177 ANTHOCYANIN ISOLATED FROM PURPLE SWEET POTATO IMPROVES DEVELOPMENT AND REDUCES OXIDATIVE STRESS OF BOVINE PRE-IMPLANTATION EMBRYOS EXPOSED TO HEAT SHOCK

Abstract

Development of cleavage-stage pre-implantation embryos is disrupted by exposure to heat shock. Heat shock also increases intracellular reactive oxygen species (ROS) in pre-implantation embryos. Therefore, reduction of intracellular ROS levels might improve the development of heat-shocked embryos. Recently the antioxidative activities of polyphenols have been widely reported to reduce the oxidative stress. In this study, we investigated the effect of purple sweet potato anthocyanin, a kind of polyphenol that is a strong ROS scavenger, on development and intracellular redox status of bovine pre-implantation embryos exposed to heat shock. Experiment 1: In vitro-produced 8-16-cell-stage embryos on Day 2 after fertilization were exposed to 41.5°C for 6 h in CR1aa containing 0, 0.1, 1, and 10 ¼g/mL anthocyanin at 5% CO₂, 5% O₂, and 90% N₂. After heat shock, embryos were cultured at 38.5°C at 5% CO₂, 5% O₂ until Day 8. On Day 8, the proportion of embryos developing to the blastocyst stage was evaluated. Blastocyst total cell number and the ratio between inner cell mass and tropheoderm were evaluated by differential staining. The experiment was replicated five times with more than 70 embryos used in each treatment. Experiment 2: Heat shock treatment of in vitro-produced 8-16-cell-stage embryos was carried out as described in experiment 1. After heat shock, intracellular ROS and glutathione (GSH) levels were measured in individual 8-16 cell stage embryos with fluorescent probes (22,72-dichlorodihydrofluorescein diacetate for ROS and CellTracker" Blue (Invitrogen Japan K. K., Tokyo, Japan) for GSH). The fluorescence emissions of each treatment were normalized to those of 8-16 cell stage embryos cultured at 38.5°C without anthocyanin to obtain the relative fluorescence emission. This experiment was replicated four times. Embryos treated with heat stress without anthocyanin (0 ¼g/mL) showed low development (14.6 ± 3.6%) and blastocyst total cell number (88.2 ± 9.4). However, embryos treated with 0.1 ¼g/mL anthocyanin improved development (31.7 ± 4.5%, P < 0.05) and increased the total cell number (96.5 ± 11.3). The higher concentrations of anthocyanin (1 and 10 ¼g/mL) did not affect development and cell number. The intracellular ROS levels in heat-shocked embryos were significantly reduced by all concentrations of anthocyanin (P < 0.05). In addition, anthocyanin increased GSH levels at all doses tested (P < 0.05). These results indicate that an appropriate concentration of anthocyanin improves development by regulating intracellular redox balance in bovine embryos exposed to heat shock.