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Optimized vitrification, cell cycle compatibility and volume of cytoplasts for bovine embryonic cloning

Cell transfer cloning is a promising method for multiplying elite alleles in a population. Together with gene editing, it can increase the genetic gain of livestock and support the adaptation of cattle to climate change. Embryos with superior genetics, including edited genes, can be expanded in vitro, and each single embryonic donor cell can be used in cell transfer to produce new embryos with a copy of those genes. Enucleated oocytes in metaphase II (MII cytoplasts) serve as recipient cells for the embryonic donors, supporting their reprogramming and development. Currently, the rate of healthy calves that can be produced from cloning with somatic cell donors is about 5% and the technology is associated with placental, fetal and neonatal abnormalities, referred to as the cloning syndrome. This is largely due to incomplete epigenetic reprogramming in somatic cells. The genome of a pluripotent embryo-derived donor is postulated to require less reprogramming to reach an embryonic state than a somatic donor, which could lead to improved embryo formation, increased numbers of healthy offspring and reduced animal welfare problems. These donors are also hypothesised to be more amenable to DNA editing. While the conditions for cytoplasts that receive somatic donors are well-researched, cytoplast conditions for embryo-derived donors remain to be optimised. The primary aim of this research project was to further the knowledge on bovine cell transfer methodology by trialling different treatments of cytoplasts that receive embryo-derived donors. Blastocysts were produced through in vitro fertilization and plated to generate embryo-derived outgrowths as a source of donors for cloning. The zona pellucida was removed with assisted hatching techniques before plating. The rate of embryo-derived outgrowth formation after natural hatching without assistance was 80%. This was raised to 88% when mechanical zona pellucida dissection was performed on blastocysts before plating, while enzymatically removing the zona pellucida lowered the production of primary embryo outgrowthsto 36%. Donor outgrowths were arrested into mitosis by incubation in 500 nM nocodazole overnight before fusion with cytoplasts and activation with ionomycin and cycloheximide. Such chemically activated reconstructs had a 22% pseudo-polar body extrusion rate and blastocyst development was 7%. This was lower than blastocyst development in reconstructs without nocodazole-synchronization and with ionomycin and 6-dimethylaminopurine activation (13%). Non-synchronized donors were fused to cytoplasts that had received various treatments: aging and cooling, volume increases, and vitrification. Reconstructs were activated with ionomycin and 6-dimethylaminopurine. Embryos constructed with cytoplasts that had been aged and cooled to alter their MII state had lowered blastocyst development (4%) compared to embryos constructed with cytoplasts that were not aged and cooled (15%). Increasing the volume of a MII cytoplast with embryo-derived donors did not significantly affect total blastocyst development (6%) compared to in control cytoplasts without altered volume (11%). Vitrified oocytes had a high survival rate after thawing (97%). Artificially activated zona-free vitrified oocytes were able to support development to the blastocyst stage, although this was at a lower rate (3%) than artificially activated fresh oocytes (22%). Vitrified cytoplasts had a high survival rate after thawing (92%) and were able to produce a blastocyst after cell transfer with somatic donors (2%). However, this blastocyst development occurred at a lower rate than with fresh cytoplasts and somatic donors (8%). The ability of vitrified cytoplasts to support the development of the embryo-derived donors to the blastocyst stage after cell transfer could not be proven. Before proceeding to in vivo cloning trials, confirmation of blastocyst ploidy, and continued refinement of vitrification protocols, through trialling different reagents and concentrations, is required. Further data on the use of vitrified cytoplasts in double ECT is required to elucidate the role of reprogramming factors in cytoplasts on embryo-derived donors. Embryo transfer of the produced blastocysts will be the final measure of the effect of the cytoplast conditions trialled on overall cloning efficiency. Successful embryo transfer would accomplish the ultimate research aim of this project: to establish that bovine embryonic pluripotent stem cells can be successfully reprogrammed during cloning to produce blastocysts and then live offspring.
Type of thesis
The University of Waikato
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