Transcriptomic Profiles of Bovine Mammary Epithelial Cells Under Heat Stress and During Subsequent Recovery

Heat stress (HS) poses a significant challenge to the dairy industry as it negatively affects milk production. However, the molecular mechanism behind mammary gland responses to HS and recovery remains poorly understood. This study aimed to determine the transcriptomic profile and uncover the underlying mechanism using RNA-sequencing and functional analysis in bovine mammary epithelial (MAC-T) cells. MAC-T cells were cultured in 6 groups: 1) basal (undifferentiated, unDiff) medium or 2) lactogenic (differentiated, Diff) medium for four days. Differentiated cells were subject to either 3) non-HS (37?) or 4) HS (42?) for 1hr. Two HS groups were further allowed to recover at 37? for 2 hr (D2R) and 6 hr (D6R). Among six group comparisons, 1,668 differentially expressed genes (DEGs) were identified. HS vs. D6R groups yielded the highest number of DEGs, followed by HS vs. non-HS, and Diff vs. unDiff groups. Functional analysis suggested that differentiated cells exhibited upregulated genes associated with milk lipid synthesis, indicating their lactogenic state. Moreover, HS suppressed cellular differentiation and activated several heat shock protein (HSP) genes. Additionally, several transcription factors were identified as potential positive or negative regulators of HS response. During recovery, chaperon-mediated protein folding remained elevated and apoptosis regulation was induced after 2 hr. After 6 hrs, response to oxidative stress and cellular homeostasis regulation were enriched. Overall, differentiated MAC-T cells enriched genes in lipid synthesis activities. HS triggered heat shock response and protein misfolding while impaired cellular differentiation in differentiated MAC-T cells. Furthermore, MAC-T cells demonstrated dynamic recovery responses, including regulations on apoptosis and hypoxia Overall design: RNA-seq and functional analyses were used to detect the differentially expressed genes and pathways in MAC-T cells under differentiation, heat stress, and during recovery