In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres

Abstract 

This study investigated the effect of culture media and gas atmospheres on the development of porcine nuclear transfer embryos. Oocytes derived from a local abattoir were matured for 42–44h and enucleated. Fetal fibroblasts were prepared from a Day 35 porcine fetus. Confluent stage fetal fibroblasts were introduced into the perivitelline space of enucleated oocytes. Fusion and activation were induced simultaneously with two direct current (1.2kV/cm for 30μs) in 0.3M mannitol medium. For parthenogenetic activation, the same pulses were used. In Experiment 1, parthenogenetically activated oocytes were cultured in North Carolina State University-23 (NCSU-23), Porcine Zygote Medium-3 (PZM-3), or Beltsville Embryo Culture Medium-3 (BECM-3). Parthenogenetically activated oocytes cultured in PZM-3 had a higher (P<0.05) developmental rate to the blastocyst stage (15.2% versus 3.7–9.6%) as compared to BECM-3 or NCSU-23. The number of nuclei in Day 6 blastocysts was higher (P<0.05) in PZM-3 (23.6) and NCSU-23 (21.4) than BECM-3 (14.2). In Experiment 2, parthenogenetically activated oocytes were cultured in NCSU-23 under a gas atmosphere of 5% CO₂ in air for 6 days (T1), 5% CO₂, 5% O₂, 90% N₂ for 6 days (T2), 5% CO₂ in air for 3 days, then 5% CO₂, 5% O₂, 90% N₂ for 3 days (T3), or 5% CO₂, 5% O₂, 90% N₂ for 3 days, then 5% CO₂ in air for 3 days (T4). Blastocyst formation rates were not different among treatments (12.9±3.6%, 13.5±4.2%, 10.8±2.4%, and 12.6±2.7%, respectively). However, T2 (36.7±2.9) and T3 (33.8±3.0) resulted in more nuclei per blastocyst than T1 (23.2±2.1) or T4 (26.0±2.1). In Experiment 3, reconstructed porcine nuclear transfer (NT) embryos were cultured in NCSU-23 or PZM-3 under a gas atmosphere of 5% CO₂ in air or 5% CO₂, 5% O₂, 90% N₂. Developmental rates to blastocyst stage for porcine NT embryos cultured in NCSU-23 under a gas atmosphere of 5% CO₂ in air or 5% CO₂, 5% O₂, 90% N₂ were 7.2±1.4% and 12.3±1.4%, and the number of nuclei was 12.2±0.8 and 19.4±1.0, respectively. NT embryos cultured in PZM-3 under a gas atmosphere of 5% CO₂ in air or 5% CO₂, 5% O₂, 90% N₂ had developmental rates to blastocyst stage of 18.8±1.9% and 17.8±3.0%, and the nuclei number was 20.9±1.9 and 21.9±3.3, respectively. NT embryos cultured in NCSU-23 had a higher developmental rate to the blastocyst stage in 5% CO₂, 5% O₂, 90% N₂ than in 5% CO₂ in air (P<0.05). Regardless of gas atmospheres, NT embryos cultured in PZM-3 had a higher developmental rate (18.3±1.7% versus 9.7±1.4%) and nuclei number (21.4±1.8 versus 16.9±1.2) than in NCSU-23 (P<0.05). In conclusion, a gas atmosphere of 5% CO₂, 5% O₂, 90% N₂ supported a higher development rate of porcine NT embryos than 5% CO₂ in air when the porcine NT embryos were cultured in NCSU-23. Furthermore, regardless of atmosphere, PZM-3 supported a higher development rate of porcine nuclear transfer embryos than NCSU-23.

Keywords:  Porcine, Nuclear transfer, Culture medium, Gas atmosphere, Developmental rate, Number of nuclei