A Compound Feed Additive Improves Saline–Alkaline Stress Tolerance in Nile Tilapia (Oreochromis niloticus) Through Regulation of Hepatic Metabolism, Osmoregulation, and Intestinal Health

Saline–alkaline aquaculture is a promising strategy to alleviate freshwater shortages; however, such environments severely impair fish growth and physiological homeostasis. Nutritional regulation has been proposed to improve stress tolerance, yet the benefits of single additives are often limited under the multifactorial challenges imposed by saline–alkaline conditions. Therefore, a compound feed additive (CFA) consisting of glutamate, cholesterol, β-glucan, myo-inositol, zinc methionine, and curcumin was developed and evaluated in Nile tilapia (Oreochromis niloticus). To assess the robustness and practical applicability of this nutritional strategy, three independent feeding trials were conducted using different commercial basal diets as validation systems. Within each dietary system, fish were reared under freshwater (FW), saline–alkaline water (SAW), or saline–alkaline water supplemented with CFA (SAW+CFA). Saline–alkaline stress significantly reduced WG and SR, increased FCR, and elevated VSI and HSI, indicating impaired growth performance and metabolic burden. These changes were accompanied by increased serum glucose and ion concentrations (Na+, K+, Cl−), elevated ammonia levels, and reduced crude protein content. Dietary CFA improved growth and feed utilization under saline–alkaline conditions. It enhanced hepatic glycogen content and reduced serum glucose levels. Meanwhile, it downregulated glycolysis-related genes (hk, pfk1, pk) and upregulated genes involved in gluconeogenesis and the pentose phosphate pathway (g6pase, pc, g6pdh), indicating altered glucose metabolism and improved energy homeostasis. Saline–alkaline stress induced oxidative stress, apoptosis, and histological damage in the liver, whereas CFA alleviated these alterations by reducing MDA levels, enhancing antioxidant enzyme activities (CAT, GSH-Px, T-SOD) and suppressing apoptosis-related genes (caspases, p53, c-myc). In addition, CFA alleviated saline–alkaline stress-induced gill structural damage and reduced serum ion concentrations while modulating ion transport-related gene expression, suggesting improved osmoregulatory capacity. It also enhanced ammonia metabolism and transport, as reflected by reduced serum ammonia levels and altered expression of related genes. Furthermore, Saline–alkaline stress impaired intestinal structure and function, whereas CFA improved intestinal villus structure, increased digestive enzyme activities (amylase, trypsin, lipase), and suppressed pro-inflammatory genes (il-1β, il-8). Importantly, similar beneficial response patterns were observed across the three independently analyzed dietary systems. Overall, CFA improved saline–alkaline adaptability of Nile tilapia and was associated with improvements in energy metabolism, oxidative homeostasis, osmoregulation, ammonia detoxification, and intestinal function, providing a practical nutritional strategy for saline–alkaline aquaculture.