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CHAPTER 3.2 90 3.2.3.2. Quality analysis Objective motility assessment was performed using CASA (Hoogewijs et al., 2010). Briefly, a Ceros 12.3 CASA system (Hamilton Thorne Inc. Beverly, MA, USA) was used. The chambers were loaded with semen and maintained at 37 °C using a Tokai Hit thermoplate (Tokai Hit CO., Ltd, Shizoka-ken, Japan). Five randomly selected microscopic fields, evenly distributed over the slide, were scanned 5 times each, obtaining 25 scans of every semen sample. The mean of the 25 scans for each microscopic slide was used for the statistical analysis. The software settings of the HTR 12.3, based on Loomis and Graham (2008), are summarized in Table 2. Following parameters recorded by CASA were used for statistical analysis: concentration (CONC), percentage progressive motility (PM), percentage rapid sperm cells (Rapid), average pathway velocity (VAP, µm/s), straight line velocity (VSL, µm/s), curved linear velocity (VCL, µm/s), amplitude of the lateral head displacement (ALH, µm), beat cross frequency (BCF, Hz), straightness (STR, %), and linearity (LIN, %). Every sample was analyzed using ten different chambers. To avoid a possible influence from an increased incubation time, the first chamber used for analysis changed in alternating way for the different samples, so every chamber was equally used as a first and as a consecutive chamber. After each analysis in any type of chamber, concentration was recorded and an additional dilution was performed if this concentration obtained with CASA was higher than 35 × 10 6 sperm /mL, in order to obtain a concentration within the 25 – 35 × 10 6 sperm /mL range for all motility analyses. Table 2. Software settings of the Hamilton Thorne Ceros 12.3 used in this study. Parameter Value Frames acquired 30 Frame rate (Hz) 60 Minimum contrast 60 Minimum cell size (pixels) 6 Minimum static contrast 25 Straightness cut-off (%, STR) 75 Average-path velocity cut-off PM (µm/s,VAP) 50 VAP cut-off static cells (µm/s) 20 Cell intensity 100 Static head size 0.55 – 2.04 Static head intensity 0.45 – 1.70 Static elongation 11 - 99
3.2.3.3. The different chambers and loading technique CHAPTER 3.2 The different types of counting chambers with their specific characteristics used in this study are presented in Table 3. Disposable chambers are marked with a “d” following the depth in µm while reusable chambers are marked with an “r”. The ISAS20r chamber was filled both with capillarity (C) as well as using the drop filled cover slide technique (M). A detailed description of the loading technique per chamber is presented in addendum II. Briefly, chambers filled with capillarity (Leja10d, Leja12d, Leja20d, ISAS12d, ISAS16d, ISAS20d and ISAS20rC) were loaded by placing an abundance of semen in the loading area. When the semen reached the opposite air valve, the excess of semen was carefully removed by means of a Kimtech Science ® tissue (Kimberly-Clark, Surrey, UK). Drop filled chambers (ISAS20rM, Makler10r and WHO) were filled by placing the exact volume on the designated place, followed by immediate application of the coverslide. All chambers were left to rest (at 37°C) prior to analysis until drifting of the cells ceased (approximately 30s). Table 3. Characteristics of the different counting chambers used to analyze equine semen with CASA. Name Chambers/ slide Depth (µm) Volume/ chamber (µL) Way of filling Volume loaded (µL) Reusable - Disposable Leja10d 4 10 1 capillarity 2.5 disposable Leja12d 2 12 3 capillarity 5 disposable Leja20d 4 20 2 capillarity 5 disposable ISAS12d 4 12 - capillarity 5 disposable ISAS16d 4 16 - capillarity 5 disposable ISAS20d 4 20 - capillarity 5 disposable ISAS20rM 2 20 - drop filled 5 reusable ISAS20rC 2 20 - capillarity 5 reusable Makler10r 1 10 - drop filled 5 reusable WHO 1 20.6 - drop filled 10 reusable 3.2.3.4. Experimental design The fifty straws were thawed individually, concentration was determined using the NucleoCounter and the semen was diluted within the above mentioned range and loaded in a chamber. A random rotation with the 10 viewing chambers was done to avoid time effects. After every scan with the CASA system, the playback function was used to ensure the correct reading of the sample. If a given sample loaded in a certain chamber yielded a concentration out of range for that chamber, an additional dilution was prepared based on the later results to ensure a motility analysis within the designed concentration range. 91
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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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Page 56 and 57: CHAPTER 1.2 48 Cryopreservation - t
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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
Page 88 and 89: CHAPTER 3.1 a CEROS (Hamilton-Thorn
Page 90 and 91: CHAPTER 3.1 Progressive Motility CA
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
Page 111 and 112: CHAPTER 3.2 sperm (tail) which coul
Page 113 and 114: CHAPTER 3.2 Hoogewijs M.K., Govaere
Page 115: CHAPTER 3.2 Spizziri B.E., Fox M.H.
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.
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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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