141 Antioxidant approach to mitigate the impact of thermal stress on bovine granulosa cell function

The detrimental impacts of heat stress (HS)-induced oxidative stress on dairy cattle fertility have been extensively studied. HS disrupts the proper redox homeostasis in the ovary, which is crucial for producing the viable gametes necessary for fertilization and successful pregnancy establishment. Phytochemicals acting as antioxidants have been reported to mitigate HS-induced oxidative stress, but knowledge about the protective mechanisms on follicular cells is limited. Oocyte maturation relies on bidirectional communication amid intrafollicular granulosa cells (GCs), events that are vital for embryonic developmental competence. In the face of HS, GCs employ various protective mechanisms, such as activating heat shock proteins (HSPs) and glucose-regulated proteins (GRPs). Most importantly, the Nrf2/Keap1/ARE pathway, a key antioxidant response system, is triggered to reduce reactive oxygen species (ROS) accumulation. Nevertheless, this pathway becomes ineffective once the cellular antioxidant capacity is overwhelmed. Phytochemicals such as quercetin (Que), carnosol (Car), and sulforaphane (SFN) have been reported to activate the Nrf2 pathway, improving antioxidant capacity. Therefore, we hypothesized that phytochemicals can reduce cellular damage caused by HS in bovine GCs. We aimed to investigate the impact of Que, Car, and SFN and their combinatorial effects on bovine GCs under HS conditions. Initially, bovine GCs aspirated from small follicles (3–8 mm) were cultured until subconfluency, followed by supplementation with 5 µM Que, 10 µM Car, 1 µM SFN, and a mix. The GCs were then cultured, either under normal temperature (NT; 38.5°C) for 24 h, or under HS (42°C) for 8 h, followed by 38.5°C for 16 h. A vehicle (DMSO) and a nontreated group were used as controls. Data were analyzed using a two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons tests. An ROS accumulation assay was performed using 2ʹ,7ʹ-DCFH-DA dye, and ROS was found to be reduced in every antioxidant-treated group compared with the control group (P < 0.05) under HS. Moreover, a mitochondrial membrane potential (MMP) assay using JC-1 staining revealed increased MMP in every antioxidant-treated group under HS conditions compared with the control group (P < 0.05). Moreover, qRT-PCR was performed to quantify expression of selected stress-related genes Nrf2, SOD1, HO1, CAT, PRDX1, HSP70, HSP90, GRP78, and GRP94. Results showed that the expression pattern of Nrf2 was significantly enhanced in all antioxidant-supplemented groups under HS conditions (>1.71–14.5-fold increase). Similarly, the expression pattern of genes downstream of Nrf2, namely, SOD1, HO1, CAT, and PRDX1, was increased in all antioxidant-treated groups under HS (>1–50-fold increase). No significant difference existed between treatment and control under the HS condition on GRP78, GRP94, HSP70, and HSP90 expression. Further experiments will investigate the role of antioxidant supplementation during oocyte maturation under HS conditions.