Exploring methods to understand bovine embryo competency in vitro


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Virginia Tech


The development of a preimplantation embryo is a stepwise process consisting of morphological, biochemical, and genomic changes. Much remains unknown about the attainment of embryo competency to develop and establish pregnancy. To investigate this, we compared methods of selection at the oocyte or embryo level for improved blastocyst production. Brilliant cresyl blue staining was used to sort oocytes by their growth status (not fully grown vs. fully grown) and the timing of the first embryonic cell division to sort embryos. We found that an embryo's cleavage kinetics are more indicative of their competency than the growth status of the oocyte that gave rise to that embryo. We further investigated the cryopreservation survival of embryos with fast or slow cleavage kinetics and found no significant differences in their ability to hatch post-thawing. Next, we used the complete sequence of the cattle Y chromosome to identify oligonucleotides for efficient sexing of samples. These materials may be used to understand sexual dimorphism as a biological factor in future experiments. Finally, we designed a new method to induce targeted DNA sequence deletions and mRNA cleavage in zygotes using CRISPR-Cas. We targeted the gene OCT4, since the literature shows variable knockout outcomes. Our method improved deletion efficiency while accounting for preexisting or maternally inherited mRNA of the target gene. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes.



Oocyte, blastocyst, developmental competence, gene editing, CRISPR-Cas