Effects of Licorice Stem and Leaf Semi-Dry Silage Instead of Alfalfa Hay on In Vitro Rumen Fermentation Characteristics and Dynamic Changes of Rumen Microbial Community in Holstein Cows

This experiment aimed to investigate the effects of replacing alfalfa hay with Glycyrrhiza stem and leaf silage (moisture content: 45%) on rumen in vitro fermentation parameters, nutrient digestibility, and dynamic changes of microbial community composition. In vitro fermentation was conducted with 0% (control group G0A100), 50% (G50A50), and 100% (G100A0) alfalfa hay replaced by semi-dry silage of Glycyrrhiza stems and leaves with 45% moisture content for 72 h. Cumulative gas production (GP), fermentation parameters, microbial community composition at different time points, and post-fermentation nutrient digestibility were determined, with comprehensive evaluation by principal component analysis (PCA) and gray relational analysis (GRA). Results showed that GP of G50A50 and G100A0 was significantly higher than G0A100 at 3 h (<i>p</i> < 0.05), and that of G50A50 was significantly higher than the other two groups at 24 h (<i>p</i> < 0.05). pH of G50A50 was significantly lower than the other two groups at 3 h (<i>p</i> < 0.05). In vitro dry matter digestibility (IVDMD) at 24 h and 72 h, in vitro neutral detergent fiber digestibility (IVNDFD) at 12 h, and in vitro acid detergent fiber digestibility (IVADFD) at 12, 24 and 72 h of G0A100 and G50A50 were significantly higher than G100A0 (<i>p</i> < 0.05). PCA comprehensive scores ranked as G0A100 (0.170) > G50A50 (0.141) > G100A0 (-0.311). GRA comprehensive scores ranked as G50A50 (0.792) > G0A100 (0.756) > G100A0 (0.681). LEfSe analysis indicated distinct microbial biomarkers at 72 h, and KEGG functional profiles were highly consistent among groups. Under the experimental conditions, 50% Glycyrrhiza stem and leaf silage is recommended to replace alfalfa hay in dairy cow diets.