EBOOK - Laboratory production of cattle embryos 2nd Edition (I. Gordon) | Cộng đồng Kỹ thuật cơ điện Việt Nam EBOOKBKMT

*3,000 new references added since the first edition

*Gives information necessary to produce embryos totally through in vitro techniques

*Shows commercial applications of embryo and oocyte research

Cattle remain at the forefront of many new developments in reproductive technology and what can be done for the cow today will later be applicable to other farm livestock and perhaps humans. This new edition reviews the considerable advances and issues in embryo production technology, based on reports since the first edition in 1994. This is a must-have volume for those who own the first edition, and an incredibly informative text.

CONTENTS:

1 Developments in Embryoin VitroProduction (IVP) Technology1

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 22

1.6.3. Pigs 26

1.6.4. Sheep and goats 28

1.6.5. Deer 30

1.6.6. Camelids 31

1.7. Humanin VitroFertilization 32

1.7.1. Historical aspects 32

1.7.2. Establishment of pregnancy by embryo transfer 33

1.7.3. Ovarian stimulation regimens for IVF 33

1.7.4. Recovery of human oocytes 34

1.7.5.In vitromaturation of human oocytes 34

1.7.6. Intracytoplasmic sperm injection (ICSI) 36

1.7.7. Early embryo culture 37

1.7.8. Assessing embryo quality 38

1.7.9. Cryopreservation of embryos and oocytes 39

Oocyte preservation 40

1.7.10. Gender preselection 40

2 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 46

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 55

2.3.1. Gonadotrophin release 55

2.3.2. Intraovarian events 56

2.4. Synchronizing Oestrus 56

2.4.1. Treatment regimens 56

2.5. Prenatal Development of the Bovine Ovary 58

2.5.1. Migration of primordial germ cells 59

2.5.2. Formation of oogonia 59

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 of the zona pellucida 62

2.5.7. Development of growing follicles 63

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 65

Granulosa cells 66

Thecal cells 67

Basement membrane 68

vi Contents

2.6.4. 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 73

2.7. Induction of Multiple Ovulations in the Cow 73

2.7.1. Gonadotrophins 74

2.7.2. Control of follicle growth 74

Controlling ovulation 75

2.7.3. Animal and environmental effects 75

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 of preovulatory follicles and oocytes after superovulation 78

3 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 83

Other farm animals 83

3.1.4. Transillumination-aspiration ovary (TAO) 83

3.2. Abattoir Ovaries 83

3.2.1. Ovary storage: temperatures and time-limits 83

3.2.2. Ovary storage to enhance oocyte quality

3.2.3. Temperature sensitivity of oocytes

3.2.4. Follicle size and quality

3.3. Recovering Oocytes: Live Cattle 85

3.3.1. Advantages and alternatives 86

Mares 87

Buffaloes and pigs 87

3.3.2. Laparoscopic methods of follicular aspiration 87

3.3.3. Ultrasonic methods of follicular aspiration 88

Developments in ultrasound technology 88

Ultrasound in research and practice 88

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 93

Retinol 93

3.3.6. Oocytes from pregnant cattle 93

FSH stimulation 94

Contents vii

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 95

3.4.1. Recovering secondary oocytes 96

3.4.2. Enhancing quality of primary oocytes 96

3.4.3. Oocyte transportation 97

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

Lipid 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

4 Maturing the Bovine Oocyte 112

4.1. Oocyte Maturationin 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 ofin vitromaturation in cattle 120

4.3.In VitroMaturation (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

viii Contents

4.3.8. Temperature, gas phase and toxic factors 125

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 IVMmedia 128

Heat treatment of serum 128

4.3.10. Bovine follicular fluid 129

Inhibitory action of follicular fluid 130

Follicular fluid composition 130

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 132

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 147

Contents ix

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

Mitochondrial distribution 156

4.12.3. Morphological assessment and staining methods 157

4.12.4. Gene expression and oocyte competence 157

5 Capacitating Bovine Sperm 158

5.1. Introduction 158

5.1.1. Historical 158

5.1.2. The capacitation process 159

5.1.3. Hyperactivation 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 eventsin 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

x 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 173

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

6 In VitroFertilization 176

6.1. Introduction 176

6.1.1.In vitromaturation 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. Lifespan 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 forin VitroFertilization 182

6.3.1. Use of fresh bull semen 182

6.3.2. Assessing the quality of frozen–thawed semen 183

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. Percoll 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 VitroFertilization Culture Systems 195

Contents xi

6.6.1. The fertilization medium 195

TALP medium 195

SOF medium 196

Fert-CDMmedium 196

6.6.2. Protein supplementation 196

6.6.3. Gas phase considerations 197

6.6.4. Temperature, light and osmolarity 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-b-cyclodextrin 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

xii Contents

6.12. Variability in Bull Fertility 215

6.12.1. Effect of bull on IVF outcome 215

6.12.2.In vitrofertilization 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 vivoculture systems 220

7.1.3.In vitroculture 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 VivoCulture 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 VitroCulture 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 xiii

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 and cytokines 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 260

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 264

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

xiv 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 270

Lipid droplets 270

Interferon-tau secretion 270

Golgi apparatus 271

Proliferating cell nuclear antigen 271

Embryo cryosurvival 271

Proteins involved in embryo developmental competence 271

7.6.5. mRNA expression 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

8 Preservation of Embryos and Oocytes 277

8.1. Introduction 277

8.1.1. Embryo cryopreservation: past and present 277

In vitro-produced embryos 278

Vitrification 279

Advances in other farm mammals 280

8.1.2. Advantages 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 to cooling 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 290

Delipidizing the pig embryo 291

Contents xv

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 Vivo-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

9 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 vivoembryos 309

In vitroembryos 310

Operator skill 310

9.4. Donor–Recipient Synchrony 311

9.4.1. Importance of synchronization 311

Accuracy of oestrus detection 312

xvi 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- andin 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 333

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 334

Post-mortem use of valuable genetic material 335

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 xvii

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 vitrofertilization 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 of AI 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. Fluorescencein situhybridization 354

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

xviii 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 vitroculture and evaluation of nuclear-transfer embryos 369

Ploidy analysis 369

Ribosomal RNA gene activation 369

Apoptosis 370

ICMand TE cells 370

Gene expression patterns 370

Mitochondrial heteroplasmy 370

Telomerase activity 370

10.6.7. Gestational and perinatal losses 371

Neonatal care 372

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 379

Predicting transgene integration 379

Preserving embryos 379

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

thein VitroProduction and Transfer of Cattle Embryos 394

References 396

Index

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