Prairie vole (Microtus ochrogaster) is a highly social animal and is a commonly used animal model for neuropsychopharmacological and psychiatric studies. To date, only a few reports on the development of transgenic prairie voles which was primarily due to the suboptimal development of assisted reproductive technology (ART) in prairie voles. Limitations in ART further hinder the development of genetically modified prairie voles such as the application of conventional gene targeting technologies using embryonic stem (ES) or induced pluripotent stem (iPS) cells to generate chimeric prairie voles. Moreover, recent advancement in genome-editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas technology provide an unprecedented opportunity to create gene targeting animal model and the development of ART in prairie voles is necessary for future development of novel transgenic prairie vole model. We have established efficient method for in vitro embryo culture and sperm cryopreservation with high fertilization rate. In G-1 PLUS and G-2 PLUS sequential culture condition, 81.0% (# of Blastocysts/total n) of one-cell embryos developed to blastocysts. In contrary, no embryos were developed to blastocyst stage in KSOM medium (0/total # of embryos in culture). In vitro fertilization rate using fresh and frozen-thawed sperm was 32.6% and 29.3%, respectively. This is the first report of IVF using cryopreserved prairie vole sperm. We employed mouse IVF methods in prairie voles and optimize culture conditions using human G-1/G-2 PLUS sequential culture method that resulted in high embryonic development rate. The development in vole reproductive technology will facilitate the generation of transgenic voles in the future. (C) 2015 Elsevier Inc. All rights reserved.