OPTIMUM GAS ATMOSPHERE FOR IN-VITRO MATURATION AND IN-VITRO FERTILIZATION OF BOVINE OOCYTES

The objective of this study was to determine optimal gas atmosphere co nditions for in vitro maturation (IVM) and in vitro fertilization (IVF ) of bovine oocytes. In Experiment 1, groups of 10 to 12 cumulus-oocyt e complexes (COCs) were matured (24 h) and fertilized (18 h) under 1) 5% CO2, 5% O-2; 2) 5% CO2, 10% O-2 or 3) 5% CO2 20% O-2. The COCs were cultured in 50 mu l drops of maturation medium (TCM-199 + 10% bovine calf serum + oLH, oFSH and estrogen) or fertilization medium (TALP + s wim-up separated spermatozoa +1 mu g/ml heparin sulfate) under a layer of 10 ml paraffin oil at 39 degrees C with saturated humidity. Half o f the oocytes in each drop were assigned randomly for maturation scori ng and the remainder were inseminated. Reduced atmospheric O-2 drastic ally decreased proportions of oocytes reaching MII (71.4, 26.9 and 9.3 % with 20, 10 and 5% O-2, respectively; P<0.05). The percentages of to tal fertilization in 10 and 20% Oz were similar and considerably highe r than in 5% Oz (80.3, 87.0 and 53.1%, respectively; P<0.05). In addit ion, the percentage of polyspermy markedly increased when IVF was cond ucted in reduced O-2 (26.6 and 28.8% in 5 and 10% O-2 vs 15.4% in 20% O-2; P<0.05). Experiment 2 was similar to Experiment 1 except that CO2 was the variable: 1) 2.5% CO2 in air, 2) 5% CO2 in air and 3) 10% CO2 in air. The proportion of MII oocytes did not differ across treatment s (64.9, 68.9 and 61.9%, respectively; P>0.05). Although the percentag es of total fertilization among treatments were not different (75.4, 8 0.9 and 76.1%, respectively), the proportion of normal fertilization w as significantly reduced in 10% CO2 (55.1%) when compared with that of either 2.5% CO2 (62.7%) or 5% CO2 (68.7%; P<0.05). This study indicat es that low O-2 is detrimental for IVM/IVF of bovine oocytes and that optimal atmospheric conditions are either 2.5 or 5% CO2 and 20% O-2.