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Centrifugation technique 188 5.2 Centrifugation is one of the common procedures when processing equine semen. Currently, centrifugation is mandatory when subjecting an equine ejaculate to cryopreservation. Alternative approaches such as fractionated collection are not as successful (Sieme et al., 2004) or are still in its infancy (sperm filters – Alvarenga et al., 2010). The influence of centrifugation on spermatozoa is not completely understood, or at least the principle(s) by which spermatozoa are influenced due to centrifugation remain(s) unclear. A first theory is based on alterations in the shape of cells submitted to centrifugation. Using an atomic force microscope, fitted in a large centrifuge, Van Loon et al. (2009) were able to visualize mouse osteoblasts during centrifugation. The osteoblasts were markedly reduced in height during centrifugation at only 3 × g. Although the structure of a spermatozoon is completely different from an osteoblast, especially because of the dramatic reduction in the cytoplasm of a mature sperm cell, it is likely that centrifugation at much higher speeds also affect the structure of a sperm cell. Further refinement of this technique by Van Loon et al. (2009) and research on different cell types is necessary in order to accurately describe what happens with spermatozoa during centrifugation. Another explanation could act at the level of the sperm membrane. Like any plasma membrane, a sperm membrane consists of a liquid phospholipid bilayer (Amann and Pickett, 1987). Different domains of the plasma membrane all contain different concentrations and distributions of intramembranous particles, with different, specific functions in the process of fertilization (Flesch and Gadella, 2010). These domains undergo redistribution following capacitation (Gadella et al., 1994). Perhaps centrifugation acts on this liquid lipid layer and disturbs as such its function by rearranging the distribution of the intramembranous particles. Excessive centrifugation forces induce even more damage, and provoke a compact sperm pellet that is difficult to resuspend (Webb and Dean, 2009). In order to identify the effects of centrifugation force on sperm damage, we investigated the impact of increasing forces (centrifugation protocols from 600 × g to 2400 × g) on stallion semen for a reduced period of time (5 min) (Chapter 4.1). Additionally, we investigated sperm loss when aspirating 90% of the supernatant following centrifugation using the different forces. Not all the spermatozoa were pelleted at the bottom of the tube, but a part remained in suspension in the
CHAPTER 5.2 supernatant allowing sperm yield to be calculated. This technique of centrifugation only results in separation of the spermatozoa from the seminal plasma. Alternatively, the forces originating during centrifugation can also be used to select the superior sperm fraction from a sample when low g- forces are combined with a colloid (Chapter 4.2). 5.2.1. Centrifugation to concentrate equine semen samples Much uncertainty exists on how spermatozoa react on a cellular level to centrifugation. The influence of centrifugation on sperm yields and semen characteristics is controversial as well. However, much of the variation in study results can be explained by differences in experimental design. Extender type (Cochran et al., 1984), volume per centrifugation tube (Cochran et al. 1984, Webb et al., 2009), dimensions of the centrifugation tube (Webb and Dean, 2009), and of course centrifugation time and force (Cochran et al., 1984; Weiss et al 2004; Len et al. 2008, 2010; Webb et al., 2009; Hoogewijs et al., 2010) are all factors influencing yield and quality of equine semen after centrifugation. Our findings (Chapter 4.1) on sperm yields were consistent with the general trends in literature. Using the standard protocol (600 × g for 10 min) 78% of the initial number of spermatozoa were maintained. Sperm quality was not influenced largely. Immediately following centrifugation motility and membrane integrity were only slightly reduced compared with non-centrifuged samples. The use of higher forces for a shorter period of time reduced sperm losses without affecting the in vitro sperm quality following cooled storage or cryopreservation. When looking at the variations in the above mentioned studies on sperm losses, it is obvious there might be a stallion effect as well. Some stallions have semen that sediments easier during centrifugation, and some have spermatozoa that are more prone to damage caused by centrifugation compared to other stallions (personal observations). So individual centrifugation protocols might be necessary for some stallions, and if corrections for sperm loss following centrifugation are made, it is important to re-assess the actual sperm number following centrifugation and not to use a fixed correction factor since sperm loss is influenced by so many parameters. 189
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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
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CHAPTER 3.2 sperm (tail) which coul
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CHAPTER 3.2 Hoogewijs M.K., Govaere
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CHAPTER 3.2 Spizziri B.E., Fox M.H.
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3.3.1. Abstract CHAPTER 3.3 Routine
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CHAPTER 3.3 morphological abnormali
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CHAPTER 3.3 The extended semen was
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CHAPTER 3.3 Table 1: Average values
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Repeatability Agreement SQA-Ve PM (
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References CHAPTER 3.3 Arunvipas P.
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3.4.1. Abstract CHAPTER 3.4 In this
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CHAPTER 3.4 therefore in these expe
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CHAPTER 3.4 The averages obtained a
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3.4.4.2. Experiment 2 CHAPTER 3.4 F
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References CHAPTER 3.4 Arunvipas P.
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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 162 and 163: CHAPTER 4.1 Love and coworkers (200
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: CHAPTER 5.1 treatment strategy for
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
Page 212 and 213: SUMMARY from stallions previously c
Page 214 and 215: SAMENVATTING beweeglijkheid (Hoofds
Page 216 and 217: SAMENVATTING de spermacellen gesele
Page 219 and 220: DANKWOORD Het obligatoire dankwoord
Page 221: DANKWOORD dankzij jou en ze zijn er
Page 225: CURRICULUM VITAE Maarten Hoogewijs
Page 228 and 229: BIBLIOGRAPHY Govaere J., Ducatelle
Page 230 and 231: BIBLIOGRAPHY ABSTRACTS AND PROCEEDI
Page 233 and 234: FUNDAMENTAL ASPECTS OF CRYOPRESERVA
Page 235 and 236: ADDENDUM I presence of these CPAs a
Page 237 and 238: ADDENDUM I Fig. 3. Theoretical curv
Page 239 and 240: ADDENDUM II DETAILED LOADING TECHNI
Page 241: In m’n hoofd is alles heel eenvou
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