150 The protective role of quercetin supplementation in bovine granulosa cells against thermal stress

Heat stress (HS) adversely affects the reproductive performance of cattle through impairments to the proper ovarian function needed to facilitate competent gametes for fertilization. Granulosa cells (GCs) provide a critical microenvironment mediating intrafollicular communication with the oocyte. Mammalian cells differentially respond to HS through various pathways, including through the activation of heat shock proteins (HSPs) and antioxidative responses like NRF2, a master regulator of the cellular response to environmental stress. Polyphenols like quercetin (3,3′4′,57-pentahydroxyflavone) have been found to induce cytoprotective effects through their reaction with free radicals, metal chelation, and its interactions with lipid membranes and proteins. The present study explores the role of quercetin during in vitro supplementation in bovine GCs to regulate the oxidative stress response under thermal stress conditions. For this, bovine GCs were cultured for 72 h, with the addition of quercetin after the first 24 h at various doses (0.2, 0.5, 0.7, 1, 5, 10, 20, 50, 100, and 200 μM), and control groups being a vehicle (DMSO) and a nontreated group. Heat exposure started after 48 h of culture and remained in place for 24 h, consisting of culture under basal (38.5°C) for one group and HS (42°C) conditions for the other group. Quercetin supplementation’s impact on granulosa cells’ viability was then evaluated using the Cell Counting Kit 8 CCK-8 (Dojindo Molecular Technology, Japan) in quadruples. Additionally, real-time quantitative reverse transcription PCR (qRT–PCR) was performed for selected stress-related genes NRF2, SOD1, HSP70, HSP90, GRP78, and GRP94. Data were analysed using a two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Contrary to the lower concentrations, the cell viability assay revealed levels of toxicity in concentrations equal to and higher than 20 μM quercetin. Supplementation in microdose amounts (0.2, 0.5, 0.7, and 1 μM) showed increases in cell viability under HS conditions compared with the control group under thermoneutral conditions, with an increase at 0.5 μM dose (P < 0.05). Additionally, qRT–PCR results showed that the expression pattern of NRF2 was enhanced in granulosa cells supplemented with 0.2, 1, and 5 μM quercetin under HS conditions, 1 μM being the highest but not statistically significant (>1.41; 1.72; 1.40-fold increase). Similarly, 1 μM quercetin showed the highest expression of SOD1 under normal and HS conditions compared with control groups (>1.71; 1.33-fold increase). GRP78 and GRP94 expression was significantly increased in all groups under HS versus normal temperature. However, HSP70 expression was increased in the control groups under HS compared with normal temperature and reduced in the 1 μM group under HS. In conclusion, results from this study validate that microdoses of quercetin supplementation play a protective role in mitigating the negative impact of HS on bovine granulosa cell function.