Effects And Regulatory Mechanisms Of Dietary Selenium On Fish Muscle Growth

Selenium(Se)is an essential trace element for fish growth and performs its physiological functions mainly through incorporating into selenoproteins.Both Se deficiency and excess are adverse to fish growth.However,the regulatory mechanisms of Se on fish growth are still unclear.Thus,this study aimed to address this issue.Fish skeletal muscle,which is predominantly composed of white muscle,is the largest tissue in fish and represents 40%-60% of fish body mass.It is well known that white muscle growth makes a great contribution to fish somatic growth.Given that,we propose a hypothesis whether Se controls fish somatic growth by regulating the white muscle growth.Skeletal muscle derives from the somites formed from the paraxial mesoderm in a stereotypic rostral to caudal progression.Its basic unit is muscle fiber which contains bundles of myofibrils.Muscle formation,including development in embryonic phase and growth in postembryonic phase,is a complex dynamic process involving both the recruitment of new muscle fibers(hyperplasia)and the subsequent enlargement of existing fibers(hypertrophy).In varius fish species,there exist two growth patterns: indetermined growth and determined growth.The determinate growth type fish present a finite size while the indeterminate growth type fish present no fixed size.The primary difference between these two growth patterns is the postembryonic muscle growth patterns.In indeterminate growth type fish,skeletal muscle presents not only a hypertrophic growth but also a persistence of hyperplastic growth throughout the larval,juvenile even adult stages.However,in determinate growth type fish,skeletal muscle primary presents the hypertrophic growth with very little muscle fiber hyperplasia at postembryonic phase.In this study,rainbow trout(Oncorhynchus mykiss),an indetermined growth type fish,and zebrafish(Danio rerio),a determined growth type fish,have been selected to explore the effects and regulatory mechanisms of Se on fish muscle growth.The main contents and results are as follows: 1.Effects and regulatory mechanisms of dietary Se on muscle growth in rainbow troutWe firstly evaluated the effects of dietary Se level on the growth performance and Se status of rainbow trout by feeding the fish with a basal diet unsupplemented or supplemented with 2,4 and 6 mg/kg Se as Se yeast(dietary Se levels are 1.01,2.58,4.36 and 6.26 mg/kg,respectively).After 10 weeks of feeding,fish growth performance,whole body Se deposition,oxidative status,glutathione peroxidase(GPX)activities and the expression of 28 selenoprotein genes in tissues have been analyzed.Results suggest that the optimal level of Se supplementation(as Se yeast)in rainbow trout diet is 4 mg/kg.At this level,rainbow trout presented the maximum growth rate,lowest oxidative status and optimal Se status.We further explored the effects and regulatory mechanisms of nutritional level of dietary Se on muscle growth in rainbow trout.Fish were fed a basal diet unsupplemented or supplemented with 2 and 4 mg/kg Se(as Se yeast)for 10 weeks.Results showed that trout growth performance and muscle growth were significantly enhanced by dietary Se supplementation and exhibited significant correlation with each other(P < 0.05).Histological characterization of whole body cross section at the vent level showed that trout muscle growth throughout the whole feeding trial was implemented by both hyperplasia(contributed 53.77%-56.56%)and hypertrophy(contributed 43.44%-46.23%)of muscle fibers.Although both hyperplasia and hypertrophy of white muscle fibers were accelerated by dietary Se supplementation,the elevated contribution of hypertrophy to white muscle growth in trout fed diets with Se supplementation suggestes a greater influence of dietary Se on hypertrophic growth than hyperplastic growth of trout white muscle.Molecular results showed that dietary Se supplementation exerted no influences on protein synthesis and autophagy-mediated proteolysis in trout white muscle,whereas,it led to the inhibition of calpain-and ubiquitin-mediated proteolysis and the accelerated fusion of myoblasts into the existed muscle fibers.Furthermore,dietary Se supplementation did not affect the levels of thyroxine(T4)and 3,5,3?-triiodothyronine(T3)in plasma while increased their levels in white muscle.However,no significant differences were observed in the levels of growth hormone(GH)and insulin-like growth factor 1(IGF1)in both plasma and muscle.Additionally,dietary Se supplementation upregulated the expression of 5 selenoprotein(Sel)genes in trout white muscle,and the expression of Sel K and Sel Wl showed significant correlations with all the indicators or factors related to fish somatic growth,muscle growth,muscle protein degradation,and fusion of myoblasts with the existed fibers.Given all this,this study demonstrates that nutritional level of dietary Se inhibits calpain-and ubiquitin-mediated proteolysis and promotes the fusion of myoblasts into the existed muscle fibers to promote hypertrophic growth of trout white muscle,thereby accelerating trout somatic growth.Besides,Sel K and Sel W might be two key selenoproteins involving in the regulation of these processes.Our previous study investigated the effects of nutritional level of dietary Se on protein deposition in trout muscle at the basic metabolic level(fasted for 48 h),however,protein synthesis and degradation in fish tissues are much sensitive to amino acid level and present dymatic changes after a meal.This study aimed to investigate the effects of nutritional level of dietary Se on the postprandial protein deposition in trout white muscle.Fish were fed a basal diet unsupplemented or supplemented with 4 mg/kg Se(as Se yeast).After 6 weeks of feeding,trout fed diet with Se supplementation exhibited a significantly larger body size(weight and length)and a significantly higher protein content of white muscle compared to those fed the basal diet(P < 0.05).Subsequently,trout were fasted for 48 h to obtain the basal levels of metabolites in tissues,and then received a single meal.Protein synthesis and degradation were analyzed before refeeding and 4,8,12 and 24 h after refeeding.Results showed that dietary Se supplementation exerted no significant influences on postprandial translational capacity and translation elongation in trout white muscle,however,it led to a continuously higher phosphorylation levels of translation initiaion related factors including ribosomal protein S6(postprandial 8-12 h)and eukaryotic translation initiation factor 4E binding protein 1(4E-BP1)(postprandial 4-24 h).Furthermore,postprandial changes of factors related to the three main proteolysis systems(calpain system,ubiquitin-proteasome system and autophagy-lysosome system)in trout white muscle exhibited no significant differences between fish fed diet with Se supplementation and the basal diet.The results of the present study suggest that nutritional level of dietary Se promotes and prolongs the target of rapamycin complex 1(TORC1)pathway-mediated protein synthesis,resulting in an accelerated protein deposition in trout white muscle.2.Effects and regulatory mechanisms of dietary Se on muscle growth in zebrafishWe firstly evaluated the effects of dietary Se level on the growth performance and Se status of zebrafish by feeding the fish with a basal diet unsupplemented or supplemented with 0.10,0.20,0.30,0.40 and 0.50 mg/kg Se as Se yeast(dietary Se levels are 0.10,0.22,0.34,0.44,0.57 and 0.69 mg/kg,respectively).After 30 days of feeding,fish growth performance,Se deposition,oxidative status,GPX activity and the expression of 37 selenoprotein genes in whole body have been analyzed.Results suggest that the optimal dietary Se level based on Se yeast for on-growing zebrafish is 0.32-0.37 mg/kg.At this level,zebrafish presented the maximum growth rate,lowest oxidative status and optimal Se status.When dietary Se level was lower than 0.32 mg/kg and higher than 0.44 mg/kg,zebrafish presented Se deficiency and excess,respectively.Se deficiency and excess led to a suppressive growth performance and an elevated oxidative stress for zebrafish.We further explored the effects and regulatory mechanisms of Se status on zebrafish muscle growth.In this study,zebrafish was fed with different dietary Se levels(0.10,0.22,0.34,0.44,0.57 and 0.69 mg/kg)for 30 days,and then muscle growth,muscle growth pattern and muscle growth-related biological processes have been analyzed.Results showed that zebrafish exhibited the maximum rate of muscle growth under the optimal Se status(dietary Se level was 0.32-0.37 mg/kg),while exhibited the declined muscle growth rate under the status of Se deficiency and excess(dietary Se level was lower than 0.32 mg/kg and higher than 0.44 mg/kg,respectively).Correlation analysis showed that the growth rate of zebrafish was positively correlated with the muscle growth rate(P < 0.01).Histological characterization of whole body cross section at the vent level showed that,throughout the whole feeding trial,zebrafish muscle presented a hypertrophic growth(contribution rate of hypertrophy was 78.48%-85.51%)and exisisted less muscle fiber hyperplasia(the total number of muscle fibers increased by 11.63%-15.81%,the contribution rate of hyperplasia was 14.49%-21.52%).Se status exerted no effects on the fusion of myoblasts into the existed muscle fibers,whereas it promoted protein deposition in zebrafish muscle.Compared with the optimal Se status,Se deficiency resulted in the suppressive protein synthesis and elevated autophagy activity.Furthermore,analysis of the hormone levels of T4-T3-GH-IGF1 axis in zebrafish muscle showed that the changes of T4,T3,GH and IGF1 levels in zebrafish muscle under different Se status were not consistent with the growth of zebrafish muscle.Analysis of the expression of 37 selenoprotein genes in zebrafish muscle showed that the increase of dietary Se level significantly increased the expression of 4 selenoprotein genes(GPX1a,GPX4 a,Sel U1 a and Sel W1).Correlation analysis showed that the expression of Sel W1 was significantly correlated with the somatic growth and muscle growth of zebrafish(P < 0.05)when fish were fed nutritional level of Se(dietary selenium level was 0.10-0.34 mg/kg).The present results suggest that Se status is closely related to muscle growth in zebrafish,both Se deficiency and excess are adverse to muscle growth.In addition,this study preliminarily revealed the regulatory mechanism of nutritional level of dietary Se on muscle growth in zebrafish: upregulating the expression of Sel W in muscle,promoting protein synthesis and inhibiting autophagy-mediated protein degradation,thereby promoting the hypertrophic growth of zebrafish muscle,thus resulting in an enhanced growth performance of zebrafish.Furthermore,the present results suggest that Se exess inhibits the muscle growth in zebrafish by inhibiting the hypertrophic growth.However,the underlying regulatory mechanisms are still unclear.In summary,this study preliminarily demonstrates a general regulatory mechanism that nutritional level of dietary Se regulates fish muscle growth by upregulating the expression of Sel W,accelerating the protein deposition,thereby promoting the hypertrophic growth of fish muscle.The results of this study could enrich our understanding of the nutritional function of Se in fish,and provide an important foundation for the further studies about the regulation mechanisms of Se on fish growth performance.