79 REACTIVE OXYGEN SPECIES LEVEL IN CULTURED PORCINE EMBRYOS AFTER HIGH HYDROSTATIC PRESSURE

Abstract

Our recent study (Romek et al., Proc. of 29th Scientific Meeting of A.E.T.E., 2013, 196) demonstrated that high hydrostatic pressure (HHP) decreased the potential of the inner mitochondrial membrane in porcine embryos from morula to blastocyst stage. Therefore, the aim of this study was to find out if HHP treatment of cultured porcine embryos has an effect on production of reactive oxygen species (ROS) in these cells. Gilts were superovulated and inseminated using standard methods. Then zygotes were surgically collected after flushing the oviducts of the donors gilts 22 to 24 h after insemination. Obtained zygotes were cultured in NCSU-23 (North Carolina State University-23) medium up to the blastocyst stage, in an atmosphere containing 5% CO₂ in air at 39°C. In the experimental group, embryos at zygote, 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages were treated with 20 MPa of hydrostatic pressure (HHP100, Cryo-Innovation Ltd., Szeged, Hungary) for 60 min at 39°C with an interval of 60 min between HHP treatment and subsequent embryo staining. For the control group of embryos at the same stage of development, the HHP treatment was omitted. An additional group of blastocysts derived after culture was analysed 4 h after the HHP treatment. ROS level was measured using 5 μM CM-H₂DCFDA fluorescent dye (Molecular Probes Inc., Eugene, OR, USA). Embryos from the experimental and control groups were stained for 30 min at 39°C and then analysed under a Nikon Eclipse microscope equipped with a CCD camera. The total amount of fluorescence emitted from each individual embryo was measured. The data (in arbitrary unit) were analysed using one-way ANOVA and post-hoc Tukey test. After HHP zygote treatment, the percentage of obtained blastocysts was 67.01, whereas in control group it was 63.95 (P < 0.05). ROS level proportional to the measured amount of fluorescence (mean ± standard error of the mean) was 9.15 ± 2.70 (n = 15), 7.11 ± 2.46 (n = 18), 8.67 ± 2.4 (n = 19), 11.47 ± 1.94 (n = 29), and 54.74 ± 2.28 (n = 21) for zygote, 2- to 4-cell, 8- to 16-cell, morula, and blastocyst stage of the control group, respectively. After HHP treatment, the ROS level was 7.39 ± 2.4 (n = 19), 6.66 ± 2.28 (n = 21), 9.14 ± 2.61 (n = 16), 7.23 ± 2.28 (n = 21), 33.06 ± 2.4 (n = 19) for zygote, 2- to 4-cell, 8- to 16-cell, morula, and blastocyst stage, respectively, and 35.57 ± 2.4 (n = 10) for blastocyst 4 h after HHP treatment. In conclusion, (1) HHP treatment of porcine zygotes improve embryo developmental potential; (2) ROS level in both control and experimental groups remained unchanged up to morula stage, whereas at the blastocyst stage, after HHP treatment ROS level significantly decreased (P < 0.05) in comparison with nontreated blastocysts; (3) HHP treatment on porcine blastocysts resulted in a lowered level of ROS that remained unchanged for 4 h. These results suggest that HHP treatment could improve the quality of cultured porcine blastocysts.

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