In order to mimic the maternal oviductal environment, we evaluated the effect of oviductal fluid (OF) and/or uterine fluid (UF) supplementation on in vitro embryo development and quality. In vitro-produced zygotes were cultured with 1.25% OF from Day 1 to Day 4 after insemination (OF group), 1.25% OF from Day 1 to Day 4 followed by 1.25% UF from Day 4 to Day 9 (OF+UF group) or 1.25% UF only from Day 4 to Day 9 (UF group). Control groups were cultured in the presence of synthetic oviduct fluid (SOF) supplemented with 3mgmL-1 bovine serum albumin (BSA) or 5% fetal calf serum (FCS). Supplementation of the culture medium with OF and/or UF (both at 1.25%) supported embryo development (Day 9 blastocyst rate 28.2-30.6%). At 72h after vitrification-warming, the survival of blastocysts from the OF and OF+UF groups was similar to that of blastocysts in the SOF+BSA group (61.0±5.7% and 62.8±6.4% vs 64.8±6.4% respectively), but significantly higher than that of blastocysts from the SOF+FCS group (31.6±4.9%; P<0.001). Blastocysts from the OF group exhibited upregulation of epigenetic genes (i.e. DNA methyltransferase 3α (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R)), compared with expression in the SOF+FCS group (P<0.05). Whereas those from OF+UF and UF groups exhibited downregulation of oxidative stress genes compared to SOF+BSA and OF groups for glutathione peroxidase (GPX1) and to SOF+FCS, SOF+BSA and OF groups for chloride intracellular channel 1 (CLIC1) (P<0.05). In addition, accumulation of reactive oxygen species was lower in blastocysts from the OF, OF+UF and UF groups. In conclusion, the use of low concentrations of OF and UF in in vitro serum-free culture supports embryo development, with OF providing a better control of embryo methylation, whereas UF may have antioxidant activity.