Cryopreservation of spermatozoa is a strategy that has been used to conserve the sperm of animal species and animal strains that are valuable for biomedical research. A simple method for preserving spermatozoa after application of intracytoplasmic sperm injection (ICSI) is much needed. It has been shown previously that spermatozoa frozen at 20°C can activate oocytes and support full-term embryo development. However, epigenetic reprogramming could be affected by the environment and by the in vitro manipulation of gametes. Here, we investigated embryo epigenetic reprogramming including DNA methylation and histone modification, in embryos derived from sperm preserved at 20°C without cryoprotectants. The results showed that although both fertilization and embryo developmental competence were decreased, the dynamic epigenetic reprogramming of embryos derived from frozen sperm was similar to the reprogramming of embryos derived from fresh sperm. The results reported in this study indicate that sperm frozen without cryoprotectant is epigenetically safe for ICSI. spermatozoa freezing, ICSI, epigenetic, histone modification, DNA methylation Citation:Chao S B, Li J C, Jin X J, et al. Epigenetic reprogramming of embryos derived from sperm frozen at 20°C.