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CHAPTER 1.1 al., 2002), boars (Maes et al., 2003; Vyt et al., 2004) rams (Fukui et al., 2004), bulls (Hoflack et al., 2005), and mice (Tayama et al., 2006)]. The newest version of the SQA (version V) was initially developed for human samples as well and later on adapted for different animal species, namely bulls, boars, stallions and turkeys. Specific devices for rams and roosters are in development. The SQA is ready to use after delivery and does not require (or allow) user specific settings, as such reducing a large potential source of bias (Hoflack et al., 2005). 14 Objective motility analysis: Computer Assisted Sperm Analysis The best known way to objectively analyze sperm motility is analysis by means of a CASA system. The first CASA system was introduced over 30 years ago, and has been used ever since in human and veterinary andrology laboratories. By equipping a microscope with a camera, the sperm cells are visualized and the actual sperm tracks can be analyzed after sperm cell recognition. The latter can be achieved either based on the number of pixels and intensity or, in newer versions, by means of fluorescent dyes. This technique facilitates sperm recognition and allows CASA analysis of (post thaw) samples containing egg yolk particles (Tardif et al., 1998) or debris for example after obtaining epididymal sperm in cats (Filliers, personal communication). The major advantages of CASA systems are, amongst others, the good repeatability and the absence of subjectivity. Nevertheless, standards for analysis need to be established first, since different technical settings and sample preparations have been shown to influence the outcome of CASA analysis (Table 1). The impact of different technical settings (frame rate and number of frames analyzed) and procedures (temperature, concentration, diluents and chamber) have been studied for different species (Contri et al., 2010; Lenz et al., 2011; Rijsselaere et al., 2003). Also, the motility settings may have an influence on the CASA outcome as well. Based on low and medium average pathway velocity (VAP) cut-off values and on straightness (STR), spermatozoa are graded. In literature, a plethora of settings is used (Table 2). Not only is there a lack of agreement in the cut-off values used by different research groups, also the description of these values is not uniform for CASA systems from different companies. A comparable lack of standardization can be found in the chambers used to analyze the motility of a semen sample. Although the effect of the counting chamber was already described in 2001 (Iguer-ouada and Verstegen, 2001), different brands and types of chambers are used. The most frequently used chambers are a 20 µm deep Leja chamber (Waite et al., 2008; Ortega-Ferrusola et al., 2009; Len et al., 2010), a 20 µm deep Cell-Vu (Almeida and Ball, 2005; Glazar et al., 2009; Spirizzi et al., 2010) and the 10 µm deep reusable Makler chamber (Kavak et al., 2003; Johannisson et al., 2009).
CHAPTER 1.1 These chambers are frequently loaded with a different volume of sperm, sometimes only the depth of the chamber is mentioned without the brand name (Pagl et al., 2006; Aurich and Spergser, 2007), or only the volume used is mentioned (Quintero-Moreno et al., 2003; Price et al., 2008) and occasionally nothing is mentioned at all (Love et al., 2004; Macia-Garcia et al., 2009; Ponthier et al., 2009). In three recent studies, the influence of different chambers on motility was evaluated. For bull semen, the Makler chamber resulted in higher total and progressive motility compared to 20 µm deep Leja chambers (Contri et al., 2010; Lenz et al., 2011) but results were not different compared to these obtained with the WHO prepared slide (Lenz et al., 2011). The effect of different counting chambers when analyzing equine semen needs to be analyzed. Table 1. Influence of technical settings and sperm preparation on outcome motility parameters generated with a CASA system (tested variables are listed between brackets). Setup Rijsselaere et al., 2003 Contri et al., 2010 Species Dog Cattle Type of CASA Hamilton-Thorne CEROS 12.1 Frame rate Influence (15-30-60Hz) Number of frames Limited influence (30-60) Concentration Influence (100 – 50 – 25 × 10 6 ) Advised concentration: 50 × 10 6 Diluent Influence (physiological saline – prostatic fluid – Hepes-TALP-medium – egg-yolk-Tris extender) 1.1.6. Morphology Hamilton-Thorne IVOS 12.3 Influence (30-60Hz) No influence (30-45) Influence (100 – 50 – 30 – 20 – 10 – 5 × 10 6 ) Advised concentration: 20 × 10 6 Influence (physiological saline – PBS – Bio-excell) In human andrology, three different stainings are recommended by the WHO (2010), namely the Papanicolaou (PAP), the Shorr and the Diff-Quick stain which can all be interpreted using brightfield optics. With these staining procedures, the sperm head is stained pale blue in the acrosome region and dark blue in the post-acrosomal region. The midpiece may show some red staining and the tail is stained blue or reddish. Excess residual cytoplasm is stained pink or red using the PAP stain or reddish-orange in case of the Shorr stain (Boersma et al., 2001; WHO, 2010). The use of rapid staining methods such as eosin-nigrosin staining, is not recommended by the WHO because 15
Page 1 and 2: AUTOMATED AND STANDARDIZED ANALYSIS
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Page 7 and 8: A Area AI Artificial Insemination A
Page 9 and 10: PREFACE The first recorded case of
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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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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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