| Tea tree,also known as Melaleuca ahemifolia,belongs to the myrtle family.The tea tree oil extracted from its fresh branches and leaves,which has a special aroma.The tea tree oil is rich in terpene substances and it also has a variety of biological activities.So it is widely used in various industries such as medicine and food.However,because of its poor water solubility and easy decomposition,the use of tea tree oil is limited.Nanoemulsification technology is one of the important methods to solve this problem.Its small particle size can not only improve the stability of essential oil but also enhance its biological activity.Procambarus clarkii,also known as freshwater crayfish,is an important freshwater economic shrimp in China.Recently,with the increasing scale of farming and the increasing frequency of disease outbreaks,the search for green antibiotic alternatives is urgent.At present,the studies of tea tree oil on poultry,aquatic animals and other animals show that tea tree oil has antioxidant,antibacterial and growth promotion effects,and has the potential as a feed additive.Therefore,this paper explored the emulsification of tea tree oil and its addition in the feed of Procambarus clarkii,in order to provide experimental basis for the application of tea tree oil in the compound feed of Procambarus clarkii.The main research contents are as follows:(1)Formulation screening of tea tree oil nanoemulsionThe surfactant,cosurfactant,Km and the ratio of oil phase and water phase in tea tree oil nanoemulsion formula were screened by emulsion particle size,PDI,Zetapotential value and emulsion particle shape.The results showed that the suitable tea tree oil nanoemulsion formula was 2.5% soybean lecithin as surfactant,2.5% n-propanol as cosurfactant,10% tea tree oil,85%water.The particle size of tea tree oil nanoemulsion was 102.931±1.606 nm,the PDI was 0.135±0.005,and the Zeta potential was-53.5±1.809 m V.The nanoemulsion particles were uniform,the type was oil-in-water,and the temperature stability and centrifugal stability were good.(2)Antibacterial performance and antioxidant capacity of tea tree oil nanoemulsion in vitroAfter treatment at different temperatures(25,65,105 ℃),the antibacterial and antioxidant properties of tea tree oil and nanoemulsion were determined.The results showed that the tea tree oil nanoemulsion was superior to the tea tree oil in terms of antibacterial circle,minimum antibacterial concentration and minimum bactericidal concentration against Aeromonas hydrophila and Vibrio cholerae at the same temperature after being treated at different temperatures.With the increase of treatment temperature,the antibacterial properties of tea tree oil and nanoemulsion decreased to different degrees.However,the performance of tea tree oil nanoemulsion treated at105 ℃ was still higher than that of tea tree oil stock treated at 25 ℃.In the test of antioxidant capacity,the antioxidant capacity of tea tree oil and nanoemulsion decreased to different degrees after treatment at 65 and 105 ℃.But tea tree oil nanoemulsion was superior to tea tree oil stock solution in terms of total antioxidant capacity,hydroxyl radical inhibition ability and DPPH free radical scavenging ability under different temperatures.(3)Effects of dietary tea tree oil and astaxanthin on growth performance,antioxidant capacity and immune-related gene expression of Procambarus clarkiiTo investigate the effects of tea tree essential oil and astaxanthin on growth performance,antioxidant capacity and immune-related gene expression of Proambarus clarkii,six isonitrogenic and isoenergy diets were designed,which were basal diet(CT),astaxanthin 50 mg/kg group(AS50),50 mg/kg astaxanthin and 50 mg/kg tea tree oil group(AS50+AST50),50 mg/kg astaxanthin and 100mg/kg tea tree oil group(AS50+AST100),50 mg/kg astaxanthin and 200 mg/kg tea tree oil group(AS50+AST200),50 mg/kg astaxanthin and 400 mg/kg tea tree oil group(AS50+AST400)respectively.An 8-week culture experiment was conducted.After astaxanthin was added,the feed conversion ratio of AS50 was significantly decreased(P<0.05),the final average weight,weight gain rate and specific growth rate with the increased trend were no significant differences with the CT group(P>0.05),and the total antioxidant capacity(T-AOC)in hemolymph and intestinal tissue were significantly increased compared with the CT group(P<0.05).The content of malondialdehyde(MDA)in hemolymph and intestinal tissue was significantly decreased(P<0.05).The expression of Crustin,Astacidin,Cu Zn SOD and HSP70 genes was significantly increased compared with the CT group(P<0.05).After tea tree essential oil and astaxanthin was added,the final body weight,weight gain rate,specific growth rate and T-AOC in AS50+AST100 were significantly higher than those in AS50 and CT group(P<0.05).The intestine MDA content was significantly lower than that in AS50 and CT group(P<0.05).The expression levels of Crustin and Astacidin genes in intestinal tissues increased with the increase of tea tree essential oil content,and the combination group of AS50 and AST50 was significantly lower than the other groups(P<0.05).The expression level of Cu Zn SOD was firstly increased and then decreased with the increase of tea tree oil concentration,and the group of AS50+AST50 and AS50+AST100 were significantly higher than those in other groups(P<0.05).The expression of HSP70 were significantly higher than those in CT group(P<0.05).The results showed that adding combination of 50 mg/kg astaxanthin and 100 mg/kg tea tree oil in the diet could improve the antioxidant capacity,immune capacity,and promote the growth of Procambarus clarkii.In conclusion,the diet supplemented with 50 mg/kg astaxanthin and 100 mg/kg tea tree oil significantly improved growth performance,antioxidant capacity and immune-related gene expression of Procambarus clarkii. |
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