| Thiram is a low toxicity and broad-spectrum fungicide widely used in agricultural practices to store grain and feed to prevent fungal diseases.However,drug residues of thiram have triggered adverse effects and posed severe threats to the physiological health of animals,human food safety and the stability of the ecosystem,causing metabolic disorders,dysbiosis of the intestinal flora and skeletal diseases,such as tibial dyschondroplasia(TD),a group of metabolic cartilage disorders that commonly occur in birds.Among them,broilers are formed during their rapid growth and development stages.Its clinical manifestations are tibial deformity,lameness of both legs,and pathological features of thickened,non-vascular,non-mineralized,dark white cartilage embolism in the growth plate of the tibial epiphysis,whose excessive incidence leads to heavy economic losses in the broiler industry.Therefore,the research for effective drugs to replace antibiotics for TD control has attracted attention throughout the industry.Existing studies have indicated that the gut microbiota and its metabolites affect the host’s biological activity,nutritional status,immune homeostasis and organismal metabolism,which mediates bone mass regulation.Sodium butyrate(SB)is a green feed additive fermented by intestinal microorganisms to produce short-chain fatty acids,essential in controlling intestinal pathogens,reducing oxidative stress and inducing chondrocyte differentiation.Therefore,this study was conducted to investigate the therapeutic effects of different doses of SB on TD broilers by growth performance,tibial parameters,pathological characteristics,and antioxidant properties.Based on the Wnt/β-catenin signaling pathway,we investigated the mechanism of regulating osteogenic gene expression by different concentrations of SB.16 S rRNA sequencing was used to examine the effect of SB on the intestinal flora of TD broilers and its association with bone.During the study,200 one-day-old broilers’ chicks were randomly selected and equally divided into five groups: Control group,TD group,300 mg/kg SB group,500 mg/kg SB group,and 700 mg/kg SB group.The animal experiment duration was 18 days and the results of the study were as follows.1.Effect of sodium butyrate on the growth of TD broilersThe growth and tibial development characteristics were recorded after TD induction in broilers by thiram.It was found that TD broilers had feeding difficulties,weight loss,lameness,swollen knee joints,and splitting of both legs.The tibia index of TD broilers was measured,showing a significant reduction in tibia weight,a thickening of the width of the growth plate,and a significant decrease in bone density and mineral content(P<0.05).Moreover,the formation of white cartilage plugs in the growth plate without obvious vascular invasion was also noticed.At the same time,the chondrocytes at the growth plate were noticed to be accumulated in the resting area without differentiation,and the cell arrangement was disordered and irregular.A large number of nuclei had solidification,lysis,and vacuolation phenomena.In contrast,broilers treated with SB showed significant improvement(P<0.05)in standing ability,feed intake,body weight,tibia morphology,bone mass,rich vascularity,and infiltration at the growth plate with full chondrocyte morphology and orderly arrangement.The 500 mg/kg SB group observed the best growth promotion effect.Hence,these results showed that SB could improve the feed reward ratio,growth performance,and bone development level of TD broilers.2.Effect of sodium butyrate on the antioxidant performance of TD broilersSerum was collected to determine the biochemical and antioxidant properties of broilers.It was found that TD broilers showed disorders of calcium(Ca)and phosphorus(Pi)metabolism and impairment of the body’s defense system,exhibiting low calcium(Ca),high phosphorus(Pi),and low alkaline phosphatase(AKP)levels(P<0.05).Also,total antioxidant capacity(T-AOC)and total superoxide dismutase(T-SOD)decreased significantly(P<0.001),while malondialdehyde(MDA)levels increased significantly(P<0.001).After treatment with SB,the TD group’s biochemical levels and antioxidant capacity returned to the control group’s level,exhibiting high calcium and antioxidant levels(P<0.001).Among them,the improvement effect of different doses of SB was good,and there was no significant difference(P>0.05).These results demonstrated that SB could restore the bone metabolic balance in TD broilers,promote bone mineralization,inhibit lipid peroxidation,and improve antioxidant capacity,thus maintaining the physiological health of the organism.3.Effect of sodium butyrate on bone formation genes and proteins in TD broiler chickensThe expression levels of relevant osteogenic genes and proteins on the Wnt/β-catenin signaling pathway were detected by RT-qPCR,immunofluorescence,and Western Blot techniques.The abnormal expression of β-catenin in the TD group was significantly higher than that in the SB and control groups(P<0.001)and inhibited the expression levels of the downstream osteogenic genes Runx2 and OPN(P<0.001).In contrast,after treatment with SB,Wnt signaling was activated in the tibia tissue of TD broilers,which upregulated the expression of Wnt-5α(P<0.05),inhibited the degradation activity of GSK-3β complex,which in turn prevented β-catenin phosphorylation and upregulated its expression,and finally promoted the normal accumulation of β-catenin(P<0.001),and increased the expression of osteogenic genes,i.e.,Runx2 and OPN(P<0.05).The expression of these osteogenic genes was increased.Meanwhile,the trend of protein expression results of each group in the experiment was almost consistent with the gene expression results,indicating the stability of the experimental model.Notably,the gene and protein expression levels in the 300 mg/kg SB group were close to those in the 500mg/kg SB group,but the expression was unstable during the test period,while the β-catenin protein overexpression in the 700 mg/kg SB group was even higher than that in the TD group(P<0.001),and the osteogenic protein Runx2 and OPN were lowly expressed or even sometimes lower than that in the TD group(P<0.001).These results suggest that SB regulates gene expression in the Wnt/β-catenin signaling pathway in a dose-dependent manner and that an appropriate dose of SB is required to activate Wnt signaling and promote the average accumulation of β-catenin,which in turn induces chondrocyte maturation and regulates osteoblast differentiation,ultimately promoting bone formation.Conversely,high doses of SB induce cytotoxicity,inhibit cell proliferation and differentiation,and aggravate bone damage.4.Effect of sodium butyrate on the intestinal flora of TD broiler chickensThe differences in species diversity and community composition among the control,TD,and 500 mg/kg SB groups were investigated by high-throughput sequencing.The sequencing results showed that the differences in Alpha diversity among the three groups were not significant(P>0.05),but the differences in Beta diversity were substantial,indicating that the samples in the TD group were dispersed.The species composition differed from the other two groups,while the species in the SB group were concentrated and similar to the control group.Analysis of the combined Metastats and LEf Se data showed that the TD group significantly increased the abundance of the pathogenic groups,i.e.,Proteobacteria,Subdoligranulum,Erysipelatoclostridium and Ruminococcaceae UCG-005(P<0.05),which induced the development of inflammation in the organism and led to bone loss.In contrast,the SB group was significantly enriched with numerous beneficial bacteria,including Bacteroidetes,Verrucomicrobia,Faecalibacterium,Butyricoccus,Collinsella,Flavonifractor,Barnesiella,and Rikenella,which improved the immunity and growth performance of the organism and promoted bone deposition.In conclusion,this study systematically evaluated the therapeutic effect of SB on TD in broiler chickens and briefly elucidated the mechanism of action of SB in promoting bone growth.We revealed that SB alleviated the intestinal ecological dysregulation of acute thiram exposure.It also suggested that SB regulated bone metabolism in a dosedependent manner,which provides a reference value for the prevention and treatment of skeletal diseases in the broiler industry. |
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