Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin-specific monoallelic expression. Follicle culture under oil overlay has been associated with altered imprinting establishment in mouse oocytes. We previously demonstrated normal imprinting establishment at four key imprinted genes in mouse oocytes grown and matured in a long-term in vitro follicle culture system without oil overlay. Ammonium (300 μM) has been linked to aberrant imprinting in in vitro preimplantation embryo culture. Compared to culture without oil, mineral oil overlay during follicle culture led to a dramatic increase in ammonia levels in culture medium: mean ammonia levels were, respectively, 39 and 290 μM at Day 4 of culture, 73 and 465 μM at Day 8, and 101 and 725 μM at Day 12 (P < 0.0001). Mineral oil overlay and high ammonia levels (comparable to the follicle culture system for which aberrant imprinting was previously described) during follicle culture did not affect follicle survival, metaphase II (MII) rate, or MII oocyte diameter. Bisulphite sequencing revealed that high levels of ammonia and mineral oil overlay during follicle culture did not alter the methylation status of differentially methylated regions of three key imprinted genes (Snrpn, Igf2r, and H19) in MII oocytes. In the current culture setup, ammonium accumulation and mineral oil overlay during follicle culture do not induce aberrant imprinting establishment at the studied regulatory sequences in mouse oocytes.