PS5-3. In Vitro Digestibility of Sorghum Sudangrass Baleage and Dried Distillers Grains Using Rumen Inoculum from Cattle.

Abstract Maintaining livestock productivity is critical for food security, yet production systems are increasingly challenged by environmental and biological factors. Infectious diseases can compromise both animal health and reproductive performance. Uterine epithelial cells line the uterine lumen and form a specialized mucosal immune barrier that balances reproductive processes, including maternal recognition of pregnancy, with protection against pathogens. However, antiviral innate immune responses in bovine uterine epithelial cells remain poorly characterized. This study aimed to establish a novel serum-free bovine uterine epithelial cell culture system that provides a physiologically relevant in vitro model for studying pathogen-epithelial interactions. Using this model, we characterized epithelial innate immune responses to viral stimuli and explored potential mechanisms by which highly pathogenic avian influenza (HPAI) H5N1 virus may influence reproductive tract immunity. To mimic viral double-stranded RNA, cells were exposed to polyinosinic:polycytidylic acid [poly(I:C)], a synthetic ligand recognized primarily by TLR3. Poly(I:C) exposure resulted in activation of canonical antiviral signaling pathways, including phosphorylation of downstream signaling intermediates associated with TLR3 activation. This response was accompanied by robust induction of interferon-stimulated genes (ISGs), consistent with activation of type I interferon signaling through the interferon alpha/beta receptor (IFNAR). To further characterize interferon responsiveness independently of viral mimic stimulation, epithelial cells were treated with multiple interferon subtypes, including IFN-α, IFN-β, IFN-λ, and IFN-ω. These treatments confirmed that uterine epithelial cells are competent to mount interferon-dependent antiviral responses, although differences in responsiveness among interferon classes were observed. We additionally assessed the potential susceptibility of uterine epithelial cells to HPAI H5N1 virus. Lectin-based immunofluorescence staining demonstrated the presence of cellular glycoprotein receptors associated with HPAI H5N1 viral entry on uterine epithelial cells. Using a lentiviral approach, we further demonstrated that HPAI H5N1 viral envelope proteins and glycoprotein receptors can mediate binding and entry into these cells, suggesting that uterine epithelium could represent appropriate physiological conditions. Ongoing work is focused on determining how specific HPAI H5N1 viral proteins modulate innate immune signaling pathways in uterine epithelial cells and whether such interactions influence reproductive pathways mediated by maternal recognition of pregnancy signals. Collectively, these findings establish a tractable epithelial cell model for studying antiviral innate immunity in the reproductive tract and provide a framework for investigating how emerging viral pathogens may influence reproductive health in livestock. For image description, please refer to the figure legend and surrounding text.