Validation and Procedural Refinement of a Breed-specific Ovum Pick-Up Protocol in Tharparkar Cows

Ovum pick-up (OPU) is a transvaginal ultrasound-guided technique used for repeated follicular aspiration and in vitro embryo production in cattle. Procedural variables such as needle gauge, aspiration pressure, flow rate, follicle size, synchronization strategy, and temperature influence oocyte recovery and cumulus–oocyte complex (COC) integrity. This study describes validation of a standardized OPU preset in cyclic, non-stimulated Tharparkar (Bos indicus) cows. Clinically healthy cyclic cows were screened by gynaecological examination and ultrasonography. Dominant follicle ablation (≥9 mm) was performed to synchronize a new follicular wave. OPU was conducted on Days 4–7 post-ablation during early follicular recruitment. Procedures were standardized, including uniform restraint, caudal epidural anesthesia (4–5 mL of 2% lignocaine), aseptic preparation, and single-operator execution. Follicles measuring 3–9 mm were aspirated using an 18-gauge disposable needle connected to a regulated vacuum system. Refinement observations identified an optimal aspiration pressure of 80–85 mmHg with a controlled flow rate of ~15 mL/min. Higher pressures (85–90 mmHg) increased COC denudation, whereas lower pressures (70–80 mmHg) reduced follicular evacuation efficiency. Collection media were maintained at 38.5°C throughout. A total of 210 follicles yielded 118 oocytes, with an overall recovery rate of 56.08 ± 3.27%. Mean follicles aspirated and oocytes recovered per session were 21.0 ± 0.81 and 11.8 ± 0.87, respectively. Morphological grading revealed 32.09 ± 4.27% Grade A, 27.77 ± 3.90% Grade B, 17.15 ± 3.52% Grade C, and 23.00 ± 5.24% Grade D oocytes, with 59.86 ± 3.11% classified as acceptable (Grade A+B). The defined preset enabled consistent follicular aspiration and stable oocyte recovery, supporting a reproducible, breed-adapted OPU protocol for indigenous Bos indicus cattle."