The Analysis Of Effects And Mechanisms Of Dietary ARA On Sex Steroid Hormone Regulation Of Stage Ⅱ Female Chinese Sturgeon

Sturgeons of the order Acipenseriformes are the only extant large-size Chondrostei species in Osteichthyes.They have the characteristics of large individuals,long lifespan,and long development cycle.Sturgeon fossils have been found in the late Jurassic or early Cretaceous strata in northern Hebei,China,which indicates that some physiological characteristics of sturgeon may have been maintained for more than 100million years,which is of great scientific research value.Most of the 27 species of sturgeon in the world are endangered at different levels due to the superimposed interference of various activties such as hydroletric project,overfishing,shipping and pollution.However,Chinese sturgeon,known as“giant panda in water”,has become an extremely dangerous species.During the 5-year,no natural reproduction behavior has been detected.Five years of scientific research survey,no natural reproduction behavior has been detected,and less than 50 spawning populations have migrated to the dam,and the wild population is on the verge of extinaction.A number of Chinese sturgeon populations have been conserved under artificial ex situ protection measures.The artificial propagation of the Chinese sturgeon has alreay been successful,but with limited population of females for propagation,which hampers the speed of populations recover.Based on this,this paper creates abiotic environmental conditions for the life of Chinese sturgeon in the Yangtze River and constructs suitable water environmental conditions under artificial environmental conditions.The F₂ generation Chinese sturgeon with fourth-year-old were fed with experimental diets in a controlled micro-flow aquaculture pond for 22 weeks to investigate whether arachidonic acid could regulate the synthesis of steroid hormones in Chinese sturgeon and the possible regulatory mechanism,and whether it has any damage to the growth,survival,body safety and health of the cultured Chinese sturgeon.Further investigate whether arachidonic acid has the effect of regulating oocyte viability,hormone synthesis and steroid hormone metabolism pathway-related gene transcription under in vitro conditions,and the possible regulatory mechanism.The results of this paper organically integrated the abiotic environment,water environment and Chinese sturgeon safety.The results will be helpful to understand the endocrine regulation mechanism of gonad development of Chinese sturgeon,and provide theoretical basis and technical support for the regulation of sturgeon ovary development.The main results of this paper are su mmarized as follows:1.An 22-week feeding trial was performed to evaluate the effects of dietary ARA on the growth performance,morphological parameters,nutritional composition,tissue ARA metabolism enzyme activities,tissue ARA metabolites,tissue sex steroid hormones and vitellogein contents in F₂generation Chinese sturgeon.Four diets with different ARA levels（0.0%,0.5%,1.0%and 2.0%in the diets）were formulated.The results showed that dietary ARA did not affect weight gain rate（WGR）,feed conversion ratio（FCR）,survival rate（SR）,hepatosomatic index（HSI）,viscerosomatic index（VSI）,condition factor（CF）and gonadalsomatic index（GSI）（P>0.05）.The contents of crude protein,crude lipid,crude ash and and moisture in muscle were not affected by dietary ARA（P>0.05）.The contents of crude lipid and moisture in liver were not affected by dietary ARA（P>0.05）,crude protein in the 2.0%ARA diet was significantly higher than that the 0.0%ARA diet（P<0.05）,but no differences were observed among the ARA-supplemented diets（P>0.05）.The contents of ARA in liver,muscle and ovary increased significantly with increasing dietary ARA（P<0.05）.In the liver,the activities of cyclooxygenase-1（COX-1）,cyclooxygenase-1（COX-2）,lipoxygenase（LOX）and cytochrome P450(CYP₄₅₀)of the 0.0%ARA group were significantly lower than the 2.0%ARA group（P<0.05）.In the ovary,the activities of COX-1,COX-2,LOX and CYP₄₅₀of the 0.0%ARA group were significantly lower than those of the 1.0%and 2.0%ARA groups（P<0.05）.In the muscle,the activities of COX-1,COX-2,LOX of the 1.0%ARA group were significantly higher than the 2.0%ARA group（P<0.05）;and also the CYP₄₅₀ activity of the 1.0%ARA group was significantly higher than that of the 0.0%ARA group（P<0.05）.In the liver and ovary,the metabolites of prostaglandin E₂（PGE₂）and 5-hydroxyeicosatetraenoic acid（5-HETE）increased significantly with increasing dietary ARA（P<0.05）.In the muscle,the contents of the two metabolites in the 1.0%ARA diet were significantly higher than that the 0.0%and 2.0%ARA diets（P<0.05）.Significantly decreased liver and ovary E₂ and T levels were found in the 0.0%ARA diet compared with 2.0%ARA diet（P<0.05）.In the ovary,the contents of vitellogen（Vtg）increased significantly with increasing dietary ARA（P<0.05）.In the liver,the contents of E₂,T and Vtg in the0.0%ARA group were significantly lower than that of the 1.0%and 2.0%ARA groups（P<0.05）.The E₂ contents in the 1.0%ARA group was significantly higher than that of the 0.0%ARA group（P<0.05）.The serμM contents of T were not affected by the dietary ARA（P<0.05）.Vtg contents in the serμM of 0.0%ARA group was significantly lower than that of the supplementation with ARA groups（P<0.05）.The above results showed that the supplementation of ARA to the diet had significant effects on the ARA deposition in different tissues,the activity of ARA metabolic enzymes in different tissues,the content of ARA metabolites in different tissues,the synthesis of hormones and the content of Vtg in different tissues of the F₂ generation Chinese sturgeon;Considering the ARA roles and economic cost,it is suggested that including 1%ARA to the diet is the most appropriate.2.In order to explore the response mechanism of the F₂ generation female Chinese sturgeon to different ARA diets after 22 weeks of feeding four ARA supplemented diets.Transcripts in ovary,lipid metabolites in serum and liver lipid metabolism enzymes were analyzed of the F₂ generation female Chinese sturgeon.A total of 1018differentially expressed genes（DEGs）,853 DEGs,2680 DEGs,303 DEGs,1185 DEGs and 409 DEGs were identified in the 0.5%ARA group versus 0.0%ARA group,1.0%ARA group versus 0.0%ARA group,2.0%ARA group versus 0.0%ARA group,1.0%ARA group versus 0.5%ARA group,2.0%ARA group versus 0.5%ARA group and2.0%ARA group versus 1.0%ARA group comparisions,respectively.Gene ontology（GO）showed that the DEGS between the ARA-supplemented diet group and the unsupplemented diet group were enriched in regulation of cell development process,cell process regulation,signal transduction,multicellular biological process,multicellular organ development,biological process regulation and reproduction.Four KEGG pathways involved in the steroid hormone biosynthesis and cholesterol biosynthesis in lipid metabolism and ovarian steroidogenesis as well as the nuclear-initiated estrogen signaling pathway in endocrine system were enriched.Furthermore,weighted correlation network analysis（WGCNA）identified six hub genes that regulate gonad development and cholesterol synthesis,namely,mitogen-activated protein kinase（MAPK14）,cyclin-dependent kinase 2（CDK2）,heat shock protein 8（HSPA8）,two forkhead boxes（FOXP3 and FOXJ3）and cytochrome P₄₅₀ sterol 14α-demethylase（CYP51A1）.Blood lipid analysis showed that 8 of the 9cholesterol ester（CE）in the 2.0%ARA group were down regulated compared with the0.0%ARA group,they are CE（18:1）,CE（19:1）,CE（20:1）,CE（22:1）,CE（17:2）,CE（19:2）,CE（20:5）and CE（22:5）;one CE is up-regulated,which is CE（20:4）.Similarly,compared with the 0.0%ARA group,all eight CE are down regulated,which are CE（18:1）,CE（19:1）,CE（20:1）,CE（17:2）,CE（19:2）and CE（17:2）,CE（20:2）,CE（20:5）,CE（22:5）.The activities of hepatic lipase（HL）,lipoprotein lipase（LPL）,lipase and total lipase in the liver increased significantly with increasing dietary ARA（P<0.05）.The contents of triglycerides（TG）,total cholesterol（TC）,low density lipoprotein cholesterol（LDLC）in the blood decreased significantly with increasing dietary ARA（P<0.05）.TG contents in the liver in 2.0%ARA group were significantly higher than that of the 0.0%,0.5%and 1.0%ARA groups（P<0.05）.In conclusion,the molecular mechanism by which ARA regulates the synthesis of steroids,may be related to enhanced cholesterol ester metabolism and facilitation of steroidogenesis gene transcription,which ultimately benefits ovary development in F₂ generation Chinese sturgeon.3.In order to explore whether the organ of F₂generation Chinese sturgeon is safe,healthy,and whether intestinal microbiota is stable after 22 weeks of feeding four ARA supplemented diets in the micro flow concrete ponds.Further evaluation on blood biochemistry,morphological characteristics of intestine and liver,tissue antioxidant capacity and intestinal microorganisms of the F₂ generation Chinese sturgeon was conducted.The results showed that in fish fed with the 0.0%ARA diets,the activities of aspartate aminotransferase（AST）and alanine aminotransferase（ALT）in the blood was significantly higher than that of the 1.0%ARA diet group（P<0.05）.The contents of total protein（TP）,albumin（ALB）and globulin（GLO）in the blood in 1.0%ARA group was significantly lower than that of the 0.0%ARA group（P<0.05）.Blood of the ratio of A/G and AST/ALT as well as Glu contents were not affected by the dietary ARA（P<0.05）.Different ARA diets did not cause pathological phenomena,such as intestinal mucosa abscission,congestion and increase of inflammatory infiltrating cells in the F₂generation Chinese sturgeon,nor dit it cause pathological phenomena,such as liver cell swelling,vacuolization and increase of inflammatory cells in the F₂generation Chinese sturgeon.In the liver and muscle,the activities of superoxide dismutase（SOD）,catalase（CAT）and glutathione S-transferase（GST）were significantly increased with dieatry ARA content increasing（P<0.05）,while the contents of malonic aldehyde（MDA）was decreased with dieatry ARA content increasing.The activities of serum SOD,CAT,GST as well as the contents of MDA were not affected by the dietary ARA（P<0.05）.The Chao1 index,ACE index,Shannon index and observed-species number of the 0.0%ARA group were significantly higher than those of the 0.50%ARA group（P<0.05）,and there were no significant differences among the 0.0%,1.0%and 2.0%ARA groups（P>0.05）.Simpson index and PD-Whole tree index were not affected by the dietary ARA（P>0.05）.Fusobacteriota,Firmicutes and Proteobacteria are the dominant bacteria under the different ARA diets;CetobacteriμM,RμMinococcus＿gnavus＿group and Streptococcus may be the dominant bacteria under the different ARA diets.The results showed that ARA included to the diets had significant effects on the serμM biochemical indexes,tissue antioxidant capaity and intestinal microbialαdiversity as well as intestinal microbial communities of the F₂ generation Chinese sturgeon;however there was no pathological damage to liver and intestine,which may be indicating that ARA level was safe and healthy for the F₂ generation Chinese sturgeon.Considering the above parameters,the inclusion of ARA at the level of 1.0%or above could enhanced the antioxidant capacity of fish tissues and regulated intestinal microbialαdiversity.4.An 22-week feeding results showed that ARA could enhance the expression of transcription level of related genes in steroid hormone synthesis pathway,which was conducive to hormone synthesis.Based on this,ARA was used to induced and incubated F₂ generation Chinese sturgeon oocytes with four years and had gonads in development stage II.To investigate the effects of ARA on cell viability,main metabolites production,steroid hormone synthesis,steroid production gene expression levels in steroid hormone synthesis pathway as well as ARA metabolism and lipid-related genes at different times,so as to elucidate the differences between ARA in vitro and in vivo.The results showed that under different ARA concentration for 24 h incubation,the viability of oocytes induced by 100μM ARA concentration was the best,which was significantly higher than that of 0,200 and 400μM ARA concentration（P<0.05）.Under different ARA concentration for 48 h,the viability of oocytes induced by100μM ARA concentration was still the best,but there was no significant difference between 100μM ARA concentration and 200μM ARA concentration（P>0.05）.The oocytes viability induced by 400μM ARA was the worst,and oocytes viability were lower than 0,100 and 200μM ARA（P<0.05）.After 24 h ARA induction,PGE₂content in 200μM ARA concentration group was significantly higher than that in 400μM ARA concentration group（P<0.05）.The 5-HETE content in 100 and 200μM ARA concentration groups was significantly lower than that in 0 and 400μM ARA concentration groups（P<0.05）.After 48 h ARA induction,PGE₂ content in 0 and 100μM ARA concentration groups was significantly lower than that in 400μM ARA concentration group（P<0.05）.No significant difference were observed in 5-HETE content among all groups（P>0.05）.After 24 h and 48 h ARA induction,E₂ and T contents had no significant difference among all groups（P>0.05）.After 24 h induction and incubation,the expression levels of steroid production genes,including Cyp17a1,Cyp17a2,Cyp19a1,Hsd17b,Ldlr,Adcy4 and Fshr were not affected by different ARA concentrations（P>0.05）,only Cyp11a1 gene was significantly affected by ARA,and400μM ARA significantly reduced the expression of this gene.ARA and lipid metabolism related genes,such as Cbr1,Hrasls2,Adh5 and Cpt1a,were not affected by ARA concentration（P>0.05）.The expression of Pla2g4 gene in 400μM ARA group was significantly lower than that in control group（P<0.05）.After 48 h induction and incubation,the expression levels of steroid production genes,including Cyp17a1,Cyp17a2,Cyp19a1,Hsd17b,Ldlr,Adcy4 and Fshr were not affected by ARA concentration stimulation（P>0.05）,only Cyp11a1 gene was significantly affected by ARA,and 400μM ARA significantly reduced the expression of this gene.ARA and related genes in lipid metabolism,such as Cbr1,Pla2g4,Hrasls2 and Adh5,were not affected by ARA concentration induced stimulation（P>0.05）.Cpt1a gene expression in 400μM ARA group was significantly lower than that in control group（P<0.05）.In conclusion,under in vitro incubation conditions,certain concentration of ARA significantly regulated the activity of the F₂ generation Chinese sturgeon oocytes,the production of main alkanates and sterologenic gene Cyp11a1 as well as ARA and lipid metabolism genes such as Pla2g4 and Cpt1a,but did not significantly regulated hormone synthesis.5.In order to further evaluate whether ARA and HCG play a synergic role and PGE₂acting alone in regulating oocyte activity,production of main metabolites,steroid hormone production,steroid production gene expression levels in steroid hormone synthesis pathway and ARA as well as lipid-related gene expression levels,and whether INDO induction can inhibit the above effects.This chapter intends to carry out the research on the co-induction and regulation of ARA,HCG and INDO,as well as the single regulation of PGE₂.The results showed that the oocyte viability at 100μM ARA and 100 IU/m L HCG concentration was the best,which was significantly higher than that in the control group（P<0.05）.Oocyte viability was the best at 1μM/m L PGE₂concentration,which was significantly higher than that in control group（P<0.05）.Oocyte viability decreased with the increase of INDO concentration,and oocyte viability at 100μM ARA and 100μM/m L INDO concentration was significantly lower than that in control group（P<0.05）.When HCG was combined with 100μM ARA,the content of PGE₂ in HCG group was significantly higher than that in 0 IU/m L HCG group（P<0.05）,but no significant difference were observed among HCG supplemental groups.The 5-HETE content of 10 and 100 IU/m L HCG group was significantly lower than that of 1 IU/m L HCG group（P<0.05）,no significant difference were observed between 0 IU/m L HCG group and 10 IU/m L HCG group（P>0.05）.No significant differences were observed in E₂and T contents among all groups（P>0.05）.When PGE₂ single induction,the content of PGE₂in 10 and 100μM groups was significantly higher than that in 0μM PGE₂group（P<0.05）,but there was no significant difference among 1,10 and 100μM PGE₂ groups（P>0.05）.The production of 5-HETE was not affected by PGE₂ content（P>0.05）.E₂ content at 100μM/m L PGE₂concentration was significantly lower than that in control group（P<0.05）,and T content was not affected by PGE₂ treatment（P>0.05）.When INDO was combined with 100μM ARA,the content of PGE₂ in INDO group was significantly higher than that in 0 IU/m L INDO group（P<0.05）,but there was no significant difference between INDO supplementation groups（P>0.05）.The content of E₂ in control group was significantly higher than that in 10μM/m L INDO group（P<0.05）,and the content of T was not affected by INDO treatment（P>0.05）.The expression levels of steroid production genes,Cyp17a1,Cyp17a2,Hsd17b,Ldlr and Fshr were not affected by ARA concentration induced by 100μM ARA combined with HCG（P>0.05）.The expression levels of Cyp19a1 and Adcy4 in 100μM HCG induction group were significantly higher than those in 10μM HCG induction group（P<0.05）.The expression level of Cyp11a1 gene was significantly lower than that of 1 and 10μM HCG induction groups（P<0.05）.ARA and related genes in lipid metabolism,such as Cbr1,Hrasls2,Pla2g4,Adh5 and Cpt1a were not affected by ARA and HCG concentration induced stimulation（P>0.05）.Single induction of PGE₂,the expression levels of steroid production genes,Cyp11a1,Cyp17a1,Cyp19a1,Cyp17a2,Hsd17b,Ldlr,Fshr and Adcy4 were not affected by PGE₂ concentration induced stimulation（P>0.05）.Similarly,ARA and related genes in lipid metabolism,such as Cbr1,Hrasls2,Pla2g4,Adh5 and Cpt1a were not affected by PGE₂ concentration induced stimulation（P>0.05）.In combination with ARA and INDO,the expression levels of steroid production genes,Cyp11a1,Cyp17a1,Cyp19a1,Cyp17a2,Hsd17b,Ldlr,Fshr and Adcy4 were not affected by ARA and INDO concentration induced stimulation（P>0.05）.Similarly,ARA and related genes in lipid metabolism,such as Cbr1,Hrasls2,Pla2g4,Adh5 and Cpt1a were not affected by ARA and INDO concentration induced stimulation（P>0.05）.In sμMmary,under the conditions of vitro incubation,co-induction of 100μM ARA with a certain concentration of HCG and INDO,as well as PGE₂ single induction could regulate the activity of oocyte,the production of main alkanoic acids and the synthesis of steroid hormones,and regulate the Cyp11a1,Cyp19a1,Adcy4 genes induced by 100μM ARA and HCG.6.1-year ARA feeding had no significant effects on final body weight,hepatosomatic ratio,viscerosomatic ratio,condition factor and gonadalsomatic index of the F₂generation female Chinese sturgeons（P>0.05）.The levels of E₂,T and Vtg in serμM and the synthesis of E₂and Vtg in ovary were not significantly affected by ARA（P>0.05）.The T content in 2.0%ARA group was significantly higher than that in 0.0%ARA group,but there was no significant difference between 2.0%ARA group and1.0%ARA group（P>0.05）.The contents of PGE₂ and 5-HETE in muscle and liver were not significantly affected by dietary ARA（P>0.05）.The contents of PGE₂and5-HETE in 1.0%ARA group were significantly higher than those in 0.0%ARA group（P<0.05）,but there was no significant difference between 1.0%ARA group and 2.0%ARA group（P>0.05）.The ARA feeding results at 22 weeks showed that the diameter of oocytes increased with the increase of dietary ARA level,and the diameter of oocytes in 2.0%ARA group was the largest,but there was no significant difference among all treatment groups（P>0.05）.With the extension of feeding time,the oocytes of the F₂ generation Chinese sturgeon were still developing.At different time points,the diameter of oocytes under the same ARA treatment was significantly increased（P<0.05）.In conclusion,after 1 year of feeding,the development of oocytes in the F₂generation Chinese sturgeons was not inhibited by 1%or 2%dietary ARA,and oocytes were still developing.