发酵菌糠替代白酒糟对育肥牛瘤胃发酵参数、微生物菌群结构和功能的影响

Effects of Replacing Distiller’s Grains with Fermented Spent Mushroom Substrate on Rumen Fermentation Parameters,Microbial Community Structure and Function of Fattening Cattle

本试验旨在研究发酵菌糠替代白酒糟对育肥牛瘤胃发酵参数、微生物菌群结构和功能的影响。选取18月龄、体重[(350±30)kg]接近和健康状况良好的育肥牛(西门塔尔牛×本地黄牛)24头,随机分为3组,每组8头。对照组粗饲料以白酒糟、稻草和全株玉米青贮组成,记为白酒糟组(DG组),试验Ⅰ和Ⅱ组分别用发酵菌糠替代对照组50%和100%白酒糟(干物质基础),分别记为白酒糟-发酵菌糠组(DG-FSMS组)和发酵菌糠组(FSMS组)。预试期14 d,正试期60 d,于正试期第60天晨饲前采集各组瘤胃液,提取总DNA后采用16S rRNA通用引物扩增,并用Illumina HiSeq平台测序。结果表明:1)3组育肥牛瘤胃挥发性脂肪酸(VFA)和氨态氮(NH 3-N)含量均无显著差异(P>0.05)。2)共获得有效序列1629727条,聚类后得217个操作分类单元(OTU),3组间的共享OTU有215个。3)微生物组成分析发现,在门水平上,3组优势菌门均为拟杆菌门和厚壁菌门,其中DG-FSMS组TM7相对丰度显著低于FSMS组(P<0.05);在属水平上,3组优势菌属均为普雷沃氏菌属,DG组瘤胃球菌属相对丰度显著高于DG-FSMS和FSMS组(P<0.05)。DG组假丁酸弧菌属相对丰度显著低于DG-FSMS组(P<0.05)。DG组脱硫弧菌属相对丰度极显著高于DG-FSMS组(P<0.01),FSMS组显著高于DG-FSMS组(P<0.05)。4)多样性分析发现,3组间微生物α多样性指数差异不显著(P>0.05),β多样性仅DG组和DG-FSMS组主成分2(13.48%)差异显著(P<0.05)。5)通过重建未观测状态(PICRUSt)分析发现,在第2层级中有4条代谢通路相关功能基因丰度有显著差异(P<0.05),发酵菌糠替代白酒糟降低了脂类代谢和遗传信息处理代谢通路相关功能基因丰度,提高了其他次生代谢产物的生物合成和其他氨基酸代谢通路相关功能基因丰度。在第3层级中发现3组共有24条代谢通路相关功能基因丰度有显著差异(P<0.05),发酵菌糠替代白酒糟主要降低了脂肪酸合成、脂肪酸代谢、糖酵解和糖质新生、丙酮酸代谢、丙酸酯代谢、丁酸酯代谢、苯甲酸酯降解、磷酸戊糖途径、氨酰基-tRNA生物合成、蛋白质翻译代谢通路、赖氨酸降解、缬氨酸、亮氨酸和异亮氨酸生物合成、氯烷和氯烯降解、四环素生物合成和RNA聚合酶相关功能基因丰度,同时提高了氨基糖和核苷酸糖代谢、光合作用蛋白、光合作用、硫代谢、氰氨基酸代谢、鞘脂代谢、苯丙烷生物合成、硒化合物代谢和其他离子耦合转运蛋白代谢通路相关功能基因丰度。综上所述,饲喂发酵菌糠替代白酒糟饲粮对育肥牛瘤胃发酵参数、菌群多样性、优势菌门和菌属相对丰度均无不良影响,仅影响部分代谢通路相关功能基因和非优势菌属相对丰度;本研究用发酵菌糠替代50%白酒糟饲喂育肥牛对瘤胃微生物菌群结构影响更小,建议生产中发酵菌糠替代50%白酒糟效果更佳。

This experiment was conducted to investigate the effects of replacing distiller’s grains with the fermented spent mushroom substrate on rumen fermentation parameters,microbial community structure and function of fattening cattle.For this purpose,24 healthy fattening cattles with similar body weight[(350±30)kg]and similar age(18 months of age)were selected and divided into 3 groups(DG,DG-FSMS and FSMS groups)with 8 cattle in each group.The roughage of the control group was consisted of distiller’s grains,rice straw and whole-plant silage corn,and in diets for cattle in the other two groups,50%and 70%fermented spent mushroom substrate(dry matter base)were included to replace part of distiller’s grains in the basal diet.The pre-feeding period was 14 days and the normal feeding period was 60 days.Before morning feeding on day 60 of the normal feeding period,the total DNA of rumen content and universal prokaryote primers were used to hypervariable region of 16S rRNA,finally the products were sequenced on Illumina HiSeq sequencing platform.The results showed as follows:1)there was no significant difference in rumen volatile fatty acids(VFA)and ammonia nitrogen(NH 3-N)contents of fattening cattle among 3 groups(P>0.05).2)A total of 1629727 sequences across all rumen content and fecal samples were generated,and the total number of operational taxonomic units(OTU)detected by the cluster analysis reached 217,and there were 215 shared OTU among 3 groups.3)Firmicutes and Bacteroidetes were the most abundant in rumen under phyla level,and the relative abundance of TM7 in the DG-FSMS group was significantly lower than that in the FSMS group(P<0.05).Prevotella was the most abundant in rumen under genus level,and the relative abundance of Ruminococcusin in the DG group was significantly higher than that in the DG-FSMS and FSMS groups(P<0.05).The relative abundance of Pseudobutyrivibrioin in the DG group was significantly lower than that in the DG-FSMS group(P<0.05).The relative abundance of Desulfovibrio in the DG group was extremely significantly higher than that in the DG-FSMS group(P<0.01)and it in the FSMS group was significantly higher than that in DG-FSMS group(P<0.05).4)There was no significant difference in microbial Alpha diversity index among 3 groups(P>0.05),and Beta diversity was significantly different only between DG and DG-FSMS groups(13.48%)(P<0.05).5)There were significant differences in the abundance of related functional genes in 4 metabolic pathways at level 2(P<0.05).The substitution of fermented spent mushroom substrate for distiller’s grains decreased the abundance of functional genes related to lipid metabolism and genetic information processing metabolic pathways,and increased the abundance of functional genes related to biosynthesis of other secondary metabolites and metabolism of other amino acids pathways.There were significant differences in the abundance of related functional genes in 24 metabolic pathways at level 3(P<0.05).The substitution of fermented spent mushroom substrate for distiller’s grains decreased the abundance of functional genes related to fatty acid biosynthesis,fatty acid metabolism,glycolysis and gluconeogenesis,pyruvate metabolism,propanoate metabolism,butanoate metabolism,benzoate degradation,translation proteins,pentose phosphate pathway,aminoacyl-tRNA biosynthesis,lysine degradation,valine,leucine and isoleucine biosynthesis,chloroalkane and chloroalkene degradation,tetracycline biosynthesis and RNA polymerase metabolic pathways,and increased the abundance of functional genes related to amino sugar and nucleotide sugar metabolism,photosynthesis proteins,photosynthesis,sulfur metabolism,cyanoamino acid metabolism,sphingolipid metabolism,phenylpropanoid biosynthesis,selenocompound metabolism and other ion-coupled transporters metabolic pathways.In conclusion,there are no adverse effects on rumen microflora diversity,relative abundance of dominant bacteria and genera,but only some metabolic pathway related functional genes and relative abundance of non-dominant bacteria in fattening cattle fed the diet replacing distiller’s grains with fermented spent mushroom substrate.Under the conditions of this study,feeding fattening cattle with 50%distiller’s grains replaced by fermentation fungus chaff has less effect on rumen microflora structure,and it is suggested that 50%of distiller’s grains should be replaced by fermented spent mushroom substrate.