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CHAPTER 5.2 5.2.2. Selection of spermatozoa 192 The possibility to perform sperm selection using a single layer centrifugation (Morrell et al., 2008) and the adaptation to scale up the volume per centrifugation tube (Morrell et al., 2009) extends its practical use in equine andrology. This technique was used to select a superior sperm population prior to cryopreservation and obtained post-thaw samples with increased overall sperm quality (Chapter 4.2). The sole intention of the cryopreservation protocol we used was to analyze the effect of cryopreservation of a superior sperm population. It is not surprising to find an overall increased sperm quality after cryopreservation. Dead, damaged, or spermatozoa with a dysfunctional motility are very unlikely to survive the freeze-thaw cycle, or to have a sufficient post-thaw quality. Until recently, it was accepted that reactive oxygen species (ROS) in (human) semen were almost exclusively produced by leucocytes. This is true for fertile men, although in oligozoospermic patients the spermatozoa themselves were identified as a second major source of ROS (Aitken et al., 1992). Equine semen is normally devoid of leucocytes in contrast to human semen, but equine spermatozoa are equally capable of generating ROS, since damaged spermatozoa or morphological abnormal spermatozoa generated significantly greater amounts of ROS than live, morphologically normal spermatozoa (Ball et al., 2001). The presence of ROS impairs in vitro motility of equine spermatozoa (Baumber et al., 2002) and promotes DNA fragmentation of equine spermatozoa ( Baumber et al., 2003). We hypothesize that the increased post-thaw sperm quality we have noticed following pre- freeze SLC selection could perhaps also be partially attributed to reduced levels of ROS. The use of Androcoll-E might remedy these ROS related problems by working on two levels. First, due to the selection procedure, the few leucocytes that are present are being held back from the sperm pellet. Second, fewer abnormal spermatozoa (dead spermatozoa or with excess residual cytoplasm) are present in the sperm pellet. As such the ROS content in the selected subpopulation of spermatozoa should be lower than in the “full” sperm pellet, reducing the ROS related sperm damage. The effect of sperm selection prior to cryopreservation could even be further increased if stallion-adapted cryopreservation protocols would be used, fitting specific needs . Additionally, sperm selection using Androcoll-E and subsequent washing of the sperm pellet removes all seminal plasma. In our study seminal plasma from the same stallion and the same ejaculate was added back to rule out any possible effect of presence or absence of seminal plasma on the post-thaw results. Seminal plasma from stallions with poor freezing sperm was shown to play an important role in the poor freezability (Aurich et al., 1996). The use of seminal plasma of stallions with good freezability improved the post-thaw quality of stallions with poor freezability (Aurich et al., 1996). The
CHAPTER 5.2 components of the seminal plasma that are associated with fertility are not yet completely categorized. Jobim et al. (2005) described the absence of protein 19 and higher levels of protein 17 in the seminal plasma of low fertile stallions. The involvement of other seminal plasma components on fertility requires further research. The use of heterologous seminal plasma implies some risks. First of all the seminal plasma should be guaranteed free of spermatozoa, to avoid faulty paternity. A combination of high speed centrifugation (1000 × g for 10 min) of raw semen followed by passage of the supernatant through tandem 5 µm and 1.2 µm nylon syringe filters should be a safe procedure to avoid spermatozoa contamination (Rigby et al., 2001). Equally important are the risks of disease transmission through the seminal plasma of the donor. This donor should at least have the same sanitary status as the stallion whose spermatozoa should be frozen, and even so legislative limitations might occur. A modified protocol in which the best diluter for a given poor freezing stallion is used in combination with seminal plasma from a good freezing stallion might further increase post-thaw semen quality, and enable us to freeze semen from stallions previously deemed unfreezable. Further determination of the different components of the seminal plasma and the identification of their function might enable researchers to isolate those proteins which positively influence cryopreservation. These proteins could then be added to extenders to form an optimized defined cryopreservation diluter. Applying sperm selection prior to cryopreservation offers great advantages compared to post-thaw selection of semen, as is frequently done in combination with assisted reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection. First of all, the ROS are removed prior to freezing, reducing the exposure of spermatozoa to the toxic influence. Besides that, application of sperm selection techniques after thawing requires some level of experience and the presence of laboratory facilities in order to process the semen, which factors are very often not present in practice. Selection prior to cryopreservation bypasses these problem since it delivers ready-to-use frozen semen. 193
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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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4.1 Influence of different centrifu
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CHAPTER 4.1 4.1.2. Introduction 140
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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
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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: CHAPTER 5.2 such keeping the pellet
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
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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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