复合抗菌肽对山羊瘤胃菌群结构的影响

Effects of composite antimicrobial peptide on rumen bacteria community structure of goat

试验旨在探讨复合抗菌肽对山羊瘤胃菌群结构的影响,以期为抗菌肽应用于反刍动物提供理论依据。选取24只4月龄川中黑山羊,分为4组,每组6只,正常精料组(A),正常精料+抗菌肽组(C),双倍精料组(D),双倍精料+抗菌肽组(E)分别饲喂精料300、300、600和600 g·d^(-1)·只^(-1),C与E组饲喂3 g·d^(-1)·只^(-1)复合抗菌肽。于21 d晨饲前每组随机选取3只采集瘤胃液,提取样品总DNA,扩增16S rRNA V4区,扩增产物采用Miseq平台测序。结果表明:1)共获得高质量序列629 634条,97%相似度下聚类后共获得13227个OTU。2)所得OTU经物种注释后,门水平上,除D组最优势菌门为厚壁菌门(Firmicutes)(34.30%)外,其余各组最优势菌门均为拟杆菌门(Bacteroidetes)(38.52%~43.68%),且添加复合抗菌肽显著降低变形菌门(Proteobacteria)与螺旋菌门(Spirochaetes)含量(P<0.05);增加精料量显著增加厚壁菌门与螺旋菌门含量(P<0.05),显著降低拟杆菌门与变形菌门含量(P<0.05)。3)属水平上,普雷沃菌属(Prevotella)为所有样品中最优势菌属(25.54%~33.88%),添加抗菌肽后,琥珀酸菌属(Succiniclasticum)、瘤胃球菌属(Ruminococcus)、假丁酸弧菌属(Pseudobutyrivibrio)、脱硫弧菌属(Desulfovibrio)显著或极显著增加(P<0.05或P<0.01),琥珀酸弧菌属(Succinivibrio)极显著降低(P<0.01),且不受精料量影响,但月形单胞菌属(Selenomonas)与密螺旋体(Treponema)的变化趋势与精料量相关,正常精料组显著降低(P<0.05),而双倍精料组无显著变化(P>0.05);增加精料量显著增加普雷沃菌属、密螺旋体等含量(P<0.05),降低琥珀酸弧菌属等含量(P<0.05)。4)各样品在alpha多样性Chao、ACE、Shannon和Simpson指数上,差异不显著(P>0.05)。表明添加复合抗菌肽降低变形菌门和螺旋菌门的相对含量,提高部分降解纤维菌属的含量;增加精料量可增加厚壁菌门和螺旋菌门相对含量,降低拟杆菌门和变形菌门的相对含量,增加普雷沃菌属相对含量,降低琥珀酸弧菌属相对含量;瘤胃细菌多样性并不受复合抗菌肽及精料量的影响。

The aim of this study was to evaluate the effects of composite antimicrobial peptide（ AMP） on rumen bacteria community structure by Mi Seq high throughput sequencing technology,which could provide theoretical basis forthe application of antimicrobial peptide in ruminant. Twenty-four male goats（ 4-month-old） were randomly divided into 4 groups according to the similar weight. Four groups were normal concentrate group A（ concentrate 300 g·d-1·goat-1）,normal concentrate ＋ AMP group C（ concentrate 300 g·d-1·goat-1＋ AMP 3 g·d-1·goat-1）,double concentrate group D（ concentrate 600 g·d-1·goat-1）,double concentrate ＋ AMP group E（ concentrate 600 g·d-1·goat-1＋ AMP 3 g·d-1·goat-1）. The rumen fluid samples were collected after 20 d,and then the total DNA were extraced for amplification 16 S rRNA（ 520 F-802 R）,sequencing by Mi Seq Illumina250. Results showed as follows：1） A total of 629 634 valid 16 S r DNA sequences and 13 227 operational taxonomic unit（ OTU） across 12 samples were obtained. 2） At phylum level,Bacteroidetes was the most abundant phyla in A,C and E groups（ 38. 52%-43. 68%）,except for the D group（ Firmicutes was the most abundant phyla,34. 30%）,and Proteobacteria and Spirochaetes were significantly decreased when adding AMP（P〈0.05）; Firmicutes and Spirochaetes were significantly increased in D group compared with A group（ P〈0. 05）,while Bacteroidetes and Proteobacteria were significantly decreased（ P〈0.05）.3） At genus level,Prevotella was the most abundant genus in all samples（25.54%-33.88%）,Succiniclasticum,Ruminococcus,Pseudobutyrivibrio and Desulfovibrio were significantly increased when adding AMP（P〈0.05 or P〈0.01）,while Succinivibrio was extremely significantly decreased（ P〈0.01）,and it was not affected by the amount of concentrate; But the change tendency of Selenomonas and Treponema were affected by the amount of concentrate when adding AMP,significantly decreased in C group compared with A group（ P〈0.05）,but no significant difference between D and E groups（P〈0.05）. Prevotella and Treponema were significantly increased in D group compared with A group（P〈0.05）,while Succinivibrio was significantly decreased（P〈0.05）.4） There were no significant differences（P〈0.05） between groups on alpha diversity index（ Chao,ACE,Shannon and Simpson）. In conclusion,Proteobacteria and Spirochaetes were decreased when adding complex antibacterial peptide,while some genus of cellulolytic were increased; Firmicutes and Spirochaetes were increased when the amount of concentrate changed from 300 to 600 g·d-1·goat-1,while Bacteroidetes and Proteobacteria were decreased,and Prevotella was increased,while Succinivibrio was decreased. Moreover alpha diversity index was not affected by complex antibacterial peptide or the amount of concentrate changed from 300 to 600 g·d-1·goat-1.