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5.3.5 The development of SCNT embryos reconstructed using TI enucleated oocytes treated with caffeine The developmental potential of SCNT embryos to blastocyst stage using TI enucleated oocytes treated with 5 mM caffeine at 168 hpa was recorded. Three replicates were carried out. The frequency of blastocyst formation was 8.8 ± 0.7 % (3 blastocysts out of 34 couplets) and total cell number was 29.7 ± 0.9 (Figure 5.7). " *ý ýý I Figure 5.7 Hatching porcine SCNT blastocyst (black arrow) TI arrested oocytes were enucleated at 36 - 38 hpm, treated with 5 mM caffeine, cell-transferred and fused by 2 consecutive 50-psec DC pulses of 1.4kV/cm at 42 hpm, activated by 2 consecutive 60-psec pulses of 1.2 kV/cm DC at 44 hpm, and diploidy was induced by treatment for 6h with 7.5 mg/ml CB before in vitro culture for 7 days. Scale bar = 200 pm. 104
5.4 DISCUSSION In the first experiment, I further developed an MPF/MAPK double kinase assay protocol. MPF and MAPK are thought to be important to oocytes and nuclear reprogramming of the NT embryos. Also, MPF and MAPK double kinase assay gives an overview about MPF and MAPK activities in hourly intervals during in vitro maturation. Naito and Toyoda (1991) found repeated freezing and thawing reduced the kinase activities and hence in this experiment all oocytes were subjected to snap-freeze-thawing only once for 30 sec, as was different from Lee and Campbell (2006). Oocytes were stored at -80°C to break the membrane until snap-freeze-thawing. This was also based on Naito and Toyoda (1991), which was also different from Ye et al. (2003) and Lee and Campbell (2006). In addition, the recipe of the kinase assay buffer was slightly different from Ye et al. (2003) and Lee and Campbell (2006). Caffeine treatment of TI enucleated oocytes may contribute to the efficiency of nuclear transfer. In experiments two and three, the effects of different concentrations of caffeine was examined. Early MII oocytes were chosen for these two experiments to help select the caffeine concentration because TI arrested oocytes progressed to MII within 30 min after denuding the cumulus cells. As seen in the second experiment, 5,10 or 20 mM caffeine did not increase either of MPF and MAPK activities of porcine oocytes selected at TI and early MII stage statistically after 6 h. However, Kikuchi et al. (2000) reported that treatment of 72 h-cultured aged oocytes with 5 mM caffeine (last 10 h of culture) elevated MPF activity. Kawahara et al. (2005) found that the addition of 2.5 mM caffeine in the maturation medium of 44 h-matured oocytes for 8h significantly increase the MPF level. The reason for the results observed in these studies could be that those oocytes 105
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The University of -210 Nottingham M
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treated oocytes was recorded at 38
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TABLE OF CONTENTS ABSTRACT ........
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2.5.1.2 Preparation of enucleation/
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CHAPTER 6 .........................
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LIST OF FIGURES Figure 1.1 Process
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LIST OF ABBREVIATIONS A23187 AC AI
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MgSO4.7H20 MAPK MAPKK MAPKKK MBP mg
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CHAPTER 1 LITERATURE REVIEW The aim
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The production of multiple offsprin
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cytoplasm which contained the nucle
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pluripotency, as demonstrated by th
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gene-targeting via somatic cell clo
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conventional breeding and genetic m
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Oocyte maturation is generally cont
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ooýcýýommý MPF Time Figure 1.3
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not obtain clones by SCNT using bra
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A Nuclear NEBD PCC reformation DNA
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1.2.3.2 MPF, MAPK, NEBD and PCC in
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Little is known about the mechanism
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development of porcine embryos prod
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short-wavelength, UV excitable fluo
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The procedure is to expel the PBI a
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The majority of successful porcine
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Until 1997, the established dogma i
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deacetylases (HDACs). Trichostatin
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Figure 1.5 presents the general exp
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CHAPTER 2 General materials and met
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2.3 Oocyte staining and assessment
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holding pipette enucleation pipette
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! 00-0 !".. ww "'') ^ýýe ;ý ý
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,rjý. 7!: rY Is All Pi D r. Figure
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were washed three times in embryo c
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Three replicates of batches of oocy
Page 69 and 70: fertilisation, the occurrence of po
Page 71 and 72: 3.2 MATERIALS AND METHODS 3.2.1 Exp
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Page 75 and 76: 3.3 RESULTS 3.3.1 Conventional matu
Page 77 and 78: ýIC M 00 N N N N 0 öa oÜd U 0 0
Page 79 and 80: Meiotic stage in CAMP treated oocyt
Page 81 and 82: 3.4 DISCUSSION The development of N
Page 83 and 84: 44 h. It was comparable to that (93
Page 85 and 86: oocyte (Chen et al., 1990). In the
Page 87 and 88: CHAPTER 4 Parthenogenetic developme
Page 89 and 90: mechanical and chemical procedures.
Page 91 and 92: COCs were collected and oocytes wer
Page 93 and 94: f. " ý1403: 0.71 14 - 'Ir I A Figu
Page 95 and 96: 100% 90% 80% 70% d 60% 50% 0 40% 30
Page 97 and 98: 4.4 DISCUSSION For successful porci
Page 99 and 100: In addition to the listed benefits
Page 101 and 102: CHAPTER 5 Effect of caffeine treatm
Page 103 and 104: in vitro substrates. In pigs, Naito
Page 105 and 106: 5.2 MATERIALS AND METHODS 5.2.1 Exp
Page 107 and 108: 5.2.2 In vitro maturation of porcin
Page 109 and 110: Blastocysts at day 7 were stained a
Page 111 and 112: Figure 5.2 MPF and MAPK activities
Page 113 and 114: A 4500000 N 4000000 3500000 Z 30000
Page 115 and 116: e ýo; +ý \.. 4.0 ob "i "s NW Ir
Page 117 and 118: 5.3.4 Effects of caffeine treatment
Page 119: Figure 5.6 Effects of 5 mM caffeine
Page 123 and 124: In the final experiment, developmen
Page 125 and 126: CHAPTER 6 General discussion 7.1 Ma
Page 127 and 128: cumulus cells could be observed spe
Page 129 and 130: parthenogenetic development excludi
Page 131 and 132: demonstrated treatment of the TI en
Page 133 and 134: REFERENCES Abeydeera, L. R., Wang,
Page 135 and 136: Bromhall, J. D. (1975) Nuclear trna
Page 137 and 138: Christmann, L., Jung, T. and Moor,
Page 139 and 140: and human ovarian oocytes. Nature,
Page 141 and 142: Funahashi, H., Cantley, T. C. and D
Page 143 and 144: Hartwell, L. H. (1973) Three additi
Page 145 and 146: Jelinkovä, L., Kubelka, M., Motlik
Page 147 and 148: Kubelka, M., Anger, M., Pavlok, A.,
Page 149 and 150: Le Bourhis, D., Beaujean, N., Ruffi
Page 151 and 152: Liu, L., Oldenbourg, R., Trimarchi,
Page 153 and 154: Minshull, J., Blow, J. J. and Hunt,
Page 155 and 156: Oback, B. and Wells, D. N. Cloning
Page 157 and 158: A. E., Garst, A. S., Moore, M., Dem
Page 159 and 160: Rossomando, A. J., Payne, D. M., We
Page 161 and 162: Spemann, H. Embryonic development a
Page 163 and 164: Development. 55(2), 121-127. Törne
Page 165 and 166: Production of the first cloned came
Page 167 and 168: oocytes. Reproduction, 125(5), 645-
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