Review: In vitro and ex vivo models advancing the study of host-microbiota interactions across organ systems in farm animals

In farm animals, the gut microbiota plays a pivotal role in early life development, immunity, resistance to infections, growth, and overall welfare. Emerging evidence also suggests that functions of non-digestive organs, such as the mammary gland, lungs, and reproductive tract, are also influenced by their microbiota. A deeper understanding of how the microbiota interacts with host cells is crucial to develop novel strategies for improving animal health and productivity. While correlative analyses between microbiota composition, metabolic activity, and animal phenotypes are common, they do not establish causality. Furthermore, ethical considerations and compliance with the 3Rs principles (Replacement, Reduction, Refinement) are increasingly encouraging the reduction of the use of animals for in vivo experimentations. In this context, in vitro and ex vivo models offer powerful, species-specific, and tissue-specific tools to investigate the causality of host-microbiota interactions in farm animals and underlying mechanisms. In this cross-organ review, we first focus on the gut to illustrate how in vitro (e.g., immortalised cell lines, organoids) and ex vivo (e.g., primary cell cultures, tissue explants) models from farm animals have been used to study the effects of microbiota-related treatments, including commensal bacteria, probiotics, bacterial metabolites, and extracellular vesicles, on intestinal and rumen cell functionality. Secondly, we examine the development of advanced in vitro and ex vivo models of non-digestive organs, focusing mainly on the mammary gland as an example of tissue for which existing models have the potential to elucidate interactions with the microbiota. Looking forward, we discuss the potential of organ-on-chip technologies, which more closely replicate physiological microenvironments through dynamic microfluidic systems. The implementation of such systems in farm animal species, particularly with microbiota co-culture, could significantly enhance the physiological relevance of in vitro studies. Finally, we explore the potential of multiorgan-on-chip platforms to model inter-organ microbial communication, such as the entero-mammary pathway or gut-lung axis, whose roles in the health of farm animals are increasingly recognised. We also emphasise the value of in vitro and ex vivo models for investigating microbiota-induced imprinting of host cells, which may pave the way for innovative strategies to program animal phenotypes for greater robustness and resilience.