For in vitro maturation (IVM), bovine ovaries, collected from a local slaughterhouse, were kept in normal saline and transported to the laboratory within 2 h. Once in the laboratory, ovaries were thoroughly washed with 70% ethanol and PBS solution to avoid contamination. Subsequently, cumulus-oocyte complexes (COCs) with a diameter of about 100–130 μm and a central oocyte surrounded by at least three layers of cumulus cells were collected according to the department's protocols (32 (link)).
After collection, COCs were transferred to 500 μl of IVM droplets (40–50 COCs per droplet) covered by mineral oil and cultured for a subsequent 24 h in a CO2 incubator (38.5°C and 5% CO2). IVM medium applied in this study was TCM-199 medium supplemented with 0.3 mM sodium pyruvate, 1 μg/ml β-2-oestradiol, 2 mM GlutaMAX, 10 ng/ml EGF, 10% fetal bovine serum, 10 U/ml FSH, 10 U/ml LH and 100 μM cysteamine (67 (link)–69 (link)).
After collection, COCs were transferred to 500 μl of IVM droplets (40–50 COCs per droplet) covered by mineral oil and cultured for a subsequent 24 h in a CO2 incubator (38.5°C and 5% CO2). IVM medium applied in this study was TCM-199 medium supplemented with 0.3 mM sodium pyruvate, 1 μg/ml β-2-oestradiol, 2 mM GlutaMAX, 10 ng/ml EGF, 10% fetal bovine serum, 10 U/ml FSH, 10 U/ml LH and 100 μM cysteamine (67 (link)–69 (link)).
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