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CHAPTER 3.2 102 In veterinary medicine, a number of studies have focussed on counting chambers in combination with CASA devices. Discrepancies between CASA estimates and the Bürker haemocytometer have been described for different species (Rijsselaere et al., 2003; Vyt et al., 2004). To obtain a sharp image of the sperm cells when performing the motility analysis, CASA devices are primarily used in combination with shallow chambers to keep the moving sperm within focus plane of the microscope. Therefore CASA systems are frequently used in combination with 20 µm deep disposable counting chambers. These chambers are most often loaded by capillary force, and such a capillary flow in a 20 µm chamber follows the laminar Poiseuille flow (Douglas-Hamilton et al., 2005a). This leads to a transverse lifting force on suspended particles and results in an unevenly concentration throughout the sample as described by Segre and Silberberg and is, as such, known as the SS effect (Douglas-Hamilton et al., 2005b). The influence of this SS effect on the distribution throughout the slide depends on the viscosity of the sample. Correction factors are available and are based on the time it takes for a chamber to be filled, and as such determined by viscosity of the loaded sample (Douglas-Hamilton et al., 2005a). Although the SS effect is well documented in literature, interpretation of the results is not always performed correctly. For example, in a recent study (Maes et al., 2010), two types of 20 µm Leja slides were compared, where one of the slides was especially developed to be able to correct for the SS effect. Unfortunately, the results obtained with the two chambers were compared as such, without applying the correction factor. Therefore it was faulty concluded that the newly shaped counting chamber was not able to correct for the SS effect. CASA instruments were developed to provide a solution for problems linked with subjective motility analysis. Indeed, subjective assessment of motility is known to be inaccurate, imprecise and subject to variability (Davis and Katz, 1993; Matson, 1995). However, in order for CASA systems to contribute to a standardized and objective analysis, uniformity in protocols and settings is required. In literature, a large variation in technical settings can be found as well as a lack in uniformity for preparing a sample for motility analysis. It has been demonstrated that the large variety in motility settings used influences motility outcomes significantly (Hoogewijs et al., 2009). The chambers used in combination with CASA are very diverse and quite often not clearly described. As such, it is nearly impossible to compare results between different studies. The big difference in loading technique and motility outcomes may be attributed by physical forces on the spermatozoa during loading as postulated by Lenz et al. (2011). When a semen sample is placed on the loading area of a capillarity filled chamber, the flow that occurs might damage the
CHAPTER 3.2 sperm (tail) which could result in decreased motility, however, these hypotheses remain yet to be tested. The pressing question for standardization becomes more alive when one considers the approval and distribution of equine semen by the dose and not by the pregnancy. Indeed, the widespread use of frozen-thawed semen resulted in increased trading of semen. Mare owners buy semen without the assurance of obtaining a pregnancy, so they tend to make demands on semen quality. When using the following criteria as a minimum for post-thaw equine semen; a post-thaw PM ≥30% and a minimum dose of 240×10 6 progressive motile spermatozoa per insemination dose (Loomis, 2001), the insemination dose from the same ejaculate analyzed in this study can vary from 2 straws (CASA analysis using a Makler chamber) to 10 straws (CASA analysis using Leja10). Comparable results can undoubtedly also be found for other species, where the economic consequences are even more important. This might encourage researchers to get together and start developing a comparable manuscript as the “WHO laboratory manual for the examination and processing of human semen”, so that semen samples from the different domestic species can be analyzed using the standards for that given species. In conclusion, automated semen analysis using highly specialized CASA systems does not produce easily comparable motility outcomes. The influence of the chamber used for analysis on concentration and motility is not minor. As such a complete and accurate description of the “materials and methods” used is of utmost importance, and without it, the correctness of a semen report can at least be considered as questionable. 103
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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
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
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
Page 95 and 96: 3.2.1. Abstract CHAPTER 3.2 Sperm m
Page 97 and 98: CHAPTER 3.2 depth but not the chamb
Page 99 and 100: 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
Page 107 and 108: 60 PM Rapid 50 40 30 20 10 0 CHAPTE
Page 109: Table 5. Averages (± standard erro
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.
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 152 and 153: CHAPTER 4.1 144 In the experiment 3
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 (
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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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