58 HANDMADE CLONING IN TRANS-SPECIES NT: CULTURE MEDIUM HAS AN EFFECT ON THE ABILITY OF RECONSTRUCTED BOVINE-MURINE EMBRYOS TO DEVELOP BEYOND THE 8-CELL STAGE

Abstract

The objectives of studies of trans-species nuclear transfer (NT) include epigenetic reprogramming and stem cell technology. The present study evaluated the effect of culture media on the development of reconstructed bovine-murine embryos. Bovine NT served as a technical control. The NT embryos were produced with the hand made cloning (HMC) technique (Vajta G et al. 2003 Biol. Reprod. 68, 571–578), and to our knowledge, this is the first report on the application of HMC in trans-species NT. Abattoir-derived bovine oocytes were matured for 21 h and enucleated by hand as described (Vajta G et al. 2003). Bovine cytoplasts were fused with either bovine granulosa cells or murine fetal fibroblasts. The bovine NT embryos were cultured for 7 days in modified SOFaaci (Holm P et al. 1999 Theriogenology 52, 683–700) containing either 5% FBS (8 trials) or 4 mg mL⁻¹ fatty acid free albumin (FAFBSA, 6 trials). Bovine-murine NT embryos were cultured for 4.5 days in SOFaaci + FAFBSA (5 trials), or for the first 12 h in SOFaaci + FAFBSA and until 4.5 days in KSOMaa (Biggers JD 1991 J. Reprod. Fertil. 91, 543) containing 1 mg mL⁻¹ embryo-tested BSA (4 trials). The results are shown in Table 1. In bovine NT embryos, both cleavage (day 2) and day 7 blastocyst rates were significantly improved was SOFaaci + FAFBSA was used as culture medium. Culture medium did not affect the cleavage rate of bovine-murine NT embryos at 12–16 h after start of culture. The development of reconstructed bovine-murine embryos beyond the 8-cell stage was significantly improved when SOFaaci + FAFBSA was replaced with KSOMaa + BSA after 12 h culture. Fourteen of a total of 464 (3.0%) Day 4.5 bovine-murine reconstructed embryos reached early morula stage with signs of compaction. The study showed that the development of the reconstructed NT embryos was significantly affected by the culture medium. Contrary to earlier findings (Park SH et al. 2004 Mol. Reprod. Dev. 68, 25–34), the bovine-murine reconstructed embryos developed beyond 8-cell stage, even until early compaction. The gene expression of species-specific and development-related genes of the reconstructed embryos is under characterization.

Table 1.
Effect of culture medium on cleavage and development of reconstructed NTt embryos

Professor Gabor Vajta is greatly acknowledged for his contribution in establishing the HMC technique in our laboratory.