Effects Of Bacillus Subtilis On Production Performance,Bone Physiological Property In Laying Hens And Its Mechanism

Due to long raising period and large demand of egg production,laying hens have high risk osteoporosis.Accordingly,the osteoporosis is prone to decreased egg produc-tion of laying hens and eggshell quality,get fracture,paralysis and even death,which has a negative influence on hens’welfare.Probiotics as a new type of non-toxic,non-residue,non-pollution green and safe mircroecological agents can maintain host intes-tinal health,improve immunity,growth and production performance.Thereby,it has been an effective alternative to antibiotics.Recent research has shown that probiotics can also affect bone metabolism in humans and animals,like enhancing bone density and mineral content in the legs as well prevent bone loss.Therefore,this study was to investigate the effect and mechanism of Bacillus subtilis on production performance and bone physiological property of laying hens,which can provide some experimental basis for extension of it in practical application.Twenty-four 48-week-old layers were randomly divided into two groups（n=12）,including control group（Cont,feeding basic diet）and Bacillus subtilis group（B0.5,basic diet with 0.5 g/kg Bacillus subtilis）for 60 days.Water and feed were offered ad libitum.In order to analyze the effects of Bacillus subtilis on production performance,in the experiment,the number of egg production and soft breaking eggs rate were rec-orded every day.Egg qualities including egg weight,hartz unit,height of albumen,yolk color,eggshell thickness and strength were tested every ten days by Egg Analyzer,Egg Force Reader and Egg Shell Thickness gauge.At the 30^th and 60^th day,the fresh faeces were collected to measure the colonization efficiency of Bacillus subtilis.To verify the effect of Bacillus subtilis on bone metabolism of laying hens and to explore its mecha-nism.At the 30^th and 61^st day,blood samples were collected from each chicken,the level of 1,25-（OH）₂D₃,PTH,CT,E2,IL-1,IL-6,TNF-α,LPS,D-LA,Ca,P,ALP,AST and TRACP in blood were measured by ELISA kits or an automatic biochemistry ana-lyzer.At the 61^st,the femur,tibia,the intestinal segments and their contents samples of hens were collected for further detection.Bone strength and density were analyzed via Universal Test Machines and In Alyzer,the morphological of tibia and intestinal seg-ments were observed by microscope with HE staining.The Ca,P and Mg content dis-tribution in femur,eggshell and excretion were examined by plasma-optical emission spectrometer.Meanwhile,the relative gene expression of femur bone including OPG,SOST,COL1A2,RANK and RANKL,and the main intestinal segments affected by Bacillus subtilis was determined by analyzing the tight junction protein of different intestinal segments including OCLN and JAM-A through Real-time PCR and the pH value of different intestinal segments vis using pH meter.The duodenum was selected to detect and transcriptome sequencing to analyze the changes of gene in duodenum,while the immune-related genes of TF,CYBB,P2RX7 and PRKCB,and the inflamma-tory genes of IL-1,IL-6 and TNF-αwere further screened.The oxidative stress index SOD,MDA and GSH-Px of duodenum were analyzed by kits.16s RNA was used to analyze the diversity of cecal microflora of laying hens.ExperimentⅠ:The results showed the rate of soft breaking eggs（P<0.05）and eggshell thickness（P<0.05）in B0.5 group were decreased and increased,respectively,meanwhile maximum femur load（P=0.06）,maximum stress（P=0.01）,stiffness（P<0.01）,Young’s modulus（P<0.01）were elevated,and femur maximum strain（P=0.06）was suppressed.At the 61^th day,compared with Cont group,serum P content（P<0.01）of B0.5 group was decreased but femur P was significantly increased（P<0.05）,Mg of bone was reduced（P=0.04）but excreted Mg had a increased tendence（P=0.09）.In addition,plasma E2 concentration（P=0.01）and OPG mRNA（P<0.05）expression were promoted and plasma IL-1（P<0.01）and TNF-α（P<0.01）concentration were declined by 0.5 g/kg Bacillus subtilis.ExperimentⅡ:The results showed that the values of pH of the duodenum,ileum and cecum（P>0.05）were not changed,but pH value of jejunum（P<0.01）was ele-vated in B0.5 group.Compared with Cont group,the relative expression of OCLN mRNA（P<0.05）in duodenum of laying hens was increased but had no change in other segments.According to the results of PCR,the duodenum samples were selected to analyze by transcriptome sequencing,there was 397 up-regulated genes and 501 down-regulated genes,in total of 898 differential genes.These genes were majorly riched in inflammatory and immune-related GO functions.The CYBB（P<0.01）,TF（P<0.05）,P2RX7（P=0.07）,PRKCB（P=0.09）and TNF-αmRNA（P<0.01）was down-regu-lated,which were consistent with the transcriptome results.Feeding Bacillus subtilis could significantly reduce content of MDA in duodenum,but was unable to change the activity of SOD and GSH-p X（P>0.05）.16s RNA analysis showed that,compared with Cont group,Bacillus subtilis could significantly suppressed the relative abundance of Cyanobacterium（P=0.035）and Fusobacteria（P=0.098）on phylum level.Compared with Cont group,the relative abundance of Eubacterium＿hallii＿group（P=0.04）,Mail-hella（P=0.04）,norank＿f＿＿norank＿o＿＿Saccharimonadales（P=0.02）,Lachnospi-raceae＿FCS020＿group（P=0.03）,norank＿f＿＿norank＿o＿＿Gastranaerophilales（P=0.04）,Oxalobacter（P=0.03）,Hydrogenoanaerobacterium（P=0.05）,Ruminiclostridium（P=0.04）,Lachnospirace-ae＿UCG-010（P<0.05）,Ruminococcus＿torques＿group（P=0.06）,CHKCI001（P=0.07）,Fusobacterium（P=0.10）and Bilophila（P=0.08）decreased as well as Weissella（P=0.08）increased on genus level in B0.5 group.The above results indicated that Bacillus subtilis could decrease the harmful pro-inflammatory bacteria to adjust intestinal microbial barrier.In addition,Bacillus subtilis could protect the immune barrier and physical barrier of the duodenum by inhibition oxidative damage and inflammation and increasing the expression of OCLN.These was helpful to reducinge systemic inflammatory response,incrasing the secretion of estro-gen,and changing the metabolism of calcium and phosphorus for protecting bone qual-ity as well improving bone strength and eggshell quality.