Optimised CO(2)-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO(2) incubator

Conventional in vitro culture and manipulation of mouse embryos require a CO(2) incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO(2) incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO(2) gas-generating agent, to increase the CO(2) partial pressure of CZB medium to 4%–5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO(2) incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO(2) incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO(2) incubator: 34.3%). This study demonstrates that CO(2) incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.