92 Culture method for long-distance transport of bovine embryos derived from IVF before blastulation using microtubes
Authors: Yamanouchi T. , Matsuda H. , Ogata K. , Hashiyada Y. 
Published: December 2018

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: 25mM Hepes-CR1aa (H-CR1)+5% CS or 25mM 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 48h 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 1mL 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 1mL of CR1 or H-CR1 in microtubes in a portable incubator set at 38.5°C for 48h. The CR1 was pre-equilibrated in an incubator in 5% CO2 for 24h before use. Embryos were harvested from microtubes after 48h 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 1000km over a period of 44h 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&gt;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&gt;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&gt;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 48h after culture. In Exp. 4, blastocyst development at 8 dpi was not affected (P&gt;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).