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CHAPTER 4.1 4.1.2. Introduction 140 Numerous studies have demonstrated the beneficial effect of diluting raw semen with an appropriate extender. As such, the negative influence of seminal plasma (SP) on the preservation of equine sperm can be reduced. Centrifugation of diluted equine semen and subsequent resuspension of the sperm pellet in fresh extender can even further reduce the amount of SP in stored samples. A small proportion of SP in semen improves sperm motility after cooling and storage (Jasko et al., 1991). However, centrifugation itself may act like a double edged sword exerting both beneficial and deleterious effects on sperm motility (Martin et al., 1979). Although the number of sperm cells can be maximized by increasing centrifugation time and force, physical damage is exerted on the sperm cells at the same time. The direct negative outcome of centrifugation on semen quality is demonstrated by the decrease in motility and velocity in contrast to uncentrifuged controls (Jasko et al., 1991) which indicates that the centrifugation protocol (CP) may play an important role in the quality of the inseminate. After 24h of cooled storage the harmful effect of centrifugation is no longer present (Jasko et al., 1991). A CP of 600 × g for 10 min is commonly used for equine semen (Ecot et al., 2005; Knop et al., 2005; Vidament et al., 2000; Weiss et al., 2004). In literature, data on the amount of sperm loss after using this protocol are contradictory and vary from 1.9% (Weiss et al., 2004) to 25% (Ecot et al., 2005; Knop et al., 2005). Nowadays, it is generally accepted that sperm losses will vary around 25% when diluted semen is centrifuged at 400-600 × g for 10-15 min (Aurich, 2008; Loomis, 2006). The importance of an appropriate CP has been clearly demonstrated for human sperm. The duration of centrifugation was shown to be more important than the centrifugation force for causing iatrogenic sperm membrane injuries which resulted in an increased formation of reactive oxygen species (Shekarriz et al., 1995). In boars, similar findings were described: a CP with a high g-force for a short time (2400 × g for 3 min) was used without detrimental effects on sperm yield compared to the standard regime (800 × g for 10 min) (Carvajal et al., 2004). Additionally, a positive effect on semen quality after cryopreservation was detected when using high g-forces for a shorter period of time (Carvajal et al., 2004). Experiments with equine semen have been performed where semen was centrifuged at 400 × g for an increasing period of time. Sperm loss was approximately 20%, if samples were centrifuged for 10 min or longer, while no adverse effects on motility immediately after centrifugation were present, unless semen was centrifuged for 20 min. However, effects on preserved by cooling or freezing were not determined (Heitland et al., 1996). A high g-force for a
CHAPTER 4.1 short time was not detrimental for porcine sperm (Carvajal et al., 2004) and prolonged centrifugation of equine sperm negatively influenced sperm quality (Heitland et al., 1996). The aim of the present study was to compare high speed CPs with the standard protocol on cooled and cryopreserved equine semen. The impact of CP on the subsequent number and quality of sperm cells was assessed daily during 3 consecutive days of cooled preservation. Additionally, the effect of different CPs on the quality of frozen-thawed equine sperm was investigated. 4.1.3. Materials and methods 4.1.3.1. Stallions and semen collection For experiments 1 and 2, five ejaculates from each of five experienced Shetland ponies (n=25), aged 3 to 6 years, were collected between August and October 2007. For experiment 3, one ejaculate from 4 of these ponies was used. The ponies were housed in groups in a straw bedded stable and were fed good quality hay ad libitum. Prior to the onset of the experiment, the extra- gonodal sperm reserves were depleted with 3 collections every other day for one week. For the experiment, the semen was collected twice a week. Semen was collected on a teaser mare using standard procedures with a custom made open ended artificial vagina based on the Colorado State University model as described by Pickett (1993). After collection, semen was filtered through a sterile gauze to remove the gel fraction and debris. The gel-free volume was noted and the sperm concentration was determined using a Bürker haemocytometer after 1:9 dilution with HCl 1M. 4.1.3.2. Media Fresh-cooled semen was processed and stored in Gent Extender (Minitüb, Landshut, Germany), which is a skimmed milk based diluter containing 5 % clarified egg yolk and gentamycin (1 mg/mL). If the semen was to be cryopreserved, a second dilution was performed in Gent Extender for Freezing (Minitüb, Landshut, Germany) which has the same composition as the Gent Extender plus 5 % glycerol. For the fluorescent staining procedures, the samples were diluted in a HEPES buffered solution containing 0.04% BSA. 141
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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
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 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.
Page 145: 4.1 Influence of different centrifu
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
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Page 160 and 161: CHAPTER 4.1 152 4.1.4.2. EXPERIMENT
Page 162 and 163: CHAPTER 4.1 Love and coworkers (200
Page 164 and 165: CHAPTER 4.1 Love C.C., Brinsko S.P.
Page 167 and 168: 4.2.1. Abstract CHAPTER 4.2 The inc
Page 169 and 170: 4.2.3. Materials and methods 4.2.3.
Page 171 and 172: CHAPTER 4.2 For the SLC group, 15 m
Page 173 and 174: CHAPTER 4.2 adding 600 µL of AO st
Page 175 and 176: 4.2.3. Results 4.2.3.1. Sperm quali
Page 177 and 178: CHAPTER 4.2 The aforementioned “T
Page 179 and 180: CHAPTER 4.2 Sperm yields markedly d
Page 181 and 182: 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
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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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