Novel insights in commercial in vitro embryo production in cattle Maaike Catteeuw Promoter: Prof. dr. Ann Van Soom Copromoters: Prof. dr. Joris Vermeesch, Dr. Katrien Smits Dissertation submitted to Ghent University in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Veterinary Sciences 2018 Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine Members of the examination committee Prof. dr. Herman Favoreel Chairman – Faculty of Veterinary Medicine, Ghent University, Belgium Prof. dr. Bjorn Heindryckx Faculty of Medicine and Health Sciences, Ghent University, Belgium Prof. dr. Luc Peelman Faculty of Veterinary Medicine, Ghent University, Belgium Prof. dr. Geert Opsomer Faculty of Veterinary Medicine, Ghent University, Belgium Dr. Erik Mullaart CRV BV, the Netherlands Dr. Karen Goossens Research institute for Agriculture, Fisheries and Food (ILVO), Belgium Funding Project G039214N: Chromosomal instability during early embryonic development: elucidating key mechanisms in a bovine model Printed by University Press, Zelzate TABLE OF CONTENTS LIST OF ABBREVIATIONS CHAPTER 1 GENERAL INTRODUCTION 7 1.1 ASSISTED REPRODUCTIVE TECHNOLOGIES IN CATTLE 9 1.2 COMMERCIAL IN VITRO EMBRYO PRODUCTION 11 1.3 QUALITY ASSESSMENT OF EMBRYONIC DEVELOPMENT 25 1.4 GENETIC DISORDERS AND CHROMOSOMAL ABNORMALITIES IN CATTLE 34 1.5 CHROMOSOMAL ABNORMALITIES IN HUMAN EMBRYOS AND THE BOVINE MODEL 35 1.5 REFERENCES 37 CHAPTER 2 AIMS OF THE STUDY 51 CHAPTER 3 HOLDING IMMATURE BOVINE OOCYTES IN A COMMERCIAL EMBRYO HOLDING MEDIUM 55 CHAPTER 4 IN VITRO PRODUCTION OF BOVINE EMBRYOS DERIVED FROM INDIVIDUAL DONORS 75 IN THE CORRAL® DISH CHAPTER 5 DEVELOPMENTAL COMPETENCE OF EARLY, INTERMEDIATE AND LATE CLEAVING 91 EMBRYOS USING TIME-LAPSE IMAGING CHAPTER 6 CHROMOSOME INSTABILITY IN IN VIVO AND IN VITRO BOVINE EMBRYOS 107 CHAPTER 7 GENERAL DISCUSSION 129 7.1 INFLUENCE OF THE DONOR ANIMAL 132 7.2 THE INTRINSIC OOCYTE QUALITY 134 7.3 EMBRYO DEVELOPMENTAL COMPETENCES 137 7.4 GENERAL CONCLUSIONS 144 7.5 FUTURE PERSPECTIVES 145 7.6 REFERENCES 146 CHAPTER 8 SUMMARY – SAMENVATTING 153 DANKWOORD 163 BIBLIOGRAPHY 167 CURRICULUM VITAE 171 APPENDIX 173 LIST OF ABBREVIATIONS AC Apoptotic cell ACR Apoptotic cell ratio AI Artificial insemination AMH Anti-Muellerian Hormone ART Assisted reproductive technologies BAF B allele frequency BMP15 Bone morphogenetic protein 15 BoHV1 Bovine Herpes Virus 1 BSA Bovine serum albumin cAMP Cyclic AMP CIDR Controlled internal drug release CIN Chromosomal instability CN Copy number COCs Cumulus oocyte complexes CR1aa Charles Rosenkrans 1 amino acids dpi Days post insemination eCG Equine chorionic gonadotropin EGA Embryonic genome activation EGF Epidermal growth factor EHM Embryo holding medium FCS Fetal calf serum FISH Fluorescent in situ hybridization FSH Follicle stimulating hormone GC Guanine Cytosine GDF9 Growth differentiation factor 9 GEO Gene Expression Omnibus GnRH Gonadotrophin releasing hormone h Hours i.m. Intramuscularly ICM Inner cell mass IETS International Embryo Technology Society ITS Insulin-Transferrin-Selenium IVC In vitro culture IVF In vitro fertilization IVM In vitro maturation IVP In vitro production LH Luteinizing hormone LogR Log R ratio LOS Large offspring syndrome LSM Least square means MDA Multiple displacement amplification MEM Minimal essential medium MET Maternal-embryonic transition MOET Multiple ovulation and embryo transfer MPF Maturation promoting factor NA Not available NGS Next generation sequencing OCS Oestrus cow serum OMIA Online Mendelian Inheritance in Animals OPU Ovum pick-up OR Odds ratio PBS Phosphate-buffered saline PCR Polymerase chain reaction PGD Preimplantation genetic diagnosis PGF Prostaglandin PGS Preimplantation genetic screening PVA Polyvinyl alcohol PVP Polyvinyl pyrrolidone qPCR Quantitative polymerase chain reaction rob Robertsonian translocation RT Room temperature SE Standard error SEM Standard error of the mean SNP Single polynucleotide polymorphism SOF Synthetic oviductal fluid TALP Tyrode's albumin-pyruvate-lactate TCM-199 Tissue Culture Medium 199 TCN Total cell number TE Trophectoderm TLC Time-lapse cinematography WGA Whole genome amplification WOW Well-of-the-well CHAPTER 1 GENERAL INTRODUCTION Chapter 1 1.1 Assisted reproductive technologies in cattle Today, assisted reproductive technology (ART) refers to all fertility treatments in which human or animals’ eggs, semen and embryos are being manipulated. Primarily, ART is used to overcome infertility and to help couples to fulfil their desire to have children. In cattle, ART is mainly performed to improve reproductive results but it is also intensively used to improve genetic selection in valuable individuals or herds. Furthermore, ART can also be a part of the preservation of endangered species. ART can be as simple as artificial insemination (AI) in which sperm cells from a male animal of interest is manually deposited in the reproductive tract of the female. This allows the use of genetic material of superior males, the import of semen to introduce new genetic material without the need to transport live animals, the use of frozen semen long after the animals’ dead and the risk reduction of spreading transmittable sexual diseases (Foote 2002). Generally, AI techniques have been standardized for many species and AI is performed globally in more than 100 million cattle every year (Boa-Amponsem and Minozzi 2006). Pregnancy rates after AI however can vary between 30 and 70% depending on several factors such as timing (Lamb et al. 2010), parity (Pursley et al. 1997) and number of AI performed (Chebel et al. 2004). Due to its massive worldwide application, the genetic impact from the male side increased enormously. Some bulls have over hundred thousands of descendants and have therefore a remarkable impact on the genetic pool of a breed (Thibier 2005; Goovaerts et al. 2007). Less evident is however producing more offspring from genetically valuable females to increase their genetic impact on the selection process. Starting from the mid-seventies, female donors are routinely treated with hormones to induce superovulation and are subsequently inseminated to recover embryos by uterine flushing. These in vivo derived embryos are then transferred to synchronized recipients (multiple ovulation and embryo transfer – MOET). Although annually more than 500,000 in vivo derived embryos are transferred worldwide to increase the genetic impact from the female side, it is not successful at all times due to the unpredictable response to the hormonal treatment between cows (Hasler et al. 1995; Van Wagtendonk-de Leeuw 2006). Nonetheless, the embryo transfer technique proved to be very useful in the transfer of in vitro produced embryos (IVP) (Goovaerts et al. 2007), in which immature oocytes are removed from the ovaries and are fertilized with semen in a dish. During culture, the fertilized oocytes or zygotes will start cleaving and when the blastocyst stage is reached, transfer of the embryo to the uterus of a recipient donor is possible. As some disadvantages of MOET, such as the unreliable hormonal response, can be overcome by IVP, it gained worldwide interest and nowadays more than 660,000 IVP embryos are produced each year. Since the first in vitro produced calf, including in vitro maturation of the oocyte, was born in 1990 (Fukuda et al. 1990), tremendous progress has been made 9 Chapter 1 not only in the oocyte retrieval process (ovum pick-up – OPU) but also in the in vitro procedures (maturation, fertilization and culture) and cryopreservation techniques. Other ART are part of fundamental or biomedical research, such as the production of cloned embryos by somatic cell nuclear transfer or the production of transgenic embryos. However, cloned and genetically engineered animals have already raised interest in the meat and dairy industry, for application in breeding programs, such as the production of hornless offspring or improved food products from animals (Foote 2005). Until now, European legislation only recommends cloning or producing genetically modified animals by novel techniques such as Crispr-Cas for research purposes as the European Group on Ethics is still in doubt whether using cloned or genetically modified animals for food supply is ethically justified (European legislation COM/2010/0585). 10 Chapter 1 1.2 Commercial in vitro embryo production In order to produce bovine embryos in vitro from follicular oocytes, it is required to perform a series of essential techniques following a strict timing. After oocyte collection, which can be performed in live animals by ovum pick-up (OPU) or by follicular aspiration of slaughterhouse ovaries, three subsequent phases can be distinguished: in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) (Figure 1). When all consecutive steps are carried out punctually, viable embryos will be the result. In commercial settings, this will be the higher goal of IVP as these viable embryos will be transferred to recipient animals which will hopefully become pregnant and deliver a healthy calf. 1.2.1 Oocyte collection Ovum pick-up in live donor animals Repeated oocyte collection by transvaginal ultrasound-guided follicular puncture (OPU) followed by routine IVP has become an important alternative to MOET in cattle. Adapted from human reproduction, OPU in cattle was performed for the first time in 1988 (Pieterse et al. 1988). It can be considered to be more advantageous than MOET as it can also be used in acyclic