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Table 2. Type of counting chamber used and technical settings [average pathway velocity (VAP) and straightness (STR)] in combination with computer assisted sperm analysis in horses as reported in literature, blank fields indicate that the information was not available in the paper. STR (%) medium VAP cut-off (µm/s) low VAP cutoff (µm/s) Frames acquired Frame rate (Hz) Counting Chamber Study Albrizio et al., 2010 Leja (20 µm) 60 45 20 50 75 Aurich and Spergser, 2007 20 µm Ball and Vo, 2001 Baumber et al., 2002 Brinsko et al., 2000a Glazar et al., 2009 Cell-Vu (20 µm) 60 45 20 50 75 Kavak et al., 2003 Makler (10 µm) 32 15 10 25 Loomis and Graham, 2008 60 40 20 50 75 Macias Garcia et al., 2009 Leja (20 µm) 10 15 45 Melo et al., 2007 Makler (10 µm) 60 30 30 70 80 Miro et al., 2005 25 16 Nascimento et al., 2008 Leja (20 µm) 80 30 20 70 60 Ortega-Ferrusola et al., 2009 Leja (20 µm) 25 10 15 45 Pagl et al., 2006 20 µm Papa et al., 2008 Squires et al., 2004 Cell-Vu (20 µm) 20 20 25 80 Taberner et al., 2010 10 90 75 Vidament et al., 2009 60 30 20 40 80 Waite et al., 2008 Leja (20 µm) 60 45 15 30 50 Webb et al., 2009 Leja (20 µm) 60 30 20 50 75
CHAPTER 1.1 it is not possible to observe the details necessary for the morphological classification when spermatozoa are not equally distributed using the smearing technique (WHO, 2010). The PAP stain is almost a synonym for sperm morphology assessment according to WHO standards. However, the major downside of this stain, is the time consuming procedure, which involves 20 processing steps using more than 12 different chemical solutions. Due to the long staining procedure, the PAP staining is not frequently used for morphological analysis of animal semen. However, a rapid PAP stain procedure has been developed which has been used in combination with automated sperm morphology assessment (ASMA) (Boersma et al., 2001). The importance of the staining procedure for morphology analysis is well known. Staining techniques affect the morphometric dimensions of the sperm head, most likely due to differences in osmolarity in fixatives and staining solution. Most of these substances are not iso-osmotic in relation to the sperm (Marree et al., 2010). For instance, after staining sperm with PAP, the heads were shrunk when compared to fresh sperm. A new developed stain, SpermBlue®, is iso-osmotic in relation to human semen (van der Horst and Maree, 2009) and has been demonstrated not to influence the morphometric dimensions of the human sperm head (Marree et al., 2010). Additionally, the entire fixation and staining process requires only 25 min. Animal semen samples are often analyzed as wet mounts without staining (Estrada and Samper, 2007), after fixing in buffered formol saline or buffered glutaraldehyde solution (Brinsko et al., 2011). Depending on the laboratory, animal semen is stained with specific sperm stains [e.g., Williams stain (Williams, 1950) and Casarett stain (Casarett, 1953)], general purpose stains (e.g., Wright’s, Giemsa, Hematoxylin-Eosin) or background stains (e.g., eosin-nigrosin, India ink) (Barth and Oko, 1998; Varner, 2008). Despite the already mentioned associated disadvantages, eosin-nigrosin is probably the most frequently used staining method for animal semen because of its simplicity. Besides, this staining method gives good results for routine morphology evaluations. The eosin component of the stain will penetrate the damaged sperm membrane, causing the cell to turn pink, while sperm with an intact sperm membrane will remain white against the dark background provided by the nigrosin (Barth and Oko, 1989). Based on this principle, the eosin-nigrosin stain can also be used to assess the percentage of live, acrosome intact sperm, since stained sperm are considered to be dead or to have lost the acrosome (Brinsko et al., 2011). However, artefactual changes can occur simply from cold and osmotic shock which will lead to an increased percentage of stained sperm. Therefore, the 17
Page 1 and 2: AUTOMATED AND STANDARDIZED ANALYSIS
Page 3: AUTOMATED AND STANDARDIZED ANALYSIS
Page 7 and 8: A Area AI Artificial Insemination A
Page 9 and 10: PREFACE The first recorded case of
Page 11: GENERAL INTRODUCTION CHAPTER 1 3
Page 15 and 16: 1.1.1. Introduction CHAPTER 1.1 The
Page 17 and 18: 1.1.4. Concentration CHAPTER 1.1 Ac
Page 19 and 20: Fluorescent Activated Cell Sorter C
Page 21 and 22: CHAPTER 1.1 preparation (D, µm) is
Page 23: CHAPTER 1.1 These chambers are freq
Page 27 and 28: CHAPTER 1.1 overview of common abno
Page 29 and 30: CHAPTER 1.1 head, (B5) pyriform hea
Page 31 and 32: CHAPTER 1.1 The acrosome can be eva
Page 33 and 34: CHAPTER 1.1 The sperm chromatin str
Page 35: Collection and processing of equine
Page 38 and 39: CHAPTER 1.2 30 (a) Colorado State U
Page 40 and 41: CHAPTER 1.2 can be induced pharmaco
Page 42 and 43: CHAPTER 1.2 centrifugation time and
Page 44 and 45: CHAPTER 1.2 36 → Colloid centrifu
Page 46 and 47: CHAPTER 1.2 of a stallion stored fo
Page 48 and 49: CHAPTER 1.2 1.2.4. Cryopreservation
Page 50 and 51: CHAPTER 1.2 0.5 mL straws, and glyc
Page 52 and 53: CHAPTER 1.2 determined if the final
Page 54 and 55: CHAPTER 1.2 46 Chemically defined e
Page 56 and 57: CHAPTER 1.2 48 Cryopreservation - t
Page 59 and 60: 1.3.1. Standards for equine AI dose
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
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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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