The OCCs were then washed in modified TCM 199 without FSH or LH and matured for an additional 20 h. Matured oocytes were stripped of cumulus cells and fertilized in 50-|xl drops of modified Tris-buffered medium consisting of 113.1 mM NaCl, 3 mM KCL, 7.5 mM CaCl2, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, 2 mM caffeine, and 0.2% (w/v) BSA.
Cryopreserved boar semen was thawed in 10 ml of Dulbecco PBS (Gibco) supplemented with 0.1% (w/v) BSA, and spermatozoa were washed two times by centrifugation (1000 X g for 4 min) and added to fertilization drops to a final concentration of 5 X 105 spermatozoa/ml.
Porcine Embryo Heat Shock Treatments
Oocyte collection, maturation, and fertilization were performed as described above with the following modifications: First, oocytes were matured in the presence of gonadotropins (LH and FSH; concentrations as described above) for the entire 40- to 44-h maturation period. Second, 1 X 106 spermatozoa/ml were used for the production of in vitro fertilized embryos. Embryos were removed from the fertilization microdrops and immediately placed in North Carolina State University-23 (NCSU-23) embryo culture medium at 5% CO2 for approximately 15 h at 39°C, then half the embryos were heat shocked at 42°C for 9 h while the remaining half were maintained at 39°C. After the 9-h heat shock period, all embryos were incubated at 39°C for continued culture.