Effects Of Stocking Density And Hypoxia On The Welfare Of Amur Sturgeon Acipenser Schrenckii And Its Mechanism

Stocking density and hypoxia are considered priority issues in aquaculture research. In this study, two experiments were carried out in order to investigate the effects of chronic stress (stocking density) and acute stress (hypoxia) on the growth, metabolism, stress response and immune physiology responses of juvenile Amur sturgeon (Acipenser schrenckii). In the chronic stress study, three triplicate groups of Amur sturgeon (42.0 ± 2.3g) were reared in nine square concrete ponds (4.4 x 4.4 x 0.45 m3) at three stocking densities (3.7,6.9 and 9.0 kg/m3) for 60 days. In the acute stress study, three triplicate groups:normal group (7mg/l), hypoxia group 1 (5mg/l) and hypoxia group 2 (3mg/l) were used in nine 100L indoor tanks. The low-oxygen conditions was kept stable with nitrogen. Sampling was performed at the end of the stocking density experiment (60 days) and at 0,0.5,1.5,3 and 6h after hypoxia stress.1. Effect of stocking density on the survial, growth and ingestion of Amur sturgeonTo evaluate effects of stocking density on welfare of Amur sturgeon (Acipenser schrenckii), a stocking density experiment was designed for 60 days. The mortality, as well as feed conversion rate (FCR), was not affected by stocking density. However, the specific growth rate (SGR), final weight and weight gain in the HSD group were significantly lower than in the LSD and MSD groups. The hepatosomatic (HSI) varied inversely with regard to stocking density. Serum hormone FT3, FT4 and IGF-1 showed significant decrease with the increase of the stocking density at 40 days and 50 days of the experiment. The result of this study revealed that the fish in the low stocking density could gain better welfare.2. Effect of stocking density on the nutrition and metabolism of Amur sturgeonStocking density did not affect crude protein levels in fish. In contrast, the total lipid level was significantly higher in the LSD group compared to the MSD and HSD groups. This reduction seems to be related to an increasing lipid mobilization to cope with the increasing energy demand caused by the crowding environment. In this study, ARA increased in fishes in the HSD group. The result was in accordance with the good survival rate of the HSD group. DHA contents in our research showed a decline in the MSD and HSD groups, the result indicated relatively higher energy utilization at high stocking density. Differences between treatments were not registered for albumin. But serum total bilirubin and urea in HSD group were significantly higher than in the LSD group while serum triglycerides showed opposite tendencies. The result of this study revealed that stocking density influenced the nutritional status and substance metabolism process of Amur sturgeon.3. Effect of stocking density on the stress response and physiology of Amur sturgeonIn this study, the cDNA sequences of HSP70 and HSP90 were isolated from the special chonrdr-ganoid scale fish, Amur sturgeon, for the first time. Homology analysis indicated that amino acid senquences of HSP70 and HSP90 shared high identity with other species (82.68-99.07% and 90.19-98.07%, respectively). The tissue expression analysis showed that the asHSP70 and asHSP90 mRNA were ubiquitously expressed in all the examined tissues under unstressed condition. The expression pattern of HSP70 and HSP90 under crowding stress were examined by q-PCR in liver, spleen and kidney. Results showed that stocking density could significantly influence the expression of HSP70 and HSP90 levels in liver and spleen and kidney. The assessment of asHSP70 and asHSP90 mRNA levels under crowding stresses indicated that asHSP70 and asHSP90 gene might be good indicators of stressful situations for Amur sturgeon. However, the serum cortisol and glucose showed no significant difference among different stocking. This result could be related to the capability of Amur sturgeon to adapt to intensive culture.4. Effect of stocking density on the immunology response of Amur sturgeonIn this study, the complete coding sequence of Amur sturgeon CYP 1A was cloned for the first time. The tissue-specific expression and stress-induced expression of CYP IA were also measured. CYP 1A mRNA in liver showed over expressions in crowding condition. The same trend was also found in spleen and kidney which may provide evidence that CYPIA could serve as a good indicator of immune response in Amur Sturgeon. This results also showed that increased stocking density reduced the morphological indexes (hepatosomatic index, spleen-somatic index and kidney-somatic index). In addition, the contents of hemoglobin increased significantly in the stressed group. This response may be related to the increase in energy demand imposed by high stocking density.5. Effect of hypoxia on the growth and metabolism of Amur sturgeonIn the hypoxia experiment, no fish was death in the normal group and hypoxia 1 group. In hypoxia 2 group there was also no fish death at 0.5,1 and 3h, but all the fish were dead at 6h after hypoxia 2 stress. This result suggested that Amur sturgeon could not survived at the extremely hypoxia condition. The levels of serum FT3 was significantly influenced by hypoxia stress. Serum FT3 showed a positively correlated with hypoxia (P<0.05). On the contrary, differences between treatments were not registered for serum FT4. Serum IGF-1 showed a significant increase at 1h after hypoxia stress. Serum ALT showed an increasing trend after hypoxia stress, but it showed no significant difference with the control group.6. Effect of hypoxia on the stress response and physiology of Amur sturgeonThe levels of HSP70 transcript in H1 and H2 group indicated a remarkable increase in spleen after hypoxia stress. The levels of HSP70 transcript in liver were similar to those in spleen. In kidney, HSP70 also showed a significant increase in H1 and H2 group. The levels of HSP90 transcript in H1 and H2 group showed significant increases in all the three tissues after hypoxia stress except the transcript of liver in H2 group 6h after hypoxia. We supposed that the up-regulated mRNA expression of asHSP70 and asHSP90 under hypoxia stress indicated that the asHSPs were inducible and involved in the immune response of Amur sturgeon. The levels of cortisol, glucose and hematological parameters elevated significantly after this stress. In our study. fish under crowding and hypoxia stress (6h after hypoxia stress) showed a significant increase in the levels of the serum total protein. It is speculated that fish may increase specific proteins such as lysozyme or complement to enhance the immunity level to cope with stress7. Effect of hypoxia on the immunology response of Amur sturgeonSpleen-somatic index showed a decline after hypoxia though H1 group returned to the normal level at 3h and 6h after hypoxia stress. HSI showed a significant increase at 3h after hypoxia in H2 group. As for white blood cells, an increase trend was observed after hypoxia stress in this study which might be associated with an enhanced immune response. Red blood cells and haemoglobin in our study also significantly elevated after hypoxia stress. The increase in the number of red blood cells may raise blood oxygen capacity and improves oxygen delivery to the tissues. Similar to the result in the stocking density experiment, the increase of the levels of CYP 1A mRNA expression indicated that this gene could function as an unspecific biomarker.