In Vitro Embryo Production
Cumulus-oocyte complexes (COC) were aspirated, matured, and fertilized in vitro as described by Farin and Farin. Briefly, ovaries from Holstein cattle were collected at a local abattoir and held in saline with 0.75 ^g/ml penicillin for 4-6 h at ambient temperature. COC were aspirated from 2- to 7-mm follicles and washed five times in modified Tyrode’s medium (TL-Hepes; ). COC were matured in groups of 20-30 for approximately 22 h in 1 ml TCM-199 with 10% heat-inactivated estrous cow serum (ECS), 10 ^g/ml LH, 5 ^g/ml FSH, 1 ^g/ml estradiol, 200 ^M pyruvate sodium salt, and 50 ^g/ml gentamicin. Cultures were maintained in an atmosphere of 5% CO2 in air with 100% humidity.
At the end of the 22-h maturation period, COC were washed once and placed in fertilization medium consisting of heparin-supplemented TALP medium with 6 mg/ ml fatty acid-free BSA. Thawed frozen semen from the same Holstein sire was used for production of all in vivo and in vitro embryos. Motile spermatozoa were collected by swim-up procedure and used at a final concentration of 1 X 106 spermatozoa/ml. Gametes were coincubated for 18-20 h, after which time presumptive zygotes were washed six times in TL-Hepes and randomly distributed to one of three culture treatments. These treatments were 1) IVPS (in vitro produced with serum): TCM-199 with 10% ECS and 50 ^g/ml gentamicin; 2) IVPSR (in vitro produced with serum restriction): TCM-199 with 10 mg/ml BSA and 50 ^g/ml gentamicin until 72 hpi followed by TCM-199 with 10% ECS and 50 ^g/ml gentamicin from 72 hpi to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): synthetic oviductal fluid with 6 mg/ml BSA, 1% v: v MEM nonessential amino acids, 0.5 mM sodium citrate, 1.5 mM glucose, 0.33 mM pyruvate sodium salt, and 50 ^g/ml gentamicin (modified from Tervit et al. and Lee and Fukui ). All embryos were cultured in wells containing 1 ml treatment medium in an atmosphere of 5% CO2 in air with 100% humidity. Culture media were changed at 48-h intervals throughout the 144-h culture period. Grade 1 compact morulae (n = 5 per treatment) were harvested at 144 hpi, fixed in McDowell’s and Trump’s fixative, and held at 4°C until processed for transmission electron microscopy.