Packing density has a significant influence on the outcome of ensiling forage. In this study, we aim to investigate the effect of packing density on the ensiling properties, microbiome, and in vitro digestibility of barley silages. Barley was ensiled in polyethylene drum silos (30 L) with respective densities of 600, 650, 700, and 750 kg/m
3 fresh matter (FM), and stored for 60 days. The bacterial communities, fermentation quality, and in vitro digestibility were analyzed. Fresh barley had a low count of lactic acid bacteria (LAB, 10
4 cfu/g of FM), and
Lactobacillus was nearly undetectable (<1%). Increasing the packing density decreased the pH and the content of ammonia nitrogen (NH
3-N), ethanol, neutral detergent fiber (NDF), and acid detergent fiber (ADF) of barley silage (
p < 0.05), and increased in vitro digestibility of dry matter, NDF, ADF, and DM recovery (
p < 0.05). A higher packing density decreased the abundances of
Enterobacter (from 47.4% to 35.4%) and
Clostridium (from 13.5% to 3.8%), and increased the abundance of
Lactobacillus (from 1.8% to 17.0%). Thus, packing density positively correlated with
Lactobacillus (
p < 0.05) but negatively correlated with
Enterobacter (
p < 0.05). The pH and the content of ethanol were positively correlated with
Enterobacter (
p < 0.05) but negatively correlated with
Lactobacillus (
p < 0.05). In conclusion, the density of 750 kg/m
3 FM resulted in the highest silage quality of the densities tested.
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