Supplying sufficient nutrients, such as dietary energy and protein, has a great effect on the growth and rumen development of ruminants. This study was conducted to evaluate the effects of dietary energy and protein levels on growth performance, microbial diversity, and structural and physiological properties of the rumen in weaned lambs. A total of 64 two-month-old Hu lambs were randomly allotted to 2 × 2 factorial arrangements with four replicates and with four lambs (half male and half female) in each replicate. The first factor was two levels of dietary metabolizable energy (ME) density (ME = 10.9 MJ/Kg or 8.6 MJ/Kg), and the second factor was two levels of dietary crude protein (CP) content (CP = 15.7% or 11.8%). The trial lasted for 60 days. A low dietary energy level restrained the growth performance of lambs (p < 0.05). The ruminal concentration of acetate and the ratio of acetate to propionate increased but the propionate concentration decreased significantly with the low energy diet. However, the rumen morphology was not affected by the diet energy and protein levels. Moreover, a low energy diet increased ruminal bacterial diversity but reduced the abundance of the phylum Proteobacteria (p < 0.05) and genus Succinivibrionaceae_uncultured (p < 0.05), which was associated with the change in ruminal fermentation phenotypes. By indicator species analysis, we found three indicator OTUs in the high energy group (Succinivibrionaceae_uncultured, Veillonellaceae_unclassified and Veillonellaceae_uncultured (p < 0.01)) and two indicator OTUs in the low energy group (Bacteroidales_norank and Lachnospiraceae_uncultured (p < 0.01)). In conclusion, these findings added new dimensions to our understanding of the diet effect on rumen microbial community and fermentation response, and are of great significance for establishing the optimal nutrient supply strategy for lambs.

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