饲粮中性洗涤纤维水平对山羊瘤胃细菌结构及组成的影响

Effects of Dietary Neutral Detergent Fibre Level on Structure and Composition of Rumen Bacteria in Goats

本研究旨在利用高通量测序技术研究饲粮中性洗涤纤维(NDF)水平对山羊瘤胃细菌结构及组成的影响。选用6只山羊进行3×3拉丁方试验,依据饲粮NDF水平分为低(35.01%,LN组)、中(40.10%,MN组)和高NDF水平组(45.16%,HN组),每组2只。分3期进行饲养试验,每期试验20 d,其中预试期14 d,正试期6 d。正试期结束后采集山羊瘤胃内容物,提取细菌总DNA后,用细菌通用引物对16S rRNA的V4区进行PCR扩增,扩增产物用Illumina Hi Seq250PE测序平台进行高通量测序,测序结果用QIIME 1.8.0等生物信息学软件进行分析。结果表明:1)HN组的瘤胃液氨态氮(NH3-N)浓度极显著低于LN组和MN组(P<0.01);LN组瘤胃液乙酸/丙酸显著低于HN组(P<0.05),但MN组与其他2组无显著差异(P>0.05)。2)各组间Chao1指数和Shannon指数的差异均不显著(P>0.05);LN组observed species指数显著高于其他2组(P<0.05),其他2组间则无显著差异(P>0.05)。3)在门水平上,3组间所有细菌的相对丰度差异均不显著(P>0.05);在属水平上,HN组普雷沃氏菌科UCG-001(Prevotellaceae UCG-001)、普雷沃氏菌科UCG-003(Prevotellaceae UCG-003)和瘤胃球菌科UCG-014(Ruminococcaceae UCG-014)的相对丰度显著高于其他2组(P<0.05);HN组瘤胃球菌科NK4A214(Ruminococcaceae NK4A 214 group)、瘤胃球菌科UCG-005(Ruminococcaceae UCG-005)的相对丰度显著高于LN组(P<0.05);HN组SP3-e08和Lachnoclostridium 10的相对丰度显著低于其他2组(P<0.05);LN组解琥珀酸菌属(Succiniclasticum)相对丰度显著高于其他2组(P<0.05);MN组赖氨酸芽孢杆菌属(Lysinibacillus)、芽孢杆菌属(Bacillus)和叶杆菌属(Phyllobacterium)的相对丰度显著高于其他2组(P<0.05),食物谷菌属(Victivallis)的相对丰度极显著高于其他2组(P<0.01)。综合得出,饲粮NDF水平在35.01%~45.16%变化时,显著影响山羊瘤胃液NH3-N浓度和乙酸/丙酸,显著影响瘤胃Prevotellaceae UCG-001、Prevotellaceae UCG-003等多种菌属的相对丰度。

This study was conducted to investigate the effects of dietary neutral detergent fibre(NDF)level on structure and composition of rumen bacteria in goats using high throughput sequencing technique.Six goats were used in a 3×3 Latin square design and divided into 3 groups according to dietary NDF level,which was low(LN group,35.01%),medium(MN group,40.10%)and high NDF level groups(HN group,45.16%),and each group had 2 goats.The feeding trial lasted for 3 stages with 20 d per stage,and each stage consisted of 14 d of pre-trial period and 6 d of trial period.After the trial period,rumen contents were collected to extract bacterial total DNA.The bacteria universal primers were used to amplify the V4 region of bacteria 16S rRNA by PCR.The sequencing of the amplified products of bacteria was performed using the Illumina HiSeq 250PE sequencing platform,and the results were analyzed by bioinformatics software such as QIIME 1.8.0.The results showed as follows:1)rumen fluid ammonia nitrogen(NH 3-N)concentration of HN group was significantly lower than that of LN and MN groups(P<0.01).Rumen fluid acetate/propionate of LN group was significantly lower than that of HN group(P<0.05),but there was no significant difference between MN group and other groups(P>0.05).2)The differences of Chao1 and Shannon indexes among groups were not significant(P>0.05);observed species index of LN group was significantly higher than that of the other two groups(P<0.05),but there was no significant difference between MN group and HN group(P>0.05).3)At phylum level,the relative abundance of all bacteria of three groups was not significant(P>0.05);at genus level,the relative abundances of Prevotellaceae UCG-001,Prevotellaceae UCG-003 and Ruminococcaceae UCG-014 of HN group were significant higher than those of the other two groups(P<0.05);the relative abundances of Ruminococcaceae NK4A214 group and Ruminococcaceae UCG-005 of HN group were significant higher than those of LN groups(P<0.05);the relative abundances of SP3-e08 and Lachnoclostridium 10 of HN group were significantly lower than those the other two groups(P<0.05);the relative abundance of Succiniclasticum of LN group was significantly higher than that of the other two groups(P<0.05);the relative abundances of Lysinibacillus,Bacillus and Phyllobacterium of MN group were significantly higher than those of the other two groups(P<0.05),and the relative abundance of Victivallis of MN group was extremely significantly higher than that of the other two groups(P<0.01).These results suggest that when dietary NDF level changing at 35.01%to 45.16%,NDF level significantly affects NH 3-N concentration and acetate/propionate in rumen fluid,and the relative abundance of various bacterial genera,such as Prevotellaceae UCG-001 and Prevotellaceae UCG-003 in rumen of goats.