100 Morphokinetic features of in vitro-produced equine embryos during fertilization and in early development observed with time-lapse monitoring

Time lapse monitoring (TLM)-equipped incubators enable nondisruptive study of in vitro-produced embryo development, revealing diverse morphokinetic features unique to each mammalian species. Various studies on embryo TLM technology in equine established the normal mitotic timeline for in vitro-produced embryos (Lewis et al. 2019 Reprod. Fert. Dev. 31(12), 1851–1861; Meyers et al. 2019 Reprod. Fert. Dev. 31(12), 1874–1884; Brooks et al. 2019 Reprod. Fert. Dev. 31(12), 1840–1850). However, such studies have mostly focused on the morphokinetic analysis of the embryo development starting from the first cleavage, and few studies to date have closely examined events that occur around the time of fertilization. Our study provides a detailed morphokinetic description of early equine in vitro embryonic development, particularly focusing on events after fertilization and before the first cleavage. This investigation was performed on equine oocytes collected via ovum pickup (OPU) from live donor mares, matured in vitro, and fertilized by intracytoplasmic sperm injection (ICSI). Immediately after ICSI, the oocytes were incubated in a self-contained TLM incubator (Geri®, Genea Biomedx) that performed sequential imaging every 5 min, allowing continuous monitoring. Preliminary results were obtained considering the morphokinetic parameters from 181 injected oocytes that developed to blastocyst stage of freezing or embryo transfer grade. The results indicated that second polar body extrusion occurred at 2.5 ± 1.7 h (mean ± SD) after ICSI. A frequent and moderate cytoplasmic fragmentation noticeable at 17.9 ± 3.9 h after ICSI occurred before the first cleavage in 80.6% of cases, with debris accounting for 5%–25% of the cellular volume. The first cleavage occurred at 22.7 ± 4.4 h of culture, and it was possible to classify different cleavage modalities according to the dynamic of the cellular division, such as normal cleavage, direct cleavage, chaotic cleavage, and reverse cleavage (Lewis et al. 2019), observed at rates of 51.7%, 21.1%, 24.4%, and 2.8%, respectively. Ongoing analysis involves studying the same morphokinetic parameters in zygotes that reached the blastocyst stage but did not meet the morphological criteria for ET or freezing, as well as those that ceased development before blastocyst formation. So far, no significant correlation has been found between cleavage modalities and blastocysts developmental rate (one-way ANOVA; P < 0.05). Nevertheless, similar cleavage modalities have been identified across different preimplantation embryos obtained from the same donor mare. In conclusion, our data suggest that abnormal cleavage is compatible with the development of blastocysts of freezable or transferrable grade in equine species. This study could shed light on how the timing of these early meiotic and mitotic events and different embryo cleavage behaviors might affect the preimplantation potential and ultimately the developmental competence in establishing healthy pregnancies in recipient mares.