| Blunt snout bream(Megalobrama amblycephala,Yih),commonly known as Wuchang fish,is a kind of hypoxia-sensitive fish.Compared to carp(Cyprinus carpio)and crucian carp(Carassius auratus),pomfrets often face hypoxia during culture,which seriously limits the sustainable development of the local culture economy.To address the above problems,this study investigated the performance indexes of hypoxia tolerance,redox homeostasis,morphological structure of gill tissues and apoptosis mechanism of the gill under hypoxic environment by hypoxic stress,with a view to providing theoretical references for the healthy development of the gill breeding industry of blunt snout bream.The main findings are as follows:1.In this study,in order to evaluate the hypoxia tolerance of blunt snout bream,the surface respiration(ASR)tolerance of blunt snout bream with a body mass of 50 ±0.5 g and 10 ±0.5 g was investigated and measured.The results showed that ASR measured at25.0 ± 0.1°C was significantly(P < 0.05)lower for the group bream with a body mass of50 g(1.31 ± 0.10 mg/L)than for the group bream with a body mass of 10 g(1.66 ± 0.06mg/L).It was shown that the larger the body mass,the better the tolerance to low oxygen.In order to study the effects of hypoxic stress and reoxygenation on redox homeostasis of blunt snout bream,the young fish of blunt snout bream "Pujiang No.2" with a body mass of 10 ± 0.5 g were stressed for 0,3,6,12 and 24 hours under hypoxia(2.0 ± 0.1 mg/L),and then recovered for 3 hours under normoxia(7.9 ± 0.1 mg/L).The detection results showed that the level of ROS in the gill tissue of the blunt snout bream significantly(P<0.05)increased after hypoxia stress.After 3 hours of reoxygenation,the ROS activity in the gill tissue decreased,and there was no significant difference compared to 12 hours of hypoxia.The results showed that acute hypoxia stress induced excessive ROS production in the gill tissue of the blunt snout bream.The antioxidant level and oxidative stress level of gill,heart,kidney,liver and intestinal tissues were detected,and the test results showed that the activity of SOD and CAT and the content of MDA in tissues increased significantly(P < 0.05)during hypoxic stress.After brief reoxygenation,compared with 24 hours of hypoxia stress,the content of SOD,CAT,and MDA in the heart and gill tissues significantly decreased(P<0.05).The results showed that acute hypoxic stress induced oxidative stress in the blunt snout bream.After a short period of reoxygenation,the content of MDA decreased but was still higher than0 h.Hypoxia stress would cause significant oxidative damage to the juvenile fish.2.In this study,we observed their gill tissues by tissue sectioning and Hoechst staining and measured the mitochondrial membrane potential and Caspase-3 activity in gill tissues.During hypoxic stress,the Lamellar respiratory surface area increased significantly(P < 0.05),the volume of interlaminar cell mass(ILCM)decreased significantly(P < 0.05),and the water-blood diffusion distance(P < 0.05)decreased.After reoxygenation,gill morphology gradually recovered.The results of morphological studies showed that under hypoxia stress,the blunt snout bream could induce gill remodeling in gill tissue.Under hypoxic stress,the mitochondrial membrane potential decreased significantly(P < 0.05),and the level of Caspase-3 in the gills of the blunt snout bream showed an upward trend.After 3 hours of reoxygenation,there was no significant difference in Caspase-3 levels in gill tissue compared to 12 hours of hypoxia,but they were still significantly higher than 0 h(P < 0.05).Hoechst staining results show that Hoechst-stained apoptotic cells increased significantly(P < 0.05)in gill tissues with the prolongation of hypoxic stress time.After reoxygenation for 3 hours,the apoptotic situation basically returned to the level of 12 hours of hypoxia.Hoechst staining structure analysis showed that hypoxia stress induced apoptosis in gill tissue of blunt snout bream.The analysis of mitochondrial membrane potential and Caspase-3 activity showed that hypoxia stress induced apoptosis in the gill tissue of blunt snout bream mediated by mitochondrial pathway.3.The anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax in the Bcl-2 family gene were clonely identified and sequenced.The changes of apoptosis-related genes in gill tissue were detected by real-time fluorescence quantitative(q-PCR)technique.The cloning,identification and sequence analysis of Bcl-2 and Bax genes showed that the sequence was highly evolutionarily conservative.The expression levels of PHD1 and Bcl-2,as well as the ratio of Bcl-2/Bax in the gill tissue of blunt snout bream was significantly(P < 0.05)decreased with the prolongation of hypoxia stress time.The expression of Hif-1α,Bax and Caspase-8 was significantly(P < 0.05)regulated in the gill tissue of blunt snout bream with the prolongation of hypoxic stress time.The analysis of Bcl-2,Bax expression level and Bcl-2/Bax m RNA ratio showed that hypoxia stress induced apoptosis mediated by mitochondrial pathway in the gill tissue of blunt snout bream.Analysis of caspase-8 expression level showed that hypoxia stress induced apoptosis of death receptor pathway in the gill tissue of blunt snout bream.Analysis of Hif-1α and PHD1 expression changes in gill tissues showed that PHD1 reduced the inhibition of Hif-1α and enhanced Hif-1α during this process expression.This reduces the damage caused by hypoxic stress to the gill tissue of the blunt snout bream. |
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