Four well dish

COCs were maturated in 500 μL of pre-IVM culture medium containing TCM-199 (Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 0.1% (v:v) PVA in a four-well dish (Nunc, Roskilde, Denmark). The 60 COCs isolated from SF were cultured in mediums either with or without 1 μM PACAP (Pre-SF[−]PACAP group or Pre-SF[+]PACAP group) and incubated with 5% CO₂ in air at 39°C for 18 hours (the pre-IVM period). After the pre-IVM period, COCs were rinsed twice with the IVM composed of TCM 199 added with 0.91 mM sodium pyruvate, 0.6 mM cysteine, 75 μg/mL kanamycin, 10 ng/mL epidermal growth factor (EGF) and 1 μg/mL insulin. Then, 60 of the COCs derived from MF, SF and pre-matured groups were cultured in a medium containing with 10 IU/mL hCG and 10 IU/mL eCG at 39°C with 5% CO₂. These groups were then maturated in a medium with hormones. After 22 hours, the COCs were rinsed twice and cultured in an IVM medium for another 20 to 22 hours. Experimental design is expressed through illustration (Figure 1A).

All protocols performed were approved by the Animal Care and Use Committee of Nanjing Agriculture University and were performed in accordance with Animal Research Institute Committee guidelines. Porcine ovaries were collected from the local slaughterhouse and then transported to laboratory within 3 h in sterile saline (0.9% NaCl) containing 0.03 g/mL of penicillin and 0.03 g/mL of streptomycin at 37°C. Cumulus-oocyte complexes (COCs) were extracted from 3 to 6 mm follicles of ovaries by aspirating with a 20-gauge needle attached to a 5-ml disposable syringe. Oocytes with compact cumulus cells and a uniform ooplasm were selected for in vitro maturation (IVM). The COCs was washed three times with IVM medium [TCM199 (St. Louis, MO, United States,# M2154) supplemented with 75 μg/ml of penicillin, 50 μg/ml of streptomycin, 0.5 μg/ml of LH, 0.5 μg/ml of FSH, 10 ng/ml of epidermal growth factor (mouse EGF, Sigma, #E4127), and 0.57 mM cysteine] and cultured in each well of a four-well dish (Nunc, Roskilde, Denmark) containing 500 μl of IVM medium covered with 200 μl mineral oil at 38.5°C in a humidified atmosphere of 5% CO₂ incubator for IVM. After 42–46 h cultivation, COCs were transferred to 0.1% hyaluronidase (w/v) for 3 min at 38.5°C. After three to four rinses with TCM199, matured MII oocytes were collected for next treatment.

Follicular aspirates were collected into 14 mL Falcon tubes (Falcon, Code 352001), which were kept at 37 °C and filtered using a strainer (Falcon-70 µm pore size-, Code 352350). Under a stereomicroscope (Olympus SZ 61, Shibuya, Tokyo, Japan), oocytes were isolated into the perimeter of a Falcon center-well dish containing flushing medium without heparin and subsequently incubated for two to three hours in IVM media.
Upon completion of oocyte collection, the final medium was prepared using commercially available IVM medium (Vial 2 Medi-Cult IVM System), 0.5 mL of patient serum, 50 µL FSH prepared from a stock solution of 0.075 IU/mL (75 IU GONAL-f was diluted with 10 mL IVM medium and 5 µL hCG, and then incubated at 37 °C and 6% CO₂ and 5% O₂. After incubation in LAG medium, oocytes were placed equally in the wells of a four-well dish (Nunc, Roskilde, Denmark) containing 0.6 mL of final IVM medium covered with liquid paraffin and incubated for 26-28 hours. Nuclear maturity of oocytes was not assessed any further.

Pig oocyte collection and in vitro maturation (IVM) were performed following previously described procedures [12 (link)]. Briefly, porcine ovaries were obtained from a local slaughterhouse (Nonghyup Moguchon, Gimje, Korea), immediately transported to a laboratory in normal saline solution supplemented with penicillin, and maintained in a thermos at 30–35 °C. Cumulus-oocyte complexes (COCs) were collected and washed in Tyrode’s lactate-Hepes containing 0.1% (w/v) polyvinyl alcohol. Oocytes with several layers of cumulus cells were selected and washed three times in TCM-199 (GIBCO, Grand Island, NY, USA) supplemented with 0.1% polyvinyl alcohol (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 µg/mL luteinizing hormone, 0.5 µg/mL follicle stimulating hormone, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid (pFF), 75 µg/mL penicillin G, and 50 µg/mL streptomycin. For IVM, 50 COCs were transferred into 500 µL of maturation medium in a four-well dish (Nunc, Roskilde, Denmark). The oocytes were matured for 40–42 h at 38.5 °C in an incubator with 5% CO₂.