Methods for oocyte collection, maturation, and somatic cell preparation are described in other chapters of this book as well as in our earlier publications (8,12). Briefly, 25 oocyte-cumulus cell complexes were matured in each well of a 4WD filled with 400 |L of bicarbonate buffered TCM 199 medium with additives, hormones, and 15% cattle serum, covered with 400 |L of oil. Fibroblasts were cultured in wells of a 4WD containing 400 |L of Dulbecco's modified Eagle's medium supplemented with additives and 10% cattle serum (see Note 1) and covered with 400 |L of oil for 3 to 5 d to reach almost total confluence. For one routine HMC program, 150-200 oocytes and one well of somatic cell culture were used.
This chapter deals in detail only with procedures that are specially performed in bovine HMC, that is, (1) cytoplast preparation, (2) fusion, (3) activation, and (4) embryo culture. Except where otherwise indicated, all manipulations were performed on 39°C heated stages and plates using minimum background light.
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