| Vitamin E is an essential nutrient for most aquatic animals.It cannot be synthesized independently in the body and must be supplemented by exogenous sources.Studies have shown that vitamin E deficiency or excess in feed formula will cause adverse effect to some fish.It is of great significance to study the vitamin E requirement of aquatic animals so as to benefit the development of healthy and sustainable aquaculture.Epinephelus fuscoguttatus?×Epinephelus lanceolatus?was selected as the object to investigate the effects of vitamin E on the growth and immunity of aquatic animal based on levels of physiological and biochemical,histological observation and transcriptome.The results were below:(1)Six diets with different levels of vitamin E(actual vitamin E content measured by high performance liquid chromatography were 12.10,32.15,45.17,75.72,135.78,263.37 mg/kg,respectively)were designed to carry out a 84-day feeding trial(feeding trial for short).The results showed that appropriate amount of vitamin E could significantly improve the fish(average initial weight:20.48±0.20 g)weight gain,survival rate,growth and development of immune organs,promote muscle quality,maintain the dynamic balance of vitamin E content in liver,muscle and serum and meet the demand of body growth,reduce the content of blood fat to maintain the body health,enhance the immunity and antioxidant defences of serum,head kidney,spleen and liver,improve the absorption of nutrients in intestines and liver,reduce lipid peroxidation reaction.Significant difference of growth performance and immunity was found between in the control group and 135.78 mg/kg dietary vitamin E group.Meanwhile,compared with other tissues,the liver and foregut were the two most sensitive tissues response to vitamin E in fish digestion and the head kidney was the most sensitive tissue in that of immunity.Weight gain as an indicator to assess the optimal supplemental amount of vitamin E was 130.13 mg/kg.(2)Dietary vitamin E with 135.78 mg/kg could maintain the structure and function of liver cells,facilitate the growth of intestinal tissue and maintain its integrity,increase the secretion of immune-related substances in the head kidney to elevate the body's immunity,and improve the non-specific immunity of fish by regulating the number of lymphocytes in the spleen.(3)Two groups consisted of control group and the optimal supplemental vitamin E group(actual vitamin E content:10.38 and 132.15 mg/kg,respectively)were set and the fish(average initial weight:80.48±3.30 g)were fed with the corresponding diets for 70 days,at last,all fish were infected with half lethal dose(8.86×10~6CFU/ml)of Vibrio harveyi(bacterial infection trial for short).Results showed that vitamin E could improve the antioxidant capacity and disease resistance of body according to the change of immunity and antioxidant capacity between-in the experimental group and the control group before and after bacterial infection in serum,liver,head kidney and spleen.Histological observation further suggested that vitamin E played an important role in protecting the liver,head kidney and spleen against bacterial infection.(4)The mRNA analysis results of the feeding trial showed that vitamin E induced 366,69and 9023 differentially expressed genes in liver,foregut and head kidney,respectively.These differentially expressed genes can be mainly enriched in the pathways related to apoptosis,metabolism and immunity in liver,metabolism in foregut,while immunity in head kidney.(5)The mRNA analysis of the bacterial infection trial showed that fish intaking appropriate amount of vitamin E could resist against the invasion of Vibrio harvey by improving the metabolic compensation in the liver.However,the inflammation and injury of vitamin E-deficient fish would be further aggravated after this invasion.The immune related pathways of head kidney in fish fed with appropriate amount of vitamin E could be highly activated once infected with Vibrio harvey,and then played an important role in the immune defense system,while fish with vitamin E deficiency would be probably induced autoimmune disease after Vibrio harvey infection,resulting in pathological increase of some enzymes secretion and certain genes expression.(6)Results of miRNA analysis in the feeding trial showed that vitamin E might affect metabolism and immunity of liver mainly by regulating the expression of mi R-34-x.The mi R-122-x together with mi R-735-x could be regulated to maintain the intestinal health so as to provide a good internal environment for the normal digestion and absorption of nutrients.Immune and antioxidant capacities of the head kidney were regulated by mi R-1357-x and miRNA-31-x.The miRNA analysis of the bacterial infection trial showed that vitamin E might regulate the different expression of mi R-1246-x,mi R-1260-x,mi R-7133-x(and y),mi R-29-x(and y),mi R-122-x,etc to affect the immune capacity of the head kidney against the bacterial infection.(7)The miRNA-mRNA network analysis of the feeding trial showed that vitamin E could up regulate the expression of mi R-551-x,mi R-3604-x and mi R-193-y,as well as down regulate the expression of mi R-883-y,novel-m0222-3p and mi R-34-x to maintain liver cell function and improve metabolism and immune capacity.The immunity and antioxidant capacitiy of the head kidney were affected by down regulating the expression of mi R-31-x,mi R-735-x,mi R-1357-x,etc,as well as some novel miRNAs,such as novel-m0200-5p,novel-m0183-3p,novel-m0184-5p,etc.The result of miRNA-mRNA network analysis in bacterial infection trial revealed that vitamin E might mainly regulate the anti-invasion of Vibrio harvei in the head kidney by up regulating the expression of mi R-1260-x,mi R-7133-x(and y),mi R-1246-x,etc.and down regulating the expression of mi R-129-x,mi R-122-x,mi R-735-x,etc.In conclusion,vitamin E plays an important role in regulating growth and immunity of Epinephelus fuscoguttatus?×Epinephelus lanceolatus?.To some extent,this study revealed the functional essence of vitamin E in promoting growth performance and immunity and enriched the bioinformatics data of this fish. |
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