Regulation Of Lipid Metabolism And Ovarian Development By Myo-inositol In Chinese Mitten Crab (Eriocheir Sinensis)

Myo-inositol,as a vitamin-like functional nutrient,has been widely used in aquatic feeds.Numerous studies have shown that myo-inositol has important regulatory effects on growth,antioxidant capacity,immunity and lipid metabolism in aquatic animals.In addition,metabolites of myo-inositol such as inositol triphosphate can be involved in the process of ovarian development of animals.However,the physiological functions of myo-inositol in crustaceans at different growth stages have been rarely reported.Therefore,the present study was conducted to systematically investigate the effects of dietary myo-inositol on lipid metabolism and ovarian development in E.sinensis,an economically important crustacean in China,through nutritional,histological and molecular biological methods.Firstly,a gradient nutrient addition experimental design was used to determine the appropriate proportion of myo-inositol in the diet of E.sinensis and the negative effects of dietary myo-inositol deficiency on various physiological functions of juvenile E.sinensis.Subsequently,based on the basic physiological functions of myo-inositol and the appropriate addition level of myoinositol in the diet,the study was conducted to investigate the regulatory effects of myoinositol on lipid metabolism of E.sinensis under different dietary lipid levels or lipid sources.Finally,considering the physiological functions of myo-inositol and the active lipid metabolism of the female crab at the fattening stage,the study was conducted to investigate the effects and possible regulation of dietary myo-inositol on ovarian development and lipid metabolism of female crab.The main findings and conclusions are as follows.1.The optimum dietary myo-inositol level and its physiological functions in Eriocheir sinensisThis study was conducted to evaluate the optimum myo-inositol(MI)level in the diets and its potential physiological functions in juvenile Chinese mitten crab(Eriocheir sinensis).Crabs was fed with six diets supplemented with graded levels of MI(0,204,406,810,1650,and 3211 mg/kg)for 8 weeks.The results showed that dietary MI significantly affected the growth performance of juvenile E.sinensis,and the maximum values of weight gain and specific growth rate were found in the group with 1650 mg/kg MI in the diet.The crabs fed with diets containing 1650 and 3211 mg/kg MI had significantly lower total lipid content in the whole body and significantly higher MI level in the hepatopancreas than those fed diets without supplemented MI.The crabs fed 1650 mg/kg MI had significantly higher total antioxidant capacity and glutathione peroxidase activity in hepatopancreas than those fed diets without supplemented MI.However,the crabs fed diets without supplemented MI had the highest malondialdehyde level in the hepatopancreas compared with those fed with MI supplementation.The highest serum alkaline phosphatase activity was observed in crabs fed 1650 mg/kg MI.Total protein and hemocyanin contents in the hemolymph were significantly increased with increasing dietary MI levels.Dietary 1650 and 3211mg/kg MI significantly decreased the triglyceride contents in the hepatopancreas compared with dietary 0 mg/kg MI group.Results of gene expression analysis revealed that dietary MI reduced hepatic triglyceride accumulation probably through inhibiting lipid synthesis related genes and promoting lipid export related genes at the transcriptional level.Broken-line analysis of WG,SGR and MI levels in the hepatopancreas against dietary MI levels indicates that the optimal MI requirement for juvenile crabs ranged from 1613 to 1707 mg/kg.The dietary MI supplementation can improve growth performance,antioxidant capacity and nonspecific immunity and reduce hepatopancreatic triglyceride accumulation in juvenile crab.2.Effects of myo-inositol on growth performance and lipid metabolism of juvenile E.sinensis fed different percentage of lipidPrevious results showed that myo-inositol(MI)was closely associated with the lipid metabolism of E.sinensis.In order to investigate whether MI can improve the lipid utilization when dietary lipid level was increased and the regulatory effect of MI on lipid metabolism.Juvenile crabs were fed four diets containing two lipid levels(7%and 13%)and two MI levels(0 and 1600 mg/kg)for 8 weeks.The results showed that dietary MI significantly increased the growth performance when dietary lipid level was13%.The dietary MI could reverse the reduction of MI content in the hepatopancreas,and lipid accumulations in the whole-body,hepatopancreas and muscle as a result of feeding on the high dietary lipid(13%)in crabs.Dietary MI significantly increased the antioxidant capacity,and reduce lipid peroxidation in the hepatopancreas of crabs when dietary lipid level was 13%.Moreover,dietary MI enhanced the expression of genes involved in lipid oxidation and exportation,yet reduced lipid absorption and synthesis genes expression in the hepatopancreas of crabs fed diet with 13% lipid.Meanwhile,the protein expression analysis revealed that IP3R/Ca MKK?/AMPK signalling pathway may be involved in the regulation of lipid metabolism by MI in E.sinensis.This study demonstrates that MI could increase lipid utilization and reduce lipid deposition in the hepatopancreas of E.sinensis fed a high lipid diet.3.Effects of myo-inositol on growth performance and lipid metabolism of juvenile E.sinensis fed different lipid sourcesVegetable oils are widely used in the aquatic feed industry because of their stable source and low price,but they can cause a series of adverse effects on aquatic animals.Myo-inositol(MI)plays a vital role in regulating the health and lipid metabolism of aquatic animals.However,it remains unclear whether MI has a regulatory role on the utilization of different vegetable oils.Juvenile E.sinensis were fed diets with four oil sources,including palm oil(PO),olive oil(OO),safflower oil(SO),and perilla oil(PFO)and two MI levels(0 and 1600 mg/kg)for 8-week to investigate the effects of MI on the growth and lipid metabolism of crabs under different lipid sources.The results found that the best growth performance and the lowest lipid levels in the whole body,hepatopancreas and muscle were observed in crabs fed the PFO diet without MI supplementation.Dietary MI markedly reduced the lipid deposition in the hepatopancreas when crabs were fed the PO and OO diets.Moreover,dietary MI increased the expression of genes related to the uptake and ?-oxidation of fatty acid when crabs were fed the PO and OO diets.The highest malondialdehyde content was observed in the PFO group regardless of the MI level.Dietary MI could decrease malondialdehyde content in the hepatopancreas when crabs were fed the PO and PFO diets.However,the lowest inflammation-related genes expression was observed in crabs fed the PFO diet without MI supplementation.Dietary MI could decrease the m RNA levels of inflammation-related genes when crabs were fed the PO and OO diets.This study indicates that MI is a functional nutrient to reverse the low lipid utilization and inflammatory responses caused by dietary palm oil and olive oil in E.sinensis.4.Regulation of ovarian development by myo-inositol in female E.sinensisThe previous results showed that myo-inositol(MI)plays an important role in the regulation of lipid metabolism and antioxidant capacity in E.sinensis.The ovarian development of animals is generally accompanied by an extremely active lipid metabolism process.Therefore,considering the physiological functions of MI,it may be able to promote ovarian development in crustaceans.Female E.sinensis before reproductive molting were fed diets with 0,1600 and 3200 mg/kg MI supplementation for 13 weeks.The results showed that dietary MI supplementation significantly increased the gonadal index and the accumulation of yolk granules in the ovary of female crabs compared with the group without MI supplementation.Also,dietary MI supplementation significantly enhanced the vitellogenin synthesis in the hepatopancreas,the content of vitellogenin in the hemolymph and the vitellogenin receptor m RNA level in the ovary.According to the results of ecdysteroid content in hemolymph,cyclic adenosine monophosphate(c AMP)content in hepatopancreas,and gene expression of retinoid X receptor(rxr),protein kinase A(pka)and ecdysteroid receptor(ecr)in the hepatopancreas,it can be speculated that MI may promote the synthesis of vitellogenin through c AMP/PKA and Ec R/RXR pathways by affecting ecdysteroid content in the hemolymph.Meanwhile,MI may promote yolk protein deposition in the ovary by up-regulating the expression of vitellogenin receptor.In addition,the present study found that dietary MI supplementation significantly increased the lipid and protein content in the ovary and the protein content in the muscle.The neutral lipid content in the hepatopancreas tended to decrease as the dietary MI level increased,while the polar lipid content in the hepatopancreas and the neutral and polar lipid contents in the ovary tended to increase.Dietary 1600 mg/kg MI supplementation significantly decreased the triglyceride content in the hepatopancreas of female crabs compared with the group without MI supplementation.Similarly,Dietary MI supplementation significantly reduced the cholesterol content in the hepatopancreas.However,dietary MI supplementation significantly increased triglyceride and cholesterol levels in the hemolymph and ovary.The results of lipid metabolism-related gene expression analysis showed that dietary MI supplementation significantly enhanced the fatty acid synthesis in the hepatopancreas,but did not significantly affect the expression level of gene related to triglyceride synthesis.Also,dietary MI supplementation significantly enhanced lipid catabolism,lipid absorption and transport in the hepatopancreas,and those related to lipid absorption in the ovary.In addition,dietary MI increased the proportion of highly unsaturated fatty acids in the hepatopancreas and the proportion of C22:6n-3(DHA)in the ovary.These results suggested that MI may provide energy for vitellogenesis by regulating the mobilization of triglyceride in the hepatopancreas of female crabs,and it may promote the transfer of lipids from the hepatopancreas to the ovary,providing nutrients for ovarian development and enhancing the nutritional value of the edible parts(hepatopancreas and ovary)of female E.sinensis.