| The operations of modern aquaculture often expose fish to a variety of acute stressors,which have a negative impact on fish growth,survival and animal welfare.Anesthetics can relieve fish's pain,induce sedation,and reduce the effects of the operation stress on fish.Although anesthetics can reduce the response that stress caused to the body by human operation,it are also a source of stress.Anesthetics have been widely reported as secondary stressors and gills are the main target organs of anaesthetic stress injury in fish.This study takes Chinese sea bass(Lateolabrax maculatus),an important economic fish species in southern China,as the research object.First,this study compared that the efficacy of eugenol and tricaine methanesulphonate as anaesthetics for juvenile L.maculatus during simulated transport.Second the effects of two anesthetics on the anesthesia injuring of juvenile L.maculatus were evaluated,and the mechanism of anesthesia stress injury was preliminary explored.Finally,the effects of transdermal enhancers to reduce the use of anesthetic of the juvenile L.maculatus and the protective effect of teprenone on the anesthesia stress injury of juvenile L.maculatus were analyzed respectively.The specific results are as follows:1. The efficacy of eugenol and tricaine methanesulphonate as anaesthetics for juvenile L.maculatus during simulated transportThe purpose of this study was to evaluate the anaesthesia effects of eugenol and MS-222 sedatives applied on juvenile L.maculatus during simulated transport.In experiment 1,the juveniles were divided into two groups,with seven concentrations tested on each group(eugenol[4,6,8,10,12,14 and 16 mg/L]and MS-222[20,30,40,50,60,70 and 80 mg/L]).Induction and recovery times were recorded.The time for anaesthesia was shortened,and the time for complete recovery was prolonged as the anaesthetic concentration increased.The optimal transport concentration for each anaesthetic tested was 6 mg/L of eugenol and 30 mg/L for MS-222.In experiment 2,the5 h simulated transport test showed that the survival rate of L.maculatus juveniles with anaesthesia was 100%,and without anaesthesia,survival was 60%.After 24 h of recovery following transport,the fish showed 100%survival for the group with added anaesthetic and 40%for the group without added anaesthetic.Compared to the non-anaesthetized groups,the anaesthetized transport groups showed significant increases in the levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)(p<0.05).The levels of AST,ALT and alkaline phosphatase(AKP)were significantly higher in the MS-222 transport group than in the eugenol transport group(p<0.05).The levels of AKP were significantly higher in the non-anaesthetized transport group than in the anaesthetized group(p<0.05).According to the present experiment results,eugenol was an efficient anaesthetic in L.maculatus,and this study recommend eugenol instead of MS-222 as an anaesthetic for the short-time transport of L.maculatus.2. Study on the anaesthetic injury of eugenol and MS-222 on juvenile L.maculatusIn this study,the impacts of tricaine methanesulphonate(MS-222)(60 mg/L)and eugenol(12 mg/L)on the physiological and immune in L.maculatus were investigated.The immune assays results revealed that MS-222 and eugenol decrease total immunoglobulin content and increase alkaline phosphatase activity in skin mucus and gills.In addition,eugenol increase lysozyme activity,while MS-222 is the opposite.Anaesthetic exposure also promoted expression of Cytochrome C,Cysteine-aspartic proteases-9 and Cysteine-aspartic proteases-3 in the gills,which suggested that MS-222and eugenol exposure induced apoptosis in the gills.The stress assays results showed that the anaesthetic treatment remarkably altered the content of the Heat Shock Protein70 and the activity of Na~+/K~+-ATPase.Additionally,the activities of superoxide dismutase,catalase and peroxidase,and the contents of glutathione and malondialdehyde were promoted in gills at 3-6 h,which indicating that anaesthetic induced oxidative stress and lipids and proteins injuring of gills tissue.These results may further enriches the data of immune and stress impacts of anaesthetic to fish,which may be useful for the risk assessment of anaesthetic.3. Study on the anaesthetic-penetration effect of Acorus tatarinowii and azone on juvenile L.maculatusThis study compared the penetration effects of two common penetration enhancers(PE)(Acorus tatarinowii essential oil and water soluble Azone)with different mass fraction(1%,4%,7%,10%,and 1%,3%,5%,10%)on the juvenile L.maculatus absorbing eugenol and MS-222.This study found that:1.PEs can significantly reduce the time of anaesthesia and recovery,and reduce the dosage of anesthetics to achieve similar anesthetic effects.2.With the increasing of the mass fraction of PE,the penetration effect is first enhanced and then weakened.3.The optimum mass fraction of penetration of A.tatarinowii essential oil and water soluble azone on eugenol or MS-222 are 7%and 3%,respectively.Determining the antioxidant index of the gills tissue,this study found that:1.After soaking the fish in the anesthetics,the activities of peroxidase(POD),superoxide dismutase(SOD),catalase(CAT)and the concentrations of malonaldehyde(MDA),glutathione(GSH)are increasing in the gills tissue of L.maculatus(P<0.05).2.The activities of SOD,CAT and the concentrations of MDA,GSH are increasing,which manifests that PE reduce the dose of anesthetics to decrease the oxidative stress of gills,when the anesthetized control group compare with the PE anesthetics group,significantly.(P<0.05).The results of this study provides experimental basis for the application of PE in aquaculture,and also enriches data reference for improving the effect of the anesthetics.4. Protective effects of teprenone on gills tissue anesthesia stress injury in juvenile L.maculatusIn this experiment,the mechanism of anesthesia injury of MS-222 and eugenol and the protective effect of feeding teprenone in advance on anesthesia injury of L.maculatus were studied.1)Both MS-222 and eugenol decrease Na~+-K~+-ATPase(NKA)activity and induce heat shock protein 70(HSP70)expression in gills tissue.Feeding teprenone in advance significantly increased NKA activity and HSP70 expression(p<0.05).2)MS-222 and eugenol significantly increased the activity of apoptotic factors.Feeding teprenone in advance significantly decreased the activity of apoptotic factors(p<0.05).3)The morphology of gill tissue in the control group was normal.After treatment with MS-222 and eugenol,the gill morphology showed that the gill lamella(GL)were not arranged neatly,GL base volume increases,the blood channel is widened and the internal red blood cells are swollen,mitochondrion-rich cell has come off,and its overall structure deviates from the normal shape.The gill tissue structure tended to be normal after feeding teprenone in advance and then anesthesia.In conclusion,(1)eugenol and MS-222 are well used for anesthesia and anesthesia transport of juvenile L.maculatus.(2)Eugenol and MS-222 as a source of stress can easily cause anesthesia stress injuring in the gills tissue of juvenile L.maculatus,mainly manifested as:causing oxidative injuring to the gills tissue,reduce osmotic pressure regulation of gill cells,change non-specific immune enzyme activity and related protein expression,and promote apoptosis factor activity,and the resuscitation rate decreases with increasing anesthetic concentration.(3)By using a transdermal enhancer to reduce the dose of anesthetic,the oxidative injuring caused by anesthetic stress can be reduced.(4)Feeding teprenone in advance improve HSP70 expression,which can protect subsequent anesthesia stress injury. |
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