80 REACTIVE OXYGEN SPECIES LEVEL IN PIG EMBRYOS CULTURED IN PRESENCE OF HYALURONAN

Abstract

It has been reported that during in vitro embryo culture reactive oxygen species (ROS) are generated and are detrimental to embryo development. A recent study (Smorag et al., Proc. 9th ICPR, 2013, 110) demonstrated that an addition of 1 mg mL^–1 of hyaluronan (HA) to porcine embryo culture medium improves the development of zygote to blastocyst stage and the quality of produced embryos. Moreover, the embryos cultured with HA showed lower inner mitochondrial membrane potential (Romek et al., 2015 Proc. Symp. Progress in Cell Biology: Mitochondria and Chloroplast, Krakow, 31). Based on the beneficial effect provided by supplementation of HA during embryo culture, we investigated the ROS level in porcine embryos cultured with HA. Porcine zygotes were obtained surgically after flushing the oviducts of superovulated and inseminated gilts. In the experimental group, zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 1 mg mL^–1 of HA (CROMA, Pharma GmbH, Leobendorf, Austria), in an atmosphere containing 5% CO₂ in air, at 39°C. In the control group, HA supplementation was omitted. To measure ROS level, embryos at the stages 2–4 and 8–16 cell, morula, and blastocyst (experimental group) and zygote, 2–4 and 8–16 cell, morula, and blastocyst (control group) were labelled with 5 μM CM-H₂DCFDA dye (Molecular Probes Inc., OR, USA) for 30 min at 39°C. Labelled embryos were then examined under a Nikon Eclipse microscope with a CCD camera. The total amount of fluorescence emitted from each individual embryos and proportional to the ROS level was measured in arbitrary units. The data were analysed using one-way ANOVA and post-hoc Tukey test. ROS level (mean ± standard error of the mean) in the experimental group was 8.21 ± 2.65 (n = 25), 10.31 ± 3.13 (n = 18), 9.08 ± 2.89 (n = 21), and 20.45 ± 2.38 (n = 31) for 2–4 cell, 8–16 cell, morula, and blastocyst, respectively, whereas in the control group was 9.15 ± 3.43 (n = 15), 7.11 ± 3.13 (n = 18), 8.67 ± 3.04 (n = 19), 11.47 ± 2.46 (n = 29), and 54.74 ± 2.89 (n = 21) for zygote, 2–4 cell, 8–16 cell, morula, and blastocyst, respectively. For experimental and control groups, ROS levels remained unchanged up to morula. On the contrary, at the blastocyst stage from the experimental group ROS level decreased significantly (P ≤ 0.05) in comparison with blastocysts from the control group. In conclusion, porcine blastocysts derived from zygotes cultured with supplementation of 1 mg of HA possess a significantly lower ROS level than blastocysts cultured without HA. This suggests that HA supplementation in culture medium can reduce the ROS level in porcine cultured blastocysts.

The project was funded by the National Science Center based on decision number DEC-2012/07/B/NZ9/01326.