Causes Of Keel Bone Damage And Its Impact On Welfare In Laying Hens

With the transformation of laying hens from conventional cage to alternative housing systems as furnished cage and cage-free housing systems（aviary,floor,outdoor,and organic systems）,keel bone damage has become a severe problem influencing welfare and health of hens.Studies reported that the percentage of keel bone damage of laying hens is higher in alternative housing systems than in conventional cage,and keel bone damage（especially fractures）negatively affects production performance,egg quality,mobility,health and welfare.However,study on the causative factors of keel bone damage is limited.Therefore,this study primarily aimed to explore behavioral and bone-biological factors causing keel bone damage based on the behavior performance and keel bone development and metabolism character of laying hens housed in furnished cages.Secondly,to investigate the incidence of keel bone damage in two strains of laying hens at different laying stages and its impact on behavior and welfare.The results of the present study could provide new insights into the research of keel bone damage in laying hens.Section 1:Screening of characteristic behaviors causing keel bone damage in laying hensTotal one hundred and twenty Hy-line Brown laying hens at 18 weeks of age（WOA）were studied and housed in 12 furnished cages with 10 birds per cage.Keel bone status of laying hens was assessed at 39,42 and 45 WOA,respectively,and these hens were divided into the birds with normal keel（NK）,deviated keel（DK）and fractured keel（FK）bones based on the presence or absence of keel bone damage（deviation and fracture）at each time-point.After the assessment of keel bone damage at 42 and 45 WOA,new DK and new FK hens were recorded and selected as focal animals to observe their behaviors during 39-42 and 42-45 WOA.The results showed that the incidence of keel bone damage was increased with age of hens.Compared to NK laying hens,the time of walking and the numbers of siting and comforting behaviors in DK hens,as well as the numbers of siting,comforting and preening behaviors in FK hens were significantly reduced（P<0.05）,while the numbers of posture-change and walking in FK laying hens was significantly increased（P<0.05）.Additionally,the correlation analysis between keel bone status and behaviors displayed that keel bone damage was negatively related to siting time,comforting and preening behaviors（P<0.05）,but it was positively related to walking number（P<0.01）in laying hens.Therefore,the results of this study indicated that behavioral changes in sitting,walking,comforting and preening may be associated with keel bone damage of laying hens housed in furnished cages.Section 2:Bone-biological factors causing keel bone damage in laying hensExperiment 1:Total eighty healthy 18-week-old Hy-line Brown laying hens were studied and housed in eight furnished cages with 10 birds per cage.Keel bone status of all laying hens were assessed at 18,22,25,and 29 WOA using the X-ray method,and the serum samples of laying hens with NK,DK and FK that occurred at 29 WOA were collected across all the time-points.Subsequently,the serum samples were used to measure markers related to the metabolism of Ca and P and activities of osteoblast and osteoclast.The results showed that the concentration of serum P was significantly decreased（P<0.05）and that of serum 25-OHD₃ and 1,25-（OH）₂D₃ was significantly increased（P<0.05）in FK laying hens,and the concentrations of serum 1,25-（OH）₂D₃,PTH,and CT were significantly increased（P<0.05）and that of serum 25-OHD₃ was significantly decreased（P<0.05）in DK laying hens when compared to NK laying hens.Furthermore,laying hens with keel bone damage had higher levels of serum ALP,OC,OPG,TRAP,and CORT than NK laying hens（P<0.05）.However,the levels of serum Ca,P,PTH,ALP,TRAP,OPG,OC,and CORT in laying hens fluctuated with the age of the birds.Generally,the results of this study indicated that keel bone damage,especially fractures,could be associated with abnormal bone metabolism in laying hens.Experiment 2:Total one hundred and twenty healthy Hy-line Brown laying hens were used and housed in twelve furnished cages with 10 birds per cage for study from 18 to 45 WOA.Keel bone status of each laying hen was assessed by an X-ray machine at 45 WOA,and then the birds were divided into NK and FK laying hens according to the absence or presence of bone fractures.Subsequently,10 NK birds and 10 FK birds were randomly selected to collect blood samples for the determination of serum Ca and P metabolism,and osteoblasts and osteoclasts activity-related markers.Finally,these selected laying hens were slaughtered to collect keel bone samples for osteopath-ology staining,microstructural measurement,and RNA-seq analysis.The results showed that FK laying hens had evidently lower levels of Ca,P,CT,ALP,TRAP,and OC in serum（P<0.05）and had higher levels of PTH,OPG and COTR in serum（P<0.01）than those in NK laying hens.Bone H&E staining displayed that FK had obviously reduced trabeculae density and number（Tb.N）and elevated trabeculae separation（Tb.Sp）and osteoclasts number compared to NK.Micro-CT analysis showed that the Tb.N and BMD of FK were significantly reduced and Tb.Sp was significantly elevated（P<0.05）compared with NK.Additionally,bone RNA-seq found that there was 214 differently expressed genes（DEGs）（|log2Fold-Change|>1,P<0.05）between both NK and FK,including 88 upregulated DEGs and 126 downregulated DEGs.The q TR-PCR was applied to verify 14 DEGs of RNA-seq enriched into focal adhesion and calcium signaling KEGG pathway,and the result of q RT-PCR verification was similar to RNA-seq result.Taken together,the results of this study indicated abnormal bone metabolism may be a primary factor causing keel bone fractures in laying hens.Section 3:Effect of keel bone damage on intestinal health in laying hensTotal two hundred and forty 17-week-old Hy-line Brown laying hens and Lindian chickens（native breed）（120 birds/strain）were used and housed in 24 furnished cages（12 cages/strain）with 10 birds per cage.Keel bone status of laying hens was assessed every 4-5 weeks during entire experiment period from 17 to 45 WOA.At 45 WOA,10 NK,10 DK,and 10 FK laying hens each strain were randomly selected and slaughtered to collect duodenum,jejunum and cecum tissues and cecum contents for histopathology and morphological observation,intestinal physical barrier and Ca and P absorption-related indexes,as well as bacterial abundance and function analysis.The results of intestinal tract H&E staining showed that villus structure disorder and breakage,and villus height decreased in jejunum and caecum of laying hens with keel bone damage,and this pathologic change was the most obvious in intestine of FK hens.Intestinal ultrastructure observation showed that nucleus shrinkage,chromatin condensation and partial mitochondrial crest disappearance in jejunum and caecum of FK laying hens.Compared to NK laying hens,the m RNA and protein levels of Ca and P absorption-related genes TRPV-6,Ca BP-D28k,NCX-1,ATP2B1,ATP2B2,SLC34A1,SCL34A2,and VDR,as well as intestinal physical barrier-related genes Claudin-1,Claudin-3,Claudin-4,Occludin,TJP-1,TJP-2,TJP-3,Mucin-2,E-cadherin,and TFF-2 in intestinal tract of laying hens with keel bone damage were reduced（P<0.05）.Intestinal microflora alpha diversity indexes like Observed＿otus and Chao1 and the abundance of Firmicutes were decreased（P<0.05）,and the abundance of Bacteroidetes was increased（P<0.05）in FK laying hens when compared to NK laying hens,while these indexes was not significantly different between NK and DK hens（P>0.05）.Functional analysis of the microbial composition showed that the changes induced by keel bone fractures mainly altered the abundance of microbial pathways for amino acid,fatty acid and biotin biosynthesis（P<0.05）.In addition,the expression of majority of Ca and P absorption and intestinal physical barrier-related genes,and composition and function of intestinal flora were evidently different in both Hy-line Brown laying hens and Lindian chickens（P<0.05）.Overall,keel bone fractures impaired intestinal morphology and structure,affected nutrient absorption and transport,and altered microbial composition and function,damaging intestinal health of laying hens.Otherwise,strain effect of laying hens had significant impacts on intestinal health and function.Section 4:Effect of keel bone damage on welfare in laying hensTotal two hundred 18-week-old healthy Hy-line Brown laying hens and Lindian chickens（100birds/strain）were used and housed in 20 furnished cages（10 cages/strain）with 10 birds per cage.Keel bone status of laying hens was assessed at 25,32,39,and 45 WOA,and egg production and egg quality of NK,DK and FK laying hens were measured at each time-point.At 32 WOA,the blood of NK,DK and FK hens was collected to determine the concentration of serum markers in relation to stress and emotion,including CORT,serotonin,interleukin-1βand interleukin-6.Furthermore,fearful responses of NK,DK,and FK laying hens to certain fear tests,such as human approach test（HAT）,novel object test（NOT）,open filed test（OFT）,inversion（INV）,isolation（ISO）and tonic immobility（TI）tests,were measured at 32 and 45 WOA.The results showed that Hy-line Brown laying hens had significantly higher laying rate and lower percentage of keel bone damage than Lindian chickens（P<0.05）throughout the entire experiment period.Compared to NK laying hens,the laying rate,eggshell breaking strength,eggshell thickness,eggshell weight,and blood serotonin content were reduced（P<0.05）,but blood COTR,interleukin-1βand interleukin-6 contents were increased（P<0.05）in FK laying hens.Besides,FK laying hens had a longer TI-duration and NOT-latency time（P<0.05）,and had lesser INV-flapping wings number（P<0.05）than NK hens.However,keel bone fractures had no significant effects on feather cover quality and footpad and toe health（P>0.05）.Additionally,the level of serum serotonin,ISO-distressed vocalization number,NOT-latency time,HAT-score,and INV-flapping wings number and duration were increased（P<0.05）,while body weight,egg weight,egg quality,serum interleukin-6 content and TI-duration were decreased（P<0.05）when compared to Hy-line Brown laying hens.Therefore,keel bone fractures caused stress and fear response,impairing hen welfare;and strain of laying hens also influenced welfare and health status.