Effects Of α-lipoic Acid On Growth,antioxidant And Carbohydrates Metabolism In Oriental River Prawn Macrobrachium Nipponense

Protein is the most expensive nutrient in feed,while carbohydrate is the most economical energy source.Improving the utilization efficiency of carbohydrate can not only reduce feed cost and save protein,but also reduce the pollution of aquaculture water.The aerobic oxidation efficiency of carbohydrate is closely related to the activity of rate-limiting enzymes during carbohydrate metabolism.Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexare are rate-limiting enzymes for the oxidative decomposition of glycolytic pyruvate into mitochondria.As the cofactor of these two rate-limiting enzymes,alpha lipoic acid（α-LA）can catalyze oxidative decarboxylation and play an important role in carbohydrate metabolism.Oriental river prawn Macrobrachium nipponense is one of the main freshwater economic aquaculture species in China and some Asian countries,which has a high demand for fish meal protein and limited ability to use carbohydrate.Therefore,the objective of this study was to investigate the effects of α-LA on the efficiency of carbohydrate metabolism in Macrobrachium nipponense.The methods of nutrition,physiology,biochemistry and molecular biology were used in the study.Requirement of α-LA,effects of lipoic acid on growth,antioxidant activity,carbohydrate metabolism,hepatopanpancreatic structure and intestinal microflora of Macrobrachium nipponense at two carbohydrate levels,and the effects of lipoic acid on growth and energy metabolism of Macrobrachium nipponense at different protein-to-carbohydrate ratios were systematically studied.The main results and conclusions are as follows:1.Dietary α-LA requirement and its mediating antioxidant status,carbohydrate metabolism,and intestinal microflora in oriental river prawn Macrobrachium nipponense.α-LA is not only a natural antioxidant,but also a key cofactor for the enzymes pyruvate dehydrogenase and α-ketoglutarate dehydrogenase.The purpose of this study was to investigate the effects of dietary α-lipoic acid on the growth,antioxidant status,carbohydrate metabolism,and intestinal microflora of prawn,Macrobrachium nipponense.A total of 1250 juvenile M.nipponense with an average body weight of 0.050±0.003 g were randomly divided into five groups（50 prawns per tank）for an 8-week feeding trial.The groups were fed with basal semi-purified diets containing α-lipoic acid at five different concentrations: 0 mg/kg（LA1）,500mg/kg（LA2）,1000 mg/kg（LA3）,2000 mg/kg（LA4）and 4000 mg/kg（LA5）.The results are as follows:（1）The weight gain（WG）,specific growth rate（SGR）and the survival rate were significantly lower in LA5 than in other groups（P<0.05）.（2）Dietary α-lipoic acid at a concentration below 2000 mg/kg（LA4）significantly reduced the malondialdehyde（MDA）content and increased the glutathione（GSH）content in the hepatopancreas of M.nipponense,and significantly increased the activities of glutathione reductase（GR）,glutathione peroxidase（GSH-Px）,and superoxide dismutase（SOD）（P<0.05）.However,the MDA content in the hepatopancreas was higher in LA5 than in LA1（P<0.05）.（3）The prawns in the LA3 group showed the highest activities of isocitrate dehydrogenase（ICDH）and key enzymes in glycolysis（pyruvate kinase,PK;6-phosphofructokinase,PFK;Hexokinase,HK）,but lower activity of lactate dehydrogenase（LDH）compared with those in LA1 and LA2（P<0.05）.（4）Analyses of gut microflora at the operational taxonomic unit（OTU）level indicated that abundance（Chao index）and diversity（Simpson index）were lower in LA5 than in LA1 and LA3（P<0.05）.The Shannon index’s index was highest in LA5.The intestinal flora of M.nipponense comprised five main phyla and five main bacteria.The proportions of Proteobacteria and Citrobacter were higher in LA3 and LA5 than in LA1.The proportions of Actinobacteria,Bacteroidetes,Aeromonas,Tyzzerella＿3 and Mycobacterium were significantly higher in LA5 than in LA3 and LA1（P<0.05）.In summary,dietary α-lipoic acid at an appropriate level can improve the antioxidant capacity and carbohydrate metabolism of M.nipponense and modulate its intestinal microbiota without reducing growth performance.Excessive α-lipoic acid（4000 mg/kg）negatively affected the growth performance and antioxidant activity of M.nipponense.Calculations based on SOD and WG indicate that the optimal addition amount of α-lipoic acid for juvenile M.nipponense is and995.83 mg/kg 1354.8 mg/kg,respectively.2.Modulation of growth,antioxidant status,hepatopancreas morphology,and carbohydrate metabolism and intestinal microflora mediated by α-LA concentration in oriental river prawn Macrobrachium nipponense under two dietary carbohydrate levels.In order to further verify the effect of α-LA on improving the carbohydrate utilization efficiency of Macrobrachium nipponense,six diets were formulated using casein and fish meal as the protein sources,fish oil and soybean oil as the lipid sources,and corn starch as the carbohydrate source,at two carbohydrate levels（15% and 30%）and three concentrations of α-LA supplementation（0,700,and 1400 mg/kg）.Each experimental diet was fed to prawns（mean weight 0.050 ± 0.003 g）,twice daily to apparent satiation,in five replicates.The feeding trial lasted for 8 weeks.The results are as follows:（1）In the 15%（low）-carbohydrate groups,weight gain was significantly reduced in prawns fed 1400 mg/kg α-LA compared with 0 or 700 mg/kgα-LA（P<0.05）,but there was no significant effect in the 30%（high）-carbohydrate groups（P>0.05）.Survival was significantly higher among prawns fed 700 mg/kg α-LA compared with 0or 1400 mg/kg α-LA regardless of the carbohydrate level（P<0.05）.（2）The levels of dietary carbohydrate and α-LA also significantly affected the activities of key enzymes involved in glycolysis and the citric acid cycle（P<0.05）.The activities of hexokinase and pyruvate kinase activities were highest in prawns fed l400 mg/kg α-LA,in both the low-and high-carbohydrate groups.Supplementation with 700 or 1400 mg/kg α-LA significantly increased the activities of6-phosphofructokinase,pyruvate dehydrogenase,and succinate dehydrogenase regardless of the carbohydrate level（P<0.05）.（3）The level of malondialdehyde decreased significantly with increased α-LA supplementation（P<0.05）;in contrast,the levels of superoxide dismutase,glutathione peroxidase,and glutathione were significantly higher in prawns fed 700 or 1400mg/kg α-LA when compare with those fed no α-LA supplement（P<0.05）.（4）Dietary α-LA did not affect the overall morphology of the hepatopancreas,but 1400 mg/kg α-LA reduced the number of B（blister-like）cells.（5）At 30% carbohydrate level,Chao index and Ace index increased significantly with the increase of α-LA（P<0.05）,and Shannon index was the lowest in prawn fed 700 mg/kg α-LA（P<0.05）.Thus,700 mg/kg α-LA supplementation under either dietary carbohydrate level could improve the antioxidant status,carbohydrate metabolism,morphology of the hepatopancreas and stability of intestinal florain of M.nipponense.We conclude that α-LA supplementation can be used to improve the efficiency of carbohydrate utilization when a 30% carbohydrate diet is used for this prawn.3.Effect of dietary α-lipoic acid modulate growth,antioxidant abilities,hepatopancreas morphology and energy metabolism in oriental river prawn Macrobrachium nipponense under three protein-to-carbohydrate ratiosThe aim of this study was to verify the protein saving effect after the improved carbohydrate metabolic efficiency of α-LA of Macrobrachium nipponense.Six isolipid and isoenergetic semi-purified diets were formulated at two α-lipoic acid levels（0 mg/kg and 1300 mg/kg）and three protein-to-carbohydrate ratios（P41/C18,P37/C24 and P33/C30）.The prawns with initial body weight of（0.11±0.01 g）were tested.The feeding trial lasted for 50 days.The results are as follows:（1）α-LA significantly improved the survival of Macrobrachium nipponense（P<0.05）.At P41/C18 and P37/C24 groups,the weight gain and specific growth rate of prawn fed 1300 mg/kgα-LA were significantly lower than those fed no α-LA supplement（P<0.05）,while there were no significant differences in weight gain and specific growth rate between 0 and 1300 mg/kg α-LA in P33/C30 group（P>0.05）.（2）Compared with the groups of no α-LA supplement,1300 mg/kgα-LA increased the activities of HK,PK and PDH significantly（P<0.05）.（3）Protein-to-carbohydrate ratios and α-LA supplement had significant interaction with the gene expression of adenine ribonucleotide dependent protein kinase（AMPK α,AMPK β）,glycolysis key enzyme（HK）,pentose phosphate pathway key enzyme（glucose-6-phosphate dehydrogenase,G6PDH）,tricarboxylic acid circulating malate dehydrogenase（MDH）,key enzymes of fat synthesis（acetyl-Co A carboxylase,ACC）and fatty acid transport key protein（fatty acid binding protein,FABP 10）in energy metabolism（P<0.01）.The expression of AMPK α,AMPK β,COX,HK,PEPCK,G6 PDH and MDH genes in hepatopancreas of juvenile prawns fed 1300 mg/kg α-LA was significantly higher than that fed 0 mg/kg α-LA in group P33/C30（P<0.01）.In high protein-to-carbohydrate ratios group,α-LA increased ACC gene expression involved in lipid metabolism significantly（P<0.05）;（4）（4）α-LA was found had a peroxidation effect,the structure of hepatopancreas was destroyed and the number of B cells decreased at P41/C18 and P37/C24.However,at P33/C30,α-LA enhanced the antioxidant capacity,improved the hepatopancreas structure and increased the number of B cells.In conclusion,under the protein-to-carbohydrate ratios of P33/C30 with 1300 mg/kg α-LA supplementation has no significant effect on growth performance,but can be used to improve the survival,antioxidant capacity,carbohydrate metabolism and energy metabolism,morphology of the hepatopancreas of Macrobrachium nipponense.We conclude that diets with α-LA supplementation can be used to coordinate metabolism and spare protein.