【作者】 张燕；

【导师】 李家奎；

【作者基本信息】 华中农业大学 ， 临床兽医学， 2023， 硕士

【摘要】 福美双是一种低毒广谱杀菌剂,在农业生产实践中被广泛用来储存谷物和饲料,以防止真菌疾病的发生。然而,福美双的药物残留可引发不良效应,并对动物的生理健康、人类的食品安全以及生态环境系统的稳定等造成严重威胁,引发了动物机体代谢紊乱、肠道菌群生态失调和骨骼疾病等,例如胫骨软骨发育不良(Tibial Dyschondroplasia,TD),它是普遍发生于禽类中的一类代谢性软骨疾病。其中肉鸡在其快速生长发育阶段形成TD,临床表现为胫骨畸形、双腿跛行,病理特征是胫骨干骺端生长板出现增厚的、无血管的、无矿化的暗白色软骨栓塞,其过高的发病率导致肉鸡产业经济损失惨重。因此,寻找有效药物防治TD引起了整个行业的关注。现有研究表明,肠道微生物群及其代谢产物影响着宿主的生物活性、营养状况、免疫稳态以及机体代谢等,进而介导骨量的调节。丁酸钠(Sodium Butyrate,SB)是一种绿色饲料添加剂,入肠后发酵为短链脂肪酸,在控制肠道病原体、降低氧化应激以及诱导软骨细胞分化等方面起着重要作用。故而,本研究通过分析肉鸡的生长性能、胫骨参数、病理特征、抗氧化性能等,探究不同剂量SB对TD肉鸡的治疗作用;基于Wnt/β-catenin信号通路探究不同剂量SB对TD肉鸡体内成骨基因表达的调节;基于16S rRNA测序技术探究SB对TD肉鸡肠道菌群的影响及其与TD的联系。本试验随机选取1日龄肉鸡200只并平均分为五组:Control组、TD组、300mg/kg SB组、500mg/kg SB组和700mg/kg SB组。动物试验周期持续18天,研究结果如下:1.丁酸钠对TD肉鸡生长情况的影响通过福美双诱导肉鸡发生TD并记录其生长状况和胫骨发育特征。研究发现TD肉鸡表现为采食困难、体重下降、跛行、膝关节肿胀和双腿劈叉等。测量TD肉鸡胫骨指数可见胫骨重量显著减轻、生长板宽度增厚以及骨密度与骨矿物质含量显著下降(P<0.05),生长板形成白色软骨栓且无明显血管入侵。同时生长板处的软骨细胞堆积在静止区不分化,且细胞排列紊乱不规则,大量细胞核存在固缩、溶解和空泡现象。而经SB治疗后的肉鸡,站立能力、采食量和体重明显恢复至Control组水平(P<0.05),胫骨形态发育良好且骨量显著增加(P<0.05),生长板处血管丰富且浸润明显,软骨细胞形态饱满且排列相对规则有序。其中500mg/kg SB组的促生长效果最佳。这些结果表明了SB能提高TD肉鸡的料肉比、生长性能和骨骼发育水平。2.丁酸钠对TD肉鸡抗氧化性能的影响收集血清测定肉鸡的生化、抗氧化性能。研究发现TD肉鸡呈现钙磷代谢紊乱和机体防御体系受损,表现出低钙(Ca)、高磷(Pi)和低碱性磷酸酶(AKP)水平(P<0.05),且机体的总抗氧化能力(T-AOC)水平、总超氧化物岐化酶(TSOD)水平显著下降(P<0.001),而丙二醛(MDA)水平明显提升(P<0.001)。在经SB治疗后,TD组生化水平和抗氧化能力显著恢复至Control组水平,表现为高钙和高抗氧化水平(P<0.001)。此外,对比不同剂量SB组的改善效果,发现治疗组之间不存在显著差异(P>0.05)。这些结果表明了SB能恢复TD肉鸡的骨代谢平衡,促进骨钙化,并通过抑制脂质过氧化来提高其抗氧化能力,进而维持机体的生理健康。3.丁酸钠对TD肉鸡成骨基因和蛋白的影响通过RT-qPCR、免疫荧光和Western Blot技术检测Wnt/β-catenin信号通路上的相关成骨基因和蛋白的表达水平。研究发现TD组的β-catenin异常表达,显著高于SB组和Control组(P<0.001),并抑制了下游成骨基因Runx2和OPN的表达水平(P<0.001)。而经SB治疗后,TD肉鸡胫骨组织的Wnt信号被激活,上调了Wnt-5α的表达(P<0.05),抑制了GSK-3β复合物的降解活性,进而阻止β-catenin磷酸化并上调其表达(P<0.001),最终促进β-catenin的正常积累,并进一步地结合转录因子,显著提高了成骨基因Runx2和OPN的表达(P<0.05)。同时,试验中各组蛋白表达结果与基因表达结果的趋势几乎一致,表明了试验模型的稳定性。其中值得注意的是,300mg/kg SB组的基因和蛋白表达水平与500mg/kg SB组的接近,但在试验期间,其表达不稳定。而700mg/kg SB组的β-catenin蛋白过表达甚至高于TD组(P<0.001),成骨蛋白Runx2和OPN低表达甚至有时低于TD组(P<0.001)。这些结果表明了SB以剂量依赖性的方式调节Wnt/β-catenin信号通路上的基因表达,适当剂量的SB才能激活Wnt信号,促进β-catenin正常积累,进而诱导软骨细胞成熟,并调节成骨细胞分化,最终促进骨形成。相反地,高剂量的SB诱导细胞毒性,抑制软骨细胞的增殖和分化,加重骨骼损伤。4.丁酸钠对TD肉鸡肠道菌群的影响通过高通量测序探究Control组、TD组和500mg/kg SB组的组间物种多样性和群落组成差异。测序结果显示,三组间的Alpha多样性差异不显著(P>0.05);但Beta多样性差异显著,表现为TD组组内样品离散分布且物种组成与另外两组差异大,而SB组组内物种集中分布且与Control组相似程度高。综合Metastats和LEf Se数据进行组间差异分析,结果显示TD组显著增加了致病菌群Protobacteria、Subdoligranulum、Erysipelatoclostridium和Ruminococcaceae UCG-005的丰度(P<0.05),进而诱发机体炎症的发生,导致骨质流失。而SB组显著富集了众多有益的细菌包括Bacteroidetes、Verrucomicrobia、Faecalibacterium、Butyricicoccus、Collinsella、Flavonifractor、Barnesiella和Rikenella等,提高了机体免疫能力和生长性能,促进骨沉积。综上所述,本研究系统评估了SB对肉鸡TD的治疗效果,简单阐明了SB促进骨骼生长的作用机制,初步揭示了SB缓解福美双诱导的急性肠道生态失调,并提示SB以剂量依赖性地方式调节骨代谢,为肉鸡产业中骨骼疾病的防治提供了参考价值。更多还原

【Abstract】 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.更多还原