Effect And Mechanism Of Clostridium Butyricum On Bone And Immune Function Of Laying Hens

Bones are important immune and calcium storage organs in laying hens.Strong bones are the basis of healthy and productive laying hens.Osteoclasts,which are responsible for resorbing bone tissue,and osteoblasts,which deposit bone tissue,work together to maintain the balance of bone metabolism in laying hens.Imbalances in metabolism will lead to bone diseases in laying hens,such as osteoporosis,which seriously affect laying hen production.The immune system and the bone system are closely related,and there are many shared functional cells and cytokines between them,such as Treg cells,IL-1 and IL-10,which act on the RANKL/OPG,RUNX2 and WNT signaling pathways to regulate bone metabolism and maintain bone stability.Besides,gut microbiota can also adjust bone metabolism.On the one hand,intestinal bacteria directly participates in the regulation of bone metabolism by changing the level of short-chain fatty acids.On the other hand,the intestinal flora is closely related to immunity,affecting bone metabolism by regulating the body immune function.Gut microbes may be potential targets for addressing bone disease in laying hens.Clostridium butyricum,a kind of butyrate-producing gut commensal bacteria,is widely used as probiotics in the poultry meat breeding industry for its effects on maintaining the homeostasis of intestinal flora and regulating immune functions.There are few studies on the application of Clostridium butyricum in laying hens,and no one has explored the effects and mechanisms of Clostridium butyricum on the bones of laying hens before.Therefore,we carried out the research on the effects and mechanisms of Clostridium butyricum on the bones and immune functions of laying hens.1.The effects of Clostridium butyricum on the bones and production performance of laying hens during the brooding periods,the early laying periods,the peak laying periods,and the late laying periods.It is the basis of all research to have a positive effect on the production of laying hens.The study explored the effects of Clostridium butyricum supplementation on the development and performance of laying hens by adding Clostridium butyricum to the laying hen feed.As the result showed,Clostridium butyricum had no significant effects on the body weight of laying hens during the brooding periods.However,the shin width of the group of laying hens fed on1×10~9 CFU/kg Clostridium butyricum(hereinafter referred to as 1×10~9 CFU/kg Clostridium butyricum treatment)increased at the 5th experimental week(P<0.05)by decreasing the feed intake of laying hens during the brooding period from the 21st day of feeding(P>0.05).Both Clostridium butyricum and butyrate treatments significantly increased the bone indexes of the tibia and femur of the laying hens during brooding periods(P<0.05).For laying hens during the early laying periods,the egg production rate decreased in the 1×10~7 CFU/kg Clostridium butyricum treatment(P<0.05),while that in the 1×10~8 CFU/kg Clostridium butyricum treatment increased(P<0.05).The average egg weight of the two Clostridium butyricum treatment groups both increased(P<0.05).For the 1×10~8 CFU/kg Clostridium butyricum treatment,the tibial index increased(P>0.05),while the tibial strength decreased(P<0.05).When it came to laying hens during the peak laying periods,for 1×10~8 CFU/kg Clostridium butyricum treatment,the egg production rate slightly increased(P>0.05)and the eggshell weight and egg average weight significantly increased(P<0.05).However,the eggshell index and eggshell strength decreased in the 6th and 8th experimental week(P<0.05).There was no significant effect on the flexural strength of the tibia(P>0.05).While the flexural strength of the femur increased to a certain extent(P>0.05),and serum calcium(P>0.05)and phosphorus levels rose(P<0.05).The treatment of Clostridium butyricum had slight effects on laying hens during the late laying periods(P>0.05),only slightly increasing the egg production rate and eggshell index(P>0.05).As the research shows,adding Clostridium butyricum to the feed can improve the egg production rate,average egg weight and eggshell quality of laying hens,and can also significantly promote skeletal development of laying hens during the brooding period.2.The regulatory effects of Clostridium butyricum on the gut microbes of laying hensThe intestinal flora can be roughly divided into probiotics,neutral bacteria and harmful bacteria.This study explores the regulatory effects of Clostridium butyricum on intestinal flora from the three aspects above.As the results showed,the treatment of Clostridium butyricum and butyrate could increase the richness and genetic diversity of the gut microbiota and could significantly increase the relative abundance of short-chain fatty acid-producing genera Faecalibacterium and Subdoligranulum in the intestines,and up-regulate the acetic,propionic,isobutyric,n-butyric,isovaleric and n-valeric acids in cecal contents.The abundance of butyrate-producing bacteria in the gut microbiome increased in both Clostridium butyricum and butyrate treatments.The relative abundance of Subdoligranulum?variabile mainly increased in Clostridium butyrate treatment,while the relative abundance of Faecalibacterium?prausnitzii increased in butyrate treatment.Using Salmonella enteritidis as the representative intestinal harmful bacteria,the study found that the number of Salmonella decreased in the liver(P<0.05)and spleen(P>0.05)infected by 1×10~6 CFU Salmonella 6 days later with the pre-treatment of Clostridium butyricum.In conclusion,the above results show that Clostridium butyricum can positively regulate the intestinal flora of laying hens,improve the flora richness and genetic diversity,increase the abundance of beneficial bacteria,up-regulate the content of short-chain fatty acids in the intestine,and can effectively resist the invasion of Salmonella enteritidis.3.The effects and mechanisms of Clostridium butyricum on the bones of laying hensThe study explores the mechanisms of the positive effects of Clostridium butyricum on skeletal development of laying hens through detecting the effects of Clostridium butyricum on skeletal development,calcium and phosphorus content,bone structure and bone cell metabolism of laying hens.The results showed that Clostridium butyricum and butyrate treatments had no significant effects on the body weight of laying hens(P>0.05).The tibial index,tibial ash and tibial calcium content of laying hens significantly increased and the phosphorus content in serum significantly decreased with the administration of Clostridium butyricum(P<0.05),while with the treatment of Butyrate,only tibia ash increased(P<0.05).Neither Clostridium butyricum nor butyrate treatment significantly affected the flexural strength of tibia and femur(P>0.05).Further analysis found that Clostridium butyricum treatment mainly increased the BMD at both ends of the tibia(P<0.05),and increased the number and thickness of the trabecular bone at the upper end of the tibia.The content of serum osteogenic marker ALP in the Clostridium butyricum treatment group significantly increased(P<0.05).The ALP and TRAP staining results of tibial tissue sections showed that treatment with Clostridium butyricum and butyrate could enhance the activity of osteoblasts and reduce the number of osteoclasts.Clostridium butyricum significantly up-regulated the relative expressions of BMP2 in cecum and femur,and RUNX2 in tibia(P<0.05),and down-regulated the ratio of RANKL/OPG in each tissue.In a word,these studies show that with the treatment of the Clostridium butyricum,expression of BMP2 and RUNX2 was upregulated,the RANKL/OPG ratio has been lowered,increased osteoblast activity and decreased osteoclast bone count,the calcium content of the tibia and the bone density at both ends of the tibia can increase and the bone development of laying hens.4.Effects and mechanisms of Clostridium butyricum on immunity and osteoblast function of laying hensThe study comprehensively analyzed the effects and mechanisms of Clostridium butyricum on immunity and bones of laying hens from the following three aspects,including examining the effects of Clostridium butyricum on cytokine expressions and lymphocyte subset ratio changes in the immune system in vivo,the effects of Clostridium butyricum culture supernatant(CBCS)on lymphocyte differentiation and cytokine expressions in vitro,and the effects of CBCS treatment Effects of culture supernatants after lymphocytes on bone formation and cytokine expressions of osteoblasts.As the test showed,Clostridium butyricum could promote the development of the spleen,and significantly increase spleen IL-2,IL-10,cecal IL-10,NF-?B,tibia IL-10 and in vitro cultured lymphocytes IL-1,IL-10 relative expression(P<0.05).CD4⁺in spleen,CD4⁺CD25⁺(Tregs)in spleen and peripheral blood,and the proportion of CD8⁺and CD4⁺CD25⁺T cells in cultured lymphocytes in vitro were significantly up-regulated with the treatment of Clostridium butyricum(P<0.05).CBCS also up-regulated the ratio of P-JNK/JNK and P-P38/P38 in lymphocytes(P<0.05),and the butyrate control group(SASB)up-regulated the ratio of P-ERK/ERK(P<0.05).Through the detection of alizarin red staining and ALP activity of osteoblasts,using CBCS and SASB alone had no significant effects on the mineral nodule formation ability of osteoblasts(P>0.05),while lymphocyte culture products,especially with the treatment of CBCS and SASB,had significant osteoblast-inducing activity,and significantly up-regulated OPG,BMP2,RUNX2 and Wnt3a in osteoblasts,and down-regulated the relative expressions of RANKL and Wnt5a in osteoblasts(P<0.05).Clostridium butyricum can activate the MAPK signaling pathways and increase the proportion of Tregs and the expressions of IL-10.With the mediation of lymphocytes,Clostridium butyricum can also regulate the BMP2/RUNX2,RANKL/OPG and WNT pathways and promote the bone formation of osteoblasts.In conclusion,Clostridium butyricum positively regulates gut microbiota and immune function in laying hens and benefits bone development.