Lipopolysaccharide (LPS) is one of the main factors inducing endometritis in dairy cows. However, the specific pathogenesis of LPS-induced endometritis in dairy cows is not fully understood. The objective of this study was to establish an in vitro endometritis model using LPS-induced bovine endometrial epithelial (BEND) cells. BEND cells were treated with LPS of different concentrations and times. The cell-counting kit-8 (CCK-8) was used to detect the cell survival rate after LPS treatment, and quantitative real-time PCR (RT-qPCR) was used to detect the expression of control group and LPS-treated group of inflammatory factors interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). The results showed that the survival rate of endometrial epithelial cells stimulated by 5 μg/mL LPS for 6 h was 75.13%, and the expression of inflammatory factors was significantly increased. Therefore, 5 μg/mL LPS for 6 h could be selected as a suitable model for the study of inflammation. In addition, miRNA sequencing and target gene prediction was performed on normal and LPS-treated BEND cells. Among twenty-one differentially expressed miRNAs, six miRNAs were selected and their expression levels were detected by RT-qPCR, which were consistent with the sequencing results. Twenty-one differentially expressed miRNAs collectively predicted 17,050 target genes. This study provides a theoretical basis for further investigation of the pathogenesis of endometritis.

Heat stress is a critical environmental factor that compromises reproductive performance in dairy cattle, primarily through deleterious effects on ovarian follicular cells. Granulosa cells (GCs), which provide essential nutritional and hormonal support for oocyte maturation, are particularly vulnerable to hyperthermic insult. However, the early molecular and subcellular events that precede overt cellular dysfunction remain inadequately characterized. This study aimed to investigate the temporal dynamics of mitochondrial morphological alterations and transcriptomic reprogramming in bovine GCs subjected to acute in vitro heat stress. Bovine GCs were exposed to 43 °C for 10, 20, 30, or 40 min, with untreated cells serving as controls. Heat shock protein expression (HSP90 and HSPA1A) was evaluated by qRT-PCR. Intracellular calcium concentration was measured using the Fluo-3 AM fluorescent probe. Mitochondrial ultrastructure was examined via transmission electron microscopy (TEM). Global transcriptional changes were profiled by RNA sequencing (RNA-seq) at 20 and 40 min post-treatment, followed by bioinformatics analysis. Nine selected differentially expressed genes (DEGs) were validated by qRT-PCR. HSP90 and HSPA1A expression was significantly upregulated at 30 and 40 min of heat exposure (p < 0.05). Intracellular Ca²⁺ concentration exhibited a biphasic pattern—markedly elevated at 20 min but significantly reduced at 40 min. TEM revealed progressive mitochondrial swelling and cristae disruption at both time points, with more severe damage at 40 min. RNA-seq identified 185 DEGs at 20 min and 831 DEGs at 40 min, with 285 genes commonly dysregulated across both time points. Functional enrichment analysis demonstrated that the 20-min response was dominated by heat shock protein-related pathways, whereas the 40-min response expanded to encompass apoptosis, inflammation, steroidogenesis, and ciliogenesis pathways. qRT-PCR validation confirmed the reliability of the RNA-seq data. These findings provide a mechanistic framework for understanding heat stress-induced ovarian dysfunction and may inform strategies to preserve fertility in dairy cattle during thermal challenge.