On random days of the estrous cycle, cumulus-oocyte
complexes (COCs) were collected using transvaginal
ultrasound-guided oocyte collection with a 5-MHz sectorial
transducer equipped with a needle guide and attached to
an Aloka 900 ultrasound scanner (Aloka, Tokyo, Japan).
Cumulus-oocyte complexes were aspirated using a 17-ga
needle and a vacuum pump set to a pressure of 70 mm
Hg. Cumulus-oocyte complexes were collected from all
visible follicles 3 mm. The contents of each follicle were
collected into a 50 mL conical tube with 10 mL of oocyte
collection medium. All COCs were washed in TCM-199
maturation medium for oocytes with HEPES supplemented
with 10% (vol/vol) bovine fetal serum (Gibco,
Langley, OK, USA), 2 mg/mL pyruvate, and 75 mg/mL
gentamicin (wash media), and then placed in oocyte
maturation medium for transport back to the laboratory in
a portable incubator. The COCs were divided into four
categories based on the compactness of the cumulus cells
and the homogeneity and transparency of the ooplasm