Effect Of Vitamins A,Vitamin D And Vitamin E On Growth,Immunity And Intestinal Health For Juvenile Hybrid Grouper(Epinephelus Fuscoguttatus ♀ × Epinephelus Lanceolatus ♂)

Vitamins A,D and E are essential nutrients for fish and play an important role in promoting growth and maintaining intestinal health.In this thesis,the juvenile hybrid grouper(Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂),an important mariculture fish in China,was used as a research model.This object was to investigate effect of vitamins A,D and E on growth,immunity and intestinal health for juvenile hybrid grouper,and to determine the appropriate supplemental levels of three fat-soluble vitamins for formulating reasonably commercial aquatic feeds for grouper.The main results are as follows:1.This study was carried out to investigate the effects of dietary vitamin A on growth,antioxidant capacity,digestion,intestinal immune response,mRNA expression of intestinal tight junction proteins and microbiota structure for juvenile hybrid grouper.Six isonitrogenous and isolipidic experimental diets were formulated to obtain vitamin A levels(317,1136,2038,4142,7715,15204 IU/kg diet,respectively).The triplicate groups of fish(average weight of 9.01 ± 0.27 g)were fed twice daily(8:00 and 16:00)for 49 d.Based on the broken-line analysis model of weight gain(WG)and lysozyme(LYZ)activity in intestine,the dietary vitamin A requirement of hybrid grouper were estimated to be 2688.58 and4096.36 IU/kg diet,respectively.The vitamin A concentration in the liver increased with increasing dietary vitamin A level to 7715 IU/kg and then plateaued(P<0.05).These results showed that vitamin A deficiency or excess could reduce WG,specific growth rate(SGR),and protein efficiency ratio(PER),and increase feed conversion ratio(FCR)and hepatosomatic index(HSI)(P<0.05).In addition,vitamin A deficiency could reduce the serum activities of acid phosphatase(ACP),superoxide dismutase(SOD),and total antioxidant capacity(T-AOC)and increase the malondialdehyde content(MDA)(P<0.05).Vitamin A deficiency also could increase the lipid content of the liver,and reduce intestinal activities of ACP,alkaline phosphatase(AKP),LYZ,complement 3(C3),complement 4(C4)contents,and activities of alpha-amylase,lipase,and trypsin(P<0.05).Meanwhile,vitamin A deficiency could reduce villus height(VH)in proximal intestine(PI)and mid intestine(MI),as well as muscle thickness(MT)in PI and distal intestine(DI)(P<0.05).Moreover,vitamin A deficiency could down-regulated antimicrobial peptides(β-defensin,hepcidin [not in MI and DI],epinecidin),anti-inflammatory cytokines(interleukin 10 and transforming growth factor β1 [not in DI]),tight junction proteins(occludin and claudin3)mRNA levels in the PI,MI and DI,and up-regulated pro-inflammatory cytokines(tumor necrosis factor α[not in MI] and interleukin 1β [not in MI]),signaling molecules c-Rel and P65(P<0.05).By analysis of microbiota of distal intestine,vitamin A deficiency decreased the number of operational taxonomic units(OTUs),Chao1 and Ace indices.At the phylum level,the Proteobacteria and Firmicutes were the dominant phylum of the intestinal microbiota.Appropriate vitamin A level in diet increased the abundance of Lachnospiraceae and Muribaculaceae.Collectively,vitamin A deficiency could reduce growth,because of a negative effect on intestinal health by increasing liver lipid deposition and depressing antioxidant capacity in the serum,digestive abilities,intestinal morphology,immunity,tight junction function and abundance of beneficial bacteria in the intestine.2.The study was carried out to investigate the effects of dietary vitamin D on growth,intestinal morphology,immune response and intestinal microbiota structure for juvenile hybrid grouper.Six isonitrogenous and isolipidic experimental diets were formulated with different vitamin D levels of 282,772,1250,2440,4640 and 8790 IU/kg,respectively.Three replicates of each group(mean body weight 13.53±0.34 g)were fed twice daily(8:00 and16:00)for 56 d.The results showed that compared with the control group,WG,SGR and HSI of fish fed 1250-8790 IU/kg diet were significantly higher,and FCR of fish fed 4640IU/kg diet was significantly lower(P<0.05).With increasing dietary vitamin D level,hepatic vitamin D content first increased and then kept stable.The vitamin D supplementation in the diet significantly reduced liver lipid content and increased intestinal AKP and LZY activities,C3 and C4 content,upregulated mRNA levels of antimicrobial peptide(epinecidin),antiinflammatory cytokines(IL-10 and TGF-β1)and signaling molecules(IκBα),and downregulated mRNA levels of pro-inflammatory cytokines(TNF-α and IL-1β)and signaling molecules(c-Rel)in DI(P<0.05).In addition,vitamin D deficiency and excess in the diet reduced intestinal VH,villus width(VW)and MT(P<0.05).By analysis of microbiota of distal intestine,the 8790 IU/kg group was significantly lower in the number of OTUs and significantly higher in both Shannon and Simpson,compared with that in control group(P<0.05).Meanwhile,the abundance of Cyanobacteria,Bacteroidetes at the phylum level and many bacteria families at the family level,were the lowest in 8790 IU/kg group.Based on the broken-line analysis model of WG and LYZ activity in intestine,the vitamin D requirement of juvenile hybrid grouper was estimated to be 2485.76 and 2027.89IU/kg diet,respectively.In conclusion,appropriate vitamin D in the diet could improve liver lipid metabolism,the structural morphology of the intestine and enhance intestinal immunity,which will have a beneficial effect on the regulation of the intestinal microbiota and thus improve growth.3.The present study was conducted to investigate the effect of dietary vitamin E on the growth,antioxidant responses,lipid metabolism of liver,intestinal immunity and microbiota structure for juvenile hybrid grouper.The 540 fish(initial weight 8.79 ± 0.06 g)were distributed into six groups with three replicates and fed isonitrogenous and isolipidic diets with different levels(11,43,98,193,404,and 789 mg/kg)of vitamin E for 49 d.Results showed that the WG,SGR,PER,and condition factor of the fish significantly promoted with increasing dietary vitamin E levels from 11 to 193 mg/kg(P<0.05).The vitamin E concentration in the liver increased with increasing dietary vitamin E level to 404 mg/kg and then plateaued(P<0.05).Compared with the control group,the serum triglyceride levels of the treatment groups were significantly lower.The dietary supplementation of 98–404 mg/kg vitamin E significantly improved the activities of hepatic SOD and catalase(CAT)and the T-AOC.In addition,the diet with vitamin E supplementation significantly improved the mRNA expression levels of hepatic copper/zinc superoxide dismutase,CAT,and nuclear factor erythroid-2-related factor 2(P<0.05).The number and size of lipid droplets and the content of MDA in the liver significantly decreased with increasing dietary vitamin E level(P<0.05).The diet with vitamin E supplementation decreased the activity and mRNA level of fatty acid synthase and increased the activity and mRNA level of hormone sensitive lipase in the liver(P < 0.05).The mRNA level of hepatic lipase increased,and the mRNA levels of peroxisome proliferator activated receptor-γ and carnitine palmitoyl transferase-1decreased in the liver of the treatment group(P<0.05).In addition,the mRNA level of hepatic lipoprotein lipase decreased in the groups fed 98 and 193 mg/kg diet(P<0.05).Meanwhile,the diet with vitamin E supplementation significantly increased ACP,AKP,LYZ activities and C3 and C4 contents(P<0.05),and the VH of PI,MI and DI,and MT of PI(P<0.05).By analysis of microbiota of distal intestine,the number of OTUs,Chao1 and Ace were highest in the group fed 98 mg/kg diet.At the phylum level,the Firmicutes was the dominant phylum of microbiota.At the family level,the highest abundance of Lachnospiraceae in the group fed 193 mg/kg diet and Fusobacteriaceae in the group fed 404mg/kg diet were observed.Based on the broken-line analysis model of WG and LYZ activity in intestine,the dietary vitamin E requirement of hybrid grouper were estimated to be 133.45 and 209.42 mg/kg diet,respectively.Overall,appropriate vitamin E in diet could enhance the antioxidant and intestinal immunity,promote the development of intestinal structure morphology,increase the abundance of beneficial bacteria in intestine,and reduce liver lipid deposition of hybrid grouper,thus improving its growth.