Oocyte cryopreservation remains a challenge in most mammalian species because of their sensitivities to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw viability and pre-implantation embryo development. The aim of this study was to investigate the effect of cytochalasin B (CB) pre-treatment before vitrification on viability, frequencies of in vitro fertilisation (IVF) and subsequent development of ovine cumulus-oocyte complexes (COCs) vitrified at the germinal vesicle (GV) stage using cryoloop. COCs obtained at slaughter were randomly divided into two groups and incubated with or without 7.5µg/mL CB for 60 min. Oocytes from each group were then vitrified using a cryoloop or used as toxicity and controls. Oocytes were then matured, fertilised, and cultured in vitro for 7 days. Viability following vitrifiaction and warming, fertilisation events following IVF and subsequent pre-implantation embryo development were evaluated. No significant differences were observed in survival rates between CB treated and non-treated oocytes in both vitrified and toxicity groups. Frequencies of fertilisation were increased in CB-vitrified group (oocytes pre-treated with CB before vitrification) than those vitrified without CB pre-treatment (57.0% vs 40.7%). Cleavage was significantly lower (P < 0.05) in vitrified and CB-vitrified oocytes at both 24 hpi (12.5% vs 9.1%) and 48 hpi (25.0% vs 16.2%) than in other groups. Based on the numbers of cleaved oocytes, (48 hpi), 16.1% and 18.8% of the cleaved embryos developed to blastocysts in both vitrified and CB-vitrified groups. These values did not differ significantly from those obtained in CB-control group (37.8%). No significant differences were observed in mean cell numbers per blastocyst between all groups. In conclusion, pre-treatment of ovine GV oocytes with cytochalasin B as cytoskeleton stabilizer before vitrification increased frequencies of in vitro fertilisation and subsequently resulted in production of good quality late stage pre-implantation embryos following IVF.