RNA-Seq技术在瘤胃微生物研究中的应用进展

反刍动物瘤胃内多种多样的微生物组成一个复杂的微生态系统,它们在瘤胃内物质消化过程中发挥重要作用。RNA-Seq技术的出现为反刍动物瘤胃微生物的研究提供了前所未有的机遇。本文主要从新一代高通量测序技术出发,阐述RNA-Seq技术在瘤胃微生物代谢酶特性、瘤胃微生物的多样性、瘤胃微生物新功能基因方面的应用,以期为瘤胃微生物的研究提供参考。     

Effect of Megasphaera elsdenii NCIMB 41125 dosing on rumen development,volatile fatty acid production and blood β‐hydroxybutyrate in neonatal dairy calves

Thirty calves were randomly assigned to two treatments and fed until weaning [42 days (d) of age]. Treatments were a control group (n = 15), which did not receive Megasphaera elsdenii (Me0) and a M. elsdenii group, which received a 50‐ml oral dose of M. elsdenii NCIMB 41125 (10⁸ CFU/ml) at day 14 day of age (Me14). Calves were given colostrum for the first 3 day followed by limited whole milk feeding. A commercial calf starter was offered ad libitum starting at day 4 until the end of the study. Fresh water was available throughout the study. Feed intake and growth were measured. Blood samples were collected via jugular venipuncture to determine β‐hydroxybutyrate (BHBA) concentrations. Fourteen male calves (seven per group) were euthanised on day 42 and digestive tracts harvested. Reticulo‐rumen weight was determined and rumen tissue samples collected from the cranial and caudal sacs of the ventral and dorsal portions of the rumen for measurements of papillae length, papillae width and rumen wall thickness. Dosing with M. elsdenii NCIMB 41125 improved starter dry matter intake (DMI), weaning body weight (BW) and tended to improve average daily gain. Calves in Me14 group had greater plasma BHBA concentration than Me0‐calves during the last 3 weeks of the trial and had at day 42 greater reticulo‐rumen weight, papillae width and papillae density compared to Me0. No differences in rumen wall thickness or papillae length were observed between the two groups. Total volatile fatty acids, acetate and propionate production did not differ between treatments, but butyrate production was greater in Me14 than Me0. Dosing M. elsdenii NCIMB 41125 showed benefit for calves with improved feed intake and rumen development suggesting increased epithelium metabolism and improved absorption of digestive end products.     

Effects of different forms and origins of oilseeds on dynamics of ruminal biohydrogenation of long‐chain fatty acids in vitro

Dietary unsaturated fatty acids (FA) are intensively hydrogenated in the rumen, resulting in reduced amount of poly‐unsaturated fatty acids (PUFA) and accumulation of several biohydrogenation (BH) products. In this study, BH of PUFA originating from different oilseeds (linseed, soya beans, sunflower seed and rapeseed) present in crushed oilseeds or their free oils were assessed in vitro. The assay substrates were incubated in buffered rumen fluid for 0, 6, 12 and 24 h. After incubation, the FA pattern of the incubated samples was analysed using gas chromatography. Biohydrogenation is defined as disappearance of double bonds (DB) calculated from the contents of unsaturated FA. After 24‐h incubation, the DB contents of all oilseeds were reduced (p < 0.001) by 40–60%. The reduction was higher (p < 0.001) for the crushed form compared with the oil form. In addition, linseed and sunflower seed known as oilseeds with high contents of linolenic acid C18:3 c9,12,15 (LNA) and linoleic acid C18:2 c9,12 (LA), respectively, showed a higher (p < 0.001) accumulation of the BH intermediates conjugated linoleic acid (CLA, isomer C18:2 c9t11) and vaccenic acid (C18:1 t11) for the crushed form, when compared with the oil. These results suggest an inherent effect of the physical form of the assay oilseeds on in vitro BH. Changes in FA pattern during BH in vitro can be attributed to both source and physical form of the assay oilseeds. However, further investigations are warranted to ensure whether the observed in vitro effects on ruminal BH can be confirmed in vivo.     

The fate of glycerol entering the rumen of dairy cows and sheep

This study investigated the fate of glycerol entering the rumen, in particular whether glycerol could be absorbed across the rumen epithelium. Three non‐lactating rumen‐fistulated cows were used to calculate the overall disappearance rate of glycerol in vivo and evaluate the rate of ruminal glycerol absorption. Rumen epithelial tissues isolated from sheep were used to characterise glycerol transport properties. The rate of rumen microbial degradation of glycerol was then studied in an in vitro system under anaerobic and thermo‐regulated conditions. The results showed that glycerol can be absorbed from the rumen in significant amounts. The fractional rate of absorption of glycerol was not affected by variations in glycerol concentration in the buffer solution in the in vivo study. The glycerol absorption apparently occurred largely by passive diffusion and was probably not facilitated by carriers. Glycerol also disappeared via microbial digestion and outflow from the rumen through the omasal orifice.     

Changes in the predicted function of the rumen bacterial community of Japanese Black beef cattle during the fattening stages according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses

We investigated changes in the predicted functions of the rumen bacterial community in Japanese Black beef cattle during fattening. Nine cattle were fed a high-concentrate diet during the early, middle, and late fattening stages consecutively (10–14, 15–22, and 23–30 months of age, respectively). The rumen fluid and solid samples collected at each stage were subjected to sequencing analyses. The sequencing results were clustered and classified into operational taxonomic units (OTUs). Representative sequences and a raw counting table for each OTU were submitted to the Piphillin website. The predicted functions were revealed by the Kyoto Encyclopedia of Genes and Genomes database as the ratio of the total sequence. In the early stage, “Biosynthesis of secondary metabolites” was significantly higher in the fluid fraction than in the solid fraction. “Two-component system” in the middle stage was significantly lower and “Purine metabolism” in the late stage was significantly higher in the fluid fraction than those in the solid fraction. The fluid fraction was significantly correlated with acetic acid, propionic acid, and bacterial metabolism, such as “Biosynthesis of secondary metabolites” and “Sugar metabolism.” Moreover, the solid fraction was correlated with “Purine metabolism” and “Biosynthesis of secondary metabolism”. These results suggest that the rumen bacterial community in Japanese Black beef cattle adapts to changes in rumen conditions by altering their functions in response to a long-term high-grain diet.     

基于16S rRNA基因克隆文库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性

本研究旨在利用16S rRNA基因克隆库技术分析广西富钟水牛瘤胃产甲烷菌组成及多样性。选取3头体况基本一致的健康雌性富钟水牛作为试验动物,采用机械破壁法提取瘤胃内容物总DNA,采用产甲烷菌引物Met86F/Met1340R扩增16S rRNA基因,构建16S rRNA基因克隆文库。结果表明,本试验共获得93个非嵌合体16S rRNA序列,按照97%的相似性划分为39个分类操作单元（OTU）。其中,60个序列（15个OTU）与已培养细菌16S rRNA序列相似性≥97%,占总序列的64.5%;32个序列（23个OTU）与已培养菌16S rRNA序列相似性处于90%^<97%;仅有1个序列与Methanomassiliicoccus luminyensis相似性<90%。系统发育树分析表明,98.9%的序列均属于甲烷杆菌目（Methanobacteriales）,部分序列与Methanobacteriales中任何已知相似序列都相隔较远,它们可能代表Methanobacteriales中新的属或种。由上述结果可见,富钟水牛瘤胃产甲烷菌以Methanobacteriales为优势菌群,其中有许多未知的产甲烷菌需进一步分离培养并对其功能进行分析。     

Determination of bacteria constituting ruminal fibrolytic consortia developed on orchard grass hay stem

To determine the relationship between Fibrobacter succinogenes and other rumen bacteria, the bacterial community structure on fiber was analyzed by using two different materials. These were ruminally incubated orchard grass hay stems without and with preincubation with F. succinogenes (natural and artificial consortia, respectively). The natural consortium mainly consisted of Firmicutes (56.6%) and Bacteroidetes (33.1%), while the artificial consortium showed a significantly higher proportion of Firmicutes (85.5%) and a lower proportion of Bacteroidetes (4.6%). At species or genus level, Butyrivibrio fibrisolvens, the U2 group, Ruminococcus albus and Lachnospiraceae incertae sedis made up a higher proportion in the artificial consortium. The most dominant bacterial group was the Butyrivibrio‐Pseudobutyrivibrio‐Lachnospiraceae incertae sedis group, which accounted for 19.7% in the natural and 29.5% in the artificial consortium. Within the genus Butyrivibrio, the phylogenetic groups SA and VA2 and phylogeny‐undefined Butyribivrio, but not VA1, were detected at high frequency in the artificial consortium. These results suggest that ecological and possibly functional relationships exist in the rumen among F. succinogenes, a subset of B. fibrisolvens, the U2 group, R. albus and Lachnospiraceae incertae sedis.     

Effect of increasing levels of seven tree species extracts added to a high concentrate diet on in vitro rumen gas output

This study was conducted to investigate the effects of increasing levels of extracts of Byrsonima crassifolia, Celtis pallida, Enterolobium cyclocarpum, Fraxinus excelsior, Ficus trigonata, Phoradendrom brevifolium and Prunus domestica on in vitro gas production (GP) and ruminal fermentation of a high concentrate diet. Plant extracts were prepared at 1 g dry matter (DM)/8 mL of solvent mixture (methanol : ethanol : water, 1:1:8) and added at levels of 0, 0.6, 1.2 and 1.8 mL/g DM of a high concentrate diet. In vitro GP was recorded at 2, 4, 6, 8, 10, 12, 24, 48 and 72 h of incubation. Increasing addition of extracts linearly increased (P < 0.001), the GP₂₄, GP₄₈ and GP₇₂ (mL/g DM), and linearly decreased (P < 0.001), the discrete GP lag time. Moreover, increasing extract doses linearly increased (P < 0.001) the asymptotic GP and decreased (P < 0.001) the rate of GP. GP₆ was not impacted by treatments and GP₁₂ increased linearly (P = 0.01) with increasing addition of extracts. Rumen pH declined linearly (P < 0.05) with increasing doses of extracts added. As no interactions (P > 0.05) occurred between the extracts and doses, it could be conclude that all extracts positively modified rumen fermentation at doses of 1.2 to 1.8 mL extract/g diet DM.     

半胱胺对藏绵羊断奶羔羊瘤胃pH值、总脱氢酶及TVFA的影响

试验研究了半胱胺(Cs)对藏绵羊断奶羔羊瘤胃pH值、总脱氢酶及TVFA的影响。将10只(公母各半)80日龄左右、体重平均为(16.00±0.31)kg、装有永久性瘤胃瘘管的藏绵羊断奶羔羊随机分为2组(试验组和对照组),在日粮精料中添加300 mg/(kg·BW)的Cs,隔日添加1次,试验期42 d。正式试验当日及试验第7、14、21、28、35天的7:00、9:00、11:00、13:00、15:00、17:00、19:00、21:00、23:00、1:00、3:00、5:00于颈静脉采血样10 mL,同时采集瘤胃液10 mL,测定瘤胃pH值、总脱氢酶的活性和TVFA。检测结果表明,试验组精料中添加Cs使藏绵羊断奶羔羊瘤胃液pH值升高;血清尿素氮浓度降低,与对照组相比差异极显著(P0.01);瘤胃液总脱氢酶活力提高极其明显(P0.01);整个试验期内,半胱胺对瘤胃TVFA的影响极显著(P0.01)。     

不同断奶日龄羔羊瘤胃组织显微及超微结构比较

试验通过比较不同断奶日龄的湖羊羔羊瘤胃组织结构的差异，特别是瘤胃乳头超微结构变化，旨在为羔羊适宜断奶日龄的确定提供瘤胃生长发育的依据。选择新生湖羊羔羊24只（初生重：2.80 kg±0.08 kg），随机分为3组，每组8只，分别为30日龄早期断奶组（EEW-30）、45日龄早期断奶组（EW-45）和60日龄常规断奶组（NW-60）。在各断奶日龄及75日龄时每组分别屠宰4只羔羊，测定体重及瘤胃空重，计算瘤胃相对重量；采集瘤胃组织进行显微及超微形态学测定。结果显示：①EEW-30组羔羊断奶体重极显著低于NW-60组（P<0.01），而EW-45组与NW-60组断奶体重无显著差异（P>0.05）；75日龄时3组羔羊的体重无显著差异（P>0.05）。各组羔羊瘤胃相对重量与其体重变化规律相同。②光镜下可见：断奶时，EEW-30组瘤胃壁厚度极显著低于NW-60组（P<0.01），EW-45组与NW-60组无显著差异（P>0.05）；75日龄时，EW-45组羔羊瘤胃壁厚度显著高于NW-60组（P<0.05）。断奶时EEW-30组瘤胃乳头较短小，极显著短于NW-60组（P<0.01），EW-45组也显著短于NW-60组（P<0.05）。断奶时瘤胃乳头宽度和面积在3组间差异均不显著，在75日龄时也无显著组间差异（P>0.05）。③扫描电镜观测结果显示：EEW-30组断奶时单位面积瘤胃微乳头（ruminal micro-papillae）数量极显著低于NW-60组（P<0.01），而EW-45与NW-60组间无显著差异（P>0.05）；75日龄时，3组羔羊单位面积瘤胃微乳头数量无显著差异（P>0.05）。上述试验结果显示，断奶时羔羊瘤胃形态结构在EEW-30与NW-60组间差异明显，而EW-45与NW-60组较为接近，瘤胃微乳头状结构也呈相同变化趋势。提示45日龄时断奶较为适宜。