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CHAPTER 4.1 Love and coworkers (2005) where increased DNA damage immediately after centrifugation was reported. These authors showed that complete removal of SP after centrifugation and subsequent resuspension of the sperm pellet in fresh diluter resulted in better sperm quality (motility and DNA) compared to samples in which SP was still present after simple dilution of the semen. Therefore the impact of the centrifugation on DNA integrity needed to be further examined. In our study, no difference in DNA damage was present immediately after centrifugation which might be explained by the method used to evaluate DNA integrity. The study of Love et al. (2005) evaluated DNA integrity using the sperm chromatin structure assay (SCSA). In comparison to the TUNEL assay, with the SCSA a larger number of sperm cells is analyzed by a flow cytometer based on the colour of the metachromatic dye acridine orange (Evenson and Wixon, 2006). Therefore it might be advisable to measure the direct impact of different CPs with flow cytometry (SCSA or TUNEL) to get a more accurate impression of the possible side effect of centrifugation on DNA integrity. 154 Cushioned centrifugation techniques have previously been described as an answer to reduce sperm damage while maximizing the sperm harvest. A variety of dense solutions acting as a cushion have been described (Cochran et al., 1984). The frequently used iodixanol was first reported for stallion sperm cells in 1997 (Revell et al., 1997). Very high recovery rates have been described (Ecot et al., 2005; Knop et al., 2005) using such a commercially available cushion, when centrifuging at 1000 × g for 20 min. However, the most critical step is carefully removing the cushion material after centrifugation. Indeed, it is critical to aspirate as much cushion fluid as possible without aspirating sperm cells from the adjacent band to avoid possible deleterious effects of the iodixanol solution (Waite et al., 2008). The use of very small amounts (30 µL) of cushion without removal was proven to be harmless. However, these small volumes require specially designed, custom-made nipple centrifugation tubes which are not readily available (Waite et al., 2008). Because of these important drawbacks, cushioned centrifugation is not commonly used in practice. Therefore, the impact of CP on sperm quality remains an important subject to examine. In summary, sperm losses can be reduced substantially by centrifuging stallion semen for 5 min at 1800 or 2400 × g without a cushion and without impairing the in vitro sperm function after cooled or frozen storage. This leads to an increase in the number of AI doses produced per ejaculate. It can easily result in an additional two AI doses from an average ejaculate of 10 billion sperm. Nevertheless, the effects of these CPs on DNA damage requires further investigation as well as the impact on in vivo fertility.
References CHAPTER 4.1 Aurich C. 2008. Recent advances in cooled-semen technology. Animal Reproduction Science 107:268- 75. Barth A.D., Oko R.J. 1989. Abnormal morphology of bovine spermatozoa. Ames, Iowa: Iowa State University Press. Carvajal G., Cuello C., Ruiz M., Vázquez J.M., Martínez E.A., Roca J. 2004 Effects of centrifugation before freezing on boar sperm cryosurvival. Journal of Andrology 25:389-96. Cochran J.D., Amann R.P., Froman D.P., Pickett B.W. 1984. Effects of centrifugation, glycerol level, cooling to 5°C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology 22:25-38. De Pauw I.M.C., Van Soom A., Mintiens K., Verberckmoes S., de Kruif A. 2003 In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions. Theriogenology 59:1093-107. Ecot P., Decuadro-Hansen G., Delhomme G., Vidament M. 2005. Evaluation of a cushioned centrifugation technique for processing equine semen for freezing. Animal Reproduction Science 89:245-8. Evenson D.P., Wixon R. 2006. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 65:979-91. Garner D.L., Johnson L.A. 1995. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biology of Reproduction 53:276-84. Gil M.C., García-Herreros M., Barón F.J., Aparicio I.M., Santos A.J., García-Marín L.J. 2009. Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Theriogenology 71:254-63. Heitland A.V., Jasko D.J., Squires E.L., Graham J.K., Pickett B.W., Hamilton C. 1996 Factors affecting motion characteristics of frozen-thawed stallion spermatozoa. Equine Veterinary Journal 28:47-53. Jasko D.J., Moran D.M., Farlin M.E., Squires E.L. (1991) Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Theriogenology 35:1059-67. Knop K., Hoffmann N., Rath D., Sieme H. 2005. Effects of cushioned centrifugation technique on sperm recovery and sperm quality in stallions with good and poor semen freezability. Animal Reproduction Science 2005;89:294-7. Loomis P.R. 2006. Advanced methods for handling and preparation of stallion semen. Veterinary Clinics of North America: Equine Practice 22:663-76. Loomis P.R., Graham J.K. 2008. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Animal Reproduction Science 105:119-28. 155
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AUTOMATED AND STANDARDIZED ANALYSIS
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AUTOMATED AND STANDARDIZED ANALYSIS
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A Area AI Artificial Insemination A
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PREFACE The first recorded case of
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GENERAL INTRODUCTION CHAPTER 1 3
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1.1.1. Introduction CHAPTER 1.1 The
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1.1.4. Concentration CHAPTER 1.1 Ac
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Fluorescent Activated Cell Sorter C
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CHAPTER 1.1 preparation (D, µm) is
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CHAPTER 1.1 These chambers are freq
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CHAPTER 1.1 it is not possible to o
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CHAPTER 1.1 overview of common abno
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CHAPTER 1.1 head, (B5) pyriform hea
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CHAPTER 1.1 The acrosome can be eva
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CHAPTER 1.1 The sperm chromatin str
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Collection and processing of equine
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CHAPTER 1.2 30 (a) Colorado State U
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CHAPTER 1.2 can be induced pharmaco
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CHAPTER 1.2 centrifugation time and
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CHAPTER 1.2 36 → Colloid centrifu
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CHAPTER 1.2 of a stallion stored fo
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CHAPTER 1.2 1.2.4. Cryopreservation
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CHAPTER 1.2 0.5 mL straws, and glyc
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CHAPTER 1.2 determined if the final
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CHAPTER 1.2 46 Chemically defined e
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CHAPTER 1.2 48 Cryopreservation - t
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1.3.1. Standards for equine AI dose
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CHAPTER 1.3 The use of density grad
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CHAPTER 1 Avery B., Greve T. 1995.
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CHAPTER 1 Cheng F.-P., Fazeli A., V
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CHAPTER 1 Flipse R.J., Patton S., A
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CHAPTER 1 Kuisma P., Andersson M.,
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CHAPTER 1 Medeiros A.S.L., Gomes G.
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CHAPTER 1 Pickett B.W. 1993. Collec
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CHAPTER 1 Taberner E., Morató R.,
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CHAPTER 1 Waite J.A., Love C.C., Br
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CHAPTER 2 As evidenced in the gener
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3.1 Influence of technical settings
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CHAPTER 3.1 a CEROS (Hamilton-Thorn
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CHAPTER 3.1 Progressive Motility CA
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CHAPTER 3.1 than at least they shou
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3.2.1. Abstract CHAPTER 3.2 Sperm m
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CHAPTER 3.2 depth but not the chamb
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3.2.3.3. The different chambers and
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CHAPTER 3.2 Finally, Pearson coeffi
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90 80 70 60 50 40 30 20 10 0 drop f
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ISAS20rM Makler10r WHO 120 120 120
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60 PM Rapid 50 40 30 20 10 0 CHAPTE
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Table 5. Averages (± standard erro
Page 111 and 112: CHAPTER 3.2 sperm (tail) which coul
Page 113 and 114: CHAPTER 3.2 Hoogewijs M.K., Govaere
Page 115: CHAPTER 3.2 Spizziri B.E., Fox M.H.
Page 119 and 120: 3.3.1. Abstract CHAPTER 3.3 Routine
Page 121 and 122: CHAPTER 3.3 morphological abnormali
Page 123 and 124: CHAPTER 3.3 The extended semen was
Page 125 and 126: CHAPTER 3.3 Table 1: Average values
Page 127 and 128: Repeatability Agreement SQA-Ve PM (
Page 129: References CHAPTER 3.3 Arunvipas P.
Page 133 and 134: 3.4.1. Abstract CHAPTER 3.4 In this
Page 135 and 136: CHAPTER 3.4 therefore in these expe
Page 137 and 138: CHAPTER 3.4 The averages obtained a
Page 139 and 140: 3.4.4.2. Experiment 2 CHAPTER 3.4 F
Page 141: References CHAPTER 3.4 Arunvipas P.
Page 145: 4.1 Influence of different centrifu
Page 148 and 149: CHAPTER 4.1 4.1.2. Introduction 140
Page 150 and 151: CHAPTER 4.1 142 4.1.3.3. Semen proc
Page 152 and 153: CHAPTER 4.1 144 In the experiment 3
Page 154 and 155: CHAPTER 4.1 centrifugation (T1) and
Page 156 and 157: CHAPTER 4.1 (non CP vs CP) was sign
Page 158 and 159: 35 B 80 A 33 beat cross frequency (
Page 160 and 161: CHAPTER 4.1 152 4.1.4.2. EXPERIMENT
Page 164 and 165: CHAPTER 4.1 Love C.C., Brinsko S.P.
Page 167 and 168: 4.2.1. Abstract CHAPTER 4.2 The inc
Page 169 and 170: 4.2.3. Materials and methods 4.2.3.
Page 171 and 172: CHAPTER 4.2 For the SLC group, 15 m
Page 173 and 174: CHAPTER 4.2 adding 600 µL of AO st
Page 175 and 176: 4.2.3. Results 4.2.3.1. Sperm quali
Page 177 and 178: CHAPTER 4.2 The aforementioned “T
Page 179 and 180: CHAPTER 4.2 Sperm yields markedly d
Page 181 and 182: References CHAPTER 4.2 Barth A.D.,
Page 183: CHAPTER 4.2 Waite J.A., Love C.C.,
Page 187 and 188: CHAPTER 5 This thesis originated fr
Page 189 and 190: CHAPTER 5.1 Fig. 1. Schematic prese
Page 191 and 192: CHAPTER 5.1 higher than the current
Page 193 and 194: CHAPTER 5.1 al., 2007) showing a cl
Page 195 and 196: CHAPTER 5.1 treatment strategy for
Page 197 and 198: CHAPTER 5.2 supernatant allowing sp
Page 199 and 200: CHAPTER 5.2 such keeping the pellet
Page 201 and 202: CHAPTER 5.2 components of the semin
Page 203 and 204: Final reflections 5.4 Actual change
Page 205 and 206: CHAPTER 5 Dillon R.H., Fauci L.J.,
Page 207: CHAPTER 5 Suárez S.S., Osman R.A.
Page 210 and 211: SUMMARY technique that enables proc
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SUMMARY from stallions previously c
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SAMENVATTING beweeglijkheid (Hoofds
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SAMENVATTING de spermacellen gesele
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DANKWOORD Het obligatoire dankwoord
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DANKWOORD dankzij jou en ze zijn er
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CURRICULUM VITAE Maarten Hoogewijs
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BIBLIOGRAPHY Govaere J., Ducatelle
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BIBLIOGRAPHY ABSTRACTS AND PROCEEDI
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FUNDAMENTAL ASPECTS OF CRYOPRESERVA
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ADDENDUM I presence of these CPAs a
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ADDENDUM I Fig. 3. Theoretical curv
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ADDENDUM II DETAILED LOADING TECHNI
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In m’n hoofd is alles heel eenvou
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