Sequential refinement of ovum pick-up technical parameters improves oocyte recovery and developmental competence in Holstein cows.

Ovum pick-up (OPU) technical variability limits the efficiency and consistency of bovine in vitro embryo production (IVP). Here, we established a sequential refinement framework for OPU by systematically evaluating key technical parameters across seven cumulative experiments in Holstein cows. An 18 G aspiration needle achieved significantly higher cumulus-oocyte complex (COCs) recovery than 20 G and 22 G needles (53.05% vs. 40.03-45.79%; p < 0.05). 90° improved recovery compared with non-vertical insertion (55.64% vs. 48.86%), while insertion to one-third to one-half follicle depth and bevel-down orientation further enhanced recovery efficiency (56.52% and 57.97%, respectively; p < 0.05). Polyvinyl chloride (PVC) tubing provided superior COCs recovery (63.29%) and morphological quality, with a tubing length of 1.2-1.5 m. Vacuum pressure of 50 mmHg resulted in higher COCs recovery (70.27%) and blastocyst rate (40.65%), significantly outperforming the previously used 70 mmHg setting. In contrast, excessive aspiration pressure (80 mmHg) was associated with mitochondrial dysfunction, disrupted cortical granule distribution, and increased apoptosis (p < 0.05), indicating impaired oocyte nuclear and cytoplasmic maturation under excessive mechanical stress. Notably, recovery efficiency progressively increased from 53.05% to 70.27% throughout the sequential optimization process throughout the sequential refinement process. Collectively, this study provides a field-based refinement approach for OPU procedures and demonstrates the association between aspiration parameters and bovine oocyte developmental competence under the tested conditions.