Cryopreservation of Equine Semen with a Mechanical Control Rate Freezer

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

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?!

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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

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!)

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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

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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

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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

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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.

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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

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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,
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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 20^thcentury, 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].
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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
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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 -70^oC, 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.
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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 (nonpentrating) 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].
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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
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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
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