In Vitro Production of Pig Embryos
In vitro production of pig embryos was based on the procedures reported in our previous study. Briefly, oocytes were aspirated from antral follicles (3-6 mm in diameter) of ovaries collected from prepubertal gilts. After being washed 4 times with Hepes-buffered Tyrode’s medium containing 0.1% (w:v) polyvinyl alcohol (Hepes-TL-PVA), each group of 50 oocytes surrounded by compact cumulus was cultured in BSA-free NCSU 23 medium supplemented with 0.57 mM cysteine (Sigma Chemical Co., St Louis, MO), 10% pig follicular fluid, 10 ng/ml epidermal growth factor, 10 IU/ml eCG (Intervet America Inc., Mills-boro, DE) and 10 IU/ml hCG at 39°C and 5% CO2 in air in a 500-^l drop of the same medium. After being cultured for 22 h, oocytes were washed 3 times and then cultured in maturation medium without hormones for another 22 h. buy yasmin online
After maturation, oocytes were separated from the enclosed cumulus by pipetting in maturation medium containing 0.02% hyaluronidase (Sigma). After being washed 3 times, cumulus-free oocytes were transferred to 50 ^l of insemination medium, a modified Tris-buffered medium, consisting of 113.1 mM NaCl, 3.0 mM KCl, 7.5 mM CaCl2, 20.0 mM Tris, 11.0 mM glucose, 5.0 mM sodium-pyruvate, 2 mM caffeine, and 2 mg/ml BSA. The dishes were kept in a CO2 incubator until spermatozoa were added for insemination. For IVF, 0.2-ml frozen ejaculated semen was thawed at 39°C in 10 ml of PBS containing 1 mg/ml BSA and antibiotics.