211 Postponed in vitro fertilization timing improves cleavage competence in oocytes with slow-predicted nuclear maturation speed in Japanese Black beef heifers

As nuclear maturation is essential for oocyte development, oocytes with slow nuclear maturation speed (NMS) may not have sufficient time between nuclear maturation and fertilization, which can compromise their developmental competence. We have previously demonstrated that NMS prediction during IVM can be achieved by machine learning applications based on the morphology of bovine oocytes. However, the impact of delayed IVF timing on embryo development in oocytes with different predicted NMS has not been investigated. Therefore, this study aims to reveal the effect of postponed IVF timing on embryo development in oocytes with predicted NMS. Cumulus–oocyte complexes (COCs) were obtained from 2 to 8 mm follicles of Japanese Black beef heifers from two abattoirs. Each COC was matured individually in 10 μL of IVM medium (IVMD101) at 38.5°C under 5% CO₂ in air, and morphological features were extracted from the photographs taken at 0, 12, 15, and 18 h after IVM start. These features were applied to an established machine-learning model for NMS prediction. The COCs were randomly divided into groups to receive either 24 or 28 h of IVM, followed by 6 h of co-incubation with sperm (2 × 10⁶ sperm mL⁻¹) in 10 µL of IVF medium (IVF100) under 5% O₂, 5% CO₂, 90% N₂ at 38.5°C. Presumptive zygotes were then cultured individually in a microwell dish filled with 125 μL of IVC medium (BO-IVC™) under the same atmosphere as for IVF. The occurrence of cleavage at 30 and 48 h after IVF start was either monitored using a time-lapse camera or photographically recorded. The quality of formed blastocysts at 192 h after IVF start was evaluated by counting the total cell number in some of the blastocysts stained with Hoechst and propidium iodide. Chi-square test was used to compare cleavage and blastocyst rates, whereas a Student t-test was applied to analyse differences in cleavage timing and embryo quality. A total of 379 COCs were used in 19 replicates. Results of embryo development are presented in Table 1. Lower cleavage rates were detected in oocytes with slow-predicted NMS when matured for 24 h, but this was improved by extending IVM to 28 h. No difference was found in the first cleavage timing and blastocyst quality between NMS predictions and IVM durations. In general, extending the IVM duration from 24 to 28 h, namely postponed IVF timing, improved the cleavage rate of oocytes with slow-predicted NMS but did not enhance the efficiency of embryo development. The mechanisms of how NMS affects developmental competence and the optimal IVF timing in accordance with NMS need further examination.