Laboratory production of cattle embryos:
Gespeichert in:
1. Verfasser: | |
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Format: | Buch |
Sprache: | English |
Veröffentlicht: |
Cambridge, MA
CABI Pub.
2003
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Ausgabe: | 2. ed. |
Schriftenreihe: | Biotechnology in agriculture
27 |
Schlagworte: | |
Online-Zugang: | Inhaltsverzeichnis |
Beschreibung: | XXVIII, 548 S. Ill., graph. Darst. |
ISBN: | 0851996663 |
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245 | 1 | 0 | |a Laboratory production of cattle embryos |c I. Gordon |
250 | |a 2. ed. | ||
264 | 1 | |a Cambridge, MA |b CABI Pub. |c 2003 | |
300 | |a XXVIII, 548 S. |b Ill., graph. Darst. | ||
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490 | 1 | |a Biotechnology in agriculture |v 27 | |
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650 | 7 | |a In vitro maturation |2 cabt | |
650 | 7 | |a Cattle |2 cabt | |
650 | 7 | |a Embryonic Development |2 cabt | |
650 | 4 | |a Bétail - Embryons | |
650 | 4 | |a Bétail - Embryons - Transplantation | |
650 | 7 | |a Embrião de animal (transplante) |2 larpcal | |
650 | 7 | |a Gado |2 larpcal | |
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Datensatz im Suchindex
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adam_text | Titel: Laboratory production of cattle embryos
Autor: Gordon, Ian R.
Jahr: 2003
Contents
List of Tables and Figures
Preface xxvi
1 Developments in Embryo in Vitro Production (IVP) Technology 1
1.1. Historical Aspects 1
1.1.1. Early IVF reports 1
1.1.2. Cattle IVF 1
1.2. Cambridge Contributions 3
1.2.1. School of Agriculture 3
1.2.2. Animal Research Station 3
Embryos across the Atlantic 4
Using rabbits to good effect 4
Dawn of cattle ET industry 5
1.2.3. Cambridge, Babraham and beyond 5
1.3. Irish Contributions 6
1.3.1. Early studies in cattle 7
1.3.2. Cattle twins by embryo transfer 8
1.3.3. Low-cost embryos 8
1.3.4. Commercializing the embryo production procedure 10
1.3.5. Commercial unacceptability 10
1.3.6. Towards sexed semen on the farm 12
1.4. Developments in ET Technology 13
1.4.1. Thirty years of progress 13
1.4.2. Current cattle ET activity 14
1.4.3. Commercial advantages of cattle ET 15
1.5. Laboratory-produced Embryos 15
1.5.1. Current level of activity 16
Ovum pick-up (OPU) 16
1.5.2. Research with bovine IVP embryos 17
1.5.3. Commercial use of IVP embryos 18
1.5.4. Pathogen-free IVP embryos 19
1.5.5. Animal health and welfare considerations 20
1.6. Embryo Production in Other Farm Mammals 20
1.6.1. Buffaloes 21
1.6.2. Horses 11
Contents
1.6.3. Pigs 26
1.6.4. Sheep and goats 28
1.6.5. Deer 30
1.6.6. Camelids 31
1.7. Human in Vitro Fertilization 3 2
1.7.1. Historical aspects 32
1.7.2. Establishment of pregnancy by embryo transfer 33
1.7.3. Ovarian stimulation regimens for IVF 3 3
1.7.4. Recovery of human oocytes 34
1.7.5. In vitro maturation of human oocytes 34
1.7.6. Intracytoplasmic sperm injection(ICSI) 36
1.7.7. Early embryo culture 3 7
1.7.8. Assessing embryo quality 3 8
1.7.9. Cryopreservation of embryos and oocytes 39
Oocyte preservation 40
1.7.10. Gender preselection 40
The Bovine Oestrous Cycle and Associated Events 42
2.1. Oestrus and the Oestrous Cycle 42
2.1.1. Oestrus 42
2.1.2. Expression of heat 44
2.1.3. Aids to heat detection 44
2.1.4. Endocrine basis of oestrus 45
2.2. The Oestrous Cycle 46
2.2.1. Corpus luteum and progesterone 4 6
2.2.2. Follicular dynamics in the cow 49
Growing understanding of folliculogenesis 49
Zebu cattle 52
2.2.3. The dominant follicle 52
2.2.4. Monitoring ovarian activity 53
2.3. Endocrine Events in the Oestrous Cycle 5 5
2.3.1. Gonadotrophin release 55
2.3.2. Intraovarian events 5 6
2.4. Synchronizing Oestrus 56
2.4.1. Treatment regimens 56
2.5. Prenatal Development of the Bovine Ovary 5 8
2.5.1. Migration of primordial germ cells 5 9
2.5.2. Formation of oogonia 5 9
2.5.3. The primordial follicle 60
2.5.4. Activation of primordial follicles 61
2.5.5. Growth and development of follicles 61
2.5.6. Formation ofthezonapellucida 62
2.5.7. Development of growing follicles 6 3
2.5.8. Antral follicles 63
2.5.9. Follicular atresia 64
2.6. The Bovine Ovary in Postnatal Life 64
2.6.1. The prepubertal animal 64
2.6.2. Antral follicle population 65
2.6.3. Follicle development 6 5
Granulosa cells 66
Thecal cells 67
Basement membrane 68
Contents
2.6 A. Oocyte growth and development 68
Nucleus and nucleolus 70
Mitochondria 70
Golgi complex 70
Cortical granules 71
Ribosomes and cytoplasmic lattices 71
Biochemical aspects of oocyte growth 71
2.6.5. Endocrine events during follicle growth and development 72
Gonadotrophins 72
Oestradiol and progesterone 72
Androstenedione and testosterone 72
2.6.6. Follicular atresia 7 3
2.7. Induction of Multiple Ovulations in the Cow 7 3
2.7.1. Gonadotrophins 74
2.7.2. Control of follicle growth 74
Controlling ovulation 75
2.7.3. Animal and environmental effects 7 5
Nutritional effects 76
2.7.4. Long-range assessments and sexed semen 77
2.7.5. Recombinant bovine somatotrophin (r-BST) and follicle growth 77
2.7.6. Characteristics ofpreovulatory follicles and oocytes after superovulation 78
Recovering the Bovine Oocyte 79
3.1. Oocyte Recovery: Abattoir Ovaries 79
3.1.1. Dissecting the intact follicle 79
Sheep and cattle 79
Other farm animals 80
3.1.2. Aspiration techniques: old and new 80
3.1.3. Ovary slicing techniques 81
Slicing and aspiration 8 3
Other farm animals 8 3
3.1.4. Transillumination-aspiration ovary (TAO) 8 3
3.2. Abattoir Ovaries 8 3
3.2.1. Ovary storage: temperatures and time-limits 83
3.2.2. Ovary storage to enhance oocyte quality 84
3.2.3. Temperature sensitivity of oocytes 8 4
3.2.4. Follicle size and quality 84
3.3. Recovering Oocytes: Live Cattle 8 5
3.3.1. Advantages and alternatives 8 6
Mares 87
Buffaloes and pigs 8 7
3.3.2. Laparoscopic methods of follicular aspiration 8 7
3.3.3. Ultrasonic methods of follicular aspiration 8 8
Developments in ultrasound technology 88
Ultrasound in research and practice 8 8
3.3.4. Developments in ovum pick-up technology 89
OPU in zebu cattle 92
3.3.5. Hormonal and nutritional pretreatments 92
Influence of growth hormone 9 3
Retinol 93
3.3.6. Oocytes from pregnant cattle 93
FSH stimulation 94
Contents
3.3.7. Oocytes from post-partum cattle 94
3.3.8. Oocytes from calves and prepubertal cattle 94
3.4. Live Donors: Other Considerations 9 5
3.4.1. Recovering secondary oocytes 96
3.4.2. Enhancing quality of primary oocytes 96
3.4.3. Oocyte transportation 9 7
3.5. Factors Affecting Oocyte Quality 97
3.5.1. Age of animal 98
Oocytes from fetal ovaries 101
3.5.2. Cattle category, oestrous cycle and ovarian morphology 101
Cattle category 101
Stage of cycle 101
Determining cycle stage 102
Morphology of ovaries 102
Cystic follicles 103
3.5.3. Body condition and nutritional considerations 103
3.5.4. Reproductive status of donor 104
3.5.5. Animal factors 104
3.5.6. Environmental factors 104
3.6. Assessing Oocyte Quality 105
3.6.1. Oocyte morphology: classification schemes 105
Oocyte diameter 108
Iipid vesicles 108
Oestradiol: progesterone ratio 108
Gene expression 108
Oocytes from zebu cattle 108
3.7. Oocytes from Preantral and Early Antral Follicles 108
3.7.1. Birth of young in mice 109
3.7.2. Differences between mice and cattle follicles 109
3.7.3. Utilizing early antral follicles 110
3.7.4. Preantral follicles in humans and pigs 111
Maturing the Bovine Oocyte 112
4.1. Oocyte Maturation in Vivo 112
4.1.1. Summary of events 112
4.1.2. Events leading to ovulation 113
4.1.3. Nuclear and cytoplasmic maturation 114
4.1.4. Biochemical and physiological events during maturation 116
4.2. Oocyte Maturation in the Laboratory 118
4.2.1. Historical aspects 118
4.2.2. Current understanding of in vitro maturation in cattle 120
4.3. In Vitro Maturation (IVM) Culture Systems 121
4.3.1. Culturing intact follicles 122
4.3.2. Simple and complex maturation media 122
Tissue culture medium 199 123
4.3.3. Buffering systems, osmolarity and surface tension 123
4.3.4. Water-quality considerations 123
4.3.5. Static and flux culture systems 124
4.3.6. Effect of maturation time 124
4.3.7. Antibiotic cover and oil overlay 125
Contents
4.3.8. Temperature, gas phase and toxic factors 12 5
Temperature 125
Gas phase 126
Toxic factors - ammonium 126
4.3.9. Bovine serum and bovine serum albumin 126
Bovine serum albumin (BSA) 127
Sources of bovine serum 127
Constituents of bovine serum 127
Serum levels employed in IVM media 128
Heat treatment of serum 128
4.3.10. Bovine follicular fluid 129
Inhibitory action of follicular fluid 130
Follicular fluid composition 1 30
Hyaluronic acid as a serum substitute 131
Hyaluronan in culturing oocytes in small groups 131
4.4. Somatic-cell Support 132
4.4.1. Cumulus-oocyte complex (COC) 132
4.4.2. Connexin 43 and oocyte meiotic maturation 1 32
4.4.3. Additional cumulus/granulosa cells 133
4.4.4. Special needs of ovum pick-up (OPU) oocytes 134
4.4.5. Use of non-follicular cells 135
4.4.6. Action of theca cells 135
4.5. Hormones and Growth Factors 135
4.5.1. Hormones 136
Follicle-stimulating hormone and luteinizing hormone 136
Prolactin 137
Growth hormone (somatotrophin) 137
Steroids 137
Insulin and GH-RH 138
4.5.2. Growth factors 138
Epidermal growth factor (EGF) 139
IGF family 139
Midkine and other growth factors 140
Other farm animals 140
4.5.3. Cytokines 140
4.5.4. Oocyte-derived growth factors 140
4.6. Oocytes Cultured Singly or in Groups 141
4.6.1. Single-oocyte culture systems 141
4.7. Single-culture medium systems 142
4.7.1. Synthetic oviductal fluid (SOF) formulations 142
4.8. Chemically Defined Culture Systems 143
4.8.1. Using synthetic oviductal fluid (SOF) 143
4.8.2. TCM-199 143
4.9. Oxidative Stress in Oocyte Maturation 144
4.9.1. Role of glutathione (GSH) 144
4.10. Two-step Culture Systems 145
4.10.1. Background information 145
4.10.2. Maintenance of meiotic arrest 146
Cattle oocytes 147
4.10.3. Biological inhibitors 147
Influence of granulosa-theca cells 14/
Contents
4.10.4. Biochemical inhibitors 148
Role of cyclic 3 5 -adenosine monophosphate (cAMP) 148
Manganese 148
4.10.5. Pretreatment of donor cattle 148
4.10.6. Two-step treatment in the laboratory 149
Roscovitine 149
Butyrolactone I 150
Other farm animals 151
4.10.7. Enhancing the quality of oocytes from small follicles 152
4.10.8. Synchronizing germinal vesicle development 152
4.11. Other Factors Influencing Oocyte Maturation 153
4.11.1. Energy sources and second messengers 153
Glucose 153
cAMP and analogues 153
4.11.2. Hormones and vitamins 153
Prostaglandins and steroids 153
Retinoic acid 154
4.11.3. Opioid antagonists and chemical agents 154
Endogenous opioid peptides 154
Dimethylsulphoxide and ethanol 154
Selenium 154
4.11.4. Simplifying maturation culture systems 154
4.12. Evaluating the Maturation Process 155
4.12.1. Stages in nuclear maturation 155
4.12.2. Cumulus-cell expansion 156
Mitochondria! distribution 156
4.12.3. Morphological assessment and staining methods 157
4.12.4. Gene expression and oocyte competence 157
Capacitating Bovine Sperm 158
5.1. Introduction 158
5.1.1. Historical 158
5.1.2. The capacitation process 159
5.1.3. Hyperacti vation 161
5.1.4. The acrosome reaction 161
5.1.5. Artificial induction of capacitation 163
5.2. Capacitation in the Cow 163
5.2.1. Sperm transport 163
5.2.2. Oviductal secretory cells 164
5.2.3. Glycosaminoglycans 166
5.2.4. Simulating oviductal events in vitro 166
5.3. Capacitation Procedures 167
5.3.1. Historical 167
5.3.2. Modifying osmolarity and pH 168
pH values 168
5.3.3. Evaluating sperm-capacitation systems 168
5.4. Heparin and Heparin-like Glycosaminoglycans 169
5.4.1. Actions and interactions of heparin 169
5.4.2. Practical application of heparin treatment 170
5.5. Use of Fresh or Frozen Semen 171
5.5.1. Fresh semen 171
Contents
5.5.2. Frozen semen 171
5.5.3. Semen diluents 172
5.6. Bulls as a Source of Variability 172
5.6.1. High- and low-fertility bulls 173
5.6.2. Bull variability 17 5
5.6.3. Methods of assessing bull fertility 174
5.6.4. Enhancing semen quality 174
5.7. Efficiency of Capacitation Procedures 175
5.7.1. Staining methods 175
5.7.2. Oocyte penetration tests 175
5.7.3. Sperm-zona binding 175
In Vitro Fertilization 176
6.1. Introduction 176
6.1.1. In vitro maturation and fertilization: early reports 176
6.1.2. Chapter contents 177
6.2. Fertilization in the Cow 178
6.2.1. Oviductal environment 178
6.2.2. Iifespan of the secondary oocyte 180
6.2.3. Dispersion of cumulus cells 180
6.2.4. Fertilization rates in cattle 181
6.2.5. Fertilization rates in superovulated animals 181
Accessory spermatozoa 182
6.3. Preparing Sperm for in Vitro Fertilization 182
6.3.1. Use of fresh bull semen 182
6.3.2. Assessing the quality of frozen-thawed semen 18 3
6.3.3. Swim-up procedures 183
Swim-up and hyaluronic acid 184
Swim-up and caffeine 184
Swim-up and the sex ratio 185
6.3.3. Percoil density gradients 185
Sex-ratio deviations 186
Other farm animals 187
6.3.5. Glass-wool filtration procedures 187
6.3.6. Use of hyaluronic acid 187
6.3.7. Cell-to-cell contact 187
Cell-to-cell interactions with epididymal cells 188
6.3.8. Sperm abnormalities 188
Proximal droplets 188
Nuclear vacuoles 189
Knobbed acrosome defect 189
Robertsonian translocations 189
Hypo-osmotic swelling (HOS) as a screening assay 190
6.3.8. Sperm doses 190
6.4. Enhancing Sperm Motility 191
6.4.1. Penicillamine, hypotaurine, epinephrine (adrenalin) (PHE) 191
6.4.2. Caffeine, theophylline and pentoxifylline 191
6.5. Preparing Oocytes for Fertilization 192
6.5.1. Beneficial effects of cumulus cells 192
Cumulus-cell removal after fertilization 194
6.6. In Vitro Fertilization Culture Systems 195
Contents
6.6.1. The fertilization medium 195
TALP medium 195
SOF medium 196
Fert-CDM medium 196
6.6.2. Protein supplementation 196
6.6.3. Gas phase considerations 197
6.6.4. Temperature, light andosmolarity 197
Temperature 197
Light 197
Osmolarity 198
6.6.5. Somatic cells in the fertilization medium 198
6.6.6. Activation of COCs with calcium ionophore (A23187) 198
6.6.7. Oxidative stress in the IVF culture system 199
6.6.8. Other factors influencing efficacy of IVF system 199
Glucose 199
GH-RH 199
Methyl-fJ-cycIodextrin 200
Hyaluronic acid 200
Dimethylsulphoxide (DMSO) 200
Prostaglandins 200
Toxic factors 200
6.7. Interaction of Spermatozoon and Oocyte 200
6.7.1. Sperm-oocyte recognition mechanisms 200
Oviductal factors 202
6.7.2. Early events in the fertilization process 202
Changes in zona pellucida 203
6.7.3. Crossing the interspecific sperm barrier 203
6.7.4. Factors with a negative effect on fertilization 204
Zona hardening 204
6.8. Post-insemination Treatment of Oocytes 204
6.8.1. Effect of sperm exposure time 204
6.9. Micro-assisted Fertilization 206
6.9.1. Zona thinning 206
6.9.2. Zona drilling and partial zona dissection 206
6.9.3. Subzonal sperm insertion (SUZI) 207
6.9.4. Intracytoplasmic sperm injection (ICSI) 208
Twelve thousand years into the past 209
ICSI in cattle 209
Successful cattle ICSI without artificial activation 210
Cattle ICSI in research 211
Gender preselection in cattle by ICSI 211
ICSI in the mare 212
ICSI and factors influencing oocyte activation 213
ICSI in mice 213
6.10. Efficiency of IVF Procedures 214
6.10.1. Criteria for assessing fertilization 214
Chromosome preparation 214
6.11. Fertilization Abnormalities 214
6.11.1. Polyspermy and parthenogenesis 214
Polyspermy 214
Parthenogenesis 214
Contents
6.12. Variability in Bull Fertility 215
6.12.1. Effect of bull on IVF outcome 215
6.12.2. In vitro fertilization tests in predicting bull fertility 216
Sperm chromatin structure 217
The bovine centrosome (centriole) 217
6.12.3. Reducing bull fertility 219
7 Culturing and Evaluating the Early Bovine Embryo 220
7.1. Introduction 220
7.1.1. Historical 220
7.1.2. In vivo culture systems 220
7.1.3. In vitro culture systems 221
7.1.4. Chapter contents 222
7.2. Early Embryo Development in the Cow 222
7.2.1. The oviductal microenvironment 222
7.2.2. Cleavage of the bovine embryo 223
Duration of cell cycles 224
Steroidogenic activity of embryo 224
Nucleoli and nucleolus organizer regions in the early embryo 224
7.2.3. Compaction and cavitation 225
Hatching 226
Apoptosis 226
7.2.4. Post-hatching progress 227
7.2.5. Embryo mortality 228
Factors in embryo mortality 229
Embryo-pathogen interactions 229
Fate of the conceptus 230
Losses after embryo transfer 230
Fetal losses 230
7.3. In Vivo Culture Systems 230
7.3.1. The rabbit oviduct 230
7.3.2. The sheep oviduct 231
Elongation-stage bovine embryos 232
7.3.3. The isolated mouse oviduct 233
Using oviducts of live mice 233
7.4. Metabolism of the Early Embryo 234
7.4.1. Monitoring embryo metabolism 234
Oxygen consumption 235
Glucose utilization 236
Energy metabolism-related gene expression 236
Myo-inositol, adenylyl cyclase 236
Ultrastructural autoradiography of RNA synthesis 236
7.4.2. The development block 236
7.4.3. Activation of the bovine embryonic genome 236
7.5. In Vitro Culture Systems 238
7.5.1. Embryo culture systems: past and present 238
Serum-restricted culture systems 239
Use of commercial media 240
Towards defined culture systems 240
Sequential media 240
Microfluidic embryo manipulation 241
Contents
7.5.2. TCM-199 and SOF culture media 241
7.5.3. Co-culture with bovine oviductal cells 243
Bovine oviductal cell monolayer 243
7.5.4. Co-culture with non-oviductal cells 245
7.5.5. Serum-supplemented culture systems 247
Duration of serum treatment 248
Serum substitutes 248
Supplementation with bovine follicular fluid 249
7.5.6. Serum-free culture systems 249
7.5.7. Defined culture systems 250
Growth factors 251
Hyaluronic acid supplementation 251
7.5.8. Sequential media 251
Culturing cattle embryos 252
Pig embryos 253
7.5.9. Embryo group size 253
The WOW culture system 255
7.5.10. Gas atmosphere 255
Oxygen 255
Carbon dioxide 256
Ambient laboratory air 256
7.5.11. Temperature and light 256
7.5.12. Protection from oxidative stress 257
7.5.13. Hormones, growth factors andcytokines 258
Growth hormone and insulin-like growth factors 258
Interferon-tau/ alpha 259
Epidermal growth factor 259
Effect of cytokines 260
7.5.14. Culture media components 2 60
Antibiotics 260
Insulin 261
Amino acids 261
Heparin 261
Hexoses 262
Vitamins 262
Surface-active components 262
Mineral and silicone oils 262
7.5.15. Possible toxic agents 263
Ammonia 263
Nitric oxide 263
7.5.16. Simplifying culture systems 263
7.6. Evaluating Embryo Quality 2 64
7.6.1. Morphological and morphometric parameters 264
Variability in embryo grading 265
Ultrastructural features 265
Human embryo quality considerations 266
7.6.2. Age and developmental stage attained 266
Time of first cleavage 266
Early cleavage and pregnancy rates in human assisted reproduction 267
Assessing embryo quality at morula stage 267
Timing of blastocyst formation 268
Contents
7.6.3. Metabolic tests 268
7.6.4. Indications of embryo normality 269
Staining tests 269
Chromosomal abnormalities and cell numbers 269
Intercellular communication 2 70
Lipid droplets 2 70
Interferon-tau secretion 2 70
Golgi apparatus 271
Proliferating cell nuclear antigen 271
Embryo cryosurvival 271
Proteins involved in embryo developmental competence 271
7.6.5. mRNAexpression patterns 271
Embryos under stress 273
7.6.6. Post-hatching evaluation 273
7.6.7. Post-transfer evaluation 275
Insulin-like growth factors 275
Heavy calves 275
IVP and nuclear-transfer cattle embryos 275
Preservation of Embryos and Oocytes 277
8.1. Introduction 277
8.1.1. Embryo cryopreservation: past and present 277
In vitro-produced embryos 2 78
Vitrification 279
Advances in other farm mammals 280
8.1.2. Ad vantages of embryo storage 280
8.2. Storing the Fresh Embryo 280
8.2.1. Embryo storage at ambient temperature 280
8.2.2. Embryo sensitivity tocooling 281
IVP cattle embryos 281
8.2.3. Embryo storage at refrigerator temperature 282
8.3. Conventional Freeze-Thaw Protocols 283
8.3.1. Cryoprotectants 283
8.3.2. Two-step to one-step temperature decrease 284
8.3.3. Straws for storage 284
8.3.4. One-step thawing procedures 284
8.3.5. Ethylene glycol as the cryoprotectant 286
Prefreezing additives 287
Ultrastructural studies 287
Demi-embryos 287
Trophoblastic vesicles 287
8.3.6. Thawing and cryoprotectant removal 287
8.4. Freezing the IVP bovine embryo -288
8.4.1. Morphological and functional differences 288
Embryo ultrastructure after cryopreservation 288
8.4.2. Embryo survival and pregnancy rates 288
Glycerol 289
Ethylene glycol 289
Prefreezing additives 290
Freezing zygotes and early-cleavage embryos 290
8.4.3. Delipidizing the embryo 29°
Delipidizing the pig embryo 291
Contents
Delipidizing the IVP bovine embryo 291
Lipids and mitochondria 291
8.4.4. Effect of culture medium 292
8.4.5. Rapid freezing of IVP embryos 292
8.5. Vitrification ofin Fivo-Produced Embryos 293
8.5.1. Early studies 293
Cleavage-stage embryos 294
8.5.2. Vitrification and slow freezing as alternatives 294
8.6. Vitrification of IVP Embryos 295
8.6.1. Developing an effective vitrification procedure 295
Assisted hatching 296
Avoiding contamination of embryos 297
Factors relevant to the success of vitrification 297
8.7. Cryopreservation of the Bovine Oocyte 297
8.7.1. Factors relevant to oocyte cryopreservation 298
8.7.2. Freeze-thawing 298
Ultrastructural evaluation 299
8.7.3. Vitrification 299
Previtrification additives 301
Bovine vs. equine oocytes 301
Ultrastructural evaluation 301
8.8. Embryo Evaluation after Thawing 302
8.8.1. Evaluation of IVP embryos 302
Establishing Pregnancies with IVP Embryos 303
9.1. Introduction 303
9.1.1. Historical 303
9.1.2. Requirements for on-farm applications 304
From research to practice 305
Embryo transfer as a research tool 305
9.1.3. Welfare implications of using IVP embryos 305
9.2. Preparing Embryos for Transfer 306
9.2.1. Media employed 306
Antibiotic/antimicrobial cover 306
Serum and serum substitutes 306
Tropical environment 307
Handling cattle embryos 307
9.2.3. Protecting the embryo 307
Embryo encapsulation technology 307
Predicting embryo hatching 307
9.2.4. Number of embryos transferred 307
9.3. Surgical and Non-surgical Transfers 308
9.3.1. Surgical transfers 308
Endoscopy and tubal transfer of embryos 309
9.3.2. Non-surgical transfers 309
Factors affecting success 309
In vivo embryos 309
In vitro embryos 310
Operator skill 310
9.4. Donor-Recipient Synchrony 311
9.4.1. Importance of synchronization 311
Accuracy of oestrus detection 312
Contents
9.4.2. Synchronization in the IVP embryo context 312
9.5. Oestrus Synchronization Techniques 312
9.5.1. Protocols for synchronizing oestrus 312
Ovsynch 313
Progesterone/progestogen 313
9.6. Selection and Management of Recipients 315
9.6.1. Heifers versus cows 315
9.6.2. Factors affecting recipient suitability 316
Recipient hormone levels 316
Plasma urea nitrogen 316
Repeated transfers 316
Role of the major histocompatibility complex (MHC) 316
9.6.3. Minimizing stress in recipients 316
Tranquillization 317
Welfare concerns 317
9.6.4. Detecting early pregnancy in recipients 317
9.7. Enhancing Pregnancy Rates in Recipients 318
9.7.1. Progesterone supplementation 318
9.7.2. Hormonal therapy in early dioestrus 319
9.7.3. Hormonal therapy in late dioestrus 319
9.7.4. Use of trophoblastic vesicles 320
9.7.5. Prostaglandin inhibitors 320
9.7.6. Oral treatment with propylene glycol 320
9.7.7. Re-synchrony of non-pregnant recipients 321
10 Embryos and Oocytes in Research and Commerce 322
10.1. Introduction 322
10.1.1. From research to commercial application 323
10.1.2. Cattle products and human health 323
Population growth and food resources 324
10.2. Embryo Production Technology: Problems 325
10.2.1. Differences between IVP- and in vivo-derived offspring 326
10.2.2. The large-offspring syndrome (LOS) 327
Placental abnormalities 327
Gene expression 328
IVP embryo laboratories and LOS 328
10.2.3. Large-offspring syndrome: human implications? 329
10.3. Embryo Production Technology: Prospects 330
10.3.1. Animal-health considerations 330
Contaminated semen 332
Problems posed by IVP cattle embryos 332
Detection of viruses 3 33
Reducing infectivity associated with IVP embryos 333
10.3.2. IVP embryos in breeding-improvement programmes 333
MOET schemes 333
Open-nucleus breeding scheme 334
Reducing the generation interval 3 34
Post-mortem use of valuable genetic material 535
Future developments 335
10.3.3. Beef calves from dairy cows 335
10.3.4. Twinning by embryo transfer 336
Mechanisms controlling double ovulations 336
Contents
Embryo transfer 336
Feasibility of twinning in farming practice 338
10.3.5. Preserving genetic diversity 338
Using immature sperm cells 339
10.3.6. Embryos for tropical/subtropical regions 339
10.3.7. Bypassing heat-stress problems 339
10.3.8. Dealing with repeat breeders 341
Infertile cows 341
10.3.9. Cattle embryos and oocytes for research 342
Interspecies nuclear transfer 342
Identifying toxicants 343
Environmental pollutants 343
10.4. Sex Control by Sperm Separation 343
10.4.1. The case for semen sexing 345
10.4.2. Semen-sexing technology 346
Beltsville sexing technology 346
Other sorting studies 346
Sperm-membrane changes in sorted sperm 347
Effect of Fert Plus peptide 347
Frozen sexed semen 347
In vitro fertilization with sorted bull sperm 348
10.4.3. Alternatives to sexing by flow cytometry 349
Immunological approach 349
Separation by density gradients 349
Spermatozoal head size and volume 350
10.4.4. Effect ofAI timing on sex ratio 350
10.4.5. Sperm separation in other farm animals 351
Pigs 351
Horses 352
Sheep 352
10.5. Embryo Sexing 352
10.5.1. Sexing by polymerase chain reaction technology 353
10.5.2. Fluorescence in situ hybridization 3 54
Sexing by male-specific antigen 354
10.5.3. Sexual dimorphism 355
10.6. Cloning in Cattle: Progress and Problems 356
10.6.1. Introduction 357
Story to date 357
Normality of clones 359
Safety of food products 359
10.6.2. Embryo splitting 359
10.6.3. Essential steps in nuclear transfer 360
Quality of recipient oocytes 360
Enucleation 360
Telophase enucleation 362
Introduction of donor nucleus 362
Choice of donor cell and cell-cycle stage 363
Fetal or adult somatic cells 364
Quiescent or proliferating cells 365
Non-viable cells as donors 365
Activation 365
10.6.4. Nuclear reprogramming 366
Contents
10.6.5. Simplifying nuclear-transfer protocols 367
10.6.6. Preserving donor cells, cytoplasts and cloned embryos 368
Refrigeration 368
Freezing 368
Vitrification 368
10.6.6. In vitro culture and evaluation of nuclear-transfer embryos 369
Ploidy analysis 369
Ribosomal RNA gene activation 369
Apoptosis 3 70
ICM and TE cells 370
Gene expression patterns 3 70
Mitochondrial heteroplasmy 370
Telomerase activity 3 70
10.6.7. Gestational and perinatal losses 371
Neonatal care 3 72
Preventing LOS? 372
10.6.8. Development of clones after birth 372
10.6.9. Embryonic stem cells 373
10.7. Transgenic Cattle 373
10.7.1. Development of transgenic technology in cattle 375
10.7.2. Potential advantages of transgenic cattle 375
10.7.3. Methods of genetic modification in cattle 376
Pronuclear injection 376
Transfected cells for nuclear transfer 377
Gene targeting 378
Sperm-mediated DNA transfer 378
Retroviral infection of early embryos 379
10.7.4. Transgenic embryos in the laboratory 3 79
Predicting transgene integration 3 79
Preserving embryos 3 79
10.7.5. Losses in transgenic embryos, fetuses and calves 380
10.7.6. Transgenic cattle on the farm 380
Germ-line mosaic bulls 380
Transgenic cows 380
10.7.7. Welfare of transgenic cattle 380
Appendices
Appendix A: Embryo Production Protocols 382
Appendix B: Preparation of Culture Media 389
Appendix C: Cryopreservation Procedures 392
Appendix D: Journals, Books and On-line Sources of Information Relevant to
the in Vitro Production and Transfer of Cattle Embryos 394
References 396
Index 537
Tables and Figures
Table 1.1.
Table 1.2.
Table 1.3.
Table 1.4.
Table 1.5.
Table 1.6.
Table 1.7.
Table 1.8.
Table 1.9.
Table 1.10
Table 1.11
Table 1.12
Table 2.1.
Table 2.2.
Table 2.3.
Table 2.4.
Table 2.5.
Table 2.6.
Table 2.7.
Table 2.8.
Table 2.9.
Table 3.1.
Table 3.2.
Table 3.3.
Table 3.4.
Table 3.5.
Table 3.6.
Tables
Milestones in mammalian in vitro fertilization 2
Effect of IVP-embryo transfer on calf output per cow 12
Milestones in farm-animal embryo transfer and related techniques 13
Data for worldwide cattle ET activity in 2000 14
IVP cattle embryo production during 12 months in a German laboratory (BFZF) 16
Cattle IVP embryos transferred worldwide in 2000 16
Comparison of cloning efficiency in cattle and buffalo embryos 2 2
Equine embryo development: sheep oviduct vs. in vitro culture 24
Pregnancies after transfer of in vitro- vs. in Wvo-produced sheep embryos 29
Pregnancies after transfer of in vitro- vs. in vivo-produced goat embryos 30
Embryo transfer results from llama donors 3 2
Outcome of embryo transfers after ICSI in relation to origin of sperm 3 7
Detecting the cow in heat - signs to observe 4 3
Scoring system for heat detection - based on behavioural signs 4 3
Least-square means of corpus luteum weights and progesterone levels on
days 3-19 of the natural oestrous cycle 48
Number of primordial, primary, secondary and Graafian follicles in cattle
ovaries 59
Action of growth factors on follicular cells 64
Numbers of antral follicles in the ovaries of the cow 65
Size of follicles in the ovaries of Japanese black cattle 66
Relationship between oocyte diameter and maturation rate 70
Superovulatory responses with or without CIDR-P4-OB 7 5
Bovine oocyte quality and aspiration vacuum pressure 82
Bovine oocytes obtained by slicing or aspiration 82
Follicle size and oocyte development 8 5
Comparisons between problem, pregnant and cyclic cows in embryo
production 86
Ovum pick-up in Indian cattle 8 7
Oocyte recovery in cattle and African buffaloes using a new device 90
Tables and figures
Table 3.7. Oocytes from FSH-treated post-partum beef cattle 94
Table 3.8. Treatment to obtain in vivo-matured bovine oocytes 96
Table 3.9. Comparison of calf vs. cow oocytes in blastocyst yield 99
Table 3.10. Appearance of bovine corpus luteum at different stages of the cycle 102
Table 3.11. Genetic merit and embryo yield in dairy cattle 104
Table 3.12. Criteria used in assessing bovine oocyte quality 105
Table 3.13. Beltsville criteria for bovine oocyte selection 106
Table 3.14. Characterizations of nine morphological groups used to classify bovine oocytes 106
Table 3.15. Development of in vifro-grown bovine oocytes 1 1 1
Table 4.1. Towards an understanding of mammalian oocyte maturation 120
Table 4.2. Effect of maturation time on developmental competence of the bovine oocyte 124
Table 4.3. Effect of temperature gradients on bovine oocyte maturation 126
Table 4.4. Amino acid concentrations in sera of clinically normal cows 12 8
Table 4.5. Development of bovine oocytes in HA-supplemented medium 131
Table 4.6. Effect of hyaluronic acid in a serum-free maturation medium on blastocyst
yield 132
Table 4.7. Developmental capacity of cattle oocytes matured with and without
granulosa-cell supplementation 1 34
Table 4.8. SOF formulation employed as a single-culture system for bovine embryo
production 143
Table 4.9. Culturing cattle oocytes under roscovitine meiotic inhibition 1 50
Table 4.10. Effect of retinoic acid on meiotically inhibited bovine oocytes 150
Table 4.11. Stages of germinal vesicle development based on changes in the nuclear
morphology of bovine oocytes 152
Table4.12. Assessing bovine oocytes after maturation 156
Table 5.1. Approaches to the artificial capacitation of bovine sperm in vitro 163
Table 5.2. Effect of pH on capacitation and fertilization in cattle 168
Table 5.3. Summary of molecules proposed as markers of sperm fertility 169
Table 5.4. Effect of sperm collection method on IVF in cattle 172
Table 6.1. Amino acids in bovine oviductal and uterine fluids 179
Table 6.2. Reports dealing with loss of cumulus cells from bovine oocytes shortly after
ovulation 181
Table 6.3. Accessory sperm in superovulated and single-ovulating cows 182
Table 6.4. Comparison of Percoll and swim-up techniques for sperm selection 186
Table 6.5. Sperm abnormalities and fertility in bulls 189
Table 6.6. Effect ofpentoxifylUne on cattle IVF 192
Table 6.7. Effect of denudation method on IVF and embryo development in cattle 193
Table 6.8. Citrate cleaning of cattle oocytes 195
Table 6.9. Effect of granulosa cells on bull sperm motility 196
Table 6.10. Effect of oxygen level on cattle IVF 197
Table6.11. Effect of co-culture ofbull sperm with BRL cells 198
Table6.12. Effect ofglutathione(GSH) in fertilization medium on blastocyst yield 199
Table 6.13. Developmental stages of male and female pronuclei 203
Table 6.14. Proposed sequence of events in human ICSI 208
Table 6.15. Factors affecting the success of ICSI 209
Table 6.16. Cattle ICSI in combination with oocyte activation 210
Table 6.17. Use of sperm from fertile and infertile stallions by ICSI 212
Table 6.18. Effect of bull on cleavage rate and blastocyst yield 216
Tables and Figures
Table 7.1. Development of early bovine embryos on an oviductal-cell monolayer 221
Table 7.2. Summary of data on cattle embryo yields after culture in the sheep oviduct 232
Table 7.3. ATP content oflVP cattle embryos at different stages of development 235
Table 7.4. Growth factors related to reproduction in the cow 239
Table 7.5. Composition of synthetic oviductal fluid (original formulation and a modified
SOF formulation) 242
Table 7.6. Development of in vitro- or in vivo-produced zygotes in a bovine oviductal cell
co-culture system 245
Table 7.7. Development of IVF bovine embryos co-cultured with different cell types 245
Table 7.8. Effect of B2 and TCM-199 media on IVP bovine embryo development 246
Table 7.9. Culture of IVP cattle embryos in different media + 5% FCS 248
Table 7.10. Effect of serum concentration in SOFaa medium on IVP embryo development 248
Table 7.11. Effect of changing KSOM + BSA on day 4 to SOFaaci + 5% FCS 248
Table 7.12. Effect of hyaluronic acid on development of IVP embryos 251
Table 7.13. Composition ofhuman embryo culture media-Gl and G2 252
Table 7.14. IVP embryo production with single or group embryo culture 254
Table 7.15. Embryo production in relation to group size 255
Table 7.16. Embryo development in different culture systems and group sizes 256
Table 7.17. Effect of heparin supplementation on blastocyst yield and cell number 262
Table 7.18. Embryo grading scheme (International Embryo Transfer Society Manual) 264
Table 7.19. Volume density of cellular components in cattle blastocysts produced under
different culture conditions 266
Table 7.20. Time of first cleavage and bovine embryo development 267
Table 8.1. Milestones in the birth of young after transfer of frozen-thawed embryos 278
Table 8.2. Milestones in vitrification of mammalian embryos 279
Table 8.3. Development and survival of vitrified bovine embryos produced under
different culture conditions 2 79
Table 8.4. Media employed by 26 commercial cattle ET companies in the USA 281
Table 8.5. Development of cattle embryos chilled at different cleavage stages 282
Table 8.6. Pregnancy rates after transfer of cooled and transported horse embryos 282
Table 8.7. Evaluation of pig embryos stored at different temperatures and in different
media 283
Table 8.8. Freezing protocol for bovine embryos using glycerol as cryoprotectant 284
Table 8.9. Pregnancy rates after transfer of embryos frozen in glycerol or ethylene glycol 286
Table 8.10. Survival of bovine blastocysts developed from delipidated zygotes 291
Table 8.11. Comparison of conventional freezing and vitrification 296
Table 9.1. First records of successful embryo transfers 303
Table 9.2. Dulbecco s phosphate-buffered saline (D-PBS) 306
Table 9.3. Pregnancy rates in recipients after transfer of half-embryos, embryos or
two embryos 308
Table 9.4. Pregnancy rates after transfer of fresh or frozen in vivo and IVP embryos into
Friesian heifer recipients at one location 310
Table 9 5. Effect of embryo-recipient synchrony on pregnancy rates 312
Table 9.6. Effect of low-dose eCG (PMSG) treatment on pregnancy rates in embryo
recipients 314
Table 9.7. Effect of timing of PG dose in progesterone-synchronized recipients on
pregnancy rate 315
Table 9.8. Methods of pregnancy diagnosis and times at which they can be used during
pregnancy 317
Tables and Figures
Table 9.9. Progesterone levels and pregnancy rates in recipients orally dosed with
propylene glycol 521
Table 10.1. Potential applications of cattle IVP technology in commercial practice and
research 324
Table 10.2. Birth weights and gestation lengths in Japanese Black cattle after transfer of
IVP embryos or after artificial insemination 326
Table 10.3. Fetal, placental and other measures of IVP-derived and AI calves 329
Table 10.4. Assessment of risk of transmission of infectious diseases by in vivo-derived
cattle embryos 3 32
Table 10.5. Twin ovulations and twin births in British and Irish cattle 3 36
Table 10.6. Twinning by embryo transfer to bred recipient cattle 3 37
Table 10.7. Development of bovine oocytes injected by various types of male gametes 340
Table 10.8. Possible value of sex control in cattle fanning 345
Table 10.9. Effects of Fert Plus on cleavage and blastocyst production in cattle 348
Table 10.10. Pregnancy rates using sexed and unsexed frozen-thawed semen 348
Table 10.11. Effect of time of AI after the onset of oestrus on the sex ratio in cattle 350
Table 10.12. Zimmermann mammalian cell-fusion medium formulation 362
Table 10.13. Pregnancies in recipients that received three different clone types and IVP
embryos 365
Table 10.14. Pregnancy rates after transfer of fresh or vitrified cloned embryos 369
Table 10.15. Tissue characteristics of cloned and control fetuses 371
Table Bl. Preparation of phosphate-buffered saline (PBS) 389
Table B2. Preparation of oocyte washing medium (modifiedHEPES-buffered Tyrode s
medium) 389
Table B3. Preparation of sperm capacitation medium (modified Ca2+-free Tyrode s
medium) 390
Table B4. Preparation of fertilization medium (modified Tyrode s medium) 3 90
Table B5. Preparation of motility-stimulating mixture: penicillamine-hypotaurine-
epinephrine (adrenalin) (PHE) 390
Table B6. Procedures for fixation and staining of oocytes and embryos 391
Table B7. Salt concentrations in CR1 and CR2 media 391
Figures
Fig. 1.1. Beef cattle twins produced by totally in vitro procedures 2
Fig. 1.2. Steps in Lu s IVP embryo production process 9
Fig. 1.3. Cattle twins born after one-embryo transfer to a bred recipient 10
Fig. 1.4. Production ofcattlelVP embryos in Ovamass laboratories in 1989-1991 11
Fig. 1.5. Foal born in 1984 at University College Dublin after non-surgical embryo
transfer 23
Fig. 2.1. Standing heat in the heifer and cow 42
Fig. 2.2. Aids to oestrus detection in the cow 44
Fig. 2.3. Control of steroidogenesis in preovulatory follicles 46
Fig. 2.4. Changing hormone concentrations during the bovine oestrous cycle 47
Fig. 2.5. The bovine corpus luteum 4 8
Fig. 2.6. Schematic diagrams of two patterns of follicular development 49
Fig. 2.7. Effects of nutrition on folliculogenesis in the cow 51
Tables and Figures
Fig. 2.8. Possible sequence of events during selection of a dominant follicle 54
Fig. 2.9. Two approaches to oestrus control in the cow 5 7
Fig. 2.10. Timing and purpose of hormone injections in synchronization protocol 5 8
Fig. 2.11. Schematic representation of folliculogenesis in the cow 60
Fig. 2.12. Stages of follicle development in the bovine ovary 62
Fig. 2.13. Follicle size categories in the bovine ovary 67
Fig. 2.14. Ultrastructural changes during follicle growth 68
Fig. 2.15. Relationship between oocyte diameter and follicle size 69
Fig. 3.1. Bovine oocyte recovery by follicle dissection 80
Fig. 3.2. Bovine oocyte recovery by follicle aspiration 81
Fig. 3.3. Cytochemical and ultrastructural differences between calf and cow oocytes 100
Fig. 3.4. Progress in the manipulation of oocytes in preantral follicles in mice and farm
animals 109
Fig. 4.1. Bovine follicle at the time of ovulation 113
Fig. 4.2. Stages in meiosis and oocyte maturation 115
Fig. 4.3. Nucleolar proteins during bovine oocyte meiosis and mitosis 117
Fig. 4.4. Chronology of events during oocyte maturation in the cow 118
Fig. 4.5. Germinal-vesicle-stage bovine oocyte and germinal-vesicle breakdown (GVBD) 119
Fig. 4.6. Before and after in vitro maturation of the bovine oocyte 120
Fig. 4.7. Forms of bovine serum employed in maturation media 127
Fig. 4.8. Bovine oocyte and associated follicular cells 133
Fig. 4.9. Interaction between granulosa and theca cells in the inhibition of meiotic
resumption 135
Fig. 4.10. Growth hormone and IGF-I effects on apoptosis in bovine cumulus-oocyte
complexes during maturation 138
Fig. 4.11. Oocyte-cumulus actions in the mouse 141
Fig.4.12. Role of glutathione (GSH) in the in vitro maturation medium 144
Fig. 4.13. Prematuration essential for developmental competence 146
Fig. 4.14. A pre-IVM treatment for improving bovine oocyte quality 146
Fig. 4.15. The mature bovine oocyte 155
Fig. 5.1. Mechanism of sperm capacitation by bovine seminal plasma proteins and
high-density lipoprotein 160
Fig. 5.2. Regulation of hyperactivation in bovine sperm 162
Fig. 5.3. Schematic drawing of the bovine acrosome reaction 162
Fig. 5.4. Sperm reservoir in the isthmus and site of fertilization in the cow 164
Fig. 5.5. Maternal and embryonic IGF circuits 166
Fig. 6.1. In vitro fertilization system for cattle (Kyoto University) 177
Fig. 6.2. IVMF-derived calves born in Ireland in 1987 177
Fig. 6.3. Swim-up technique to obtain hypermotile sperm for IVF 184
Fig. 6.4. Separation of bull sperm on a 45 and 90% Percoll gradient 186
Fig. 6.5. Hypo-osmotic swelling to screen bull sperm 190
Fig. 6.6. Denuding the mature bovine oocyte with sodium citrate 194
Fig. 6.7. Possible mechanisms whereby calcium oscillations may be generated in the
fertilization process 201
Fig. 6.8. Gamete interactions at fertilization in the cow 202
Fig. 6.9. Decondensation of sperm head and formation of pronuclei 204
Fig. 6.10. Pronuclei in the bovine oocyte: normal and abnormal 205
Tables and Figures
Four approaches to micro-assisted fertilization in the cow 206
Paternal centrosomal dynamics at fertilization 21K
Stages in the development of the bovine embryo 22 3
Events in the early life of the bovine embryo 228
Factors in embryonic mortality in the cow 229
Transferring bovine zygotes to the sheep oviduct for in vitro culture 2 31
Diagrammatic representation of the growth and development of the sheep
embryo 2 3 3
Culturing cattle embryos in the isolated mouse oviduct 2 34
From two-cell embryo to late morula in 6 days 2 37
Mouse blastocysts produced in traditional and microchannel culture systems 241
Recovering bovine oviductal epithelial cells 244
Lipid droplets in bovine embryos cultured with and without serum 2 50
Effect of BST and IGF-I on bovine embryo development 2 59
Development of in vivo vs. in vitro bovine embryos 268
Non-invasive assessment of blastocyst viability 269
Stages in the hatching of the bovine blastocyst 2 74
The plastic straw as an embryo-freezing container 285
One-step thawing of an embryo within a straw 285
Lipid classes in IVP embryos and in vivo embryos 292
Steps in the vitrification of a bovine embryo 294
Conventional straw vitrification vs. open pulled straw (OPS) 300
Non-surgical embryo transfer in the cow 304
Factors influencing success of non-surgical embryo transfer in cattle 310
Treatment protocol for fixed-time embryo transfer in cattle with an Ovsynch
programme 314
Fixed-time cattle embryo transfer protocol using progesterone device 314
Possible antiluteolytic strategies to enhance pregnancy rates in cattle 318
Hypothetical chain of infection in embryo transfer 331
Acceptable and unacceptable embryos in terms of disease control incattleET 333
Effect of environment on conception rates in cows and heifers 340
Schematic diagram of main components of a typical flowcytometer 344
Methods employed in the sexing of cattle embryos 353
Identical twins in cattle produced by embryo splitting 3 56
Nuclear-transfer calf derived from an embryonic cell 358
Chemically assisted removal of maternal chromosomes 361
Phases of the mammalian cell cycle 364
Fig. 10.10. Simplification of cloning technology in cattle 367
Fig. 10.11. Approaches to the production of transgenic sheep 3 74
Fig. 10.12. Transgenic cattle to produce recombinant protein in milk 377
Fig. Al. Acceptable-quality bovine primary oocytes 386
Fig. A2. Unacceptable-quality bovine primary oocytes 386
Fig. A3. Before and after in vitro maturation of the bovine oocyte 387
Fig. A4. Stages in the progress of the bovine blastocyst 388
Fig. A5. From bovine morula to expanded blastocyst 388
Fig. 6.11.
Fig. 6.12.
Fig .7.1.
Fig .7.2.
Fig .7.3.
Fig .7.4.
Fig .7.5.
Fig .7.6.
Fig .7.7.
Fig, .7.8.
Fig. .7.9.
Fig. . 7.10.
Fig. 7.11.
Fig. 7.12.
Fig. 7.13.
Fig. 7.14.
Fig. 8.1.
Fig. 8.2.
Fig. 8.3.
Fig. 8.4.
Fig. 8.5.
Fig. 9.1.
Fig. 9.2.
Fig. 9.3.
Fig. 9.4.
Fig. 9.5.
Fig. 10.1.
Fig. 10.2.
Fig. 10.3.
Fig. 10.4.
Fig. 10.5.
Fig. 10.6.
Fig. 10.7.
Fig. 10.8.
Fig. 10.9.
|
any_adam_object | 1 |
author | Gordon, Ian R. |
author_facet | Gordon, Ian R. |
author_role | aut |
author_sort | Gordon, Ian R. |
author_variant | i r g ir irg |
building | Verbundindex |
bvnumber | BV016516443 |
callnumber-first | S - Agriculture |
callnumber-label | SF887 |
callnumber-raw | SF887 |
callnumber-search | SF887 |
callnumber-sort | SF 3887 |
callnumber-subject | SF - Animal Culture |
classification_tum | LAN 610f LAN 681f |
ctrlnum | (OCoLC)51582540 (DE-599)BVBBV016516443 |
dewey-full | 636.2/0898178059 636.20898178059 |
dewey-hundreds | 600 - Technology (Applied sciences) |
dewey-ones | 636 - Animal husbandry |
dewey-raw | 636.2/0898178059 636.20898178059 |
dewey-search | 636.2/0898178059 636.20898178059 |
dewey-sort | 3636.2 9898178059 |
dewey-tens | 630 - Agriculture and related technologies |
discipline | Agrarwissenschaft Agrar-/Forst-/Ernährungs-/Haushaltswissenschaft / Gartenbau |
edition | 2. ed. |
format | Book |
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id | DE-604.BV016516443 |
illustrated | Illustrated |
indexdate | 2024-07-09T19:11:24Z |
institution | BVB |
isbn | 0851996663 |
language | English |
lccn | 2003043498 |
oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-010206375 |
oclc_num | 51582540 |
open_access_boolean | |
owner | DE-M49 DE-BY-TUM DE-19 DE-BY-UBM DE-188 |
owner_facet | DE-M49 DE-BY-TUM DE-19 DE-BY-UBM DE-188 |
physical | XXVIII, 548 S. Ill., graph. Darst. |
publishDate | 2003 |
publishDateSearch | 2003 |
publishDateSort | 2003 |
publisher | CABI Pub. |
record_format | marc |
series | Biotechnology in agriculture |
series2 | Biotechnology in agriculture |
spelling | Gordon, Ian R. Verfasser aut Laboratory production of cattle embryos I. Gordon 2. ed. Cambridge, MA CABI Pub. 2003 XXVIII, 548 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biotechnology in agriculture 27 Fertilization cabt In vitro maturation cabt Cattle cabt Embryonic Development cabt Bétail - Embryons Bétail - Embryons - Transplantation Embrião de animal (transplante) larpcal Gado larpcal Livestock Embryos Livestock Embryos Transplantation Rind (DE-588)4050061-5 gnd rswk-swf In vitro (DE-588)4250701-7 gnd rswk-swf Produktion (DE-588)4047347-8 gnd rswk-swf Embryo (DE-588)4014555-4 gnd rswk-swf Rind (DE-588)4050061-5 s Embryo (DE-588)4014555-4 s Produktion (DE-588)4047347-8 s In vitro (DE-588)4250701-7 s DE-604 Biotechnology in agriculture 27 (DE-604)BV004064741 27 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010206375&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
spellingShingle | Gordon, Ian R. Laboratory production of cattle embryos Biotechnology in agriculture Fertilization cabt In vitro maturation cabt Cattle cabt Embryonic Development cabt Bétail - Embryons Bétail - Embryons - Transplantation Embrião de animal (transplante) larpcal Gado larpcal Livestock Embryos Livestock Embryos Transplantation Rind (DE-588)4050061-5 gnd In vitro (DE-588)4250701-7 gnd Produktion (DE-588)4047347-8 gnd Embryo (DE-588)4014555-4 gnd |
subject_GND | (DE-588)4050061-5 (DE-588)4250701-7 (DE-588)4047347-8 (DE-588)4014555-4 |
title | Laboratory production of cattle embryos |
title_auth | Laboratory production of cattle embryos |
title_exact_search | Laboratory production of cattle embryos |
title_full | Laboratory production of cattle embryos I. Gordon |
title_fullStr | Laboratory production of cattle embryos I. Gordon |
title_full_unstemmed | Laboratory production of cattle embryos I. Gordon |
title_short | Laboratory production of cattle embryos |
title_sort | laboratory production of cattle embryos |
topic | Fertilization cabt In vitro maturation cabt Cattle cabt Embryonic Development cabt Bétail - Embryons Bétail - Embryons - Transplantation Embrião de animal (transplante) larpcal Gado larpcal Livestock Embryos Livestock Embryos Transplantation Rind (DE-588)4050061-5 gnd In vitro (DE-588)4250701-7 gnd Produktion (DE-588)4047347-8 gnd Embryo (DE-588)4014555-4 gnd |
topic_facet | Fertilization In vitro maturation Cattle Embryonic Development Bétail - Embryons Bétail - Embryons - Transplantation Embrião de animal (transplante) Gado Livestock Embryos Livestock Embryos Transplantation Rind In vitro Produktion Embryo |
url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010206375&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
volume_link | (DE-604)BV004064741 |
work_keys_str_mv | AT gordonianr laboratoryproductionofcattleembryos |