Pregnant mare serum gonadotropin

The ovaries of prepubertal gilts were obtained from a local slaughterhouse (Anyang-si, Gyeonggi-do, Korea) and transferred to the laboratory in warm saline. Cumulus-oocyte complexes (COCs) were collected by aspirating 3 to 7 mm follicles of the prepubertal gilts using a 10 mL syringe with an 18-gauge needle. Sediments were washed with TL–HEPES–PVA medium, and oocytes with compact cumulus cells and granulated cytoplasm were selected for in vitro maturation. The washed COCs were cultured in tissue culture medium (TCM-199; Life Technologies, Carlsbad, CA, USA) containing 10 ng/mL epidermal growth factor, 1 mg/mL insulin, and 10% porcine follicular fluid for 44 hours at 39°C at 5% CO₂ and 100% humidity. The COCs were matured with 10 IU/mL gonadotropin hormone, pregnant mare serum gonadotropin (Lee Biosolutions, Maryland Heights, MO, USA), and human chorionic gonadotropin for the first 22 hours. The COCs were then matured under hormone-free conditions. To generate parthenotes, cumulus-free oocytes were activated with an electric pulse (1.0 kV/cm for 60 ms) in activation medium (280 mM mannitol, 0.01 mM CaCl₂, and 0.05 mM MgCl2) using a BTX Electro Cell Manipulator (BTX, CA, USA), followed by 4 hours of incubation in PZM3 medium containing 2 mmol/L 6-dimethylaminopurine.

The ovaries of the prepubertal gilts were obtained from a local slaughterhouse (Anyang‐si, Gyeonggi‐do, Korea) and transferred to the laboratory in warm saline. Cumulus‐oocyte complexes (COCs) were collected by aspirating 3‐ to 7‐mm follicles of the prepubertal gilts using a 10‐ml syringe with an 18‐gauge needle. Sediments were washed with TL–HEPES–PVA medium, and oocytes with compact cumulus cells and granulated cytoplasm were selected for in vitro maturation. The washed COCs were cultured in tissue culture medium (TCM‐199; Life Technologies, Carlsbad, CA, USA) containing 10 ng/ml of epidermal growth factor, 1 mg/ml of insulin, and 10% porcine follicular fluid for 44 h at 39°C in 5% CO₂ and 100% humidity. The COCs were matured with 10 IU/ml of gonadotropin hormone, pregnant mare serum gonadotropin (Lee Biosolutions, Maryland Heights, MO, USA), and human chorionic gonadotropin for the first 22 h. The COCs were then matured under hormone‐free conditions. To generate parthenotes, cumulus‐free oocytes were activated with an electric pulse (1.0 kV/cm for 60 ms) inactivation medium (280 mM mannitol, 0.01 mM CaCl₂, 0.05 mM MgCl₂) using a BTX Electrocell Manipulator (BTX, CA, USA), followed by 4 h of incubation in PZM3 medium containing 2 mmol/L of 6‐dimethylaminopurine.

The ovaries of the prepubertal gilts were obtained from a local slaughterhouse (Anyang‐si, Gyeonggi‐do) and transferred to the laboratory in warm saline. Cumulus‐oocyte complexes (COCs) were collected by aspirating 3‐7 mm follicles of the prepubertal gilts using a 10 mL syringe with an 18‐gauge needle. Sediments were washed with TL‐HEPES‐PVA medium, and oocytes with compact cumulus cells and granulated cytoplasm were selected for in vitro maturation. The washed COCs were cultured in tissue culture medium (TCM‐199; Life Technologies) containing 10 ng/mL epidermal growth factor, 1 mg/mL insulin and 10% porcine follicular fluid for 44 hours at 39°C at 5% CO₂ and 100% humidity. The COCs were matured with 10 IU/mL gonadotropin hormone, pregnant mare serum gonadotropin (Lee Biosolutions) and human chorionic gonadotropin for the first 22 hours. The COCs were then matured under hormone‐free conditions. To generate parthenotes, cumulus‐free oocytes were activated with an electric pulse (1.0 kV/cm for 60 ms) in activation medium (280 mmol L⁻ mannitol, 0.01 m mol L⁻ CaCl₂ and 0.05 m mol L⁻ MgCl₂) using a BTX Electrocell Manipulator (BTX), followed by 4 hour of incubation in PZM3 medium containing 2 mmol/L 6‐dimethylaminopurine.