Regulation Mechanism Of Hypoxia And Thiram On The Tibial Vascularization And Its Effect On Tibia Growth Of Tibetan Chicken And Broiler Chicken

Tibial dyschondroplasia(TD)is a kind of nutrition metabolic disorder of poultry leg disease that is characterized by the abnormal endochondral ossification and appearance of non-vascularized and non-mineralized cartilage masses in tibial growth plates(TGPs).The disease was first discovered and reported in the United States in 1965 by Leach RM and MC Nesheim,which often occurs in poultry such as chicken,duck and Turkey.The main clinical manifestation is the reluctance to walk or stand,the deformation of the leg bone,the action is very slow often with the two wings to support the walking,the serious poultry even lost the ability to walk.Avian TD disease is widely distributed in countries around the world,and there is no regional concentration or high frequency,which causes an inestimable economic loss to the poultry industry worldwide every year.It is difficult to study the TD mechanism due to the complexity of the its pathogenesis,and the mechanisms underlying the etiology of TD in poultry remain unclear.Tibetan chicken(TBC)live mainly in 2,200~4,100 m of agriculture and animal husbandry of qinghai-tibet plateau region,is one of oldest indigenous breeds living in the high altitude hypoxia environment.Our investigation found no reports of leg disease in TBCs,which may have been associated with long-term high-altitude hypoxic conditions.There are also studies show that the occurrence of the chicken TD is related to the abnormality of the angiogenesis in the TGP,and the process of bone formation is closely related to the process of blood vessel growth.In view of this,a comparative study of high-altitude TBCs and low-altitude Arbor Acres chickens(AACs)was conducted.Moreover,to verify the effect of high altitude hypoxic environment on the growth and development of tibia in broiler chickens,further study on whether the occurrence of TD was related to the growth inhibition of TGP.While,a in-depth study was performed how the vascular growth process of the TGP is regulated and how does the decrease in angiogenesis affect the growth and development of the tibia in TD chickens.The main research contents are as follows.1.Comparative study on production performance and bone development of Tibetan chicken and broiler chickenTBCs live at high altitude hypoxic environment.TD cases have not been reported in TBCs.But it is commonly seen in commercial broiler chickens.There is still a great unknown about the skeletal growth difference between TBCs and low-altitude broiler chickens.In the same month,60 healthy high-altitude TBCs and 60 healthy low-altitude Arbor Acres chickens(AACs)were raised separately.The results showed that the body weight(BW),average daily weight gain(ADG)and average daily feed intake(ADFI),and the weight,length,growth plate width of tibia were significantly lower in TBCs than those of the low-altitude AACs(p<0.05).However,the histopathology of tibia revealed a significant increase in the distribution of blood vessels in the TGP in Tibetan chicken.q RT-PCR results showed HIF-1?,VEGFA and its receptor VEGFR2 and VEGFR1 expressions in the TGP of TBC were significantly higher than that of low-altitude of AAC(p<0.05).The results of ELISA showed similar changes.In the whole experiment,the contents of VEGFA were higher than that of low-altitude AACs.The content of VEGFR2 and IL-8 in serum was lower than that of AAC in the 7th day,and was higher in the 3rd day,10 th day and 14 th day than the AAC(p>0.05).These results suggested that the growth of the body and tibia was inhibited by high altitude hypoxia.But the TGP area of Tibetan chicken is rich in vascular distribution and the higher expression levers of angiogenesis-related genes including HIF-1?,VEGFA and its receptors,which might be TBC is not responsible for TD.2.Effect of high altitude hypoxic environment on the tibial growth plate via regulating the HIF-1?/VEGF/VEGFR signaling pathway in chickens120 healthy low-altitude AACs were transported to Tibet for breeding,which were divided into natural hypoxia group and normoxia group.The results showed an undesirable impact on the overall performance,body weight,tibia mass,tibia length,and mortality of AACs exposed to a high altitude hypoxic environment.However,AACs raised under hypoxic conditions showed an elevated number of red blood cells(RBCs)and an increase in hemoglobin(Hb)and hematocrit(Hct)values on day 14 compared to the hypobaric normoxia group.Notably,the histology analyses showed that the blood vessel density was also higher after exposure to the hypoxic environment for 14 days.q RT-PCR and Western blot results show that the AAC under the condition of high altitude hypoxia,tibial growth plate of oxygen sensitive gene HIF-1? and angiogenesis-related genes VEGFA and VEGFR1 expression level had a significant increase.ELISA detected changes in serum protein content,which further confirmed that the protein content of VEGFA,VEGFR2 and IL-8 could be increased by hypoxia.These results suggest that the low-altitude AACs living at high altitude hypoxia environment,had a up-regulation of HIF-1? and VEGFA and its receptors expression promoting distribution of the blood vessels in hypertrophic chondtrocyte zone in tibial growth plate,which may provide new insights for the therapeutic manipulation of hypoxia in poultry TD.3.Regulation of HIF-1?/VEGF/VEGFR signaling pathway in thiram-induced TD chickensThiram-induced chicken TD showed that the chicken was limping,the tibial growth plate was widened and the non-vessel area in TGP was white.Measurement of tibial morphological indexes showed that the weight and length of tibia decreased,and the width of growth plate and the width of growth plate increased.Histopathology showed no significant changes in the distribution of blood vessels in the proximal tibia and proliferation areas,while the vascular distribution in the hypertrophic chondrocytes decreased significantly and the chondrocytes died in large numbers in TD chickens.In addition,the complete blood count(CBC)also showed that there was significantly less RBC and Hb and Hct in TD chicken.Next,by RT-PCR,Western blot and ELISA method for detection of chickens angiogenesis related gene and protein expression in tibial growth plate and serum,respectively.The results showed that HIF-1?,VEGFA and its receptor gene expression and protein levels in the seventh day of TD broilers group were significantly reduced,and the gene expression level and distribution of the blood vessels of the tibial growth plate is strong positive correlation.Together,these findings suggest that the occurrence of chicken TD probably by regulating the angiogenesis related gene and protein expression(including HIF-1?,VEGFA,VEGFR1,VEGFR2,and IL-8)in tibial growth plate and serum,the distribution of blood vessels in the hypertrophic chondrocytes of the tibia was inhibited,which resulted in the inhibition of the growth and development of the tibia.Taken together,these findings reveal that the occurrence of TD is highly associated with inhibition of tibial angiogenesis through down-regulated expression of HIF-1?,VEGFA and VEGF receptors,which results in suppression of TGP development.4.Mechanism of VEGF/FGF/Ang-1/PDGF-BB and their receptors in the formation process of tibial blood vessels in TD chickensThe results showed that the expression levels of FGF2 and FGFR1 gene and protein(regulating mesoderm differentiation marker genes)were significantly reduced in the 7th day of TD chickens(p<0.05).Similarity,the serum protein concentrations of VEGFA and receptor VEGFR1\VEGR2(forming vascular island marker genes)were significantly reduced(p<0.05).The regulation of vascular island maturation related genes including angiogenin(Ang1,Ang2)and their endothelial receptor tyrosine kinase Tie2 were also inhibited.The suppression of these regulatory genes may lead to delays in vasculogenesis process.Next,the results show that the VEGFA and VEGFR1\VEGFR2 receptor gene and protein expressions in tibial growth plate are significantly lower,PDGF-BB and receptor PDGFR-? gene expression and serum protein levels are also decreased,which further lead to abnormal proliferation of tibial growth plate of the blood vessels and mature,reduction of blood vessel formation in TD broiler chicken.In summary,the inhibition of angiogenesis may be caused by inhibiting the expression of genes and proteins involved in the regulation of vasculogenesis and angiogenesis processes,thereby reducing the distribution of blood vessels on the tibial growth plate in TD chickens.5.Mechanism of OPG/RANKL signaling pathway on tibial osteogenesis of TD chickensThe results showed that compared with the normal group of AACs,the weight of the chicken,the weight of the tibia and the central diameter were significantly reduced,and the weight of the tibia was related to the weight of the tibia.Histopathological and morphological results showed a significant decrease in vascular distribution in the hypertrophy area of the proximal tibial growth plate of TD broilers.The chondrocytes showed nucleation and dissolution,and the growth plate width was increased.Serum biochemical results showed that the serum ALP activity was inhibited and the balance of calcium and phosphorus was destroyed.In addition,by RT-PCR and ELISA to detect bone formation related gene and protein in tibial growth plate and serum,the results showed that the OPG gene and protein expressions was significantly suppressed,RANKL expressions increased in TD chickens,which will promote RNAK signal activation.Moreover,the OPG level was positively correlated with the distribution of blood vessels on the tibial growth plate,and RANKL was negatively correlated.These results suggest that the reduction of proximal tibial blood vessels in TD chickens may lead to the death of chondrocytes,and dead chondrocytes accumulate in the growth plate.In addition,reduction of the vascular invasion in hypertrophic zone,the level of serum calcium phosphate imbalance will affect the calcification of cartilage matrix.The regulation of bone formation OPG/RNAKL gene balance was destroyed,which inhibit bone transformation,resulting in reduction of bone mass.In conclusion,the experimental study revealed for the first time that the physiological mechanism of TBCs not suffering from various leg diseases may be related to the adaptation of long-term high-altitude hypoxic environment,up-regulating the expression of HIF-1?,VEGF and its receptor genes,and improving the blood vessels on the growth plate area.Moreover,this mechanism of role of high-altitude hypoxic environments on broiler chickens has been further confirmed.In addition,thiram-induced TD model showed abnormal expression of VEGF and its receptors VEGFR1 and VEGFR2 genes on the growth plate of tibia.It was found that there was a strong positive correlation between the inhibition of tibial growth plates' vascularization and the occurrence of TD in broiler chickens,which process may be achieved through the abnormal expression of related regulatory genes during vasculogenesis and angiogenesis,thereby inhibiting the normal distribution of tibial vascularization;importantly,the formation of blood vessels and bone is closely coupled,the vascular distribution in the hypertrophic chondrocytes in tibial growth plates decreased may affect the expression of imbalance of OPG/RANKL signaling pathway and the abnormality of calcium-to-phosphorus ratio in serum,which may eventually affect the formation of bone,causing a reduction in the bone mass of the tibia.