In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers.

The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO(2), 5% O(2,) and 90% N(2). All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 μg/mL of estradiol-17β, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 μM of β-mercaptoethanol, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P < 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In cumulus-free oocytes collected from ovaries at estrus and/or diestrus, the proportions of M-II oocytes co-cultured with the CGML were low (3.0%) and similar (P > 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P < 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P < 0.05), and least oocyte degeneration (P < 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes.