Amino acids (AAs) are essential for the growth, development, and health of pigs, yet their direct effects on the intestinal barrier and microbiota remain poorly understood in vivo, despite extensive in vitro studies on mucosal barrier integrity. While intestinal cell lines and organoids provide valuable insights, they cannot fully capture the complexity of the in vivo intestinal mucosa, where diverse cell types interact dynamically with each other and the microbiota. To address this gap, we hypo