92 Culture method for long-distance transport of bovine embryos derived from IVF before blastulation using microtubes

Abstract

In vitro-produced (IVP) embryos are more easily damaged by cryopreservation than in vivo-derived embryos. Therefore, transportation of fresh IVP embryos in a manner that can maintain viability is necessary. This study was conducted to determine the preferable culture conditions for transport of embryos at 5 days post-insemination (dpi) in 1.5-mL microtubes. Cumulus-oocyte complexes derived from an abattoir were matured and then inseminated with frozen-thawed semen. Presumptive zygotes were cultured in mCR1aa (CR1) + 5% calf serum (CS) until use. In Exp. 1, embryos with 5 blastomeres at 5 dpi were randomly assigned to 1 of 3 groups: 25 mM Hepes-CR1aa (H-CR1) + 5% CS or 25 mM Hepes-M199 (H-M199) + 5% CS in air, or CR1 in 5% CO₂. Embryos were cultured in microdrops overlaid with liquid paraffin in a petri dish for 48 h at 38.5°C. In Exp. 2, the optimal number of embryos to culture per microtube was assessed. Presumptive zygotes were cultured in groups of 20, 40, or 80 in 1 mL of CR1 covered with liquid paraffin in microtubes in an incubator at 38.5°C in 5% CO₂ until 7 dpi. For Exp. 3, culture of embryos in microtubes in a portable incubator was tested. At 5 dpi, 5-cell embryos (n = 17 to 36 per microtube) were statically cultured in 1 mL of CR1 or H-CR1 in microtubes in a portable incubator set at 38.5°C for 48 h. The CR1 was pre-equilibrated in an incubator in 5% CO₂ for 24 h before use. Embryos were harvested from microtubes after 48 h and were then cultured in microdrops of CR1 overlaid with liquid paraffin in a petri dish in an incubator at 38.5°C in 5% CO₂ until 8 dpi. In Exp. 4, embryos (n = 29 to 39 five-cell embryos per microtube) were transported in a portable incubator by land for 1000 km over a period of 44 h using the same conditions as in Exp. 3. Control embryos were statically cultured in microdrops of CR1 in an incubator in 5% CO₂. Statistical analyses were carried out by ANOVA (Exp. 1 and 2), t-test (Exp. 3), or Fisher’s exact test (Exp. 4). In Exp. 1, there was no effect (P > 0.05) of culture medium on blastocyst development at 7 dpi (27.6 ± 2.3, 25.7 ± 7.2, and 17.3 ± 2.9% for CR1, H-CR1, and H-M199, respectively). In Exp. 2, blastocyst development at 7 dpi was not affected (P > 0.05) by the number of presumptive zygotes cultured per microtube (43.6 ± 8.3, 42.4 ± 4.0, and 39.9 ± 2.9% for 20, 40, and 80 presumptive zygotes, respectively). In Exp. 3, blastocyst development at 8 dpi was not affected (P > 0.05) by culture medium (60.7 ± 7.4 and 53.1 ± 4.4% for CR1 and H-CR1, respectively); however, the pH of CR1 changed from 7.5 to 8.1 at 48 h after culture. In Exp. 4, blastocyst development at 8 dpi was not affected (P > 0.05) by transport (57.1, 64.4, and 75.5% for CR1, H-CR1, and control, respectively). These results indicate that IVP embryos harvested at 5 dpi can be transported by portable incubator with no effect on embryo development to the blastocyst stage.

This work was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and the development for next-generation technology).