EVALUATION OF CULTURE SYSTEMS CONTAINING BOVINE OVIDUCT EPITHELIAL-CELLS OR GRANULOSA-CELLS TO MATURE AND MAINTAIN THE DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES IN-VITRO

The effects of estrous cow serum (ECS), bovine oviduct epithelial cell s (BOEC), and bovine granulosa cells (GC) on in vitro maturation (IVM) of immature oocyte-cumulus complexes (OCCs) were evaluated. Selected OCCs were cultured for 24 to 26 h in microdroplets of culture medium ( CM;TCM 199 + 25 mM HEPES + 100 pg gentamicin sulfate/ml) or in CM medi um supplemented or conditioned with 20% ECS, BOEC + 20% ECS or GC + 20 % ECS. Supplemented media were incubated for 2 h before addition of OC Cs, whereas media were conditioned by incubation with 20% ECS or BOEC +/- 20% ECS for 6 d, or with 20% ECS + GC for 24 or 48 h before additi on of OCCs. The developmental competence of oocytes after IVM was asse ssed by insemination with glass wool separated, frozen-thawed bovine s permatozoa in microdroplets of modified medium (TALP) containing hepar in (5 mu g/ml) and BOEC for 18 h. The presumptive zygotes were culture d in microdroplets of CM medium + 20% ECS + BOEC for 7 to 9 d to asses s embryo development to morula and blastocyst stages. The percentages of OCCs undergoing IVM (85 to 94%) and in vitro fertilization (IVF) (6 6 to 80%) were high, irrespective of the IVM conditions. Only after th e IVM of OCCs in CM medium alone was the percentage of oocytes undergo ing IVF significantly lower (66%; P<0.05). The proportion of IVF oocyt es developing to blastocysts with a normal complement of cells (126 to 138) increased significantly (P<0.05) when the OCCs were matured in s upplemented or conditioned CM medium containing ECS and/or somatic cel ls (18 to 28%) compared with those in CM medium alone (9%). When the C M medium was supplemented or conditioned with GC + 20% ECS, the propor tion of fertilized oocytes developing to blastocysts increased signifi cantly (28%; P<0.05). These results indicate that the potential of imm ature OCCs to be fertilized and to complete embryonic development to t he blastocyst stage in vitro is enhanced by maturation in CM medium co ntaining 20% ECS and/or BOEC or GC.