Received June 13, 2011
Accepted June 25, 2011

Objective:Although droplet method has been widely used in IVF laboratories, the toxic contamination produced by deterioration of mineral oil quality and extraction of steroids from the culture medium into the oil overlay has been associated with impaired in vitro development and freezability of oocytes and embryos.The use of washed and autoclaved oil with 0.9% saline may provide a useful tool to prevent toxic contaminants in commercial mineral oil, but its effect upon steroids extraction from the culture medium remains to be determined. Methods:This study was designed to compare the concentration of steroids in IVM droplets of cumulus oocyte complexes (COCs) cultured under two types of oil overlay:washed and autoclaved mineral oil(WMO) or mineral oil in its commercial formulation(CMO) and to evaluate its influence on subsequent embryo development.Bovine COCs were cultured at a density of 1/10 μL in TCM-199 supplemented with estradiol-17β at 38.5°C, 5% CO2 and 100% humidity during 24h. After fertilization, presumptive zygotes were co-cultured with cumulus cells until 224h post-insemination(hpi). Media were collected before and after IVM, and stored at -20°C. E2 and P4 were determined by chemiluminescence and embryo development was evaluated by cleavage, blastocyst and hatching rates at 168-224hpi. Results:E2 and P4 were dramatically reduced (P<0.01) in both oil overlay. Remaining levels of E2 in IVM microdrops were significantly higher in WMO(313.03±72.6ng/mL) than CMO(123.5±46.1ng/mL) showing lower steroid absorption by WMO overlay(P<0.01). P4 secreted by COCs was almost totally absorbed by mineral oil, irrespectively to the oil preparation. There were significantly lower hatching rates in WMO