Effects of different forage compositions on rumen microbiota in vitro using quantitative real-time polymerase chain reaction assay

反芻動物係利用瘤胃中共生微生物降解結構性或非結構性碳水化合物，以協助動物攝取飼糧所含營養分；但瘤胃微生物之菌相組成複雜，且多數難以經由分離培養方式鑑定菌種。即時定量聚合酶連鎖反應 (Quantitative real-time polymerase chain reaction, qPCR) 技術結合rRNA基因序列則可針對不易培養之微生物菌群作為一快速、準確定量之分子生物工具。本研究探討以qPCR分析反芻動物中瘤胃微生物相對含量之方法，並利用體外發酵系統 (in vitro fermentation system) 評估飼糧變化對於瘤胃微生物菌相之影響。本試驗分為方法建立與影響評估兩大部分。第一部分以逢機採樣方式，萃取瘤胃液樣品之微生物總體基因體DNA後，利用qPCR技術進行相對定量分析，分析牛隻瘤胃中不同分類之微生物相對含量。結果顯示透過qPCR技術可由屬 (Genus) 層級與目 (Order) 層級觀察瘤胃中細菌、原蟲 (Protozoa)、古生菌 (Archaea) 的相對含量；其中，樣品中細菌與古生菌的相對含量皆與前人研究結果相近。菌相相對含量結果變動幅度大，推測因受到不同因素影響其動態平衡，證明qPCR技術可做為研究瘤胃微生物之有效分析工具。第二部分則以三種比例之百慕達乾草與金針菇廢棄基質青貯料 (100:0、80:20、60:40) 作為體外發酵系統之發酵基質，混合瘤胃液與人工唾液 (Artificial saliva) 進行體外發酵培養，經0小時、24小時、48小時培養後分析其瘤胃微生物菌相之相對含量。結果顯示，在屬層級下，擬桿菌屬與普雷沃氏菌屬皆呈現培養時間增加而菌屬相對比例顯著下降之情形 (P<0.0001；P=0.0046)，顯示基質之消耗明顯造成部分細菌於培養液中的相對含量減少。另一方面，以目層級而言，甲烷桿菌目培養24小時之比例相對高於其培養48小時後之數量 (P<0.0001)，此結果與甲烷排放數據的增加量相符。由此可知應用qPCR技術可協助體外發酵試驗解析培養液中微生物菌相的相對變化量。綜上所述，qPCR技術可做為一具經濟考量與資料參考性之瘤胃菌相分析方式，而透過體外發酵系統亦可評估qPCR技術分析菌相消長變化的效果。Ruminants degrade structural or non-structural carbohydrate by the symbiotic rumen microbes for dietary nutrients. However, it is difficult to analyze rumen microbes with conventional culture-dependent assay due to the complexity and difficulty to maintain natural rumen microorganisms in laboratory. By using quantitative real-time polymerase chain reaction (qPCR) combined with 16S rRNA gene sequencing technique, it is possible to characterize rumen microbes precisely and avoid the bias introduced by bacterial culturing. The aim of this study is to validate qPCR technique for analyzing the relative quantitative ratio of rumen microbes in ruminants, and to analyze the effects of various diets on rumen microbial populations using in vitro fermentation system. The rumen fluid samples were randomly obtained, followed by total genomic DNA extrac ion from rumen fluid samples. The relative quantification analysis was performed by using qPCR to analyze the contents of rumen microbes at different taxonomic levels. Results showed that qPCR technique could help figuring out the relative content of ruminal bacteria, protozoa, and archaea by Genus and Order level of classification. The relative contents of bacteria and archaea were close to that of previous reports, suggesting that qPCR could be used as a robust tool for relative quantification of rumen microbes. In addition, different ratios of bermuda hay to spent mushroom (Flammulina velutipes) substrate (SMS) silage (100:0, 80:20, and 60:40) were used as substrates for in vitro fermentation, and microflora analysis were conducted in incubation buffer with 0 hour, 24 hour, and 48 hour incubation. The results showed that the relative ratios of Genus Bacteroides and Prevotella were significantly decreased with the increase of incubation time (P<0.0001 and P=0.0046, respectively), which suggests that substrates consumption would cause the relative reduction of certain bacteria groups. On the other hand, the relative ratio of Order Methanobacteriales with 24 hour fermentation was higher than that with 48 hour fermentation (P