After being washed 3 times, spermatozoa were resuspended with insemination medium to give a concentration of 5 X 105 cells/ml, and 50 ^l of the sample was added to 50 ^l of fertilization drops containing the oocytes. Six hours after insemination, oocytes were removed from fertilization drops and cultured in 500 ^l of culture medium (NCSU 23 containing 4 mg/ml BSA) in a four-well culture plate until examination. buy flovent inhaler
Collection of In Vivo Embryos
In vivo-derived embryos were surgically recovered from 12 naturally mated pigs on Days 2-6 (3 on Day 2, 6 on Day 4, 2 on Day 5, and 1 on Day 6) of the estrous cycle. Because experiments were focused on early embryos in the present study, more animals were used for collection of early-stage embryos. Briefly, sexually mature pigs, 6-8 mo of age and weighing 100-120 kg, were examined for signs of estrus twice daily by exposure to a sexually mature boar. On Day 0 of estrus, the animals were artificially inseminated, and oviducts or uteri were flushed with 30 ml Hepes-TL-PVA on Days 2-6 to collect embryos at various stages.