Phase separation inhibitor-mediated embryonic phase homeostasis enhancing hypoxia tolerance in grass carp (Ctenopharyngodon idella)

Hypoxia frequently restricts intensive culture of grass carp (Ctenopharyngodon idella) and causes economic losses. The establishment of phase homeostasis during the embryonic stage exerts a profound impact on subsequent stress resistance. To investigate the effect of phase homeostasis on hypoxia tolerance of C. idella, we treated fertilized eggs with a phase separation inhibitor (1% 1,6-Hexanediol) to obtain phase-homeostatic fry. A study was conducted by combining natural oxygen consumption, 9-hour acute hypoxic stress, and transcriptional analysis to compare the differences in hypoxic tolerance of C. idella between the 1,6-Hexanediol treatment group and the control group. Closed static water treatment showed that phase-homeostatic C. idella exhibited a delayed appearance of aerial surface respiration (ASR), with significantly lower DO values (0.53 mg/L vs. 0.71 mg/L) and mortality rates compared to the control group (P<0.05), indicating enhanced hypoxia tolerance. Under acute hypoxia stress (DO ≈ 0.5 mg/L), there was no significant difference in plasma cortisol and glucose concentrations between the two groups at 4.5 h. However, the treatment group showed significantly higher cortisol levels at 9 h (P<0.05), which returned to normal after 12 h of reoxygenation. Hepatic antioxidant tests revealed that the treatment group maintained significantly higher levels of malondialdehyde (MDA) and total protein (TP) than the control group at all time points, while the activity of superoxide dismutase (SOD) and other antioxidant enzymes was lower. Transcriptomic analysis indicated that the number of differentially expressed genes increased with prolonged hypoxia and decreased after reoxygenation, with 398, 680, 1083, and 586 at 0 h, 4.5 h, 9 h, and R12 h. These genes were involved in HIF signaling pathway, antioxidant defense, ubiquitination and protein homeostasis, mitochondrial function regulation, and energy metabolism reconstruction after reoxygenation. Quantitative real-time PCR verification confirmed that key genes such as hif1al, ddit3, ddit4 and foxo3b showed a sharp increase in expression from 4.5 h of hypoxia, and their expression levels in the treatment group were consistently significantly higher than those in the control group (P<0.05), indicating that 4.5 h is an important time point for C. idella to respond to hypoxic stress. This study provides a new perspective for improving hypoxia resistance in grass carp and offers a potential technical approach for breeding stress-resistant C. idella varieties.