Effect Of Stocking Density And Dietary 25-OH-D₃ Supplementation On Production Performance,egg Quality And Intestinal Health Of Laying Hens

The present study was conducted to evaluate the effect of stocking density and dietary25-hydroxycholecalciferol（25-OH-D₃）supplementation on production performance,egg quality and intestinal health of laying hens.A total of 800 45-week-old Lohmann laying hens were randomly allotted into a 2×2 factorial design with two levels of dietary25-OH-D₃（0 and 69μg/kg）and two stocking densities[506（low density）and 338（high density）cm²/hen].There were 4 treatments with 10 replicates per treatment and 4continuous cages per replicate.The experiment lasted for 16 weeks.The results show that:（1）High stocking density reduced the egg production,average egg weight（except12-16 weeks）,and average daily feed intake（except 9-12 weeks）of laying hens（P<0.05）.Dietary 25-OH-D₃ supplementation had no significant effect on egg production rate,egg weight,average daily feed intake and feed conversion ratio at each stage（P>0.05）.High stocking density reduced eggshell strength（P<0.05）,eggshell thickness（P<0.01）,and relative weight of eggshells（P<0.01）at 16 weeks,increasing the lightness（L*）value of eggshell color（P<0.05）,reducing the eggshell redness value（a*）（P<0.01）and yellowness value（b*）（P<0.05）.Dietary 25-OH-D₃supplementation significantly increased the thickness of eggshells at the 4th week（P<0.05）,and increased the relative weight of the eggshells at the 4th and 8th weeks（P<0.05）.Dietary 25-OH-D₃ significantly increased the shell thickness in layers under high stocking density（interaction,P<0.05）.25-OH-D₃significantly reduced the L*value of eggshell at 4,12 and 16 weeks（P<0.05）,increased the a*value of eggshell at 12 weeks（P=0.09）,and increased the b*value of eggshell at4,12 and 16 weeks（P<0.05）.（2）High stocking density significantly increased the levels of testosterone（P<0.01）,corticosterone（P<0.05）and endotoxin（P<0.05）in serum of laying hens.Dietary25-OH-D₃ supplementation significantly decreased the content of corticosterone and endotoxin（P<0.05）,and increased the content of carbon-substituted enzymes（P<0.01）in serum of laying hens.25-OH-D₃ increased FSH more significantly at high stocking density;25-OH-D₃ decreased diamine oxidase significantly at high stocking density;25-OH-D₃increased serum 25-OH-D₃ more significantly at high stocking density.（3）High stocking density significantly reduced the ash content and calcium content in the tibia of laying hens（P<0.05）.Dietary 25-OH-D₃ supplementation increased the strength of the tibia（P<0.05）.High stocking density significantly reduced the content of parathyroid hormone（P<0.05）and increased the content of osteocalcin（P<0.05）.25-OH-D₃ significantly reduced the content of osteocalcin（P<0.01）,which increased the content of calcitonin（P<0.01）.The medullary trabeculae in the bone marrow cavity of high stocking density were sparse and broken,the structure of tibia was poor.The medullary trabeculae were closely connected and the tibia structure was good with dietary25-OH-D₃ supplementation.（4）High stocking density significantly decreased the depth of（P<0.05）and the ratio of villus to crypt of ileum（P<0.01）;25-OH-D₃ significantly increased the villus of duodenum（P<0.05）,the villus height of jejunum（P<0.01）and the depth of crypt（P<0.05）,and significantly increased the ratio of duodenal villus to crypt（P=0.09）.At high stocking density,25-OH-D₃ significantly increased the villus height and crypt depth of ileum.High stocking density significantly increased the expression of MUC-1（P<0.05）.25-OH-D₃ significantly decreased the expression of MUC-1 and significantly increased the expression of MUC-2（P<0.05）.At high stocking density,25-OH-D₃ significantly increased the expression of Occludin.At stocking feeding density,25-OH-D₃ significantly increased the activity of T-AOC.Dietary 25-OH-D₃ supplementation significantly decreased observed species,Chao1 and ACE index of cecum（P<0.05）.At low stocking density,25-OH-D₃ would reduce the observedspeces and ACE index,indicating that25-OH-D₃ would reduce the diversity of cecal flora at low stocking density.In the composition of the flora,the dominant bacteria in the cecum were bacteroidetes and firmicutes,which account for 40.50%～60.28%and 32.26%～51.21%,respectively.High stocking density significantly reduced the ratio of bacteroidetes（P<0.05）and spirulina（P<0.01）,and also significantly increased the proportion of firmicutes（P<0.05）.25-OH-D₃ reduced the proportion of bacteroidetes at high stocking density,and increased the proportion of firmicutes.The dominant genus of the cecal was bacteroides,accounting for 16.68%～26.61%,followed by lactobacillus,faecalibacterium,and desulfovibrio.High stocking density significantly increased the proportion of Lactobacillus（P<0.05）and robusta（P<0.05）.25-OH-D₃ decreased the proportion of bacteroides at high stocking density.The results showed that high stocking density reduced egg production performance,eggshell quality（color,thickness）,bone quality（ash,calcium level）,enhances bone transformation（PTH,BGP）,and caused kidney damage（morphology,apoptosis）caused damage to the intestine（morphology,LPS,MUC-1）.25-OH-D₃ could improve eggshell quality（color,thickness）,increase bone quality（morphology,strength）,enhance bone resorption（BGP,CT）,and reduce kidney damage（morphology,apoptosis）in laying hens.It could enhance the intestinal physical barrier（MUC-1,MUC-2,Occludin）,reduce intestinal damage（morphology,LPS,DAO）,and change the composition of the intestinal flora.It indicated that supplementation with 25-OH-D₃ can alleviate the adverse effects of high stocking density on laying hens during late laying stage to some extent.