4.1. Myoblast Cell Culture

All animal experiments were approved by the Animal Care and Use Committee of Nanjing Agricultural University. Primary myoblast cells were isolated from embryonic 9-day chicken skeletal muscle according to a previous study [17]. Myoblasts were maintained and cultured in growth medium (GM) consisting of DMEM, 10% FBS, and a mixture of 1% penicillin-streptomycin (PS). When the cell confluence reached to 90%, culture medium was replaced with differentiation medium (DM) consisting of DMEM, 2% horse serum (HS, Gibco, Carlsbad, CA, USA) and 1% PS. After induction for 3 days, cells were harvested for further analysis.