BIOTECHNOLOGY IN AGRICULTURE SERIES

General Editor: Gabrielle J. Persley, Biotechnology Adviser, Environmentally Sustainable
Development, The World Bank, Washington, DC, USA.
For a number of years, biotechnology has held out the prospect for major advances in agricultural
production, but only recently have the results of this new revolution started to reach application in
the field. The potential for further rapid developments is, however, immense.
The aim of this book series is to review advances and current knowledge in key areas of
biotechnology as applied to crop and animal production, forestry and food science. Some titles focus
on individual crop species, others on specific goals such as plant protection or animal health, with yet
others addressing particular methodologies such as tissue culture, transformation or immunoassay.
In some cases, relevant molecular and cell biology and genetics are also covered. Issues of relevance
to both industrialized and developing countries are addressed and social, economic and legal
implications are also considered. Most titles are written for research workers in the biological sciences
and agriculture, but some are also useful as textbooks for senior-level students in these disciplines.
Editorial Advisory Board:
E.P. Cunningham, Trinity College, University of Dublin, Ireland.
P. Day, Rutgers, University, New Jersey, USA.
J.H. Dodds, Attorney at Law/Patent Attorney, Washington, DC, USA.
S.L. Krugman, United States Department of Agriculture, Forest Service.
I. Morrison, Institute for Animal Health, Compton, UK.
W.J. Peacock, CSIRO, Division of Plant Industry, Australia.

Titles Available:
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Beyond Mendel’s Garden: Biotechnology in the Service of World Agriculture*
G.J. Persley
Agricultural Biotechnology: Opportunities for International Development
Edited by G.J. Persley
The Molecular and Cellular Biology of the Potato*
Edited by M.E. Vayda and W.D. Park
Advanced Methods in Plant Breeding and Biotechnology
Edited by D.R. Murray
Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology
Edited by P.R. Shewry
Rice Biotechnology
Edited by G.S. Khush and G.H. Toenniessen
Plant Genetic Manipulation for Crop Protection*
Edited by A. Gatehouse, V. Hilder and D. Boulter
Biotechnology of Perennial Fruit Crops
Edited by F.A. Hammerschlag and R.E. Litz

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Bioconversion of Forest and Agricultural Plant Residues
Edited by J.N. Saddler
Peas: Genetics, Molecular Biology and Biotechnology
Edited by R. Casey and D.R. Davies
Laboratory Production of Cattle Embryos
I. Gordon
The Molecular and Cellular Biology of the Potato, 2nd edn
Edited by W.R. Belknap, M.E. Vayda and W.D. Park
New Diagnostics in Crop Sciences
Edited by J.H. Skerritt and R. Appels
Soybean: Genetics, Molecular Biology and Biotechnology
Edited by D.P.S. Verma and R.C. Shoemaker
Biotechnology and Integrated Pest Management
Edited by G.J. Persley

Biotechnology of Ornamental Plants
Edited by R.L. Geneve, J.E. Preece and S.A. Merkle
Biotechnology and the Improvement of Forage Legumes
Edited by B.D. McKersie and D.C.W. Brown
Milk Composition, Production and Biotechnology
Edited by R.A.S. Welch, D.J.W. Burns, S.R. Davis, A.I. Popay and C.G. Prosser
Biotechnology and Plant Genetic Resources: Conservation and Use
Edited by J.A. Callow, B.V. Ford-Lloyd and H.J. Newbury
Intellectual Property Rights in Agricultural Biotechnology
Edited by F.H. Erbisch and K.M. Maredia
Agricultural Biotechnology in International Development
Edited by C. Ives and B. Bedford
The Exploitation of Plant Genetic Information: Political Strategies in Crop Development
R. Pistorius and J. van Wijk
Managing Agricultural Biotechnology: Addressing Research Program Needs and Policy
Implications
Edited by J.I. Cohen
The Biotechnology Revolution in Global Agriculture: Innovation, Invention and Investment in
the Canola Industry
P.W.B. Phillips and G.G. Khachatourians
Agricultural Biotechnology: Country Case Studies – a Decade of Development
Edited by G.J. Persley and L.R. MacIntyre
Biotechnology and Sustainable Development: Voices of the South and North
Edited by I. Serageldin and G.J. Persley
Laboratory Production of Cattle Embryos, 2nd edition
I. Gordon
Intellectual Property Rights in Agricultural Biotechnology, 2nd edition
F.H. Erbisch and K.M. Maredia

*Out of print


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Biotechnology in Agriculture No. 27

Laboratory Production of Cattle Embryos
Second Edition

I. Gordon
Professor Emeritus
Department of Animal Science and Production
University College Dublin
Ireland

CABI Publishing

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CABI Publishing is a division of CAB International
CABI Publishing
CAB International
Wallingford
Oxon OX10 8DE
UK

CABI Publishing
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4th Floor

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©CAB International 2003. All rights reserved. No part of this publication may
be reproduced in any form or by any means, electronically, mechanically, by
photocopying, recording or otherwise, without the prior permission of the
copyright owners.

A catalogue record for this book is available from the British Library, London, UK.

Library of Congress Cataloging-in-Publication Data
Gordon, Ian R.
Laboratory production of cattle embryos / I. Gordon. -- 2nd ed.
p. cm.
Includes bibliographical references and index.
ISBN 0-85199-666-3 (alk. paper)
1. Livestock--Embryos. 2. Livestock--Embryos--Transplantation. I. Title.
SF887.G59 2003
636.2′0898178059--dc21
2003043498


ISBN 0 85199 666 3

Typeset by AMA DataSet Ltd, UK.
Printed and bound in the UK by Cromwell Press, Trowbridge.

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Contents

List of Tables and Figures
Preface
1

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Developments in Embryo in Vitro Production (IVP) Technology
1.1. Historical Aspects
1.1.1. Early IVF reports
1.1.2. Cattle IVF
1.2. Cambridge Contributions
1.2.1. School of Agriculture
1.2.2. Animal Research Station
Embryos across the Atlantic
Using rabbits to good effect
Dawn of cattle ET industry
1.2.3. Cambridge, Babraham and beyond
1.3. Irish Contributions
1.3.1. Early studies in cattle

1.3.2. Cattle twins by embryo transfer
1.3.3. Low-cost embryos
1.3.4. Commercializing the embryo production procedure
1.3.5. Commercial unacceptability
1.3.6. Towards sexed semen on the farm
1.4. Developments in ET Technology
1.4.1. Thirty years of progress
1.4.2. Current cattle ET activity
1.4.3. Commercial advantages of cattle ET
1.5. Laboratory-produced Embryos
1.5.1. Current level of activity
Ovum pick-up (OPU)
1.5.2. Research with bovine IVP embryos
1.5.3. Commercial use of IVP embryos
1.5.4. Pathogen-free IVP embryos
1.5.5. Animal health and welfare considerations
1.6. Embryo Production in Other Farm Mammals
1.6.1. Buffaloes
1.6.2. Horses

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1.6.3. Pigs
1.6.4. Sheep and goats
1.6.5. Deer
1.6.6. Camelids
1.7. Human in Vitro Fertilization
1.7.1. Historical aspects
1.7.2. Establishment of pregnancy by embryo transfer
1.7.3. Ovarian stimulation regimens for IVF
1.7.4. Recovery of human oocytes
1.7.5. In vitro maturation of human oocytes
1.7.6. Intracytoplasmic sperm injection (ICSI)
1.7.7. Early embryo culture
1.7.8. Assessing embryo quality
1.7.9. Cryopreservation of embryos and oocytes
Oocyte preservation
1.7.10. Gender preselection
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The Bovine Oestrous Cycle and Associated Events
2.1. Oestrus and the Oestrous Cycle
2.1.1. Oestrus
2.1.2. Expression of heat
2.1.3. Aids to heat detection
2.1.4. Endocrine basis of oestrus
2.2. The Oestrous Cycle
2.2.1. Corpus luteum and progesterone
2.2.2. Follicular dynamics in the cow
Growing understanding of folliculogenesis
Zebu cattle

2.2.3. The dominant follicle
2.2.4. Monitoring ovarian activity
2.3. Endocrine Events in the Oestrous Cycle
2.3.1. Gonadotrophin release
2.3.2. Intraovarian events
2.4. Synchronizing Oestrus
2.4.1. Treatment regimens
2.5. Prenatal Development of the Bovine Ovary
2.5.1. Migration of primordial germ cells
2.5.2. Formation of oogonia
2.5.3. The primordial follicle
2.5.4. Activation of primordial follicles
2.5.5. Growth and development of follicles
2.5.6. Formation of the zona pellucida
2.5.7. Development of growing follicles
2.5.8. Antral follicles
2.5.9. Follicular atresia
2.6. The Bovine Ovary in Postnatal Life
2.6.1. The prepubertal animal
2.6.2. Antral follicle population
2.6.3. Follicle development
Granulosa cells
Thecal cells
Basement membrane

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2.6.4. Oocyte growth and development
Nucleus and nucleolus
Mitochondria
Golgi complex
Cortical granules
Ribosomes and cytoplasmic lattices
Biochemical aspects of oocyte growth
2.6.5. Endocrine events during follicle growth and development

Gonadotrophins
Oestradiol and progesterone
Androstenedione and testosterone
2.6.6. Follicular atresia
2.7. Induction of Multiple Ovulations in the Cow
2.7.1. Gonadotrophins
2.7.2. Control of follicle growth
Controlling ovulation
2.7.3. Animal and environmental effects
Nutritional effects
2.7.4. Long-range assessments and sexed semen
2.7.5. Recombinant bovine somatotrophin (r-BST) and follicle growth
2.7.6. Characteristics of preovulatory follicles and oocytes after superovulation
3

Recovering the Bovine Oocyte
3.1. Oocyte Recovery: Abattoir Ovaries
3.1.1. Dissecting the intact follicle
Sheep and cattle
Other farm animals
3.1.2. Aspiration techniques: old and new
3.1.3. Ovary slicing techniques
Slicing and aspiration
Other farm animals
3.1.4. Transillumination-aspiration ovary (TAO)
3.2. Abattoir Ovaries
3.2.1. Ovary storage: temperatures and time-limits
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
3.3.1. Advantages and alternatives
Mares
Buffaloes and pigs
3.3.2. Laparoscopic methods of follicular aspiration
3.3.3. Ultrasonic methods of follicular aspiration
Developments in ultrasound technology
Ultrasound in research and practice
3.3.4. Developments in ovum pick-up technology
OPU in zebu cattle
3.3.5. Hormonal and nutritional pretreatments
Influence of growth hormone
Retinol
3.3.6. Oocytes from pregnant cattle
FSH stimulation

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3.3.7. Oocytes from post-partum cattle
3.3.8. Oocytes from calves and prepubertal cattle
3.4. Live Donors: Other Considerations
3.4.1. Recovering secondary oocytes
3.4.2. Enhancing quality of primary oocytes
3.4.3. Oocyte transportation
3.5. Factors Affecting Oocyte Quality
3.5.1. Age of animal
Oocytes from fetal ovaries
3.5.2. Cattle category, oestrous cycle and ovarian morphology
Cattle category
Stage of cycle
Determining cycle stage
Morphology of ovaries
Cystic follicles

3.5.3. Body condition and nutritional considerations
3.5.4. Reproductive status of donor
3.5.5. Animal factors
3.5.6. Environmental factors
3.6. Assessing Oocyte Quality
3.6.1. Oocyte morphology: classification schemes
Oocyte diameter
Lipid vesicles
Oestradiol: progesterone ratio
Gene expression
Oocytes from zebu cattle
3.7. Oocytes from Preantral and Early Antral Follicles
3.7.1. Birth of young in mice
3.7.2. Differences between mice and cattle follicles
3.7.3. Utilizing early antral follicles
3.7.4. Preantral follicles in humans and pigs
4

Maturing the Bovine Oocyte
4.1. Oocyte Maturation in Vivo
4.1.1. Summary of events
4.1.2. Events leading to ovulation
4.1.3. Nuclear and cytoplasmic maturation
4.1.4. Biochemical and physiological events during maturation
4.2. Oocyte Maturation in the Laboratory
4.2.1. Historical aspects
4.2.2. Current understanding of in vitro maturation in cattle
4.3. In Vitro Maturation (IVM) Culture Systems
4.3.1. Culturing intact follicles
4.3.2. Simple and complex maturation media

Tissue culture medium 199
4.3.3. Buffering systems, osmolarity and surface tension
4.3.4. Water-quality considerations
4.3.5. Static and flux culture systems
4.3.6. Effect of maturation time
4.3.7. Antibiotic cover and oil overlay

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4.3.8. Temperature, gas phase and toxic factors
Temperature
Gas phase
Toxic factors – ammonium
4.3.9. Bovine serum and bovine serum albumin
Bovine serum albumin (BSA)
Sources of bovine serum
Constituents of bovine serum
Serum levels employed in IVM media
Heat treatment of serum
4.3.10. Bovine follicular fluid
Inhibitory action of follicular fluid
Follicular fluid composition
Hyaluronic acid as a serum substitute
Hyaluronan in culturing oocytes in small groups
4.4. Somatic-cell Support
4.4.1. Cumulus–oocyte complex (COC)
4.4.2. Connexin 43 and oocyte meiotic maturation
4.4.3. Additional cumulus/granulosa cells
4.4.4. Special needs of ovum pick-up (OPU) oocytes
4.4.5. Use of non-follicular cells
4.4.6. Action of theca cells
4.5. Hormones and Growth Factors
4.5.1. Hormones
Follicle-stimulating hormone and luteinizing hormone
Prolactin
Growth hormone (somatotrophin)
Steroids

Insulin and GH-RH
4.5.2. Growth factors
Epidermal growth factor (EGF)
IGF family
Midkine and other growth factors
Other farm animals
4.5.3. Cytokines
4.5.4. Oocyte-derived growth factors
4.6. Oocytes Cultured Singly or in Groups
4.6.1. Single-oocyte culture systems
4.7. Single-culture medium systems
4.7.1. Synthetic oviductal fluid (SOF) formulations
4.8. Chemically Defined Culture Systems
4.8.1. Using synthetic oviductal fluid (SOF)
4.8.2. TCM-199
4.9. Oxidative Stress in Oocyte Maturation
4.9.1. Role of glutathione (GSH)
4.10. Two-step Culture Systems
4.10.1. Background information
4.10.2. Maintenance of meiotic arrest
Cattle oocytes
4.10.3. Biological inhibitors
Influence of granulosa–theca cells

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4.10.4. Biochemical inhibitors
Role of cyclic 3′5′-adenosine monophosphate (cAMP)
Manganese
4.10.5. Pretreatment of donor cattle
4.10.6. Two-step treatment in the laboratory

Roscovitine
Butyrolactone I
Other farm animals
4.10.7. Enhancing the quality of oocytes from small follicles
4.10.8. Synchronizing germinal vesicle development
4.11. Other Factors Influencing Oocyte Maturation
4.11.1. Energy sources and second messengers
Glucose
cAMP and analogues
4.11.2. Hormones and vitamins
Prostaglandins and steroids
Retinoic acid
4.11.3. Opioid antagonists and chemical agents
Endogenous opioid peptides
Dimethylsulphoxide and ethanol
Selenium
4.11.4. Simplifying maturation culture systems
4.12. Evaluating the Maturation Process
4.12.1. Stages in nuclear maturation
4.12.2. Cumulus-cell expansion
Mitochondrial distribution
4.12.3. Morphological assessment and staining methods
4.12.4. Gene expression and oocyte competence
5

Capacitating Bovine Sperm
5.1. Introduction
5.1.1. Historical
5.1.2. The capacitation process
5.1.3. Hyperactivation

5.1.4. The acrosome reaction
5.1.5. Artificial induction of capacitation
5.2. Capacitation in the Cow
5.2.1. Sperm transport
5.2.2. Oviductal secretory cells
5.2.3. Glycosaminoglycans
5.2.4. Simulating oviductal events in vitro
5.3. Capacitation Procedures
5.3.1. Historical
5.3.2. Modifying osmolarity and pH
pH values
5.3.3. Evaluating sperm-capacitation systems
5.4. Heparin and Heparin-like Glycosaminoglycans
5.4.1. Actions and interactions of heparin
5.4.2. Practical application of heparin treatment
5.5. Use of Fresh or Frozen Semen
5.5.1. Fresh semen

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5.5.2. Frozen semen
5.5.3. Semen diluents
5.6. Bulls as a Source of Variability
5.6.1. High- and low-fertility bulls
5.6.2. Bull variability
5.6.3. Methods of assessing bull fertility
5.6.4. Enhancing semen quality
5.7. Efficiency of Capacitation Procedures
5.7.1. Staining methods
5.7.2. Oocyte penetration tests
5.7.3. Sperm–zona binding
6

In Vitro Fertilization
6.1. Introduction
6.1.1. In vitro maturation and fertilization: early reports

6.1.2. Chapter contents
6.2. Fertilization in the Cow
6.2.1. Oviductal environment
6.2.2. Lifespan of the secondary oocyte
6.2.3. Dispersion of cumulus cells
6.2.4. Fertilization rates in cattle
6.2.5. Fertilization rates in superovulated animals
Accessory spermatozoa
6.3. Preparing Sperm for in Vitro Fertilization
6.3.1. Use of fresh bull semen
6.3.2. Assessing the quality of frozen–thawed semen
6.3.3. Swim-up procedures
Swim-up and hyaluronic acid
Swim-up and caffeine
Swim-up and the sex ratio
6.3.3. Percoll density gradients
Sex-ratio deviations
Other farm animals
6.3.5. Glass-wool filtration procedures
6.3.6. Use of hyaluronic acid
6.3.7. Cell-to-cell contact
Cell-to-cell interactions with epididymal cells
6.3.8. Sperm abnormalities
Proximal droplets
Nuclear vacuoles
Knobbed acrosome defect
Robertsonian translocations
Hypo-osmotic swelling (HOS) as a screening assay
6.3.8. Sperm doses
6.4. Enhancing Sperm Motility

6.4.1. Penicillamine, hypotaurine, epinephrine (adrenalin) (PHE)
6.4.2. Caffeine, theophylline and pentoxifylline
6.5. Preparing Oocytes for Fertilization
6.5.1. Beneficial effects of cumulus cells
Cumulus-cell removal after fertilization
6.6. In Vitro Fertilization Culture Systems

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Contents

6.6.1. The fertilization medium
TALP medium
SOF medium
Fert-CDM medium
6.6.2. Protein supplementation
6.6.3. Gas phase considerations
6.6.4. Temperature, light and osmolarity
Temperature
Light
Osmolarity
6.6.5. Somatic cells in the fertilization medium
6.6.6. Activation of COCs with calcium ionophore (A23187)
6.6.7. Oxidative stress in the IVF culture system
6.6.8. Other factors influencing efficacy of IVF system
Glucose
GH-RH
Methyl-b-cyclodextrin
Hyaluronic acid
Dimethylsulphoxide (DMSO)
Prostaglandins
Toxic factors
6.7. Interaction of Spermatozoon and Oocyte
6.7.1. Sperm–oocyte recognition mechanisms

Oviductal factors
6.7.2. Early events in the fertilization process
Changes in zona pellucida
6.7.3. Crossing the interspecific sperm barrier
6.7.4. Factors with a negative effect on fertilization
Zona hardening
6.8. Post-insemination Treatment of Oocytes
6.8.1. Effect of sperm exposure time
6.9. Micro-assisted Fertilization
6.9.1. Zona thinning
6.9.2. Zona drilling and partial zona dissection
6.9.3. Subzonal sperm insertion (SUZI)
6.9.4. Intracytoplasmic sperm injection (ICSI)
Twelve thousand years into the past
ICSI in cattle
Successful cattle ICSI without artificial activation
Cattle ICSI in research
Gender preselection in cattle by ICSI
ICSI in the mare
ICSI and factors influencing oocyte activation
ICSI in mice
6.10. Efficiency of IVF Procedures
6.10.1. Criteria for assessing fertilization
Chromosome preparation
6.11. Fertilization Abnormalities
6.11.1. Polyspermy and parthenogenesis
Polyspermy
Parthenogenesis

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6.12. Variability in Bull Fertility
6.12.1. Effect of bull on IVF outcome
6.12.2. In vitro fertilization tests in predicting bull fertility
Sperm chromatin structure

The bovine centrosome (centriole)
6.12.3. Reducing bull fertility
7

Culturing and Evaluating the Early Bovine Embryo
7.1. Introduction
7.1.1. Historical
7.1.2. In vivo culture systems
7.1.3. In vitro culture systems
7.1.4. Chapter contents
7.2. Early Embryo Development in the Cow
7.2.1. The oviductal microenvironment
7.2.2. Cleavage of the bovine embryo
Duration of cell cycles
Steroidogenic activity of embryo
Nucleoli and nucleolus organizer regions in the early embryo
7.2.3. Compaction and cavitation
Hatching
Apoptosis
7.2.4. Post-hatching progress
7.2.5. Embryo mortality
Factors in embryo mortality
Embryo–pathogen interactions
Fate of the conceptus
Losses after embryo transfer
Fetal losses
7.3. In Vivo Culture Systems
7.3.1. The rabbit oviduct
7.3.2. The sheep oviduct
Elongation-stage bovine embryos

7.3.3. The isolated mouse oviduct
Using oviducts of live mice
7.4. Metabolism of the Early Embryo
7.4.1. Monitoring embryo metabolism
Oxygen consumption
Glucose utilization
Energy metabolism-related gene expression
Myo-inositol, adenylyl cyclase
Ultrastructural autoradiography of RNA synthesis
7.4.2. The development block
7.4.3. Activation of the bovine embryonic genome
7.5. In Vitro Culture Systems
7.5.1. Embryo culture systems: past and present
Serum-restricted culture systems
Use of commercial media
Towards defined culture systems
Sequential media
Microfluidic embryo manipulation

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7.5.2. TCM-199 and SOF culture media
7.5.3. Co-culture with bovine oviductal cells
Bovine oviductal cell monolayer
7.5.4. Co-culture with non-oviductal cells
7.5.5. Serum-supplemented culture systems
Duration of serum treatment
Serum substitutes
Supplementation with bovine follicular fluid
7.5.6. Serum-free culture systems
7.5.7. Defined culture systems
Growth factors

Hyaluronic acid supplementation
7.5.8. Sequential media
Culturing cattle embryos
Pig embryos
7.5.9. Embryo group size
The WOW culture system
7.5.10. Gas atmosphere
Oxygen
Carbon dioxide
Ambient laboratory air
7.5.11. Temperature and light
7.5.12. Protection from oxidative stress
7.5.13. Hormones, growth factors and cytokines
Growth hormone and insulin-like growth factors
Interferon-tau/alpha
Epidermal growth factor
Effect of cytokines
7.5.14. Culture media components
Antibiotics
Insulin
Amino acids
Heparin
Hexoses
Vitamins
Surface-active components
Mineral and silicone oils
7.5.15. Possible toxic agents
Ammonia
Nitric oxide
7.5.16. Simplifying culture systems

7.6. Evaluating Embryo Quality
7.6.1. Morphological and morphometric parameters
Variability in embryo grading
Ultrastructural features
Human embryo quality considerations
7.6.2. Age and developmental stage attained
Time of first cleavage
Early cleavage and pregnancy rates in human assisted reproduction
Assessing embryo quality at morula stage
Timing of blastocyst formation

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7.6.3. Metabolic tests
7.6.4. Indications of embryo normality
Staining tests
Chromosomal abnormalities and cell numbers
Intercellular communication
Lipid droplets
Interferon-tau secretion
Golgi apparatus
Proliferating cell nuclear antigen
Embryo cryosurvival
Proteins involved in embryo developmental competence
7.6.5. mRNA expression patterns
Embryos under stress
7.6.6. Post-hatching evaluation
7.6.7. Post-transfer evaluation
Insulin-like growth factors
Heavy calves
IVP and nuclear-transfer cattle embryos
8

Preservation of Embryos and Oocytes
8.1. Introduction

8.1.1. Embryo cryopreservation: past and present
In vitro-produced embryos
Vitrification
Advances in other farm mammals
8.1.2. Advantages of embryo storage
8.2. Storing the Fresh Embryo
8.2.1. Embryo storage at ambient temperature
8.2.2. Embryo sensitivity to cooling
IVP cattle embryos
8.2.3. Embryo storage at refrigerator temperature
8.3. Conventional Freeze–Thaw Protocols
8.3.1. Cryoprotectants
8.3.2. Two-step to one-step temperature decrease
8.3.3. Straws for storage
8.3.4. One-step thawing procedures
8.3.5. Ethylene glycol as the cryoprotectant
Prefreezing additives
Ultrastructural studies
Demi-embryos
Trophoblastic vesicles
8.3.6. Thawing and cryoprotectant removal
8.4. Freezing the IVP bovine embryo
8.4.1. Morphological and functional differences
Embryo ultrastructure after cryopreservation
8.4.2. Embryo survival and pregnancy rates
Glycerol
Ethylene glycol
Prefreezing additives
Freezing zygotes and early-cleavage embryos
8.4.3. Delipidizing the embryo

Delipidizing the pig embryo

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Delipidizing the IVP bovine embryo
Lipids and mitochondria
8.4.4. Effect of culture medium
8.4.5. Rapid freezing of IVP embryos
8.5. Vitrification of in Vivo-Produced Embryos
8.5.1. Early studies
Cleavage-stage embryos
8.5.2. Vitrification and slow freezing as alternatives
8.6. Vitrification of IVP Embryos
8.6.1. Developing an effective vitrification procedure
Assisted hatching
Avoiding contamination of embryos
Factors relevant to the success of vitrification
8.7. Cryopreservation of the Bovine Oocyte
8.7.1. Factors relevant to oocyte cryopreservation
8.7.2. Freeze–thawing
Ultrastructural evaluation
8.7.3. Vitrification
Previtrification additives
Bovine vs. equine oocytes
Ultrastructural evaluation
8.8. Embryo Evaluation after Thawing
8.8.1. Evaluation of IVP embryos
9

Establishing Pregnancies with IVP Embryos
9.1. Introduction

9.1.1. Historical
9.1.2. Requirements for on-farm applications
From research to practice
Embryo transfer as a research tool
9.1.3. Welfare implications of using IVP embryos
9.2. Preparing Embryos for Transfer
9.2.1. Media employed
Antibiotic/antimicrobial cover
Serum and serum substitutes
Tropical environment
Handling cattle embryos
9.2.3. Protecting the embryo
Embryo encapsulation technology
Predicting embryo hatching
9.2.4. Number of embryos transferred
9.3. Surgical and Non-surgical Transfers
9.3.1. Surgical transfers
Endoscopy and tubal transfer of embryos
9.3.2. Non-surgical transfers
Factors affecting success
In vivo embryos
In vitro embryos
Operator skill
9.4. Donor–Recipient Synchrony
9.4.1. Importance of synchronization
Accuracy of oestrus detection

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9.4.2. Synchronization in the IVP embryo context
9.5. Oestrus Synchronization Techniques
9.5.1. Protocols for synchronizing oestrus
Ovsynch
Progesterone/progestogen
9.6. Selection and Management of Recipients

9.6.1. Heifers versus cows
9.6.2. Factors affecting recipient suitability
Recipient hormone levels
Plasma urea nitrogen
Repeated transfers
Role of the major histocompatibility complex (MHC)
9.6.3. Minimizing stress in recipients
Tranquillization
Welfare concerns
9.6.4. Detecting early pregnancy in recipients
9.7. Enhancing Pregnancy Rates in Recipients
9.7.1. Progesterone supplementation
9.7.2. Hormonal therapy in early dioestrus
9.7.3. Hormonal therapy in late dioestrus
9.7.4. Use of trophoblastic vesicles
9.7.5. Prostaglandin inhibitors
9.7.6. Oral treatment with propylene glycol
9.7.7. Re-synchrony of non-pregnant recipients
10 Embryos and Oocytes in Research and Commerce
10.1. Introduction
10.1.1. From research to commercial application
10.1.2. Cattle products and human health
Population growth and food resources
10.2. Embryo Production Technology: Problems
10.2.1. Differences between IVP- and in vivo-derived offspring
10.2.2. The large-offspring syndrome (LOS)
Placental abnormalities
Gene expression
IVP embryo laboratories and LOS
10.2.3. Large-offspring syndrome: human implications?

10.3. Embryo Production Technology: Prospects
10.3.1. Animal-health considerations
Contaminated semen
Problems posed by IVP cattle embryos
Detection of viruses
Reducing infectivity associated with IVP embryos
10.3.2. IVP embryos in breeding-improvement programmes
MOET schemes
Open-nucleus breeding scheme
Reducing the generation interval
Post-mortem use of valuable genetic material
Future developments
10.3.3. Beef calves from dairy cows
10.3.4. Twinning by embryo transfer
Mechanisms controlling double ovulations

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Embryo transfer
Feasibility of twinning in farming practice
10.3.5. Preserving genetic diversity
Using immature sperm cells
10.3.6. Embryos for tropical/subtropical regions
10.3.7. Bypassing heat-stress problems
10.3.8. Dealing with repeat breeders
Infertile cows
10.3.9. Cattle embryos and oocytes for research
Interspecies nuclear transfer
Identifying toxicants
Environmental pollutants
10.4. Sex Control by Sperm Separation

10.4.1. The case for semen sexing
10.4.2. Semen-sexing technology
Beltsville sexing technology
Other sorting studies
Sperm-membrane changes in sorted sperm
Effect of Fert Plus peptide
Frozen sexed semen
In vitro fertilization with sorted bull sperm
10.4.3. Alternatives to sexing by flow cytometry
Immunological approach
Separation by density gradients
Spermatozoal head size and volume
10.4.4. Effect of AI timing on sex ratio
10.4.5. Sperm separation in other farm animals
Pigs
Horses
Sheep
10.5. Embryo Sexing
10.5.1. Sexing by polymerase chain reaction technology
10.5.2. Fluorescence in situ hybridization
Sexing by male-specific antigen
10.5.3. Sexual dimorphism
10.6. Cloning in Cattle: Progress and Problems
10.6.1. Introduction
Story to date
Normality of clones
Safety of food products
10.6.2. Embryo splitting
10.6.3. Essential steps in nuclear transfer
Quality of recipient oocytes

Enucleation
Telophase enucleation
Introduction of donor nucleus
Choice of donor cell and cell-cycle stage
Fetal or adult somatic cells
Quiescent or proliferating cells
Non-viable cells as donors
Activation
10.6.4. Nuclear reprogramming

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10.6.5. Simplifying nuclear-transfer protocols
10.6.6. Preserving donor cells, cytoplasts and cloned embryos
Refrigeration
Freezing
Vitrification
10.6.6. In vitro culture and evaluation of nuclear-transfer embryos
Ploidy analysis
Ribosomal RNA gene activation
Apoptosis
ICM and TE cells
Gene expression patterns
Mitochondrial heteroplasmy
Telomerase activity
10.6.7. Gestational and perinatal losses
Neonatal care
Preventing LOS?
10.6.8. Development of clones after birth
10.6.9. Embryonic stem cells
10.7. Transgenic Cattle
10.7.1. Development of transgenic technology in cattle
10.7.2. Potential advantages of transgenic cattle
10.7.3. Methods of genetic modification in cattle

Pronuclear injection
Transfected cells for nuclear transfer
Gene targeting
Sperm-mediated DNA transfer
Retroviral infection of early embryos
10.7.4. Transgenic embryos in the laboratory
Predicting transgene integration
Preserving embryos
10.7.5. Losses in transgenic embryos, fetuses and calves
10.7.6. Transgenic cattle on the farm
Germ-line mosaic bulls
Transgenic cows
10.7.7. Welfare of transgenic cattle

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Appendices
Appendix A:

Embryo Production Protocols

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Appendix B:


Preparation of Culture Media

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Appendix C:

Cryopreservation Procedures

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Appendix D: Journals, Books and On-line Sources of Information Relevant to
the in Vitro Production and Transfer of Cattle Embryos

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References

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Index

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Tables and Figures

Tables

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.

Milestones in mammalian in vitro fertilization
Effect of IVP-embryo transfer on calf output per cow
Milestones in farm-animal embryo transfer and related techniques
Data for worldwide cattle ET activity in 2000
IVP cattle embryo production during 12 months in a German laboratory (BFZF)
Cattle IVP embryos transferred worldwide in 2000
Comparison of cloning efficiency in cattle and buffalo embryos
Equine embryo development: sheep oviduct vs. in vitro culture
Pregnancies after transfer of in vitro- vs. in vivo-produced sheep embryos
Pregnancies after transfer of in vitro- vs. in vivo-produced goat embryos
Embryo transfer results from llama donors
Outcome of embryo transfers after ICSI in relation to origin of sperm

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Table 2.1.
Table 2.2.
Table 2.3.

Detecting the cow in heat – signs to observe
Scoring system for heat detection – based on behavioural signs
Least-square means of corpus luteum weights and progesterone levels on
days 3–19 of the natural oestrous cycle
Number of primordial, primary, secondary and Graafian follicles in cattle
ovaries
Action of growth factors on follicular cells
Numbers of antral follicles in the ovaries of the cow
Size of follicles in the ovaries of Japanese black cattle
Relationship between oocyte diameter and maturation rate
Superovulatory responses with or without CIDR-P4-OB

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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.

Bovine oocyte quality and aspiration vacuum pressure
Bovine oocytes obtained by slicing or aspiration
Follicle size and oocyte development
Comparisons between problem, pregnant and cyclic cows in embryo
production
Ovum pick-up in Indian cattle
Oocyte recovery in cattle and African buffaloes using a new device

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Tables and Figures

Table 3.7.
Table 3.8.
Table 3.9.
Table 3.10.
Table 3.11.
Table 3.12.
Table 3.13.
Table 3.14.
Table 3.15.

Oocytes from FSH-treated post-partum beef cattle
Treatment to obtain in vivo-matured bovine oocytes
Comparison of calf vs. cow oocytes in blastocyst yield
Appearance of bovine corpus luteum at different stages of the cycle
Genetic merit and embryo yield in dairy cattle
Criteria used in assessing bovine oocyte quality
Beltsville criteria for bovine oocyte selection
Characterizations of nine morphological groups used to classify bovine oocytes
Development of in vitro-grown bovine oocytes


Table 4.1.
Table 4.2.
Table 4.3.
Table 4.4.
Table 4.5.
Table 4.6.

Towards an understanding of mammalian oocyte maturation
Effect of maturation time on developmental competence of the bovine oocyte
Effect of temperature gradients on bovine oocyte maturation
Amino acid concentrations in sera of clinically normal cows
Development of bovine oocytes in HA-supplemented medium
Effect of hyaluronic acid in a serum-free maturation medium on blastocyst
yield
Table 4.7. Developmental capacity of cattle oocytes matured with and without
granulosa-cell supplementation
Table 4.8. SOF formulation employed as a single-culture system for bovine embryo
production
Table 4.9. Culturing cattle oocytes under roscovitine meiotic inhibition
Table 4.10. Effect of retinoic acid on meiotically inhibited bovine oocytes
Table 4.11. Stages of germinal vesicle development based on changes in the nuclear
morphology of bovine oocytes
Table 4.12. Assessing bovine oocytes after maturation

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Table 5.1.
Table 5.2.
Table 5.3.
Table 5.4.

Approaches to the artificial capacitation of bovine sperm in vitro
Effect of pH on capacitation and fertilization in cattle
Summary of molecules proposed as markers of sperm fertility
Effect of sperm collection method on IVF in cattle

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Table 6.1.
Table 6.2.

Amino acids in bovine oviductal and uterine fluids
Reports dealing with loss of cumulus cells from bovine oocytes shortly after
ovulation
Accessory sperm in superovulated and single-ovulating cows
Comparison of Percoll and swim-up techniques for sperm selection
Sperm abnormalities and fertility in bulls
Effect of pentoxifylline on cattle IVF
Effect of denudation method on IVF and embryo development in cattle
Citrate cleaning of cattle oocytes
Effect of granulosa cells on bull sperm motility
Effect of oxygen level on cattle IVF
Effect of co-culture of bull sperm with BRL cells
Effect of glutathione (GSH) in fertilization medium on blastocyst yield
Developmental stages of male and female pronuclei
Proposed sequence of events in human ICSI
Factors affecting the success of ICSI
Cattle ICSI in combination with oocyte activation
Use of sperm from fertile and infertile stallions by ICSI
Effect of bull on cleavage rate and blastocyst yield

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Table 6.3.
Table 6.4.

Table 6.5.
Table 6.6.
Table 6.7.
Table 6.8.
Table 6.9.
Table 6.10.
Table 6.11.
Table 6.12.
Table 6.13.
Table 6.14.
Table 6.15.
Table 6.16.
Table 6.17.
Table 6.18.

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Table 7.1.
Table 7.2.
Table 7.3.
Table 7.4.
Table 7.5.
Table 7.6.
Table 7.7.
Table 7.8.
Table 7.9.
Table 7.10.
Table 7.11.
Table 7.12.
Table 7.13.
Table 7.14.
Table 7.15.
Table 7.16.
Table 7.17.
Table 7.18.
Table 7.19.
Table 7.20.

Tables and Figures


Development of early bovine embryos on an oviductal-cell monolayer
Summary of data on cattle embryo yields after culture in the sheep oviduct
ATP content of IVP cattle embryos at different stages of development
Growth factors related to reproduction in the cow
Composition of synthetic oviductal fluid (original formulation and a modified
SOF formulation)
Development of in vitro- or in vivo-produced zygotes in a bovine oviductal cell
co-culture system
Development of IVF bovine embryos co-cultured with different cell types
Effect of B2 and TCM-199 media on IVP bovine embryo development
Culture of IVP cattle embryos in different media + 5% FCS
Effect of serum concentration in SOFaa medium on IVP embryo development
Effect of changing KSOM + BSA on day 4 to SOFaaci + 5% FCS
Effect of hyaluronic acid on development of IVP embryos
Composition of human embryo culture media – G1 and G2
IVP embryo production with single or group embryo culture
Embryo production in relation to group size
Embryo development in different culture systems and group sizes
Effect of heparin supplementation on blastocyst yield and cell number
Embryo grading scheme (International Embryo Transfer Society Manual)
Volume density of cellular components in cattle blastocysts produced under
different culture conditions
Time of first cleavage and bovine embryo development

Table 8.1.
Table 8.2.
Table 8.3.

Milestones in the birth of young after transfer of frozen–thawed embryos

Milestones in vitrification of mammalian embryos
Development and survival of vitrified bovine embryos produced under
different culture conditions
Table 8.4. Media employed by 26 commercial cattle ET companies in the USA
Table 8.5. Development of cattle embryos chilled at different cleavage stages
Table 8.6. Pregnancy rates after transfer of cooled and transported horse embryos
Table 8.7. Evaluation of pig embryos stored at different temperatures and in different
media
Table 8.8. Freezing protocol for bovine embryos using glycerol as cryoprotectant
Table 8.9. Pregnancy rates after transfer of embryos frozen in glycerol or ethylene glycol
Table 8.10. Survival of bovine blastocysts developed from delipidated zygotes
Table 8.11. Comparison of conventional freezing and vitrification
Table 9.1.
Table 9.2.
Table 9.3.
Table 9.4.
Table 9 5.
Table 9.6.
Table 9.7.
Table 9.8.

First records of successful embryo transfers
Dulbecco’s phosphate-buffered saline (D-PBS)
Pregnancy rates in recipients after transfer of half-embryos, embryos or
two embryos
Pregnancy rates after transfer of fresh or frozen in vivo and IVP embryos into
Friesian heifer recipients at one location
Effect of embryo–recipient synchrony on pregnancy rates
Effect of low-dose eCG (PMSG) treatment on pregnancy rates in embryo
recipients

Effect of timing of PG dose in progesterone-synchronized recipients on
pregnancy rate
Methods of pregnancy diagnosis and times at which they can be used during
pregnancy

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Table 9.9.

Progesterone levels and pregnancy rates in recipients orally dosed with
propylene glycol

Table 10.1. Potential applications of cattle IVP technology in commercial practice and
research
Table 10.2. Birth weights and gestation lengths in Japanese Black cattle after transfer of
IVP embryos or after artificial insemination
Table 10.3. Fetal, placental and other measures of IVP-derived and AI calves

Table 10.4. Assessment of risk of transmission of infectious diseases by in vivo-derived
cattle embryos
Table 10.5. Twin ovulations and twin births in British and Irish cattle
Table 10.6. Twinning by embryo transfer to bred recipient cattle
Table 10.7. Development of bovine oocytes injected by various types of male gametes
Table 10.8. Possible value of sex control in cattle farming
Table 10.9. Effects of Fert Plus on cleavage and blastocyst production in cattle
Table 10.10. Pregnancy rates using sexed and unsexed frozen–thawed semen
Table 10.11. Effect of time of AI after the onset of oestrus on the sex ratio in cattle
Table 10.12. Zimmermann mammalian cell-fusion medium formulation
Table 10.13. Pregnancies in recipients that received three different clone types and IVP
embryos
Table 10.14. Pregnancy rates after transfer of fresh or vitrified cloned embryos
Table 10.15. Tissue characteristics of cloned and control fetuses
Table B1.
Table B2.
Table B3.
Table B4.
Table B5.
Table B6.
Table B7.

Preparation of phosphate-buffered saline (PBS)
Preparation of oocyte washing medium (modified HEPES-buffered Tyrode’s
medium)
Preparation of sperm capacitation medium (modified Ca2+-free Tyrode’s
medium)
Preparation of fertilization medium (modified Tyrode’s medium)
Preparation of motility-stimulating mixture: penicillamine–hypotaurine–
epinephrine (adrenalin) (PHE)

Procedures for fixation and staining of oocytes and embryos
Salt concentrations in CR1 and CR2 media

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Figures
Fig. 1.1.
Fig. 1.2.
Fig. 1.3.
Fig. 1.4.
Fig. 1.5.

Fig. 2.1.
Fig. 2.2.
Fig. 2.3.
Fig. 2.4.
Fig. 2.5.
Fig. 2.6.
Fig. 2.7.

Beef cattle twins produced by totally in vitro procedures
Steps in Lu’s IVP embryo production process
Cattle twins born after one-embryo transfer to a bred recipient
Production of cattle IVP embryos in Ovamass laboratories in 1989–1991
Foal born in 1984 at University College Dublin after non-surgical embryo
transfer

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10
11

Standing heat in the heifer and cow
Aids to oestrus detection in the cow
Control of steroidogenesis in preovulatory follicles

Changing hormone concentrations during the bovine oestrous cycle
The bovine corpus luteum
Schematic diagrams of two patterns of follicular development
Effects of nutrition on folliculogenesis in the cow

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Fig. 2.8.
Fig. 2.9.
Fig. 2.10.
Fig. 2.11.
Fig. 2.12.
Fig. 2.13.
Fig. 2.14.
Fig. 2.15.


Possible sequence of events during selection of a dominant follicle
Two approaches to oestrus control in the cow
Timing and purpose of hormone injections in synchronization protocol
Schematic representation of folliculogenesis in the cow
Stages of follicle development in the bovine ovary
Follicle size categories in the bovine ovary
Ultrastructural changes during follicle growth
Relationship between oocyte diameter and follicle size

Fig. 3.1.
Fig. 3.2.
Fig. 3.3.
Fig. 3.4.

Bovine oocyte recovery by follicle dissection
Bovine oocyte recovery by follicle aspiration
Cytochemical and ultrastructural differences between calf and cow oocytes
Progress in the manipulation of oocytes in preantral follicles in mice and farm
animals

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100

Bovine follicle at the time of ovulation
Stages in meiosis and oocyte maturation
Nucleolar proteins during bovine oocyte meiosis and mitosis
Chronology of events during oocyte maturation in the cow
Germinal-vesicle-stage bovine oocyte and germinal-vesicle breakdown (GVBD)

Before and after in vitro maturation of the bovine oocyte
Forms of bovine serum employed in maturation media
Bovine oocyte and associated follicular cells
Interaction between granulosa and theca cells in the inhibition of meiotic
resumption
Growth hormone and IGF-I effects on apoptosis in bovine cumulus–oocyte
complexes during maturation
Oocyte–cumulus actions in the mouse
Role of glutathione (GSH) in the in vitro maturation medium
Prematuration essential for developmental competence
A pre-IVM treatment for improving bovine oocyte quality
The mature bovine oocyte

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119
120
127
133

Fig. 4.1.
Fig. 4.2.
Fig. 4.3.
Fig. 4.4.
Fig. 4.5.
Fig. 4.6.
Fig. 4.7.
Fig. 4.8.

Fig. 4.9.
Fig. 4.10.
Fig. 4.11.
Fig. 4.12.
Fig. 4.13.
Fig. 4.14.
Fig. 4.15.
Fig. 5.1.
Fig. 5.2.
Fig. 5.3.
Fig. 5.4.
Fig. 5.5.

Mechanism of sperm capacitation by bovine seminal plasma proteins and
high-density lipoprotein
Regulation of hyperactivation in bovine sperm
Schematic drawing of the bovine acrosome reaction
Sperm reservoir in the isthmus and site of fertilization in the cow
Maternal and embryonic IGF circuits

Fig. 6.1.
Fig. 6.2.
Fig. 6.3.
Fig. 6.4.
Fig. 6.5.
Fig. 6.6.
Fig. 6.7.

In vitro fertilization system for cattle (Kyoto University)
IVMF-derived calves born in Ireland in 1987

Swim-up technique to obtain hypermotile sperm for IVF
Separation of bull sperm on a 45 and 90% Percoll gradient
Hypo-osmotic swelling to screen bull sperm
Denuding the mature bovine oocyte with sodium citrate
Possible mechanisms whereby calcium oscillations may be generated in the
fertilization process
Fig. 6.8.
Gamete interactions at fertilization in the cow
Fig. 6.9.
Decondensation of sperm head and formation of pronuclei
Fig. 6.10. Pronuclei in the bovine oocyte: normal and abnormal

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