Mineral oil (MO) as a barrier can minimize temperature, osmolality and pH fluctuation in the oocyte/embryo culture medium. Regardless of these advantages, MO acts as a potential source of embryo-toxic compounds which can affect embryo viability. This study was conducted to assess the signs and effects of the four-well culture dish (FW) and microdrop culture systems (MD) on reducing the harmful effects of mineral oil during the in-vitro production of sheep embryos. Oocytes retrieved from abattoir-derived ovary were in-vitro matured, fertilized and cultured in MD and FW, overlaid with two different lots of mouse embryo tested mineral oils (SMO1; nMD = 133, nFW = 103, and SMO2; nMD = 137, nFW = 144) and a clinically tested mineral oil (OMO; n = 107) as a control group. The results showed that using SMO1 and SMO2 in the MD can dramatically decrease cleavage and blastocyst rates. Although they did not show any membrane damage after IVM and 24 h after IVF, the sign of damage was appeared at 48 h after fertilization and completely destroyed the blastomere membrane at 72 h after fertilization, while the FW did not affect membrane integrity. In conclusion, FW compares to the MD, can reduce the deleterious effects of mineral oil on sheep embryos.

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