311 EFFECT OF HEAT SHOCK DURING THE FERTILIZATION AND CULTURE PERIOD ON BOVINE EMBRYO DEVELOPMENT AND THE PROTECTIVE EFFECT OF BROWN ALGAE PHLOROTANNINS

Abstract

It is widely reported that heat stress adversely affects the reproductive function of cattle, such as ovarian functions, fertilization, and embryo development. In a previous study, we reported that heat shock decreases embryo development and increases intracellular reactive oxygen species (ROS). Also some antioxidants increase embryo development under conditions of heat shock by reducing the intracellular ROS. Phlorotannins extracted from brown alga are known as a strong antioxidant. However, heat shock and the antioxidative effect of phlorotannins on fertilization and embryo development has not been carefully studied. In the present study, we investigated the effect of heat shock on fertilization and early embryo development, and the protective effect of phlorotannins on embryo development under conditions of heat shock. In all experiments, bovine oocytes were collected from the local abattoir and matured with TCM-199 (Experiment 1). Bovine sperm drops prepared by BO solution were pretreated at 41°C for 4 h with or without 100 ng mL^-1 of phlorotannins. After heat shock, oocytes were fertilized in drops at 38.5°C for 6 h. Putative zygotes were cultured with CR1 + 5% FCS at 38.5°C. The percentages of embryos cleaved and developed to blastocysts were evaluated on Days 2 and 8. The percentages of embryo division and development were compared with embryos fertilized with sperm pretreated at 38.5°C for 4 h (Experiment 2). Oocytes were fertilized at 41°C for 6 h with or without 100 ng mL^-1 of phlorotannins. Putative zygotes were cultured with CR1 + 5% FCS at 38.5°C. On Days 2 and 8, the percentages of cleaved embryos and those developed to blastocysts were evaluated and compared with embryos fertilized at 38.5°C for 6 h (Experiment 3). Oocytes were fertilized at 38.5°C for 6 h. Putative zygotes were cultured with or without 10 ng mL^-1 of phlorotannins in CR1 + 5% FCS. On Day 2, embryos were exposed to 41°C for 6 h as heat shock. After heat shock, embryos were cultured at 38.5°C to Day 8, and embryo development was evaluated. The percentages of embryo development were compared with those for embryos cultured at 38.5°C through to Day 8 without phlorotannins. Mean values were compared by Student's t-test. There were no significant differences in the percentages of embryo cleavage among all experiments. The percentages of embryo development were significantly (P < 0.05) decreased by heat shock in all experiments [Experiment 1: 45.0 vs. 29.2%; Experiment 2: 25.1 vs. 6.6%; Experiment 3: 28.6 vs. 15.3% (control vs. heat shock)]. In contrast, the addition of phlorotannins to the fertilization or culture media tended to improve the embryo development (Experiment 1: 41.9%; Experiment 2: 15.1%; Experiment 3: 22.2%). These results indicate that heat shock affects not only embryo development but also fertilization. And under conditions of heat shock, the addition of phlorotannins would be effective in improving embryo development from fertilization to development.