Integrated biochemical and transcriptomic analyses elucidate the physiological mechanisms in rainbow trout (Oncorhynchus mykiss) head kidney under hypoxia stress

Hypoxia has emerged as a critical threat to the stability of aquatic ecosystems and the sustainability of aquaculture industries. Rainbow trout (Oncorhynchus mykiss), a globally significant economic fish species, exhibits remarkable sensitivity to hypoxia. However, the physiological mechanisms of head kidney in rainbow trout under hypoxia stress, especially under acute and severe hypoxia conditions, remain largely unexplored. This research explored the transcriptomic responses of rainbow trout head kidney to moderate hypoxia (4.5 ± 0.1 mg/L) and severe hypoxia (3.0 ± 0.1 mg/L) for 12 h, alongside dynamic changes in biochemical parameters during hypoxia exposure (0, 4, 8, 12, 24 h, and 1 month) and reoxygenation (R12 h and R24 h). The results showed that compared with the control group, the activities of T-AOC, CAT, T-SOD, GOT, GPT, and ACP were decreased under moderate and severe hypoxia conditions, while the content of MDA and the activity of AKP were increased. In RNA-seq analysis, we identified several hypoxia-related, immune-related and metabolism-related genes (hif-1a, fih1, vhl, ddit4, egln1/2/3, cxcr1/3/4/5, ccr5/7, clec4e/m, tlr2, socs2/3, ighv3-43, ighv3-53, g6pc3, pk, ldha, hk2, and foxo1a) from 20,399 significantly differentially expressed genes, which were enriched in insulin signaling pathway, glycolysis/gluconeogenesis, Fc?R-mediated phagocytosis, hematopoietic cell lineage and B cell receptor signaling pathway. Additionally, the expression levels of cxcr1, ddit4, egln2, egln3, fih1 and foxo1a in rainbow trout were significantly up-regulated under hypoxia stress and returned to normal after reoxygenation. These findings provide new insights into the physiological and molecular responses of rainbow trout under hypoxia stress and contribute to developing effective strategies for fish to cope with environmental hypoxia. Overall design: Full-sib rainbow trout (average weight of 130 ± 5.0 g) were obtained from a farm located in Yongjing, Gansu Province, China. Prior to experimentation, fish were divided into three groups (normoxic control, moderate hypoxia, and severe hypoxia) and acclimatized for two weeks at the Fisheries Science Training Center of Gansu Agricultural University. During acclimatization, DO was maintained at 8.5 ± 0.1 mg/L, and water temperature was controlled at 12.0 ± 0.1°C. Following an acclimation period, the DO levels of the moderate and severe hypoxia groups were rapidly reduced to 4.5 ± 0.1 mg/L and 3.0 ± 0.1 mg/L by nitrogen gas. Sampling was conducted at specified intervals throughout hypoxia exposure (0, 4, 8, 12, 24 h, 1 month) and subsequent reoxygenation (R12 h, R24 h, DO restore to 8.5 ± 0.1 mg/L), with rainbow trout randomly sampled at each stage. Head kidney tissues from MS-222-anesthetized fish were harvested into freezing tubes and flash-frozen in liquid nitrogen for later analyses. All experimental procedures strictly adhered to the Animal Ethics Committee of Gansu Agricultural University (GSAU-Eth-AST-2021-004). **National Natural Science Foundation of China (Grant No. 32060823) and Discipline Team Project of Gansu Agricultural university (NO: GAU-XKTD-2022-23).