Effects Of Small Peptides And Vitamin D₃ On Growth,liver Metabolism And Intestinal Microorganisms Of Largemouth Bass Micropterus Salmoides

1.Altered growth performance,digestibility,liver and serum biochemical parameters of largemouth bass?Micropterus salmoides?was related to dietary small peptides supplementationTo investigate whether incorporation of small peptides into diets could change the growth performance,digestibility and liver health of the largemouth bass,Micropterus salmoides,four diets were designed:SP0⁺group?positive control group?was formulated according to the nutritional requirement of the largemouth bass;In SP0^-group?negative control group?the crude protein level was decreased by 30g/kg feed compared with SP0⁺group;SP2 group was added 2%small peptides based on the SP0^-group,and its crude protein level did not reach that of SP0⁺group.In SP6.5 group,6.5%small peptides additive was added on the basis of SP0^-group to obtain same crude protein level as SP0⁺group.Fish were fed with these four different diets for 9 weeks.Results showed that:Compared with the negative control group,the addition of 2%small peptides had no significant effect on the weight gain rate and feed coefficient of the largemouth bass;adding 6.5%small peptides could significantly increase the weight gain rate and reduce the feed coefficient?P<0.05?,both reaching the level of the positive control group.Adding 2%or/and 6.5%of small peptides could significantly increase the content of cysteine and proline,the total amino acid,essential amino acid and non-essential amino acid in the muscle?P<0.05?,as well as fatty acids,especially ARA,EPA and DHA.Adding 2%or 6.5%small peptides to the feed could increase the protease activity of the digestive system while increasing the antioxidant capacity of the liver and reducing the malondialdehyde content in the liver.The addition of small peptides to the feed could cause a marked increase in the apparent protein digestibility.Because total superoxide dismutase and catalase protected them from oxidation,muscle quality and live health were both improved,consequently enhanced transaminase in the liver and reduced transaminase in the serum were observed.All these results revealed small peptides supplementation could improved the growth and health of largemouth bass.2.Effects of feed protein level and small peptide addition on liver metabolite profiles of Micropterus salmoidesIn order to further investigate the relationship between liver metabolites and the differences in growth,digestion,and health of largemouth bass caused by feed protein levels and small peptide additions in Chapter 1,detection of liver metabolites was conducted based on GS/MC metabolomics technology.There were 15 common differential metabolites identified among SP0⁺,SP0^-,SP2 and SP6.5,and 16 common differential metabolites identified among SP0^-,SP2 and SP6.5.Metabolites mainly included alcohols,amines,organic acids,alkanes,fatty acids and sugars,.The most significantly affected metabolic pathways were Arginine and Proline metabolism Pathways,Alanine,Aspartic acid and Glutamate metabolism pathways,citric acid cycle?TCA cycle?,butyric acid metabolism,?-alanine metabolism and Purine metabolism.The experimental results indicated that the addition of small peptides in feed could significantly affect the protein metabolism,fat metabolism and carbohydrate metabolism?P<0.05?,which partly accounted for the growth and health change of the largemouth bass.3.Effects of dietary small peptides and protein levels on intestinal microorganisms of Micropterus salmoidesIn this chapter,intestinal flora of largemouth bass was investigated to explore the influences of small peptides additive and protein levels in the feed on growth and health of largemouth bass.Illumina Mi Seq high-throughput sequencing technology was used to analyze the intestinal flora of SP0⁺,SP0^-,SP2 and SP6.5.The results found that:Compared with the SP0⁺group,reduced feed protein in the SP0^-group would increase the intestinal richness of largemouth bass.SP2 group or SP6.5 group will increase the richness of intestinal flora of largemouth bass,due to the increase of dietary protein level and addition of small peptides.In terms of intestinal flora diversity,it reduced in the SP0^-group when compared with SP0⁺group,,while increased in the SP2 and SP6.5as the content of feed protein and the amount of small peptide rising,and finally reached the level of SP0⁺group.At phylum level,the dominant flora of the four groups included Proteobacteria,Tenericutes,Firmicutes and Bacteroidetes.At the genus level,the dominant flora was unclassified-c-Mollicutes,Mycoplasma,Achromobacter,Alcaligenes and Eubacterium.The Mycoplasma genus was the absolutely dominant flora of SP0⁺,SP0^-and SP6.5,while the unclassified C.permococcus was the absolutely dominant flora in the SP2group.The genus level differential bacteria research found that the intestinal flora of the four groups had significant differences in samples genus level.The abundance of Rhodopseudomonas in SP2 and SP6.5 was significantly higher than other two groups?P<0.05?,the abundance of harmful bacteria Staphylococcus and Eubacteria in SP2group was significantly lower than that in SP0⁺and SP0^-?P<0.05?.The above results indicated that addition small peptides in feed could improve the intestinal health and then improve the body health of largemouth bass by increase the abundance of beneficial bacterias in the intestine of largemouth bass and reduce the abundance of harmful bacterias in the intestine4.The optimal amount of vitamin D₃ in feed based on growth,biochemical indexes and anti-oxidation ability of Micropterus salmoidesIn order to investigate the optimal amount of vitamin D₃ in the largemouth bass feed,vitamin D₃with 0,15,30,45 and 60IU/kg dietwas added to the basic feed respectively in test 1.Five kinds of isonitrogenous and isoenergetic experimental diets feeds with vitamin D₃ contents of 1370,1385,1400,1415 and 1430IU/kg were designed.The largemouth bass with an initial body weight of?14.19±0.05g?was reared for nine weeks.In test 2,vitamin D₃ with 0,1000,2000,3000 and 4000IU/kg diet was added to the basic feed respectively.Five kinds of isonitrogenous and isoenergetic experimental diets feeds with vitamin D₃ contents of 514,1514,2514,3514and 4514IU/kg were designed.The largemouth bass with an initial body weight of?24.01±0.07?was reared for nine weeks.The results showed that different levels of vitamin D₃ in the feed significantly affected the weight gain rate,specific growth rate and feed conversion ratio of largemouth bass?P<0.05?,but had no significant effect on survival rate,condition factor and visceral somatic index?P>0.05?.Increased content of vitamin D₃ in the feed would significantly reduce the hepatic index and liver fat content of largemouth bass?P<0.05?;With the increase of vitamin D₃ content in the feed,the contents of crude ash,calcium and phosphorus and serum calcium and phosphorus in the spine of largemouth bass increased?P<0.05?.However,there was no significant effect on the content of crude protein,crude fat,crude ash and moisture in the muscle in test 1.?P>0.05?.In the second experiment,with the increase of vitamin D₃content,the calcium content in muscle,spine and serum showed a trend of increase followed by decrease.The calcium content in muscle and serum reached the maximum value in the VD3000 group,and the calcium content in the spine reached the maximum value in the VD2000 group.the phosphorus content in the muscle and serum increased with the vitamin D₃ content in the diet,while that in the spine reached a maximum in the VD3000 group.Different vitamin D₃ content in the feed had no significant effect on muscle crude protein,crude ash and moisture content?P>0.05?.However,with the increase of vitamin D₃ in the feed,the crude fat content of muscle first increased and then decrease,and reached the maximum value in the VD2000 group?P<0.05?.In test 1,the total superoxide dismutase activity,catalase activity and total antioxidant activity in the liver were significantly increased by the addition of vitamin D₃ in the feed?P<0.05?,and the activity of AST in serum was significantly decreased?P<0.05?.The anti-infection ability of the body to Aeromonas hydrophila was also enhanced with the increase of vitamin D₃ content in the feed?P<0.05?.In test 2,increased vitamin D₃ content in feed could significantly enhance total superoxide dismutase activity,catalase activity,total antioxidant capacity and alanine aminotransferase activity in liver and serum of largemouth bass?P<0.05?,and significantly reduced MDA content in liver and serum?P<0.05?.The total cholesterol and triglyceride content in the serum first increased and then decreased with the increase of the amount of vitamin D₃ in the feed?P<0.05?.On the other hand,the content of vitamin D₃ in the feed can significantly affect the serum albumin content,which obtained peak value in the VD2000 group.Based on the analysis of quadratic curve model,it was concluded that when the largemouth bass achieved maximum growth,the vitamin D₃ requirement in the feed was 3033 IU/kg,and when the calcium content in the spine reached maximum,the demand for vitamin D₃is 3550IU/kg feed.5.Effect of Vitamin D3 content in feed on intestinal microorganisms of Micropterus salmoidesIn order to understand whether dietary vitamin D₃content could affect the growth and health of largemouth bass through intestinal flora.The Illumina Mi Seq high-throughput sequencing technology was used to analyze the intestinal flora of VD0,VD2000 and VD4000.The results showed that with the content of vitamin D₃ increased in the feed,the intestinal Ace and Chao indexes of the largemouth bass first increased and then decreased.vitamin D₃ deficiency and excess would both affect the richness of intestinal flora.However,appropriate amount of vitamin D₃ could increase the richness of the intestinal flora.At the phylum level,the dominant bacteria groups in VD0,VD2000 and VD4000 are Proteobacteria,Tenericutes,Firmicutes and Bacteroidetes.The abundance of Anemone and Pachyphyta species in the intestine was positively correlated with the content of vitamin D₃ in the feed.The species abundance of Helicobacter,Acidobacteria,Bacteroides,Clostridia,and Proteobacteria was negatively correlated with the content of vitamin D₃ in the feed.The content of vitamin D₃in the feed had a relatively large effect on the distribution of the sample community in the VD0 and VD4000 groups,and small effect on the sample community distribution in the VD2000 group.At the genus level,the dominant flora was Mycoplasma,Plesiomonas,Cetobacterium,Acinetobacter,and Aeromonas.Mycoplasma and Pseudomonas were the dominant groups of VD0,VD2000 and VD4000.The abundance of Empedobacter in the VD2000 group was significantly higher than that in the VD0 group?0.001<P?0.01?.The abundance of Aeromonas,Pseudomonas,Chyseobacterium,and Stable Bacteria in VD2000 group was significantly higher than that in VD4000 group?P<0.05?.6.Effect of Vitamin D3 content in feed on liver metabolite profiles of Micropterus salmoidesIn this study,based on GS/MC metabolomics technology,metabolomics investigation was conducted to explore the association between liver metabolites and the change of growth and health resulting from vitamin D₃content in feed of Chapter 4and Chapter 5.There were 95 differential metabolites screened in the VD2000 and VD0groups,including five kinds of amino acids,twenty-one kinds of carbohydrates,two kinds of fatty acids,five kinds of nucleosides and five kinds of alcohols.114 differential metabolites screened in the VD4000 and VD0 groups,including Seventeen kinds of amino acids,fourteen kinds of carbohydrates,one fatty acid,seven kinds of nucleosides and five alcohols,and 63 differential metabolites screened in the VD4000 and VD2000groups,including seven kinds of amino acids,five kinds of carbohydrates,and five kinds of fatty acids and their combinations..The metabolic pathways significantly affected included sterol biosynthesis pathway,pentose phosphate metabolism pathway,galactose metabolism pathway,insulin signaling pathway,Fox O signaling pathway,AGE-RAGE signaling pathway,ABC transport pathway,Glutathione metabolism pathway,Arginine biosynthesis pathway,Starch and sucrose metabolism pathway,Cysteine and methionine metabolism pathway,Arginine and proline metabolism pathway,Pyrimidine metabolism and Caffeine metabolism pathway.The results showed that the increased amount of vitamin D₃ in the feed could enhance protein,lipid and carbohydrate metabolism,the defiency of vitamin D₃ inhibited nutrient metabolism.All these results revealed that different amount of dietary vitamin D₃could significantly change three nutrient-related metabolic pathways and inclusive metabolites of largemouth bass,and thereby affect the phenotypes such as the growth and health of the body.Lack of vitamin D₃ will inhibit the metabolic pathways of largemouth bass.In Chapter 4,the optimal demand obtained from the evaluation indexes of growth and spinal calcium content is not applicable to this chapter.The vitamin D₃ content of the4514IU/kg feed in VD4000 group didn't inhibit the three major nutrient metabolism pathways and enhanced the glucose metabolism pathway.