Effects Of Vitamin A On Lipid Metabolism And Ovary Development Of Chinese Mitten Crab(Eriocheir Sinensis)

Lipid metabolism has always been a hot topic in aquatic nutrition research.Vitamin A is a essential fat-soluble vitamin,which plays important roles in animals lipid metabolism.In aquatic animals,the research scope of vitamin A is mainly related to the requirement,growth and reproduction,immune enhancement,health maintenance and other aspects,and there is no systematic research on the relationship between vitamin A and lipid metabolism.The Chinese mitten crab Eriocheir sinensis is an important economic breeding species.In its growth cycle,the rapid growth of juvenile crab and the gonad development of adult crab are both important research directions in nutrition.In this study,the relationship between vitamin A supplement and lipid metabolism of crab was systematically studied in combination with field breeding experiment and laboratory indices analysis.Firstly,the optimal requirement of vitamin A in juvenile crab was obtained by gradient experiment design,and the important functions of vitamin A in lipid metabolism,immunity and antioxidant capacity of crabs were further verified by transcriptomics.Subsequently,based on the results about vitamin A requirement and problems in practice,through the studies about vitamin A addition under different lipid levels and oil sources,as well as the experimental about vitamin A supplement in diets with fish oil replaced by vegetable oil source,we preliminarily define how vitamin A improves the utilization of high-fat feeds,and the positive effect of vitamin A in improving the utilization of vegetable oil sources.Finally,we also discussed the relationship between vitamin A and the accumulation of fatty nutrients in the adult stage of crabs,and further studied the important role of vitamin A in ovarian development.The results of this study not only provide a reference for the exploitation of complete formula feeds of crabs,but also enrich the fundamental data about vitamin A in crustaceans nutriology.The main results and conclusions are as follows:1.Dietary vitamin A requirement of juvenile Eriocheir sinensisThe aim of this experiment was to investigate the effect of different levels of vitamin A on growth,antioxidant,hepatopancreas health and lipid metabolism of juvenile crabs(0.84±0.004 g).Six diets(isonitrogenous and isoenergetic)were prepared by adding vitamin A into the basic diet(the actual gradients were 0-control group,2322,4511,8478,14333,30444 IU/kg),which were fed to the juvenile crabs for 8 weeks.The results showed that vitamin A content in the whole body of crabs was increased as dietary vitamin A increased.Compared with the control group,the weight gain rate and specific growth rate were obviously improved in any vitamin A supplementation groups.Vitamin A level of 8478 IU/kg promoted the hemolymph alkaline phosphatase activity of juvenile crabs.And the activities of hemolymph alanine transaminase and aspartate transaminase significantly decreased with the increase of vitamin A concentration(linearly and quadratically changed).Vitamin A levels of 4511,8478 and 30444 IU/kg all increased the activities of antioxidant enzymes,and dietary vitamin A supplementation also significantly reduced the hepatopancreas malondialdehyde content.Hepatopancreas index,body fat,total fat content,triglyceride and free fatty acid in the hepatopancreas and triglyceride content in the hemolymph showed linear and quadratic decreasing trend with the increase of vitamin A level.Transcriptome sequencing results showed that compared with the control group,30444IU/kg vitamin A was involved in the regulation of lipid metabolism,and achieved lipidlowering effect by inhibiting lipid synthesis,promoting hydrolysis and β-oxidation.Vitamin A also upregulated the expression of some antioxidant and immune-related genes.In summary,dietary vitamin A could improve the growth,immunity,antioxidant capacity and hepatopancreas health of juvenile crabs,and reduce lipid deposition in the hepatopancreas and whole body.Based on the weight gain rate and specific growth rate,the vitamin A requirement of juvenile crabs is 5340-5540 IU/kg.2.Effects of dietary vitamin A on growth,antioxidant and lipid metabolism of juvenile crab under different lipid levelsA 2×2 two-factor design was used in this experiment,four groups of isonitrogenous and isoenergetic diets were prepared including two vitamin A levels(0and 6000 IU/kg)and two fat levels(7 and 13%),which were fed to juvenile crabs(3.95±0.12 g)for 8-week.It was showed that the interaction between vitamin A and lipid level significantly affected the weight gain rate and specific growth rate of juvenile crabs.The growth performance of juvenile crabs in 7% lipid level supplemented with vitamin A was the optimal,and there was no significant difference among this group and the high lipid level groups.Vitamin A increased the activity of relevant antioxidant indices under different lipid levels.Vitamin A can also reduce the increase of hepatopancreas malondialdehyde content caused by high fat diets.In the high fat groups,dietary vitamin A decreased hepatopancreas index,total lipid content in the whole body and hepatopancreas of crabs,hepatopancreas triglyceride and free fatty acid contents.Vitamin A alleviates the accumulation of fat in hepatopancreas by promoting the process of lipid β-oxidation and transport,and inhibiting the synthesis of lipid under high fat diets.The injection experiments in vivo also showed that retinoic acid had the ability to reduce triglyceride content in the hepatopancreas of crabs.Si RNA interference of RXR showed that the expression of genes related to lipid synthesis and decomposition was significantly down-regulated.In this study,dietary supplementation of vitamin A can alleviate lipid peroxidation induced by high fat diets and promote the utilization of high fat diets by optimizing fat metabolism in juvenile crabs.Vitamin A may be involved in the regulation of lipid metabolism through the retinoic acid-RXR pathway.3.Research about the relationship between dietary vitamin A and the utilization of vegetable oil sources3.1 Effects of dietary vitamin A supplementation under different vegetable oil sources on growth,antioxidant,inflammation and lipid metabolism of juvenile crabA 3×2 two-factor design was used in this experiment,six experimental diets were prepared with or without vitamin A supplement(6000 IU/kg)in three different vegetable oil sources(coconut oil,olive oil and perilla oil),which were fed to juvenile crabs(0.71±0.01 g)for 8 weeks.It was revealed that the interaction between vitamin A and lipid sources did not affect the specific growth rate,weight gain rate and the final body weight.The growth performance of juvenile crabs in coconut oil group was the worst,and hepatopancreas inflammation was more severe than other groups.Compared with other oil sources,the growth performance of juvenile crabs in perilla oil group was the best,and the body lipid content and hemolymph triglyceride content were the lowest.In addition,the contents of malondialdehyde and polyunsaturated fatty acid in hepatopancreas of crabs fed perilla oil were higher than others.However,vitamin A supplementation enhanced the antioxidant activity of juvenile crabs under olive and perilla oil,alleviating lipid peroxidation problems caused by perilla oil.In addition,vitamin A inhibited fat synthesis under coconut oil as well as promoted fat absorption.Supplementation of vitamin A under different oil sources improved decomposition of fat and reduce triglyceride content in hepatopancreas.It was also found that vitamin A contributed to the accumulation of n-3HUFA under all three oil sources.Supplementation of vitamin A in olive oil can reduce the total saturated fatty acid content of hepatopancreas,and supplementation of vitamin A in perilla oil can reduce the total monounsaturated fatty acid content of hepatopancreas.In summary,supplementation of vitamin A under different oil sources can improve the healthy problems of juvenile crab caused by vegetable oil and positively influence the utilization of fatty acids by optimizing fat metabolism,and improve the growth performance of juvenile crabs.3.2 Effect of vitamin A addition on growth performance,immunity,antioxidant,inflammation and lipid metabolism in juvenile crab fed diet with fish oil totally replaced by palm oilIn this study,three experimental diets were designed with fish oil,palm oil and palm oil +6000 IU/kg vitamin A were used as the main oil sources,respectively,which were fed to juvenile crabs(0.69±0.01 g)for 8 weeks to further evaluate the effect of vitamin A supplementation in vegetable oil which was used to replace fish oil.The results showed as follows: compared with fish oil group,the feed utilization efficiency and growth performance of palm oil group decreased significantly.Palm oil also significantly increased the total fat content of hepatopancreas and the whole body,and triglyceride content in hepatopancreas,and decreased the antioxidant capacity and immune enzyme activities,damaging the health of hepatopancreas.Compared with palm oil without vitamin A addition,the supplementation of vitamin A in palm oil can inhibit the synthesis of fat,promote the decomposition of fatty acids and reduce the deposition of hepatopancreas fat.Supplementation of vitamin A in palm oil significantly decreased the deposition of C16:0 and C18:1n-9 in hepatopancreas,and increased the accumulation of n-3HUFA.In addition,the supplementation of vitamin A in palm oil down-regulated the expression of Kelch-like epichlorohydrin associated protein-1 and up-regulated the expression of antioxidant genes such as catalase and cuzn-superoxide dismutase,thereby alleviating the decreased antioxidant capacity caused by palm oil.Vitamin A also upregulates the expression of immunity related genes and reduces the expression of pro-inflammatory factors through Toll2-My D88(myeloid differentiation factor)-Relish pathway.The results of this chapter indicate that vitamin A can be used as a functional nutrient to improve the utilization of vegetable oil source,alleviate the negative effects caused by vegetable oil,and promote the growth of crab.4.Effects of dietary vitamin A on gonadal development and lipid metabolism of female crabsThis study was conducted to investigate the effects of vitamin A on the growth,lipid metabolism and ovarian development of female crabs(74.44±0.81 g).Three kinds of isonitrogenous and isoenergetic diets supplemented vitamin A levels of 0(control group),8000,and 30000 IU/kg(test value-0、7878、29420),respectively,which were fed to the female crabs(74.44±0.81 g)before reproductive molting for 90 days.The results showed that 7878 IU/kg vitamin A increased the gonadosomatic index,hemolymph vitellogenin content and promoted the deposition of ovarian vitellogenin granules.Vitamin A may induce the expression of vitellogenin gene by up-regulating the transcription of key nuclear receptor factors such as ecdysone receptor,retinoid X receptor,and ecdysone early response gene E75,thus promoting vitellogenesis.In addition,vitamin A also optimized fat metabolism to promote the accumulation of lipids in tissues.It specifically promoted the absorption of fat by hepatopancreas and ovaries,enhanced the lipid synthesis in hepatopancreas and the transport of hepatopancreas fat to the ovaries,and inhibited the decomposition of hepatopancreas lipid.7878 IU/kg vitamin A also improved the hemolymph immune and antioxidant capacity and promoted the body’s health.However,the results of this study showed that 29420 IU/kg vitamin A had a negative effect on both the antioxidant system and yolk particle deposition.In conclusion,the optimal dietary vitamin A content(7878IU/kg)could optimize lipid metabolism of female crabs and promote ovary development.In conclusion,dietary proper vitamin A supplementation can improve the growth,immune and antioxidant capacity of juvenile crabs,and improve the utilization of high fat diet(13% level)and different vegetable oil sources such as palm oil,coconut oil,olive oil and perilla oil.In the female crabs,appropriate vitamin A supplementation contributes to the deposition of fat nutrients and yolk protein,promoting the ovary development.