| As one of the excellent laying hens in China,Lindian chicken has important research value in poultry breeding and germplasm protection.However,for a long time,the behavioral characterizations of Lindian chickens are still unclear.Therefore,in order to study the behavioral responses of fearfulness of Lindian chickens and the related production performance,immune ability and neurotransmitter levels,and to explore the molecular regulation mechanism of the difference in fear behavior between Lindian chickens and commercial Hy-Line Brown laying hens,80 Lindian chickens and 80 Hy-Line Brown laying hens at their 56 weeks old were selected in this experiment.Chickens were housed in traditional cages and divided into Lindian chickens group(L group)and Hy-Line Brown laying hens group(H group).The open field test(OFT),tension immobility test(TI)and Startle reflex test(SRT)for evaluating behavioral responses of fearfulness was carried out,and the production performance and egg quality of laying hens were detected.Subsequently,the levels of serum immunoglobulin Ig A and immune cytokines(IL-1β,IL-2,IL-4,IL-6,IL-8,IL-10,IL-17,TNF-α and IFN-γ)were determined.The concentrations of hypothalamus and serum neurotransmitters(5-HT,CORT and DA)were measured,and immune(IL-1β,IL-2,IL-4,IL-6,IL-8,IL-10,IL-17,TNF-α,IFN-γ)and neurotransmitter receptor-related genes(5-HT1 A,5-HT1 D,5-HT2 A,5-HT2 C,NR3C1,CRH,FKBP5,DRD1,DRD2,DRD3,DRD4 and DRD5)were determined.And the relative expression of oxidative stress factors(n NOS,e NOS,NQO1,SOD1,SOD2,SOD3,GST,GCLC,GCLM,CAT and HO-1)were detected,respectivily.Three laying hens were randomly selected from each group in L group and H group and killed for tissue samples.The hypothalamus samples were collected for histological observation(H&E staining and Nissl staining),and the transcriptome sequencing was carried out.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were performed to screen differential genes.The reliability of the sequencing results was verified by q RT-PCR.Finally,the gene correlation between the differential genes and immune cytokines,oxidative stress-related factors and neurotransmitter receptors was analyzed.The results of production performance and egg quality showed that the egg production rate of Lindian chicken was low.Compared with Hy-Line Brown laying hens,the latent time of the first movement of Lindian chickens was significantly prolonged in OFT(P < 0.01),and the number of crossing grids and the number of movement steps in the field were significantly increased for the OFT(P < 0.05).The results of TI test showed that the duration of tension immobility in Lindian chickens was significantly lower than that in Hy-Line Brown hens(P <0.01).In the SRT,when Lindian chickens were frightened,the score of stress behavior was significantly increased(P < 0.05).Therefore,the behavioral tests showed that the fear emotional responses of Lindian chickens were lower,while the vigilance behavioral responses were stronger,indicating that the unselected local varieties may have higher risk avoidance potential.ELISA results showed that the concentration of serum immunoglobulin Ig A and the levels of cytokines IL-2,IL-10 and IL-17 in Lindian chickens were significantly higher than those in Hy-Line brown laying hens(P < 0.05).The concentration of 5-HT and DA in hypothalamus was higher(P < 0.05),while the concentration of corticosterone was lower(P < 0.05).The serum DA content of Lindian chickens was significantly higher than that of Hy-Line Brown hens(P <0.01).The results of q RT-PCR showed that the m RNA expression levels of IL-1β,IL-4,IL-6,IL-8,IL-10,IL-17,TNF-α and IFN-γ in spleen of Lindian chickens were significantly higher than those of Hy-Line Brown laying hens(P < 0.05).The expression levels of n NOS,e NOS,NQO1,SOD1,SOD2,SOD3 and GST m RNA in spleen were significantly higher than tho se in Hy-line brown laying hens(P < 0.001).The m RNA expression levels of NR3C1,CRH,DRD1,DRD3 and DRD5 in hypothalamus were also significantly higher than those in Hy-Line Brown laying hens(P < 0.05).The results of Nissl staining showed that the number of Nissl bodies in the hypothalamus of Lindian chickens was significantly higher than that of Hy-line brown laying hens(P < 0.01).The results of immune,antioxidant,and neurotransmitter showed that Lindian chicken had higher immune and antioxidant levels,indicating that the tolerance of local varieties to the environment may be better than that of commercial laying hens.The results of hypothalamus transcriptome showed that a total of 231 significantly different genes were screened in Lindian chickens compared with Hy-Line Brown laying hens,including162 up-regulated genes and 69 down-regulated genes.The most significant molecular function(MF),biological process(BP)and cell component(CC)entries of GO enrichment were glutathione transferase activity,synaptic modification and cell periphery.The GO entries are closely related to the antioxidant activity,metabolism,synaptic plasticity and immune response of animals.The results of KEGG analysis showed that the main enriched pathways of differential genes in laying hens were the metabolism of exogenous substances by cytochrome P450,drug metabolism-cytochrome P450 and glutathione metabolism signaling pathways.The selected differential genes included ANGPTL2,ST6GALNAC2,GSTM2,SLC38A4,MEIOB,ABCB11 and STC2.The functions of the above genes mainly focused on production performance,immunity,antioxidant activity and disease resistance.The results of gene correlation analysis showed that ABCB11 was positively correlated with SOD2(P < 0.05),and negatively correlated with e NOS,NQO1 and GST(P < 0.05).MEIOB was positively correlated with IL-10 and CRH(P < 0.05).INSRR was negatively correlated with HO-1(P < 0.05).ANGPTL2 was positively correlated with e NOS(P < 0.05),and negatively correlated with SOD1,SOD2 and CRH(P < 0.05).SLC38A4 was positively correlated with IL-17 and CAT(P< 0.05),and negatively correlated with DRD3(P < 0.05).ST6GALNAC2 was negatively correlated with HO-1(P < 0.05).STC2 was positively correlated with IL-1β,IL-4,IL-6,IL-17 and SOD3(P < 0.05).ABCB11 was positively correlated with SOD2 and SLC38A4 in Lindian chickens(P < 0.05).MEIOB was significantly positively correlated with ST6GALNAC2(P <0.01).INSRR was negatively correlated with 5-HT1D(P < 0.05).ANGPTL2 was negatively correlated with DRD3(P < 0.05)and positively correlated with NR3C1(P < 0.01).There was a significant positive correlation between GSTM2 and HO-1(P < 0.05).The results of protein interaction network analysis showed that the main targets of differential genes were CRH,IL-1β,IL-6,HO-1 and CAT,suggesting that differential genes were closely related to antioxidant activity and immune ability.Therefore,there was a molecular basis for the differences in fear behavior,production performance,immunity and antioxidant between Lindian chickens and commercial Hy-line brown laying hens.Conclusion: Lindian chickens have lower fear and stronger alertness.Unselected local varieties may have higher potential for risk aversion.Lindian chicken has higher immune and antioxidant levels,and the tolerance of local varieties to the environment is better than that of commercial laying hens.The high secretion of excitatory neurotransmitters affected the expression of fear behavior in laying hens.The sequencing of hypothalamus transcriptome confirmed that there were molecular differences in fear behavior,production performance,immunity and oxidative stress in Lindian chickens compared from Hy-Line Brown laying hens.The 8 differentially expressed genes screened could be used as candidate genes to regulate the fear behavior of Lindian chickens. |
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