| 摘要: |
| 【目的】利用16S rDNA高通量测序技术和LC-MS/MS非靶向代谢组学技术，研究短期和长期饲喂添加枯草芽孢杆菌日粮对育肥湖羊瘤胃内容物菌群和血清代谢物的影响。【方法】选择日龄((60±10) d)相近、体质量((19.51±2.07) kg/头)接近的湖羊断奶公羔36只，随机分为3组，分别为对照组(CON)、枯草芽孢杆菌Ⅰ组(BSN)和枯草芽孢杆菌Ⅱ组(BSH)，每组12只羊。CON组饲喂基础饲粮，BSN组饲喂基础饲粮+100 mg/kg枯草芽孢杆菌，BSH组饲喂基础饲粮+200 mg/kg枯草芽孢杆菌。预试期10 d，正试期60 d，分别在正试期的第30天（短期）和第60天（长期）每组随机选择6只羊取瘤胃内容物和血清进行分析。【结果】① 短期和长期饲喂添加枯草芽孢杆菌饲粮对湖羊瘤胃微生物丰富度指数Chao 1指数和多样性指数Shannon指数、Simpson指数均无显著影响(P＞0.05)。湖羊瘤胃菌群在门水平上，短期饲喂BSN组的螺旋体菌门、迷踪菌门相对丰度较CON组显著降低(P＜0.05)，长期饲喂BSH组的变形菌门相对丰度显著升高(P＜0.05)；在属水平上，短期饲喂BSN组普雷沃氏菌属UCG-003相对丰度显著降低(P＜0.05)，而瘤胃球菌属1相对丰度显著升高(P＜0.05)。长期饲喂BSN和BSH组湖羊普雷沃氏菌属UCG-003相对丰度显著降低(P＜0.05)。② 短期饲喂BSN和BSH组湖羊血清代谢物中L-苹果酸、D-2,3-二羟基丙酸、5-氨基水杨酸、2-氮杂环丁烷羧酸、棕榈酰溶血磷脂酸、1-磷酸果糖、核黄素均较CON组显著降低(P＜0.05)；BSN组5 磷酸脱氧核糖、丙烯酸、甘氨酰异亮氨酸、2-酮丁酸、1,2,4-偏苯三酚、β-D-半乳糖、丁酸乙酯均较CON组显著降低(P＜0.05)，而3′-磷酸腺苷较CON组显著升高(P＜0.05)，差异代谢物相关通路为辅因子的生物合成、核黄素代谢、磷酸转移酶系统等。长期饲喂BSH组10E,12Z 共轭亚油酸、N4-乙酰胞嘧啶、吡咯烷酮羧酸、9,10-环氧十八烷酸、肌酸酐均较CON和BSN组显著升高(P＜0.05)，差异代谢物相关性最高的关键通路为程序性坏死、鞘脂信号通路和鞘脂代谢。③ 属水平上差异微生物和差异代谢物相关性分析发现，普雷沃氏菌属UCG-003与大豆苷显著负相关(P＜0.05)，螺旋菌属2与3′-磷酸腺苷显著负相关(P＜0.05)，而瘤胃球菌属UCG-011与对苯二甲酸、喹啉酸、5,7-二羟基-2-(4-羟基-3,5-二甲氧基苯基)-4H-色胺-4-酮显著正相关(P<0.05)。【结论】枯草芽孢杆菌短期饲喂可调节湖羊瘤胃微生态平衡，使纤维降解有关的细菌相对丰度增加，但长期饲喂不仅调节能力降低，且长期大剂量（200 mg/kg饲粮）饲喂还会引起瘤胃微生物菌群失调；同时，枯草芽孢杆菌可影响湖羊代谢过程，短期饲喂可能调控辅因子的生物合成、核黄素代谢、磷酸转移酶系统等代谢过程，长期饲喂可能调控程序性坏死、鞘脂信号通路和鞘脂代谢。 |
| 关键词: 枯草芽孢杆菌 瘤胃菌群 血清代谢物 湖羊 |
| DOI： |
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| 基金项目:国家自然科学基金项目（32002203） |
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| Effect of Bacillus subtilis on rumen microbes and metabolites in Hu sheep |
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SONG Shuzhen,LIU Junbin,LIU Lishan,et al
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| Abstract: |
| 【Objective】This study assessed the short-term and long-term effects of Bacillus subtilis in diet on rumen microbes and serum metabolites in Hu sheep using 16S rDNA amplicon sequencing technology and LC-MS/MS non-targeted metabolomics technology.【Method】A total of 36 Hu lambs with similar age of (60±10) d and body weight of (19.51±2.07) kg were randomly divided into control group (CON),Bacillus subtilis group Ⅰ (BSN) and Bacillus subtilis group Ⅱ (BSH),with 12 lambs in each group.CON was fed the basal diet,BSN was fed the basal diet with 100 mg/kg Bacillus subtilis,and BSH was fed the basal diet with 200 mg/kg Bacillus subtilis.The pretest period was 10 days and the test period was 60 days.Six sheep in each group were randomly selected to collect rumen contents and serum for analysis at days 30 and 60.【Result】① Feeding Bacillus subtilis for short-term (30 days) and long-term (60 days) had no significant effect on microbial richness index of Chao 1 and Shannon and Simpson diversity indexes of rumen contents of Hu sheep (P>0.05).At the level of phylum,relative abundances of Spirochetes and Elusimicrobia were significantly lower in group BSN for short-term feeding (P<0.05),and that of Proteobacteria was significantly higher in group BSH for long-term feeding (P<0.05).At the level of genus,relative abundance of Prevotellaceae UCG-003 decreased significantly (P<0.05),while that of Ruminococcus 1 increased significantly in group BSN for short-term feeding (P<0.05).The relative abundances of Prevotellaceae UCG-003 decreased significantly in group BSN and BSH for long term feeding (P<0.05).② Among serum metabolites,L-malic acid,D-2,3-dihydroxypropanoic acid,mesalazine,2-azetidinecarboxylic acid,lyso PA(16:0/0:0),fructose-1P and riboflavin decreased significantly in group BSN and group BSH for short term feeding (P<0.05).The deoxyribose 5-phosphate,caprylic acid,glycyl-Isoleucine,2-ketobutyric acid,benzene-1,2,4-triol,beta-D-galactose and ethyl butyrate decreased significantly (P<0.05),while 3′-AMP increased significantly in group BSN (P<0.05).The pathways related to differential metabolites were biosynthesis of cofactors,riboflavin metabolism and phosphotransferase system. The 10E,12Z-octadecadienoic acid,N4-acetylcytidine,pyrrolidonecarboxylic acid,9,10 epoxyoctadecanoic acid and creatinine increased significantly in group BSH for long-term feeding (P<0.05), and the related pathways were necroptosis,sphingolipid signaling pathway and sphingolipid metabolism.③ The correlation analysis of differential microorganisms and their metabolites at genus level showed that Prevotellaceae UCG-003 had significantly negative correlation with Daidzin,Treponema 2 had significantly negative correlation with 3′-AMP (P<0.05),while Ruminococcaceae UCG-011 had significantly positive correlation with terephthalic acid,quinolinic acid and 5,7-dihydroxy-2-(4-hydroxy-3,5-dimetHoxyphenyl)-4H-chromen-4-one (P>0.05).【Conclusion】Short term feeding with Bacillus subtilis regulated rumen micro-ecological balance and increased relative abundances of bacteria related to fiber degradation,but long-term feeding with large dose (200 mg/kg) decreased the regulation effects and caused imbalance of rumen microbial flora.Feeding with Bacillus subtilis also affected the metabolic process of Hu sheep.Short-term feeding could regulate biosynthesis of cofactors,riboflavin metabolism and sphingolipid metabolism,while long-term feeding could regulate necroptosis,sphingolipid signaling pathway and sphingolipid metabolism. |
| Key words: Bacillus subtilis rumen microbes serum metabolites Hu sheep |
