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CHAPTER 4.1 144 In the experiment 3, DNA integrity was analyzed using a Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay. The TUNEL assay was performed using the In Situ Cell Death Detection Kit (Boehringer, Mannheim, Germany) to detect the presence of free 3′- OH termini in single and double-stranded sperm DNA (De Pauw et al., 2003). In short, sperm samples were diluted with PVP solution (1 mg/mL in PBS) to a final concentration of 10 × 10 6 sperm/mL from which a 10 μL aliquot was smeared onto a poly-l-lysine-coated microslide. After fixation with 4% paraformaldehyde in polyvinyl pyrrolidone (PVP) solution (pH 7.4) and permeabilisation with 0.5% (v/v) Triton X-100 in PBS, the sperm cells were incubated with the TUNEL-mixture (fluorescein-dUTP and terminal deoxynucleotidyl transferase) for 1 h at 37 °C in the dark. Both positive (1 mg/mL DNAse I) and negative controls (nucleotide mixture in the absence of transferase) were included in each replicate. Hoechst 33342 was used to counter stain sperm DNA. The samples were examined by fluorescence microscopy (Leica DMR; magnification 400×, oil immersion). At least 200 sperm cells from each sample were analyzed randomly to evaluate the percentage of TUNEL-positive sperm cells (bright green nuclear fluorescence) (see De Pauw et al. (2003) for further details). 4.1.3.5. Centrifugation protocols After dilution of the raw semen with Gent Extender (at 37 °C), each tube, except for the uncentrifuged control, was centrifuged with a Heraeus Multifuge 3 L-R (Kendro Laboratory Products, Waltham, USA) at ambient temperature using 1 of 5 CPs. Centrifugation protocol 1 was the reference protocol being 10 min at 600 × g (Ecot et al., 2005; Knop et al., 2005; Vidament et al., 2000; Weiss et al., 2004); CPs 2, 3, 4 and 5 were 5 min at 600 × g, 1200 × g, 1800 × g and 2400 × g, respectively. The deceleration speed was 1, which was the slowest possible deceleration in order to minimize disturbance due to turbulence. Since only one centrifuge was available, the processing of each ejaculate from the same stallion started with a different CP to eliminate influence of a prolonged contact time with the SP. So for each stallion every ejaculate was centrifuged first with a different alternating protocol.
4.1.3.6.Experimental design CHAPTER 4.1 EXPERIMENT 1: influence of the centrifugation protocol on the function of fresh and cooled sperm Fresh semen was analyzed (concentration, CASA, eosin/nigrosin staining and SYBR14-PI) immediately after collection (T0). The semen was diluted to 25 × 10 6 sperm cells/mL and allocated to six 15-mL test tubes (Cellstar ® , Greiner bio-one, Germany). Five of these tubes (1 to 5) were subjected to one of the 5 CPs whereas tube 6 served as a negative control (no centrifugation). After centrifugation, the sperm concentration was determined in the aspirated supernatant. The sperm pellet was resuspended in fresh diluter and an aliquot of each test tube was analyzed (T1). The test tubes containing the processed semen were allowed to cool gradually to 5°C in 50-mL test tubes filled with water (22°C) and placed in a 5°C refrigerator. Sperm analyses were repeated at 24h (T2), 48h (T3) and 72h (T4). PSA-FITC staining was performed immediately after centrifugation and at 72h. EXPERIMENT 2: influence of the pre-freezing centrifugation protocol on the function of frozen-thawed sperm The initial semen processing was performed as described in experiment 1. After centrifugation using each of the five CP’s, the sperm pellet was resuspended in Gent extender for freezing to a final concentration of 50×10 6 sperm cells/mL. The extended semen was loaded into 0.5 mL straws (MRS1, L’Aigle Cedex, France) at ambient temperature, placed on racks and transported to the freezing chamber of an automated controlled rate freezer (IceCube 14S, Neupurkersdorf, Austria). The semen was first cooled from 22°C to 4°C in 80 min and then frozen from 4 °C to -140 °C at 60 °C/min (adapted from Vidament et al. (2000)). At -140 °C the freezing racks were removed from the freezing chamber and plunged into liquid nitrogen. Frozen semen was stored for at least 1 month before analysis. The samples were thawed by plunging into a 37 °C water bath for 30 s. Five straws from the same batch (same ejaculate, same treatment) were pooled, allowed to equilibrate for 5 min at 37 °C and analyzed once, performing the same analyses as described in experiment 1. EXPERIMENT 3: influence of the centrifugation protocol on the DNA integrity of fresh and cooled sperm From four stallions, one ejaculate was collected and diluted in Gent Extender to a final concentration of 25×10 6 sperm cells/mL. The diluted semen was subjected to three different CP’s (CP1, CP2 and CP5) whereas one tube served as negative (uncentrifuged) control. Immediately after 145
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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
Page 101 and 102: CHAPTER 3.2 Finally, Pearson coeffi
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Page 105 and 106: ISAS20rM Makler10r WHO 120 120 120
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Page 109 and 110: 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
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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 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
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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
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Page 199 and 200: CHAPTER 5.2 such keeping the pellet
Page 201 and 202: CHAPTER 5.2 components of the semin
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Final reflections 5.4 Actual change
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CHAPTER 5 Dillon R.H., Fauci L.J.,
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CHAPTER 5 Suárez S.S., Osman R.A.
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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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