UC Davis

Mammalian embryo quality is often assessed by morphology, the timing of mitotic events, and aneuploidy. These parameters are more commonly investigated in non-equid mammalian species, therefore, the parameters which indicate embryo quality in the horse remains to be fully elucidated. The overall objective of this dissertation was to optimize the in vitro production (IVP) of equine embryos by understanding the consequences of errors that can arise during pre-mitotic and post-mitotic events. This was accomplished in 3 chapters. Chapter 1 is an introduction to the dissertation. Chapter 2: Review of relevant literature provides a review of the literature pertaining to early mammalian embryo development, knowledge gaps, and implications for equine embryo quality assessment. The specific aims of the experiments conducted in Chapter 3: Pronuclear formation and cytoplasmic extrusion and Chapter 4: Aneuploidy in the early cleavage stage equine embryo were to determine the pre- and post-mitotic events in the zygote using fluorescence microscopy, and the exact percentage of aneuploid embryos in the cleavage stage equine embryo. Currently, the general timing of pronuclear (PN) apposition and fusion is unknown, and this information is critical to bridge the gap in understanding the transition from the PN phase to first cleavage. We found that in vitro PN formation is delayed by more than 6 hours compared to previous observations of PN in the in vivo equine model. The sperm head is still intact at 12 hours post intracytoplasmic sperm injection (ICSI). Additionally, cytoplasmic extrusion (CE) is a pre-cleavage event unique to the horse, and previous studies have shown that CE is a critical event in equine embryo development and occurs as it is associated with developmental success. While CE has also been described as a fragmentation event, there was little previous evidence that the extruded material contains DNA. Using fluorescence microscopy, we confirmed that no DNA was present in the extruded CE material and at this moment, the zygote was at the metaphase of mitosis. In Chapter 4, euploid and aneuploid blastomeres were determined by single-cell sequencing for the first time in the horse. To accomplish this, embryos were removed from culture between days 2 and 3 of embryo culture following ICSI. Because embryonic endpoints were unknown, a prediction model using machine learning using time-lapse data from Chapter 3 was used to determine if the embryos would have failed or survived to blastocyst stage and whether the early developmental stages were predictive of aneuploidy. According to the results from the sequencing data and prediction model, the blastocyst prediction model could not distinguish embryos with a higher percentage of euploid blastomeres in the horse. The percentage of aneuploid embryos was alarmingly high (81.25%), which could be explained by the frequent multipolar divisions observed during the early cleavage divisions. The findings from this work inform future research efforts to improve IVP practices in the horse with potential applications for improving embryo outcome predictions, selection methods as well as pregnancy outcomes.