| Effects of waterborne Fe(â…¡) and dissolved oxygen (DO) on physiological parameters such as respiratory, haematology, acid-base balance, nucleus anomaly, enzyme activities, gill and liver histology on juvenile turbot Scophthalmus maximus were studied. Six waterborne Fe(â…¡) concentration (0.01, 0.05, 0.1, 0.5, 1 and 2 mg/L) and eight DO concentration, including severe hypoxia(0.2, 1, 2 mg/L), hypoxia(3, 5 mg/L), normoxia(normal aeration as control, about 7 mg/L) and hyperoxia(11, 14 mg/L), were used for the study. The effect of DO on the expression of heme oxygenase-1 (HO-1) was also analyzed.At first, the determination method of waterborne Fe(â…¡) in the sea water was established. The surface water samples in some sites of the coast of Yellow Sea and Bohai Sea, and some sites of Yellow Sea were collected in 2009. The surface water samples of Swan lake, near shore and offshore sea area in Rongcheng bay from 2009 to 2011 were also determined. In addition, the water samples in some fish farms in Tianjin, Rongcheng and Rizhao were also analyzed. Results showed that the Fe(â…¢) and Fe(t) concentrations in most sampling site of the coast of Bohai Sea were higher than those of Yellow Sea. Fe(â…¢) and total iron (Fe(t)) concentrations in the sampling site near to East Sea were higher. In Rongcheng bay, no obvious annual variation trend of the concentration of Fe(â…¡), Fe(â…¢) and Fe(t) was found. The highest Fe(t) concentration appeared in Swan lake, and the lowest appeared in offshore site. The Fe(â…¢) concentrations in the groundwater were higher than Fe(â…¡) concentrations, but the Fe(â…¡) concentration in some groundwater was still at high level. Results about the physiological effects of waterborne Fe(â…¡) on juvenile turbot showed that the growth of juvenile turbot was not affected during the experiment period except the 2mg/L groups. Fish mortality was only observed in the 2 mg/L groups, in which one fish died on day 1 and the rest died in 7 days. Generally, the respiratory rates of fish in the experiment groups increased with increasing Fe(â…¡) concentration. The values showed no significant differences on day 1 and 7. On day 28, the 0.1 mg/L, 0.5 mg/L, and 1 mg/L groups showed a significant increase when compared with those in the blank and ascorbic acid control groups (P<0.05), respectively. No severe hematological perturbation was found, except the mean cellular hemoglobin concentration (MCHC) values in the 1mg/L groups was significantly higher than those in the two control groups on day 28 (P<0.05). Micronucleus and nucleus anomaly in erythrocytes could be observed when exposed to 0.1mg/L waterborne Fe(â…¡), and the nucleus anomaly in the 2mg/L groups was significantly higher than those in the other groups (P<0.05). On day 1, no significant difference was observed on the serum AKP activities of juvenile turbot among all the groups. The serum SOD activities in the 2mg/L groups were significant lower than those in the two control groups (P<0.05). On day 28, the serum AKP activities increased and decreased with the waterborne Fe(â…¡) concentration increasing (P<0.05). The serum SOD activities showed the same trend but no significant difference was found. A high Fe(â…¡) concentration (0.1 mg/L or higher) could cause turbot gill damage, such as vacuoles in branchial lamellae, epithelial necrosis, and hypertrophy of epithelial cells. Therefore, excess waterborne Fe(â…¡) and long-term exposure to Fe(â…¡) should be responsible for poor growth and high mortality of juvenile turbot in culture. The waterborne Fe(â…¡) firstly damage the gill structure of turbot, the efficiency of oxygen acquirement was affected. Then both the respiratory rates and the hematological parameters compensatory changed. Fe(â…¡) could also lead to nucleus anomaly. With the increasing of waterborne Fe(â…¡), the damage of gill increased, fish mortality even presented. The 0.01mg/L waterborne Fe(â…¡) didn't affect the nucleus anomaly, AKP and SOD activities, and gill structure. It could be regard as the safe concentration in turbot culture. Different DO concentrations on juvenile turbot experimental results showed that neither hypoxia nor hyperoxia affects the growth of juvenile turbot during the experiment period. Fish mortality was only observed in the 0.2, 1 and 2 mg/L groups, in which all fish died in 2h, 1 day and 2 days, respectively. With increasing of DO concentration, the respiratory rates of fish in the experiment groups increased firstly, and then decreased on 2h, day 1 and day 7. On day 28, the respiratory rates of fish in the experiment groups decreased with increasing of DO concentration (P<0.05), and no obvious difference was found after 14 days recovery. The hematological parameters, such as RBC, HGB and HCT didn't show clear changing regularity. The values of the three parameters in the 2 and 3mg/L groups were higher than those in the other groups on day 1. After 14 days recovery, the values in the hyperoxia groups were higher than those in the hypoxia groups. The effects of DO on nucleus anomalies were shown with obvious time-cumulative effects and dose-cumulative effects in the 28 days experiment. The micronuclei were first observed in the 5, 14 mg/L and control groups on day 28. It was still found in the hyperoxia and control groups after recovery for 14 days. At 2h, the blood pH in the severe hypoxia groups was significant lower than those in the hyperoxia groups (P<0.05), and no significant difference was found among all the groups on day 28. After 14 days recovery, the blood pH in the hyperoxia groups was significant lower than those in the hypoxia groups. With the DO concentration increasing, the pCO2 and plasma bicarbonate concentrations decreased firstly, and then increased at 2h. And their concentrations continue to increase on day 1, 7 and 28, respectively. The values of the two parameters of all the groups were nearly the same after 14 days recovery. The difference of SOD activities in different groups was significant at each sampling time (P<0.05). The difference in CAT activities among experiment groups was significant only after 7 days exposure (P<0.05). The MDA concentration increased with the concentration increasing at 2h. In the hypoxia and hyperoxia groups, the MDA concentration decreased with treatment time prolonging. After 14 days recovery, the MDA concentration in the hypoxia groups were significant higher compared with those in the control groups (P<0.05). The histological structure of the fish gills in the hypoxia and extreme hypoxia groups was basically unchanged during the experiment period, except mild hypertrophy in epithelial cell layer appeared in 3mg/L groups after 28 days exposure. In the hyperoxia groups, mild degenerative changes were observed in the fish gills on day 7, and vacuoles between the epithelial cell layer and the capillary vessel appeared in some branchial lamellae. On day 28, the vacuoles and hypertrophy in gill became universal. The histological structure of the turbot liver in all the groups was basically unchanged during the experiment period, except the structure of liver was looser in the hyperoxia groups compared with which in the normoxia and hypoxia groups on day 28. After 14 days recovery, the histological structure of the fish gills and liver was basically normal in all groups. In this study, we also cloned HO-1 from turbot. This cDNA with 1247bp encodes a protein of 275 amino acids. A heme oxygenase signature motif with a relatively high level of sequence conservation was found in the amino acid sequence. RT-PCR analysis indicated that HO-1 was highly transcribed in most tissues except gondal and kidney. The expression of HO-1 gene was up-regulated by either hypoxia or hyperoxia, and recovered after 14 days recovery. The above results showed that the physiological response of turbot to hypoxia and hyperoxia was different. However, either hypoxia or hyperoxia brought oxidative stress to turbot. It is worth to be mentioned that the oxidative stresses were also found in control groups, but no obvious changes on the total nucleus anomalies and other anti-oxidative index were found in 5 mg/L groups. Therefore, mild hypoxia may more suitable for juvenile turbot culture.
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