| In order to determine the appropriate dietary vitamin K3 requirement and investigate the effects of vitamin K3 on growth promotion and physiological function of largemouth bass（Micropterus salmoides）,growth performance,tissue composition,hematological indexes,antioxidant ability,digestion,immunity,metabolism and other aspects were investigated in this study.The optimal dietary vitamin K3 of largemouth bass was determined based on the growth performance and regression analysis.And the possible molecular mechanism of vitamin K3 affecting the physiological function of largemouth bass was preliminarily explored via the analysis of metabonomics and transcriptomics.1.The optimum dietary vitamin K3 requirement based on growth,blood coagulation time,haematological index and antioxidant capacity of largemouth bass（Micropterus salmoides）An 8-week feeding trail was carried out to investigate the optimum dietary vitamin K3 requirement of largemouth bass.A total of 600 healthy fish（12.96±0.07 g）were fed diets containing graded levels of vitamin K3at 0.78（K0）,5.80（K5）,10.82（K10）,15.84（K15）and 20.85（K20）mg/kg with four replicates.Results showed that dietary vitamin K3 could enhance growth performance.The weight gain rate and specific growth rate in K15 were significantly higher than K0（P<0.05）.No significant difference was observed in the content of crude protein,crude lipid,moisture,crude ash and phosphorus in muscle（P>0.05）.However,the supplementation of vitamin K3 significantly contributed to calcium accumulation in muscle and vertebrae（P<0.05）,and increased the amount of red blood cell in blood（P<0.05）.The blood coagulation time decreased significantly with increasing dietary vitamin K3 from 0.78 to 10.82 mg/kg（P<0.05）,and then tended to stable（P>0.05）.The antioxidant capacity was improved with the addition of vitamin K3,but the malondialdehyde contents of liver and serum in K20 were significantly higher than other groups（P<0.05）and the liver MK-4（a form of menaquinone）content increased and then decreased.These might be related to the toxic effects of excessive vitamin K3.Taking account of growth performance and the regression analysis of liver malondialdehyde content,serum glutamic pyruvic transaminase activity,erythrocytic number,blood clotting time and liver MK-4 content,the appropriate dietary vitamin K3 requirement was in the range of 9.93-15.22 mg/kg diet.2.Effects of graded levels of dietary vitamin K3 on digestibility,serum metabolic index,muscle amino acids and fatty acids of largemouth bassFurther tests were conducted on the experimental fish in Chapter 1,aiming to explore the effects of vitamin K3 on the digestion,metabolism,muscle amino acids and fatty acids of largemouth bass.The results showed that digestibility and the activities of digestive enzymes in the group with vitamin K3supplementation were significantly lower than K0 group（P<0.05）.The apparent digestibility and protein apparent digestibility of K10,K15 and K20 were significantly lower than K0（P<0.05）.The serum urea nitrogen content in group K20 was significantly higher than that in the other four groups（P<0.05）,but there were no significant differences in the serum total protein content,albumin content,total cholesterol content and triglyceride content among the five groups（P>0.05）.There were no significant differences in the contents of essential amino acids,non-essential amino acids and total amino acids among all groups（P>0.05）,essential amino acids index as well,but with the increase of dietary vitamin K3 content,the contents of essential amino acids and total amino acids showed an increasing trend.The contents of threonine,isoleucine and leucine in K0 group were significantly lower than those in other groups（P<0.05）.With the increase of dietary vitamin K3 content,the content of monounsaturated fatty acids showed a decreasing trend,but the polyunsaturated fatty acids showed an increasing trend.The content of monounsaturated fatty acids in group K0 was significantly higher than other groups（P<0.05）,but the content of polyunsaturated fatty acids in group K0 was lower than other groups,and was significantly lower than K15（P<0.05）.Linoleic acid content in test groups was significantly higher than K0（P<0.05）,linolenic acid content in K15 group was significantly higher than K0（P<0.05）,and linolenic acid content in the other three test groups were also higher than K0.There were no significant differences in ARA,EPA,and DHA contents among groups.3.Effect of vitamin K3 in feed on liver metabolism of largemouth bassIn order to explore the mechanism of vitamin K3 acting on largemouth bass,LC-MS metabonomic method was used to analyze the liver metabolites of the fish in K0 and K15 group according to the result of growth in chapter one.The metabolic rules of vitamin K3 on the liver were preliminarily analyzed from the perspective of small molecular differential metabolic production and metabolic pathways.The results showed that 712 differential metabolites were identified,and 326 differential metabolites upregulated in K15 group compared with K0 group.A total of 13differential metabolites were observed in KEGG database.7 differential metabolites upregulated in K15 group compared with K0 group,which were L-serine,Uridine5’-diphosphoglucuronic acid,dehydroascorbic acid,D-urobilin,D-Glucosaminide,Lyso SM（d18:1）and Lyso PC（O-18:0）.6 differential metabolites downregulated in K15 group compared with K0 group,which were folinic acid,lumichrome,Lyso PC（16:1（9Z））,Lyso PC（20:3（5Z,8Z,11Z））,Lyso PC（18:1（9Z））and Lyso PC（22:6（4Z,7Z,10Z,13Z,16Z,19Z））.These differential metabolites were involved in 23 pathways.These pathways of glycine,serine and threonine metabolism,sphingolipid metabolism,ether lipid metabolism and glycerophospholipid metabolism were integrated and the pathways of ascorbate and aldarate metabolism and amino sugar and nucleotide sugar metabolism were integrated.Differential metabolites were mainly concentrated in these two integration pathways.From the perspective of liver metabolites and metabolic pathways,appropriate amount of vitamin K3could help largemouth bass improve the capabilities of antioxidant,immune,and body recovery,thereby promote growth and maintain health.4.Liver transcriptional regulatory mechanism of dietary vitamin K3 on largemouth bassThe transcriptomics method was used to explore the possible influence mechanism of dietary vitamin K3 of largemouth bass in K0 and K15 group based on the results of growth in Chapter 1.The results showed that there were 86 differentially expressed genes between the two groups in the GO database,and 26 of the secondary functions were involved,which mainly focused on 8 functions,such as binding,membrane part,cellular process,catalytic activity,metabolic process,biological regulation,stress response and cellular component.In the KEGG database,29 genes of the two groups were involved in 46 metabolic pathways,mainly focusing on the immune system,signal transduction,signal molecules and interactions,endocrine system and lipid metabolism.The analysis of calcium signaling pathways,endocrine and other factor-regulated calcium reabsorption,osteoclast differentiation and related differential genes indicated that vitamin K3 had a good effect on the skeletal development of largemouth bass.The expression of MHCⅡin K15 group was significantly higher than K0 group（P<0.05）,and the expression of MHCⅡin K15group was significantly lower than K0 group（P<0.05）.These two genes were involved in multiple pathways in the immune system,suggesting that vitamin K3 may have a role in regulating immunity. |
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