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CHAPTER 1.2 0.5 mL straws, and glycerol concentration was not corrected for the different concentrations. Later on, Clulow et al. (2008) froze equine semen at 40 × 10 6 /mL and 400 × 10 6 /mL, and adjusted this time the glycerol levels for each group to 4%. Immediately following thawing, the low concentration yielded a superior motility, however, after incubation for 3h, motility was clearly higher for the higher concentration. Semen frozen in BotuCrio® showed better motility characteristics for semen frozen at 100 × 10 6 /mL, and 200 × 10 6 /mL was better than 400 × 10 6 /mL (Nascimento et al., 2008). In this study, no correction for cryoprotectant concentration was made. In conclusion, the influence of concentration on post-thaw semen quality is not uniformly elucidated, however, high concentrations might negatively influence the outcome of cryopreservation. Interactions between the spermatozoa and the different components of the diluter cannot be excluded and as such might be determinant for the optimal sperm concentration for cryopreservation. 42 Glycerol as cryoprotectant The first developed freezing media were rich in egg yolk or milk and contained glycerol as cryoprotective agent (CPA). After some experimental modifications, following media were developed and frequently used: (a) modified lactose EDTA (Martin et al., 1979), (b) lactose EDTA egg yolk (Tischner, 1979), and (c) INRA82 with egg yolk and glycerol (Palmer, 1984); the exact composition of these extenders is presented in table 3. Although glycerol is still the most frequently used cryoprotectant, little uniformity exists concerning the glycerol concentration which is used. The optimal glycerol concentration differs depending on the extender composition and might be influenced by the egg yolk concentration (Ecot et al., 2000). Nevertheless, glycerol has a clear negative effect on fertility, since not only post-thaw sperm motility is affected (Burns and Reasner, 1995) but it will also influence female fertility. This effect is well known in hens, although the contraceptive effect has also been documented in equine species (Bedforf et al., 1995; Vidament, 2005), and is most pronounced in asine species (Vidament, 2005). In literature, a wide variety in glycerol concentration is found in equine freezing extenders, i.e., 2.5% (Palmer, 1984; Vidament et al., 2000, 2001), 3% (Wilhelm et al., 1996a), 3.5% (Tischner, 1979), 4% (Leipold et al.,1998, Wilhelm et al., 1996b), 5% (Martin et al., 1979) and 7% (Pace and Sullivan, 1975). It is advised that glycerol concentration in frozen stallion semen should not exceed 3.5% in order to avoid negative influences on fertility (Vidament et al., 2005). However, the final glycerol concentration is often not available since the glycerol concentration is indicated on the freezing extender but not for the processed semen, where it is variable depending on the dilution ratio used to resuspend the sperm pellet following centrifugation (Vidament, 2005). The final glycerol concentration can accurately be
Table 3. Composition of different media for centrifugation, preservation and freezing of equine semen. a) modified lactose EDTA b) lactose EDTA egg yolk c) INRA82 for freezing d) Modified INRA82 (Martin et al., 1979) (Tischner, 1979) (Palmer, 1984) (Magistrini et al., 1992) Glucose 6.0 g Lactose 11 g Glucose 2.5 g Glucose 2.5 g 100 150 Na2-EDTA 370 mg EDTA Lactose Lactose 150 mg mg mg 150 Na-citrate 375 mg Na-citrate 89 mg Raffinose Raffinose 150 mg mg Na-bicarbonate 120 mg Na-bicarbonate 8 mg Na-citrate 30 mg Na-citrate (5.5 H2O) 30 mg Streptomycin 50 mg Streptomycin 50 mg K-citrate 41 mg K-citrate (H2O) 41 mg INRA82 Penicillin 50 IU Penicillin 50 IU Gentamycin 5 mg Hepes 476 mg Centrifugation medium Distilled water 100 mL Egg yolk 1.6 g Penicilin 500 IU Gentamycin 50 IU/mL Lactose (11%, w/v) 50 mL Glycerol 3.5 mL Distilled water 50 mL Penicillin 50 IU/mL Distilled water 50 mL 50 mL UHT sterilized skimmed milk 100 mL Distilled water 25 mL Centrifugation medium UHT skimmed milk 50 mL INRA82 + 2% egg yolk Centrifugation medium Freezing medium Egg yolk 20 mL pH 7.1 INRA82 + 2% egg yolk + 2.5% glycerol OrvusEs paste 0.8 mL Freezing medium Glycerol 5 mL
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 and 24: CHAPTER 1.1 These chambers are freq
Page 25 and 26: CHAPTER 1.1 it is not possible to o
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 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
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
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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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