Effects Of Vitamin D₃ On Growth Performance And Induction Of The β-defensins Expression In Silky Fowl

Avian beta defensins(AVBDs) are important effector molecules of innate immune,which play a critical role in the host against invasion of pathogenic microorganisms.Vitamin D3(VD3) as an immunomodulator,could directly induce the expression of antimicrobial peptide and related immune factors in mammalian monocytes, epithelial cells,however in poultry,lacking the 1α-hydroxylase of vitamin D3 into the 1,25 dihydroxy-vitamin D3.Thus,they were studied that of the relationship of VD3 and the expression ofβ-defensin between,and the effect of VD3 on the performance of production and immune,including the following six experiments.Exp.1 Effect of Vitamin D3 by Injection on theβ-defensins in Taihe Silky FowlThe expression of AVBDs,Toll-like receptors(TLRs) and vitamin D receptor(VDR) was studied following in vivo by injection vitamin D₃.Healthy 6w Silky Fowl were abdominally injected with vitamin D₃ or untreated.Real-time PCR analyses reveal that injection of vitamin D₃ showed significantly up-regulated the expression of TLRs (TLR2,TLR6 and TLR7),VDR,AVBDs(AVBD 1,AVBD 5,AVBD 7,AVBD 9,AVBD 10 and AVBD 12) and 24-hydroxylase(CYP24A1) in the gut(duodenum,jejunum,ileum and cecum) during different time from 8 h to 24 h postinjection,similarly the expression of TLRs(TLR2,TLR5),VDR,AvBDs(AVBD-6,GAL-1) and 24-hydroxylase(CYP24A1) in the tissues(liver,spleen and kidney).These results suggest that expression of VDR,AVBDs and TLRs seems to be induced by vitamin D₃ and was concluded that the tissues expressing TLRs and VDR responds to vitamin D₃ and in turn upregulate this tissues cellular functions to synthesize AVBDs.Intraperitoneal injection of vitamin D₃ likely resulted in enhanced the expression of AVBDs,TLRs and VDR,which provided insight into factors important to the control of the innate immune response in the chicken.Exp.2 Effect of Salmonella enteritidis by Injection on theβ-defensins in Silky FowlThe expression of AVBDs and TLRs was studied following in vivo challenge with Salmonella enteritidis.Healthy 6w Silky Fowl were abdominally injected with Salmonella enteritidis or untreated.Real-time PCR analyses reveal that Salmonella enteritidis by injection showed significantly up-regulated the expression of eight avianβ-defensins (AVBD-1,GAL-1,AVBD-5,AVBD-6,AVBD-7,AVBD-9,AVBD-10 and AVBD-12) and five TLRs(TLR1,TLR4,TLR5,TLR6 and TLR7)in the tissues(thymus,duodenum, jejunum,ileum,cecum,bursa of fabricius,liver,spleen and kidney)at 0-32 h postinjection.These results suggest that the Silky Fowl expresses at least eight isoforms of avianβ-defensins and five types of TLRs with a greater expression in the 9 tissues. Thus expression ofβ-defensins and TLRs seems to be affected by Salmonella enteritidis, and was concluded that the tissues expressing TLRs responds to Salmonella enteritidis and in turn upregulate cellular functions to synthesize avianβ-defensins.Such expression and functions of TLRs may play an essential role in Silky Fowl innate immunity for host defense.Exp.3 Effects of Different Supplemental Levels of Vitamin D in Diets onβ-defensins in Taihe Silky FowlThe expression of AVBDs,TLRs and vitamin D3 hydroxylase associated protein (VDHA) was studied following in vivo by feeding vitamin D₃.Healthy Silky Fowl were fed vitamin D₃ of 0,800,1600,3200 and 6400 IU/kg feed from 1 to 6w.Real-time PCR analyses reveal that feeding of vitamin D₃ showed significantly changed the expression of AVBDs(AVBD 1,AVBD 5,AVBD 7,AVBD 9,AVBD 10 and AVBD 12) in the thymus, duodenum,jejunum,ileum,cecum and bursa of Fabricius during different dose from 800 to 6400 IU,similarly the expression of TLRs(TLR2,TLR5),VDHA and AVBDs (AVBD-6,GAL-1) in the tissues(liver,spleen and kidney).These results suggest that expression of VDHA,AVBDs and TLRs were induced by vitamin D₃ and the genes expression levels existed a dose-dependent in same tissues of birds with fed VD3 of different dose,and the appropriate dose of VD3 can directly up-regulate the gene expression ofβ-defensins in the tissues.Exp.4 Effect of Vitamin D on the Cytokines in Taihe Silky FowlThe aim of this experiment was to research the effects of VD3 on interleukin-2(IL-2) and interleukin-18(IL-18) in Taihe Silky Fowl.Real-time PCR analyses reveal that VD3 by injection showed significantly changed the expression of IL-2 and IL-18 in the tissues (thymus,duodenum,jejunum,ileum,cecum and bursa of fabricius).Long-term feeding 6400IU of VD3 in diets was up-regulated the expression of IL-18 in thymus,bursa of Fabricius and gut of Silky Fowl.Feeding high-dose VD3 also was up-regulated the expression of IL-2 in the duodenum,the bursa of Fabricius and cecal,but down-regulated the expression in the thymus,jejunum and ileum.The results suggest that doses of vitamin D3 effect the expression change of IL-2 and IL-18 in Silky Fowl,but this cytokines had regulated different in the different tissues.Exp.5 Effects of Different Supplemental Levels of Vitamin D in Diets on Growth performance and Immune Organs in Taihe Silky FowlThe aim of this experiment was to research the effects of different supplemental levels of vitamin D in diets on growth performance and immune organs in Taihe Silky Fowl.One hundred and twenty Taihe Silky Fowl were divided into 6 treatments and each treatments contained 20 Taihe Silky Fowl.Vitamin D levels in the diets of six treatments were 0, 400,800,1600,3200 and 6400 IU/kg respectively.Other nutrient levels all reached the recommended nutrient standard of Chinese chicken.The results showed that daily gain and average weight of Taihe Silky Fowl were improved with the increase of vitamin D levels. The number of cells of the spleen and bursa were improved with the increase of vitamin D levels,while the number of cells of thymus were not affected.In conclusion,the growth performance of Taihe Silky Fowl were improved with the increase of vitamin D levels,and the the number of cells of the spleen and bursa were improved significantly at the same time.Exp.6 Cloning,Sequencing and Distribution ofβ-defensins Gene in Silky FowlWith specific primers,six chichenβ-defensin cDNA fragments,were analyzed by real-time PCR from Silky Fowl.Different mRNA distribution of genes in different tissues, indicated that AVBD-1,AVBD-5,AVBD-7,AVBD-9,AVBD-10 and AVBD-12 were widely expressed in thymus,duodenum,jejunum,ileum,caecum and bursal tissue,but there were difference in expression.AVBD-10 was amplified by reverse transcription-polymerase chain reaction(RT-PCR).After being sequenced,AVBD-10 cDNA fragments were identified and deposited to GenBank TM.The results of sequence comparison with theβ-defensin gene sequences published in GenBank TM indicated that nucleotide similarity of the AVBD-10 genes ranged from 88%to 100%.Analysis by DNAstar showed that Silky Fowl AVBD-10 by registered of the GenBank and sequenced AVBD-10 gene were not the same embranchment,which were probably caused by hybrid of species. In summary,VD3,whose function was mediated by TLRs,VDHA and Cyp24, induced the expression of defensins in Silky Fowl;the gene expression of AVBDs,IL2 and IL18 in tissue was up-regulated,and the growth performance and mass index of immune organ were improved with appropriate dose of vitamin D3...