Screening Of Differentially Expressed Genes In TD And Therapeutic Effects Of Naringin And Anacardic Acid In TD Affected Chickens

Tibial Dyschondroplasia(TD)is a nutritional metabolic disease of leg,which mainly occurs in rapidly growing chickens and turkeys.The clinical symptoms of TD mainly include walking disorder,reduced growth performance,skeletal deformities and mental malaise.According to statistics,the incidence of TD around the world is about 10%-30%,however,it is mostly manifested as subclinical symptoms,it may often be overlooked.Tibial Dyschondroplasia not only affect the growth performance of chickens,but also cause a decline in quality of meat,therefore,causing huge economic losses to the poultry industry annually.In addition,it is difficult to prevent and treat TD due to its complex pathogenesis.Previous studies have shown that TD is closely related to broiler breed,nutrition and environment.However,the most fundamental cause of TD is still unknown,and no targeted drug have been developed to prevent and treat TD.The incidence rate of TD is about 10% in China.Therefore,TD is a threat to the chicken industry of China and cannot be underestimated.A study was planned in order to investigate the etiology,clinical symptoms,pathological changes,pathogenesis of TD,and to find effective drug against TD.In the present study,thiram was used to induce TD in broiler chicken.The genes with significant changes were identified by high-throughput sequencing technology and the regulatory effects of differential genes were analyzed through in-vivo and in-vitro experiments in chondrodysplasia of tibia in chickens.It provided a premise for elucidating the pathogenesis of TD and helped in evaluating the effect of drug therapy.The main contents of this research were as follows:1.Screening and analysis of genes related to TDTD is a common skeletal disease in birds,which is characterized by the formation of white cartilage plugs in the cartilage growth plate.Previous studies have shown that TD was closely related to the abnormal expression of angiogenic factors,hypoxia-inducible factors,and various other genes.However,the specific mechanism of TD is still unknown due to the complexity of etiology.The development of bone isa complex process involving the expression of genes and proteins in multiple pathways,and the failure of any one of these pathways can lead to abnormal bone development.The purpose of this study was to screen differentially expressed genes in normal chickens(control group)and TD chickens(TD group)by high-throughput sequencing technology.Our results suggested that a total of 14,380 genes were selected from the control group and TD group,including 1128 differential genes.These genes play an important role in the process of bone formation,which suggested that the occurrence of TD may be related to these genes.The GO analysis of differentially expressed genes showed that glutamate secretion,peptide-antigen binding and MHC I-protein complex formation play important roles in the occurrence of TD.Meanwhile,we found that there were 36 differential genes in the cellular community of cellular processes,64 differential genes in the signaling molecules and interaction of environmental information processing,6 differential genes in folding,sorting and degradation of genetic information processing,71 differential genes in the global and overview maps of metabolism,21 differential genes in the endocrine system of body systems by the classification statistics of KEGG enrichment pathway.The results suggested that there were many genes interactively expressed in the occurrence of TD and multiple genes participate in the expression regulation mechanism of TD.This study analyzed the differentially expressed genes in the occurrence of TD and screen out the key target genes with significant differentially expressed genes and metabolic substances involved in the occurrence of TD by RNA sequencing technology,which laid a theoretical foundation for further study on the mechanism of TD.2.Expression of differential genes in TDEndochondral osteogenesis is a complex process,which is closely related to chondrocytes,osteoblasts and blood vessels differentiation and regulated by multiple hormones and cytokines.Ihh-PTHr P signal axis plays an important role in regulating the differentiation and maturation of growth plate chondrocytes in broiler bone development.Wnt4 and Col? have been confirmed to be involved in the proliferation,differentiation and collagen synthesis of animal bone cells.In this study,thiram was used to induce TD model and the results showed that the expression of Ihh,Wnt4 and Col? genes in the TD group was down-regulated compared with the control group,which was consistent with the results of differential gene screening in chapter 1.However,the expression of PTHr P gene in the TD group was significantly up-regulated compared with the control group in the in-vivo test.In-vitro test,the PTHr P gene was expressed both in the cytoplasm of the control and the thiram-induced TD groups,but the difference between the two groups was not significant.The results suggested that the changes in the expression levels of PTHr P,Ihh,Wnt4 and Col? genes play an important role in the occurrence of TD broilers and can be considered as important regulatory genes in TD.This study laid down the foundation for further studies on the expression regulation of the above-mentioned differential genes for the control of TD by traditional Chinese medicine.3.Effects of naringin on TD induced by thiram in chickensTD is a leg disorder and it affects the growth performance.Up till now,no targeted drug has been found to treat TD.Naringin is a traditional Chinese medicine,which is commonly used in the treatment of various bone diseases.However,it is still unclear that whether naringin supplementation has an ameliorative and protective role against TD or not.Therefore,the objective of the present study was to explore the ameliorative effects of naringin on thiram-induced TD chickens.A total of 180one-day-old Arbor Acres(AA)broiler chickens were randomly divided into three equal groups(n=60): control group(standard diet),thiram induced group(thiram 50mg/kg from day 3 to day 18),and naringin treated group(naringin 100 mg/kg from day 8 to day 18).During the 18-day experiment,the clinical symptoms,growth performance,tibial bone parameters,antioxidant property of liver and serum biochemical changes were recorded to evaluate the protective effects of naringin.Additionally,m RNA expressions and protein levels of Ihh and PTHr P genes were determined via RT-q PCR and western blot.The results indicated that naringin can significantly improve the lameness in TD affected broilers by down-regulating Ihhand up-regulating PTHr P gene and their protein expression.Chickens in naringin treated group exhibited better growth trend compared with TD group and significant difference was observed between two groups on day 10,14,and 18(p<0.05 or p<0.01).Our results suggested that there was a significant increase(p<0.05 or p<0.01)in the FCR along with significant decrease(p<0.01)in ADWG and ADFI in TD group chickens during the whole experiment as compared to control group.The ADWG and ADFI in the naringin-treated group were significantly improved(p<0.05 or p<0.01)and FCR was significantly reduced.However,administration of naringin showed significant reduction in the width of growth plate as compared with thiram-fed TD group.Simultaneously,administration of naringin regained the weight and length of the tibia to normal level on day 18.The results of serum biochemical showed that the levels of ALT and AST in TD group were significantly increased(p<0.01),while AKP level was significantly decreased(p<0.01).The ALT and AST levels of the naringin-treated were significantly decrease,while the AKP level was significantly increase as compared to TD group.The levels of liver antioxidant enzymes SOD,GSH-Px and T-AOC in TD group were significantly reduced and MDA levels was significantly increased as compared to control group(p<0.05 or p<0.01).However,naringin improved the antioxidant capacity of liver by improving the levels of GSH-Px,T-AOC and SOD and by reducing the MDA content.Additionally,the GSH-Px and SOD contents of the naringin group was recovered to the levels of the control group on day 18.The results showed that naringin treatment can promote the growth performance,restore the growth plate width and reduce the liver damage induced by thiram.Meanwhile,naringin promoted the proliferation and development of chondrocytes,improved the formation of blood vessels and restored the damage of the thiram-induced tibial dyschondroplasia by regulating the expression of Ihh and PTHr P genes.The study suggested that naringin can be a potential drug for the treatment of TD.4.Effects of anacardic acid on TD in broilers by regulating Wnt4 and Col?expressionAnacardic acid(AA)is a traditional Chinese medicine,which plays an important role in treating osteoarthritis and other bone diseases.However,no information is available about medicinal effects of anacardic acid against TD.Therefore,the aim of this study was to investigate ameliorating effects of anacardic acid in thiram induced TD.A total of 300 one-day-old AA broiler chickens were equally divided into three groups;control group,TD group and anacardic acid(AA)treatment group.After that,mortality rate,tibia bone indicators,antioxidant index of liver,angiogenesis,pathological changes of cartilage,genes and proteins expression of Wnt4 and Col? in the growth plate were examined.Our results showed that the length,width and weight of tibia were low in TD chickens,while the width of tibial growth plate was enlarged significantly(p<0.05).In the anacardic acid treatment group,the length,width and weight of tibia showed improvement and the width of tibial growth plate also tend to return to normal and the number of blood vessels in the tibia was improved.The level of GSH-Px,T-AOC and SOD were significantly decreased(p<0.05),while the MDA level and size of growth plate(GP)was increased in TD affected chickens as compared to the control group.On the other hand,the anacardic acid administration significantly improved the antioxidant capacity of the liver.Immunohistochemical analysis showed that anacardic acid significantly increased the expression of Wnt4 and Col? genes.Gene expression and western blotting analysis revealed a down-regulation of Wnt4 and Col? expression in TD chickens as compared to normal chickens,while anacardic acid treatment up-regulated Wnt4 and Col?.The gene Wnt4 and gene Col? expression results were same as the chapter 1.In conclusion,the present study demonstrates that the anacardic acid can recover growth plate size and the claudication of TD-affected chickens via regulation of the Wnt4 and Col? expression in chickens and and can stimulate the growth and re-development of chondrocytes,promote bone re-absorption and collagen formation.