Ovaries ( n=40) were collected at a local slaughterhouse
from 20 adult (1–3 years old) mixed-breed goats; we set up
a total of four replicates (five goats/replicate). Immediately
postmortem, the ovaries were washed in 70% alcohol,
followed by two rinses in MEM supplemented with
100 μg/ml penicillin and 100 μg/ml streptomycin. The
ovaries were transported in MEM at 4°C within 1 h to the
laboratory (Chaves et al. 2008), where the surrounding fat
tissue and ligaments were stripped off. Ovarian cortical
slices (1–2 mm in diameter) were cut from the ovarian
surface by using a surgical blade under sterile conditions.
Then, the ovarian cortex was placed in a fragmentation
medium, consisting of MEM plus HEPES. Secondary
follicles of approximately 200 μm in diameter were
visualized under a stereomicroscope (SMZ 645 Nikon,
Tokyo, Japan) and manually dissected from the strips of
ovarian cortex by using 26 gauge (26 G) needles. After
isolation, follicles were transferred to 100 μl drops
containing fresh medium under mineral oil to evaluate the
follicular quality further. Follicles with a visible oocyte,
surrounded by granulosa cells, an intact basement membrane and no antral cavity were selected for culture.