This study demonstrated that there was a broad range in blastocyst development between the 19 different ECT experiments, independent of the variation introduced by different treatments. The total blastocyst development in PG controls varied from 0-41% between runs and high-quality blastocyst development varied from 0-18%. The total blastocyst development in non-treated ECT controls varied from 0-25% and high-quality blastocyst development varied from 0-17%. As the conditions for controls were the same throughout experiments it would be expected that the development rates should be similar. The large range may be explained by the weekly variation in ovary quality. Ovary collections alternated between different abattoirs which caused the lengths of incubation in saline to differ by a couple of hours. Collections also varied in the different ages of mothers and the season. Summer and winter ovine oocytes can vary in the number of 2–8 mm follicles per ovary, and the cleavage and blastocyst rate following chemical activation (Zeron et al., 2001).
The average production of blastocysts from ECT with all treatments in this study was
≈ 7% out of reconstructs that went into IVC. The average production of blastocysts from ECT with standard protocol and no treatment was ≈ 9% out of reconstructs that went into IVC. Blastocyst production was lower than Bogliotti et al. (2018) who used bEPSCs as CT donors and established a blastocyst development rate of 10-20% and Zhao et al. (2021) who used bEPSCS in CT to establish a blastocyst development rate of 21%. Other studies have found that of CT embryos derived from an ES cell nucleus 10–30% reach the morula/blastocyst stage in vitro (Oback & Wells, 2002). Cloning with bovine cultured ICM donors has also been able to generate blastocysts at a rate of 15% (Sims & First, 1994). The lowered average rate of blastocyst development in this study compared to the literature can be explained by the variation between runs.
Some runs had zero development, bringing the averages down. The maximum blastocyst development from non-treated controls achieved in a CT run in this thesis was 25%, which exceeds rates in other literature with bEPSCs, meaning embryo- derived cloning protocols in this thesis can improve blastocyst development with further refinement and consistency.
As no ETs were completed with blastocysts during this thesis, their ability to produce live offspring is unknown. Although we can conclude whether a treatment has
beneficial effects on bovine blastocyst development, no conclusions on the effect on overall cloning efficiency can be made. A treatment that improves blastocyst development may not necessarily improve pregnancy and healthy birth rates. For example, the TM/blastocyst development of CT with cumulus or fibroblast nuclei (55%) is higher than with an ES cell nucleus (10–30%) while blastocysts that reach adulthood is 5–15-fold higher from ES cell nuclei than cumulus or fibroblast nuclei (Oback & Wells, 2002). High-quality blastocysts should undergo ET into recipient animals in order to calculate overall cloning efficiency. Cloning efficiency with blastocysts from double, vitrified, aged, or synchronized treatments can be compared to the cloning efficiencies of non-treated ECT blastocysts. The cloning efficiency of the ECT blastocysts can be compared to the cloning efficiency of SCT blastocysts for insight into the value of the embryo-derived donor cells.
To advance the findings of this study, testing the cells of the embryo-derived outgrowths for pluripotency is required to confidently confirm the cells are ePSCs.
Testing should begin with molecular assessment of pluripotency. This could be done through either reverse transcription quantitative real-time PCR or histological staining for pluripotency markers such as SOX2, OCT4 and NANOG can be done (De Los Angeles et al., 2015). Testing alkaline phosphatase levels which are downregulated during lineage differentiation can also illuminate the degree of pluripotency. The level of DNA methylation can be used to determine whether the cells are in a ground state (low levels of methylation) or primed state (high levels of methylation). bEPSCs and fibroblasts have comparable levels of DNA methylation which can be revealed through bisulfite sequencing (Zhao et al., 2021). Molecular tests can suggest pluripotency, but then functional assays are needed to confirm the developmental potential of the ePSCs (De Los Angeles et al., 2015). The next steps of testing for pluripotency would be functional assessments such as an embryoid body assay, teratoma formation or tetraploid formation (De Los Angeles et al., 2015; Pettinato et al., 2015). Finally, a germline chimera can be formed (De Los Angeles et al., 2015). A single suspected ePSC with can be introduced to a preimplantated host embryo and evaluated for whether it can support normal development and form all somatic cells and germline cells. This is typically measured by evaluating the somatic cells and germline cells of the chimera for expression of a target gene such as tdTomato that was in the ePSC (Zhao et al., 2021). It is important to determine pluripotency in the embryo-derived
donor cells as reducing the reprogramming required is the main motivation for using embryonic donors over somatic donors in CT.
Further steps to encourage the reprogramming of donor cells is to include treatments for epigenetic alterations. It has been observed that blastocysts from CT have incomplete reprogramming of the epigenome (Ding et al., 2008). The demethylation of some DNA regions which occurs after normal fertilization, is absent after CT fusion.
As a result, CT embryos have hypermethylation which promotes heterochromatin.
This limits the access of transcription factors which decreases the activation of gene transcription (Czernik et al., 2019). Treatments for the inhibition of DNA methylation transfer during replication have been trialled, such as 5-aza-dC, which improved in vitro development of the bovine embryos (Ding et al., 2008). Increasing acetylation loosens the chromatin configuration to allow access to the transcription factors (Czernik et al., 2019). Treatment with deacetylase inhibitors such as Trichostatin A and scriptaid have been trialled and shown to increase acetylation and improve blastocyst development after CT (Satoshi Akagi et al., 2011; Ding et al., 2008). One main epigenetic alteration noted for its resistance to reprogramming during CT is the histone methylation of H3K9me3 (Antony et al., 2013). This marker is present in CT embryos but not IVF embryos. Overexpression of the demethylase Kdm4b in donor cells has been shown to decrease H3K9me3 and H3K936me3 levels in bovine embryos (Meng et al., 2020). Kdm4E and Kdm4D used in cattle SCT can decrease methylation of H3K9me3 and improve blastocyst development (Liu et al., 2018). Incorporation of epigenetic reprogramming treatments such as KDM4D/E isoforms into ECT should be trialled to analyse its effect on embryonic donor methylation and to increase developmental potential of embryos. Especially when using trophoblast donor cells which have high levels of H3K9me3 accumulation and are highly resistant to genomic reprogramming during cell transfer (Hada et al., 2022). When Hada et al. (2022) removed H3K9me3 with Kdm4D mRNA the genome was reprogrammed and the trophoblast cells produced live offspring from ECT. This process could be crucial as the embryo-derived donor cells used by the outgrowths in this thesis are predominantly trophoblastic.