Selection of immature pig oocytes for homologous in vitro penetration assays with the brilliant cresyl blue test

Abstract

The present study was designed to evaluate the brilliant cresyl blue (BCB) test as a means of selection of immature pig oocytes to improve the performance of the homologous in vitro penetration (hIVP) assay carried out with immature oocytes to assess the penetrating ability of boar spermatozoa. Immature pig oocytes harvested from abattoir ovaries were incubated in 13 µМ BCB in phosphate-buffered saline, and selected according to their colour (blue and colourless oocytes) after 90 min incubation at 39°C in 5% CO ₂ in air. To assess the selection criteria of the BCB test (Experiment 1), the diameter and viability (FDA test) of blue and colourless oocytes were determined. Differences (P<0.01) were found in both mean diameter (113.08 ± 0.65 µm v. 100.29 ± 0.96 µm) and the viability rate (100 v. 70.71%) between blue and colourless oocytes. In Experiment 2, the selection effectiveness (healthy oocytes relative to inseminated oocytes) and penetrability (percentage of penetrated oocytes and number of sperm in penetrated oocytes) of unincubated (selected by morphological criteria) and BCB incubated oocytes were evaluated. Blue oocytes showed the best (P<0.001) selection effectiveness and the highest (P<0.01) penetrability. Finally, the effectiveness of the BCB test to reduce interassay variability in the results of hIVP assays was examined (Experiment 3). It was found that more consistent data (P<0.05) of selection effectiveness and percentage of penetrated oocytes were obtained when blue oocytes were used. In summary, these results indicate that the BCB test improves hIVP performance when used to preselect immature oocytes.