On the other hand, the development of mouse embryo banks in which the strains are cryopreserved at the embryo level have shown great promise. These embryo banks have prevented the discontinuation of strains due to genetic mutation or natural DNA/RNA Synthesis inhibitor disasters
and provide a significant cost-savings, including avoiding the need for breeding space . As the cost to maintain rat strains are even higher than that for mice, it is important to preserve rat strains by cryopreserving early-stage embryos. We planned to build a rat embryo bank by cryopreserving early rat embryos. Whittingham  modified the slow freezing method used for mouse early-stage embryos and cryopreserved two-, four-, and eight-cell stage rat embryos. In addition, Kono , Isachenko , Tada , Jiang , Anzai , and Seita  cryopreserved rat embryos using the vitrification method. Thus, methods used successfully for other animal species have been applied to rats, usually with some modification. In the present study, we determined the optimal pre-treatment for vitrification and the components of the vitrification solution using rat two-cell embryos. To facilitate manipulation of the collection and embryo transfer, two-cell stage embryos are used for cryopreservation in many mouse embryo banks, and we therefore
examined the cryopreservation of rat embryos using the two-cell stage embryos. Han et al. Han et al.  reported that embryo survival and in vivo buy SP600125 development are improved when two-cell stage rat embryos are exposed to a pretreatment solution containing a low concentration of cell-permeable cryoprotectant, and vitrification of these embryos is then conducted. Based on these findings, we investigated the vitrification method after pretreatment of two-cell stage rat embryos. For pretreatment, as it is necessary to select a cryoprotectant with low cytotoxicity and with a low risk of damaging the embryos due to osmotic expansion, we investigated the permeation rate of cell-permeable
cryoprotectants and fetal development. To prevent damage to the embryos by osmotic expansion after warming without the occurrence of freeze fractures when the vitrification solution vitrifies after cooling, we investigated different types and concentrations of cell-permeable cryoprotectants, sugars, and high molecular weight Methane monooxygenase molecules added to the vitrification solution. Using the pretreatment and vitrification solutions developed in this study, vitrification of rat two-cell stage embryos was conducted and the survival and in vivo development after warming were investigated. Rats of the BrlHan:[email protected](GALAS) strain (CLEA Japan, Inc., Tokyo Japan) were used for the experiments. The breeding conditions were as follows: room temperature, 22 ± 0.5 °C; humidity, 55 ± 5%; and lighting from 08:00 to 20:00. Rat chow (CA-1; CLEA Japan Inc.) and tap water were available ad libitum.