Effects Of Social Rank Order On Behavior,Intestinal Microflora And Hypothalamic Gene Regulation In Caged Layers

Social hierarchy of laying hens is mainly established and maintained by aggressive behavior.Aggressive pecking and threat are main patterns of avian aggressive behavior.Social rank order is related to the development of stress and mental illness,but the research on the mechanism of social rank order is very limited.Therefore,this study took Roman white layers as the test object,and discussed the effects of social rank order on the utilization of perch resources and behavior laying hens under different social groups by configuring different lengths of perch resources;At the same time,the differences of physiology,intestinal microflora composition and hypothalamic gene expression in layers of different social rank order under stable and unstable groups were compared,so as to explore the mechanism of layer social rank order and provide theoretical reference for the formation of layer social rank order.Experiment 1: This study set up different length perch resource allocation to study the use and behavior of perch resources of different social rank order laying hens.A total of 72 7-week-old Roman white laying hens were used and housed in 12 furnished cages with 6 birds per cage.Observing the four fighting behaviors(aggressive pecking,threat,substitution and chasing)of all laying hens during the experiment to determine the social rank order of laying hens by using the Clutton Brock index(CBI),and setting the perch by increasing the length to study the usage of perch(including perch behavior)of laying hens at all social rank orders.The results showed that highranked laying hens had more count and time to use perch resources than sub-ranked laying hens(P< 0.05);Correspondingly,they showed more lying behavior,preening behavior and comfort behavior on the perch(P < 0.05).The PCI was significantly correlated with the CBI index(P <0.01),and the PCI of laying hens with social rank 1 was significantly higher than that of other subranked laying hens(P < 0.05).Therefore,under different perch length resource allocation,social rank order affects the behavior of layers and the utilization of perch resources;And the perch competition index constructed in this experiment can reflect the social rank order of layers,but its effectiveness needs further verification.Experiment 2: This study investigated the effects of providing perch resources on the behavior of laying hens with high,middle and low social rank order in different group sizes.A total of 3606-week-old laying hens were randomly divided into four groups,namely 3 hens in the group(G3),6 hens in the group(G6),12 hens in the group(G12),and 24 hens in the group(G24).There are two kinds of perch resource treatment corresponding to different group sizes,one is to set up sufficient perch(15cm/hen)for laying hens,and the other is to set up limited perch(7.5cm/hen)for laying hens to observe the use of perch,perch behavior(standing,walking,lying,preening and comforting behavior)and general behavior(feeding,drinking,walking,preening and comfort behavior)of different social rank order laying hens.The results showed that under the condition of limited perch resources,the utilization count of the high-ranked layers were significantly increased in G3 and G6 groups,while in G12 and G24 groups,the utilization count of the low-ranked layers were significantly increased(P < 0.05);The perch utilization time of high-ranked layers in G3,G12 and G24 groups decreased with the increase of group size,that of medium-ranked layers in G3,G6 and G24 groups decreased with the increase of group size,and that of low-ranked layers increased with the increase of group size(P < 0.05).The standing and comforting behavior of high-ranked layers on the perch were significantly higher than that of medium-ranked layers,and the exploring behavior of high-ranked layers on the perch was significantly higher than that of low social level layers(P <0.05).The feeding behavior of middle-ranked layers was significantly higher than that of highranked and low-ranked layers,while the drinking behavior of high-ranked layers was significantly higher than that of low-ranked layers(P < 0.05).The laying hens’ staring behavior in G12 group was significantly higher than that in G6 group,and that in G6 group was significantly higher than that in G3 group(P < 0.05).The exploring behavior of layers in G12 group was significantly higher than that in G3 group,G6 group and G24 group,while that in G6 group was significantly higher than that in G3 group(P < 0.05).The comforting behavior of layers in G12 group was significantly higher than that in G3 group,G6 group and G24 group(P < 0.05),while in G3 group was significantly higher than that in G6 group(P < 0.05).The feeding behavior of layers in G12 group was significantly higher than that in G3 group(P < 0.05).The drinking behavior of layers in G3 group was significantly higher than that in G12 group and G24 group(P < 0.05).The pecking behavior of layers in G3 group,G12 group and G24 group was significantly higher than that in G12group(P < 0.05).The feather pecking behavior of layers in G3 group was significantly higher than that in G12 and G24 groups(P < 0.05).The greening behavior of layers in G6 group was significantly higher than that in G3 group,G12 group and G24 group(P < 0.05),while that in G12 group was significantly lower than that in G3 group and G24 group(P < 0.05).Under the condition of sufficient perch resources,the perch utilization count of high-ranked layers in G3 group increased significantly,while low-ranked layers in G6,G12 and G24 groups increased significantly by group size increased(P < 0.05);The perch utilization count of highranked layers in G12 group increased significantly,the medium-ranked layers in G3,G6 and G12 groups decreased with the increase of group size,and the low-ranked layers increased with the increase of group size(P < 0.05).The lying and exploring behaviors expressed on the perch by layers of high-ranked were significantly higher than those of low-ranked layers(P < 0.05).The feeding behavior of layers with medium-ranked was significantly higher than that of layers with high-ranked(P < 0.05).The lying and head shaking behavior expressed by low-ranked layers were significantly higher than those expressed by high-ranked layers(P < 0.05),and the escaping and pecking behavior expressed by high-ranked layers were significantly higher than those expressed by medium-ranked layers(P < 0.05).There were significant differences in the greening behavior expressed by layers among different social rank order,and the order from high to low was lowranked > medium-ranked > high-ranked(P < 0.05).The comforting behavior expressed by highranked layers was significantly higher than that of low-ranked layers(P < 0.05).It can be seen that social rank order affects the behavior of layers in different social groups.Experiment 3: The effects of stable population social rank order on cecal flora,intestinal immune factors and hypothalamic transcriptome in laying hens were studied.A total of 120 16-week-old laying hens were randomly divided into small group laying hens group(6/cage)and large group laying hens group(24/cage),with 4 replicates in each group.The laying hens of the highest(small group: AH,large group: BH)and the lowest social rank order(small group: AL,large group:BL)were taken from each group to collect the blood from pterygoid vein,and then the chickens were dissected and killed to collect the contents of brain,hypothalamus,duodenum,ileum,rectum and cecum.Serum,brain and intestinal samples were used to detect physiological and cytokine indexes.The contents of hypothalamus and cecum were used for transcriptome and 16 s RNA sequencing analysis respectively.The results showed that in AH group,brain CORT,duodenal Ig M and TNF-α and INOS,rectal Ig M and IL-2 were significantly higher than those in AL group(P <0.05),while ileal Ig A and rectal INF-γ in AH group and PTGES were significantly lower than those in AL group(P < 0.05).A total of 42 differential genes(|log2foldchange| > 1,P < 0.05)were obtained by hypothalamic transcriptome sequencing in AH group and AL group,including 24 upregulated genes and 18 down-regulated genes.By analyzing the GO enrichment results of differentially expressed genes,these genes are mainly related to dopamine production and immune function.We used q RT-PCR to verify 12 differentially expressed genes(DBH,LBX1,CHRNB3,NTRK1,SEMA3 A,APOD,IHH,POMC,EPX,PITX2,EMX2,TFAP2D)that are mainly related to dopamine production and immune function,and the results of q RT-PCR were consistent with the results of transcriptome sequencing analysis.Duodenal TNF-α and PTGES,ileal IL-8,rectal Ig A and IL-8 in BH group were significantly higher than those in BL group(P < 0.05),while the contents of duodenal Ig A,rectal IL-6,COX-2 and PTGES in BH group were significantly lower than those in BL group(P < 0.05).There was no significant difference in brain 5-HT,DA and CORT,serum immunoglobulin and cytokines between the two groups(P > 0.05).Compared with BL group,the abundance of Bacteroides in BH group increased significantly,while the abundance of Prevotella decreased significantly(P < 0.05).A total of 139 differential genes(|log2foldchange| > 1,P < 0.05)were obtained by hypothalamic transcriptome sequencing between BH group and BL group,including 89 up-regulated genes and 50 down-regulated genes.We used q RT-PCR to verify the 20 differentially expressed genes(DBH,APOD,VEGFD,CCDC3,SFRP2,EN1,S100 B,FZD4,TWIST1,ENPP3,SIX1,PTK2 B,POMC,CCL19,CTLA4,CCNB3,LHX8,HCRT,NKX2-1,HRH2)related to dopamine,learning and memory and immunity screened from the enrichment results of Go enrichment,and the results of q RT-PCR were consistent with the results of transcriptome sequencing analysis.To sum up,social rank order hardly affects the secretion of brain neurotransmitters,diversity of intestinal flora,inflammatory stress level in serum and immune level of intestinal tissue in layers.This study found that DBH,EMX2,TFAP2,VEGFD,CCDC3,S100 B,SFRP2,EN1,PTK2 B and HRH2 were related to cognitive ability and learning ability.It is speculated that they may participate in the formation of social hierarchy.Experiment 4: In this study,artificial induction was used to disrupt the stable laying hen group.8 inferior individuals(Z: exposed bleeding of anal skin in a short time)and 8 dominant individuals(Y: intact anal skin)in the control group were selected.A total of 16 laying hens were slaughtered.The contents of hypothalamus,duodenum,ileum,rectum and cecum were collected for hypothalamic transcriptome sequencing,cecal 16 S r RNA gene sequencing,intestinal immunity and cytokine analysis.The results showed that compared with group Y,IL-6 and TNF-α in duodenum,IL-6 and IL-1β in ileal,Ig M,IL-6 and TNF-α in rectal expression increased significantly(P < 0.05)in group Z;but the Ig A and IL-1β in duodenum,Ig A,IL-2,INF-γ,COX-2 and INOS in ileal,IL-2and IL-8 in rectal expression decreased significantly(P < 0.05).Compared with group Y,the abundance of Elusmicrobia increased significantly(P < 0.05),while the abundance of Verrucomicrobia and Prevotella decreased significantly(P < 0.05).In group Z and group Y,85 differential genes(|log2foldchange| > 1,P < 0.05)were obtained by hypothalamic transcriptome sequencing,including 32 up-regulated genes and 53 down-regulated genes.Through KEGG pathway enrichment analysis of differentially expressed genes,it was found that these genes were mainly enriched in immune related pathways such as RIG-I-like receptor,Toll-like receptor and C-type lectin receptor signal pathways,as well as ECM receptor interaction and neuroactive ligand receptor interaction pathways.Through Go enrichment function annotation,it was found that these genes were related to stress and immunity.Finally,17 differentially expressed genes related to stress and immune function(BARHL2,ONECUT1,SCRT1,PTF1 A,WNT16,RSAD,TRIM25,NM1,DHX58,IL-10,LAT2,JCHAIN,C1 S,TLR3,STAT1,IRF7,ADIPOQ)were verified by q RT-PCR,and the results of q RT-PCR were consistent with the results of transcriptome sequencing analysis.In conclusion,in unstable groups,social rank order changes the composition of intestinal flora and affects the immune function of the intestine;In addition,the stress level and immune function of laying hens with low-ranked laying hens increased,indicating that the social rank order in the unstable population affected the health and immunity of laying hens.