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CHAPTER 3.1 a CEROS (Hamilton-Thorne Inc., Beverly, USA) CASA system using two different settings (CASA1 and CASA2, respectively; see further). Settings for CASA analysis were obtained from established semen processing laboratories as routinely applied to fresh, cooled and frozen-thawed semen samples. 80 The major settings used for CASA1 were straightness (STR) threshold for progressive motility: 75%; average path velocity (VAP) threshold for progressive motility 50 µm/s; VAP threshold for static cells: 20 µm/s (Loomis and Graham, 2008). These settings were first obtained after a personal communication (Loomis P., personal communication) and settings for cell detection were adapted for optimal sperm recognition. The major settings used for CASA2 were STR threshold for progressive motility: 50%; VAP threshold for progressive motility 30 µm/s; VAP threshold for static cells: 15 µm/s, settings for cell detection are listed as well (Waite et al., 2008). Six µL of diluted semen was loaded into a 20 µm Leja counting chamber (Leja, Nieuw-Vennep, The Netherlands), placed on a minitherm stage warmer (Hamilton-Thorne Inc., Beverly, USA) during the analysis, to maintain the sample at 37.5°C. Five different fields were analyzed 4 times to obtain a total of 20 fields in order to obtain a minimum of 1500 cells analyzed. For both CASA settings this was done twice per sample (T1 and T2). distributed). The parameters analyzed were percentage total motility (TM), PM and PMS (normally To assess repeatability each sample was analyzed twice within one minute. First, it was tested whether the parameters changed over time (T1 vs. T2), using a paired-samples t-test. Second, scatter plots were produced, where the value obtained at T1 was plotted against the value obtained at T2. The line of perfect agreement (hatched line at 45° and intercept zero) was integrated in each plot as comparison to the regression line to fit the data (Arunvipas et al., 2003). Third, Bland and Altman plots were produced. This method plots the difference of the paired measurements (y-axis) against their mean (x-axis) (Bland and Altman, 1986). Finally, for each parameter, the coefficient of variation (CV) was calculated per sample and reported as mean CV over all samples. To assess agreement, the values obtained at T1 were used for comparison, and observations obtained with CASA1 were compared with these obtained with CASA2. Statistical analysis was done as described for repeatability using a paired samples t-test, scatter plots and Bland and Altman plots. Statistical analysis was done using SPSS (version 17.0, SPSS Inc., Chicago, Illinois, USA).
3.1.4. Results CHAPTER 3.1 Analyzing the semen samples using CASA1 and CASA2 was highly repeatable as the averages at T1 were not significantly different from T2 (Table 1). The scatter plots and Bland-Altman plots show a very good repeatability (Fig. 1), and the mean CVs were
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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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Page 56 and 57: CHAPTER 1.2 48 Cryopreservation - t
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Page 61 and 62: CHAPTER 1.3 The use of density grad
Page 63 and 64: CHAPTER 1 Avery B., Greve T. 1995.
Page 65 and 66: CHAPTER 1 Cheng F.-P., Fazeli A., V
Page 67 and 68: CHAPTER 1 Flipse R.J., Patton S., A
Page 69 and 70: CHAPTER 1 Kuisma P., Andersson M.,
Page 71 and 72: CHAPTER 1 Medeiros A.S.L., Gomes G.
Page 73 and 74: CHAPTER 1 Pickett B.W. 1993. Collec
Page 75 and 76: CHAPTER 1 Taberner E., Morató R.,
Page 77: CHAPTER 1 Waite J.A., Love C.C., Br
Page 81: CHAPTER 2 As evidenced in the gener
Page 85: 3.1 Influence of technical settings
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Page 92 and 93: CHAPTER 3.1 than at least they shou
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Page 109 and 110: Table 5. Averages (± standard erro
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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
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Page 125 and 126: CHAPTER 3.3 Table 1: Average values
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Page 129: References CHAPTER 3.3 Arunvipas P.
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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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CHAPTER 4.1 142 4.1.3.3. Semen proc
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CHAPTER 4.1 144 In the experiment 3
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CHAPTER 4.1 centrifugation (T1) and
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CHAPTER 4.1 (non CP vs CP) was sign
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35 B 80 A 33 beat cross frequency (
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CHAPTER 4.1 152 4.1.4.2. EXPERIMENT
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CHAPTER 4.1 Love and coworkers (200
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CHAPTER 4.1 Love C.C., Brinsko S.P.
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4.2.1. Abstract CHAPTER 4.2 The inc
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4.2.3. Materials and methods 4.2.3.
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CHAPTER 4.2 For the SLC group, 15 m
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CHAPTER 4.2 adding 600 µL of AO st
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4.2.3. Results 4.2.3.1. Sperm quali
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CHAPTER 4.2 The aforementioned “T
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CHAPTER 4.2 Sperm yields markedly d
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References CHAPTER 4.2 Barth A.D.,
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CHAPTER 4.2 Waite J.A., Love C.C.,
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CHAPTER 5 This thesis originated fr
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CHAPTER 5.1 Fig. 1. Schematic prese
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CHAPTER 5.1 higher than the current
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CHAPTER 5.1 al., 2007) showing a cl
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CHAPTER 5.1 treatment strategy for
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CHAPTER 5.2 supernatant allowing sp
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CHAPTER 5.2 such keeping the pellet
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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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