155OXYGEN MEASUREMENTS AS INDICATORS OF CULTURE CONDITIONS AND VIABILITY OF CATTLE EMBRYOS

Abstract

Experiments were performed to study oxygen and temperature conditions experienced by embryos during routine in vitro production (IVP) (Exp. 1) and to evaluate oxygen consumption of single embryos as a viability indicator (Exp. 2). In both experiments, bovine IVP embryos were used (Holm et al. 1999, Theriogenology 52: 683–700), and a combined oxygen-temperature microsensor (tip diameter approx. 50μm) was used to attain oxygen partial pressure gradients and temperature profiles. In Exp. 1, 4-well dishes with in vitro culture (IVC) medium (400-μL or 50-μL droplets) covered with 400μL oil were taken from the incubator (5% CO2 , 5% O2 , 90% N) and measured under atmospheric air conditions at room temperature (24–25°C). The profiles were determined over 2min, starting 2min after the dishes were taken out of the incubator. The 400μL medium was measured from the top layer and gradually 3/4 down into the well (seven replicates). The oxygen partial pressure decreased from 21.0±0.66% O2 at the top layer to 15.0±1.6% O2 at the bottom layer, while the average temperature increased from 27.2±3.0°C to 31.7±0.7°C. In the 50-μL droplets, the profiles were obtained in the middle of the droplet (five replicates). The oxygen partial pressure was 17.1±2.44% O2 and the temperature 31.0±1.11°C. Consequently, routine handling of culture dishes outside the incubator seems to have a strong influence on both temperature and oxygen conditions. In Exp. 2, Day 3 and Day 7 embryos were evaluated morphologically before being loaded consecutively into one of the holes in a measuring block developed for this purpose. The block was previously covered with 40mL IVC medium before being incubated for 3 days under 5% CO2 , 95% N (38.5°C, 100% humidity). The oxygen consumption of each embryo was measured from 75±5min after removal from the incubator. Each measurement lasted approx. 5min, was repeated three times for each embryo, and was performed under atmospheric conditions on a warming stage held at 37±1°C. The average oxygen consumption by Day 3 embryos was 0.25±0.14nL/embryo/h (n=20), and no clear relation between respiration rate and embryo morphology could be demonstrated. For Day 7 embryos, the average oxygen consumption was 0.90±0.56nL/embryo/h (n=22), and this varied according to their morphological quality (1.17±0.70 (n=7), 0.98±0.49 (n=6) and 0.46±0.38 (n=9) nL/embryo/h for good, fair and poor quality embryos, respectively). In conclusion, this study illustrated the changes in O2 partial pressure that embryos can temporarily be exposed to under routine handling, even for rapid procedures such as cleavage inspection. The results also show that respiration rate is lower in Day 3 than in Day 7 embryos. Furthermore, oxygen consumption values of Day 7 embryos seem to be in agreement with the morphological embryo quality, supporting the idea that oxygen consumption can be a valuable parameter for the evaluation of embryo viability.