Porcine ovaries were collected from pre-pubertal crossbred gilts (Landrace × Large White) at a local abattoir and transported to the laboratory in Dulbecco’s phosphate buffered
saline (PBS; Nissui Pharmaceutical, Tokyo, Japan) at 35oC within 1 h. Cumulus-oocyte complexes (COCs) were aspirated from 3–6 mm follicles, and cultured in groups of 40
to 50 in 500 μL of modified NCSU-37 medium [26 (link)] without oil overlay according to Kikuchi et al. [25 (link)] in 4-well dishes (Nunclon Multidishes, Nalge Nunc International, Roskilde, Denmark) for either 22 or 26 h. The IVM medium was modified by adding 10% (v/v)
porcine follicular fluid, 0.6 mM cysteine (Sigma, St. Louis, MO, USA), 50 mM β-mercaptoethanol (Axon Medchem, Groningen, Netherlands), 1 mM dbcAMP (Sigma), 10 IU/mL eCG
(Serotropin; ASKA Pharmaceutical, Tokyo, Japan), and 10 IU/mL hCG (Puberogen; Novartis Animal Health, Tokyo, Japan). The COCs were then transferred to IVM medium without dbcAMP and
hormones and cultured for another 22 or 18 h. IVM was performed in 5% CO2 and 5% O2 at 38.5oC.
saline (PBS; Nissui Pharmaceutical, Tokyo, Japan) at 35oC within 1 h. Cumulus-oocyte complexes (COCs) were aspirated from 3–6 mm follicles, and cultured in groups of 40
to 50 in 500 μL of modified NCSU-37 medium [26 (link)] without oil overlay according to Kikuchi et al. [25 (link)] in 4-well dishes (Nunclon Multidishes, Nalge Nunc International, Roskilde, Denmark) for either 22 or 26 h. The IVM medium was modified by adding 10% (v/v)
porcine follicular fluid, 0.6 mM cysteine (Sigma, St. Louis, MO, USA), 50 mM β-mercaptoethanol (Axon Medchem, Groningen, Netherlands), 1 mM dbcAMP (Sigma), 10 IU/mL eCG
(Serotropin; ASKA Pharmaceutical, Tokyo, Japan), and 10 IU/mL hCG (Puberogen; Novartis Animal Health, Tokyo, Japan). The COCs were then transferred to IVM medium without dbcAMP and
hormones and cultured for another 22 or 18 h. IVM was performed in 5% CO2 and 5% O2 at 38.5oC.