L‐Glutamine Plus L‐Glutamic Acid Enhances Antioxidant Status and Ammonia Toxicity Resilience, Upregulates Interleukin <i>IL‐10</i> Gene, and Improves Gut Microbiota and Survival in Juvenile Nile Tilapia

ABSTRACT Glutamine (Gln) and glutamic acid (Glu) are the most abundant free amino acids (AAs) in the fish body. Although classified as non‐essential AAs, their supplementation can be a strategy to optimize the growth performance and health of fish. This study aimed to investigate the effects of dietary Gln and Glu blend on growth performance, biochemical parameters, gut microbiota composition, short‐chain fatty acids (SCFAs) production, digestive enzyme activity, histomorphometry, and liver mRNA levels of glutamine synthetase ( GS ), peroxisome proliferator‐activated receptor alpha ( PPAR‐α ), anti‐inflammatory interleukin 10 ( IL‐10 ), pro‐inflammatory interleukin 1β ( IL‐1β ), and antioxidant status of juvenile Nile tilapia. Fish ( n = 216; 0.99 ± 0.01 g) were randomly allocated into eight aquariums containing 27 fish each, in a four‐replicate design. Fish were hand‐fed a Glu + Gln unsupplemented basal diet (CON) or a basal diet supplemented with 20 g kg −1 Glu + Gln (AMG) six times daily until apparent satiety for 60 days. Relative to fish fed CON diet, fish fed AMG diet exhibited enhanced feed conversion ratio (FCR; −5.6%), energy retention efficiency (+8.20%), and protein retention efficiency (+7.69%), and a trend towards a higher survival rate (+5.9%), suggesting improved nutrient utilization. Although the general structure of the microbiota of fish fed AMG diet remained similar to that of fish fed CON diet, it was observed that Gln + Glu supplementation promoted increased relative abundance of Enterococcus sp ., a potential probiotic. Notably, fish fed AMG diet showed higher SCFA production than those fed CON diet, enhancing intestinal fold development. Fish fed AMG diet also exhibited higher liver activity of superoxide dismutase (SOD) and glutathione‐S‐transferase (GST), resulting in lower malondialdehyde (MDA) concentration and indicating a healthier intestinal mucosal state. Furthermore, fish fed AMG diet showed higher mRNA expression of IL‐10 and GS , indicating enhanced anti‐inflammatory responses and ammonia metabolism, respectively. In conclusion, 20 g kg −1 dietary Gln plus Glu enhanced FCR, nutrient retention, and survival by favorably modulating the microbiota and improving intestinal function, thereby optimizing antioxidant responses and innate immunity in juvenile Nile tilapia. These findings highlight the potential of Gln and Glu blend to improve profitability and sustainability in Nile tilapia aquaculture.