194 ANTIOXIDATIVE EFFECT OF BROWN ALGAE PHLOROTANNINS ON REACTIVE OXYGEN SPECIES GENERATION AND EMBRYO DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS UNDER OXIDATIVE AND HEAT-STRESSED CONDITIONS

We investigated the antioxidative effect of brown algae phlorotannins on reactive oxygen species (ROS) generation and embryo development of parthenogenetically activated porcine embryos under oxidative and heat-stressed conditions. Cumulus–oocyte complexes (COCs) were aspirated from follicles on the surface of porcine ovaries collected from an abbattoir. COCs were matured in NCSU-23 containing 10% (v/v) porcine follicular fluid and hCG during the first 22 h, followed by an extra 22 h of culture in hormone-free NCSU-23. After 44 h of maturation, oocytes were denuded of cumulus cells and used for parthenogenetic activation. Oocytes were activated by single 100-�s pulse of 1.5 kV cm-1 DC in 1-mm electrodes. Activated oocytes were cultured for 5 h in NCSU-23 containing BSA, EGF, and 5 �g mL-1 cytochalasin B. Embryos were then cultured for 7 days in PZM-5 medium that was a slightly modified version of the PZM-4 medium reported by Yoshioka et al. (2002 Biol. Reprod. 60, 112–119). In Experiment 1, after parthenogenetic activation, embryos were cultured for 7 days at 38.5�C under 5% O2, 5% CO 2, and 90% N2 (defined as 5% O2) as a control. Embryos were also cultured under 5% CO2 in air (defined as 20% O2) with or without 100 ng mL-1 brown algae phlorotannins extracted from Ecklonia kurome. The number of embryos developed to the blastocyst stage was observed on Day 6. The total cell number of Day 7 blastocysts was counted by DAPI staining of nuclei. On Day 2, intracellular ROS levels of individual embryos were measured with fluorescent dyes (222,722-dichlorodihydrofluorescein diacetate). In Experiment 2, on Day 1 or 2, embryos cultured in 5% O2 concentration at 38.5�C were exposed to 41.5�C for 6 h with or without 100 ng mL-1 phlorotannins and cultured at 38.5�C until Day 7. After 6 h of heat-shock on Day 1 or Day 2, intracellular ROS levels were measured as described in Experiment 1. Statistical analysis was carried out by ANOVA. In Experiment 1, the rate of blastocyst formation and the total cell number were significantly decreased (P < 0.05) when embryos were cultured under 20% O2 compared to 5% O2. In contrast, addition of phlorotannins significantly increased the rate of blastocyst formation under high O2 concentration. ROS levels were also significantly increased by higher O2 concentration. In contrast, addition of phlorotannins significantly reduced the ROS levels. In Experiment 2, heat-shock to embryos on Days 1 and 2 significantly (P < 0.05) decreased the rate of blastocyst formation compared to the control. In contrast, addition of phlorotannins significantly (P < 0.05) increased embryo development and decreased the intracellular ROS levels of heat-stressed embryos. These results indicate that oxidative and heat stress conditions decrease embryo development and increase the level of intracellular ROS. However, addition of phlorotannins promotes embryo development by decreasing the oxidative stress.