Establishment of a Step-down FSH Superovulation Protocol in Microminipigs for Zygote Collection

Domestic pigs have become increasingly important models in translational research; however, their large size presents logistical and ethical challenges. Microminipigs offer a practical alternative for long-term studies. This study aimed to develop an effective superovulation protocol using a step-down follicle-stimulating hormone (FSH) regimen to improve zygote collection in microminipigs.

In experiment 1, four female microminipigs with regular estrous cycles were used in both the control and treatment conditions in a crossover design. Treatment group was received prostaglandin F2α (PGF_2α), followed by step-down FSH, and human chorionic gonadotropin (hCG). Follicle number, area, and diameter were monitored by ultrasonography from day -6 to day +1 of estrus. In experiment 2, three female microminipigs were used. Zygotes were retrieved at day 1 by oviduct flushing.

On day 0, the number of follicles was higher in the treatment group (93.0±7.74) compared to the control (46.8±5.01). Significant differences were observed on days -1 and 0, while by day+1, the number of follicles decreased in both groups. Follicle area was significantly larger in the treatment than in the control group (0.81±0.03 cm² vs. 0.63±0.04 cm²) on day -2, with no significant differences detected on days 0 and +1. Follicle diameter was also significantly greater in the treatment group compared to the control (3.4±0.1 mm vs. 2.7±0.1 mm) on day -2, while no significant differences were found on days -1, 0, and +1. In experiment 2, an average of 14.7 zygotes per animal were recovered. The cleavage and blastocyst formation rates were 62.8% and 59.4%, respectively.

The step-down FSH protocol effectively enhanced ovarian response and embryo yield in microminipigs, marking a foundational step toward the efficient reproductive engineering for this animal model that may contribute to the advancement of translational research.