device. A minimal amount of liquid nitrogen was added to the fill chamber (3L), and was delivered to the specimen chamber at a controlled rate of speed. Once frozen, the straws were also transferred to the cryotank for storage.
Semen Thawing
The samples remained frozen a minimum of two weeks prior to thawing and post-thaw analysis. On the day of thaw a transport Styrofoam® container was filled with liquid nitrogen, and straws from the three treatments were selected randomly, ensuring a cross-section of treatments being thawed at any one time.
Initial thaw attempts with the two prescribed thawing methods for Botu-
o o Crio® (thaw at 46 C for 20 seconds for optimum results or 37 C for 1 minute,
54 Texas Tech University, Sossi M. Iacovides, December 2014 using a water bath[112]) yielded suboptimal results. Observation suggested the straws were not fully thawed and therefore the procedure was modified to remove from the nitrogen and exposure to room temperature air (21-23oC) for
o 30 seconds and then plunging and holding the sample in a 37 C water bath for 2 minutes and 30 seconds. This modification yields significantly higher post-thaw motility recover and was used as the standardized procedure for the remainder of the experiment,
While the straws were thawing in the water bath, 7 mL borasiliate glass test tubes (Becton Dickerson; East Rutherford, NJ), were prepared with .5mL of warmed Ham’s F-10 media with TRIS buffers (Irvine Scientific; Santa Ana, CA).
After the thaw was complete, the straws were removed from the water bath, the heat-sealed end was cut off, and the contents of each straw were deposited into their respective test tubes.
Upon the contents being deposited, each tube was capped, and labeled with the correlating straw number. The sealed tubes were then centrifuged for 6 minutes at 1200 rpm. When the centrifugation was completed, the tubes were carefully removed so as to not disturb the semen button on the bottoms. Using
3mL transfer pipettes, all extraneous liquid (Botu-Crio®, initial .5mL Ham’s-F10) was removed to remove all exogenous cryoprotectants and a new .5mL aliquot of warmed Ham’s-F10 was added. The tubes were inverted to mix the semen back into solution, and loaded into a 37oC test tube warmer.
55 Texas Tech University, Sossi M. Iacovides, December 2014
Using a 3μl pipette, (Fisher Scientific; Waltham, MA), a sample from each test tube was placed on a Leja® 20 micron slide (Leja; Nieuw-Vennep,
Netherlands), where the sample was air-wicked across the plate via the internal vents to produce a sample of known volume and equal density across the slide.
Once the slide was prepared, it was loaded into the IVOS, where a 0hr time point analysis of 200 cells was completed. The analysis included: concentration, motility, rapid cell progression, path velocity, track speed, lateral movement, forward progression, linearity, beat frequency and elongation.
In addition to the IVOS data, slides were prepared for later morphology and acrosome analysis. Each slide was labeled with the straw ID, time point, and
M for morphology, and A for acrosome. Using the 3μl pipette, an aliquot was placed on to two different glass slides (Fisher Scientific). Using a third slide, the drop was smeared across the morphology slide. Preparation of the acrosome slide included the addition of 3μl of 3% formalin solution, which was mixed into the semen drop prior to the smear. The slides were moved to a drying area, where they remained for a minimum of 12 hrs, after which they were stored in slide boxes at room temperature until further use. The remaining sample was then transferred to a 37oC, 95% relative humidity incubator (Forma Instruments;
Marietta, OH) until the 3hr time point where the data collection process and slide preparation was repeated.
56 Texas Tech University, Sossi M. Iacovides, December 2014
Semen Morphology & Acrosomes
Morphology slides were stained using a standard DIF quick (hematoxylin and eosin) staining kit (Origio; Mt. Laurel, NJ), following the manufacturer’s staining instruction. To ensure proper staining, the stains were replaced every
100 slides. The slides were then allowed to dry a minimum of 24 hrs prior to examination. A trained observer on a Lietz (Leica Microsystems, Buffalo
Grove, IL) microsope with a ProgRes C14 Plus Camera (Jentopik; Jena, Germany) analyzed the slides using the 100X oil emersion lens. One hundred cells were observed per slide and categorized as having normal or abnormal morphology.
Cells classified as abnormal were then subcategorized for head abnormalities, midpiece abnormalities, curled tails (as a sign of freeze/thaw induced damage), and other tail abnormalities.
The state of the samples’ acrosomes at the 0 and 3 hr time points were assessed using a chlortetracycline staining technique to evaluate capacitation and acrosome like reactions [109]. In brief, a saturated solution of chlortetracycline was prepared by placing mixing approximately 5 grams of chlortetracycline with 30 mL of a phosphate buffer saline solution (PBS; Irvine).
The saturated solution was the filtered to remove particulate matter and the solution stored in a light tight container. Covering the semen smear with the chlortetracycline solution for a period of 1 minute, then rinsing the excess chlortetracycline solution off with cold PBS stained slides. Slides were allowed to dry prior to examination. Because of the light sensitive nature of the 57 Texas Tech University, Sossi M. Iacovides, December 2014 chlortetracycline solution all staining and subsequent examination of the slides was performed in a darkened room.
The slides were examined using the 100X lens on the Lietz microscope and fluorescence with the proper excitation and barrier filters. Because the chlortetracycline interacts with the acrosomal membrane and stains it more intensely then other membranes of the cell, it was possible to judge cells as acrosomally intact, partially intact, or non-intact. Using the scope’s video system,
100 cells/slide were assessed and graded for acrosome intactness.
Statistical Analysis
Resulting data were analyzed using the Statistical Package for the Social
Sciences (SPSS ver. 12; Chicago, IL). All parameters were first compared in a two- way analysis of variance (ANOVA) comparing treatment and time, and a treat by time interaction. The data were considered significantly difference for values of
P < 0.05. If the initial analysis was significant, the data was reanalyzed within a time point (0 or 3 hr) using a one-way ANOVA and Tukey’s mean separation as appropriate.
Results
A total of 15 ejaculates where obtained from 15 quarter horse stallions as part of the routine collection schedule at a production facility. In order to meet study requirement the sample had to have extended volume of 60 -100mL, allowed the creation of a minimum of 45 straws per animal (n=675). As expected the cryopreserved/thawed semen had significantly lower parameters than 58 Texas Tech University, Sossi M. Iacovides, December 2014 that seen in the fresh unextended sample (p < 0.001). However, at least two fresh treatments returned motile cells from straws in every stallion (ECRF and
MCRF). By design, semen was examined for concentration, motility, rapid cell progression, morphology and acrosomes at thaw (0 hrs) and 3 hours later. The results were compared between treatments at the separate time points and within treatment across the two time points. Results were considered different if the p-value was less than 0.05.
59 Texas Tech University, Sossi M. Iacovides, December 2014
While there appeared to be more variability in the concentration of semen in straws frozen in the MCRF (Figure 1), as expected by the design of the study, concentration was similar between the three treatments at 0 hrs (MCRF 152.44±
5.33, ECRF - 162.42± 5.51 and VM - 152.28± 5.58; p< 0.16). Further the concentration remained stable over the 3 hr post-thaw period (MCRF 144.76 ±
5.28, ECRF - 154.32 ± 5.49 and VM - 148.50 154.32 ± 5.49; p< 0.14).
P < 0.14 0 HR 3 HR 165
158.75
152.5
146.25 Concentra on M/mL 140
133.75 MCRF ECRF VM Device Used By Time
Figure 1: A Comparison recovered concentrations immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF), No differences were seen between treatments (p< 0.16) or time of measurement (p< 0.14).
60 Texas Tech University, Sossi M. Iacovides, December 2014
Inherently, while both the MCRF and ECRF had higher numerical values motility than the VM at the 0 hr time point (19.78 ± 1.83, 18.32 ± 1.89, and 16.13 ± 1.92, respectively), they were statistically similar (p< 0.29). As expected, sperm from all three methods demonstrated decreases in motility over the 3 hr period (16.05
± 1.81, 17.03 ± 1.89 and 15.96 ± 1.90, respectively. However, the drops were not significant (p< 0.54).
P < 0.29 20 0 HR 3 HR
15
10 Sperm Mo lity %
5
0 MCRF ECRF VM Device Used By Time
Figure 2: A comparison on recovered motility immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF), No differences were seen between treatments (p< 0.54 or time of measurement (p< 0.29).
61 Texas Tech University, Sossi M. Iacovides, December 2014
Rapid cell movement is thought to be an indication of those cells which have the retained the most overall activity and therefore are the most likely to be involved in fertilization. While there was no difference between treatments, at the 0hr (MCRF - 8.48 ± 9. 33, ECRF - 8.79 ± 9.20, and VM - 7.88 ± 7.75) the 3hr time points (MCRF – 6.69 ± 7.75, ECRF - 7.69 ± 8.61, and VM - 7.17 ± 7.55) there was a clear drop in the rapid cell progression in cells frozen in each treatment
(p> 0.02).
11.25 P > 0.02 0 HR 3 HR
A, X 9 A, X B, X A, X B, X B, X 6.75
4.5 Rapid Cells M/mL
2.25
0 MCRF ECRF VM Device by Time
Figure 3: A comparison of recovered rapid cell progression immediately post- thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). No difference was seen between treatments (p< 0.47) or but there was difference seen between time of measurement (p> 0. 02), and denoted by (A,X) and (B,X).
62 Texas Tech University, Sossi M. Iacovides, December 2014
The CASA system also yielded two sets of additional parameters to assess sperm movement. The first set was speed based and consisted of: path velocity, progressive velocity and track speed. All measurements were in micrometers/second (μm/s), examining various aspects of sperm cells forward cell movement. Lateral amplitude was also analyzed in micrometers (μm) to determine the rate of sidewise movement. While there was no statistical difference treatments at either treatment (p< 0.808, but by time (p> 0.050), there were some interesting trends in the data. Both path velocity and track speed trended toward increasing speeds in cells frozen in the ECRF and VM while decreasing in the new MCRF. A study by Mortimer and Mortimer[113] suggested these parameters increase with capacitation. Further, previous studies
[62] suggested that freezing damage can lead to a premature capacitation. With cells frozen in the MCRF trending toward lower forward movement speeds, it is possible the method might not cause the damage leading to premature capacitation.
63 Texas Tech University, Sossi M. Iacovides, December 2014
Table 1: With path velocity we do not see any statistical significance, as none of these treatments have any differences with the path velocity [114] total distance traveled per second (p< 0.23), and time points are not significant as well with p< 0.54. Progressive velocity [114] was not biologically significant with a p< 0.75 for treatment and p< 0.25 for time; showing the VM anomaly again in relation to motility being retained. Track speed [114] showed no significance between treatment (p< 0.15) and time (p< 0.44). Lateral amplitude [114] was the only intermediate speed parameter close to significance showing a treatment p< 0.81 that was negligible, but time (p> 0.05), showing a potential for difference amongst movement.
Path Velocity Parameter 0 HR 3 HR % Change P Value (um/s) Time: 0.54 MCRF 57.20 55.07 -3.72 Treatment ECRF 57.18 57.46 0.48 P Value Trt: VM 57.23 62.43 9.07 0.23
Progressiveness Parameter 0 HR 3 HR % Change P Value (μm/s) Time: 0.25 MCRF 40.84 38.40 -6.13 Treatment ECRF 39.85 39.14 -1.79 P Value Trt: VM 40.35 40.28 -0.16 0.75
Track Speed Parameter 0 HR 3 HR % Change P Value (μm/s) Time: 0.44 MCRF 109.75 106.15 -3.92 Treatment ECRF 110.65 120.42 8.84 P Value Trt: VM 111.90 113.40 1.34 0.15
Lateral P Value Parameter 0 HR 3 HR % Change Movement (μm) Time: 0.05 MCRF 6.707 7.579 12.99 Treatment ECRF 6.658 7.289 9.49 P Value Trt: VM 6.969 6.954 -0.21 0.81
64 Texas Tech University, Sossi M. Iacovides, December 2014
The second set of parameters generated by the CASA, describe cell movement
and include: elongation [114], linearity[114], straightness[114] which were
analyzed by (%) and beat frequency by hertz (Hz) [114]. As seen with the forward
progression parameters, there appeared to be no difference between
treatments (p< 0.06) or within treatments across the two time points (p< 0.38).
However, those parameters that might be indicative of premature capacitation
(elongation and beat frequency)[113] again treaded higher in the VM and ECRF
with being slightly decreased in the new MCRF; further suggesting the MCRF
might induce less stress during the freezing process.
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Table 2: Movement Classifications Elongation showed no difference between time (p< 0.38) and treatment (p< 0.14). The linearity model, showed no biological significance at time (p< 0.38) or at treatment (p< 0.83). With straightness we expected to see a difference between treatment, but the data did not corroborate with this as there was no biological statistical significance for treatment p< 0.49 or time p< 0.39. Beat frequency was the only parameter of this set that showed potential for a difference between treatments, p< 0.06, but clearly there was not between time (p< 0.39).
Parameter Elongation (%) 0 HR 3 HR % Change P Value MCRF 52.01 51.19 -1.57 Time: 0.38 Treatment ECRF 54.65 55.71 1.94 P Value Trt: VM 52.52 56.46 7.51 0.14
Parameter Linearity (%) 0 HR 3 HR % Change P Value MCRF 37.267 36.393 -2.34 Time: 0.38 Treatment ECRF 37.018 35.500 -4.10 P Value Trt: VM 36.367 36.722 0.98 0.83
Parameter Straightness (%) 0 HR 3 HR % Change P Value MCRF 61.49 59.58 -3.12 Time: 0.39 Treatment ECRF 62.41 60.99 -2.27 P Value Trt: VM 61.69 62.25 0.91 0.49
Beat Frequency 0 HR 3 HR P Value Parameter (Hz) % Change Time: 0.39 MCRF 32.39 32.22 -0.51 Treatment ECRF 33.56 33.88 0.95 P Value Trt: VM 33.50 35.21 5.09 0.06
66 Texas Tech University, Sossi M. Iacovides, December 2014
Normal morphology has long been recognized as a critical component of
reproductive success. Further, disruptive changed in morphology due to
cryopreservation have been well documented. In the present experiment, while
there was no difference between treatment (p< 0.81) for 0 hr (MCRF - 89.74 ±
5.07, ECRF - 90.33 ± 5.38, VM - 90.27 ± 4.48) or the 3 hr (MCRF - 89.49 ± 4.38,
ECRF - 89.36 ± 4.29, VM - 89.24 ± 4.40), there was difference between the time
points for ECRF and VM (p> 0.01; Figure 4). These data appear to support the
findings of Pukazhenthi [26, 27], who discussed damage to the sperm cells at the
3hr time point using Botu-Crio® in the tapir, as well as decreasing motility at
3hr.
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Texas Tech University, Sossi M. Iacovides, December 2014
P > 0.01 0 HR 3 HR 90.5 A, X A, X
90.125
89.75 A, X
B, X B, X 89.375 B, X
Normal Morpholgy % 89
88.625
88.25 MCRF ECRF VM Device by Time
Figure 4: A comparison of recovered normal morphology immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (VM), no difference was seen between treatments (p< 0.81) but there was biological significance seen between time of measurement (p> 0. 01), and denoted by (A,X) and (B,X).
68 Texas Tech University, Sossi M. Iacovides, December 2014
Focuisng specifically on the abnormalites, it was expected that any freezing damage was most like to manafest itself as changes in tail morphology.
However, while there were only a few head defects in any treatment, (0 hr: 7.29
± 3.35, 6.60 ± 3.54, and 7.10 ± 3.56; 3 hr: 7.45 ± 3.52, 8.01 ± 7.46, and 8.01±
3.75, MCRF, ECRF and VM respectively), These defects increased with time (p>
0.003: Figure 5) in the ECRF and VM. These findings, which were mainly an enlargement and rounding of the head shape, might indicate membrane damage and suggest a possible mechanism for the damage, seen by Pukazhenthi[26, 27] in relation to the use of the Botu-Crio® CPO. However, the differences were small, and there was no statistical difference between treatments at either time point (p< 0.75).
69 Texas Tech University, Sossi M. Iacovides, December 2014
P > 0.003 0 HR 3 HR 9 A, Y 8.25 A, Y A, X A, Y 7.5 A, X
6.75 A, X 6
5.25
4.5 3.75 3
Abnormal Head Shape % 2.25
1.5 0.75 0 MCRF ECRF VM Device by Time
Figure 5: A comparison of recovered abnormal head morphology immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). No difference was seen between treatments (p< 0.75) or but there was difference seen between time of measurement (p> 0. 003), and denoted by (A,X) and (B,X).
70
Texas Tech University, Sossi M. Iacovides, December 2014
Evaluation of abnormal midpiece demonstrated no biological or statistically significance changes by treatment (0 hr: 2.47 ± 2.37, 2.34 ± 2.45, and 2.24 ±
2.10; 3 hr: 2.58 ± 2.06, 2.71 ± 2.46, and 2.48 ± 2.04; MCRF, ECRF and VM respectively; p< 0.61) or time (p< 0.12). This might be expected, as most midpiece deformities would occur during spermatogenesis, and not during freezing.
P < 0.12 3.5 0 HR 3 HR
2.8
2.1
1.4 Abnormal Mipiece %
0.7
0 MCRF ECRF VM Device by Time
Figure 6: A comparison of recovered abnormal midpiece morphology immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). No difference was seen between treatments (p< 0.61) or time of measurement (p< 0.12).
71 Texas Tech University, Sossi M. Iacovides, December 2014
Abnormal tail defects were examined in two different ways. As curling of tails
has directly been associates with osmotic stress and such stresses have been
reported by a number of sources in cryopreserved sperm, that effect was looked
at separately (see below). All other abnormal tail morphology was examined
together. As it could be assumed that cells in all straws started with roughly the
same level of cells with abnormal tail morphology, then any differences seen
would have to be the result of freezing technique (treatment). While the overall
differences were small (range from .9 -1.8 % of the total sperm population) there
were significant differences (p> 0.001) between the treatments at both time
points (0 hr: 1.52 ± 1.75, 1.80 ± 1.71, and 1.09 ± 1.06; 3 hr: 1.52 ± 1.41, 1.58 ±
1.60, and 1.15 ± 0.92; MCRF, ECRF and VM respectively; Figure 7). Further the
differences remained stable and there were no observed differences between
the 0 and 3 hr time points for time (p< 0.69), but there was a difference by
treatments (p> 0.001; Figure 7). However, while these results suggest difference
rates of cellular damage between the treatments, this was not reflect in the
number of cells seen with curled tails (a gross expression of osmotic shock)
where less that 1% of cells in any treatment exhibited the curled tail effect at
either time point (p< 0.51) or treatment (p< 0.49) (0 hr: 0.41 ± .85, .03 ± .61, and
0.39 ± .80; 3 hr: 0.46 ± .67, .42 ± .79, and 0.33 ± .58; MCRF, ECRF and VM
respectively; Figure 8).
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Texas Tech University, Sossi M. Iacovides, December 2014
P > 0.001 2 0 HR 3 HR
B, X
A, X B, X 1.6 A, X
C, X 1.2 C, X
Abnormal Tail % 0.8
0.4
0 MCRF ECRF VM Device by Time
Figure 7: A comparison of recovered other abnormal tail morphology immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). A difference was seen between treatments (p> 0.001) denoted by (A,X) (B,X) and (C,X), but there was no difference seen between time of measurement (p< 0.69), and denoted by (A,X) and (B,X).
73 Texas Tech University, Sossi M. Iacovides, December 2014
0.6 P < 0.49 0 HR 3 HR
0.48
0.36
Curled Tail % 0.24
0.12
0 MCRF ECRF VM Device by Time
Figure 8: A comparison of recovered curled tail abnormal morphology immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). No difference was seen between treatments (p< 0.49) or between times of measurement (p< 0.58).
74 Texas Tech University, Sossi M. Iacovides, December 2014
In order to be truly viable, sperm cells must remain progressively motile and possess the biochemistry necessary to fertilize an egg. If cells go through the biochemical reactions too quickly, and capacitate and/or go through the acrosome reaction outside of the female body, they will not be able to fertilize an oocyte once introduced to the female tract. Data from the present study suggest a difference in acrosome intactness between the three treatment (0 hr:
69.88 ± 21.81, 64.35 ± 21.14, and 71.65 ± 19.33; 3 hr: 66.44 ± 23.29, 62.61 ±
22.48, and 69.79 ± 20.68; MCRF, ECRF and VM respectively; p> 0.001; Figure 9), but time also was close to significance (p< 0.08). Interesting the VM appeared to have sigificantly more acrosome cells that either the ECRF or the MCRF, a finding which would not appear to match the vast majority of the literature. However all three were within acceptable ranges. It is possible this finding was due to the
Botu-Crio®, which by design may have helped to stabilize the acrosomes. A similar pattern was seen cells with a partially intact acrosome (cells beginning the acrosome reaction). Not only did the numbers differ by treatment (0 hr:
19.32 ± 10.60, 22.91 ± 10.80, and 20.07 ± 10.68; 3 hr: 20.96 ± 10.86, 24.64 ±
10.59, and 20.57 ± 10.15; MCRF, ECRF and VM respectively; p> 0.001; Figure 10) but time as well (p= 0.05). Finally as would be expected, the number of cells going through the acrosome reaction (non-acrosome intact) increased with time
(0 hr, 13.49 ± 13.78, 15.13 ± 14.04. and 10.28 ± 10.79; 3 hr, 15.69 ± 16.08, 16.150
± 14.63, and 12.71 ± 14.07; MCRF, ECRF and VM respectively; p= 0.05; Figure 11).
Further, while limited in number (< 20% of cell population in any treatment), 75 Texas Tech University, Sossi M. Iacovides, December 2014
the number of reacted cells per treatment was a direct mirror image of the non-
reacted cells and was different between treatments (p> 0.002).
P > 0.001 0 HR 3 HR 72.5 C, X
A, X C, X 70
67.5 A, X
65 B, X
B, X Intact Acrosomes % 62.5
60
57.5 MCRF ECRF VM Device by Time
Figure 9: A comparison of recovered intact acrosomes immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). A difference was seen between treatments (p> 0.001) denoted by (A,X) (B,X) and (C,X), but there was no difference seen between time of measurement (p< 0.08).
76
Texas Tech University, Sossi M. Iacovides, December 2014 P > 0.001 31.25 0 HR 3 HR
B, Y 25 B, X A, Y C, Y C, X A, X 18.75
12.5 Par ally Intact Acrosomes %
6.25
0 IRF CRF VM Device by Time
Figure 10: A comparison of recovered partially intact acrosomes immediately post-thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). A difference was seen between treatments (p> 0.001) denoted by (A,X & A, Y) (B,X & B,Y) and (C,X & C, Y), and a difference seen between time of measurement (p= 0.05).
77 Texas Tech University, Sossi M. Iacovides, December 2014 P > 0.002 17 0 HR 3 HR B, Y A, Y B, X
A, X C, Y 12.75
C, X
8.5 Non- Inatact Acrosomes %
4.25
0 MCRF ECRF VM Device By Time
Figure 11: A comparison of recovered non-intact acrosomes immediately post- thaw and after 3 hrs of post-thaw incubation from cells frozen using an experimental mechanical controlled rate freezer, (MCRF) an electronic controlled rate freezer (ECRF) or vertical mist freezing (MF). A difference was seen between treatments (p> 0.002), and a difference seen between time of measurement (p= 0.05) denoted by (A,X & A, Y) (B,X & B,Y) and (C,X & C,Y).
78 Texas Tech University, Sossi M. Iacovides, December 2014
Given that we expect to see lower parameters over the course of time, the objective of this study was to determine if the MCRF could replicate current industry standard parameters set by the ECRF. Conclusively we are able to show that the MCRF was able to match the industry parameters set by the ECRF for not only morphology but intact acrosomes as well as decreasing the overall loss of acrosomes. The VM system was an anomaly is this trial, as it continuously demonstrated values, which would have not been expected, and will be discussed.
79 Texas Tech University, Sossi M. Iacovides, December 2014
Table 3: Quantitative comparisons of treatments. Shows the treatment ranking by parameter categories based off of percent change calculated (Appendix, ). The ranking system is based on (+) denoting a positive quality result by treatment; (0) denoting an average result; (-) denoting poor quality result. Applying Mortimer and Mortimers’ [113] sperm kinematics, sperm cells that are hyperactivated will have larger lateral movements, lower progressive velocity, lower straightness and a lower beat frequency[113]. With the objective of this study being to match the ECRF, the table below indicated that in many categories the MCRF has surpassed the acceptable margin, and provided better quality results.
Category MCRF ECRF VM FINAL Concentration (M/mL) 0 0 + VM Motility (%) - 0 + VM Rapid Cells (M/mL) - 0 + VM Path Velocity( μm/s) + 0 - MCRF Progressive Velocity (μm/s) + 0 - MCRF Track Speed (μm/s) + - 0 MCRF Lateral Movement (μm) - 0 + VM Elongation (%) + 0 - MCRF Linearity (%) 0 + - ECRF Straightness (%) + 0 - MCRF Beat Frequency (Hz) + 0 - MCRF Normal Morphology 0 0 0 ALL Abnormal Head Morph + - 0 MCRF Abnormal Midpiece Morph + - 0 MCRF Other Abnormal Tail Morph 0 + - ECRF Abnormal Curled Tail Morph 0 - + VM Intact Acrosome + 0 0 MCRF Partially Intact Acrosomes 0 0 + VM Non-Intact Acrosomes 0 + - ECRF
80 Texas Tech University, Sossi M. Iacovides, December 2014
Discussion
Artificial insemination with cryopreserved semen has revolution animal
breeding. But, stallions have been slow on the radar for a variety of reasons, the
first being that the individual variability of semen tolerance to the freezing and
thawing process. This added to the complex physiology of the mare's estrous
cycle has prevented wide spread use of these technologies to this point. While
Samper and Morris[115] illustrate the current difficulties in the successful
freezing and thawing of stallion semen, there is research on the horizon,
designed to improve recovery rates of motility, morphology, and intact
acrosome caps, so that upon insemination those sperm are actually viable and
able to fertilize the egg.
The present study describes results of working with a new mechanically
controlled rate freezer. While details of the device are under intellectual
property review and therefore cannot be presented in an open forum, the
system has been designed to bring the technology of freezing to the ranch or
farm and with reach of almost ever production facility. The goal of the study was
to mimic develop a system that would mimic the ECRF with the expense or
limiting requires of electricity and large nitrogen sources which make the ECRF
immobile. . In the current trials the MCRF successfully mimicked an ECRF;
producing samples with equal motility parameters, morphology and functional
acrosome numbers. Further the system has been shown reliably and able to
accurately reproduce freezing curves that have been shown effective in 81
Texas Tech University, Sossi M. Iacovides, December 2014 freezing stallion sperm. In addition we have been successful in our attempts to reduce osmotic shock during CP by reducing the concentration of glycerol and using alternative CPO’s has shown to be promising; with the idea of increasing permeability with the use of lower molecular weights protectants[61]. The present study has suggested that there may be merit in this approach, as all three devices tested returned reasonable semen parameters post-thaw results using the new CPO formula. As previously mentioned, the results for the VM were better than expected and somewhat an anomaly compared to earlier reports in the literature. This may be due to the formulation of the Botu-Crio®
CPO with its larger percentage of DMF to glycerol that includes a wider variety of amides. However, the data here suggests the new CPO, along with improves in freezing techniques, possibly including the new MCRF, and the implementation of improved diagnostic tools such as the flow cytometer and CASA in sperm evaluation, have, and will continue to improve, equine semen cryobiology[2].
82 Texas Tech University, Sossi M. Iacovides, December 2014
CHAPTER III
CONCLUSION
The freezing of equine semen has lagged behind other species for a number of reasons: 1) difficulties in retuning adequate number, 2) until recently moratorium on AI and embryo transfer and 3) the low concentration of males at most facilities making highly expensive CP equipment impractical for most facilities. Therefore the equine industry would be a major beneficiary of any new system that was inexpensive, potable and reliable. Currently, the most efficient method is a controlled rate electric freezer, which requires electricity, a large amount of liquid nitrogen, and therefore cannot be easily moved, which necessitates moving studs or semen to centralized facilities. Arguably, this must be a limiting factor in the expanded use of both semen CP and other ART’s in the field. The concept of the MCRF was to provide a system that was equivalent to the ECRF without the limitation, allowing it mobility and cost-effective use on all facilities. Data from the current study suggest that the MCRF was able to match the results from the industry standard of ECRF, which, if proven in breeding trials, would ultimately allow producers to have more direct access to devices capable of freezing stallion semen in a sufficient and protective manner.
The market value of equine genetics, maybe more than in any production setting, is driven by ‘the customer is always right’ mentality. In a number of species, CP has become a powerful force to save those highly sought after
83 Texas Tech University, Sossi M. Iacovides, December 2014 superior genetics from previous and current generations, and allowing the consumer to select for certain traits and characteristics, which makes the progeny more valuable to market. To achieve the same results in the equine industry, two major problems had to be overcome: (1 the unequivocal identification of superior sires and (2 the development of techniques to harvest and preserve which would assure optimal conception rates [116].
In 2004, the American Horse Council Foundation survey indicated that there were approximately 9.2 million horses in the United States, and it is estimated that the U.S. horse industry contributes approximately $101.5 billion to the country’s gross domestic product (GDP). Since its introduction over 30 years ago, artificial insemination within the horse breeding industry has grown steadily [25].Regardless of the current advancements that animal biotechnologists have made, there is always room for improvement. For most species (including humans) CP of semen has been a cornerstone to all other ART techniques, allowing the storage of male genetics until needed. In the past decade, several new ART’s have become available to the equine industry [61].
Lack of an accessible and reliable means of equine semen CP may become a limiting factor in future growth. As an example, semen CP is particularly important in facilitating the international dispersal of superior genetics. It is necessary to overcome the time delays caused by shipping regulations and to enable the long-term preservation of genetically sought after sires [61]. The ability to collect and preserve the heredities from certain animals, and to 84 Texas Tech University, Sossi M. Iacovides, December 2014 later use those same genetics into surrogates, is something that that other aspects of the livestock industry has capitalized on. This has allowed those industries to continue to breed a standard of animals that are serving societal needs more than ever before. To take advantage of global opportunities the equine industry also needs a means to successfully and cost effectively cryopreserve semen. Not only would it increase the outsourcing of genetics to other countries, it would also allow transport of more desirable bloodlines to the
United States more efficiently. Because of current limitations in equine semen
CP, genetic variation can be thought of as sequestered to respective locations.
Development of a system to overcome these limitations would allow the equine industry the chance to have the same advantages for improving herds by allowing the incorporate genetics from a worldwide source. The MCRF represents an attempt to bring such technology to the industry.
85 Texas Tech University, Sossi M. Iacovides, December 2014
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APPENDIX
Table 4: Percent retention of concentration, motility and rapid cell progression. Percent change was calculated, with red being the largest difference and blue denoting the smallest and therefore the preferred treatment method. Less than a 1% difference was considered the same, and not rated as a positive or negative effect per treatment.
Retained Retained % Retained % % Treatment Concentration Rapid Change Motility (%) Change Change % Cells (%)
MCRF 94.96 -5.04 81.15 -18.85 78.91 -21.09
ECRF 95.02 -4.98 95.16 -4.84 87.54 -12.46
VM 97.52 -2.48 98.96 -1.04 91.06 -8.94
96 Texas Tech University, Sossi M. Iacovides, December 2014
Table 5: Percent retention of morphology. Percent change was calculated, with red being the largest difference and blue denoting the smallest and therefore the preferred treatment method. Less than a 1% difference was considered the same, and not rated as a positive or negative effect per treatment.
Retained Retained Retained Retained Retained Treatment Abnormal Abnormal Abnormal Normal Curled Tail Head Midpiece Tail MCRF 99.72 102.11 104.17 100.09 114.00 ECRF 98.93 121.37 118.41 88.22 140.82 VM 98.86 112.87 110.70 105.74 84.46
Retained Retained Retained Retained Retained Normal Abnormal Abnormal Abnormal Curled Tail Treatment Head Midpiece Tail % Change % Change % Change % Change % Change MCRF -0.28 2.11 4.17 0.09 14.00 ECRF -1.07 21.37 18.41 -11.78 40.82 VM -1.14 12.87 10.70 5.74 -15.54
Table 6: Percent retention of acrosomes. Percent change was calculated, with red being the largest difference and blue denoting the smallest and therefore the preferred treatment method. Less than a 1% difference was considered the same, and not rated as a positive or negative effect per treatment.
Retained Retained Retained % % % Treatment Intact Partially Intact Non-Intact Change Change Change Acrosomes Acrosomes Acrosomes MCRF 95.07 -4.93 108.48 8.48 116.36 16.36 ECRF 97.29 -2.71 107.53 7.53 106.73 6.73 VM 97.40 -2.60 102.49 2.49 123.64 23.64
97