Effects Of Tea Tree Essential Oil Supplementation In Low Fish Meal Diet On Growth Performance And Physiological Health Of Largemouth Bass,micropterus Salmoides

In order to solve the problems of resource shortage,high price and dependence on imports of fish meal raw materials in China,a series of new non grain protein sources have been developed as substitutes for fish meal.However,excessive use will cause negative effects on the growth performance and physiological health of aquatic animals.In the face of this problem,one of the effective countermeasures is to select suitable functional feed additives to assist the efficient utilization of low fish meal diet.In this study,we investigated the effects of tea tree essential oil（TTO）on growth performance and physiological health of largemouth bass in a low fish meal diet.To provide theoretical basis and scientific guidance for the application of TTO in low fish meal diet for aquatic animals.Experiment 1:Effects of tea tree essential oil supplementation in low fish meal diet on growth,liver and intestinal health of largemouth bass.0 g/kg（Control）,0.25 g/kg（T0.25）,0.5 g/kg（T0.5）,1 g/kg（T1）,1.5 g/kg（T1.5）,2g/kg（T2）,5 g/kg（T5）and 10 g/kg（T10）TTO was added to the iso-nitrogen and iso-lipid low fish meal diet to feed largemouth bass（initial body weight 15.06±0.05 g）for 8 weeks to study the effects on the growth,digestive enzyme activities,serum biochemical indices,liver and intestinal histomorphology and intestinal flora of largemouth bass.The results showed as follows:With the increase of TTO level in low fish meal diet,the weight gain rate,specific growth rate and protein deposition rate of largemouth bass were firstly increased and then decreased,reaching the highest level in T1 group,while the feed conversion rate was significantly decreased in T1（P<0.05）.The results of the quadratic curve model showed that the weight gain rate was the highest and the feed conversion rate was the lowest when the supplemental levels of TTO were 1.33 and 1.36 g/kg.Compared with the control group,gastric lipase activities in T0.25,T0.5 and T1 groups were significantly increased（P<0.05）,and intestinal trypsin activity was significantly increased in T1 group（P<0.05）.Serum TP and ABL levels in treatment groups were higher than those in control group,while ALT and AST in T0.25,T0.5 and T1 groups were significantly lower than those in control group（P<0.05）.GLU levels in T0.5,T1and T1.5 groups was significantly lower than that in control group（P<0.05）.Liver tissue sections showed that the vacuolization of liver cells was improved in T1 and T1.5 groups,and the degree of liver cell vacuolization was aggravated in T5 and T10 groups,which compared with control group.The midgut of largemouth bass had abundant microvilli,intact brush border,numerous goblet cells and tight connection between muscle layer and lamina propria among all groups.Moreover,the villus width in T0.25 and T1 groups were significantly higher than that in the control group（P<0.05）.When the level of TTO was1 g/kg,the relative abundance of Bifidobacterium,Staphylococcus and Paracoccus were significantly increased（P<0.05）.In conclusion,supplementation of dietary TTO in low fish meal diet could elevate the digestive enzyme activities and serum biochemical indices,improve liver and intestinal tissue morphology and intestinal flora composition to promote the growth of largemouth bass.The appropriate supplementation of dietary TTO in the low fish meal diet of largemouth bass was 1.33-1.36 g/kg.Experiment 2:Effects of tea tree essential oil supplementation in low fish meal diet on lipid metabolism,antioxidant capacity and immunity of largemouth bass.On the basis of experiment 1,the effects of TTO supplementation in low fish meal diet on serum lipid metabolism,liver fatty acid synthesis andβ-oxidation,antioxidant enzyme activities in serum,liver and intestinal,Nrf2-Keap1 antioxidant signaling pathway in liver and intestine,TLR2-MYD88 signaling pathway and inflammatory factor transcription levels in head-kidney,spleen,liver and intestine were investigated.The results showed that:（1）In lipid metabolism parameters,the contents of T-CHO,TG and LDL-C significantly decreased,and the content of HDL-C significantly increased in serum by dietary TTO.The liver crude lipid content was significantly decreased in TTO treatment groups（P<0.05）.Further study showed that mRNA expression levels of ppar-αand cpt1 in TTO groups were significantly increased,while the mRNA expression level of acc-βwas significantly decreased（P<0.05）.（2）In antioxidative parameters,1-2 g/kg TTO decreased the mRNA expression level of keap1,upregulated the mRNA expression levels of nrf2,cat and sod1,and then increased the activities of T-AOC,H₂O₂ and SOD,and reduced the contents of MDA and PC in liver,intestine and serum of largemouth bass.（3）In terms of immunity,the supplementation of 1g/kg TTO in low fish meal diet significantly decreased the expression level of il-1βand up-regulated the expression of tgf-β1 in intestine of largemouth bass infected with Aeromonas hydrophila.The supplementation level of TTO above 5 g/kg significantly increased the levels of tlr2,myd88,il-1βand tnf-αin the head-kidney,liver,intestine and spleen.The expression levels of il-10 and tgf-β1 were decreased,suggesting that high supplemental level of TTO in low fish meal diet may cause tissue inflammation.To sum up,supplementation of dietary TTO in low fish meal diet could promote liver fatty acidβ-oxidation to reduce lipid deposition.Besides,dietary TTO could activate the Nrf2-Keap1 signaling pathway,enhance antioxidant genes expression and antioxidant enzyme activities to promote the antioxidant capacity.Moreover,TTO could promote the expression of anti-inflammatory cytokines,inhibit the expression of pro-inflammatory cytokines to improve the immunity of largemouth bass.Experiment 3:Effects of terpine-4-ol on lipopolysaccharide-induced inflammatory,antioxidant and apoptotic responses of largemouth bass hepatocytesAfter GC-MS detection,it was concluded that the main active component of TTO was terpine-4-ol（relative content 28.8%）.Therefore,in this study,we extracted largemouth bass primary hepatocytes and constructed an inflammation model with lipopolysaccharide（LPS,100μg/m L）to explore the molecular regulation mechanism of terpine-4-ol on antioxidant,inflammation and apoptosis of hepatocytes.The results showed as follows:（1）Terpine-4-ol decreased the mRNA expression levels of tlr2,myd88,nf-κb and il-1β,increased the mRNA expression levels of il-10 to enhanced the anti-inflammatory ability of hepatocytes.（2）Compared with LPS group,terpine-4-ol could reduce the mRNA expression level of keap1,increase the mRNA expression levels of nrf2,cat,sod1 and ho-1,and reduce the ROS content in cells to improve the antioxidant capacity of hepatocytes induced by LPS.（3）Terpine-4-ol decreased the transcription levels of proapoptotic genes fas-l,bax,caspase3,caspase8 and caspase9,and enhanced the antiapoptotic gene bcl-2 transcription level.In conclusion,the terpine-4-ol,which the main active component of TTO could enhance the anti-inflammatory,antioxidant and ability and alleviate apoptosis of hepatocyte induced by LPS.