Study Of Differentially Expressed Genes In Broiler Tibial Dyschondroplasia Induced By Thiram

Tibial dyschondroplasia(TD),characteristic of accumulated growth plate cartilage into metaphyseal region of the tibiotarsus,bone deformity and lameness is one of diseases that affect fast-growing birds worldwide.The development of TD is involved in such factors as genotype,the calcium/phosphorus ratio and acid-base balance of the diet. Although many factors that cause TD have been identified,the detailed etiology of TD is not fully understood.TD along with ascite and sudden death are regarded as the three nutritional and metabolic diseases that severely affect broiler industry.As occurrence of TD leads to big loss to broiler industry worldwide,close attentions have been paid to identify its etiolgy.Some molecular techniques,such as in situ hybridization and semi-quantitative RT-PCR have been used to investigate the molecular mechanism of this disease.The results showed that expression of some genes were disordered,or its levels were lowered, or it was absent during the development of TD.As these techniques have limitations and blindness,new methods must be used to investigate the molecular mechanisms of TD in broilers,which will provide theoretical basis to breed TD resilient broiler species and to develop medicines for treatment of TD.In this study,TD in broilers was induced by using thiram and the dynamic pathological changes were observed and compared with those occurred spontaneously in TD.Furthermore,suppression substractive hybridization was employed to identify the differentially expressed genes during the development of TD.Gene differential expression at different stages in the development of TD was validated by using in situ hybridization and immunohistochemistry.The results were as follows:1 Effects of thiram on growth performance and tibial dyschondroplasis in Broiler ChickensThiram significantly increased incidence of TD(94.44%).The pathologic symptoms in the induced TD were similar to those of spontaneous TD,but were more severe than those of spontaneous TD.The lesion composed of noncalcified,nonvascularized, accumulated cartilage that can extend from the epiphyseal growth plate to the all metaphysic.The distinct character was that of the fragile growth plate.Broiler growth was slow,body weight was decreased,feed to gain ratio(F/G) was increased,and growth performance was affected.The model of thiram inducing TD may be regarded as an ideal animal model to study the mechanisms of occurrence and the progression of this disease. 2 Dynamic changes of histopathology of tibial dyschondroplasis in broiler chickens induced by thiramThe study of typical pathohistological changes in the metaphyseal region of the tibiotarsus in experimental broilers fed diet supplemented with thiram showed that the thiram promoted chondrocyte proliferation,resulted in death of large number of cells, compressed blood vessels which led to necrosis of blood vessel,hindered synthesis of extracellular matrix,deferred endochondral calcification.As a result,it severely disturbed the functioning of regulating endochondral bone.By comparison of spontaneously occurring TD and that induced by thiram,the result demonstrated that thiram could induce tibial dyschondroplasia,led to the changes in morphology and histology of growth plates,which were similar to those of spontaneously occurring TD.Therefore the study of dynamic changes may provide important thinking and clue to studying the molecular mechanism of TD development,and to screening for the effects of different micronutrients against the pathogenesis of tibial dyschondroplasia.3 Selection and identification of differentially expressed genes of tibial dyschondroplasa in broiler chikens induced by thiramTo investigate the molecular mechanisms of broiler tibial dyschondroplasia,forward and reverse subtracted cDNA libraries were built with cDNA from tibial cartilage growth plate of thiram-treated and normal broiler chickens by suppression subtractive hybridization(SSH) technology.Three hundred and fifty-six available clones were selected randomly in the two libraries.40 differentially expressed EST were selected by means of PCR and reverse dot blotting.The sequencing results revealed that these genes represented 10 known genes using blasting on-line with the GenBank dbase.Seven genes were differentially expressed in TD and normal broilers,which were revealed by semi-quantitative RT-PCR.Typeâ…©collagen(Colâ…©),Typeâ… collagen(Colâ… ),heat shock protein 90(Hsp90) and NADH dehydrogenase(NADH DH) gene expressing levels was up-regulated.Cytochrome C oxidaseâ…¢(COXâ…¢),Matrilin-3(MATN3) and carbonic anhydraseâ…¡(CA2) gene was down-regulated.Typeâ…©collagen facilitates endochondral ossification by forming matrix vesicles,Ca²⁺ influxing into matrix vesicles and compartmentalizing matrix components.Eprolyl 3-hydroxylation of typeâ… collagen is important for bone form.CA2 is involved in skeleton development and reconstitution. Matrilin-3 can be seen as an interface component,capable of interconnecting macromolecular networks,mediating interactions between cartilage fibrils and the extrafibrillar matrix and contributing to matrical maturation by a direct interaction with collagenâ…¨and indirectly with COMP serving as an adapter.As molecular chaperones, Hsp90 had such functions as regulating protein conformation,participating protein assemble and install,involving in signal transduction,inhibiting apoptosis,delaying aging and resisting stress.NADH DH and COXâ…¢related to cell metabolism by transferring electron.This identification of these differentially expressed genes involved in the perturbation of chondrocyte differentiation in TD is significant for studying the molecular mechanisms of TD and may open the new way to the elimination of the disorder by genetic selection.4 Gene differential expression at different stages in the development of tibial dyschondroplasa induced by thiram in Broiler chikensThe up-regulated genes typeâ…©collogen(Colâ…©),typeâ… collogen(Colâ… ),heat shock protein 90(Hsp90) and down-regulated genes Chondromodulin-1(ChM-1),carbonic anhydraseâ…¡(CA2),enolase1(ENO1) were revealed by in situ hybridization.The results demonstrated that TD chondrocytes synthesized elevated levels of typeâ… collagen and heat shock protein 90 mRNA by in situ hybridization.Protein levels of Typeâ… collagen and heat shock protein 90 were also increased revealed by immunohistochemistry.So it was suggested that the perturbation of genes expression led to TD,as a result,the research provides a new direction for studying the molecular mechanisms of TD.