放牧与舍饲对彭波半细毛羊瘤胃细菌群落的生物学信息影响

Effects of Grazing and Barn Feeding on Biological Information of Rumen Bacterial Communities in Pengbo Semi-fine Wool Sheep

旨在通过不同蛋白质、非纤维性碳水化合物(NFC)和纤维素含量对瘤胃微环境细菌群落结构的影响研究,阐明瘤胃优势菌群结构的消长情况,清晰地了解瘤胃微生物对养分的依赖程度,以期为西藏传统放牧补饲及规模化养殖营养配比奠定理论基础。本研究选用年龄2岁左右,体重相近((23.77±2.34)kg)的健康彭波半细毛母羊20只,放牧组及舍饲组各10只(每组各2个重复,每个重复5只)。采用凯氏定氮法、范氏(van soest)法和差值法分别测定放牧组可饲用牧草混合样、舍饲组饲草的粗蛋白、纤维素、NFC含量。饲养半年后,抽取瘤胃液,提取总DNA,构建重组标准质粒对瘤胃细菌进行绝对荧光定量(qRT-PCR)研究。结果:1)在门水平上,发现优势菌群集中在拟杆菌门,其在放牧组的丰度((49.52±6.07)%)比舍饲组((55.52±12.18)%)低6.00%,但差异不显著(P>0.05);放牧组厚壁菌门的丰度((43.28±4.59)%)比舍饲组((36.68±9.78)%)高6.60%,两组差异显著(P<0.05);放牧组螺旋体门的丰度((1.99±1.75)%)比舍饲组((0.76±0.59)%)高1.23%,两组差异显著(P<0.05)。2)通过PLS-DA和LEfSe分析发现,两组间均差异显著的主要细菌有:纤维素或其代谢产物主要依赖菌(放牧组)为假丁酸弧菌属(Pseudobutyrivibrio)、月形单胞菌属(Selenomonas)、粪球菌属(Coprococcus)、梭菌属(Clostridium)、厌氧弧菌属(Anaerovibrio)、毛螺旋菌科下一属(Shuttleworthia)、类普雷沃氏菌科下一属(CF231)、丁酸弧菌属(Butyrivibrio)、类普雷沃氏菌科下一属(YRC22)、毛螺菌属(Lachnospira)、毛螺菌科下一属(Moryella);高蛋白和NFC或其代谢产物主要依赖菌(舍饲组)为脱硫叶菌属(Desulfobulbus)、奇异菌属(Atopobium)、韦荣球菌科下一属(p-75-a5)、普氏菌属(Prevotella)、布劳特氏菌属(Blautia)、瘤胃球菌属(Ruminococcus)和链球菌属(Streptococcus)。结果说明,合理蛋白质能量水平的饲料,不仅利于蛋白、淀粉依赖菌的增殖,也有利于主要纤维降解菌(瘤胃球菌属(Ruminococcus))和协同纤维降解菌(普氏菌属(Prevotella))的增殖。同时,舍饲组中纤维素分解菌(厚壁菌门(Firmicutes)和螺旋体门(Spirochaetes))的整体丰度值出现显著下降。

The effect of different protein, nonfibrous carbohydrates(NFC) and cellulose nutritious ingredient on bacterial community structure in rumen microenvironment was studied. The aim was to explore the fluctuation of predominant bacteria in rumens, and to understand clearly the dependence of rumen bacteria on nutrients. It provided foundation for further study on scientific nutrition ratio for supplementary feeding under the traditional grazing and large scale breeding. Experiment was divided into 2 groups under traditional grazing and TMR fodder feeding, at each of which there were 10 Pengbo semi-fine wool ewes(2-year-old, 2 repetitions in each group,(23.77±2.34) kg of weight). The Kjeldahl, van soest and difference methods were used to determine the crude protein, cellulose and NFC content of forage grass mixed forage in the grazing group and forage in the feeding group. After half a year of feeding, the rumen fluid was extracted, the total DNA was extracted, and a recombinant standard plasmid was constructed to perform absolute fluorescence quantification(qRT-PCR) study on rumen bacteria. The results showed that: 1) At the level of phylum, the preponderant population was Bacteroidetes, its abundance in grazing group((49.52±6.07)%) was 6.00% fewer than that in barn feeding group((55.52±12.18)%), but the difference was not significant(P>0.05). The abundance of Firmicutes in grazing group((43.28±4.59)%) was 6.60% higher than that in barn feeding group((36.68±9.78)%), the difference was significant(P<0.05). The abundance of Spirochaetes in grazing group((1.99±1.75)%) was 1.23% higher than that in barn feeding group((0.76±0.59)%), the difference was significant(P<0.05). 2) Analysis by PLS-DA and LEfSe revealed that the main bacteria that showed significant differences between the 2 groups were: the cellulose or its metabolites mainly depended on Pseudobutyrivibrio, Selenomonas, Coprococcus, Clostridium, Anaerovibrio, Shuttleworthia, CF231, Butyrivibrio, YRC22, Lachnospira, Moryella(in grazing group). The high-protein and NFC(in barn-feeding) or their metabolites mainly depended on Desulfobulbus, Atopobium, p-75-a5, Prevotella, Blautia, Ruminococcus and Streptococcus(in barn feeding group). The results indicated that the reasonable protein and energy levels in diet was not only beneficial to the proliferation of protein and starch-dependent bacteria, but also to the proliferation of main fiber-degrading bacteria(Ruminococcus) and synergistic fiber-degrading bacteria(Prevotella). Meanwhile, the abundance of cellulose decomposing bacteria(Firmicutes and Spirochaetes) had all declined obviously in barn feeding group.