Optimizing fish performance depends on several factors, with dietary protein levels and rearing temperature playing important roles. In this study, Nile tilapia fingerlings (Oreochromis niloticus) weighing an average of 20.00 ± 1.26 g were divided into nine groups (in three replicates). Each group was subjected to different water temperatures (26 °C, 28 °C, and 30 °C) and received one of three dietary protein levels (20%, 25%, and 30%) for two months. Our findings indicate that higher temperatures, particularly at 30 °C, increased water electrical conductivity and total dissolved salts, especially noticeable in fish fed 25% or 30% crude protein (CP). Lower total ammonia nitrogen levels were observed at 28 °C with 25% CP, 30 °C with 30% CP, and 26 °C with 30% CP. Hepatic growth hormone receptor 1 and insulin-like growth factor 1 expression gradually rose with higher dietary CP percentages in fish at 26 °C but declined in those at 30 °C, albeit remaining higher than in the 28 °C groups with 25% CP. Fish at 28 °C showed the best final body weights and growth performance when fed 20% or 25% CP, with no significant difference between these groups. Hepatic leptin expression did not differ significantly among groups, but hepatic fatty acid binding protein expression notably increased in fish fed 30% CP at both 26 °C and 30 °C compared to those at 28 °C with 25% CP. Within the same temperature group, fish fed 30% CP exhibited higher globulin levels, particularly thriving at 28 °C or 30 °C. Hepatic mucin-like protein expression significantly increased across all groups, especially in fish at 30 °C with 30% CP compared to those at 28 °C with 25% CP. Hepatic lysozyme expression also increased notably in fish at 30 °C with 30% CP. Notable changes in superoxide dismutase, catalase, and glutathione peroxidase expression were observed, with the highest serum superoxide dismutase and catalase activities recorded in fish at 30 °C with 25% CP. Overall, dietary protein levels of 25% and 30%, combined with temperatures of 28 °C and 30 °C, yielded favorable outcomes, particularly favoring 28 °C with 25% protein.
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