Effects And Regulatory Mechanisms Of Dietary Selenium Level On Autophagic Degradation Of Triploid Rainbow Trout Muscle Protein

Selenium(Se)is an essential trace element for fish,and the deficiency and excess of dietary Se will influence the growth of fish.At present,the Se requirement of many fish including diploid rainbow trout(Oncorhychus mykiss)has been widely reported,but few studies have focused on the Se requirement and response to dietary Se in triploid rainbow trout.Studies have shown that different ploidy cultivated fish may have different nutritional requirement and feed utilization.The Se requirement of the triploid rainbow trout and the effect of dietary Se level on its body growth were studied in present research.The body growth of the fish is mainly decided by muscle growth,and the deposition rate of protein in the muscle tissue may be the main factor determining the body growth of the fish.There is evidence that the autophagic degradation system of fish muscle protein is influenced significantly by dietary Se levels.Therefore,the possible mechanisms of dietary Se affecting fish growth were also explored from the perspective of autophagic degradation of muscle protein.Therefore,this study is mainly divided into two parts:(1)By supplementing 0(Se0),0.1(Se0.1),0.3(Se0.3),0.5(Se0.5),1.0(Se1.0),4.0(Se4.0)and 16.0(Se16.0)mg Se/kg diet to the low-Se basal diet using Se-yeast as source of Se,the dietary Se requirement of triploid rainbow trout and the response of Se status to dietary Se levels are comprehensively evaluated;(2)Rainbow trout in three Se status(deficient Se,adequate Se and excessive Se)were selected to explore the effect of different Se status on autophagic degradation of muscle proteins.The internal regulation mechanism of Se on the muscle growth of rainbow trout was further explored from the perspective of protein degradation.The main findings are as follows:1.Study on dietary Se requirement and Se status of triploid rainbow troutAfter a 10-week cultivating experiment,through the analysis of the growth performance,oxidation status and Se deposition of liver and muscle,muscle nutrition composition and selenoprotein m RNA level of triploid rainbow trout,it was found that the deficiency and excess of dietary Se were not conducive to its growth.The total content of Se in liver and muscle tissues was increased linearly with the dietary Se levels,and the amount of Se deposition in the liver was much higher than that in the muscle.In terms of nutrition composition in muscle,the crude protein content of rainbow trout in Se4.0 gruop was significantly higher than that in Se0,Se0.1 and Se16.0 groups.The m RNA levels of 28 selenoprotein genes in liver and muscle of rainbow trout were analyzed.It was found that dietary Se supplementation(4.0 and 16.0 mg/kg)significantly increased expression levels of selenoprotein genes in liver(10 genes)and muscle(9 genes).Based on the S-type regression analysis of the m RNA level of the selenoprotein genes differentially expressed in the liver and muscle,the minimum dietary Se requirement was 2.19 or 1.28 mg/kg when the m RNA level of all these selenoproteins reached a plateau in muscle or liver,respectively.When the dietary Se level is greater than 1.28 or 2.19 mg/kg,the requirement of selenoprotein synthesis in rainbow trout liver or muscle can be met,respectively.The growth performance is often used to assess Se requirement of fish.Results showed that the fish fed Se4.0 diet had significantly higher weight gain and lower oxidative stress compared with the fish fed Se16.0 and Se0 diets.The optimal dietary Se requirement of triploid rainbow trout was 3.53 mg/kg based on broken-line regression of growth performance.According to the results of the present study,the response of Se status of rainbow trout to dietary Se level was comprehensively determined: rainbow trout fed Se0 diet was in deficient Se status;rainbow trout fed Se4.0 diet was in adequate Se status;rainbow trout fed Se16.0 diet was in excessive Se status.2.Effects and regulatory mechanisms of Se status on autophagic degradation of rainbow trout muscle proteinAccording to the relationship between Se status of rainbow trout and dietary Se level determined by previous study,the effects of Se status on rainbow trout muscle autophagy level and its regulatory pathways were further studied.The HE sections of white muscle fibers of rainbow trout in three Se status(deficient Se,adequate Se and excessive Se)were observed.There was no significant difference in the number of myogenic fibers in rainbow trout of three Se status groups,but the diameter of rainbow trout muscle fibers in the deficient and excessive Se status groups was significantly lower than that in the adequate Se status groups.Furthermore,it was found that the dietary Se significantly affected the crude protein and total Se content in rainbow trout muscle.In addition,the rainbow trout in adequate Se status had the highest crude protein content,which indicated that the Se status might affect the muscle growth of rainbow trout by influencing protein deposition in muscle.Based on the previous research in our laboratory,the autophagy-related gene(Atg)expression and autophagy markers(microtubule-associated protein light chain 3(LC3)and P62)levels in muscle of rainbow trout in different Se status was detected.Results showed that m RNA levels of Atg4 b,Atg12l,LC3 B,Atg13,Atg7 and Atg5 in muscle of rainbow trout in deficient and excessive Se groups were increased significantly compared with adequate Se group.The level of the LC3-II in deficient and excessive Se groups was significantly induced,while the content of P62 was significantly reduced.These results indicated that autophagy level in muscle of rainbow trout in deficient and excessive Se status groups was higher than that in adequate Se group.Through the analysis of the protein kinase B-target of rapamycin(Akt-TOR)pathway,the regulatory pathway of autophagy,the deficient and excessive Se status significantly inhibited the phosphorylation of Akt and TOR,and inhibited the activity of the Akt-TOR pathway.The phosphorylation analysis of ampactivated protein kinase α(AMPKα)also showed that the AMPK activity of rainbow trout muscle was induced in deficient and excessive Se status.The results showed that the deficient and excessive Se status could inhibited muscle growth and protein deposition in rainbow trout.Combined with the research on the level of muscle autophagic degradation and related pathways in present study,we speculate that the deficient and excessive Se status may inhibite the antioxidant capacity of rainbow trout and promote the level of muscle autophagic degradation,which may be achieved through the Akt-TOR pathway and AMPK pathway.Through the analysis of the oxidation status of rainbow trout muscle,it was found that the content of malondialdehyde(MDA)and lipid peroxide(LPO)in muscle of rainbow trout in deficient and excessive Se status was increased significantly.The muscle oxidative stress is intensified,which may be the way for dietary Se to affect AktTOR and AMPK pathways.In summary,the dietary Se requirement of triploid rainbow trout(3.53 mg/kg)was obtained by broken-line regression analysis in present study.The Se requirement of triploid rainbow trout and the relationship between dietary Se level and Se status were studied comprehensively.Moreover,the effects and possible mechanisms of Se status on autophagic degradation of rainbow trout muscle protein were preliminarily revealed——The dietary Se deficiency and excess may promote the autophagic degradation of rainbow trout muscle protein via Akt-TOR and AMPK pathways.The results can enrich our instanding of the nutritional requirements of different ploidy fish and provide an important foundation for further research on the regulation mechanism of Se on fish growth.