Integrated biochemical and transcriptomic profiling reveals graded spleen responses to moderate versus severe hypoxia in rainbow trout (Oncorhynchus mykiss)

Hypoxia severely threatens the survival and growth of fish, thereby greatly restricting the development of aquaculture. Rainbow trout (Oncorhynchus mykiss) is highly sensitive to hypoxia, but the regulatory mechanism of rainbow trout spleen under different levels of hypoxia is not yet clear. In this study, we analyzed the effects of moderate hypoxia (DO: 4.5 ± 0.1 mg/L) and severe hypoxia (DO: 3.0 ± 0.1 mg/L) on the biochemical parameters of rainbow trout spleen at various time points (4, 8, 12, 24 h, 1 month, and re-oxygenation for 12 and 24 h). Additionally, we comparatively analyzed the transcriptomic changes in moderate and severe hypoxia at 12 h, as well as in normoxic controls. Antioxidant enzyme activities (T-AOC, T-SOD, GOT, GPT, CAT) rose markedly under both moderate and severe hypoxia, while MDA content peaked early and then declined. Immune-related enzymes (AKP, ACP) were elevated under moderate hypoxia but suppressed under severe hypoxia. Reoxygenation gradually restored the activities of antioxidant and immune enzymes, yet MDA surged again. Transcriptomic analysis identified 5766 DEGs. These DEGs were mainly enriched in the B cell receptor signaling, Glycolysis/Gluconeogenesis, Notch, MAPK, and PI3K-Akt pathways. The analysis of dynamic expression of hypoxia-associated genes revealed that hif-1a, acly, egln2, plpp3, fn1, and cd22 were upregulated under moderate hypoxia, whereas aldoa and ldha were downregulated, with their expression levels recovering during reoxygenation. egln1, egln2, egln3, aldoa, plpp3, pk, g6pc, and eno1a were upregulated during severe hypoxia stress, and their expression returned to baseline levels following reoxygenation. These results improve our comprehension of the molecular processes involved in hypoxia stress in rainbow trout. Fish weighing (130 ± 5.0) g were selected and temporarily reared for two weeks in a flow-through aquaculture system with DO of 8.5 ± 0.1 mg/L, water temperature of 12 ± 0.1℃. Fish were fed a commercial pellet feed twice a day. All procedures were conducted in accordance with the approval of the Animal Ethics Committee of Gansu Agricultural University and followed the protocols approved by the institutional guidelines.Healthy rainbow trout were randomly divided into three groups, including normoxia, moderate hypoxia, and severe hypoxia groups, the oxygen levels were maintained at 8.5 ± 0.1, 4.5 ± 0.1, and 3.0 ± 0.1 mg/L, respectively, and the low oxygen environment was created by mixing oxygen and nitrogen. The reoxygenation experiment was conducted immediately after 24 h of hypoxia stress and the DO level was maintained at 8.5 ± 0.1 mg/L during the experiment. Controls were divided into C (0 h control) and CM (1 month control), and the spleens of the normoxia group were collected immediately before the start of the hypoxia experiment and 1 month after the start of the experiment. After hypoxia stress, spleen tissues were collected at 4 h, 8 h, 12 h, 24 h, TMM (moderate hypoxia for 1 month), and TMS (severe hypoxia for 1 month), as well as R12 (hypoxia for 24 h + reoxygenation for 12 h) and R24 h (hypoxia for 24 h + reoxygenation for 24 h) in each group (moderate hypoxia group and severe hypoxia group), respectively. spleen samples were collected after fish MS-222 treatment. After collection, some samples were used for histological observation, other samples were frozen immediately in liquid nitrogen, and then transferred to -80℃ refrigerator for storage for biochemical parameters, gene expression and RNA-seq assays.