Impact of the arginine silicate inositol complex on bone metabolism in broiler chickens with tibial dyschondroplasia caused by manganese deficiency.

1. Tibial dyschondroplasia (TD) is a skeletal disorder in broilers that has financial implications, necessitating dietary modifications to reduce the prevalence of this disease. This study explored how arginine silicate inositol complex (ASI) supplementation affected tibial growth plate (TGP) and overall bone health in broilers with manganese (Mn) deficiency-induced TD.2. A total of 240 broiler chicks were divided into four groups, each consisting of 60 birds (15 replicates of four broilers each) as follows: i) Control, with 60 mg Mn per kg of diet; ii) ASI, with 60 mg Mn and 1 g ASI per kg of diet; iii) TD, with 22 mg Mn per kg of diet, and iv) TD+ASI, with 22 mg Mn and 1 g ASI per kg of diet.3. It was found that ASI supplementation increased tibial bone length in Mn-deficient TD broilers (p = 0.007). There was no Mn x ASI interaction for other bone morphometry variables (p > 0.05). However, both tibial bone mineral content and density were affected by Mn and ASI (p < 0.05). With ASI supplementation, serum bone-specific alkaline phosphatase and osteocalcin levels were elevated in the TD+ASI group compared to the TD group (p < 0.001). In the TD group, osteoprotegerin (OPG) levels in the TGP decreased compared to the control groups (p < 0.001).4. In contrast, ASI supplementation in the TD broilers counteracted the decrease in OPG compared to TD broilers without ASI supplementation (p < 0.001). The Mn level and ASI supplementation significantly influenced the OPG/receptor activator of the nuclear factor-κB ligand ratio (p < 0.001).5. In conclusion, the results demonstrated that inclusion of ASI in broiler diets could enhance bone formation variables by controlling OPG levels in the TGP, potentially serving as an effective method to decrease the occurrence of TD.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions.

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4-7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8-18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

Effects of cold stress on growth performance, carcass traits and tibia attributes in broiler chickens with thiram-induced dyschondroplasia.

The objective of this study was to investigate the effect of cold stress (CS) on growth performance and tibia attributes in broiler chickens with thiram-induced dyschondroplasia (TD). Four hundred 10-day-old male broilers were randomly allocated into four groups including, NT0: normal temperature (NT) without thiram; NT50: NT + thiram; CS0: CS without thiram; and CS50: CS + thiram in a completely randomised. The birds in CS groups were placed at a constant temperature of 15 ± 1°C during 11-20 days. Thiram (50 mg/kg) was added to the diet during 11-14 days to induce TD. Results showed that main effects of CS and thiram significantly decreased body weight and daily weight gain during 11-42 days (p < 0.05). Feed intake in the thiram50 group was significantly lower than the group thiram0 during 25-42 days (p < 0.05). Feed conversion ratio in CS birds was significantly more than NT group during 25-42 days (p < 0.05). On day 16, tibia width (TW) and TW to tibia length (TL) ratio were significantly higher in CS chicks compared to the NT group. TW was significantly higher in thiram50 group than thiram0 group (p < 0.05). On day 19, TL in CS chicks was significantly shorter than NT (p < 0.05). On day 23, growth plate width (GPW) in thiram50 group was significantly higher than thiram0 birds. In general, thiram increased tibial GPW and CS decreased TD severity as well as decreased growth performance in broilers.

Comprehensive analysis of differently expression mRNA and non-coding RNAs, and their regulatory mechanisms on relationship in thiram-induced tibial dyschondroplasia in chicken.

Thiram pollution is one of the main causes of tibial dyschondroplasia (TD) induced by feed sources. Several studies have speculated that miRNA, circRNA and lncRNA may have significant impact on the development of TD, however, the specific mRNAs and noncoding RNAs and their respective regulatory mechanisms and functions in the development of TD have not been explored. Therefore, in this present study, we screened the differentially expressed mRNA, miRNA, circRNA and lncRNA by whole-transcriptome sequencing (RNA-seq) and differentially expressed genes (DEGs) enrichment, as well as constructed the interaction network among the mRNA-miRNA, mRNA-lncRNA and mRNA-miRNA-circRNA. The sequencing results were verified by fluorescence real-time quantitative PCR (RT-qPCR). The results obtained in this study, revealed that the cells were atrophied and disordered in the TD group, and the expression of BMP6, TGF-ß and VEGF were significantly reduced. A total of 141 mRNAs, 10 miRNAs, 23 lncRNAs and 35 circRNAs of DEGs were obtained (p<0.05) Theses DEGs were enriched in the adhere junction and insulin signaling pathways. In addition, the mRNA-miRNA-circRNA network suggested that several pivotal ceRNA showed a regulatory relationship between the transcripts with miRNA, circRNA or lncRNA. Taken together, the results in the present study, represent an insight for further functional research on the ceRNA regulatory mechanism of TD in broilers.

Sodium butyrate ameliorates thiram-induced tibial dyschondroplasia and gut microbial dysbiosis in broiler chickens.

Thiram is a dithiocarbamate pesticide widely used in agriculture as a fungicide for storing grains to prevent fungal diseases. However, its residues have threatened the safety of human beings and the stability of the ecosystem by causing different disease conditions, e.g., tibial dyschondroplasia (TD), which results in a substantial economic loss for the poultry industry. So, the research on TD has a great concern for the industry and the overall GDP of a country. In current study, we investigated whether different concentrations (300, 500, and 700 mg/kg) of sodium butyrate alleviated TD induced under acute thiram exposure by regulating osteogenic gene expression, promoting chondrocyte differentiation, and altering the gut microbial community. According to the findings, sodium butyrate restored clinical symptoms in broilers, improved growth performance, bone density, angiogenesis, and chondrocyte morphology and arrangement. It could activate the signal transduction of the Wnt/ß-catenin pathway, regulate the expression of GSK-3ß and ß-catenin, and further promote the production of osteogenic transcription factors Runx2 and OPN for restoration of lameness. In addition, the 16S rRNA sequencing revealed a significantly different community composition among the groups. The TD group increased the abundance of the harmful bacteria Proteobacteria, Subdoligranulum, and Erysipelatoclostridium. The sodium butyrate enriched many beneficial bacteria, such as Bacteroidetes, Verrucomicrobia, Faecalibacterium, Barnesiella, Rikenella, and Butyricicoccus, etc., especially at the concentration of 500 mg/kg. The mentioned concentration significantly limited the intestinal disorders under thiram exposure, and restored bone metabolism.

Cluster of differentiation 147 (CD147) expression is linked with thiram induced chondrocyte's apoptosis via Bcl-2/Bax/Caspase-3 signalling in tibial growth plate under chlorogenic acid repercussion.

Tibial dyschondroplasia (TD) is a metabolic disease of young poultry that affects bone andcartilage's growth. It mostly occurs in broilers due to thiram toxicity in the feed. In this disease, tibial cartilage is not yet ripe for ossification, but it also results in lameness, death, and moral convictions of commercial poultry due to numerous apoptotic changes on cell level. These changes serve a cardinal role in this situation. Many potential problems indicate that chlorogenic acid (CGA) performs an extensive role in controlling apoptosis's perception. However, the actual role of CGA in TD affected chondrocytes in-vitro is still unidentified. The current study investigates the imperceptible insight of CGA on chondrocyte's apoptosis via B-cell lymphoma 2 (Bcl-2), Bcl-2 associated x-protein (Bax), and Caspase-3 with CD147 signalling. The expression of these markers was investigated by Immunofluorescence, western blot analysis, and reverse transcription-quantitative polymerase chain (RT-qPCR). Chondrocytes from the growth plate of tibia were isolated, cultured, and processed. A sub-lethal thiram (2.5 µg/mL) was used to induce cytotoxicity and then treated with an optimum dose (40 µg/ mL) of CGA. According to the results, thiram distorted chondrocyte cells with enhanced apoptotic rate. But, in case of CGA, high expression of CD147 enhanced cell viability of chondrocytes, accompanied by downregulation of Bax/Caspase-3 signalling with the upregulation of Bcl-2. The first possibility has ruled out in the present study by the observation that the cells apoptosis marker, Caspase-3 showed a significant change in CD147 overexpressing cells. Conversely, immunodepletion of CD147 with enhanced cleavage of Caspase-3, indicating the activation of apoptosis in chondrocytes cells. Therefore, these findings suggest a novel insight about CD147 in thiram induced TD about the regulation of Bcl-2/Bax/Caspase-3 apoptosis-signalling axis.

Puerarin enhance vascular proliferation and halt apoptosis in thiram-induced avian tibial dyschondroplasia by regulating HIF-1α, TIMP-3 and BCL-2 expressions.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate pesticide used for crop protection and storage. But, it's widespread utilization is associated with deleterious growth plate cartilage disorder in broilers termed as avian tibial dyschondroplasia (TD). TD results in non-mineralized and less vascularized proximal tibial growth plate cartilage causing lameness and poor growth performance. This study investigated the therapeutic potential of puerarin against thiram toxicity in TD affected chickens. One-day-old broiler chickens (n = 240) were alienated into three equal groups i.e. control, TD and puerarin (n = 80) and were offered standard feed. Additionally, TD and puerarin groups were offered thiram at 50 mg/kg of feed from 4 to 7 days for TD induction followed by puerarin therapy at 120 mg/kg to puerarin group only from 8 to 18 days for TD treatment. Thiram feeding to TD and puerarin group chickens caused lameness, mortality, and increased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels and growth plate (GP) size and upregulated HIF-1α expression. Besides, the production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and the expressions of TIMP-3 and BCL-2 were decreased (p < 0.05). Puerarin alleviated lameness, enhanced angiogenesis and growth performance and serum and antioxidant enzymes, decreased apoptosis and recuperated GP width by significantly downregulating HIF-1α and upregulating the TIMP-3 and BCL-2 mRNA and protein expressions in puerarin group chickens (p < 0.05). In conclusion, the toxic effects associated with thiram can be mitigated using puerarin.

Effect of total flavonoids of Rhizoma Drynariae in thiram induced cytotoxicity of chondrocyte via BMP-2/Runx2 and IHH/PTHrP expressions.

Tibial Dyschondroplasia (TD) is a prevailing skeletal disorder that mainly affects rapidly growing avian species. It results in reduced bone strength, lameness and an increase risk of fragility fractures. Total flavonoids of Rhizoma drynariae (TFRD) have been used as an effective treatment of different bone diseases in humans. The current in vitro study was conducted to explore the therapeutic effect of TFRD on thiram-induced cytotoxicity in avian growth plate cells via bone morphogenetic protein-2/runt related transcription factor-2 (BMP-2/Runx2) and Indian hedgehog/Parathyroid hormone-related peptide (IHH/PTHrP) expressions. Chondrocytes were isolated, cultured and refined from chicken's tibial growth plates in a special medium. Then chondrocytes were treated with sublethal thiram having less concentration (2.5 µg/mL) to induce cytotoxicity of chondrocyte, and then treated with providential doses (100 µg/mL) of TFRD. Thiram caused distorted morphology of chondrocytes, nuclei appeared disintegration or lysed along with decreased expressions of BMP-2/Runx2 and IHH/PTHrP. TFRD administration not only enhanced the viability of chondrocytes by itself, but also well restored the damage caused by thiram on growth plate chondrocytes by significantly up-regulating the expressions of BMP-2/Runx2 and IHH/PTHrP. Therefore, this study provides a novel insight into the further treatment of TD and other skeletal ailments and lays the foundation for prevention and treatment.

Identification of differentially expressed MiRNAs profile in a thiram-induced tibial dyschondroplasia.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate, which is widely used on seeds and storing food grains. The incorporation of thiram into the food chain could be a risk for both human beings and animals. Thiram-contaminated feed has been considered a common cause of tibial dyschondrolplasia (TD) in many avian species. The molecular mechanism of action of thiram on TD 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). RT-qPCR was used to confirm the validity as compared with sequencing data. The results showed that the marked alterations in the growth plate of the TD chickens were noticeable, with shrinking cells and irregular chondrocyte columns as compared with control group. In this study, we identified total 375 (p < 0.1), 340 (p < 0.05) and 266 (p < 0.01) significant DEGs between the TD and control groups. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that the target miRNAs were significantly enriched in different treatment groups, such as apoptosis, mRNA surveillance pathway, mitophagy-animal, etc. This study provides theoretical basis for in-depth understanding the pathogenesis of thiram-induced TD and explore the new insights towards the proposed molecular mechanism of specific miRNA as biomarkers for effective gene diagnosis and treatment of TD in broilers.

Recovery of chicken growth plate by Tanshinoneâ ¡A through wnt/ß-catenin pathway in thiram-induced Tibial Dyschondroplasia.

Tibial Dyschondroplasia (TD), a metabolic disease of fast growing poultry birds that effects the growth of bone and cartilage, is characterized by anorexia, mental depression and lameness. Wnt/ß-catenin pathway can mediate the occurrence of TD, and previous study showed the therapeutic effect of TanshinoneⅡA to TD Broilers. However there is no report about the effect of TanshinoneⅡA treating TD broiler chicken through wnt/ß-catenin pathway. The objective of this study was to explore the potential mechanism of how Tanshinone II A treats TD. Hematoxylin and eosin staining was used to study histologic pathology of growth plates. Key gene expressions were tested by western blot and reverse transcription quantitative real-time PCR. Results compared with control groups, showed the TD broilers' growth plate performed significantly better by treating with TanshinoneⅡA. After chickens treated by TanshinoneⅡA, the gene and protein expression of WNT5α and BMP-2 were increased (P < 0.05), but the ß-catenin were decreased (P < 0.05), which are all key genes expressed in wnt/ß-catenin pathway. Therefore, TanshinoneⅡA can potentially treat TD by affecting the expression of genes in wnt/ß-catenin pathway and it has availability to use as treatment for TD broilers.

Ligustrazine recovers thiram-induced tibial dyschondroplasia in chickens: Involvement of new molecules modulating integrin beta 3.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate, which is extensively used in agriculture as pesticide and fungicide for treating grains intended for seed purposes and also for storing food grains. One of the most evident and detrimental effect produced by thiram is tibial dyschondroplasia (TD) in many avian species, by feeding diets containing thiram, a growth plate cartilage disease. TD is characterized by the lack of blood vessels and impaired vascular invasion of the hypertrophic chondrocyte resulting in the massive cell death. This study investigated the effects of ligustrazine on the treatment and control of thiram induced-TD. A total of 210 chicks were divided into three equal groups (n = 70): control group (received standard diet), TD group (feed on thiram containing diet from day 3-7), and ligustrazine group (feed on thiram containing diet from day 3-7 and after that ligustrazine @ 30 mg/kg from day 8 to day 18). During the experiment, the lameness, production parameters, tibia bone indicators, pathological index changes and integrin beta 3 (ITGB3) expressions were examined. The results reveal that ligustrazine plays an important role in improving angiogenesis and decreasing chondrocytes damage in TD chicks via a new molecule modulating ITGB3. So, the administration of ligustrazine can be an important way to cope with the losses and costs associated with TD in commercial poultry farming and animal welfare issue due to environmental contamination of thiram.

Chlorogenic Acid Alleviates Thiram-Induced Tibial Dyschondroplasia by Modulating Caspases, BECN1 Expression and ECM Degradation.

Chlorogenic acid (CGA) is a widely applied traditional Chinese medicine ingredient which can be used for the treatment of osteoporosis. In this experiment, we investigated the potential therapeutic effect of chlorogenic acid on thiram-induced tibial dyschondroplasia (TD) and explored the underlying mechanisms that have been rarely mentioned by others yet. Performance indicator analysis and tibial parameter analysis showed that CGA exhibited a definite positive effect on thiram-induced TD chickens. In order to further explore the mechanisms underlying the positive actions of CGA, apoptotic, autophagic genes and MMPs involved in matrix mineralization of growth plate were evaluated in this study. The results showed that CGA decreased the expression of pro-apoptotic genes caspases-3 and caspases-9, leading to the reduction of apoptotic cells accumulated in growth plate. In addition, CGA also increased the level of BECN1, an important gene involved in autophagy, which benefits the survival of abnormal cells. Furthermore, CGA also increased the expression of MMP-9, MMP-10, and MMP-13, which can directly affect the ossification of bones. Altogether, these results demonstrate that CGA possesses a positive therapeutic effect on thiram-induced TD via modulating the expression of caspases and BECN1 and regulating the degradation of ECM (extracellular matrix).

Icariin: a Potential Compound for the Recovery of Tibial Dyschondroplasia Affected Chicken Via Up-Regulating BMP-2 Expression.

BACKGROUND: Tibial dyschondroplasia (TD) is a skeletal disease of fast growing chicken and other avian species. It is characterized by an avascular and non-mineralized growth plate, which leads to a deformed tibial bone and lameness. Unfortunately, this disease is not only responsible for causing huge economic losses but also raises animal welfare concerns. Icariin is a flavonoid, which is isolated from Epimedium pubescens herb, and it has been used to cure different diseases including bone fractures and osteoporosis. RESULTS: We designed this experiment to use icariin for the treatment of TD affect chickens; for this purpose, a total of 180 chicks were equally divided into three groups: control, TD and icariin. All the three groups were offered ad libitum same normal standard diet with an addition of thiram (50 mg/kg) from 3rd day to 7th day in TD and icariin group in order to induce TD in chickens. After the induction of TD, the chickens in icariin groups were fed standard diet with an addition of icariin at the rate of 10 mg/kg in drinking water to check the therapeutic effect of this flavonoid on TD. Our results showed that the icariin helped in restoring the TD lesion into a normal structure with significantly (P < 0.05) up-regulating the bone morphogenetic protein-2 (BMP-2) expression in the tibial growth plates (GP). CONCLUSIONS: Icariin increased the vascular area in the growth plate and decreased the average TD score. In conclusion, this study shows that icariin is a potential compound for the recovery of TD affected chickens via up-regulating the BMP-2 expression without posing a threat of ingestion of toxic veterinary drug residues to human beings upon the consumption of treated chickens.

Effect of graded calcium supplementation in low-nutrient density feed on tibia composition and bone turnover in meat ducks.

Both genetic selection and increasing nutrient density for improving growth performance had inadvertently increased leg problems of meat ducks, which adversely affects animal welfare. We hypothesised that slowing weight gain with improving tibia quality probably enhanced tibial mechanical properties and alleviated leg deformities. Therefore, the present study aimed to evaluate the effect of graded Ca supplementation in a low-nutrient density (LND) diet on tibia composition and bone turnover in meat ducks. A total of 720 15-d-old male meat ducks were randomly assigned and fed a standard nutrient density positive control (PC) diet containing 0·9 % Ca, and four LND diets with 0·5, 0·7, 0·9 and 1·1 % Ca, respectively. Ducks fed the 0·5 % Ca LND diet and the PC diet had higher incidence of tibial dyschondroplasia (TD). When compared with the 0·5 % Ca LND diet, LND diets with ≥0·7 % Ca significantly improved tibia composition, microarchitecture and mechanical properties, and consequently decreased the incidence of TD. Furthermore, LND diets with ≥0·7 % Ca increased osteocyte-specific gene mRNA expression, blocked the expression of osteoblast differentiation marker genes including osteocalcin, collagenase-1 and alkaline phosphatase (ALP), and also decreased the expression of osteoclast differentiation genes, such as vacuolar-type H+-ATPase, cathepsin K and receptor activator of NF-κB. Meanwhile bone markers such as serum ALP, osteocalcin (both osteoblast markers) and tartrate-resistant acid phosphatase (an osteoclast marker) were significantly decreased in at least 0·7 % Ca treated groups. These findings indicated that LND diets with ≥0·7 % Ca decreased bone turnover, which subsequently increased tibia quality for 35-d-old meat ducks.

Clinical efficiency and safety of Hsp90 inhibitor Novobiocin in avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a bone defect of broilers and other poultry birds that disturbs growth plate and it causes lameness. Previously we evaluated differential expression of multiple genes involved in growth plate angiogenesis and reported the safety and efficacious of medicinal plant root extracted for controlling TD. In this study, clinical and protective effect of an antibiotic Novobiocin (Hsp90 inhibitor) and expression of Hsp90 and proteoglycan aggrecan was examined. The chicks were divided into three groups; Control, thiram-induced TD, and Novobiocin injected TD. After the induction of TD, the Novobiocin was administered through intraperitoneal route to TD-affected birds until the end of the experiment. The expressions and localization of Hsp90 were evaluated by qRT-PCR, immunohistochemistry (IHC) and western blot, respectively. Morphological, histological examinations, and serum biomarker levels were evaluated to assess specificity and protective effects of Novobiocin. The results showed that TD causing retarded growth, enlarged growth plate, distended chondrocytes, irregular columns of cells, decreased antioxidant capacity, reduced protein levels of proteoglycan aggrecan, and upregulated in Hsp90 expression (p < 0.05) in dyschondroplastic birds as compared with control. Novobiocin treatment restored growth plate morphology, reducing width, stimulated chondrocyte differentiation, sprouting blood vessels, corrected oxidative imbalance, decreased Hsp90 expressions and increased aggrecan level. Novobiocin treatment controlled lameness and improved growth in broiler chicken induced by thiram. In conclusion, the accumulation of the cartilage and up-regulated Hsp90 are associated with TD pathogenesis and irregular chondrocyte morphology in TD is along with reduced aggrecan levels in the growth plate. Our results indicate that Novobiocin treatment has potential to reduce TD by controlling the expression of Hsp90 in addition to improve growth and hepatic toxicity in broiler chicken.

Hsp90 inhibitor celastrol reinstates growth plate angiogenesis in thiram-induced tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is an important long bone defect of broiler chickens that disturbs the proximal growth plate and is characterized by non-vascularized cartilage, a distended growth plate and lameness. Celastrol, a medicinal root extract from the plant Tripterygium wilfordii, is reported widely as a well-known heat-shock protein 90 (Hsp90) inhibitor. Recently, Hsp90 inhibition in chondrocyte differentiation and growth-plate vascularization were effective in restoring the morphology of the growth plate. The present study was aimed at investigating Hsp90 inhibition in TD using celastrol. The broiler chicks were divided into three groups; Control; TD induced (40 mg/kg thiram) and celastrol treatment. Hsp90, vascular endothelial growth factor and Flk-1 expressions were evaluated by quantitative real-time polymerase chain reaction and the protein levels of Hsp90 were measured by Western blot analysis. Antioxidant enzymes were determined to assess the liver damage caused by thiram and the protective effects of the medicine were evaluated by levels of serum biomarkers. The expression levels of Hsp90 and vascular endothelial growth factor mRNA transcripts were increased while Flk-1 receptor was decreased in TD-affected chicks. Celastrol therapy inhibited Hsp90 mRNA and protein levels and up-regulated the expressions of receptor Flk-1 in TD-affected tibial growth plates significantly (P < 0.05) in addition to rectifying the damaging effects of thiram on the liver by decreasing the levels of aspartate aminotransferase, alanine aminotransferase and malondialdehyde and correcting the oxidative imbalance. In conclusion, administering celastrol to dyschondroplastic chicks prevented un-vascularized growth plate, lameness and reinstated angiogenesis. Celastrol may be efficacious for the treatment of TD through the inhibition of Hsp90 expression and limiting the liver damage caused by thiram in broiler chickens.

The autophagy-mediated mechanism via TSC1/mTOR signaling pathway in thiram-induced tibial dyschondroplasia of broilers.

Thiram is a member of the dithiocarbamate family and is widely used in agriculture, especially in low-income countries. Its residues lead to various diseases, among which tibial dyschondroplasia (TD) in broiler chickens is the most common. Recent studies have also demonstrated that thiram residues may harm human health. Our previous study showed that the activity of the mTOR (mammalian target of rapamycin) signaling pathway has changed after thiram exposure. In the current study, we investigated the effect of autophagy via the mTOR signaling pathway after thiram exposure in vitro and in vivo. Our results showed that thiram inhibited the protein expression of mTOR signaling pathway-related genes such as p-4EBP1 and p-S6K1. The analysis showed a significant increase in the expression of key autophagy-related proteins, including LC3, ULK1, ATG5, and Beclin1. Further investigation proved that the effects of thiram were mediated through the downregulation of mTOR. The mTOR agonist MHY-1485 reverse the upregulation of autophagy caused by thiram in vitro. Moreover, our experiment using knockdown of TSC1 resulted in chondrocytes expressing lower levels of autophagy. In conclusion, our results demonstrate that thiram promotes autophagy via the mTOR signaling pathway in chondrogenesis, providing a potential pharmacological target for the prevention of TD.

Thiram exposure induces tibial dyschondroplasia in broilers via the regulation effect of circ_003084/miR-130c-5p/BMPR1A crosstalk on chondrocyte proliferation and differentiation.

Thiram, an agricultural insecticide, has been demonstrated to induce tibial dyschondroplasia (TD) in avian species. Circular RNA (circRNAs), a novel class of functional biological macromolecules characterized by their distinct circular structure, play crucial roles in various biological processes and diseases. Nevertheless, the precise regulatory mechanism underlying non-coding RNA involvement in thiram-induced broiler tibial chondrodysplasia remains elusive. In this study, we established a broiler model of thiram exposure for 10 days to assess TD and obtain a ceRNA network by RNA sequencing. By analyzing the differentially expressed circRNAs network, we id entify that circ_003084 was significantly upregulated in TD cartilage. Elevated circ_003084 inhibited TD chondrocytes proliferation and differentiation in vitro but promote apoptosis. Mechanistically, circ_003084 competitively binds to miR-130c-5p and prevents miR-130c-5p to decrease the level of BMPR1A, which upregulates the expression of apoptosis genes Caspase 3, Caspase 9, Bax and Bcl2, and finally facilitates cell apoptosis. Taken together, these findings imply that circ_003084/miR-130c-5p/BMPR1A interaction regulated TD chicken chondrocyte proliferation, apoptosis, and differentiation. This is the first work to reveal the mechanism of regulation of circRNA-related ceRNA on thiram-induced TD, offering a key reference for environmental toxicology.

miR-206a-3p suppresses the proliferation and differentiation of chicken chondrocytes in tibial dyschondroplasia by targeting BMP6.

The poultry skeletal system serves multiple functions, not only providing structural integrity but also maintaining the balance of essential minerals such as calcium and phosphorus. However, in recent years, the consideration of skeletal traits has been overlooked in the selective breeding of broilers, resulting in an inadequate adaptation of the skeletal system to cope with the rapid increase in body weight. Consequently, this leads to lameness and bone diseases such as tibial dyschondroplasia (TD), which significantly impact the production performance of broilers. Accumulating evidence has shown that microRNAs (miRNA) play a crucial role in the differentiation, formation, and disease of cartilage. However, the miRNA-mediated molecular mechanism underlying chicken TD formation is still poorly understood. The objective of this study was to investigate the biological function and regulatory mechanism of miRNA in chicken TD formation. Based on transcriptome sequencing of tibial cartilage in the healthy group and TD group, miR-206a-3p was found to be highly expressed in TD cartilage. The function of miR-206a-3p was explored through the transfection test of miR-206a-3p mimics and miR-206a-3p inhibitor. In this study, we utilized qRT-PCR, CCK-8, EdU, western blot, and flow cytometry to detect the proliferation, differentiation, and apoptosis of chondrocytes. The results revealed that miR-206a-3p suppressed the proliferation and differentiation of TD chondrocytes while promoting their programmed cell death. Furthermore, through biosynthesis and dual luciferase assays, it was determined that BMP6 was the direct target gene of miR-206a-3p. This finding was further supported by rescue experiments which confirmed the involvement of BMP6 in the regulatory pathway governed by miR-206a-3p. Our results suggest that miR-206a-3p can inhibits the proliferation and differentiation promote apoptosis through the target gene BMP-6 and suppressing the Smad2/3 signaling pathway in chicken TD chondrocytes.

Transcriptome analysis reveals the pathogenesis of spontaneous tibial dyschondroplasia in broilers.

Tibial dyschondroplasia (TD) is a severe bone disease that affects fast-growing broiler chickens and causes economic loss. Despite previous studies, the regulatory mechanism of TD remains unclear and is thought to be primarily based on thiram induction, which may differ from that of naturally occurring diseases. To better understand TD, a digital X-ray machine was used in the present study to determine its incidence in four hundred yellow-feathered broiler chickens. The results showed that the incidence of TD was 22% after 6 weeks and gradually decreased after 8 and 10 weeks. The body weight of broilers with TD decreased significantly compared to that of NTD broilers. In addition, the length and density of the tibia were reduced after eight and 10 weeks, and the density of the tibia was reduced after 6 weeks compared with the NTD chickens. This study also examined tibial quality parameters from TD (n = 12) and NTD broilers (n = 12) and found that bone mineral content, bone mineral density, bone ash content, calcium content, and phosphorus content were significantly reduced in TD broilers. Transcriptome analysis revealed 849 differentially expressed genes (DEGs) in the growth plate between TD (n = 6) and NTD groups (n = 6). These genes were enriched in ECM-receptor interaction, cytokine-cytokine receptor interaction, calcium signaling pathway, and TGF-ß signaling. Genes encoding the alpha chain of type XII collagen, that is, COL1A1, COL5A1, and COL8A1) were identified as critical in the regulatory network of TD. Gene set enrichment analysis (GSEA) revealed that the pathways of cartilage development, circulatory system development, and nervous system development were changed in the growth plates of TD birds. In the blood transcriptome, 12 DEGs were found in TD (n = 4) and NTD chickens (n = 4), and GSEA revealed that the pathways from TD broilers' blood related to the phagosome, linoleic acid metabolism, monoatomic ion homeostasis, and calcium ion transport were downregulated. This study provides a comprehensive understanding of TD, including its effects on tibial quality, tibial changes, and the circulatory system, along with identifying important genes that may lead to the development of TD.