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 conditions for in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes. In Experiment 1, groups of 10 to 12 cumulus-oocyte 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 + swim-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 of the oocytes in each drop were assigned randomly for maturation scoring and the remainder were inseminated. Reduced atmospheric O-2 drastically 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 total fertilization in 10 and 20% Oz were similar and considerably higher than in 5% Oz (80.3, 87.0 and 53.1%, respectively; P<0.05). In addition, the percentage of polyspermy markedly increased when IVF was conducted 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 treatments (64.9, 68.9 and 61.9%, respectively; P>0.05). Although the percentages of total fertilization among treatments were not different (75.4, 80.9 and 76.1%, respectively), the proportion of normal fertilization was 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 indicates 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.