77 PRODUCTION OF BAN MINIPIG EMBRYO BY SOMATIC CELL NUCLEAR TRANSFER

Abstract

Ban pig is a one of the wild miniature pig breeds of the Vietnamese highland characterized by small body size and adult weight from 40 to 50 kg. SCNT has been applied to this breed as a tool for its conservation to maintain biodiversity as well as to multiply the minipigs as models for research and application in human xenotransplantation. In this paper we present the results of experiments on the production of Ban minipig embryos by transfer of their somatic cell nuclei into enucleated oocytes prepared from domestic pig (Landrace). Skin explants were collected from a young Ban male. After disinfection, the samples were cut into small pieces and cultured for 7-10 days in DMEM medium supplemented with 10% heat treated fetal bovine serum at 39°C under 5% CO₂. The cultures resulting in cell confluence were used for further passage and cryopreservation by rapid freezing in medium containing 10% DMSO and 0.1 M sucrose. The ovaries collected from prepubertal Landrace gilts from a slaughterhouse were transported to the laboratory in PBS at 30°C. Cumulus-oocyte complexes were aspirated from follicles of 3-6 mm in diameter using an 18-gauge needle. They were washed in Hepes-buffered TCM-199, in vitro matured (IVM) in a modified North Carolina State University-37 medium (NCSU-37) containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 1 mM dibutytyl cyclic AMP (dbcAMP), 10 IU/mL eCG, and 10 IU/mL hCG for 22 h, and then held for an additional 14 h in the modified NCSU-37 without dbcAMP, eCG, and hCG. The mature oocytes were separated from cumulus cells by vortexing in 0.1% hyaluronidase in TCM-199. Metaphase II oocytes showing a clear first polar body (PB) were selected for enucleation in the presence of cytochalasin B. For nuclear transfer, fibroblasts were induced to quiescent state by culture in a medium with a low fetal bovine serum (FBS) concentration, and a single cell was transferred into each enucleated oocyte. Fusion was induced with needle electrodes and two direct current (DC) pulses of 30 V for 25 µs in Zimmerman medium with 0.3 M mannitol. The fused oocytes were activated with 7% ethanol in HEPES-TCM-199 for 5 min and cultured in the medium containing cyclohexamide for 6 h before further culture in vitro. The percentage of reconstructed oocytes fused, cleaved, and developed to the morula and blastocyst stages was assessed under a stereomicroscope at Days 2, 3, and Day 6-7, after nuclear transfer. The results showed that the rate of oocytes with a clear PB, obtained after IVM for 36 h, was 52.8% (n = 500). From 294 reconstructed oocytes, 192 (72.7%) fused; a total of 156 (81.2%) cleaved to the 4-8-cell stage, 96 (50%) developed to the morula stage, and 24 (12.5%) developed to the blastocyst stage at Days 2-3, 5-6, and 7 after nuclear transfer, respectively. A karyotype study showed a normal distribution of chromosome number (2n = 38) for the cell population after passage and for reconstructed embryos. These results suggest that IVM oocytes prepared from domestic pig can be successfully used for the production of the SCNT BAN minipig embryos.

This work was supported by grant from the JSPS-VAST Joint Research Project for FY 2004 and by Project 82.