Puberogen

The maturation culture medium was a modified NCSU-37 solution [27 (link)]
containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 50 μM β-mercaptoethanol, 1 mM dibutyryl cAMP
(dbcAMP; Sigma), 10 IU/ml eCG (Serotropin; ASKA Pharmaceutical, Tokyo, Japan) and 10 IU/ml hCG (500 units;
Puberogen, Novartis Animal Health, Tokyo, Japan). Maturation was performed in 4-well dishes (Nunc MultiDishes,
Thomas Scientific) in 500-µl droplets of IVM medium without oil coverage for 22 h in an atmosphere of 5%
CO₂, 5% O₂ and 90% N₂ at 39 C. The COCs were subsequently cultured in the
maturation medium without dbcAMP and hormones for an additional 22 h under the same atmosphere. Forty to 50
COCs were cultured in each droplet.

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 35^oC 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 38.5^oC.

The maturation medium was porcine oocyte medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan) supplemented with 1 mM dibutyryl cAMP (dbcAMP; Sigma-Aldrich), 10
ng/ml epidermal growth factor (EGF, Sigma-Aldrich), 10 IU/ml eCG (Serotropin; ASKA Pharmaceutical, Tokyo, Japan) and 10 IU/ml hCG (500 units; Puberogen, Novartis Animal Health, Tokyo,
Japan). Forty to fifty COCs were cultured in each well of 4-well dishes (Nunc MultiDishes, Thermo Fisher Scientific, Waltham, MA, USA) in 500 -µl droplets of IVM medium without oil coverage
for 22 h in an atmosphere of 5% CO₂, 5% O_2, and 90% N₂ at 38.5°C. Thereafter, COCs were cultured in maturation medium without dbcAMP for an additional 22−24
h under the same atmosphere. At the end of IVM, COCs were denuded by treatment with 0.1% (w/v) hyaluronidase in collection medium for 2 min, followed by gentle pipetting through a
narrow-bore glass capillary tube in collection medium without hyaluronidase. Thereafter, oocytes were investigated under a stereo microscope. Those with an intact oolemma, a normal spherical
shape, a smooth surface, and a dark and evenly granular cytoplasm were considered to be alive. Oocytes with membrane damage and a brownish faded cytoplasm were considered dead. Survival
rates are expressed as the living oocytes on the total number of oocytes.