Procedure used for denuding pig oocytes influences oocyte damage, and development of in vitro and nuclear transfer embryos

• The denuding procedure could affect oocyte damage and the spindle pattern.
• Oocyte enucleation was found to be more efficient using pre-denuding method.
• For PA or IVF, vortexing or pipetting methods were found to be more efficient.

The effects of different denuding procedures used during the in vitro culture of porcine embryos on oocyte damage and aspects of porcine embryo development were investigated in a series of studies. Oocytes were denuded by vortexing or pipetting after 44 h in vitro maturation (IVM) or pre-denuded after 22 h IVM. The total oocyte death rate was significantly (P < 0.05) higher for pre-denuded (27.3 ± 1.4%) than for vortexed (20.3 ± 1.2%) or pipetted (16.2 ± 2.2%) oocytes. There was no significant difference between the treatments in the percentage of oocytes that extruded the first polar body. The type I cortical granule distribution (reflecting complete maturity) and normal spindle formation rates were significantly lower in the pre-denuding than in the vortexing and pipetting treatments. Blastocyst formation rates were significantly lower for the pre-denuding treatment in PA (25.7 ± 4.5%) and IVF (6.1 ± 1.5%) culture than in the vortexing (PA 42.0 ± 4.5%; IVF 11.2 ± 0.5%) and pipetting (PA 43.4 ± 3.1%; IVF 9.4 ± 1.6%) treatments. The proportion of oocytes developing to blastocysts in SCNT culture was not significantly different between treatments ranging from 9.9 ± 1.8% for pre-denuding to 12.3 ± 2.7% for vortexing. No significant differences in apoptosis or embryonic fragmentation were observed. This study shows that the denuding procedure used for porcine oocytes during the in vitro production of embryos can significantly affect oocyte damage, spindle patterns, oocyte maturation, embryo development but not embryonic apoptosis or the frequency of fragmentation.