Although assisted reproductive technology (ART) currently exists, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to hold embryos at room temperature. The basic medium for embryo holding for a short period of time at 4 °C, 10 °C and 20 °C consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum. To maintain survival and prevent damage during embryo incubation, three candidate small molecules were selected-CHIR99021, Y-27632 and Thiazovivin-and their concentrations were optimized. The viability and hatching rate of embryos incubated at 10 °C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after incubation at 20 °C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The pregnancy rate of embryos incubated at 20 °C was also greater in the CHIR99021+Y-27632-BNC group compared to that in the frozen group. The mechanism by which small molecules enhance survival of embryos during incubation was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.