The objective of the current study was to investigate the effects of the addition of oviduct co-culture to synthetic oviduct fluid (SOF) and Menezo B2 media used in an in vitro study of cows, and the effects of different gas atmospheres (5% CO2 or 5% CO2, 5% O-2, and 90% N-2) on in vitro cultures. Oocytes obtained through aspiration from the ovaries of slaughtered Holstein cows were washed and cultured within a TCM-199 maturation medium at 38.8 degrees C for 23 h. Then the matured oocytes and thawed semen, prepared in accordance with the swim-up method, were incubated for fertilization within in vitro fertilization (IVF)-Tyrode's albumin lactate pyruvate (TALP) medium with an atmosphere including the combination of 5% CO2, 5% O-2, and 90% N-2 gas, at 38.8 degrees C for 18-24 h. Fertilized oocytes were distributed into 3 main groups: Group I: B2 (5% CO2), Group II: B2 medium (5% CO2, 5% O-2, and 90% N-2), and Group III: SOF (5% CO2, 5% O-2, and 90% N-2), and each group was divided into 2 subgroups, with and without oviduct co-culture cells, and were cultured for 9 days. The percentage of embryos that reached the blastocyst stage was 34.0%, 20.0%, and 32.3% in the co-culture group and 26.7% and 23.3% in the without co-culture group, respectively. Group III showed the highest development of expanded blastocyst stage (P < 0.01). In conclusion, the addition of co-culture to B2 (5% CO2 atmosphere) and SOF (5% CO2, 5% O-2, and 90% N-2 atmosphere) media increased the rate of transferable embryos.