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ADDENDUM I a. b. Fig. 1. (a) Schematic presentation of the plasma membrane with the different components; (A) phosholipid, (B) transmembrane protein, (C) hydrophobic region, and (D) hydrophilic region in random arrangement in the classical lamellar bilayer, and (b) the different presentations as response to cooling; (b1). phospholipids in crystalline domains, (b2). Hexagonal-II inverted micelle, and (b3) aggregated proteins (adapted from Amann and Pickett, 1987). 226 When spermatozoa in a suspension are cooled below 0°C, ice crystals start to form. Initially, ice crystals are formed in the extracellular fluid. Due to these ice crystals, the relative amount of free extracellular water diminishes, resulting in increased concentration of salts and other osmotic active compounds (such as sugars) and as such, resulting in a hypertonic extracellular environment. Water within the spermatozoa does not freeze at this stage but will be transported extracellular which will lead to a progressive dehydration (Amann and Pickett, 1987; Muldrew et al., 2004). If cooling proceeds at a constant slow rate, the spermatozoa will remain close to the osmotic equilibrium by losing water at the same rate that water is lost by the extracellular fluid by its conversion to ice. In contrast a faster cooling rate will cause extracellular water to crystallize more rapidly than the cell can lose water, resulting in an increased osmotic gradient. Cells are damaged at rapid and slow cooling rates, so the optimal cooling rate should be in between these extremes (Mazur, 1984). Slow cooling will induce “solution-effects” injury, caused by exposure to high concentrations of solutes (hyper-osmolarity). Rapid cooling injury is associated with the formation of intracellular ice crystals. Slow cooling injuries can be avoided by increasing the cooling rate, and vice versa (Mazur et al., 1972). The addition of cryoprotectant agents (CPAs) to the freezing extender will increase the survival following freezing and thawing substantially. The CPAs used to cryopreserve semen are penetrating CPAs, they diffuse through the plasma membrane and equilibrate in the cytoplasm. The
ADDENDUM I presence of these CPAs affects the freezing point, and more importantly, they lower the concentration of salts normally found in physiological solutions for a given temperature (Muldrew et al., 2004). They do so by lowering the amount of ice present at a given temperature (less ice equals more water), and they act as secondary solvents for the salts (Pegg, 1984). Penetrating CPAs can greatly reduce slow-cooling injury, however, they provide little protection against rapid cooling injury. Fig. 2. Schematic representation of the physical changes in equine spermatozoa and surrounding extender during freezing. The effect of various cooling rates on the formation of ice crystals or microcrystals (large or small stars) and the movement of solvents and penetrating solutes (heavy or light arrows) are shown (Figure from Amann and Picket, 1987; Hammerstedt et al., 1990). 227
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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
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1.3.1. Standards for equine AI dose
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CHAPTER 1.3 The use of density grad
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CHAPTER 1 Avery B., Greve T. 1995.
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CHAPTER 1 Cheng F.-P., Fazeli A., V
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CHAPTER 1 Flipse R.J., Patton S., A
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CHAPTER 1 Kuisma P., Andersson M.,
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CHAPTER 1 Medeiros A.S.L., Gomes G.
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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.,
Page 183: CHAPTER 4.2 Waite J.A., Love C.C.,
Page 187 and 188: CHAPTER 5 This thesis originated fr
Page 189 and 190: CHAPTER 5.1 Fig. 1. Schematic prese
Page 191 and 192: CHAPTER 5.1 higher than the current
Page 193 and 194: CHAPTER 5.1 al., 2007) showing a cl
Page 195 and 196: CHAPTER 5.1 treatment strategy for
Page 197 and 198: CHAPTER 5.2 supernatant allowing sp
Page 199 and 200: CHAPTER 5.2 such keeping the pellet
Page 201 and 202: CHAPTER 5.2 components of the semin
Page 203 and 204: Final reflections 5.4 Actual change
Page 205 and 206: CHAPTER 5 Dillon R.H., Fauci L.J.,
Page 207: CHAPTER 5 Suárez S.S., Osman R.A.
Page 210 and 211: SUMMARY technique that enables proc
Page 212 and 213: SUMMARY from stallions previously c
Page 214 and 215: SAMENVATTING beweeglijkheid (Hoofds
Page 216 and 217: SAMENVATTING de spermacellen gesele
Page 219 and 220: DANKWOORD Het obligatoire dankwoord
Page 221: DANKWOORD dankzij jou en ze zijn er
Page 225: CURRICULUM VITAE Maarten Hoogewijs
Page 228 and 229: BIBLIOGRAPHY Govaere J., Ducatelle
Page 230 and 231: BIBLIOGRAPHY ABSTRACTS AND PROCEEDI
Page 233: FUNDAMENTAL ASPECTS OF CRYOPRESERVA
Page 237 and 238: ADDENDUM I Fig. 3. Theoretical curv
Page 239 and 240: ADDENDUM II DETAILED LOADING TECHNI
Page 241: In m’n hoofd is alles heel eenvou
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