41 EFFECT OF CELL TYPES AND PASSAGES ON DEVELOPMENT AND APOPTOSIS OF PORCINE CLONED EMBRYOS

Abstract

The purpose of this study was to improve the efficiency of somatic cell nuclear transfer (SCNT) in pigs by assessing the development, cell numbers and apoptosis when using different cell types as nuclear donors and different numbers of passages. Primary cultures of the donor cells, porcine fetal fibroblasts (pFFF) from a female fetus at ∼30 days of gestation and adult female ear skin cells (pAESC), were established in DMEM + 15% FCS. For nuclear donor, cells at different passages were cultured for 5 days until confluent. Cumulus-oocyte complexes were matured and fertilized in vitro as controls by the following methods (2000 Theriogenology 54, 787–797). Following enucleation, oocytes were reconstructed by transfer of donor cells and fusion with two DC pulses (1.4 kV/cm, 50 μs) in 0.28 M mannitol containing 0.01 mM CaCl₂ and MgCl₂. Eggs were then cultured in NCSU23 + 1.9 mM 6-dimethylaminopurine for 3 h. SCNT and IVF embryos were cultured in NCSU23 for 54 h and subsequently in the same medium with 5.55 mM glucose for 90 h at 38.5°C in 5% CO₂ in air. In Experiment 1, when the rates of development between IVF and SCNT embryos constituted with cells at 5–7 passages were compared, no significant (P < 0.05) differences were observed in the cleavage rates. The rates of blastocyst formation were significantly (P < 0.05) higher in IVF than in SCNT embryos with pFFF and pAESC (21% vs. 15% and 10%), but it did not differ between SCNT embryos. Total cell numbers in IVF blastocysts (35.4 ± 12) were significantly (P < 0.05) higher than in SCNT blastocysts with pFFF and pAESC (28.4 ± 8 and 26.2 ± 10, respectively). The apoptosis signal by TUNEL was initiated at Day 3 in IVF and SCNT embryos. Apoptosis rates in SCNT blastocysts with pFFF and pAESC (13.1 ± 2.5 and 16.6 ± 4.3, respectively) were significantly (P < 0.05) higher than in IVF embryos (3.6 ± 1.4). As the embryos developed, the rates of apoptosis were increased. On Day 6, the rates of apoptosis in IVF (4.8%) were significantly (P < 0.05) lower than those in SCNT embryos with pFFF (13.1%) and pAESC (16.6%). However, both total cell number and apoptosis in SCNT embryos with pFFF and pAESC revealed no significant differences. In Experiment 2, SCNT embryos with pFFF in different cell passages were compared for the development and apoptosis. No significant (P < 0.05) differences were observed in the cleavage rates of SCNT embryos among different cell passages. The rates of blastocyst formation were significantly (P < 0.05) higher in SCNT embryos with 5–7 passages than those with other numbers of passages (14% vs. 6–8%, respectively). Although total cell numbers of SCNT blastocysts did not differ among different cell passages, apoptosis rates were significantly (P < 0.05) higher when the number of cell passages was increased. These results suggest that fetal fibroblasts at 5–7 passages are ideal nuclear donor cells for obtaining high-quality porcine SCNT embryos.

This work was supported by grant No. 1000520040020000 from Biogreen 21, Republic of Korea.