This study systematically compared the effects of dietary supplementation with glutamine (Gln) and its precursors, including glutamic acid (GA) and α-ketoglutarate (AKG), on growth performance, serum antioxidant and immune parameters, and multi-region gastrointestinal microbiota in suckling lambs. Forty healthy suckling Hu lambs with similar body weight (7.37 ± 1.18 kg) and age (7 ± 0.8 d) were selected and randomly allocated into four groups (
n = 10 per group): a control group (CON, without additive), and three treatment groups (GA, AKG, and Gln), each receiving 2 g per animal per day of the corresponding additive. The experimental period lasted for 42 d. All three additives showed a tendency to increase the final body weight (
p = 0.056) and significantly increased the average daily gain (ADG) of lambs (
p < 0.05). GA supplementation increased the dry matter intake throughout the entire trial (
p < 0.05), whereas the addition of AKG and Gln increased the dry matter intake only during the later period (d 21–42) (
p < 0.05). The feed-to-gain ratios did not differ among all groups (
p > 0.05). Compared with the CON group, all three treatment groups showed elevated serum activities of catalase, glutathione peroxidase, and total antioxidant capacity, as well as increased IgA and IgG contents (
p < 0.05). In addition, malondialdehyde concentration was decreased in all three treatment groups (
p < 0.05). Moreover, GA supplementation reduced the ruminal alpha diversity while increasing the abundance of butyrate-producing bacteria (
Ruminococcaceae UCG-014) (
p < 0.05). All three interventions consistently decreased the abundance of the intestinal pathogen
Escherichia-Shigella in the ileum (
p < 0.05). Correlation analyses showed that ruminal
Treponema 2 abundance was negatively correlated with ADG, whereas jejunal
Methylobacterium and ileal
[Eubacterium] coprostanoligenes group were positively correlated with final body weight or ADG. In conclusion, glutamine and its precursors play an important role in modulating gastrointestinal bacterial diversity and composition, enhancing antioxidant and immune functions, and improving the growth performance of suckling lambs.
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