| Tibial dyschondroplasia(TD)in broilers is relatively a common skeletal disease occurring in fast growing broilers and turkeys.TD is characterized by avascular and non-mineralized growth plate,with tibial bone deformation,gait disorders,standing difficulty,less growth performance and lameness.Studies have shown that the occurrence of TD in chickens mainly involves two aspects,the blockage of angiogenesis and vascular development.It make osteoblast,osteoclast and mesenchymal stem cells to have no enough nutrients requirement,which cannot complete bone sedimentary process,and finally lead to form the white cartilage suppository in tibial growth plate(GP).Normally,the clinical and subclinical rate of TD is 10%-30 % in chickens,which result in decline in disease resistance and production performance,induce breast cyst and osteomyelitis.Moreover,it leads to cruelty and animal welfare issues and reduces the carcass quality in chickens,which cause huge losses to the world each year.The incidence of TD in broiler is 10% in China and caused huge economic losses to our country in broiler industry.In order to identify the pathogenesis,pathological changes,molecular mechanism and therapeutic measures of TD in broiler chickens in China,the thiram was used to induce TD in chickens as an animal model.The growth performance,incidence of TD,tibia index,serum biochemistry,antioxidant level,histopathological observation,expression of related factors in Wnt/β-catenin signal pathway,screening of differential expression miRNAs and its target genes were analyzed to evaluate the occurrence and development of TD and the therapeutic mechanism of icariin on TD broilers.The main research contents are as follows: 1.Investigation of TD morbidity and diagnosis in some areas of ChinaTibial dyschondroplasia has become one of the most widely disease in China in recent years and causes significant economic losses to poultry industry.In present study,a survey was conducted for the first time to investigate the morbidity of TD in major chickens breeding provinces in China.At the same time,the pathological autopsy,and histopathology were used to initially establish the diagnosis of TD for broiler chickens.The result showed that the morbidity of TD was high in Hebei province,followed by Shanxi,Henan,Jiangsu,Shandong and Liaoning.The results showed that the incidence rate of TD in broiler chickens was less than 5% in China.According to the size,distribution area and area of cartilage plug,the incidence of TD can be divided into 5 levels according to the area ratio of "white cartilage suppository" with metaphysis.This study will provide reference for the incidence rate,quantification of pathogenesis and diagnostic methods of TD in broiler chickens.2.Effect of tetramethyl thiuram disulfide(thiram)in relation to tibial dyschondroplasia in chickensTetramethyl thiuram disulfide(Thiram)is one of the important fungicides with lipotropism that can be combined with cell membrane and cause membrane damage,bone morphogenic inactivation and inhibit angiogenesis.Thiram has been commonly used to induce tibial dyschondrolplasia(TD)in various avian species.However,there is no systematic study on the changes of the correlation indexes with toxic effect of the thiram in chickens.Therefore,the toxic effects of thiram on growth performance of chickens,viscera index,TD parameters,tissue pathological changes,and genes expression related to osteoblasts differentiation,vascular and tibia bone development were evaluated.The result presented that thiram group chickens were looking unhealthy,lazy and showing clinical symptoms like lameness.Thiram significantly reduced the performance of chickens and tibia length compared with control group.Thiram toxic effect significantly increased the visceral organs index,tibia index,TD score,TD incidence,and TD severity.Thiram increased serum Ca and P concentration and decreased tibia density than control chickens but the difference was not significant.The histopathology of tibia showed that thiram decreased blood version numbers and increased the accumulation of lesions in tibial GP.Immunohistochemistry analysis revealed that HIF-1α and WNT4 antibody expressions were decreased and VEGF localization was increased significantly in thiram group chickens.The results showed that thiram could promote the apoptosis of chondrocytes,induce the injury of vascular endothelial cells,reduce the vascular distribution and increase the accumulation of a large number of dead chondrocytes in tibial growth plate to form "white cartilage mass",and lead to osteogenesis failure.3.Screening and correlation analysis of key factors involved in microRNAs with Wnt/β-catenin/Runx2 pathway in tibial dyschondroplasiaTibial dyschondroplasia(TD)is one of the most common leg problems in fast-growing birds in the poultry industry,but its molecular mechanism involving microRNA(miRNA)is not fully understood.For this purpose,the morbidity and pathologic changes were evaluated to understand the TD,and high-throughput RNA sequencing(RNA-Seq)was performed to explore the differentially expressed miRNAs(DEGs).The functional of differentially expressed miRNAs were annotated.In this study,the key genes based on the Wnt/β-catenin/Runx2 pathway and miRNAs affecting the TD pathogenesis were further identified via the combined analysis of differentially expressed miRNA and differentially expressed genes,and the pathogenesis of TD was further evaluated.In this study,a total of 375,340 and 266 DEGs were identified between the TD and control groups with p-value 0.1,0.05 and 0.01 respectively,including 168,166 and 141 up-regulated,and 207,174 and 125 down-regulated unigenes,respectively.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of DEGs showed that carbon metabolism,valine-leucine and isoleucine degradation,cell cycle,etc.played an important role in the occurrence of TD.The results showed that a total of 15 differentially expressed miRNAs were predicted in this study,among which 5 Wnt proteins(Wnt4,Wnt6,Wnt8 c,Wnt9A and Wnt11)involved in the regulation were preliminary screened.It is suggested that the differential expression of miRNAs in TD chickens may affect the occurrence of TD by regulating the expression of Wnt/β-catenin pathway.This study provide theoretical basis for further understanding the pathogenesis of TD,and explore the new insights towards the screening specific miRNA as biomarkers and effective gene diagnosis and treatment of TD in broilers.4.The effect of key factors from the Wnt/β-catenin signaling pathway on the occurrence of tibial dyschondroplasiaWnt/β-catenin signaling pathway plays an important regulatory role in growth plate chondrogenesis in mammalian.There is no report whether the Wnt/β-catenin signaling pathway have same function in bone disease in poultry or not.In this study,the expressions of key genes and their target genes related to the Wnt/β-catenin signal pathway in the tibial growth plate were detected by RT-qPCR,immunohistochemistry and western blot.The results showed that the expressions of Wnt5 a and PKC in the tibial growth plate of broiler chickens decreased significantly in TD group,while the GSK-3β protein and p-beta-catenin showed an increasing trend,accompanied by down-regulation of Axin and APC.At the same time,the expressions of key factors(Dvl,CK1,TCF and Cdk8)in Wnt/β-catenin signal pathway were decreased during the occurrence of TD in broilers,and the expressions of sFRP were increased.In addition,the downstream target genes of Wnt/β-catenin signal pathway(Runx2,Sox9,MMP9 and BMP2)showed decreasing trend in TD group in broiler chickens.The results showed that Wnt/β-catenin signal pathway and its downstream related major factors play an important role in the occurrence of TD in chickens.5.Mechanism research of icariin ameliorate thiram induced Tibial dyschondroplasiaIcariin(Epimedium sagittatum)is a traditional Chinese medicine,which is commonly practiced in the treatment of various bone diseases.Recently,many researcher reports about the beneficial effects of icariin in relation to various types of bone conditions but no report is available about promoting effect of icariin against TD.Therefore,current study was conducted to explore the ameliorating effect of icariin on thiram induced TD chickens.A total 180 broiler chicks were equally distributed in three groups: Control group,TD group(induced by 50 mg/kg thiram)and icariin group(induced by 50 mg/kg thiram from day 3 to 7,and then treated with icariin @10 mg/kg/day).Additionally,all chicken groups were administered with normal standard diet ad libitum regularly until the end of experiment.The wingless type member 4(WNT4),vascular endothelial growth factor(VEGF)and P2RX7 genes and proteins expression were analyzed by quantitative real-time polymerase chain reaction and western blot analysis respectively.Tibial bone parameters,chicken growth performance,chondrocyte development and angiogenesis were determined to assess advantage and protective effect of the medicine in broiler chicken.Icariin treatment up-regulated WNT4 and P2RX7,and down regulated VEGF gene and protein expressions significantly(P < 0.05),restored the growth plate width,reduced chondrocyte damage and "white cartilage mass",promoted the development of blood vessels in growth plates,increased growth performance,and mitigated the lameness in broiler chickens.In conclusion,icariin administration recovered growth plate size,normalized performance and prevented lameness significantly.Therefore,this study provide a reference for the prevention and control of TD for future. |
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