25 PORCINE OOCYTES SELECTION USING BRILLIANT CRESYL BLUE AND EMBRYO DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER

Abstract

The efficiency of animal cloning through somatic cell nuclear transfer (SCNT) technology is affected by numerous factors such as oocyte quality and donor cell type. Among the factors, oocyte quality can be enhanced by identification and selection of developmentally competent oocytes before in vitro maturation (IVM). Morphological criteria following homogeneous ooplasm, more than 3 layers of cumulus cells, and oocyte diameter have been used. However, the criteria vary between examiners and even within individual. In contrast, use of Brilliant cresyl blue (BCB), a marker of oocyte growing status related to activity of glucose-6-phosphate dehydrogenase (G6PD), can be a more objective selection method for the detection of fully grown oocytes in bovine, equine and porcine. To our knowledge, BCB has been used to select oocytes in parthenogenesis, intracytoplasmic sperm injection (ICSI), and IVF, excluding SCNT in porcine. The aim of this study was to investigate whether oocytes selected by BCB have better ability to develop into blastocysts than oocytes selected by morphological criteria. After aspirated cumulus–oocyte complexes (COC) from porcine ovaries were washed in HEPES-buffered TCM-199 2 times, the COC were selected through morphological criteria and divided randomly into 2 groups. Group 1 (control; 304 COC) was directly transferred into IVM medium and Group 2 was incubated in TALP supplemented with 26 μM BCB for 90 min at 38.5°C in air. Then, COC of the second group were washed twice in TALP, and COC displaying a blue-coloured ooplasm (BCB+) were selected. The 342 BCB+ COC also were transferred into IVM medium and cultured with 5% CO₂ in air at 38.5°C, with hormonal supplementation for 22 h and without the hormones for another 22 h. After denudation, the rate of degenerated oocytes and maturation rate were determined. The matured oocytes were used for SCNT and the development and total cell number of blastocysts were observed. Each experiment was repeated at least 3 times. Data were analysed by unpaired Student's t-test using Graphpad Prism (GraphPad, San Diego, CA, USA). No difference was observed between Groups 1 and 2 in the rate of degenerated oocytes (9.13 ± 0.47% and 10.68 ± 2.73%, respectively), metaphase II rate (94.60 ± 2.30% and 89.26 ± 3.76%, respectively), cleavage rate (77.02 ± 1.56% and 80.05 ± 2.31%, respectively), or blastocyst formation rate (9.74 ± 1.91% and 10.74 ± 1.30%, respectively). However, total cell number of blastocyst showed significant difference between Groups 1 and 2 (57.67 ± 1.76 and 77.50 ± 1.50, respectively; P < 0.01). In conclusion, selection of oocytes through BCB staining does not improve their developmental ability with respect to cleavage and blastocyst formation rate, but enhances embryo quality by means of increased total cell number per blastocyst in porcine SCNT.

This study was supported by IPET (#311011-05-2-SB010), MKE (#10033839-2012-21) and TS Corporation.