| Previous studies of our research group showed that glucosamine(GlcN)can be used as a feed attractant,but the regulatory mechanism of GlcN on feed intake of laying hens is still not clear.Therefore,objective of the study was to elucidate signal pathway via fluctuation of serum biochemicals,gut microbes flora and screening differential expression gene in hypothalamus and gut,and provide theoretical basis for the rational application of GlcN in laying production.The study includes three trials:Experiment 1: The effect of GlcN on performance and blood biochemical in laying hens.A total of 432 healthy Lohman hens aged 290-day-old,with similar laying rate were selected and randomly divided into 4 groups with 6 repetitions in each treatment and 18 hens per repettion.Hens consumed 0%,0.4%,0.6% and 0.8%GlcN diets,respectively.7 day for acclimation and 60 day for feeding trial.The results showed that: 1)there was quadratic curve relationship between GlcN and average daily feed intake(ADFI)in layers,hens comsumed maxiium amount feed when GlcN inclusion was 0.45%.GlcN had no significant effect on the laying rate and egg weight in hens(P > 0.05),feed/egg were increasd,and abnormal egg rate were reduced(P <0.05).2)GlcN adjusted metabolism of hens.Serum glucose,TG and LDL improved by GlcN addition(P <0.05),but there was no significant difference in blood glucose levels between 0.4% and 0.8% GlcN(P> 0.05).HDL content changed with GlcN level in quadratic curve(P> 0.05).serum Ig Y from 0.4% GlcN were highest than others(P <0.05),serum insulin of 0.6%-0.8% group were higher than 0.4% and control group.(P <0.05).The change patterns of Leptin were in a quadratic curve when the GlcN increased.The 0.8% GlcN group was significantly higher than the control group by 30.08%(P <0.05).GlcN promoted NO secretion,and there was no significant difference in NO content between 0.4% and 0.8% GlcN groups(P> 0.05).But the higher NO in the examined group compared to the control group(P <0.05).GlcN levels above 0.6% significantly inhibited the secretion of serum IL-6 and TNF-α(P <0.05).Experiment 2: The effect of GlcN on the gut morphology and microbia flora of laying hens.The experiment design was same as trial 1.6 healthy hens with similar body weight were selected from each group at 30 d and 60 d,respectively.duodenum and jejunum samples were collected and structure of intestine were measured.Cecum digesta of 3 hens were sampled from the 0.0% GlcN group and0.4% group,respectively,and gut microflora were determined with high-throughput sequencing analysis.The results revealed that: 1)Adding 0.4% and 0.6% GlcN significantly increased the height of the duodenum villi and V/C(P <0.05),but had no significant effect on the crypt depth(P> 0.05).Villi height and V/C of duodenal in0.8% GlcN group were significantly lower than that in the control group at 60 d;inclusion of 0.4% to 0.6% GlcN significantly increased villi height of jejunal,and V/C of jejunal from 0.4 % GlcN group were the highest,but jejunum villi height and crypt depth at 60 days were no different among groups,however,V/C of jejunum was significantly reduced in 0.8% GlcN group compared with the control.2)the Firmicutes is the most dominant phylum in each group,with a relative profile above44%.Bacteroides,Phytobacteriaceae＿RC9,Rumincoccus,Faecalibacterium,Unclassified＿f＿Lachnospiraceaeh and Lactobacillus were dominant genus,and relative profile of Lactobacillus has increased significantly with GlcN(P <0.05).Experiment 3: Modulation of GlcN on feed intake and pathway analysis in layers.The design of trial is same as Experiment 1.9 healthy hens with similar body weight were selected from the 0.0% GlcN,0.4% GlcN and 0.8GlcN groups,respectively.Total RNA were extracted and gene expresson of duodenum and gland stomach were measured by RT-PCR,and hypothalamus tissues were collected and screend differentially expressed genes by transcriptome technique,performd functional annotation and enrichment analysis with GO and KEGG,and to get singal pathway key genes that relative feed intake affected by GlcN.Then 3 hens were used for immunohistochemical test and RT-PCR to verified.Transcriptomics analysis results indicated that: 0.4% GlcN group compared with 0.0% GlcN group,a total of 588 differential genes were screened,of which 328 genes were up-regulated,260 differential genes were down-regulated,and the differential gene functions were distributed in 102 pathways after annotation;Comparison of 0.8% GlcN group with0.0% GlcN group,displayed that a total of 445 differential genes were screened,ofwhich 236 genes were up-regulated,209 differential genes were down-regulated,and the differential gene functions were distributed in 87 pathways after annotation.Cluster analysis showed that neuroactive ligand-receptor interaction pathway and the adipokine signal pathway were influenced eith GlcN addition,and Insulin signaling pathway and MAPK signal pathway in adipokine signal pathway were two key pathways.Compared with the 0.0% GlcN,gene POMC was down-regulated in 0.4%GlcN,and NPY was up-regulated in 0.8% GlcN group,which was verified by immunohistochemistry test and RT-PCR.In addition,0.4% GlcN significantly reduced the expression of Ghrelin m RNA in the glandular stomach(P <0.01),while Ghrelin m RNA expression increased significantly by the GlcN were 0.8%(P <0.01)and GlcN had no significant effect on the expression of CCK m RNA in the duodenum(P> 0.05).Conclusions:(1)There was quadratic curve relationship between GlcN and ADFI,hens comsumed maxiium amount feed when GlcN inclusion was 0.45%,and abnormal egg rate were reduced.(2)GlcN improved serum IL-6 and TNF-α content,and increased Ig Y,NO,Leptin and insulin,enhance the immunity of layers.(3)Adding GlcN could improve the intestinal morphology of the laying hens,and increase profile of lactobacillus in cecum.(4)The transcriptional sequencing showed that adipocytokine signaling pathway and neuroactive ligand-receptor interaction pathway are regulated by GlcN,moreover,Ghrelin gene expresson also is modunal lated by GlcN.(5)Signal pathway related to feed intake regulated by GlcN :1)GlcN stimulates the secretion of Leptin by increasing the levels of Gluose and TG.Leptin bind to leptin receptor distributed in the hypothalamus,then up-regulate NPY/Ag RP expression and down-regulate POMC / CART expression,inhibit the secretion of gastrointestinal peptide Ghrelin,thereby enhancing the appetite of layer hens.2)GlcN increases the profile of Lactobacillus in the cecum,activates the layer’s immune system,reduces the secretion of IL-6 and TNF-α,enhances immunity,and thus increases feed intake. |
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