Background:Heat stress is known to affect follicular dynamics,oocyte maturation,and fertilization by impairing steroidogenic ability and viability of bovine granulosa cell (bGCs).The present study explored the physiological and molecular response of bGCs to different heat stress intensities in-vitro.We exposed the primary bGCs to heat stress (HS) at 39 ℃,40 ℃ and 41 ℃ along with control samples (38 ℃) for 2 h.To evaluate the impact of heat stress on bGCs,several in vitro cellular parameters including cell apoptosis,intracellular reactive oxygen species (ROS) accumulation and HSP70 kinetics were assessed by flow cytometry,florescence microscopy and west blot,respectively.Furthermore,the ELISA was performed to confirm the 17β-estradiol (E2) and progesterone (P4) levels.In addition,the RNA sequencing (RNA-Seq) method was used to get the molecular based response of bGCs to different heat treatments.Results:Our findings revealed that the HS significantly decreased the cell viability,E2 and P4 levels in bGCs,whereas,increased the cellular apoptosis and ROS.Moreover,the RNA-Seq experiments showed that all the treatments (39 ℃,40 ℃ and 41 ℃) significantly regulated many differentially expressed genes (DEGs) i.e.BCL2L1,STAR,CYP11A1,CASP3,SOD2,HSPA13,and MAPK8/P1 and pathways associated with heat stress,apoptosis,steroidogenesis,and oxidative stress.Conclusively,our data demonstrated that the impact of 40 ℃ treatment was comparatively detrimental for cell viability,apoptosis and ROS accumulation.Notably,a similar trend of gene expression was reported by RT-qPCR for RNA-seq data.Conclusions:Our study presented a worthy strategy for the first time to characterize the cellular and transcriptomic adaptation of bGCs to heat stress (39,40 and 41 ℃) in-vitro.The results infer that these genes and pathways reported in present study could be useful candidates/indicators for heat stress research in dairy cattle.Moreover,the established model of bGCs to heat stress in the current study provides an appropriate platform to understand the mechanism of how heat-stressed bGCs can affect the quality of oocytes and developing embryo.