Cryopreservation of Domestic Cat Epididymal Spermatozoa

Transcription

1 Louisin Stte University LSU Digitl Commons LSU Doctorl Disserttions Grdute School 2015 Cryopreservtion of Domestic Ct Epididyml Spermtozo Jesse Ry Senz Louisin Stte University nd Agriculturl nd Mechnicl College, Follow this nd dditionl works t: Prt of the Animl Sciences Commons Recommended Cittion Senz, Jesse Ry, "Cryopreservtion of Domestic Ct Epididyml Spermtozo" (2015). LSU Doctorl Disserttions This Disserttion is brought to you for free nd open ccess by the Grdute School t LSU Digitl Commons. It hs been ccepted for inclusion in LSU Doctorl Disserttions by n uthorized grdute school editor of LSU Digitl Commons. For more informtion, plese contct gcoste1@lsu.edu.

2 CRYOPRESERVATION OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA A Disserttion Submitted to the Grdute Fculty of the Louisin Stte University nd Agriculture nd Mechnicl College in prtil fulfillment of the requirements for the Degree of Doctor of Philosophy in The School of Animl Sciences by Jesse Ry Senz B.S., New Mexico Stte University, 2003 M.S., Louisin Stte University, 2007 My 2015

3 ACKNOWLEDGMENTS First I would like to thnk my two mjor professors Dr. Robert A. Godke nd Dr. Chrles E. Pope. Without their ptience, persistence nd finncil support this disserttion would not hve been possible. Also, I would like to thnk both Dr. Godke nd Dr. Pope for their dvice, mentorship, guidnce nd bove ll their friendship. I wnt to thnk Dr. Godke for continuing to work with me nd guide me while writing my disserttion even while he ws in retirement. Also, I hve to thnk Dr. Pope for llowing me to work in his lb nd providing me with priceless experience nd guidnce in the discipline of niml reproduction. Upon completing my PhD reserch from world renowned progrm I hve gined first clss eduction nd priceless experience in the field of niml reproduction. I need to give n individul nd very hertfelt thnk you to Mrs. Jckie Coulon Pope. Mrs. Jckie ws very instrumentl in getting me involved nd enrolled t Louisin Stte University in 2004 when I strted my Msters degree. Without Mrs. Jckie s contcts nd emils I most certinly would not be where I m t tody. I cnnot tlk bout my success in this progrm without mentioning the support nd guidnce I received from everybody tht mkes this progrm so successful. I would like to give specil thnks to my dditionl committee members (Dr. Stnley Leibo, Dr. Glen Gentry nd Dr. Collin Mitchell) for their suggestions on completing my experiments, s well s, their revisions on my disserttion. I would like to extend specil thnks to Sue Prestenbck, Dr. Rollie Norris nd Dr. Sophie Hudson for their help in sving nd collecting domestic ct testis nd ovries from their dily spys nd neuters t the Ark Animl Hospitl nd the New Orlens SPCA. I hve to give specil thnks to Sue for llowing me into her home nd helping me to experience true southern cooking nd hospitlity. Although she is now retired, I need to thnk Mrs. Slly Turner for lwys being willing to go out of her wy to help me send fx, send pckge, sign up for clsses. Also quick thnk you is due to Mrs. Cssndr for her willingness to print documents nd nswer question these lst few semesters. For their priceless friendships nd fmily like loylty I hve to thnk Dr. Mrvin Moncd, Dr. Crlos Guerrero, Irm M. Moncd nd Nthli Guerrero, long with their compnionship nd encourgement they mde completing this disserttion possible nd dy-to-dy life mngeble. Thnks to ll the fculty nd stff t the Audubon Center for Reserch of Endngered Speices (Dr. Mrth Gomez, Cherie Nobles, Json Gliguis, Anne Bullington, Monic Bincrdi ii

4 nd mny others) s well s fellow grdute student (Robin Powell). For ssistnce in completing this doctorl reserch s well s compny nd encourgement during the long work weeks nd lte nights. Thnk you ll so much Finlly I would like to thnk my loving mother nd fther (Mrth nd Rymond Senz) for the love, pryers nd finncil support tht hs lwys been there whenever I needed it, lso my sisters (Genevieve, Donnlis, Corin, Dell nd Rchel) my nieces nd nephews (Smnth, Eric, Victori, Rebecc, Gilbert, Aline, Smuel, Jcob, Isc, Johnny Ry, Nico, Rymond Roy, Len, Cody, Kyden nd Mily Re) for the never ending support nd concern. And lst but probbly most importnt I would like to give very big thnk you to my wife, my friend nd my bbies momm (Meliss P. Senz). I thnk you so very much for being so ptient, tolernt nd forgiving of me during this difficult journey. Most of ll I would like to thnk you for the birth of our first child Crson Edwrd Senz who truly hs brought n unexpected joy nd sense of pride into my life. Thnks to everyone tht hs helped me long the wy, nd to the friends I hve cquired there relly re too mny to mention. iii

5 TABLE OF CONTENTS ACKNOWLEDGMENTS.ii LIST OF TABLES...vi LIST OF FIGURES ix ABSTRACT...xiii CHAPTER I INTRODUCTION...1 CHAPTER II LITERATURE REVIEW... 4 Bsic Principles of Cooling nd Freezing Cells Historicl Perspective on Methods for Preserving Spermtozo...5 Erly Attempts t the Cryopreservtion of Spermtozo.6 Cryoprotectnts 7 Cool Storge of Spermtozo.10 Freeze-Drying of Spermtozo Semen Extenders Without Egg Yolk 16 Use of Bovine Serum Albumin (BSA) in Semen Extenders...16 Use of Low Density Lipoproteins (LDL) in Semen Extenders 18 Use of Soyben Extrct Lecithin in Semen Extenders 19 The Use of Pentoxifylline to Stimulte Sperm Motility...19 Feline Epididyml Spermtozo in Assisted Reproduction..20 Collection of Feline Epididyml Spermtozo..20 Comprison of Feline Ejculted nd Epididyml Spermtozo..21 Feline Sperm Assessments. 22 Epididyml Ct Spermtozo in Assisted Reproductive Techniques (ART)...24 CHAPTER III GENERAL EXPERIMENTAL PROCEDURES FOR ALL EXPERIMENTS..26 Tissue Collection nd Trnsport...26 Sperm Collection nd Designting Tretments.. 26 Sperm Anlysis 27 Cryopreservtion nd Thwing of Sperm Tretments In Vitro Production Medium (IVF/IVC).. 29 Oocyte Collection nd Mturtion.30 In Vitro Fertiliztion nd Culture Sttisticl Anlysis CHAPTER IV SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION IN EXTENDERS WITH OR WITHOUT EGG YOLK..32 Introduction...32 Mterils nd Methods Experimentl Design 33 Experimentl Procedures...34 Results..36 Experiment Discussion iv

6 CHAPTER V EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION 49 Introduction...49 Mterils nd Methods Experimentl Design 50 Experimentl Procedures 51 Results..51 Experiment Discussion CHAPTER VI EFFECT OF PENTOXIFYLLINE CONCENTRATIONS AND TIME OF EXPOSURE ON THE POST-THAW SURVIVAL OF DOMESTIC CAT EPIDIDYMAL SPERM...64 Introduction...64 Mterils nd Methods...65 Experimentl Design 65 Experimentl Procedures.66 Results..68 Experiment Experiment Discussion CHAPTER VII SURVIVAL OF DOMESTIC CAT EPIDIDYMAL SPERM AFTER TEMPORARY COOL STORAGE OR CRYOPRESERVATION IN DEFINED EXTENDERS 91 Introduction...91 Mterils nd Methods Experimentl Design 92 Experimentl Procedures 94 Results Experiment Experiment Experiment Discussion..121 CHAPTER VIII SUMMARY AND CONSLUSION..130 LITERATURE CITED.133 APPENDIX..156 VITA..159 v

7 Tble 2.1. Tble 4.1. Tble 4.2. Tble 4.3. Tble 4.4. Tble 5.1. LIST OF TABLES A list of some exotic mmmlin species in which pregnncies nd/live offspring hve been produced fter the use of rtificil insemintion procedures with cryopreserved spermtozo 8 Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of nine collection dys...37 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing...37 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing...41 Effect of egg yolk (Test) vs. BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing...41 Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of ten collection dys..53 Tble 5.2. Percentge of sperm motility (men±sem) before cooling, post-cooling (4 C), 0 hour nd 3 hour post-thw for ct epididyml sperm frozen in n extender contining either 2%, 5% or 10% egg yolk. 53 Tble 5.3. Tble 5.4. Tble 6.1. Tble 6.2. Percentge of sperm with intct membrnes (membrne integrity) (men±sem) before cooling, post-cooling (4 C), 0 hour nd 3 hour post-thw for ct epididyml sperm frozen in n extender contining either 2%, 5% or 10% egg yolk 57 Percentge of ct epididyml sperm with intct crosomes (crosoml sttus) (men±sem) before cooling, post-cooling (4 C), 0 hour nd 3 hour post-thw for ct epididyml sperm frozen in n extender contining either 2%, 5% or 10% egg yolk 57 Post-thw dilution tretments nd the formuls of how the tretments were constructed on ech dy the strws were thwed..67 Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of four collection dys...70 Tble 6.3. Percent motility (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline vi

8 Tble 6.4. Tble 6.5. Tble 6.6. Percent membrne integrity (men±sem) of initil, post-cool (4 C), 0 hour postthw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline..74 Percent crosome integrity (men±sem) of initil, post-cool (4 C), 0 hour postthw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline..74 Results for dte s epididyml sperm ws collected, the number of testes processed ech dy, the totl number of cells collected nd how mny strws were frozen per dy with their corresponding strw concentrtions. 80 Tble 6.7. Percent motility (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline...80 Tble 6.8. Tble 6.9. Tble 7.1. Tble 7.2. Tble 7.3. Tble 7.4. Tble 7.5. Percent intct membrnes (men±sem) of initil, post-cool (4 C), 0 hour postthw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline..84 Percent crosome integrity (men±sem) of initil, post-cool (4 C), 0 hour postthw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline..84 Experimentl design for experiment 7.2. This tble illustrtes wht redings were recorded nd t wht time point they were recorded for this experiment Experimentl design for experiment 7.2. This tble illustrtes wht redings were recorded nd t wht time point they were recorded for this experiment...95 Results for dte s epididyml sperm ws collected, the number of testes processed ech dy, the totl number of cells collected, nd how mny cells per tretment Percent motility (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders 98 Percent membrne integrity (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.101 vii

9 Tble 7.6. Tble 7.7. Tble 7.8. Tble 7.9. Tble Tble Tble Tble Tble Percent crosoml sttus (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.101 Dtes epididyml sperm ws collected/frozen, lso the number of testes processed ech dy the totl number of cells collected nd how mny strws were frozen per tretment with their corresponding strw concentrtions.108 Percent motility (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 2% egg yolk or BioXCell extenders 108 Percent membrne integrity (men±sem) of post-cool, 0 hour nd 3 hours postthw for pooled domestic ct epididyml sperm tht were extended nd frozen in 2% egg yolk or BioXCell extenders.111 Percent crosoml sttus (men±sem) of post-cool, 0 hour nd 3 hours postthw for pooled domestic ct epididyml sperm tht were extended nd frozen in 2% egg yolk or BioXCell extenders.111 Dtes epididyml sperm were collected/frozen. The number of testes processed ech dy the totl number of cells collected nd how mny strws were frozen per tretment with their corresponding strw concentrtion Percent motility (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 0 or 2% egg yolk extender; nd frozen from either room temperture or fter cooling to 4 C Percent membrne integrity (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 0 or 2% egg yolk extender; nd frozen from either room temperture or fter cooling to 4 C..122 Percent crosoml sttus (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 0 or 2% egg yolk extender; nd frozen from either room temperture or fter cooling to 4 C..122 viii

10 LIST OF FIGURES Figure 4.1. Figure 4.2. Figure 4.3. Figure 4.4. Figure 4.5. Figure 4.6. Figure 4.7. Figure 5.1. Figure 5.2. Figure 5.3. Figure 5.4. Figure 5.5. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing. 38 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing. 39 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.42 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.43 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.44 Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.46 IVF percentges when oocytes were fertilized with domestic ct epididyml sperm frozen in HSPM extender(tretment A) or TesT extender (Tretment C)..47 Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing. 54 Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing...55 Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing 58 Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing 59 Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.60 ix

11 Figure 5.6. Figure 5.7. Figure 6.1. Figure 6.2. Figure 6.3. Figure 6.4. Figure 6.5. Figure 6.6. Figure 6.7. Figure 6.8. Figure 6.9. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing 61 IVF percentges when oocytes were fertilized with domestic ct epididyml sperm frozen in TesT buffer extender contining 2% egg yolk or HSPM extender (from Experiment 4.1)...62 Percent motility (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours postthw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...71 Percent motility (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours postthw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution..72 Membrne intct percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...75 Membrne intct percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...76 Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...77 Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...78 Motility percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...81 Motility percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...82 Membrne integrity percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...85 x

12 Figure Figure Figure Figure 7.1. Figure 7.2. Figure 7.3. Figure 7.4. Figure 7.5. Figure 7.6. Figure 7.7. Figure 7.8. Figure 7.9. Membrne integrity percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...86 Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...87 Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution...88 Percent motility (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders..99 Percent motility (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders..100 Membrne intct percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.103 Membrne intct percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders..104 Acrosoml sttus percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.105 Acrosoml sttus percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.106 Percent motility (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders..109 Percent motility (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders..110 Membrne intct percentge (men±sem) of post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders..113 xi

13 Figure Figure Figure Figure Figure Figure Figure Figure Figure Membrne intct percentge (men±sem) of post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders..114 Acrosoml sttus percentge (men±sem) of post-cool (4 C), post-thw (PT), t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders..115 Acrosoml sttus percentge (men±sem) of post-cool (4 C), post-thw (PT), t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders..116 Percent motility (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed in n extender contining 0 or 2% egg yolk; nd frozen from either room temperture or fter cooling to 4 C..119 Percent motility (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed in n extender contining 0 or 2% egg yolk; nd frozen from either room temperture or fter cooling to 4 C..120 Membrne intct percentge (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed in n extender contining 0 or 2% egg yolk; nd frozen from either room temperture or fter cooling to 4 C.123 Membrne intct percentge (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed in n extender contining 0 or 2% egg yolk; nd frozen from either room temperture or fter cooling to 4 C.124 Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed in n extender contining 0 or 2% egg yolk; nd frozen from either room temperture or fter cooling to 4 C.125 Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), post-thw (PT) t 0 (h) nd 3 (h) for pooled domestic ct epididyml sperm tht were processed in n extender contining 0 or 2% egg yolk; nd frozen from either room temperture or fter cooling to 4 C.126 xii

14 ABSTRACT The primry lines of defense in preventing ny exotic species from becoming endngered or extinct should be the protection of their hbitt nd from poching. For wildlife conservtionists, these primry mesures re often not fesible, nd thus cnnot be the only mens to prevent extinction. Additionl steps to prevent species from becoming extinct re the use of ssisted reproductive techniques (ART). The objective of this reserch ws to identify semen extender tht contined little to no egg yolk nd could still effectively freeze domestic ct epididyml spermtozo, for the purpose of sex sorting, using flow cytometry. In the first experiment we showed no significnt difference mong ny of the tretments when mesuring membrne integrity or crosoml sttus. There ws significnt difference when compring the pre-cool motility vlue between the control nd the humn sperm preservtion medium extender (HSPM), but this difference ws not detected in either of the post-thw evlutions. In the second experiment, it ws determined tht even t the lowest concentrtion, 2% egg yolk ws not significntly different from the 10% or the 5% when compring motility, membrne integrity nd crosoml sttus. In the third series of experiments it ws determined tht there ws no difference in post-thw sperm motility, membrne integrity or crosoml sttus when the epididyml sperm were exposed to the pentoxifylline both before nd fter freezing. When spermtozo were exposed to pentoxifylline during post-thw only, the 1mM tretment, sperm hd significntly greter motility t the 3 hour post-thw ssessment time thn the control tretment. In the lst series of experiments gin, the inbility to detect difference between the extenders gives further evidence tht non-egg yolk extenders such s BioXCell mybe n effective replcement for the commonly used egg yolk-bsed extenders. Finlly there ws no difference between the two TesT extenders frozen fter cooling to 4 C for ny of the prmeters mesured. The TesT without egg yolk, frozen from room temperture, consistently hd lower sperm motility nd membrne integrity then sperm frozen in one or both of the extenders frozen fter cooling to 4 C. xiii

15 CHAPTER I INTRODUCTION The Interntionl Union for Conservtion of Nture (IUCN) clssifies plnts nd nimls into one of seven ctegories to designte their thret of extinction, which include: lest concerned, ner thretened, vulnerble, endngered, criticlly endngered, extinct in wild nd finlly, extinct. Of the 5,500 species of mmmls in the world, 13% re listed s endngered, criticlly endngered, extinct in the wild or extinct (IUCN 2012). A recent txonomy publiction lists s mny s 40 felid species (Wozencrft, 2005), while the IUCN lists 36, seven of which re listed s either endngered or criticlly endngered (Sunquist nd Sunquist, 2002; IUCN 2012). A potentil option of enhncing the propgtion of endngered species in the future is by the ppliction of ssisted reproductive technologies (ART). Artificil insemintion (AI), the lest invsive nd oldest of the ART methods, ws first reported in the dog by Lzzro Spllnzni in 1780 (see Senger, 1997). In vitro fertiliztion (IVF) nd the birth of offspring fter embryo trnsfer ws first reported in the rbbit (Chng, 1959). This occurred fter the importnce of spermtozo cpcittion hd been discovered erlier in the decde by Chng (1951) nd Austin (1951). Embryo trnsfer (ET) ws first ccomplished in the domestic rbbit in Englnd by Wlter Hepe (1891). Successful repetble cryopreservtion of spermtozo ws first reported in 1949, with the finding tht the ddition of glycerol to the extender solution llowed rooster spermtozo to survive the freezing process (Polge et l., 1949). The ART methods listed bove hve been used cross vrious exotic species. For exmple, Pope et l. (1984) reported the birth of live bboon following the trnsfer of n embryo tht hd been cryopreserved nd subsequently thwed. More thn decde lter, Pope et l. (1997) reported the birth of Western Lowlnd gorill resulting from the trnsfer of n embryo tht hd been produced using IVF. Artificil insemintion hs lso been successfully used to produce offspring in vrious exotic species. Exmples of some of the more difficult species where pregnncies nd prturition were finlly chieved fter decdes of effort include the elephnt (see review Hermes et l., 2007) nd more recently the rhinoceros (Hildebrndt et l., 2007). There re lso exmples of rtificil insemintion being used in exotic cnine nd feline species. Thomssen nd Frstd (2009) reported the birth of live pups when AI ws used to breed three Mexicn Gry wolves in One yer lter, As et l., (2006) reported the birth of live pups fter rtificilly inseminting single Gry wolf. Swnson et l. (1996) reported the 1

16 birth of single mle ocelot kitten fter femle ocelot ws lproscopiclly inseminted with frozen thwed semen. ART methods hve lso been used, with some success, in exotic ungulte species. Dresser et l. (1985) nd Pope et l., (1991), showed tht in vivo-produced embryos could be nonsurgiclly flushed nd nonsurgiclly trnsferred, in the bongo ntelope nd the Scimitr- Horned oryx, respectively, resulting in live helthy clves. Furthermore, Dresser et l. (1985) lso showed tht in vivo-produced bongo ntelope embryos could be trnsferred into elnd ntelope recipient (interspecies embryo trnsfer) resulting in live helthy bongo clf, which ws then rised by the elnd recipient. Over time, new methodologies hve been dded to the list of ssisted reproductive techniques. Promising exmples include intrcytoplsmic sperm injection (ICSI) nd sex sorted spermtozo. As before, the new techniques were first developed in the domestic species nd then were eventully ttempted in exotic species. When performing ICSI, single spermtozoon is physiclly microinjected into the cytoplsm of n oocyte to chive fertiliztion. This technique ws first described in the hmster, where the outcome ws the formtion of both mle nd femle pronuclei (Uehr nd Yngimchi, 1976). Ten yers lter, Mnn (1988) produced the first offspring using ICSI in the mouse. To our knowledge, no mmmlin exotic species hve been born from the ICSI procedure. To dte, there hve been only few reports of ICSI-derived embryos produced from exotic species. Pope et l. (1998) reported the trnsfer of 10 ICSI-produced Jgurundi embryos into domestic ct recipient but no kittens resulted from the trnsfer. Dmini et l. (2004) lso reported the use of ICSI to produce lion embryos using spermtozo spirted from the epididymis of vsectomized mle but gin, no offspring were born following trnsfer of the embryos to recipient femle fter they hd been frozen/thwed. Also, predetermined gender offspring hve now been produced using flow cytometry to seprte X-bering spermtozo from the Y-bering spermtozo prior to insemintion in both domestic nd exotic species (O Brin et l., 2009, Grner 2006). The first offspring produced from sex sorted spermtozo occurred >20 yers go fter sorted sperm were surgiclly inseminted in to the uterus of rbbit (Johnson et l., 1989). To dte, offspring from other domestic species hve been born including cttle (Crn et l., 1993), pigs (Johnson 1991), sheep (Ctt et l., 1996), horses (Buchnn et l., 2000), dogs (Meyers et l., 2008) nd cts (Pope et l., 2009). The gol in the present reserch experiment ws to determine suitble nonegg yolk, semi-trnsprent semen extender tht would effectively cryopreserve domestic ct epididyml 2

17 spermtozo. The hope is to eventully use flow cytometry to sort the X nd Y spermtozo in previously frozen ct sperm smples. Due to the opque coloring nd slight viscosity of egg yolk, semen smples extended nd/or frozen in n egg yolk-bsed extender must be wshed nd centrifuged to clrify the smple so tht spermtozo cn be sorted more effectively by flow cytometry. Accordingly the primry objective ws to use non-egg yolk-bsed extender to freeze feline sperm for lter use in the sex sorting procedure. The long term objective of our experiments ws to develop procedure for cryo-storge of feline sperm tht would llow for efficient gender sorting nd subsequent production of offspring of predetermined gender. 3

18 CHAPTER II LITERATURE REVIEW Bsic Principles of Cooling nd Freezing Cells The bility to preserve or suspend cells in perpetul frozen stte nd lter thw those cells unhrmed hs long been of interest to scientists. Mzur et l. (1972) hypothesized tht two fctors ffect the cryosurvivl of cells during the cooling, freezing nd thwing process: (1) intrcellulr nd extrcellulr exposure to precipitted nd concentrted solutes, ph chnges nd dehydrtion ll which occur during conversion of wter to ice nd, (2) formtion of intrcellulr ice during freezing nd recrystlliztion of the ice t thwing. Both of these fctors re believed to be dependent on cooling rte, nd my interct with ech other to influence the survivl of cells during freezing. This hypothesis generlly referred to s the two fctor hypothesis (freezing injury), is now widely recognized nd ccepted. The formtion of intrcellulr ice, which cuses substntil mount of cell dmge, is the primry problem (Ashin, 1965; Leibo nd Mzur, 1971; Merymn, 1974; Griffiths et l., 1979). Intrcellulr ice formtion is cused by fst cooling rte tht does not llow intrcellulr wter to exit the cell before freezing occurs. Furthermore, optiml cooling rtes differ mong cell types. The optiml cooling rte of lrge sphericl cells, which hve low surfce re to volume rtio, such s hmster oocytes (Griffiths et l., 1979) nd mrrow stem cell suspension (Leibo et l., 1970) is ~1 C/minute. In contrst, smll flt cells, such s erythrocytes or spermtozo tht hve high, surfce re to volume rtio, cooling rte of 100 C/minute is more effective nd less likely to llow the formtion of intrcellulr ice thn the slower rtes of ~1 C/minute. (Leibo et l., 1970; Miller nd Mzur 1976). More recently, it hs been suggested tht the formtion of intrcellulr ice during rpid cooling is not the primry cuse of sperm cell dmge, but rther it is the osmotic imblnce creted during thwing (Morris et l., 2007). Mzur nd Koshimoto (2002) reported tht the ctul formtion of intrcellulr ice in mouse spermtozo did not follow the models predicting tht intrcellulr ice should form only t cooling rtes of 1000 C/minute. They indicted tht intrcellulr ice occurred t cooling rtes of 130 to 261 C/minute. Cell injury nd/or deth during cryopreservtion is lso due to solution effect injury tht cuses dmge either by dehydrtion nd shrinkge or by exposure to high concentrtions of solutes (Lovelock, 1953). During slow cooling, intrcellulr nd extrcellulr wter is removed in the form of ice. While ice is being formed, wter remining becomes incresingly concentrted with solutes, usully electrolytes. Although the exct mechnism is not 4

19 understood, it is widely ccepted tht exposure to elevted concentrtions of slts is lethl to cells (Lovelock, 1953). Cell shrinkge nd dehydrtion lso hve been hypothesized to cuse dmge during slow cooling (Muldrew et l., 2004). It hs been reported tht cell dmge during freezing is not cused by shrinkge but by high intrcellulr concentrtions of electrolytes tht cn result in complete dehydrtion. Then, presumbly, t thwing, rehydrtion occurs so rpidly tht the cell membrne is lysed from over expnsion (Lovelock, 1953; Zde-Oppen, 1968; Frrnt nd Woolgr, 1972). Cryodmge cused by intrcellulr ice formtion nd slow cooling injury is reduced when glycerol, cryoprotective gent (CPA), is present in both the intr- nd extrcellulr enviorment (Lovelock, 1953b). A cryoprotectnt, s defined by Krow (1969), is clss of drugs tht cts specificlly to protect nd mintin the vibility of frozen niml cells. Cryoprotectnts re divided into two groups () permeting nd (b) nonpermeting tht protect cells during freezing by two different mechnisms, lthough both rely on membrne diffusion nd osmosis. Permeting CPAs re chrcteristiclly low moleculr weight, nonionic compounds tht re highly soluble in wter t low tempertures nd hve low cellulr toxicity (Muldrew et l., 2004). An intrcellulr CPA protects the cell in two wys (1) both intr- nd extrcellulr presence eventully reduces the mount of intrcellulr wter nd thus, reduces the mount of intrcellulr ice being formed t specific temperture (Leibo nd Mzur, 1971) nd, (2) by cting s secondry solvent for intrcellulr slts, nd exposure to lethl levels of electrolytes is reduced (Pegg 1984). Usully, nonpermeting CPAs hve higher moleculr weights thn their permeting counterprts, nd re long-chined polymers (sucrose being n exception). Also, their osmotic coefficients re considered to be lrge, which llows them to increse the osmollity much higher thn their molr concentrtion (Muldrew et l., 2004). Since nonpermeting CPAs do not pss through the cell membrne, intrcellulr wter is trnsported out of the cell so s to yield equl intrcellulr nd extrcellulr osmollity. The loss of wter dehydrtes the cell, thereby reducing or preventing intrcellulr ice formtion. Historicl Perspective on Methods for Preserving Spermtozo There re three mjor methods used to perverse spermtozo (1) cryopreservtion in liquid nitrogen (-196 C), (2) short-term storge 4 C to 15 C for nd (3) freeze drying. Since Polge et l. (1949) reported tht spermtozo could be cryopreserved successfully t sub-zero tempertures by the ddition of glycerol, cryopreservtion hs become the most widely used method of mmmlin sperm storge (see reviews by Krow, 1969; Foote, 1982; Wtson, 5

20 1995; Holt, 2000). Also, storge in the liquid stte between 4 to 10 C is used predominntly for short-term mintennce of mmmlin spermtozo (Pope et l., 1989; Goodrowe et l., 1993; Axner, 2012). Freeze drying sperm is nother pproch tht hs long been of interest nd the subject of numerous studies. Recently, the technology of freeze drying spermtozo hs been revisited (Wrd et l., 2003; Kwse et l., 2005; Kneko nd Nkgt 2006) nd live mouse offspring hve been born following the trnsfer of ICSI derived embryos (Wrd et l., 2003; Kneko nd Nkgt 2006) Erly Attempts t the Cryopreservtion of Spermtozo Glycerol ws first used s cryoprotectnt for freezing plnt cells nd tissues by Mximov in Lter, Bernstein nd Petropvlovsky (1937) hd miniml success using glycerol (9.2%) s cryoprotectnt for sperm of the rbbit, guine pig, bull, rm, bor, stllion nd duck, but only for cooling spermtozo to -21 C. It ws lso reported tht 18% glycerol ws toxic to spermtozo (see review Slmon nd Mxwell, 1995). However, the first successful results fter freezing spermtozo ws performed in Cmbridge Englnd by Polge et l. (1949). The post-thw success with vin spermtozo ws the result of mislbeling glycerol s sugr. The first offspring born using glycerol for freezing semen were chickens, only three live chicks were produced from 600 eggs (Smith nd Polge, 1950). The first pregnncies in cttle with frozen-thwed semen resulted from the use of rtificil insemintion (AI) nd were reported by Stewrt (1951; one bull clf born) nd Polge nd Rwson (1952; 30 out of 38 cows were pregnnt six weeks fter insemintion). Subsequently, spermtozo from both domestic nd some nondomestic species hve been frozen using glycerol, ech with vrible rtes of success. Smith nd Polge (1950) ttempted to freeze equine spermtozo, but the first fol from AI using semen frozen with glycerol ws not reported until 7 yers lter (Brker, 1957). Emmens nd Blckshw (1955) were mong the first investigtors to use rm semen tht hd been frozen to -79 C to produce pregnncies from AI in sheep. Grhm et l. (1971) reported the birth of five piglets from single litter fter inseminting eight sows with semen tht ws frozen in pellet form. The first cnine pups born using frozen-thwed semen ws reported by Seger (1969). Pltz et l. (1978) reported the first kittens born using frozen-thwed semen. The first gorill produced using frozen-thwed semen nd AI ws reported by Douglss nd Gould (1981). Pope et l. (1997) used frozen semen to produce IVF-derived embryos tht resulted in the first gorill born from embryo trnsfer. However, due to lck of ccessibility nd inconsistent qulity of thwed semen from exotic species, routine freezing of semen is done only in few species. 6

21 Since the first clf, using frozen-thwed semen ws produced in the erly 1950s, frozen semen hs become the minstrem in the North Americn nd Europen diry cttle industries. Thibier nd Wgner (2002) reported, tht >252 million doses of bovine semen, from 648 semen collection centers were frozen worldwide in Over 43 million doses were produced in North Americ from 69 semen collection centers. In comprison, 136 million doses originted in Europe from 285 semen collection centers. More recently, similr results hve been reported by the Food nd Agriculture Orgniztion (FAO) (Rodriguez-Mrtinez, 2012). The number of nondomestic mmmlin species where offspring hve been produced using AI with cryopreserved spermtozo hs lso continued over the decdes (see exmples in Tble 2.1). A more comprehensive tble listing >55 mmmlin species where vrious ART technigues were successful resulting in births of offspring hs been compiled by Wirtu (2004). Cryoprotectnts Cryoprotectnts re divided into two ctegories, bsed on their bility to pss through the sperm membrne (permeting) or those tht do not pss through the sperm membrne (nonpermeting). Both types of cryoprotectnts involve osmosis nd depend on the bility of wter to follow the concentrtion grdient nd move freely cross the cell membrne. Since the discovery of glycerol s n effective permeting cryoprotective gent, other chemiclly similr (low moleculr weight, extremely low freezing temperture) compounds hve been evluted in ttempts to further improve spermtozo cryo-survivl. Other widely used permeting cryoprotectnts include dimethyl sulfoxide (DMSO) (Lovelock nd Bishop, 1959), ethylene glycol, propylene glycol nd other neutrl solutes (Lovelock, 1954). DMSO nd is possibly the most verstile cryoprotectnt with its bility to successfully preserve vriety of cell types (Krow 1969). One exmple is DMSO s effectiveness in cryopreserving mouse bone mrrow (Ashwood, 1961), s well s DMSO s bility to produce better post-thw sperm motility vlues over other cryoprotectnts such s glycerol, formmide, propnediol nd donitol when freezing mouse semen (Sztein et l., 2001). For exmple, Loskutoff et l. (1996) showed tht Springbok epididyml spermtozo frozen in DMSO hd greter post-thw motility thn did spermtozo frozen in glycerol, ethylene glycol or propylene glycol. Ejculted spermtozo from the Africn elephnt exhibited significntly greter motility when incubted for 60 minutes with 0.5M DMSO or 0.5M ethylene glycol compred with incubtion with 0.5M glycerol (Gilmore et l., 1998). Correspondingly, Gilmore et l. (1998) nd Loskutoff et l. (1996) (30-32 C) both reported tht DMSO ws less toxic to impl epididyml sperm during holding periods of 24 to 60 hours respectively. 7

22 Tble 2.1. A list of some exotic mmmlin species in which pregnncies nd/live offspring hve been produced fter the use of rtificil insemintion procedures with cryopreserved spermtozo Assesment of Species Method of fertiliztion fertiltiy Reference Addx AI t the os cervix Live offspring Densmore et l Alpc AI Live offspring Brvo et l Americn bison AI Live offspring Dorn 1995 Bctrin cmel AI Live offspring Zho 1994 Bnteng AI Live offspring Johnston et l Blckbuck Trnscervil IUI Live offspring Holt et l Blck-footed ferret Lproscopic IUI Live offspring Howrd et l Bottlenose dolphin Trnscervil IUI Live offspring Robeck et l Cheeth Lproscopic IUI Live offspring see Howrd et l Chimpnzee Trnscervil IUI Live offspring Gould 1989 Common mrmoset AI intrcerviclly Live offspring Morrell et l Common elnd Trnscervil IUI /intrcervicl Live offspring Brtels et l Eld s deer Lproscopic IUI Live offspring Monfort et l Fllow deer Lproscopic IUI Live offspring Mulley et l.1988 Gur AI Live offspring Junior et l Gint pnd Intrcervicl Live offspring Moore et l Leoprd ct Lproscopic IUI Pregnncy see Howrd et l Mohor gzelle Lproscopic IUI Live offspring see Roldn et l Ocelot Lproscopic IUI Live offspring Swnson et l Pcific white-sided dolphin Trnscervil IUI Live offspring Robeck et l Red deer AI nd Lproscopic IUI Live offspring Asher et l Rhesus monkey Trnscervil IUI Live offspring Snchez-Prtid et l Scimitr Horned oryx Trnscervil IUI Live offspring Morrow et l Spnish ibex Lproscopic IUI Life offspring Sntigo-Moreno et l Suni ntelope Trnscervil IUI Pregnncy Rphel et l White rhinoceros Trnscervil IUI Live offspring Hermes et l White-tiled deer AI t the os cervix Live offspring High 1984 AI = rtificilly inseminted nonsurgiclly with no description of the loction where sperm were deposited. IUI = intruterine insemintion; 8

23 Ethylene glycol hs been used s cryoprotectnt for mny cell types including rt liver cells (Mglhes et l., 2012), bovine fetl nd dult crtilge cells (Cetinky nd Art, 2011), freezing nd vitrifiction of mouse nd rt embryos (Miymoto nd Ishibshi 1977; Mochid et l., 2011) nd for freezing bovine embryos (Voelkel nd Hu, 1992; Mssip, 2001). Due to its low moleculr weight, cells re more permeble to ethylene glycol thn lrger compounds such s glycerol, nd thus, osmotic shock is reduced. For this reson, ethylene glycol hs been evluted s cryoprotectnt for preservtion of spermtozo. Epididyml spermtozo from blesbok nd impl ntelope hd equl post-thw motility vlues when frozen with ethylene glycol or glycerol (Loskutoff et l., 1996). Equine semen used for AI fter freezing in 6% ethylene glycol resulted in pregnncies of 3 of 5 mres (Squires et l., 2004). Alvreng et l. (2000) reported tht crosoml sttus nd motility were similr fter cryopreservtion of stllion semen in either ethylene glycol or glycerol. In theoreticl simultion ethylene glycol ws compred with glycerol, DMSO nd propylene glycol, to determine memebrne permebility nd volume excursions induced during cryoprotectnt ddition nd removl (Gilmore et l., 1997). From the results of the computergenerted simultion, it ws hypothesized tht ethylene glycol ws the most permeble, nd ws therefore, experimentlly compred with glycerol. After tretment with ethylene glycol (1M), humn spermtozo exhibited higher percentge of motility thn spermtozo treted with 1M glycerol when the cryoprotectnts were dded nd removed t 22 C. Similrly when ethylene glycol or glycerol were dded to sperm smples t 22 C nd the smples cooled to - 80 C (progrmmble freezer), plunged in liquid nitrogen nd lter thwed t 22 C (bench top method), motility ws higher in ethylene glycol treted smples thn I ws in glycerol treted smples. Another permeting cryoprotectnt tht hs been used with some success for freezing spermtozo is propylene glycol. Vrisli et l. (2009) showed tht motility nd Acrosoml sttus of ejculted nd epididyml rm spermtozo, following one-step ddition nd removl, were similr in ll four of the 1M cryoprotectnts evluted (propylene glycol, glycerol, DMSO nd ethylene glycol). The osmotic tolernce nd membrne permebility of rhesus monkey nd chimpnzee spermtozo hve been mesured (Agc et l., 2005, 2005b). The results showed tht chimpnzee nd rhesus monkey spermtozo re more permeble to propylene glycol nd ethylene glycol thn they re to glycerol or DMSO, with ethylene glycol being the most permeble nd DMSO the lest permeble. In contrst, when red deer (Cervus elphus hispnicus) epididyml spermtozo were frozen in glycerol, ethylene glycol nd propylene 9

24 glycol, motility index, nd crosome nd membrne integrity were lower in propylene glycol smples s compred with those frozen in glycerol nd ethylene glycol (Fernndez-Sntos et l., 2006). Cool Storge of Spermtozo Extended storge of spermtozo is most often ccomplished by some form of cryopreservtion. Effective cryoprotective extenders llow spermtozo to survive t sub-zero tempertures. Storge in liquid nitrogen is the method used universlly for long-term storge (-196 C), which hs llowed bovine spermtozo to retin vibility for t lest 47 yers nd possibly indefinitely (Crwell, 2008). However, due to the risk of reduced motility nd possible crosome dmge in some mmmls (Lengwint nd Blottner, 1994), cryopreservtion is not lwys the best method for storing spermtozo. When spermtozo re used within dys following collection, temporry storge t supr-zero tempertures (4 to 15 C) hs been nother option to freezing. Cool Storge of Bovine Spermtozo The use of egg yolk s cryoprotectnt in semen extenders ws first reported by Phillips (1939) nd Phillips nd Lrdy (1940). It ws reported tht fter 150 hours of storge, spermtozo extended in n egg yolk extender (10 C) exhibited higher motility thn spermtozo in the control which ws not exposed to n egg yolk extender. Willett et l. (1940) showed tht bull semen stored in phosphte-egg yolk diluent t 5 C, could be held for 4 dys nd still result in pregnncy rte (fter AI) equl to tht of fresh semen. Diry cows inseminted with semen tht ws extended in citrte-egg yolk diluent before cooling hd significntly lower 60-to-90 dy non-return rte thn cows inseminted with semen extended fter cooling (Foote nd Brtton, 1949). De Puw et l. (2003), compred, cooling nd/or holding bull versus freezing bull semen. Ejcultes held t room temperture hd higher percentge of spermtozo with intct membrnes nd mitochondril function t ph 6, thn t lower (4 or 5) or higher (7 or 8) ph. In ddition, the percentge of spermtozo with intct membrnes ws significntly higher in isotonic medium (300 mosm/kg) s compred with those held in either hypotonic or hypertonic solution. Also, De Pw et l. (2003) showed tht the timing of dding of the egg yolk extenders influenced the optiml sperm concentrtion for holding smples. Membrne integrity ws improved in spermtozo stored t concentrtions between 100 X 10 6 nd 100 x 10 7 /ml fter extension in Trildyl -egg yolk-glycerol nd removl by wshing fter 5 minutes. When 10

25 spermtozo were not exposed to the Trildyl extender, membrne integrity ws greter when spermtozo were stored t lower concentrtions (100 X 10 5 nd 500 X 10 6 /ml). The consistently high post-thw motility vlues chieved from frozen bull semen, hs llowed the cttle industry to depend hevily on cryopreserved spermtozo. Furthermore, offspring from numerous different mmmlin species hve been born using AI nd cryopreserved spermtozo (see Wirtu, 2004). However, Wtson (2000) emphisized tht postthw vibility of cryopreserved spermtozo in the mjority of mmmlin species hs been inconsistent nd the development of species-specific cryotechniques is necessry if cryopreserved spermtozo re to be used routinely in nondomestic species. Cool Storge of Equine Spermtozo In study evluting the functionl vibility of frozen equine semen, Cochrn et l. (1983) found 26% decrese in pregnncy rte following AI of mres with frozen-thwed compred with fresh semen. Also, Cochrn et l. (1983) reported tht only 62% of frozen ejcultes resulted in post-thw progressive motility vlue of 35%. Similrly, Loomis et l. (1983) reported tht only 31% (17/54) of frozen-thwed stllion ejcultes hd 50% progressive motility nd the pregnncy rtes fter AI ws lower thn tht obtined using fresh semen. Loomis (2001) reported tht cooled stllion semen used for AI during the first estrus of the seson resulted in significntly greter pregnncy rte thn did frozen semen (59 % nd 51 %, respectively). More recently, Bckmn et l. (2004) showed tht cooled (5 C) horse semen held for 18 hours, before freezing, could be thwed nd used for AI to produce pregnncies (70%). Also, they reported tht there ws no significnt difference in pregnncy rtes when spermtozo were frozen t different concentrtions (200 x 10 6 or 400 X 10 6 sperm/ml). Cooling horse semen for the purpose of AI is prcticl lterntive to freezing, however, the fcility tht collected nd shipped the semen is nother fctor tht my influence the qulity of the semen fter shipment. When horse semen ws cooled nd shipped breeding fcilities, Heckenbichler et l. (2011) reported 67% pregnncy rte (86 mres) for single breeding seson. They noted tht processing differences between six different collection centers hd significnt effects on semen volume, rrivl temperture, ph, concentrtion of sperm, totl number of sperm, percentge of sperm with intct membrnes, morphologicl defects, totl motility, progressive motility nd totl number of progressively motile spermtozo. When stllion semen ws extended in skim milk sugr-sline extender, cooled (8 to 12 C), nd shipped to be used for AI in 56 mres, the foling rte ws 50%, with some of this 11

26 semen being used 24 hours fter collection (Rot et l., 2004). In n erlier study, Dougls- Hmilton et l. (1984) reported tht 13 of 14 mres estblished pregnncy fter AI with stllion semen extended in skim-milk extender nd cooled to 6 to 8 C for 12 to 23 hours. Recently, it ws shown tht spermtozo could be collected from stllion epididymides ndheld t 4 C 72 hours post-cstrtion. Furthermore, the recovered spermtozo were cpble of fertilizing oocytes in vitro (Vieir et l., 2013) fter cryopreservtion. Brun et l. (1994) reported tht dding 25% seminl plsm to equine epididyml spermtozo resulted in significnt increse in sperm motility t 0 hours: however motility t 24, 48 nd 72 hours ws not significntly incresed. Cool Storge of Ovine Spermtozo Purdy (2006) hs found tht rm semen could be held for up to 48 hours t 5 C without significnt decrese in totl motility, progressive motility, membrne integrity nd crosoml sttus. Previously, Slmon et l. (1979) reported tht there ws no difference in erly pregnncy rtes (dy 18 or dy 19) when ewes were inseminted with semen tht hd been cooled to 5 C nd held for 0, 2, 4, 6 nd 8 dys, lthough pregnncy rtes, lter in gesttion, did significntly decrese fter AI with semen held for 4 dys. Lter, Slmon et l. (1979) did report the birth of live lmb using semen stored t 5 C for 9 dys. Cool Storge of Swine Spermtozo Bor semen ws frozen with some success in the erly 1970s (Pursel nd Johnson, 1971), lthough bor spermtozo re less cryo-tolernt thn some other widely studied species. It hs been reported tht frrowing rtes nd litter size decresed 20% to 30% nd 2 to 3 piglets per liter, respectively when cryopreserved semen ws used to AI sows (cited by Biley et l., 2008). Due to poor post-thw survivl the use of four strws/femle, rther thn one or two strws ws recommended. The necessity of using multiple strws for AI is one disdvntge of using frozen-thwed bor semen (Css et l., 2012). Artificil insemintion is widely used in swine breeding, nd 99% of ll insemintions (19 million worldwide) re done with extended liquid semen (Johnson et l. 2000). In some Europen countries, 90% of pigs re rtificilly inseminted, s compred with 45 to 50% in the United Sttes (Johnson et l. 2000). Most insemintions re performed 24 hours fter semen collection but semen cn be used for up to 5 dys post-collection when held between 8 C nd 17 C (Pursel et l., 1973; Wberski et l., 1994; Wberski et l., 1994b; Althouse et l., 1998; Severo et l., 2011). Funhshi nd Sno (2005) showed tht bor spermtozo stored t 10 C for 29 dys could penetrte oocytes in vitro when treted with the ntioxidnt, cysteine. 12

27 Cool Storge of Cnine Spermtozo Cool storge of liquid dog semen hs some dvntges over freezing. For exmple, fresh ejcultes of dog semen re simple nd esy to cquire. In ddition, the cooled smples cn be stored for 10 dys before losing vibility (Rijsselere et l., 2011). Insemintions with dog semen cooled for 24 or 48 hours resulted in pregnncy rtes nd litter sizes tht were similr to those obtined with fresh semen (Pinto et l., 1999). Furthermore, Fromn et l. (1984) stted tht frozen-thwed dog spermtozo penetrted the zon pellucid (IVF study) of only 5% (6 of 120) of oocytes, s compred to 70% (84 of 120) penetrtion rte by fresh spermtozo. They found the lck of zon penetrtion my hve been due to the erly ctivtion of vitl zon penetrting enzyme (crosine), or the complete loss of the enzyme due to crosoml dmge. Rodens et l. (2014) cooled dog semen extended in 20% egg yolk extender t 2.25, 0.9, 0.45 nd 0.2 C/minute from 23 C to 5 C, nd found no difference between cooling rtes in sperm motility nd vibility t 0, 24, 48, 72 nd 96 hours of storge. In n effort to extend this life of cooled semen Verstegen et l. (2005) reported tht cooled cnine semen (4 C) extended in 20% egg yolk-bsed extender could retin motility up to 27 dys when extender ws replced with fresh extender on dys 11, 21 nd 27. Motility significntly incresed from 68% to 95.6% t the dy 11 extender-exchnge nd from 19.9% to 73.0% t the dy 21 exchnge. Extenderexchnge on dy 27 did not improve motility. The positive effects provided to sperm by egg yolk during cooling is well estblished; however, under certin conditions cooled epididyml (nd ejculted) sperm cn be mintined without egg yolk in the extender. Yu nd Leibo (2002) showed tht cnine epididyml spermtozo held t 4 C within epididymides for up to 8 dys were cpble of binding in vitro to dog zone pellucide. Motility decresed significntly fter 5 hours t 4 C but there ws no significnt decrese in their bility to bind with the zon until fter 48 hours. While the benefits of chilling cnine semen re evident, it hs been shown tht dog semen cn be extended, cooled to 4 C nd held for 2 dys nd then frozen with no detrimentl effects on post-thw prmeters due to the holding period (Hermnsson nd Forsberg, 2006). Cool Storge of Feline Spermtozo Studying methods of cryo-storging ct spermtozo is good model for determining its potentil for propgting endngered felid species (Pukzhenthi et l., 2006; Cocchi et l., 2010). Pope et l. (1989) showed tht domestic ct semen stored in TesT egg yolk buffer for 20±2 hours (2 C) resulted in significntly greter clevge rte in vitro thn did freshly collected 13

28 semen. Furthermore, there were two live kittens s result of trnsferring embryos tht were in vitro fertilized with the chilled domestic ct semen. Filliers et l. (2008) showed tht the percentge of motile, progressively motile nd rpid or sttic moving epididyml ct spermtozo, ws significntly reduced fter 1 dy of storge t 4 C when compred with fresh epididyml spermtozo. However, verge sperm velocity did not decrese significntly until dys 3 nd 7 of storge. Similrly, Villverde et l. (2006) reported tht ct epididyml sperm motility nd vigor decresed significntly from dy 0 to dy 3 of storge t 5 C. Also, the percentge of morphologiclly norml spermtozo decresed significntly during the first dy of cool storge. Hrris et l. (2001) demonstrted the resilience of cooled ct ejculted nd epididyml spermtozo fter it ws extended nd held t 4 C for up to 23 dys. During the 23 dy storge period, sperm motility rnged from 66.2 (dy 0) to 21.2% (dy 23) nd 79.3 (dy 0) to 31.2 (dy 23) for ejculted nd epididyml spermtozo, respectively. During tht sme time period, the percentge of spermtozo with intct membrnes, rnged from 91.0 to 65% for ejculted nd 95 to 59% for epididyml spermtozo. Additionlly Siemieniuch nd Dubiel (2007) lso reported tht ct epididyml spermtozo held t 4 C decresed in progressive motility from 73.7% t 24 hours of cooled storge to, 65.0 nd 52.5% t dys 2 nd 3 of storge, respectively. Even though progressive motility decresed by 20%, due to the ese of cooled storge compred with freeze-thwing, using 3-dy cooled spermtozo (52.5%) ws proposed s prcticl lterntive to using frozen-thwed smples in prticulr circumstncessubstitute over frozen-thwed smple. When feline semen ws exposed to repeted rpid cooling cycles (3 cycles) in ice wter (0 C), motility significntly decresed from one cycle to the next, but the presence of 20% egg yolk minimized the decrese in motility between cycles when compred with non-egg yolk smples (Glover nd Wtson, 1985). Furthermore, Tittrelli et l. (2006) reported tht feline spermtozo recovered from epididymides held t 4 C in medium contining egg yolk hd significntly greter motility, membrne integrity nd sperm velocity thn sperm recovered from epididymides held in isotonic sline solution, for 24, 48 nd 72 hours. Mrtins et l. (2009) showed tht epididyml ct sperm could be held t 5 C in n egg yolk-bsed extender for 24 hours before freezing without ny significnt difference in totl motility, progressive motility, membrne integrity or morphology s compred to sperm cooled for 60 minutes before freezing. Jimenez et l. (2013) tested three commercilly vilble extenders (Trildyl, Andromed nd Gent), for their efficcy in freezing domestic ct epididyml sperm. They fout tht smples frozen in Trildyl, n egg yolk-bsed extender, hd greter post-thw motility, vigor, 14

29 morphology, crosome sttus, membrne integrity nd DNA integrity when compred with Andromed nd Gent, neither of which contins egg yolk. Domestic ct epididyml spermtozo held in the epididymides overnight t 5 C hve been shown to be cpble of zon binding nd sperm hed nucler de-condenstion, when incubted with zon free hmster oocytes (Goodrowe nd Hy, 1993). Furthermore when compred with freshly collected epididyml sperm, the cooled overnight tretment hd greter percentge of spermtozo tht ttched to the zon. Freeze-Drying of Spermtozo Freeze-drying spermtozo is the lest used method for preserving spermtozo nd will only be mentioned here briefly. Freeze-drying (lyophiliztion) procedures re widely used for storge of food (Sos et l., 2012) nd vccines (Wng et l., 2011; Okd et l., 2012). The process of freeze-drying differs from conventionl dehydrtion. Dehydrtion is the removl of wter by evportion, while freeze-drying involves the removl of wter from specimen while it is in frozen stte (Merymn, 1960). The process of freeze-drying depends on the direct trnsition from solid (ice) into gs, which now is known s the sublimtion phenomenon (Hochi et l., 2011). Freeze-drying of spermtozo ws first ttempted in the mid 20th century (see Hochi et l., 2011). Shermn (1954) unsuccessfully ttempted to freeze-dry humn spermtozo. Bily nd Smith (1957) lso reported negtive results in their efforts to freeze-dry bull spermtozo in the presence of glycerol. In more recent study Kuskbe et l. (2001) reported tht the pregnncies could be produced in mice fter trnsfer of embryos produced by ISCI of oocytes with reconstituted freeze-dried spermtozo. Furthermore, Kuskbe et l. (2008) demonstrted tht humn spermtozo were cpble of undergoing freeze-drying without chromosome dmge. In combintion with ICSI, freeze-dried spermtozo hve been shown to be vlid source of mle genetics. Keskintepe et l. (2002) reported the formtion of bovine blstocysts from embryos generted by ICSI of oocytes with spermtozo tht hd been freeze-dried for 3 months. Abdll et l. (2009) showed tht injection of freeze-dried bull sperm-heds into mouse nd bovine oocytes induced clcium oscilltions nd resumption of meiosis, respectively, but t lower rtes thn in oocytes injected with control sperm (not freeze-dried). Recently, Hr et l. (2011) reported tht when freeze dried spermtozo re used with ICSI, the resulting impired formtion of microtubule-orgnizing center nd reduced embryo development my be result of the ICSI process, not result of using freeze-dried spermtozo. 15

30 The first (confirmed) live offspring (mice) from use of freeze-dried spermtozo were produced using ICSI to fertilize the oocytes (Wkym nd Yngimchi 1998). Six yers lter, rbbits were born fter trnsfer of embryos produced by ICSI with freeze-dried spermtozo (Liu et l., 2004). Pig embryos produced by injecting freeze-dried spermtozo into oocytes were cpble of developing into blstocysts (Kwon et l., 2004). Furthermore, results were improved when only the sperm heds were injected rther thn intct spermtozo. Semen Extenders Without Egg Yolk In the context of sperm preservtion, extender is defined s solutions/medi/diluents contining components tht re beneficil sperm vibility nd extend the smple volume. Most extenders consist of five components (1) buffer, such s 2-[[1,3-dihydroxy-2- (hydroxymethyl)propn-2-yl]mino]ethnesulfonic cid (Tes) nd/or tris(hydroxymethyl)minomethne (Tris) to prevent ph shifts, (2) n energy source, such s glucose or fructose, (3) protein source such s egg yolk or milk, to protect spermtozo from cold shock nd provide cryoprotection, (4) ntibiotics such s gentmicin, penicillin or streptomycin nd (5) cryoprotectnt, such s glycerol (Mitchell nd Dok, 2004). The ltter component is usully not included in extenders used only for temporrily mintining sperm smples in liquid stte (>0 C). Milk nd egg yolk hve proven to be very effective gents for preventing cold shock (Thcker nd Almiquist, 1951; Phillips nd Lrdy, 1940). However, for the purpose of consistency nd prevention of disese trnsmission, the development of synthetic extenders (without niml protein) hs long been n re of high interest (Choong nd Wles, 1963). Furthermore, Roy (1957), nd more recently Cbrer et l. (2005), reported tht the bulbourethrl glnd of the got releses n enzyme (phospholipse) nd protein (BUSgp60) (Pellicer-Rubio et l., 1997) into the ejculte tht rects with egg yolk or skim milk. The result is conversion of egg yolk phospholipids into lysophospholipids, such s lysolecithins, clss of detergent-like compounds tht cn be toxic to spermtozo. The lipse ctivity of the BUSgp60 protein on skim milk negtively ffect got spermtozo (Pellicer-Rubio et l., 1997). Use of Bovine Serum Albumin (BSA) in Semen Extenders As n lterntive to milk nd egg yolk-bsed extenders, bovine serum lbumin (BSA) hs been used with some success in extending longevity of motility of ejculted bovine semen (Breddermn nd Foote, 1971). For exmple, Mtsuok et l. (2006) showed tht rm spermtozo hd significntly greter post-thw motility fter freezing in n extender with 10% nd 15% BSA compred with freezing in extenders contining egg yolk with fructose. Also, 16

31 Fukui et l. (2007) reported no differences in dy 60 pregnncy rtes or lmbing rtes fter AI of 60 ewes with semen frozen either in 15% egg yolk extender or 10% BSA extender. Got spermtozo held t 15 C in two commercilly vilble extenders contining BSA [Zorlesco nd Androhep (Minitube of Americ, Inc., Veron, WI, USA)] hd greter percentges of motility t 4 nd 6 dys thn spermtozo held in two other synthetic extenders tht did not contin BSA (Xu et l., 2009). Furthermore, spermtozo held for 3, 5 nd 7 dys t 5 C in either of two dditionl synthetic extenders (mza nd mzap, contining BSA nd polyvinyl lcohol, respectively) mintined greter motility thn did sperm in the Androhep extender. Xu et l. (2009) lso reported 34% progressive sperm motility in mza extender on dy 9 of storge. In ddition, five does delivered kids when inseminted with spermtozo held for 7 dys in mzap extender. A commercilly vilble extender, Androhep is widely used in the swine industry for long-term ( 5 dys) liquid storge of bor semen (see Johnson et l., 2000). Wberski et l. (1994) reported bor semen stored for 4 dys in Androhep (with BSA nd HEPES buffer) produced significntly greter pregnncy rtes in comprison with semen stored in Kiev porcine medium (without BSA nd HEPES buffer). Fntinti et l. (2009) compred four commercil extenders, including Androhep, to n extender [swine fertiliztion medium (SFM)], contining BSA, ethylene dimine tetr-cetic cid (EDTA) but no HEPES, milk or egg yolk. On dys 3, 6 nd 9 of storge, spermtozo held in SFM hd significntly greter motility thn did spermtozo in the other extenders. Also, SFM ws the only tretment in which progressive motility ws 60% t dy 12 of storge. Therefore, it ws concluded tht, if bor semen is to be stored for more thn 4 dys, SFM ws more economicl thn commercil extenders, nd produces comprble results when properly prepred. An opticlly-cler extender (without milk or egg yolk) hs other prcticl dvntges in ddition to lowering the risk of disese trnsmission. The equine industry cn benefit gretly from sex-sorting of stllion spermtozo. However, sex-sorting of spermtozo requires the use of lsers during flow cytometry to recognize Hoeschst stined spermtozo. Gibb et l. (2011) showed tht the X nd Y split rtio improved in efficiency from 0.08 to 0.17 when stllion semen ws sorted in n opticlly cler diluent (1% BSA) when compred with smples extended in skim milk. Also, fter 18 hours of storge in the cler extender (contining BSA) sperm motility ws greter thn tht of spermtozo stored in skim milk (74% vs. 64%, respectively). 17

32 Although the incidence of milk or egg yolk trnsferring hrmful microbes to semen is low there is possibility of microbil contmintion nd exposure of semen to infectious niml borne diseses. For this reson the development of synthetic extenders, void of niml protein, is priority. In pig in vitro fertiliztion study, Jng et l. (2011) reported tht using semen frozen in egg yolk resulted in contmintion (Enterobcter cloce) during IVF, however, contmintion did not occur in the fresh semen tht ws not exposed to the egg yolk extender. Use of Low Density Lipoproteins (LDL) in Semen Extenders The use of n niml by-product, such s egg yolk or milk in semen extenders, does not llow control of stndrdiztion due to the presence of unidentified molecules tht my hve negtive effects on spermtozo (Hinsch et l., 1997 reviewed by Moustcs et l., 2011). Myer nd Lsley (1945) were the first to identify the low density lipoprotein (LDL), which is the specific frction of egg yolk tht provides the cryoprotective benefits when cooling spermtozo. The subsequent discovery of the cryoprotective properties of glycerol (Polge et l., 1949) resulted in dely in further studies on the cryoprotective nture provided by LDL. Subsequently, Pce nd Grhm (1974) confirmed nd extended the erlier study of Myer nd Lsley (1945), to show tht even in the bsence of glycerol, the low density frction ids in the survivl of spermtozo during cryopreservtion. Shortly therefter, other reports (Wtson nd Mrtin, 1975; Wtson, 1981; Grhm nd Foote, 1987, Grci nd Grhm, 1987) provided further informtion relting to the effects of lipid extrcts nd frctions on survivl of bull nd rm spermtozo during cold storge. Mouss et l. (2002) considered previous methods of purifying LDL to be indequte nd imprcticl. Accordingly they developed n improved economicl method to extrct nd purify LDL, nd reported tht post-thw motility nd sperm movement of bull spermtozo were significntly greter when spermtozo were cryopreserved in n extender contining 8% LDL when compred with extenders contining egg yolk. More recently, Ver-Munoz et l. (2009) lso showed tht replcing egg yolk with 8% LDL in Trildyl ws significntly more effective in mintining post-thw bovine sperm motility nd membrne integrity when compred with both the soy lecithin-bsed nd egg yolk-bsed extenders BioXCell nd Trildyl, respectively. Similrly, Jing et l., (2007) showed extender contining 9% LDL ws significntly more effective t protecting bor sperm DNA from dmge during cryopreservtion when compred with tht contining 0, 6, 7 nd 8% LDL. Got sperm motility, stright line velocity (VSL), mplitude of lterl hed displcement (ALH) nd Acrosoml sttus were greter for spermtozo frozen with LDL compred with egg yolk (Al Ahmd et l., 2008; Moustcs et l., 2011). 18

33 Benchrif et l. (2010) reported tht dog semen cryopreserved in commercilly vilble extender contining 6% LDL (CANIXL freeze, IMV, Aigle, Frnce) hd numericlly higher sperm motility, verge pth of velocity (VAP) nd stright line velocity (VSL) vlues, while curviliner velocity (VCL) nd lterl hed displcement (ALH) vlues were significntly greter thn sperm cryopreserved in egg yolk (20%) extender (Equex STAMP, Nov Chemicl Sles, Scitute Inc., MA, USA). Semen frozen in extender contining 6% LDL ws used to inseminte six bitches, with ll of the femles becoming pregnnt nd delivering totl of 16 pups (Benchrif et l., 2010). Use of Soyben Extrct Lecithin in Semen Extenders Further ttempts to remove niml proteins from semen extenders hve included investigtions of soy-derived lecithin. Cows inseminted with semen frozen in n extender contining soyben extrct (Biociphos-Plus IMV, Frnce) hd significntly lower 56-dy nonreturn rtes thn those inseminted with semen frozen in 20% egg yolk-bsed extender (vn Wgtendonk-de Leeuw et l., 2000; Thun et l., 2002). In contrst, Forouznfr et l. (2010) reported tht post-thw sperm motility, vibility nd clevge rte fter IVF with rm semen frozen in 1% (w/vol) soyben lecithin-bsed extender were similr to those of spermtozo frozen in 20%, but not 15% egg yolk-bsed extender. Angor buck semen frozen in nother soyben lecithin-bsed semen extender (BioXCell, IMV Technologies, L Aigle, Frnce), exhibited greter motility (CASA), verge sperm pth velocity (VAP), sperm stright liner velocity (VSL), sperm linerity (LIN = VSL/VCL), fewer crosoml bnormlities nd greter pregnncy rtes s compred with Tris + egg yolk extender (Sriozkn et l., 2010). Likewise, bufflo (Bublus bublis) semen frozen in BioXCell showed similr post-thw chrcteristics (motility, vibility, membrne integrity, norml picl ridge, bnormlities) to those of smples frozen in Tris-citric cid egg yolk extender (Akhter et l., 2010). The Use of Pentoxifylline to Stimulte Sperm Motility Pentoxifylline is smll molecule with moleculr compound (M.W. = C 13 H 18 N 4 O 3 ) belonging to clss of drugs clled hemorrheologic gents. Cffeine, chemiclly relted molecule, is lso used to increse nd/or improve sperm motility. In the bufflo, cffeine incresed post-thw motility when dded to spermtozo before freezing nd it further incresed motility when present in the thwing medium (Fttouh nd Abdou, 1991). Hicks et l. (1972) stted tht one contributing fctor involved in sperm motility ws the presence of intrcellulr camp. When humn spermtozo were incubted with pentoxifylline sperm intrcellulr camp incresed t ll time points (Clogero et l., 1998). Also, pentoxifylline 19

34 dded fter thwing, improved motility chrcteristics (totl motility, progressive motility nd motility velocity) of rm spermtozo (Mxwell et l., 1995). Post-thw ddition of pentoxifylline stimulted totl nd progressive motility of stllion spermtozo. Pre-freeze ddition produced post-thw decrese in totl nd progressive motility when compred with control smples (Grdil nd Bll, 2000). Cffeine ws shown to increse motility of equine epididyml, but not ejculted spermtozo. Also, cffeine hd no effect on the crosoml sttus of either epididyml or ejculted spermtozo (Weston et l., 2005). In contrst, Ortgies et l., (2012) reported tht ddition of pentoxifylline to ejculted stllion spermtozo produced no significnt difference in the motility pttern but did increse the percentge of live-cpcitted spermtozo. When dministered s n orl tretment 400 mg of pentoxifylline significntly improved sperm concentrtion, sperm motility nd spermtozo with norml morphology from infertile men dignosed with idiopthic oligosthenotertozoospermi (Sfrinejd et l., 2011). Tretment of frozen-thwed cnine spermtozo, with pentoxifylline (2.5, 5 nd 7.5 mm, 120 minutes) significntly incresed totl sperm motility (Milni et l., 2010). In ddition, tretment with pentoxifylline t high concentrtions (10 mm nd 100 mm, 1 hour of incubtion) incresed the cpcittion rte nd the percentge of crosome rected cnine spermtozo (Mirshokrei et l., 2011). Effect of pentoxifylline hs lso been studied in the domestic ct. Post-thw exposure of ct epididyml nd ejculted spermtozo, to pentoxifylline hs been shown to increse motility prmeters (s mesured by CASA) without ffecting longevity of motility (Stchecki et l., 1994). The effects of different concentrtions of pentoxifylline will be investigted in this reserch study. Feline Epididyml Spermtozo in Assisted Reproduction Collection of Feline Epididyml Spermtozo There re t lest five methods of collecting epididyml spermtozo, three of which re ex situ collections (flushing, mincing, incisions), either post-mortem or post-cstrtion. The other two methods [percutneous epididyml sperm spirtion (PESA)], microsurgicl epididyml sperm spirtion (MESA)] re in situ procedures performed on nesthetized mles. The in vitro fertilizing cpbility of nonejculted domestic ct (Felis ctus) spermtozo ws first reported by Bowen (1977). Using spermtozo from minced vs deferens, Bowen reported clevge rte of 79%, including four embryos tht developed into the first in vitroderived blstocysts. Bowen (1977) pointed out tht, unlike ejculted spermtozo in the ct (Hmner et l., 1970) nd epididyml spermtozo in the rbbit (Ogw et l., 1972), 20

35 spermtozo from the vs deferens of the ct did not need to undergo cpcittion in vivo to fertilize oocytes in vitro. Using epididyml sperm collected by the flushing method, Niw et l. (1985) showed tht sperm penetrted the zon pellucid by 30 minutes fter insemintion nd the first observtion of mle nd femle pronuclei were noted within 4 to 5 hours of co-incubtion (Niw et l., 1985). Pope et l. (1993), using ejculted sperm, confirmed the chronology of erly fertiliztion events found by Niw et l. (1985) with epididyml sperm. In the lter report they observed tht both mle nd femle pronuclei were completely developed by 6 hours post insemintion. Mincing of the epididymides is widely used method for obtining domestic ct spermtozo (Filliers et l., 2010). Becuse of tissue nd blood contmintion of the sperm smples obtined by mincing, centrifugtion over density grdient to remove the blood nd tissue cells is necessry. Additionlly, centrifugtion produces highly concentrted sperm pellet tht cn then be diluted to the desired concentrtion (Lengwint nd Blottner, 1994; Tsutsui et l., 2003; Chtdrong et l., 2010). Lengwint nd Blottner (1994) demonstrted tht fresh nd cryopreserved epididyml spermtozo could be used to fertilize oocytes nd produce feline 8-cell embryos in vitro. Tsutsui et l. (2003) reported the first births of domestic kittens produced fter AI using epididyml spermtozo (27.3% pregnncy rte). Comprison of Feline Ejculted nd Epididyml Spermtozo Vrious studies hve been conducted to compre the fertilizing bility freezing nd cooling bility, nd morphology of ejculted nd epididyml ct spermtozo (Goodrow, 1992; Axner et l., 1998; Hrris et l., 2001; Tebet et l., 2006; Hermnsson nd Axner, 2007; Filliers et l., 2010). Goodrowe (1992) reported tht cpcittion times of epididyml spermtozo nd ejculted spermtozo were similr. Also, t 3 nd 4 hours fter in vitro insemintion, there were more epididyml spermtozo ttched to the zon pellucid thn ejculted spermtozo (9.6 vs. 5.2 nd 12.0 vs. 5.6, respectively). In ddition, Goodrowe (1992) compred collection of epididyml spermtozo immeditely fter cstrtion (fresh) compred with epididymides mintined overnight t 5 C. Cooled smples were found to hve lower motility nd lower proportions of bnorml spermtozo post-thw thn did fresh smples. Even with reduced motility nd n increse in bnormlities, cooled spermtozo hd higher occurnce of zon ttchment thn did fresh spermtozo (112.2 vs. 84.5, respectively) but both groups hd higher post-thw zon ttchment, thn did their pre-freeze counterprts. 21

36 Axner et l. (1998) compred epididyml nd electro-ejculted spermtozo from the sme mle, pre-cstrtion nd post-cstrtion. The comprison ws ccomplished by performing unilterl cstrtion before electroejcultion ws done nd then removing the second testicle. They found no significnt difference in totl proportions of bnorml spermtozo between the two types of sperm; however, the incidence of specific til bnormlities ws greter in ejculted spermtozo thn in epididyml spermtozo. This could suggest tht the bnormlities in ejculted semen resulted from the seminl fluid or from the process of ejcultion. Also, Axner et l. (1999) used nother method of determining the origin of feline tertospermi. They found tht epididyml spermtozo gin initil motility, where the cput epididymis trnsitions into the corpus portion, with the highest motility found in the cud portion or the lst one third of the epididymis. Hed bnormlities, detched sperm heds, midpiece bnormlities nd crosoml defects/bnormlities decresed significntly when the first nd lst regions were compred, but there were no decreses between djoining regions, suggesting grdul decrese long the length of the epididymis. In contrst, til bnormlities significntly incresed (lthough still reltively low) between the efferent duct nd cud portion of the epididymis (Axner et l., 1999). Thus, the ct epididymis ppers to serve s filter to eliminte spermtozo with specific bnormlities during trnsit but the mjority of til bnormlities seem to occur in the epididymis. Epididyml nd ejculted ct spermtozo hve been compred for their bility to be stored in the liquid (cool, > 0 C) nd frozen (liquid nitrogen -196 C) stte. Hrris et l. (2001) showed tht fter 23-dy storge t 4 C in TEST yolk buffer, epididyml nd ejculted spermtozo mintined similr motility nd membrne integrity prmeters. Hermnsson nd Axner (2007) showed tht neither electro-ejculted nor epididyml spermtozo were susceptible to cold shock when cooled slowly (0.5 C/minute) or rpidly (3 C/minute) from room temperture (+20 C) to 4 C. Also, they noted tht 20% egg yolk in the extender helped mintin sperm motility nd crosoml sttus but it hd negtive effect on mintennce of membrne integrity. Furthermore, ejculted nd epididyml spermtozo from the sme mle cts showed no significnt difference in survivl prmeters, both pre-freeze nd post-thw (Tebet et l., 2006). Feline Sperm Assessments Motility is generlly considered good indictor of the bility of sperm to fertilize n oocyte (Linford et l., 1976). Morphology is nother importnt chrcteristic tht is widely used for ssessing sperm qulity nd potentil fertilizing bility. A common technique for ssessing 22

37 ct sperm morphology is by counting 500 spermtozo on crbol fuchsine-stined smer using light microscopy t mgnifiction of 1000X (see Axner et l., 1998; 2004). An intct crosome is essentil for sperm to penetrte nd fertilize n oocyte, however, dmge or loss of the crosome cn occur premturely due to physicl stress or chemicl induction. Pope et l. (1991) developed simple single step crosoml ssy for feline spermtozo by using 1% fst green, 1% rose bengl nd 40% ethyl lcohol in 0.1M citric cid nd 0.2M disodium phosphte buffer. A common method for evluting Acrosoml sttus is the use of fluorescent-conjugted lectins clled FITC-PNA (fluorescent isothiocynte conjugted penut gglutinin) (Silv nd Gdell, 2006). Since the crosome contins hydrolytic enzymes nd crbohydrte moieties of glycoproteins, the fluorescently tgged lectin binds specificlly to the glycoproteins. Determining sperm membrne integrity (the percentge of live vs. ded sperm) is nother method to ssessing spermtozo. Grner et l. (1994) developed competitive binding dul-stin using SYBR 14 nd propidium iodide (PI). Although PI hs higher ffinity for DNA thn does SYBR 14, it is lso lrger molecule nd in contrst to SYBR 14 cnnot pss freely through n intct membrne (Grner nd Johnson 1995). So, sperm with vible or intct membrne show green fluorescence (SYBR 14), while those with dmged or ruptured membrne will fluoresce red (PI). The combintion of SYBR 14 nd PI is commercilly vilble s kit (Live/Ded Sperm Vibility Kit, Moleculr Probes, Inc., Eugene, OR, USA), nd its effectiveness hs been shown with spermtozo from vrious mmmlin species (Grner nd Johnson 1995) including the domestic ct (Filliers et l., 2008). Recently, the Live/Ded Kit ws used to determine plsm membrne integrity of domestic ct cryopreserved epididyml spermtozo (Kshiwzki et l., 2005). Siemieniuch nd Dubiel (2007) showed tht SYBR 14/PI could be used in conjunction with flow cytometry for efficient nd objective nlysis of ct spermtozo, however, mjor disdvntge to the method ws tht high concentrtions of 1 x 10 6 feline spermtozo were required. When eosinnigrosin stining ws used to compre nd confirm the SYBR14/PI dul stining in domestic ct sperm, Filliers et l. (2008) noted tht the results for the two methods were similr, lthough SYBR 14/PI consistently resulted in lower vlues. The uthors ttributed the consistently lower vlues, to the higher sensitivity of SYBR 14/PI nd its bility to detect minor membrne ruptures with rpid response thus, mking it more ccurte indictor of membrne integrity. Computers cn lso be used to ccurtely nd objectively ssess sperm smples. Computer ssisted sperm nlysis (CASA) is currently the most objective nd ccurte wy to determine motility chrcteristics nd ptterns. Mesurements, such s verge curviliner 23

38 velocity (VCL), linerity of the cells swim pth (LIN), stright line velocity (VSL) nd mplitude of lterl hed displcement (ALH) re clculted in mtter of seconds mesuring thousnds of cells simultneously (Stchecki et l., 1993; Filliers et l., 2008). Stchecki et l. (1993) were the first to report results on sperm motion prmeters derived by CASA in the domestic ct. Initilly, they used the CASA prmeters set for humn spermtozo nd slowly djusted the settings until the CASA system ws more specificlly clibrted to more ccurtely mesure ct spermtozo prmeters. Filliers et l. (2008) considered tht the CASA prmeters they described for domestic ct spermtozo were suitble reference point for future feline sperm studies. Siemieniuch nd Potock (2008) proposed tht their CASA progrm more ccurtely mesured motility of feline spermtozo thn did trditionl subjective methods. Their prmeters were modified from those used for evluting cnine spermtozo rther thn those using the few feline sperm studies done t the time (Siemieniuch nd Potock, 2008). Epididyml Ct Spermtozo in Assisted Reproductive Techniques (ART) Assisted reproductive techniques (ART) such s rtificil insemintion (Sojk et l., 1970; Tsutsui, 2006; Chtdrong et l., 2007), in vitro fertiliztion (Hrris et l., 2002), embryo trnsfer (see Kremer et l., 1979; Pope et l., 1993; Pope, 2000; Gomez et l., 2004), intrcytoplsmic sperm injection (Pope et l., 1998; Gomez et l., 2000) nd use of sexed sorted semen (Spinci et l., 2006; Pope et l., 2009) hve been pplied to the domestic ct. Most of these techniques hve been used with vrying success using domestic ct epididyml spermtozo. Bowen (1977) demonstrted tht spermtozo collected from the vs deferens were cpble of fertilizing feline oocytes in vitro nd tht resulting embryos could develop to the blstocyst stge in vitro. Niw et l. (1985) showed tht fresh epididyml spermtozo were cpble of penetrting ct oocytes. They reported the presence of n enlrged sperm hed with ttched til, ppernce of the first polr body nd ctivted chromosomes s soon s 30 minutes fter IVF. Goodrowe nd Hy (1993) reported tht ct epididymides could be held t 5 C overnight without ffecting sperm progressive motility or number of norml spermtozo when compred with spermtozo recovered from fresh epididymides. Furthermore, fertiliztion of zon-free hmster ov nd ttchment to the zone of feline oocytes were higher using cooled sperm s compred with fresh spermtozo. Lengwint nd Blottner (1994) found tht the clevge rte of ct oocytes fter IVF with cryopreserved epididyml spermtozo (25%, 43/170) to be lower thn tht obtined with fresh spermtozo. 24

39 Ct epididyml spermtozo hve lso been used for intrcytoplsmic sperm injection (ICSI) (Gomez et l., 2000). Bogliolo et l. (2001) reported tht 7 of 129 in vitro mtured oocytes developed to the blstocyst stge when the oocytes were injected with ct epididyml spermtozo. More recently, fter ICSI, there ws no significnt difference in clevge rtes fter using spermtozo held for 72 hours t 4 C in the epididymis or in sperm TALP medium when compred with the control group using fresh spermtozo (Ringleb et l., 2011). Since seven of 36 feline species re endngered or criticlly endngered, the development of optiml protocols for ssisted reproductive techniques, such s sperm cryopreservtion, is n importnt priority. Due to the similrity in the genome of ll felids, the domestic ct is n excellent model for developing these ssisted reproductive techniques. Using combintion of ssisted reproductive techniques nd gene bnking, cryopreserved ct spermtozo could help insure the long-term survivl of ll species of cts. 25

40 CHAPTER III GENERAL EXPERIMENTAL PROCEDURES FOR ALL EXPERIMENTS Tissue Collection nd Trnsport Testes nd ovries from the domestic ct (Felis ctus) were collected from routine ct neuters/spys performed t locl veterinry clinics in New Orlens, Louisin. Tissues were collected yer round nd were used rndomly cross seven different experiments. Immeditely fter removl, the reproductive tissue (testes with epididymides nd vs deferens nd ovries with oviducts nd ovrin burs) were plced in 150 ml sterile vil contining ~25 ml of HEPES-buffered sline solution (UltrSline A Solution, Bio-Whittker, Wlkersville, MD) with 50 µg/ml of Gentmicin,(Sigm #G1272) t mbient temperture (~27 C). Reproductive tissue ws trnsported within 120 minutes to the Audubon Center for Reserch of Endngered Species (ACRES) lbortory in New Orlens, Louisin. Tissues (epididymides, vs deferens nd ovries) were trimmed nd wshed in HEPES-199 medium before gmetes were hrvested. Sperm Collection nd Designting Tretments After removl from the testes, epididymides nd vs deferens were rinsed, nd further processed in HEPES-199 medium (t ~22 C). The vs deferens ws dissected wy from the epididymis so tht it ws stright duct, open t both ends. Ech vs deferens ws then squeezed nd milked in mnner tht removed the spermtozo (sperm) from the lumen. The epididymides were sliced repetedly nd minced nd incubted in HEPES-199 medium for 30 minutes to llow sperm to migrte from the epididyml ducts. Before being ssigned to tretment, the sperm suspension (contining HEPES-199 medium, minced tissue nd sperm) ws filtered through 40 μm cell striner (Flcon 40 µm Cell Striner, Corning, Inc., Corning, NY, USA) into 50 ml plstic conicl centrifuge tube. The sperm suspension ws then lyered gently over two-lyer (45% upper/90% lower) density grdient column (#99264, Isolte, Irving Scientific, Snt An, CA, USA), tht ws prepred in 15 ml conicl centrifuge tube, nd centrifuged t 650 g for 20 minutes. After centrifugtion, the sperm pellet ws removed nd plced into 1.6 ml conicl liquot tube (Thoms Scientific #2591K99). Before tretment, n initil ssessment of sperm motility, membrne integrity nd crosoml sttus ws recorded (see below for detils). Following the initil ssessment, sperm smples were divided into one of three tretments in ech experiment. Ech of the three frctions of the sperm pellet ws plced into 1.6 ml liquot tube nd clculted volume of extender without glycerol, ws dded to ech frction of the sperm pellet. The desired sperm concentrtion per strw, determined the totl 26

41 volume of extender to be dded to ech sperm frction. After ech tube ws extended to the desired concentrtion, they were plced in ~200 ml room temperture wter bth (22 C) nd grdully (~3 h) cooled to 4 C±1 C in wlk-in cooler. After cooling (4 C/post-cooling reding), the three tretments were gin ssessed for sperm motility, membrne integrity nd Acrosoml sttus. Following the post-cool (4 C) sperm ssessment, extenders with glycerol, were dded to tretments. Smples were diluted 1:1 with extender contining 12% glycerol (Prt B) in four steps: 15%, 20%, 25% nd 40% of the totl tht ws dded to the mixture (modified from Go et l. 1995) there ws pproximtely 10 minutes between the four dilution steps. Sperm Anlyses To ssess sperm motility 1 µl of the sperm pellet ws diluted in 20 µl of HEPES-199 medium. Then, 10 µl liquot ws tken nd plced on wrm pre-clened glss microscope slide, covered with wrm 18 x 18 mm glss cover slip nd plced on heted microscope stge (37 C). Motility ws determined by clculting the verge totl sperm motility in three seprte fields on ech slide. In Experiments 6.1, 6.2, 7.1, 7.2 nd 7.3, sperm motility ws determined using 1 µl of rw sperm pellet diluted in 20 µl of HEPES-199 medium. Three micoliters of this 1:20 mixture ws then plced on wrmed Lej 4 chmbered slide nd plced on the heted microscope stge (37 C) of n Olympus CX41 microscope (Olympus Americ, Inc. Center Vlley, PA, USA) equipped with Hmilton Thorne CASA (computer ssisted sperm nlysis) Helos TM system nd used with the Ceros softwre (Hmilton Thorne Reserch, Beverly, MA, USA). The motility of these smples ws determined using the CASA system, in which three fields on ech slide were digitlly nlyze. To determin membrne integrity LIVE/DEAD vibility kit (Moleculr Probes, Inc., Eugene, OR, USA) ws used. To view cell stining, light microscope equipped with ultrviolet florescence nd flourescien isothiocynte filter ws used. The LIVE/DEAD kit incorported two stins, SYBR 14, which stins the DNA of membrne intct cells, green (vible/live), nd Propidium Iodide (PI), which stins the DNA of the membrne ruptured cells, red (nonvible/ded). An liquot of 1 µl of sperm ws diluted with 4 µl of SYBR 14 nd 5 µl of PI. Also 5 µl of forml citrte ws dded to immobilize sperm without dmging intct membrnes (Dott nd Foster, 1975). Smples were protected from light nd llowed to incubte with stin for 5 minutes before evlution. Determining the percentge of membrne intct (vible) sperm ws ccomplished by rndomly counting 200 sperm using fluorescent luminescence (40X). The 27

42 number of sperm with green fluorescence ws divided by the totl count of 200 sperm to determine the percentge of live sperm. Acrosoml sttus ws evluted using FITC-PNA (see Silv nd Gdell, 2006) stining (1 mg of lectin, FITC from penut in 2 ml of HEPES-199 medium). The stock solution ws then liquoted into 0.6 ml tubes (30 ul/tube) nd frozen t -80 C until use. An liquot of 1uL sperm smple ws diluted in 5 ul of FITC-PNA stin long with 5 µl of forml citrte to immobilize sperm. The sme fluorescent microscope to evlute membrne integrity ws gin used to determine crosoml sttus. A totl of 200 sperm were rndomly counted nd the percentge of nonrected sperm ws divided by the totl number counted to clculte the percentge of intct crosomes. Sperm concentrtion ws determined either by using spectrophotometer designed specificlly for mesuring sperm concentrtion (1:5 dilution; SpermCue, Minitube of Americ, Veron, WI, USA) or by using stndrd hemocytometer (Bright-Line, Husser Scientific, Horshm, PA, USA; 1:1,000 dilution; Experiments 6.1,6.2, 7.1, 7.2, nd 7.3). Smples were djusted to the desired concentrtion by dding pproprite mounts of the extenders with nd without glycerol. Cryopreservtion nd Thwing of Sperm Tretments After loding, semen strws (0.25 or 0.5 ml) were seled with n ultrsound seling unit (UltrSel 21, Minitube of Americ, Veron, WI, USA) nd plced for 20 minutes on block of dry ice (-78.5 C) in smll Styrofom box or in -80 C freezer. After plcing strws on the dry ice block, the lid ws plced on the box. After 20 minutes, strws were plunged into 13 mm goblet/cne tht ws submerged in liquid nitrogen. The cne with strws ws then held in 35 liter liquid nitrogen storge tnk for up to 3 months before thwing. Strws were removed from liquid nitrogen nd exposed to ir (~22 C) for 5 (0.25 ml) or 10 (0.5 ml) seconds nd then immeditely immersed in 60 C wter bth for 5 seconds. Once removed from the wter bth, contents of strws were emptied into pre-wrmed (37 C) 1.6 ml liquot tube. Glycerol ws removed from the sperm using seven step dilution of HEPES-199 medium with pentoxifylline, there ws pproximtely 10 minutes between ech step to void osmotic shock to the sperm. The volumes of the first four dilution steps of HEPES pentoxifylline were 15, 20, 25 nd 40% of the thwed smple. The volumes of the lst three dilution steps were 33, 66, nd 200% of the smple fter the 40% ddition from the fourth step. The seven step solution ws then centrifuged t 200 x g for 10 minutes, nd the resulting sperm pellet ws removed nd resuspended in 20 µl of HEPES-199 medium nd the 1.6 ml tube contining the smple ws plced in 37 C wter bth nd incubted t 37 C for 3 hours. 28

43 Motility, membrne integrity nd crosoml sttus were evluted fter 0 nd 3 hours incubtion, s described previously for the initil nlysis. For IVF, sperm strws were thwed s described bove; then fter centrifugtion 3 to 6 µl of sperm pellet ws diluted with 35 µl of HEPES Tyrode s blnced slt solution. In Vitro Production Medium (IVF/IVC) The three blnced slt solutions (BSS) used to prepre the IVF/IVC medi were Tyrode s blnced slt solution, TCM-199 medium (with NHCO 3 but without glutmine) nd TCM-199 medium (without glutmine or NHCO 3 ). Additionl supplements were dded to ech BSS to crete in vitro mturtion (IVM), in vitro fertiliztion (IVF), in vitro culture-i (IVC-I), in vitro culture-ia (IVC-IA) nd in vitro culture-ii (IVC-II) medium. Tyrode s blnced slt solution (Irving Scientific, Snt An, CA #9298) ws the bse solution for IVF nd IVC medi. Tyrode s BSS ws lso used to prepre HEPES-buffered medium, used for diluting nd mintining sperm used in IVF. TCM-199 medium with NHCO 3 but without glutmine (Sigm #9120) ws the bse medium used for IVM of oocytes. TCM-199 medium without NHCO 3 or glutmine (Sigm #3769) ws the bse for the HEPES-buffered holding medium used for mintining oocytes nd embryos during hndling in tmospheric ir outside of CO 2 incubtor. The 100X supplement dded to TCM-199 for IVM medium (see Appendix III) ws prepred by dding g glutmine, g cysteine, g sodium pyruvte, g clcium lctte nd g gentmicin to 10 ml of Tyrode s BSS. The 100X solution ws filter sterilized nd liquoted (100 ul) into sterile 1.6 ml tubes for storge t -80 C until use. Also, equine chorionic gondotropin (ecg; Clbiochem, Sn Diego, CA #36722) nd humn chorionic gondotropin (hcg; Pregnyl Orgnon, Inc., West Ornge NJ) were used in the IVM medium. The gondotropins rrive, in powder form, were diluted in sterile Tyrode s BSS, liquoted into sterile 1.6 ml tubes nd stored t -80 C until use. They were diluted to 100X finl concentrtion (hcg = 100 IU/mL; ecg = 50 IU/mL). Also Epiderml Growth Fctor (EGF, Sigm #9644) ws dded to the IVM medium. The EGF, in powder form, ws diluted with Tyrode s BSS to 1,000 ng/ml (100X finl concentrtion), filtered into 1.6 ml sterile tubes nd stored t -80 C until use. Finlly bovine serum lbumin (BSA; Frction-V #82047, Serologicl Proteins, Inc., Knkkee, IL, USA) ws dded to the IVM medium (0.3g/100 ml). The 100X supplement for IVF nd IVC medi ws prepred by dding g of glutmine, g of sodium pyruvte, g of clcium lctte, nd g of gentmicin to 10 ml of Tyrode s solution. Then, the supplement ws filter sterilized, liquoted into 1.6 ml tubes nd stored t -80 C until use. The IVF nd IVC-I medi contined 0.6 g nd 0.3 g/100 ml 29

44 BSA (Frction-V), respectively. The MEM Nonessentil mino cids solution (100X; Sigm#M7145) ws dded to the IVC-I, IVC-IA nd IVC-II medi to give finl concentrtion of 1%. Upon rrivl the NEAA solution ws liquoted into 1.6 ml tubes nd store t -80 C. Finlly, the IVC-IA nd IVC-II medi contined MEM essentil mino cids (50X; EAAs) nd either BSA (IVC-IA) or fetl bovine serium (IVC-II: FBS #30070; Hyclone, Inc., Logn, UT, USA). Upon rrivl the NEAA nd the EAAs were liquoted into 1.6 ml sterile tubes (1 ml/tube) nd stored in -80 C freezer. The IVC-II medium contined 10% FBS insted of BSA. Upon rrivl FBS ws thwed, liquoted into 15 ml tubes (4.5 ml/tube) nd stored t -80 C until use. After thwing, before ddition to IVC-II medium, FBS ws het-treted t 56 C for 30 minutes. Aliquotes of frozen supplements were thwed weekly nd dded to the Tyrode s BSS, TCM-199 medi. Ech week IVM/IVC medi were djusted to 285 to 295 mosm nd 7.7 to 7.8, respectively. After steriliztion by filtrtion (0.22 µ) into 15 ml sterile centrifuge tubes (5 ml/tube) ech tube ws gssed with 5% CO 2 in ir (or 5% CO 2, 5% O 2, 90% N 2 ) for sec before seling. Then, tubes were plced in tilt rck (~30 ) ngle nd stored t 4 C. until use within 7 dys. Oocyte Collection nd Mturtion Upon rrivl t the lbortory (see Tissue Collection nd Trnsport), ovries were trimmed of excess tissue, rinsed once in Phosphte-buffered sline, rinsed three times in TL- HEPES nd minced in HEPES-199 medium (He199). Cumulus-oocyte complexes were wshed three times in HEPES-199. Cumulus-oocyte complexes (COC) with uniformly drk, finely grnulted ooplsm surrounded by severl lyers of cumulus cells nd COC with less drkly pigmented, not uniformly nd densely grnulted ooplsm surrounded by severl lyers of cumulus cells were rinsed three times in Hi-199 nd plced in fresh He-199 medium t 35 C to 38 C. Oocyte recovery, wshing, nd grding were performed under lminr flow hood using stereomicroscope with heted stge (38 C). For in vitro mturtion (IVM), COC were cultured for 24 hours, in 4-well culture dish (Nunclon, # Nunc, Denmrk) contining 500 µl of Modified TCM-199 medium (IVM medium, Appendix III). The 4-well dish contining the mturing oocytes long with 60 x 15 mm Petri dish contining 7 ml of sterile wter, were plced on n inverted lid from micro titer plte. Severl smll holes were drilled in the lid of 60 mm Petri dish. This rrngement of dishes ws then plced inside 16.5 x 20.3 cm plstic bg (Kpk SelPAK pouch, #402, Kpk Corportion, Minnepolis, MN) nd filled with 5% O 2, 5% CO 2 nd 90% N 2. During the 30

45 filling/seling process the bg ws plced on heting block (38 C). The inflted bg nd its contents were plced in 38 C incubtor (Form Scientific, Model 3130, Mriett, OH). In Vitro Fertiliztion nd Culture After 24 hours IVM culture, COC were rinsed in He199 nd plced in 40 µl droplets of IVF medium under 3 ml of pre-equilibrted sterile minerl oil (ART ; Copper surgicl, Turnbull, CT USA) in 35 mm sterile petri dish (#1008, Flcon). The desired concentrtion of motile sperm ws ~1 million sperm/ml. IVF droplets contining oocytes, sperm nd surrounded by minerl oil were then plced into 38 C incubtor with 5% CO 2 in ir. At 4-6 hours post-insemintion, oocytes were rinsed four times in He199 nd then trnsferred into 500 µl droplets of modified Tyrode s contining nonessentil mino cids nd BSA (IVC-I) in four-well dish. The dish ws plced in the closed bg system s described for IVM culture nd cultured t 38 C. On Dy 2, the number of embryos per well ws recorded, uncleved oocytes removed, nd embryos were plced into fresh IVC-I medium contining 2% essentil mino cids (IVC-IA). On Dy 5, embryos were plced into fresh IVC-I medium contining 2% essentil mino cids in which BSA ws replced with 10% FBS (IVC-II medium). Presumptive zygotes nd embryos were cultured in humidified tmosphere of 5% CO 2, 5% O 2, nd 90% N 2 t t 38 C. On Dy 8, embryos were evluted visully to determine the rte of development to the blstocyst stte (% blstocysts/embryos X100). Sttisticl Anlyses Sigm Stt Version 3.0 ws used to nlyze the dt set of ech prmeter. Student t- test ws the sttisticl test used to compre the initil, pre-freeze, nd 0 nd 3 h post-thw vlues for sperm motility, membrne integrity nd crosoml sttus. The sttisticl nlysis compred tretments within given time point s well s tht of individul tretments cross times. These prmeters (totl sperm motility, membrne integrity nd crosoml sttus) re reported s men±sem/tretment group. A P vlue <0.05 ws used to determine significnce differences. 31

46 CHAPTER IV SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION IN EXTENDERS WITH OR WITHOUT EGG YOLK Introduction Currently, niml by-products, such s milk nd egg yolk re mjor components in extenders used to cool store nd freeze mmmlin semen. These niml by-products hve been dded to semen extenders since 1856 nd their use incresed in the erly 1900s. Kolliker (1856) reported the use of milk in bull semen extender, while Milovnov nd Selivnov (1932) were the first to report the use of egg yolk lecithin nd its beneficil effect on bull spermtozo (sperm) (see Slisbury nd VnDemrk 1961). The presence of these niml byproducts re potentil sources for microbil contmintion nd trnsfer of zoonotic gents (Wrthll, 2000). Also, the unknown composition of these niml by-products produces inconsistencies mong semen extenders. Furthermore, the presence of egg yolk or milk dds dditionl chllenges when spermtozo re to be sex sorted with flow cytometer. These concerns hve prompted reserchers to serch for synthetic or defined components to replce niml by-products in semen extenders, while mintining cryoprotective properties. Soy lecithin hs been investigted s potentil lterntive to egg yolk nd milk-bsed semen extenders (Gil et l., 2003; Aires et l., 2003; Vick et l., 2011). Vick et l. (2012) reported tht ct sperm frozen in soy lecithin-bsed extender hd higher percentge of motile sperm t 3, 6 nd 24 hours post-thw, thn did sperm frozen in egg yolkbsed extender. In ddition, soy lecithin tretment hd lower percentge of spermtozo tht hd undergone cpcittion when compred with the sperm frozen with egg yolk. Bovine serum lbumin (BSA) is nother niml protein tht cn be sterilized, monitored for consistency/qulity nd purchsed commercilly. Hrrison et l. (1978) found tht BSA helped mintin sperm motility in wshed bor, rbbit, rm nd stllion semen fter 1, 3, nd 5 hours incubtion t 30 C. The primry benefit of BSA supplementtion ws reduction in the rte of decline in sperm motility. Also, in more recent study, Xu et l. (2009) showed tht the presence of BSA in got semen extender ws beneficil to sperm motility, membrne integrity, crosoml sttus nd cpcittion in buck semen held t 5 C up to 7 dys of storge. When equine semen ws incubted t 38 C in sucrose medium (with or without BSA) percentges of motile sperm nd tht of those in forwrd movement were higher in 1% or 3% BSA s compred with the control (0% BSA) (Klem et l., 1986). Furthermore, Nng et l. (2012) reported tht 1 mg/ml BSA significntly improved bull sperm vibility during storge 7 dys t 4 C. 32

47 Mtsuok et l. (2006) concluded, bsed on post-thw sperm motility nd vibility, tht 10% nd 15% BSA were suitble substitutes for egg yolk for freezing rm semen. Also, Fukui et l. (2007), from the sme lbortory, reported tht fter AI of synchronized ewes with rm semen frozen in BSA extender, 60-dy pregnncy rtes nd lmbing rtes were equivlent (not significntly different) to those of semen frozen in n egg yolk extender. Similrly, Sriozkn et l. (2009) reported tht bull semen ws frozen in the BioXCell extender nd supplemented with either oxidized glutthione, reduced glutthione or BSA. Pregnncy rte ws higher in the BSA group (72%) thn tht of the other two tretments (68.4 nd 53.2%), nd the control (BioXCell with no ntioxidnts; 54.2%) but ws only significntly higher thn the oxidized glutthione nd control tretments. There re mny studies on evlution of components to replce milk nd/or egg yolkbsed semen extenders tht produce cceptble sperm motility, membrne integrity nd crosoml sttus vlues. However, few hve investigted the possible benefits of BSA s replcement for egg yolk in the freezing of feline epididyml spermtozo. In the present study, we compred commercilly vilble egg yolk-bsed extender, tht hs been used previously for freezing feline spermtozo with two BSA-bsed extenders. We evluted sperm post-thw motility, membrne integrity nd crosoml sttus. In ddition we compred the bility of sperm frozen in egg yolk extender vs. BSA extender to fertilize oocytes in vitro s well s subsequent developmentl competence in vitro of resultnt embryos. Mterils nd Methods Experimentl Design Experiment 4.1 A totl of 55 sets of domestic ct (Felis silvestris ctus) testes were collected on 9 dys during Februry through April. Testes were processed nd epididyml spermtozo (sperm) were nlyzed nd cryopreserved on the dy of hrvest. From 3 to 15 sets of testes were processed/dy (Tble 4.1). Sperm collected ech dy were pooled nd divided cross three tretment groups (extenders). Tretment A, commercilly vilble extender, TesT egg yolk buffer (TesT) (Refrigertion Medium #9972, Irving Scientific, Snt An, CA). TesT consists of: Tes (2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)(mino)ethnesulfonic cid), Tris (Tris(hydroxymethyl)mino methne), dextrose nd 20% egg yolk. Tretment B (Humn Sperm Preservtion Medium; HSPM) ws prepred in the lbortory using the method s described by Mhdevn nd Trounson (1983). The regents were dded t 28 C s follows: sodium dihydrogen phosphte, clcium chloride, potssium 33

48 chloride, mgnesium chloride, sodium hydrogen crbonte, sodium chloride, sodium lctte, HEPES (C 8 H 18 N 2 O 4 S), sucrose, glycine, glucose nd bovine serium lbumin (BSA). In Tretment C, Tris citrte BSA (TCBSA), ws prepred in the lbortory. This extender consists of Tris, citric cid, fructose, glucose, BSA nd trehlose. After ech smple ws pooled, wshed nd centrifuged, pre-cool initil nlysis ws done. Then the sperm smple ws divided into three 1.6 ml liquot tubes contining one of the three extenders (Tretments A, B nd C). Ech sperm liquot (smple) ws then plced in seprte 125 ml plstic bottles filled with 22 C wter nd plced into 4 C wlk-in cooler. After grdul cooling to 4 C±1 C, sperm in ech tretment underwent second nlysis (postcool to 4 C). Then extender with glycerol ws dded to ech tube nd the smples were loded into 0.25 ml plstic strws nd frozen on block of dry ice. To further sses the vibility of the cryopreserved epididyml sperm, dditionl strws from ech of the three tretments were thwed nd the sperm were incorported into n in vitro fertiliztion IVF protocol to produce of feline blstocysts. Cumulus oocyte complexes (COC) from domestic cts were plced in modified TCM-199 (Irving Scientific, Snt An, CA) nd cultured for 24 hours in in vitro mturtion medium (IVM) (see Appendix III) in humidified tmosphere of 5% O 2, 5% CO 2 nd 90% N 2 t 38 C (IVM). For IVF, COC were co-incubted with sperm frozen in either the TesT or HSPM extenders (1 million sperm/ml), under 5% CO 2 in ir t 38 C. After 4-6 hours, oocytes were rinsed nd cultured using 3-step system (Pope et l., 2006). On dy 2 uncleved oocytes were removed nd number of embryos ws determined. On IVC dy 8 the number of embryos developing into blstocysts ws ssessed. Experimentl Procedures Bsic Procedures The stndrd procedures used for Experiment 4.1 were s follows: ovrin nd testes tissue collection nd trnsport, sperm collection nd tretment designtion, sperm nlysis, cryopreservtion nd thwing of sperm, in vitro embryo production medium, oocyte collection nd in vitro mturtion, in vitro fertiliztion nd culture, sttisticl nlyses. Detils of the stndrd procedures re described in Chpter 3. Semen Extender Preprtion (Appendix I nd II) TesT Yolk Buffer ws purchsed commercilly (Irving Scientific, Snt An, CA) nd shipped frozen on dry ice nd rrived in two boxes ech contining 20 x 5 ml vils. One box contined TesT Yolk Buffer vils without glycerol (Refrigertion Medium/Prt A). The second box contined vils of TesT Yolk Buffer with 12% glycerol (Freezing Medium/Prt B). The 34

49 regents nd specific molrities for the two extenders prepred in the lbortory re listed in Appendix I. The suppliers nd product numbers for ech regent re listed in Appendix II. Extenders were prepred using purified, Type I wter nd checked for desired osmolrity (310 to 330 osmoles) nd ph of 7.0 to 7.5. After djusting to the desired osmolrity nd ph, ech extender ws split eqully into two tubes. One of the hlves (Prt B) received specific mount of glycerol to mke finl concentrtion of 12% glycerol. Both Prt A nd Prt B for ll extenders (including TesT Yolk Buffer), were liquoted into 1.6 ml liquot tubes nd frozen t -80 C until use. Sperm Collection nd Designting Tretments Once removed from the testes epididymides nd vs deferens were rinsed, nd minced in HEPES-199 medium t lbortory temperture (~22 C) with number 12 sclpel blde. Before being plced into tretments, the suspension of minced tissue nd sperm ws filtered through 40 μm cell striner (BD Biosciences Discovery Lbwre, Frnklin Lkes, NJ) into 50 ml conicl centrifuge tube. The filtered sperm suspension ws then gently lyered onto 45/90 density grdient column (Isolte, Irving Scientific, Snt An, CA), in 15 ml conicl centrifuge tube, nd centrifuged t 600 g for 20 minutes. After centrifugtion, the sperm pellet ws removed nd plced into 1.6 ml conicl liquot tube. Before dividing the dily sperm pellet into the designted extenders, n initil ssessment of sperm motility, membrne integrity nd crosoml sttus ws recorded (see below for specific nlyses). Following the initil ssessment, sperm smples were divided into three tretments (extenders) in 1.6 ml liquot tubes nd clculted volume of the Prt A (no glycerol) extender ws dded. The desired sperm concentrtion/strw, determined the volume of extender dded to the pellet. After dding the volume of extender needed to produce the correct rtio of sperm/extender the 3 liquot tubes were plced in 125 ml plstic bottle filled with ~22 C wter nd plced in 4 C wlk-in refrigertor nd llowed to cool down to 5 C±1 C over period of ~3 hours. After cooling to 4 C, motility, membrne integrity nd crosoml sttus were evluted for ech tretment. Following the pre-freeze (4 C) sperm ssessment, Prt B (with glycerol) of the extenders ws dded to their respective tretments in four steps (15%, 20%, 25% nd 40% of initil volume) t intervls of 20 to 30 seconds nd 10 minutes between steps modified the fixed molrity methods by Go et l. (1995). 35

50 Results Experiment 4.1 Testes Collection, Epididyml Sperm Concentrtion nd Strws Frozen Over period of three months, 3 to 15 sets of testes were processed from 9 different collection dys (n = 9) for totl of 52 usble sets of testes. Routinely, 3 to 5 sets were processed in dy; however, there ws one dy with 9 usble sets nd nother dy with 15 usble sets hrvested. The verge totl number of pooled sperm collected ech dy ws 113 x 10 6, with rnge from 43 x 10 6 to 221 x Totl dily number of sperm ws divided evenly cross the three tretments nd depending on the totl sperm count, rnge of 1 to 6 (0.25 ml) strws were frozen/tretment/dy. On two dys, the totl number of sperm collected resulted in the freezing of extr 0.25 ml strws. These extr strws llowed for two thw replictes for these two dys (see tble 4.1). Motility The men (±SEM) initil motility for the pooled sperm smples ws 67.7±4.2% (Control). The men post-cool (4 C) vlues for sperm frozen in HSPM (Tretment A), TCBSA (Tretment B) nd TesT (Tretment C) were:, 53.2±4.9%, 54.1±6.2%, nd 66.8±4.6%, respectively. Sperm motility t 0 hour post-thw in HSPM, TCBSA nd TesT tretments were: 42.3±4.8%, 40.5±4.5%, nd 53.2±7.1%, respectively. After 3 hour post-thw incubtion, motility in HSPM, TCBSA nd TesT ws 20.4±6.2%, 25.0±6.2% nd 31.8±5.0% respectively (Tble 4.2). Motility fter cooling ws not different from initil motility in TesT, nd TCBSA smples, but motility ws lower in the HSPM group (P<0.05). After thwing, motility t 0 hour ws reduced in the non-egg yolk tretment groups (HSPM nd TCBSA) (P<0.05), but not in the TesT group, s compred to initil motility. At 3 hour fter thwing motility in ll three groups ws lower thn initil pre-freeze motility (P<0.05) (Tble 4.2). Motility t 0 hour fter thwing ws not different from tht recorded fter cooling in ny of the tretment groups. In the HSPM nd TesT groups, motility t 3 hour fter thwing ws lower thn t post-cooling nd t 0 hour post-thwing (P<0.05). In the TCBSA group, motility t 3 hour fter thwing ws lower thn fter cooling (P<0.05), but there ws no difference in motility t 0 hour versus 3 hour post-thwing (Figure 4.1). After cooling, motility in the TesT group ws higher thn in the HSPM group; but, motility in the TCBSA group ws not different from tht of the other two groups. After thwing, there were no differences in motility mong the three groups either t 0 hour or 3 hour (Figure 4.2). 36

51 Tble 4.1. Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of nine collection dys. No. of strws per Thw dte Collection dte Sets processed Totl no. extender/per dy 3/29/2008 3/24/ x /30/ Rep x /7/2008 4/2/ x /31/2008 3/31/ x /1/ Rep x /3/ Rep x /7/ Rep x /7/2008 2/27/2008? 43 x /8/2008 4/8/ x /8/2008 4/9/ x /8/2008 4/13/ x Dily verge (no.) x Totl no The lst two rows show the men vlues for ll the collection dys nd the overll totl vlues for the experiment. Tble 4.2. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing. Initil (Control) Post-cool 0 hour Post-thw 3 hour Post-thw HSPM (Trt A) 67.7± ±4.9 b 42.3±4.8 b 20.4±6.2 b TCBSA (Trt B) 67.7± ± ±4.5 b 25.0±6.2 b TesT (Trt C) 67.7± ± ± ±5.0 b Trt = Tretment;,b Superscripts only designte significnt difference between initil motility nd the vlue for ech extender t ech time period (P<0.05). 37

52 ,b b b b - Figure 4.1. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 38

53 b,b - Figure 4.2. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 39

54 Membrne Integrity The men initil percentge of sperm with intct membrnes ws 75.3±2.8%. After cooling to 4 C the percentges of sperm with intct membrnes were: 72.0±2.7%, 71.4±2.6% nd 76.8±3.0% for the HSPM, TCBSA nd TesT tretments, respectively. At 0 hour nd 3 hour fter thwing, membrne integrity percentges were 58.8±5.3% nd 52.7±2.7%, 53.6±2.8% nd 58.2±3.0%, nd 58.4±2.4% nd 51.2±2.3% in the HSPM, TCBSA nd TesT tretments, respectively. In ll three tretments, the initil percentge of sperm with intct membrnes ws higher thn the percentges seen t 0 hour nd 3 hour fter thwing (P<0.05), but not fter cooling to 4 C (Tble 4.3). There ws no difference between initil membrne integrity nd fter cooling to 4 C. However, the initil membrne integrity ws significntly higher thn t 0 hour nd 3 hour postthwing in ll tretments. Within tretments nd cross time periods, membrne integrity in the non-egg yolk extenders (HSPM nd TCBSA) ws higher t post-cool thn tht seen fter thwing (0 hour nd 3 hour). However, compring between 0 hour nd 3 hour fter thwing, sperm membrne integrity ws not different in either of the non-egg yolk extenders. In contrst, membrne integrity in the egg yolk extender (TesT), before cooling ws higher thn fter thwing, both t 0 hour nd 3 hour. Also, membrne integrity in the TesT tretment ws higher t 0 hour fter thwing tht t 3 hour (Figure 4.3). Across the three tretments there ws no differences in membrne integrity t the three time periods--fter cooling nd t 0 hour nd 3 hour fter thwing (Figure 4.4). Acrosoml Sttus Initilly 76.6±3.6% of epididyml sperm hd intct crosomes. After cooling the percentge of sperm with intct crosomes in HSPM, TCBSA nd TesT extenders ws 74.6±5.2%, 72.0±4.0% nd 76.5±2.6%, respectively. At 0 hour fter thwing 58.8±2.4%, 63.2±2.7% nd 56.2±2.8%, of sperm in HSPM, TCBSA nd TesT, respectively hd intct crosomes. At 3 hour fter thwing, 55.4±3.6%, 55.0±3.2% nd 51.6±2.0% of sperm in the HSPM, TCBSA nd TesT, respectively hd intct crosomes. These results re shown in Figure 4.5. There ws no difference between the pre nd post-cool percentge of sperm with intct crosomes in ny of the tretments. However, there ws higher percentge of sperm tht hd intct crosomes before cooling thn fter thwing in ll tretments (0 h nd 3 h fter thwing), (Tble 4.4). 40

55 Tble 4.3. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing. Initil (Control) Post-cool 0 hour post-thw 3 hour post-thw HSPM (Trt A) 75.3± ± ±5.3 b 52.7±2.6 b TCBSA (Trt B) 75.3± ± ±2.8 b 58.2±3.0 b TesT (Trt C) 75.3± ± ±2.4 b 51.2±2.3 b Trt = Tretment;,b Superscripts only designte significnt difference between initil membrne integrity nd individul vlues for ech extender t ech time period (P<0.05). Tble 4.4. Effect of egg yolk (Test) vs. BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing Initil (Control) Post-cool 0 hour post-thw 3 hour post-thw HSPM (Trt A) 76.6± ± ±2.4 b 55.4±3.6 b TCBSA (Trt B) 76.6± ± ±2.7 b 55.0±3.2 b TesT (Trt C) 76.6± ± ±2.8 b 51.6±2.0 b Trt = Tretment;,b Superscripts only designte significnt difference between initil membrne integrity nd individul vlues for ech extender t ech time period (P<0.05). 41

56 b b b b b c - Figure 4.3. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues within tretments but cross time with different superscripts re significntly different (P<0.05). 42

57 - Figure 4.4. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues cross tretments but within time with different superscripts re significntly different (P<0.05). 43

58 b,b b b b b - Figure 4.5. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues within tretments but cross time with different superscripts re significntly different (P<0.05). 44

59 The percentge of sperm in HSPM nd TesT extenders with intct crosomes ws lower (P<0.05) t 0 hour fter thwing s compred to tht of sperm fter cooling. However, there ws no significnt difference between the post-cool nd 0 hour post-thw vlues in TCBSA tretment. Also, there ws no significnt difference between the 0 nd 3 hour post-thw vlues within the three tretments. The post-cool vlues for ll three tretments were significntly higher thn t 3 hour post-thw (Figure 4.5). When compring cross tretments but within time periods, there ws no significnt difference (P>0.05) mong the tretments t the three ssessment times (Figure 4.6). In Vitro Fertiliztion Due to oocyte vilbility we were only ble to evlute sperm frozen in TesT nd HSPM extenders. Clevge frequency of 203 in vitro-mtured oocytes ws 36% nd 33% for the TesT nd HSPM tretments, respectively. The rte of in vitro embryo development to the blstocyst stge for TesT nd HSPM tretments were 50% nd 44%, respectively (Figure 4.7). Discussion Knowledge gined from reproductive reserch in the domestic ct could ply mjor role in the understnding of exotic feline reproduction for the purpose of conservtion. The proper techniques nd medi used to collect, process, store nd cryopreserve feline epididyml sperm hs mjor implictions in the propgtion of genetics from vluble mles tht otherwise my be lost. Over the lst decde, there hve been severl studies evluting domestic ct epididyml sperm. Axner (2004b) compred chrcteristics of ejculted nd epididyml sperm. Others hve nlyzed the post-thw survivl of epididyml ct sperm fter ex situ storge in the epididymis (Chtdrong et l., 2009; Gnn et l., 2009) or fter cool storge in n egg yolk-bsed extender (Mrtins et l., 2009) before cryopreservtion. In efforts to find replcements for egg yolk in semen extenders other components, including bovine serum lbumin hs been used in bors, bulls, buck rbbits, rms nd stllions (Hrrison et l., 1978). Soy lecithin extrct hs been tested in severl species including the ct (Vick et l., 2012) the dog (Beccgli et l., 2009; Ksimnickm et l., 2012), the bull (Aires et l., 2003) the buck (got) (Slmni et l., 2014). Low-density lipoproteins hve been evluted in the bull (Mouss et l., 2002), Red deer (Mrtinez-Pstor et l., 2009), dog (Benchrif et l., 2010), mle got (d Silv et l., 2014) nd the Brown ber (Rodriquez et l., 2012). The mjority of studies on ct epididyml sperm do not report concentrtions or totl number of sperm recovered. However, Zmbelli et l. (2008) did report the concentrtion of sperm collected by urethrl ctheteriztion (21.0±18 x 10 6 ) nd electro-ejcultion (33.6±34.5 x 10 6 ) of 11 toms. The dily numbers of sperm recovered in our study were considerbly higher 45

60 - Figure 4.6. Effect of egg yolk (TesT) versus BSA-bsed (HSPM nd TCBSA) extenders on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues cross tretments but within time with different superscripts re significntly different (P<0.05). 46

61 19 (50%) 14 (44%) 39 (36%) 32 (33%) Figure 4.7. IVF percentges when oocytes were fertilized with domestic ct epididyml sperm frozen in HSPM extender(tretment A) or TesT extender (Tretment C). Blstocysts percentges were derived from the number of embryos produced (Dy 2) not from the totl number of oocytes plced into IVF (Blstocysts% = no. of blstocysts/no. of embryos X 100). 47

62 thn those noted by Zmbelli et l. (2008) becuse we pooled testes from multiple individuls. Frnc nd Godinho (2003) mesured dily sperm production per grm of testis. They found tht domestic ct testis produce 15.7±1.6 x 10 6 of sperm/grm of testis. Averge testis weight of 13 toms ws 1.17±0.07 grms. Without knowing the storge cpcity of the ct epididymis it is not possible to know if our sperm recovery rtes re within the pooled men. One could mke the rgument tht since re results re bsed on pooled sperm; our recovery numbers should be significntly greter thn n individul mle sperm production. Furthermore, the upper rnge of our sperm recovery number is not so high to be considered inccurte, nd therefore our epididyml sperm recovery vlues fll within the domestic ct rnge. Our results showed no mjor differences between the egg yolk extender (TesT) nd BSA-bsed extenders (HSPM nd TCBSA), nd lthough they did not mesure sperm motility Fukui et l. (2007) similrly reported no significnt difference between pregnncy nd lmbing rtes when rm semen ws frozen in either 10% bovine serum lbumin (BSA) or 15% egg yolk-bsed extender. Cryopreservtion cuses incresed stress nd hs dmging effects on sperm membrnes tht result in n expected decrese in post-thw sperm vlues of most mmmlin species (Prks nd Grhm 1992; Medeiros et l., 2002). This could help explin the significnt decrese we reported between our pre-freeze nd post-thw vlues. Swnson et l. (1996) lso showed tht freezing resulted in significnt decrese in the percentge of intct crosomes jgur nd cheeth sperm fter thwing. In ddition, Pukzhenthi et l. (1999) proposed tht in the domestic ct, crosoml dmge ws cused prior to freezing when ejculted sperm were rpid cooled (~4 C/minute) nd ultr-rpid cooled(~14 C/minute). Domestic feline epididyml sperm hs been shown to be cpble of producing embryos in vitro when used for in vitro fertiliztion (Lengwint nd Blottner, 1994) nd offspring when used with rtificil insemintion (Tsutsui et l., 2003). We report tht ct epididyml sperm frozen in n extender contining bovine serum lbumin results in post-thw sperm vlues tht re not significntly different from sperm frozen with trditionl egg yolk-bsed extender. Furthermore, we showed tht sperm frozen in BSA extender cn be used to produce IVFderived embryos. 48

63 CHAPTER V EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION Introduction TesT buffered extender contining 20% egg yolk is commercilly vilble extender nd is commonly used in the cryopreservtion of domestic ct spermtozo (sperm) (Pope et l. 1998; Hrris et l., 2001); however, the presence of egg yolk concentrtions 20% presents obstcles tht possibly could be voided by reducing the mount of egg yolk in the extender. Extenders contining 20% egg yolk re widely used for cryo-storge of sperm in severl mmmlin species. However, in the 1950s nd 1960s egg yolk ws commonly used t concentrtion of 50% in bull semen extenders (Stewrt et l., 1950; Foote et l., 1960). There is some evidence tht sperm from some species, including the ct, could survive cryopreservtion with n egg yolk concentrtion 20% (Glover nd Wtson, 1987). The lower concentrtion of egg yolk in feline extenders is not necessrily used for the purpose of improving post-thw sperm survivl but rther for when sperm re used in IVF or ICSI, where egg yolk needs to be removed by dilution nd centrifugtion. Furthermore, nother reson for exmining the effects of lower concentrtions of egg yolk on ct sperm functions, is the importnce of using cler extender for sperm smples to be seprted into X nd Y popultion by flow cytometry. The presence of egg yolk tends to interfere with the efficiency of the sorting process so the sperm smple needs to be wshed prior to sorting. It should be noted tht Gibb et l. (2011) hs reported tht the efficiency of sex sorting stllion sperm ws improved when sperm were extended in cler nonegg yolk extender. Also, Pope et l., (2009) showed tht ct ejculted sperm stored nd shipped in nonegg yolk medium could be sex sorted nd result in gender specific kittens when used for IVF t 48 hours fter collection. Sntigo-Morno et l. (2006b) showed tht Spnish ibex epididyml sperm frozen in 6% egg yolk extender hd greter fertility rte then 20% egg yolk extender when sperm were inseminted into domestic gots. Ritr nd Slmon (1991) reported tht depending on the month, Angor got ejculted sperm displyed greter motility fter 6 hours incubtion in extenders contining 1.5% or 6% egg yolk s compred to 12% egg yolk. Webb et l. (2011) found tht fter freezing stllion semen using fst freeze (no cooling or freezing rtes) post-thw motility ws greter in 22% or 19% egg yolk s compred with 16% nd 13% egg yolk. They lso reported egg yolk concentrtion did not ffect post-thw motility of stllion semen frozen by slow freeze method. 49

64 Smith et l. (1979) compred different egg yolk concentrtions for freezing beef bull semen. It ws reported tht when freezing bull sperm in mpules, the low (1% or 2%) nd high (16% or 32%) concentrtions of egg yolk should be voided due to reduced motility nd crosome integrity. However, when freezing bull sperm in strws, there were no differences in motility mong 4, 8, 16 or 32% egg yolk. The purpose of the present study ws to determine if freezing domestic ct epididyml sperm in n extender contining n egg yolk concentrtion s low s 2% would lter sperm survivl fter thwing s compred to tht of sperm frozen in n extender with 20% egg yolk. Sperm survivl s determined by in vitro vibility, sperm motility, membrne integrity nd crosoml sttus were evluted t different time points: pre- nd post cooling, nd t 0 hour nd 3 hour fter thwing. Mterils nd Methods Experimentl Design Experiment 5.1 The objective of this experiment, ws to compre in vitro survivbility nd functionlity of domestic ct (Felis ctus) epididyml spermtozo (sperm) tht ws cryopreserved in TesT buffer (TB) extender contining 2% (Tretment A), 5% (Tretment B) or 10% (Tretment C) egg yolk (EY). For this experiment, domestic ct testes were collected on 10 seprte dys over 3 month period (n = 10) nd epididyml sperm were recovered, processed nd frozen on the sme dy testes were collected. On the 10 dys, rnge of 1 to 10 sets of testes were processed/dy for totl of 50 sets of testes. For ech dy, sperm collected from ll testes ws pooled nd eqully divided cross the three tretments. The three tretments ll contined the Tes-Tris buffer (TesT) with the concentrtion of egg yolk vrying from 2, 5 or 10%. After sperm smples were, wshed nd centrifuged they underwent the initil nlysis (pre-cool, control). Then the sperm smples were divided into three 1.6 ml liquot tubes contining one of the three egg yolk tretment extenders (Prt A). Ech tube ws plced in its own 125 ml plstic bottle wter bth filled with 22 C wter, nd then plced into 4 C wlk-in cool room nd grdully cooled to 4 C±1 C. After cooling, second sperm evlution (prefreeze/4 C) ws recorded for ech of the tretments. Following the second evlution, smples were extended 1:1 with TesT yolk buffer (2, 5, or 10%) contining 12% glycerol (Prt B) to give finl glycerol concentrtion of 6%. Then, smples were loded into 0.25 ml strws nd frozen on block of dry ice for 20 minutes before plunging inot liquid nitrogen for storge. To further sses the functionlity, cryopreserved epididyml spermtozo from ech tretment were thwed nd used for in-vitro fertiliztion protocol. Cumulus oocyte complexes 50

65 (COC) were plced in modified TCM-199 with NHCO 3 (Irving Scientific, Snt An, CA) nd cultured for 24 hours in 5% O 2, 5% CO 2, nd 90% N 2 t 38 C (IVM). For IVF, COC were coincubted with sperm frozen in either TYB or HSPM (1 million sperm/ml), under 5% CO 2 in ir t 38 C. After 18 hours, oocytes were rinsed nd cultured using 3-step system (Pope et l. 2006). Clevge frequency nd blstocyst development were ssessed on IVC dys 2 nd 8, respectively. Experimentl Procedure Bsic Procedures The bsic procedures used for Experiment 5, unless otherwise specified were: tissue collection nd trnsport, sperm collection, sperm nlysis, cryopreservtion nd thwing of sperm tretments, in vitro production medium, oocyte collection nd mturtion, in vitro fertiliztion nd culture nd sttisticl nlyses. Full detils for ech procedure were provided in Chpter 3. Sperm Extender Preprtion The bse extender, which ll three extenders were mde from, ws the commercilly vilble extender, TesT Yolk Buffer (Refrigertion Medium, Irving Scientific, Snt An, CA). This commercilly vilble extender is sold s 20% egg yolk-bsed extender tht contins Tes (176 mm), Tris (80 mm), dextrose (9 mm), penicillin-g (1,000 units/ml), streptomycin sulfte (1,000 mcg/ml) nd egg yolk (20% v/v). An identicl extender, but without egg yolk, prepred in the lb. To generte the three tretment extenders with the desired egg yolk concentrtions of 2, 5 nd 10%, the two bse extenders (the commercil nd the lb preprtions) were mixed in proportions to reduce the 20% egg yolk extender, down to 10, 5, 2% egg yolk. For exmple, 1 prt 20% egg yolk ws mixed with 1 prt 0% egg yolk to produce the 10% egg yolk extender; nd 1 prt 10% egg yolk ws mixed with 1 prt 0% egg yolk to generte the 5% egg yolk extender. For ech of the three extenders there were two prts, Prt A (without glycerol) nd Prt B (with 12% glycerol). Results Experiment 5.1 Testes Collection, Epididyml Sperm Concentrtion nd Strws frozen Routinely, three sets were processed ech dy, except for two seprte dys when only one set ws processed nd produced enough sperm to be divided cross the three tretments. When the epididyml sperm ws pooled, the verge totl number of cells collected for ech dy ws 187 x 10 6, with rnge from 83 x 10 6 to 441 x

66 The totl number of epididyml sperm ws divided evenly cross the three tretments nd depending on the sperm concentrtions rnge of 1 to 4 (0.25 ml) strws were frozen per tretment/dy, with concentrtion rnging from 19 x 10 6 to 35 x 10 6 /strw (Tble 5.1). Motility The men (±SEM) initil motility for the dily pooled epididyml sperm, ws 67.0±3.7% (Control). Men motility of sperm in 2% egg yolk fter cooling (4 C), nd t 0 hour nd 3 hour fter thwing ws 63.0±7.1%, 44.5±4.1% nd 31.5±3.3%, respectively. Men motility of sperm in 5% egg yolk fter cooling nd t 0 hour nd 3 hour fter thwing ws 75.5±2.0%, 50.0±4.0% nd 34.5±3.2%, respectively. The equivlent motility vlues in 10% egg yolk were 79.0±2.3%, 50.5±3.7% nd 31.5±3.0%, respectively (Tble 5.2) There were no significnt differences (P>0.05) between initil motility nd tht of sperm in the three tretments fter cooling /freezing. However, motility t 0 hour nd 3 hour post-thw motility ws lower (P < 0.05) thn the initil Control vlue (Tble 5.2). Sperm motility fter cooling ws significntly greter thn tht of sperm t 0 hour nd 3 hour post-thw. Similrly, motility t 0 hour post-thw ws significntly greter thn t 3 hour postthw in ll three groups (Figure 5.1). When compring cross tretments for the post-cool ssessment point, the fter cooling men motility of sperm in 2% egg yolk ws significntly less (P < 0.05) thn tht of sperm in 10% egg yolk but motility in 5% egg yolk ws not significntly different (P > 0.05) tht of the other two tretments. There ws no significnt difference in motility mong tretments t either the 0 hour or the 3 hour post-thw (Figure 5.2). Membrne Integrity The men (±SEM) percentge of sperm with n intct membrne t the initil evlution (pre-cool) ws 81.3±2.6% (Control). After cooling to 4 C, membrne integrity percentges were: 79.5±1.7%, 76.9±1.7% nd 82.5±2.8% for the 2, 5 nd 10% egg yolk tretments, respectively. Men membrne integrity t 0 hour post-thw ws 53.4±3.1%, 53.1±2.0% nd 56.5±1.4% for sperm in 2, 5 nd 10% egg yolk, respectively. After 3 hour of post-thw incubtion t 37 C, membrne integrity of sperm frozen in 2%, 5% nd 10% egg yolk ws 52.6±3.1%, 53.6±1.7% nd 53.7±2.3% (Tble 5.3). The percentge of membrne intct sperm t the initil evlution (pre-cool) ws not significntly different (P>0.05) from tht of cooled sperm in the three tretments. However, it ws significntly greter (P<0.05) thn the 0 nd 3 hour post-thw vlues in ll three tretments (Tble 5.3). 52

67 Tble 5.1. Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of ten collection dys. Thw dte Collection dte Sets processed Totl Cell Count No. of strws per extender/dy Concentrtion per strw 10/7/2008 8/4/ x x /8/2008 8/18/ x x /21/ /10/ x x /22/ /17/ x x /4/ /24/ x x /4/ /30/ x x /5/ /31/ x x /9/ /7/ x x /10/ /12/ x x /12/ /13/ x x 10 6 Dily Averge (no.) x x 10 6 Totl no The lst two rows show the men vlues for ll the collection dys nd the overll totl vlues for the experiment. Tble 5.2. Percentge of sperm motility (men±sem) before cooling, post-cooling (4 C), 0 hour nd 3 hour post-thw for ct epididyml sperm frozen in n extender contining either 2%, 5% or 10% egg yolk. Initil (Control) Post-cool 0 hour post-thw 3 hour post-thw 2% EY 67.0± ± ±4.1 b 31.5±3.3 b 5% EY 67.0± ± ±4.0 b 34.5±3.2 b 10%EY 67.0± ± ±3.7 b 31.5±3.0 b EY = egg yolk, Trt = (Tretment);,b Superscripts designte significnt difference only between initil motility nd individul vlues for ech extender t ech time period (P<0.05). 53

68 b b b c c c Figure 5.1. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 54

69 ,b b Figure 5.2. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm motility before (pre- nd post-cool) nd fter (0 h nd 3 h)freezing.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 55

70 Membrne integrity fter cooling ws significntly greter thn fter thwing, both t 0 nd 3 hours post-thw in ll three tretments; however, the 0 nd 3 hours post-thw vlues were not significntly different in ny of the three tretments (Figure 5.3). Lstly, there were no significnt differences in sperm membrne integrity mong the three tretments for ny of the three ssessment times (Figure 5.4). Acrosoml Sttus For this experiment, the men initil vlue for epididyml sperm with intct crosomes ws 85.6±1.6% (Control). After cooling to 4 C, 85.7±2.2% nd 85.9±1.9% of sperm in 2 nd 5% egg yolk extender hd intct crosomes, respectively. The 10% egg yolk extender resulted in sperm with slightly lower men intct crosoml vlue of 82.1±2.2%. At 0 hour post-thw, the percentge of sperm with intct crosomes in 2, 5 nd 10% egg yolk extender were: 62.5±2.5%, 60.1±3.1% nd 59.1±2.4%, respectively. Of the three tretments, the 2% egg yolk extender hd the highest men percentge of sperm with intct crosomes t 3 hour post-thw (56.3±2.5%). The 10% egg yolk tretment hd the lowest vlue (52.8±3.0%), while the 5% egg yolk tretment hd 56.1±2.3% of sperm with intct crosomes (Figure 5.5). The percentge of sperm with intct crosomes before cooling ws not different from tht of the three post-cool percentges; however, the pre-cool percentge ws significntly different (P < 0.05) from both the 0 hour nd 3 hour post-thw vlues in the three tretments (Tble 5.4). After cooling the percentges of sperm with intct crosome were significntly greter thn both the 0 hour nd 3 hour post-thw vlues within the three tretments. There ws no significnt difference in crosoml sttus between the 0 hour nd 3 hour evlutions within the three tretments. There were no significnt differences mong the three tretments t ny of the ssessment times (Figure 5.6). In Vitro Fertiliztion Due to limited vilbility of oocytes, we exmined in vitro functionlity of sperm frozen in 2% egg yolk extender s compre to tht of non-egg yolk extender (Humn Sperm Preservtion Medium, HSPM) evlution in n erlier experiment (Experiment 5.1). Clevge frequency of 283 in vitro-mtured oocytes ws 76% in the TesT Buffer extender with 2% egg yolk, versus 73% in HSPM extender. The percentge of embryos tht developed to the blstocysts stge fter IVF with sperm frozen in 2% egg yolk extender ws 42%, versus 38% in HSPM extender (Figure 5.7). 56

71 Tble 5.3. Percentge of sperm with intct membrnes (membrne integrity) (men±sem) before cooling, post-cooling (4 C), 0 hour nd 3 hour post-thw for ct epididyml sperm frozen in n extender contining either 2%, 5% or 10% egg yolk. Initil (Control) Post-cool 0 hour post-thw 3 hour post-thw Trt 2% EY 81.3± ± ±3.1 b 52.6±3.1 b Trt 5% EY 81.3± ± ±2.0 b 53.6±1.7 b Trt 10%EY 81.3± ± ±1.4 b 53.7±2.3 b EY = egg yolk, Trt = Tretment;,b Superscripts designte significnt difference only between initil membrne integrity nd individul vlues for ech extender for ech time period (P<0.05). Tble 5.4. Percentge of ct epididyml sperm with intct crosomes (crosoml sttus) (men±sem) before cooling, post-cooling (4 C), 0 hour nd 3 hour post-thw for ct epididyml sperm frozen in n extender contining either 2%, 5% or 10% egg yolk. Initil (Control) Post-cool 0 hour post-thw 3 hour post-thw Trt 2% EY 85.6± ± ±2.5 b 56.3±2.5 b Trt 5% EY 85.6± ± ±3.1 b 56.1±2.3 b Trt 10%EY 85.6± ± ±2.4 b 52.8±3.0 b EY = egg yolk, Trt = Tretment;,b Superscripts designte significnt difference only between initil crosoml sttus nd individul vlues for ech extender for ech time period (P<0.05). 57

72 b b b b b b Figure 5.3. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 58

73 Figure 5.4. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm membrne integrity before (pre- nd post-cool) nd fter (0 h nd 3 h),b freezing. Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 59

74 b b b b b b Figure 5.5. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 60

75 Figure 5.6. Effect of different concentrtions of egg yolk extenders (2%, 5% nd 10%) on ct epididyml sperm crosoml sttus before (pre- nd post-cool) nd fter (0 h nd 3 h) freezing.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 61

76 (53%) 73 (52%) 2% EY HSPM % Developed (42%) 28 (38%) 10 0 Cleved Blstocysts Figure 5.7. IVF percentges when oocytes were fertilized with domestic ct epididyml sperm frozen in TesT buffer extender contining 2% egg yolk or HSPM extender (from Experiment 4.1). Blstocysts percentges were derived from the number of embryos produced (Dy 2) not from the totl number of oocytes plced into IVF (Blstocysts% = no. of blstocysts/no. of embryos X 100). 62

77 Discussion Currently, with the use of biologicl stins (Mito trcker green, SYBR 14/PI, FITC/PNA, Hoechst 33342), flow cytometry is used to nlyze mitochondril function, membrne integrity, crosoml sttus nd for sex sorting sperm (Hossin et l., 2011). However, in the presence of extenders contining egg yolk or other prticle mtter from niml products (egg yolk or milk) sperm concentrtion nd percentge of non-vible sperm my be over-estimted by flow cytometry (Petrunkin nd Hrrison, 2010). Reducing the mount of egg yolk in semen extenders would llow for more ccurte sperm nlysis. Mechnicl nlysis nd ctegoriztion of sperm during sorting is improved in cler extender (Gibb et l., 2011). However, for reduced egg yolk extender to be prcticl it needs to protect sperm s effectively s extenders contining niml proteins. The lck of sttisticl difference when compring cross tretments for the post-thw vlues reported in our results were not in greement with those from recent report on the cryopreservtion of bor ejcultes (Blnch et l., 2014). A significnt decrese between 0 hour post-thw motility ws noted when bor semen ws frozen in 20% egg yolk extender when compred with 10% egg yolk extender (Blnch et l., 2014). Unlike our findings, Cbrer et l. (2005) reported tht frozen-thwed Cnry buck semen diluted in 12% egg yolk extender resulted in significntly higher sperm motility, membrne integrity nd crosoml sttus thn 6% egg yolk; nd likewise the 6% egg yolk hd significntly higher vlues thn the 1.5% egg yolk. Ritr nd Slmon (1991), lso showed difference between post-thw survivl rtes when ngor buck semen ws frozen in extenders contining different concentrtions of egg yolk. However, it ws noted tht the egg yolk concentrtion tht produced the highest post-thw vlues vried by month. When bucks were collected in Mrch sperm in the 12% egg yolk extender hd the gretest post-thw survivl. Between the months of April nd June, the 6% egg yolk produced the best post-thw vlues. In July, 1.5% egg yolk ws the superior concentrtion nd by August 0% egg yolk yielded the best post-thw survivl. No significnt difference mong the post-thw sperm vlues long with the IVF/IVC dt showing the vibility of sperm tht ws frozen-thwed in 2% egg yolk extender to produce in vitro-derived ct embryos, leds us to conclude tht 2% egg yolk extender would be n cceptble replcement for the more commonly used 20% egg yolk extender when freezing domestic ct epididyml sperm. The present results re supported by those of Mulley et l. (1988) who reported the birth of Fllow deer fwn, fter 3 does were rtificilly inseminted with ejculted semen, frozen in 2.25% egg yolk extender. 63

78 CHAPTER VI EFFECT OF PENTOXIFYLLINE CONCENTRATIONS AND TIME OF EXPOSURE ON THE POST-THAW SURVIVAL OF DOMESTIC CAT EPIDIDYMAL SPERM Introduction The bility to increse sperm motility my hve benefits in ssisted reproductive techniques. An increse in motility could men n increse in the totl number of spermtozo (sperm) tht rech the site of fertiliztion following rtificil insemintion. Incresed sperm motility could lso enhnce in vitro fertiliztion rtes. For the purpose of this study, stimulting sperm motility could increse the number nd qulity of sperm recovered in n isolte type of grdient fter centrifugtion. The incresed vible sperm number would likely trnslte into n incresed recovery rte when sperm where sex sorted with flow cytometer. Vrious gents hve been used to stimulte sperm motility in different mmmlin species. Stchecki et l. (1994; 1995) showed tht the stimulting gents pentoxifylline, cffeine nd 2 -deoxydenosine ll incresed the post-thw motility of domestic ct ejculted nd epididyml sperm. In n extensive study in the dog, Milni et l. (2010) found tht pentoxifylline, cffeine nd 2 -deoxydenosine ll hd positive effect on post-thw sperm motility but t different time points, relting to thwing rtes nd stimulnt concentrtion. Mirshokrei et l. (2011) reported tht when ejculted dog sperm were incubted for 2 hours with extender plus pentoxifylline, sperm overll motility ws incresed over the control smples. Also, significnt increse in crosome rected nd sperm cpcittion over tht of the controls ws reported when dog sperm were exposed to higher concentrtions (10 mm nd 100 mm) of pentoxifylline. Recent studies hve investigted the effects of pentoxifylline on equine epididyml sperm prmeters. In one exmple, Gusti et l. (2013) reported tht using pentoxifylline in flushing medium when collecting equine epididyml sperm, incresed motility, progressive motility, stright line velocity nd curviliner velocity. The number of rpid moving sperm were lso nlyzed immeditely fter collection nd gin fter 15 minutes of incubtion with pentoxifylline. Subsequently, sperm were wshed nd pentoxifylline removed, difference in the motility chrcteristics between the pentoxifylline tretment nd the control were no longer significntly different t the pre-freeze or post-thw evlutions. Furthermore, it ws reported tht when flushing equine epididyml sperm with medium contining pentoxifylline there ws no negtive post-thw effects on the sperm membrne integrity during the 0 hour post-thw evlutions. 64

79 Stephens et l. (2013) showed tht post-thw exposure of stllion ejculted sperm to pentoxifylline resulted in n increse of sperm overll motility, progressive motility, curviliner velocity, verge pth velocity nd stright-line velocity. Furthermore, the sperm treted with the pentoxifylline tretment hd vlues tht were significntly greter thn the cffeine-treted or turine-treted sperm. Also, the pentoxifylline-treted sperm exhibited n increse in in-vitro longevity tht ws greter thn the other two tretments. In the current study, the effect of three concentrtions of pentoxifylline on feline epididyml sperm ws evluted before nd fter cryopreservtion. Sperm motility, membrne integrity nd crosoml sttus were ll mesured nd these results would help to determine if it would be more beneficil to expose feline epididyml sperm to pentoxifylline before centrifugtion nd/or during the thwing process. Mterils nd Methods Experimentl Design Experiment 6.1 In n effort to increse the post-thw motility of domestic ct (Felis ctus) epididyml sperm, frozen semen strws were thwed nd then diluted with HEPES-199 contining different concentrtions of pentoxifylline (0mM (Tretment A), 1mM (Tretment B) or 5mM (Tretement C)). This experiment consisted of collecting domestic ct testes over one month period, from locl veterinry clinic in the New Orlens, LA re. A totl of 16 sets of domestic ct testes were collected nd processed to fill nd freeze totl of 16 (0.5 ml) strws. Epididyml sperm were frozen in 2% egg yolk-bsed extender, nd 11 of 16 strws were used in this experiment. Epididyml sperm were recovered nd frozen on the sme dy tht the testes were collected. From three to five sets of testes were processed/dy on the four collection dys nd sperm collected from ll the testes ws pooled nd frozen. Experiment 6.2 The second experiment ws designed to increse post-thw motility of domestic ct (Felis ctus) epididyml sperm using different concentrtions of pentoxifylline. The collecting, processing, freezing, nlyzing nd thwing of the epididyml sperm for this second experiment ws the sme s in Experiment 6.1, however, unlike the previous experiment the sperm were not pre-exposed to pentoxifylline for the first centrifugtion stge nd only exposed to pentoxifylline for the post-thw tretments. Collection of testes from locl veterinry clinics in the New Orlens re occurred dily Mondy thru Fridy, however, the processing nd freezing of the epididyml sperm for this experiment took plce on three seprte dys over one week 65

80 time period. During tht week, the processing of 13 set of testes resulted in 12 (0.5 ml) strws nd ten (n = 10) of those strws were thwed for use in this experiment. Experimentl Procedure Bsic Procedures Experiments 6.1 nd 6.2 used the following bsic procedures, unless otherwise stted: tissue collection nd trnsport, sperm collection, sperm nlysis, cryopreservtion nd sttisticl nlysis. Full detils for ech bsic procedure were provided in Chpter 3. Preprtion of Semen Extender nd Pentoxifylline Tretment Solutions The extender, which ll epididyml sperm ws frozen, ws 2% egg yolk-bsed extender which ws mde by diluting commercilly vilble 20% egg yolk extender (Irving Scientific, Snt An, CA). This commercilly vilble extender is sold s 20% egg yolkbsed extender tht contins Tes (176 mm), Tris (80 mm), dextrose (9 mm), penicillin-g (1,000 units/ml), streptomycin sulfte (1,000 mcg/ml) nd egg yolk (20% v/v). The formultion of the 2% egg yolk extender ws ccomplished by mking seprte extender contining the sme concentrtions of Tes, Tris, dextrose, penicillin-g nd streptomycin sulfte but no egg yolk (0% egg yolk). The 20% egg yolk extender ws then diluted with the 0% egg yolk extender until the extender ws down to the desired concentrtion of 2% egg yolk. For exmple, 1 prt 20% egg yolk mixed with 9 prts 0% egg yolk would generte the 2% extender. There ws lso 12% glycerted portion of the extender (Prt B) which lso hd 2% egg yolk nd ws mde in the sme mnner s bove. Before use in the experiments, two stock solutions of pentoxifylline (PX) were mde for ech of the 1mM nd 5mM tretments. The 1mM nd 5mM stock solutions were mde by dding nd grms of powdered pentoxifylline respectively to 10 ml of Tyrodes buffer. The two stock solutions were then liquoted, lbeled nd plced in -80 C freezer until use. Two thw solutions were mde for ech of the pentoxifylline dilution tretments, the mkeup nd volumes of the pentoxifylline stocks vs. He-199 re given in Tble 6.1. Pre-freeze Exposure, Thwing nd Sperm Dilution with Pentoxifylline After pooled sperm ws collected nd wshed sperm ws exposed to 1mM concentrtion of pentoxifylline with hopes tht the exposure would increse sperm pellet size during centrifugtion in sperm density grdient. This 1mM dose of pentoxifylline pplied before the first centrifugtion did not occur for experiment 6.2. Following centrifugtion, pelleted sperm were evluted for initil pre-cool vlues. Strws were removed from liquid nitrogen nd exposed to ir (22 C) for 10 seconds nd then immeditely immersed in 60 C wter bth for 5 seconds. Once removed from the wter bth, strw contents were emptied into pre-wrmed 66

81 Tble 6.1. Post-thw dilution tretments nd the formuls of how the tretments were constructed on ech dy the strws were thwed. 0mMPentoxifylline Pentoxifylline Stock 0mM (µl) HEPES-199 (µl) Thw Solution I Thw Solution II mMPentoxifylline Pentoxifylline Stock 1mM (µl) HEPES-199 (µl) Thw Solution I Thw Solution II mMPentoxifylline Pentoxifylline Stock 5mM (µl) HEPES-199 (µl) Thw Solution I Thw Solution II

82 (37 C) 1.6 ml tubes. The volume of the thwed sperm ws mesured nd divided eqully into 3 (1.6 ml) micro centrifuge tubes nd diluted with HEPES mM(Tretment A), 1mM(Tretment B) or 5mM(Tretment C) of pentoxifylline in 7 step fixed molrity method modified from Go et l. (1995). After dilution with HEPES pentoxifylline, smples were centrifuged nd supernte ws removed. The sperm pellet ws resuspended with HEPES-199 nd post-thw nlysis ws recorded, smples were plced in 37 C incubtor for three hours nd finl post-thw evlutions were recorded. Thw Solution I from Tble 6.1 ws dded to smples in 4 steps, the first ddition ws 15% of the originl thw volume, second step ws 20% of the originl thw volume, this continues for the third nd fourth steps with 25 nd 40% of the originl thw volume, respectively (~10 minutes between steps). At the completion of step 4 the volume of the smples ws 2X tht of the initil volume. Steps 5 thru 7 used the Thw Solution II from Tble 6.1 nd were dded to the smples t 33, 66 nd 200% of the volume fter step 4. After ll the dilutions re dded the finl volume of ech smple is ~8 times tht of the initil volume t thwing. The fully diluted smples re then centrifuged t 200 x g for 10 minutes then, supernte ws removed nd sperm pellets were resuspended in 20 µl of HEPES-199 nd plced in 37 C incubtor for 3 hours. At 0 nd 3 hours post-thw incubtion motility, membrne integrity nd crosoml sttus were nlyzed s previously described. Results Experiment 6.1 Testes Collection, Epididyml Sperm Concentrtion nd Strws frozen For this experiment, three to five sets of testes were processed per dy, for totl of t lest 16 sets of testes. Epididyml sperm from the 16 pirs of testes produced totl of ml strws which were frozen nd 11 of those strws were lter thwed for use in this experiment (n=11). When the epididyml sperm ws pooled, the verge totl number of cells collected for ech dy ws x 10 6, with rnge from 108 x 10 6 to 295 x Totl number of epididyml sperm determined how mny strws would be loded nd frozen for tht dy, three to five strws were frozen for ech of the four processing dys. Concentrtion of sperm per ech of the 16 strws rnged from 59 X 10 6 to 44.7 x 10 6 cells per strw (Tble 6.2). Motility The men (±SEM) motility for the epididyml sperm t the initil nd post-cool (4 C) time periods were 76.3±2.4% nd 72.5±1.4%, respectively. Post-thw epididyml sperm tht ws diluted with 0mM(Tretment A), 1mM(Tretment B) nd 5mM(Tretment C) of pentoxifylline, 68

83 resulted in men 0 hour post-thw motility vlues of 55.5±4.1%, 58.2±3.0% nd 65.5±3.1%, respectively. After 3 hour of post-thw incubtion t 37 C, totl sperm motility for the 0mMtretment ws 36.4±2.4%, while the other two tretments (1mM nd 5mM) hd identicl 3 hour post-thw motility vlues of 38.2±1.8 nd 38.2±2.6%, respectively. (Tble 6.3). No significnt difference ws detected (P>0.05) when compring between the initil motility nd the motility of the sperm t the post-cool ssessment. Predictbly, there ws significnt decrese (P<0.05) between the initil motility vlue nd both the 0 hour nd 3 hour post-thw vlues of ll three thwing tretments (Tble 6.3). There ws no significnt difference between the post-cool vlue nd the 0 hour postthw motility vlue for the 5mMtretments. However, there ws significnt difference between the post-cool vlue nd the 0 hour post-thw vlues for both the 0mMnd 1mMtretments. Also, ll the 3 hour post-thw motility vlues were significntly less thn their 0 hour counterprts for ll three tretments (Figure 6.1). When compring cross the three tretments for the 0 hour post-thw ssessment, no significnt difference ws detected. Similrly, there ws no significnt difference when compring cross tretments for the 3 hour post-thw evlution time (Figure 6.2). Membrne Integrity The men percentge of epididyml sperm with intct membrnes t the initil ssessment ws 83.8±1.8%, fter llowing smples to cool to 4 C (post-cool), the men percentge ws 84.5±2.7%. When evluting the membrne integrity percentges of the epididyml sperm for the 0 hour post-thw tretments, the 0mMtretment hd vlue of 64.3±1.5% while the 1 nd 5mMtretments hd vlues of 63.4±1.4 nd 62.7±1.4%, respectively. At 3 hours post-thw the 0nM, 1mMnd 5mMtretments hd membrne integrity vlues of 55.7±2.0, 51.8±1.8 nd 53.1±1.6, respectively (Tble 6.4). There ws no significnt difference (P>0.05) between the initil nd post-cool vlues when mesuring percentge of intct membrnes. However, the initil membrne integrity vlue ws significntly greter thn the ll three of the 0 hour nd 3 hour post-thw tretments (Tble 6.4). The post-cool vlue ws lso significntly greter thn ll the 0 hour nd 3 hour postthw vlues. Also, there ws significnt decrese when compring within tretments but cross time, between the 0 hour nd 3 hour post-thw vlues for ll tretments (Figure 6.3). There ws no significnt difference detected when compring mong the three tretments for neither the 0 hour or 3 hour post-thw time periods (Figure 6.4). 69

84 Tble 6.2. Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of four collection dys. Collection dte Sets processed Totl Cell Count No. frozen per dy Concentrtion per strw 2/12/ x x /19/ x x /3/ x x /4/ x x 10 6 Dily verge vlues x x 10 6 Totl no The lst two rows show the men vlues for ll the collection dys nd the overll totl vlues for the experiment. Tble 6.3. Percent motility (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline. Initil (Control) Post-cool 0 hour post-thw 3 hourss post-thw 0mMPX (Trt A) 76.3± ± ±4.1 b 36.4±2.4 b 1mMPX (Trt B) 76.3± ± ±3.0 b 38.2±1.8 b 5mMPX (Trt C) 76.3± ± ± ±2.6 b PX = pentoxifylline, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility to the post-cool nd individul vlues for ech pentoxifylline thwing tretments t ech post-thw time period (P<0.05). 70

85 b b c c c Figure 6.1. Percent motility (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours postthw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 71

86 b b b Figure 6.2. Percent motility (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours postthw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 72

87 Acrosoml Sttus The initil men vlue for feline epididyml sperm with intct crosomes ws 83.0±3.4%.After the smple ws extended nd cooled to 4 C the percent of intct crosomes ws 89.0±2.8%. The 0 hour post-thw percentge of intct crosomes for epididyml sperm tht were treted with the 0nM, 1mMnd 5mMpentoxifylline tretments were 68.9±1.8, 63.4±2.0 nd 64.7±3.1%, respectively. While, the results for the 3 hour post-thw ssessment were 59.1±1.6, 51.6±1.5 nd 53.5±1.2% for the 0nM, 1mMnd 5mMof pentoxifylline tretments, respectively (Tble 6.5). There ws no significnt difference (P>0.05) when compring the crosoml sttus between the initil nd 4 C vlues There ws however, significnt difference (P<0.05) between the initil crosoml sttus nd ll of the 0 hour nd 3 hour post-thw vlues (Tble 6.5). There ws significnt decrese between the single post-cool vlue nd ll of the 0 hour post-thw vlues. Acrosoml sttus continued to significntly decrese when compring within the tretments, between the 0 hour nd 3 hour post-thw vlues for ll tretments (Figure 6.5). There ws no significnt difference detected when compring cross tretments for neither the 0 hour nor the 3 hour post-thw vlues (Figure 6.6). Experiment 6.2 Testes Collection, Epididyml Sperm Concentrtion nd Strws Frozen Sperm ssessment vlues for this experiment re from thwing 9 strws (n= 9) frozen on the three seprte dys over the one week period. During tht week, totl of 13 sets of testes were processed with rnge of three to seven sets processed per dy. The verge number of cells collected for ech dy ws x 10 6, with rnge from 108 x 10 6 to 284 x Totl number of epididyml sperm determined how mny strws would be frozen for tht dy, 2 to 5 strws were frozen for ech of the three processing dys. Concentrtion of sperm per strw rnged from 53 x 10 6 to 55 x 10 6 cells (Tble 6.6). Motility The men (±SEM) initil nd 4 C (post-cool) motility vlues of the epididyml sperm for this experiment ws 80.0±2.9 nd 83.3±3.3, respectively. Motility vlues for the 0mM (Tretment A), 1mM(Tretment B) nd 5mM(Tretment C) tretments, t the 0 hour post-thw evlution were 60.0±2.5, 59.5±2.6 nd 63.0±2.6, respectively. Totl sperm motility fter being held in 37 C incubtor for 3 hours post-thw ws 33.0±3.0, 44.0±2.7 nd 39.0±2.3% for the 0nM, 1mMnd 5mMtretments, respectively (Tble 6.7.). 73

88 Tble 6.4. Percent membrne integrity (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline. Initil (Control) Post-cool 0 hours post-thw 3 hours post-thw 0mMPX (Trt A) 83.8± ± ±1.5 b 55.7±2.0 b 1mMPX (Trt B) 83.8± ± ±1.4 b 51.8±1.8 b 5mMPX (Trt C) 83.8± ± ±1.4 b 53.1±1.6 b PX = pentoxifylline, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility to the post-cool nd individul vlues for ech pentoxifylline thwing tretments t ech post-thw time period (P<0.05). Tble 6.5. Percent crosome integrity (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline. Initil (Control) Post-cool 0 hour post-thw 3 hours post-thw 0mMPX (Trt A) 83.0± ± ±1.8 b 59.1±1.6 b 1mMPX (Trt B) 83.0± ± ±2.0 b 51.6±1.5 b 5mMPX (Trt C) 83.0± ± ±3.1 b 53.5±1.2 b PX = pentoxifylline, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility to the post-cool nd individul vlues for ech pentoxifylline thwing tretments t ech post-thw time period (P<0.05). 74

89 b b b c c c Figure 6.3. Membrne intct percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 75

90 b b b Figure 6.4. Membrne intct percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 76

91 b b b c c c Figure 6.5. Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 77

92 b b Figure 6.6. Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 78

93 There ws no significnt difference (P>0.05) when compring between the initil nd 4 C ssessments. However, there ws significnt difference (P<0.05) when compring the initil motility vlue to the 0 hour nd 3 hour post-thw vlues for ll three tretments (Tble 6.7). The 4 C post-cool vlue ws significntly greter thn ll three tretments t the 0 hour nd 3 hour post-thw ssessments. Also, when compring within tretments ll three 0 hour post-thw vlues were significntly different from their 3 hours post-thw counterprts (Figure 6.7). When compring mong tretment groups, there ws no significnt difference t the 0 hour post-thw ssessment time. However, t the 3 hour post-thw ssessment, there ws significnt difference between the 0mMnd 1mMtretments, but the 5mMtretment ws not significntly different from either the 0 or 1mMtretments (Figure 6.8). Membrne Integrity The men (±SEM) vlue for the initil membrne integrity ssessment of the feline epididyml sperm ws 83.0±3.8%. After extending the smple nd llowing it to cool to 4 C, the post-cool evlution hd vlue of 86.7±1.2%. The membrne integrity t the 0 hour post-thw period for the 0nM, 1mMnd 5mMpentoxifylline tretments were 66.6±2.3, 69.7±2.0 nd 68.4±1.6%, respectively. Following the 0 hour post-thw redings, tretments were plced into 37 C incubtor nd llowed to sit for 3 hours, t the completion of the three hours the membrne integrity vlues for the 0nM, 1mMnd 5mMtretments were 60.0±1.8, 56.8±1.8 nd 54.9±1.6%, respectively (Tble 6.8). There ws no significnt difference (P>0.05) between the initil nd post-cool vlues. In contrst, ll of the 0 hour nd 3 hour post-thw vlues were significntly lower (P<0.05) thn the initil membrne integrity vlue (Tble 6.8). The post-cool vlue ws significntly higher thn the 0 hour nd the 3 hour post-thw vlues for ll three tretments. Furthermore, ll three of the 0 hour post-thw vlues were significntly greter thn their 3 hour post-thw counterprts for ll three tretments (Figure 6.9). Although, there ws no significnt difference cross tretments for the 0 hour post-thw ssessment, t 3 hours post-thw the percentge of membrne intct cells for the 0mM tretment ws significntly different (P<0.05) from the 5mM tretment. However, the 1mM tretment ws not significntly different from either the 0mM or 5mM tretments (Figure 6.10). 79

94 Tble 6.6. Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of three collection dys. Collection dte Sets processed Totl Cell Count No. frozen per dy Concentrtion per strw 4/1/ x x /2/ x x /8/ x x 10 6 Dily verge vlues x x 10 6 Totl no The lst two rows show the men vlues for ll the collection dys nd the overll totl vlues for the experiment. Tble 6.7. Percent motility (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline. Initil (Control) Post-cool 0 hour post-thw 3 hours post-thw 0mMPX (Trt A) 80.0± ± ±2.5 b 33.0±3.0 b 1mMPX (Trt B) 80.0± ± ±2.6 b 44.0±2.7 b 5mMPX (Trt C) 80.0± ± ±2.6 b 39.0±2.3 b PX = pentoxifylline, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility to the post-cool nd individul vlues for ech pentoxifylline thwing tretments t ech post-thw time period (P<0.05). 80

95 b b b c c c Figure 6.7. Motility percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 81

96 b,b Figure 6.8. Motility percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 82

97 Acrosoml Sttus The men Acrosoml sttus vlues for the initil nd post-cool (4 C) ssessments were 93.0±2.1 nd 93.7±1.9%, respectively. After thwing nd diluting smples with the pentoxifylline tretments the 0 hour post-thw Acrosoml sttus vlues were 73.1±2.1, 71.7±1.9 nd 68.3±2.1% for the 0nM, 1mMnd 5mMtretments, respectively. The 3 hour post-thw ssessment vlues for the 0nM, 1mMnd 3mMtretments were 62.2±2.9, 61.3±2.0 nd 58.8±2.4, respectively (Tble 6.9). There ws no significnt difference (P>0.05) between the initil nd the post-cool vlues. However, the initil crosoml sttus percentge ws significntly greter (P<0.05) thn ll the 0 hour nd 3 hours post-thw vlues (Tble 6.9). Like the initil vlue for crosoml sttus, the post-cool vlue ws significntly better thn the three 0 hour nd the three 3 hours post-thw vlues. When compring within the tretments, ll the 0 hour post-thw tretments were significntly different from ll the 3 hours post-thw vlues (Figure 6.11). The percentges of intct crosomes cross tretments t 0 hour nd 3 hours postthw were not significntly different (Figure 6.12). Discussion Pentoxifylline is smll molecule used to increse sperm motility nd is used most often in humn ssisted reproductive lbortories (Clogero et l., 1998). Some exmples of its use in domestic species re Stchecki et l. (1994), Stchecki et l. (1995) in the domestic ct, Mxwell et l. (1995) in the got nd Milni et l. (2010) in the dog. There re certin time points when it would be beneficil to expose sperm to pentoxifylline, for instnce, when collecting epididyml sperm it is often necessry to wsh nd centrifuge the sperm smple through density grdient (Filliers et l., 2008). This density grdient seprtes live motile sperm from blood, tissue cells nd immobile sperm (Phillips et l., 2012). Theoreticlly one could increse the number of sperm recovered the density grdient, by incresing the percentge of motile sperm going into the grdient. Holt et l. (1997) hve reported tht bor sperm motility (velocity nd durtion) were good in vitro indictors of bor fertility. Another crucil time to stimulte sperm motility could be during post-thw evlution to record n estimtion of mle fertility before rtificil insemintion or when incubting sperm with oocytes during in vitro fertiliztion. For these two experiments the two most notble events for feline sperm motility ws in Experiment 6.1, when the men 0 hour post-thw motility vlue for sperm treted with 5mMpentoxifylline did not decrese enough, even with the stress of cryopreservtion, to be 83

98 Tble 6.8. Percent intct membrnes (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hour post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline. Initil (Control) Post-cool 0 hours post-thw 3 hours post-thw Trt 0mMPX 83.0± ± ±2.3 b 60.0±1.8 b Trt 1mMPX 83.0± ± ±2.0 b 56.8±1.8 b Trt 5mMPX 83.0± ± ±1.6 b 54.9±1.6 b PX = pentoxifylline;,b Superscripts designte significnt difference only between initil motility to the post-cool nd individul vlues for ech pentoxifylline thwing tretments t ech post-thw time period (P<0.05). Tble 6.9. Percent crosome integrity (men±sem) of initil, post-cool (4 C), 0 hour post-thw nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were frozen, thwed then diluted with He-199 medium contining either 0, 1, or 5 nnomoles of pentoxifylline. Initil (Control) Post-cool 0 hours post-thw 3 hours post-thw Trt 0mMPX 93.0± ± ±2.1 b 62.2±2.9 b Trt 1mMPX 93.0± ± ±1.9 b 61.3±2.0 b Trt 5mMPX 93.0± ± ±2.1 b 58.8±2.4 b PX = pentoxifylline;,b Superscripts designte significnt difference only between initil motility to the post-cool nd individul vlues for ech pentoxifylline thwing tretments t ech post-thw time period (P<0.05). 84

99 b b b c c c Figure 6.9. Membrne integrity percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b,c Men vlues within tretments but cross time with different superscripts re significntly different (P<0.05). 85

100 ,b b Figure Membrne integrity percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b Men vlues cross tretments but within time with different superscripts re significntly different (P<0.05). 86

101 b b b c c c Figure Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b,c Men vlues within tretments but cross time with different superscripts re significntly different (P<0.05). 87

102 b b b Figure Acrosoml sttus percentge (men±sem) of initil, post-cool (4 C), 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed, frozen nd lter thwed with 0, 1 or 5 nnomolr pentoxifylline solution.,b Men vlues cross tretments but within time with different superscripts re significntly different (P<0.05). 88

103 significntly different from the initil or the post-cool motility vlues. The second event occurred in Experiment 6.2 when the 1mM pentoxifylline tretment hd significntly greter sperm motility thn the 0mM tretment for the 3 hour post-thw ssessment. In both experiments, feline sperm smples were not divided into tretments until the 0 hour post-thw ssessment, so s result there is only one initil nd one post-cool motility vlue. There ws no significnt difference between the initil nd post-cool motility vlues for either experiment. Also, in both experiments there ws no significnt difference mong tretments for ny time point when mesuring sperm motility. Silv et l. (2009) lso showed tht pentoxifylline hd no ffect on the post-thw motility of frozen-thwed ejculted equine sperm. However, our results contrdict the findings by Stchecki et l. (1994) who reported tht lthough pentoxifylline hd no effect on the motility of freshly collected epididyml ct sperm, there ws significnt increse in post-thw sperm motility prmeters when pentoxifylline ws used to tret thwed ct epididyml sperm. When evluting the sperm membrne integrity results, the only significnt difference ws noted in Experiment 6.2 t the 3 hour post-thw ssessment when the 0mMpentoxifylline tretment hd membrne integrity vlue tht ws significntly greter thn the 5mMpentoxifylline tretment. The membrne integrity results of our study correlte with the results reported by Ortgies et l. (2012), who reported tht the membrne integrity of equine ejculted sperm ws not ffected by pentoxifylline or ny of the other sperm stimulnts evluted. Other thn the expected decline in overll crosoml sttus resulting from cryopreservtion, there ws only one significnt difference when compring mong tretments (0nm, 1mMnd 5nm) in both experiments. Our crosoml sttus results in the ct, contrdict the findings by Ortgies et l. (2012) who reported tht pentoxifylline ws the only gent tested tht incresed the percentge of live-cpcitted equine sperm when compred with control sperm. A possible explntion for the discrepncies between our results nd other reports on the effects of pentoxifylline on domestic ct epididyml sperm is the exposure time tht we llowed pentoxifylline to be in contct with the sperm. Differences in published results on the sme species but from different lbs re not uncommon. Stephens et l. (2013) reported tht pentoxifylline improved equine post-thw vlues for motility, progressive motility, semen curviliner velocity, verge pth velocity, stright line velocity, nd sperm longevity over the untreted control. Gusti et l. (2013) reported contrdicting results, noting tht lthough pentoxifylline incresed the pre-freeze equine sperm vlues for motility, progressive motility, 89

104 stright line velocity, curviliner velocity, nd percentge of rpid sperm. The pentoxifylline hd no significnt effect on ny the post-thw sperm motility prmeters. It my hve my hve been beneficil if we would hve nlyzed the post-thw sperm before introducing pentoxifylline. However, given tht our results re similr to wht ws noted in the stllion, we were initilly stisfied with our findings. Nevertheless, due to the difference in sperm motility response compred with other feline ejculted nd epididyml sperm studies further reserch would be encourged. Since our results showed no negtive effects of pentoxifylline nd there is possible benefit to its use with feline spermtozo, it is recommended to use pentoxifylline in future experiments. 90

105 Introduction CHAPTER VII SURVIVAL OF DOMESTIC CAT EPIDIDYMAL SPERM AFTER TEMPORARY COOL STORAGE OR CRYOPRESERVATION IN DEFINED EXTENDERS The bility to sex sort spermtozo (sperm) could id in the reproductive mngement of geneticlly vluble exotic species tht re in cptive breeding progrms. Since portion of sperm re lost during the flow cytometry process (Evns, 2009; Seidel, 2009), the more live motile sperm tht enter the sorting procedure, should result in higher overll sperm recovery rte. Usully sperm sorting is performed on freshly collected or extended nd cooled sperm tht hve not yet been frozen. Pope et l. (2009) collected ejculted sperm from domestic tom ct, extended the semen nd shipped it overnight to sperm sorting fcility. Subsequently, they reported the first kittens to be born from sex gender sorted sperm (Pope et l., 2009). In species, such s bovine (Bker et l., 1953), ovine (Lezm et l., 2001) or equine (Pickett et l., 1975) ejculted sperm concentrtions re gret enough tht sperm loss due to the sorting process cn be tolerted. In contrst, when collecting feline epididyml or even ejculted sperm the totl volumes re in the microliters nd the sperm concentrtions re below 5 million/ml (unpublished dt). Therefore, ctions must be mde to ensure tht the highest possible number of live sperm re entering the sorting process. A possible solution for low sperm numbers would be to combine sperm collections from different dys. However to ccomplish this, sperm collected from erlier dys would either need to be cryopreserved or stored t cooled temperture in n extender tht could mintin sperm vibility for up to week. The benefits egg yolk provides to mmmlin sperm during the cooling nd freezing process re undenible. However, finding n effective extender tht does not contin egg yolk would eliminte the need to remove the egg yolk prticles before sperm sorting. The elimintion of this step would lso men decrese in the stress tht centrifugtion imposes on the sperm nd thereby, llowing more sperm to be vilble for the sorting process. Furthermore, the survivl of sperm nd other cell types is not solely dependent on the presence of egg yolk in freezing medium. This point is demonstrted by the recent successful freezing nd thwing of other feline gmetes (Gliguis et l., 2014) nd embryos (Pope et l., 2012). The objectives of the following studies were: (1) to evlute two commercilly vilble, cler extenders tht re mrketed for the cool storge of semen, on their bility to mintin the vibility of domestic ct epididyml sperm when stored t 4 C nd then compred with 2% egg yolk extender, (2) to determine the effectiveness of these cool storge extenders on their 91

106 bility to protect domestic ct epididyml sperm during cryopreservtion nd (3) to determine the effectiveness of semen extender tht contined only slts, buffers nd glycerol (no egg yolk), when freezing sperm from room temperture, nd fter cooling to 4 C nd how they compred with sperm frozen in conventionl 2% egg yolk extender tht ws frozen fter cooling to 4 C. Mterils nd Methods Experimentl Design Experiment 7.1 The objective of this experiment ws to determine which of three semen extenders (2% Egg Yolk TesT (Tretment A), Biolife (Tretment B) nd BioXCell (Tretment C)), mintined the highest epididyml sperm survivbility when used to store domestic ct sperm t 4 C for 72 h. Testes were collected dily Mondy thru Fridy from locl veterinry clinics in the New Orlens re. Epididymides where then processed nd the epididyml sperm tht ws recovered ws extended nd cooled ll on the sme dy tht they were collected from the epididymides. Over tht 4 month period only 5 dily collections resulted in enough epididyml sperm to be spred sufficiently cross the three tretments. A totl of 24 sets of domestic ct testes were used cross the 5 different processing dys (n = 5) reported in this expermiment. During those 5 dys, rnge of 2 to 9 sets of testes were processed per dy, nd on ech of the 5 processing dys, ll sperm collected ws pooled nd eqully divided cross the three different semen extenders mentioned bove. After the pooled epididyml sperm ws collected, wshed nd centrifuged they were evluted for initil pre-tretment nlysis nd divided into 3 seprte 1.6 ml liquot tubes nd the 3 tretment extenders were dded. Ech liquot ws plced in its own 22 C wter bth, nd plced into 4 C wlk-in cooler nd llowed to cool for 24 h. Future sperm evlutions were recorded t 24, 48 nd 72 hours from when smples were plced in the 4 C. Experiment 7.2 This experiment ws to test the two most effective extenders from the previous experiment on their freezing protection of domestic ct epididyml sperm (2% Egg Yolk TesT (Tretment A), BioXCell (Tretment B)). Testes were collected dily Mondy thru Fridy from locl veterinry clinics in the New Orlens re, enough dily epididyml sperm ws collected to disperse cross the tretments on only frction of those dys. Dt for this experiment were derived from processing 36 sets of testes on six different dys over 3 month period. A totl of 22 (0.25 cc) nd 2 (0.5 cc) strws were frozen with rnge of two to eight frozen per dy, nd from 24 frozen strws 20 (n = 10) were thwed for this experiment (ten strws per tretment). 92

107 Sperm were collected from minced epididymides nd then wshed, centrifuged, nlyzed nd divided into either TesT extender with 2% egg yolk (Tretment A) or into the commercil extender BioXCell (Tretment B). Before dividing into tretments n initil motility ws tken to confirm smple ws good enough to continue with experiment, then smple ws split into tretments. Once divided nd extended to the desired concentrtions, smples were set into seprte but equl lbortory temperture (22 C) wter bths nd plced in 4 C wlkin refrigertor nd llowed to rech 4 C. After smples reched 4 C second motility ws recorded s well s the first membrne integrity nd crosoml sttus reding. Upon completion of the 4 C redings (post-cool), smples were further extended to their finl concentrtions, loded into strws nd frozen in -80 C freezer for 20 min before being plunged nd held in liquid nitrogen until thwed. Two more ssessments were recorded for motility, membrne integrity nd crosoml sttus t 0 nd 3 hours post-thw. Tble 7.1. Experimentl design for experiment 7.2. This tble illustrtes wht redings were recorded nd t wht time point they were recorded for this experiment. Initil Post-Cool 0 hour PT 3 hours PT Pre Trt. Mot % EY -- Mot, MI, AS Mot, MI, AS Mot, MI, AS BioXCell -- Mot, MI, AS Mot, MI, AS Mot, MI, AS Mot = Motility, MI = Membrne integrity, AS = Acrosoml sttus Experiment 7.3 Relizing tht the TesT extender with 2% egg yolk (Tretment A) ws the most ffective extender from the previous two experiments, but still wnting n extender with no egg yolk we decided to compre the TesT 2% egg yolk extender versus the TesT extender with 0% egg yolk (Tretment B). Also third tretment ws dded which involved using the TesT extender with 0% egg yolk but freezing from room temperture (22 C) (Tretment C), insted of cooling sperm to 4 C then freezing. Domestic ct testes were hrvested from locl veterinry clinics in the New Orlens re for two month period, however only 10 of those dys resulted in enough epididyml sperm to divide cross the three tretments. For those 10 processing dys totl of 60 sets of testes were collected resulting in totl of 33 (0.25 cc) frozen semen strws. From the 33 totl strws, 30 were thwed for this experiment 10 from ech tretment (n = 10). 93

108 Epididymides were minced nd epididyml sperm were collected, wshed nd centrifuged to cquire clen nd highly concentrted sperm pellet tht ws divided eqully into the three tretments (2%EY (Tretment A), 0%EY (Tretment B), RT 0%EY (Tretment C)). Before dividing the pellet into tretments n initil motility, membrne integrity nd crosoml sttus were obtined. After the smple ws nlyzed nd extended, the 0 nd 2%EY tretments were plced in 22 C wter bths nd then llowed to cool to 4 C in wlkin cooler. The RT 0%EY tretment ws then further extended 1:1 with the second glycerted prt of RT 0%EY extender contining 12% glycerol. The fully extended smple ws loded into 0.25 cc strws nd frozen on dry ice block for 20 minutes. After cooling to 4 C the remining two tretments (0% nd 2%EY) were dditionlly extended 1:1 with their perspective second prts contining 12% glycerol. These smples were lso loded into 0.25 cc strws frozen on dry ice block, plunged nd held in liquid nitrogen until being thwed. One strw from ech tretment ws thwed on the sme dy (n=10) nd evluted for motility, membrne integrity nd crosoml sttus t 0 nd 3 hours post-thw. The 2%EY tretment ws derived in the lbortory from commercilly vilble TesT buffer extender contining 20% egg yolk (Test Yolk Buffer). The 0%EY tretment ws the sme TesT buffer extender s the 2% EY tretment except insted of modifying the egg yolk percentge down to 2%, no egg yolk ws dded t ll. The RT 0%EY tretment ws the exct sme extender, but the difference between the tretments ws tht the 0%EY nd the 2%EY tretments were cooled down to 4 C before being frozen while RT 0%EY ws frozen stright from room temperture. Experimentl Procedure Bsic Procedures The bsic procedures used for Experiment 7.1, 7.2 nd 7.3, unless otherwise specified were: tissue collection nd trnsport, sperm collection, sperm nlysis, cryopreservtion nd thwing of sperm tretments, in vitro production medium, oocyte collection nd mturtion, in vitro fertiliztion nd culture, sttisticl nlyses. Full detils for ech procedure were provided in Chpter 3. Semen Extender Preprtion The TesT buffered 2% EY extender tht ws used in ll three experiments ws modified from the commercilly vilble extender, TesT Yolk Buffer (Irving Scientific, Snt An, CA). This commercilly vilble extender is 20% egg yolk-bsed extender tht contins Tes (176 mm), Tris (80 mm), dextrose (9 mm), penicillin-g (1,000 units/ml), streptomycin sulfte (1,000 mcg/ml) nd egg yolk (20% v/v). A second TesT extender ws formulted using the recipe 94

109 Tble 7.2. Experimentl design for experiment 7.2. This tble illustrtes wht redings were recorded nd t wht time point they were recorded for this experiment. Initil Post-Cool 0 hour PT 3 hours PT Pre Trt. Mot, MI, AS C-2%EY -- Mot Mot, MI, AS Mot, MI, AS 4 C-0%EY -- Mot Mot, MI, AS Mot, MI, AS 22 C-0% EY Mot, MI, AS Mot, MI, AS Mot = Motility, MI = Membrne integrity, AS = Acrosoml sttus bove with the bsence of egg yolk. To generte the 2% egg yolk concentrtion the 20% egg yolk extender ws diluted with the identicl TesT extender contining 0% egg yolk. For exmple, 1 prt 20% egg yolk mixed with 9 prts 0% egg yolk would generte the 2% extender. For experiments 7.2 nd 7.3 when sperm freezing ws involved there ws lso 12% glycerted portion of the TesT extenders which hd the mtching concentrtion of egg yolk depending on which TesT extender it ws used with. BioXCell (IMV Technologies, Mple Grove, MN) nd Biolife (BioLife Solutions, Bothell, WA) re both commercilly vilble synthetic extenders contining no niml protein. The Biolife rrives redy to use while the BioXCell needs to be diluted with ultrpure wter. Also BioXCell is mrketed nd used for cool storge (>4 C) or for freezing sperm, so when used in Experiment 7.2 it ws used s single step extender nd hs no second glycerted prt to be dded. Method of Applying Tretment Extenders Following the initil ssessment, sperm smples were divided into 1.6 ml conicl centrifuge tubes contining one of the specified semen extenders. For Experiment 7.1, 200 µl of ech extender ws dded to its respective tretment smple. In Experiments 7.2 nd 7.3 the volume of extender dded ws clculted depending on the desired sperm concentrtion per strw. In Experiment 7.2, since BioXCell ws one step extender, the totl volume desired ws ll dded t the sme time, before the smple ws cooled to 4 C. For Experiment 7.2 nd 7.3 the TesT extenders hd two prts, Prt A (sperm + extender) nd Prt B (glycerted prt). The extender portion of Prt A ws dded before cooling, nd Prt B ws dded fter cooling. For the RT 0%EY tretment (which ws never cooled) both prts were dded seprtely, t room temperture, but one right fter the other. Once tretments hd the correct rtio of sperm/extender smples (except RT 0%EY) were plced in 22 C wter-bth nd plced in 95

110 4 C wlk-in cooler nd llowed ~3 hours to cool to 4 C±1 C. The volumes of Prt A nd B were identicl nd ech one ws exctly hlf of the finl volume. To void osmotic injury Prt B ws dded to ll tretments in step wise mnner in four steps; 15%, 20%, 25% nd 40% of the totl Prt B to be dded (modified from Go et l. 1995). After Prt B (glycerted prt) of the extenders ws dded to their pproprite tretments, smples were loded into (0.25 cc) strws nd frozen on block of dry ice for 20 minutes before being plunged in liquid nitrogen. All smples were held in liquid nitrogen until thwed, no longer thn 3 months fter being frozen. Results Experiment 7.1 Testes Collection nd Epididyml Sperm Concentrtions For this experiment, two to nine sets of testes were processed on five (n = 5) different dys for totl of 24 sets of testes. When the epididyml sperm ws pooled, the verge totl number of cells collected for ech dy ws 187 x 10 6 with rnge from 83 x 10 6 to 441 x Ech collection dy, the totl number of epididyml sperm ws divided evenly cross the three tretments (Tble 7.3). Motility The men (±SEM) initil motility for the pooled epididyml sperm, before being split into tretments ws 71.0±3.4%. The men 24 hours vlues for epididyml sperm held t 4 C in extenders 2% EY (Tretment A), BioLife (Tretment B) nd BioXCell (Tretment C) ws 69.6±2.9%, 55.0±3.0% nd 64.2±1.1%, respectively. At 48 hours of cold storge, the men motility for the 2% EY, BioLife nd BioXCell extenders ws 67.4±4.8%, 50.4±6.3% nd 55.4±4.0% respectively. After cooling in 4 C for 72 hours (3 dys) the 2% EY tretment mintined epididyml sperm motility t 66.4±5.2% while the sperm motility in the BioLife nd BioXCell tretments decresed from their previous vlues, to 40.0±5.5% nd 49.4±4.8% respectively (Tble 7.4). The only tretment tht showed significnt decrese (P<0.05) in motility between the initil pre-cool nd the 24, 48 nd 72 hour ssessments ws the BioLife tretment. At 48 nd 72 hours, the BioXCell motility vlues hd become significntly less thn the initil pre-cool motility vlue. The 2% EY extender ws the only tretment tht never differed significntly from the pre-cool vlue even out to dy 3 of the experiment (Tble 7.4). The 2% EY tretment ws the only tretment tht did not show ny significnt difference (P>0.05) when compring cross the three time points. Likewise, the BioLife nd BioXCell tretments did not differ significntly between the 24 nd 48 hour or between the 48 nd 72 96

111 hour ssessments; but there ws significnt difference within both the BioLife nd BioXCell tretments when compring the 24 to the 72 hour vlues (Figure 7.1). At the 24 hour ssessment the men motility vlue for sperm held in the BioLife extender ws significntly less thn the 2% EY nd BioXCell tretments. There ws no significnt difference in motility when compring cross tretments t the 48 hours ssessment. At 72 hours the 2% EY tretment hd significntly greter motility vlue thn both the BioLife nd the BioXCell extenders (Figure 7.2). Membrne Integrity The initil men vlue for epididyml sperm with intct membrnes ws 75.0±5.2%. After dividing the epididyml sperm into equl prts, extended nd llowed to cool to 4 C, the 24 hours membrne integrity ssessments were 76.6±1.5%, 80.0±2.8% nd 80.2±4.5% of intct membrnes for the 2% EY, BioLife nd BioXCell extenders, respectively. After 48 hours of cold storge t 4 C, sperm diluted in the 2%EY extender hd 76.0±5.7% intct membrnes, the BioLife extender produced membrne integrity vlue of 69.8±3.7% nd the BioXCell vlue dropped to 68.6±5.4%. Following the 3 dys of cold storge t 4 C, the 2% EY, BioLife nd BioXCell extenders reported membrne integrity vlues of 66.2±4.5, 58.6±5.3, nd 60.6±3.3%, respectively (Tble 7.5). There ws no significnt difference (P>0.05) when compring the initil membrne integrity vlue to the 24 hour or the 48 hour vlues of ll three tretments. The 72 hour vlues were significntly different (P<0.05) from the initil membrne integrity for ll three tretments (Tble 7.5). There ws no significnt difference within ny of the tretments when compring between the dy 1 nd dy 2 vlues or between the dy 2 nd dy 3 vlues. However, there ws significnt difference when compring the dy 1 to dy 3 vlues within ll three tretments (Figure 7.3). There ws no significnt difference when compring cross ny of the three tretments for ny of the three time points mesured (Figure 7.4). Acrosoml Sttus The men vlue of epididyml sperm with intct crosomes before being divided into tretments ws 86.4±4.3%. After dividing into their respective tretments nd llowed to cool in 4 C for 24 hours the tretments mintined crosoml sttus vlues of 87.0±3.2%, 92.6±1.5% nd 91.2±3.1% for the2% EY, BioLife nd BioXCell tretments, respectively. The 2% EY, BioLife nd BioXCell tretments reported 48 h, men crosoml vlues of 84.4±4.3%, 95.2±1.1% nd 80.4±3.8%,respectively. The lst ssessment recorded for ech tretment ws 97

112 Tble 7.3. Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of five collection dys. Collection dte Sets processed Totl Cell Count No. of sperm per ext. 11/2/ x x /14/ x x /15/ x x /22/ x x /1/ x x 10 6 Dily verge no x x 10 6 Totl no The lst two rows show the verge vlues for ll the collection dys, nd the totl number of processed testes for the experiment. Tble 7.4. Percent motility (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders. Initil (Control) 24 hours 48 hours 72 hours 2% EY (Trt A) 71.0± ± ± ±5.2 BioLife (Trt B) 71.0± ±3.0 b 50.4±6.3 b 40.0±5.5 b BioXCell (Trt C) 71.0± ± ±4.0 b 49.4±4.8 b EY = egg yolk, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility vlue nd individul vlues for ech extender t ech time period (P<0.05). 98

113 ,b,b b b Figure 7.1. Percent motility (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 99

114 b b b Figure 7.2. Percent motility (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 100

115 Tble 7.5. Percent membrne integrity (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders. Initil (Control) 24 hr 48 hr 72 hr 2% EY (Trt A) 75.0± ± ± ±4.5 b BioLife (Trt B) 75.0± ± ± ±5.3 b BioXCell (Trt C) 75.0± ± ± ±3.3 b EY = egg yolk, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility vlue nd individul vlues for ech extender t ech time period (P<0.05). Tble 7.6. Percent crosoml sttus (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders. Initil (Control) 24 hr 48 hr 72 hr 2% EY (Trt A) 86.4± ± ± ±3.3 BioLife (Trt B) 86.4± ± ± ±1.2 BioXCell (Trt C) 86.4± ± ± ±2.1 b EY = egg yolk, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility vlue nd individul vlues for ech extender t ech time period (P<0.05). 101

116 fter 72 hours cold storge, the 2% EY extender exhibited vlue of 89.0±3.3%, while the BioLife extender recorded the highest vlue for this time point of 95.8±1.2%, nd the BioXCell reported the lowest crosoml vlue of 79.0±2.1% (Tble 7.6). When compring the initil crosoml sttus vlue to the 24, 48 nd 72 hour vlues for ll three tretments, the only significnt difference is between the initil vlue nd the 72 hour vlue of the BioXCell tretment (Tble 7.6). When compring within tretments there ws no significnt difference when compring the 24 nd 48 hour or the 48 nd 72 hour ssessments for ny of the tretments. BioXCell ws the only tretment tht showed significnt decrese within tretment when compring between dy 1 nd dy 3 vlues (Figure 7.5). There ws no significnt difference cross ny of the tretments when compring their crosoml sttus for the 24 hours ssessment. However, t the 48 hour ssessment the BioLife crosoml sttus vlue ws significntly greter thn both the 2% EY nd the BioXCell tretments, but the 2% EY nd the BioXCell tretments were not significntly different from ech other. Likewise for the 72 hour ssessment, BioXCell hd significntly lower crosoml sttus vlue thn both the 2% EY nd BioLife tretments, but the 2% EY nd BioLife tretments were not significntly different from ech other (Figure 7.6). Experiment 7.2 Testes Collection, Epididyml Sperm Concentrtion nd Strws frozen For this experiment, two to eight sets of testes were processed on six different dys (n = 6) for totl of 36 sets of feline testicles. When the epididyml sperm ws pooled, the verge totl number of cells collected for ech dy ws x 10 6 with rnge from 77 x 10 6 to 336 x 10 6 (Tble 7.7). The totl number of dily epididyml sperm ws divided evenly cross two tretments nd depending on the sperm concentrtions rnge of 1 to 4 (0.25 cc) strws were frozen per tretment, per dy. The concentrtion of the strws rnged from 26 X 10 6 to 67.5 X 10 6 per strw (Tble 7.7). Motility The initil men (±SEM) motility for the epididyml sperm before being eqully divided into two tretments ws 75.8±3.6%. The men motility vlue for epididyml sperm post-cool (4 C) in the 2% EY extender ws 76.5±3.5%, while the sperm in the BioXCell extender hd men motility of 72.7±5.2%. The men 0 hour post-thw motility vlues for the 2% EY nd the BioXCell extenders were 58.9±3.1% nd 50.9±2.3%, respectively. After incubting t 37 C for 102

117 ,b,b,b b b b Figure 7.3. Membrne intct percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 103

118 Figure 7.4. Membrne intct percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 104

119 ,b b Figure 7.5. Acrosoml sttus percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 105

120 b b b Figure 7.6. Acrosoml sttus percentge (men±sem) of initil, 24, 48 nd 72 hours of cold storge t 4 C for pooled domestic ct epididyml sperm tht were extended nd held in 2% egg yolk, BioLife nd BioXCell extenders.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 106

121 3 hours, the epididyml sperm in the 2% EY extender hd motility vlue of 36.5±1.5% while the BioXCell extender produced sperm with motility of 35.4±2.3% (Tble 7.8). In both tretments there ws significnt decrese (P<0.05) in motility from the postcool to the 0 hour post-thw nd gin from the 0 hour to the 3 hour post-thw ssessments (Figure 7.7). However, there ws no significnt difference when compring between tretments t ny of the ssessment times (Figure 7.8). Membrne Integrity After splitting the smple into equl tretments, the extended sperm ws plced in wlk-refrigertor to cool to 4 C, the first membrne integrity vlue for the 2% EY tretment ws 81.8±3.5% while the BioXCell tretment hd 86.0±2.2% intct membrnes. After thwing strws from both tretments, the percent of intct membrnes t the 0 hour post-thw time point were 63.2±2.3% nd 57.2±2.2% for the 2% EY nd BioXCell tretments, respectively. Following 3 hours incubtion in 37 C incubtor, thwed epididyml sperm smples were evluted for finl ssessment. The epididyml sperm frozen in the 2% EY extender hd men vlue of 58.6±2.3%, while the BioXCell tretment recorded men vlue of 52.0±1.9% (Tble 7.9). The post-cool vlues for both tretments were significntly greter (P<0.05) thn both the 0 hour nd 3 hour ssessments. However there ws no significnt decrese (P>0.05) for either tretment when compring between the 0 hour nd 3 hour ssessments (Figure 7.9). There ws no significnt difference when compring between tretments for the post-thw or the 0 hour ssessments. However, the 2% EY tretment did hve significntly higher percentge of intct membrnes thn did the BioXCell tretment for the 3 hour post-thw ssessment time (Figure 7.10). Acrosoml Sttus Like the membrne integrity evlution the first crosoml sttus reding ws tken fter the smple hd been divided into tretments nd cooled to 4 C. The first crosome vlues were 92.0±2.5% for the 2% EY tretment nd 94.7±1.7% for the BioXCell tretment. The men 0 hour post-thw crosoml sttus vlues for the 2% EY nd the BioXCell tretments were 73.5±2.0% nd 72.9±3.1%, respectively. The lst reding for ech strw thwed ws fter being held in 37 C incubtor for 3 hours post-thw. The two men vlues for the 3 hours post-thw crosoml ssessment were virtully identicl with the 2% EY tretment giving vlue of 62.8±1.6% nd the BioXCell reported 62.3±1.8% (Tble 7.10). 107

122 Tble 7.7. Summry of the numbers of sets of testes processed, number of sperm recovered nd number of strws frozen on ech of six collection dys. Collection dte Sets processed Totl Cell Count No. of strws per ext per dy Concentrtion per strw 3/17/ x x /24/ x x /31/ x x /1/ x x /12/ x x /16/ x x 10 6 Dily verge no x x 10 6 Totl no The lst two rows show the verge vlues for ll the collection dys, nd the overll totl vlues for the entire experiment. Tble 7.8. Percent motility (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 2% egg yolk or BioXCell extenders. Initil (Control) Post-cool 0 hour post-thw 3 hours post-thw 2% EY (Trt A) 75.8± ± ±3.1 b 36.5±1.5 b BioXCell (Trt B) 75.8± ± ±2.3 b 35.4±2.3 b EY = egg yolk, Trt = Tretment;,b Superscripts designte significnt difference only between initil motility vlue nd individul vlues for ech extender for ech time period (P<0.05). 108

123 b b c c Figure 7.7. Percent motility (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 109

124 Figure 7.8. Percent motility (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 110

125 Tble 7.9. Percent membrne integrity (men±sem) post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 2% egg yolk or BioXCell extenders. Postcool 0 hour post-thw 3 hours post-thw 2% EY (Trt A) 81.8± ± ±2.3 BioXCell (Trt B) 86.0± ± ±1.9 EY = egg yolk, Trt = Tretment; Tble Percent crosoml sttus (men±sem) post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were extended nd frozen in 2% egg yolk or BioXCell extenders. Post-cool 0 hour post-thw 3 hours post-thw 2% EY (Trt A) 92.0± ± ±1.6 BioXCell (Trt B) 94.7± ± ±1.8 EY = egg yolk, Trt = Tretment; 111

126 When compring within tretments, the crosoml sttus vlues for both the 2% EY nd the BioXCell tretments decresed significntly (P<0.05) from the post-cool to the 0 hour nd from the 0 hour to the 3 hour ssessments (Figure 7.11). There ws no sttisticl difference (P>0.05) when compring between the 2% EY nd BioXCell tretments for the post-cool, 0 hour post-thw or the 3 hour post-thw evlution times (Figure 7.12). Experiment 7.3 Testes Collection, Epididyml Sperm Concentrtion nd Strws frozen Testes were collected over six week period from veterinry clinics in New Orlens. Testes were processed on the dy of collection nd sperm were pooled. For the present experiment three to eight sets of testes were processed cross 11 dys for totl of 55 sets of feline testicles. After pooling n verge of x 10 6 /dy, with rnge of 94 x 10 6 to 260 x 10 6 (Tble 7.11). The totl number of sperm/dy ws divided evenly cross three tretments nd depending on concentrtion, rnge of 1 to 2 (0.25 cc) strws were frozen/tretment/dy. The concentrtion of the strws rnged from 31.5 X 10 6 to 54.0 X 10 6 /strw (Tble 7.11). Motility The initil motility for the epididyml sperm before being divided into the three tretment extenders ws 66.4±1.0%. The men motility vlue for epididyml sperm fter cooling to 4 C in the 2% EY (Tretment A) nd 0% EY (Tretment B) extenders ws 61.4±2.6% nd 60.2±3.5%, respectively. The 22 C-0%EY tretment (Tretment C) ws never cooled nd ws frozen from room temperture so there is no 4 C vlue for tht tretment. The men 0 hour post-thw motility vlues for epididyml sperm frozen in the 4 C-2% EY tretment ws 31.4±2.2%, the 4 C-0% EY tretment hd post-thw motility vlue of 26.0±3.1% nd the sperm in the 0% EY extender, frozen from room temperture (22 C-0% EY) hd motility of 22.5±3.0%. After incubting t 37 C for 3 hours post-thw, the epididyml sperm in the 4 C-2% EY tretment hd motility vlue of 17.9±2.0% while the 4 C-0% EY tretment resulted in sperm with motility of 15.3±2.5% nd the 22 C-0% EY tretment hd the lowest motility vlue of 9.4±1.2% (Tble 7.12). Neither of the two post-cool vlues ws significntly different from the initil motility reding. At the 0 hour post-thw ssessment ll three 0 hour post-thw vlues were significntly less the initilly motility vlue. Likewise the 3 hour post-thw vlues were significntly less thn the initil motility vlue (Tble 7.12). 112

127 b b b b Figure 7.9. Membrne intct percentge (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders.,b Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 113

128 b Figure Membrne intct percentge (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 114

129 b b c c Figure Acrosoml sttus percentge (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders.,b,c Men vlues within tretments but cross time intervls with different superscripts re significntly different (P<0.05). 115

130 Figure Acrosoml sttus percentge (men±sem) of initil, post-cool, 0 hour nd 3 hours post-thw for pooled domestic ct epididyml sperm tht were processed nd frozen in 2% egg yolk nd BioXCell extenders.,b Men vlues cross tretments but within time intervls with different superscripts re significntly different (P<0.05). 116