Rumen Archaea Research Articles

Multi-breed host rumen epithelium transcriptome and microbiome associations and their relationship with beef cattle feed efficiency

Understanding host-microbial interactions in the rumen and its influence on desirable production traits may lead to potential microbiota manipulation or genetic selection for improved cattle feed efficiency. This study investigated the host transcriptome and its correlation with the rumen archaea and bacteria differential abundance of two pure beef cattle breeds (Angus and Charolais) and one composite beef hybrid (Kinsella) divergent for residual feed intake (RFI; low-RFI vs. high-RFI). Using RNA-Sequencing of rumen tissue and 16S rRNA gene amplicon sequencing, differentially expressed genes (FDR ≤ 0.05, |log2(Fold-change) >|2) and differentially abundant (p-value < 0.05) archaea and bacteria amplicon sequence variants (ASV) were determined. Significant correlations between gene expression and ASVs (p-value < 0.05) were determine using Spearman correlation. Interesting associations with muscle contraction and the modulation of the immune system were observed for the genes correlated with bacterial ASVs. Potential functional candidate genes for feed efficiency status were identified for Angus (CCL17, CCR3, and CXCL10), Charolais (KCNK9, GGT1 and IL6), and Kinsella breed (ESR2). The results obtained here provide more insights regarding the applicability of target host and rumen microbial traits for the selection and breeding of more feed efficient beef cattle.

Maternal rumen and milk microbiota shape the establishment of early-life rumen microbiota in grazing yak calves

Early-life gut microbial colonization and development exert a profound impact on the health and metabolism of the host throughout the life span. The transmission of microbes from the mother to the offspring affects the succession and establishment of the early-life rumen microbiome in newborns, but the contributions of different maternal sites to the rumen microbial establishment remain unclear. In the present study, samples from different dam sites (namely, oral, rumen fluid, milk, and teat skin) and rumen fluid of yak calves were collected at 6 time points between d 7 and 180 postpartum to determine the contributions of the different maternal sites to the establishment of the bacterial and archaeal communities in the rumen during early life. Our analysis demonstrated that the dam's microbial communities clustered according to the sites, and the calves' rumen microbiota resembled that of the dam consistently regardless of fluctuations at d 7 and 14. The dam's rumen microbiota was the major source of the calves' rumen bacteria (7.9%) and archaea (49.7%) compared with the other sites, whereas the potential sources of the calf rumen microbiota from other sites varied according to the age. The contribution of dam's rumen bacteria increased with age from 0.36% at d 7 to 14.8% at d 180, whereas the contribution of the milk microbiota showed the opposite trend, with its contribution reduced from 2.7% at d 7 to 0.2% at d 180. Maternal oral archaea were the main sources of the calves' rumen archaea at d 14 (50.4%), but maternal rumen archaea became the main source gradually and reached 66.2% at d 180. These findings demonstrated the potential microbial transfer from the dam to the offspring that could influence the rumen microbiota colonization and establishment in yak calves raised under grazing regimens, providing the basis for future microbiota manipulation strategies during their early life.

The Potential of Novel Gene Editing-Based Approaches in Forages and Rumen Archaea for Reducing Livestock Methane Emissions

Rising emissions of anthropogenic greenhouse gases such as carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) are a key driver of climate change, which is predicted to have myriad detrimental consequences in coming years if not kept in check. Given the potency of CH4 in terms of trapping heat in the atmosphere in the short term, as well as the fact that ruminant production currently contributes approximately 30% of anthropogenic emissions, there is an impetus to substantially decrease the generation of ruminant-derived CH4. While various strategies are being assessed in this context, a multi-faceted approach is likely required to achieve significant reductions. Feed supplementation is one strategy that has shown promise in this field by attenuating methanogenesis in rumen archaea; however, this can be costly and sometimes impractical. In this review, we examine and discuss the prospect of directly modulating forages and/or rumen archaea themselves in a manner that would reduce methanogenesis using CRISPR/Cas-mediated gene editing platforms. Such an approach could provide a valuable alternative to supplementation and has the potential to contribute to the sustainability of agriculture, as well as the mitigation of climate change, in the future.

A Review of 3-Nitrooxypropanol for Enteric Methane Mitigation from Ruminant Livestock.

Simple SummaryEnteric methane (CH4) from the anaerobic fermentation of feed carbohydrates in ruminant livestock accounts for 3 to 5% of global greenhouse gas emissions. Among the different CH4 mitigating approaches evaluated to decrease enteric CH4 emissions from ruminants, the feed additive 3-nitrooxypropanol is effective with a mean reduction in CH4 of 30%, depending on animal type, diet and dose. 3-nitrooxypropanol is chemically synthesized and studies show low safety risk with no detrimental effects to animals and humans. 3-nitrooxypropanol was recently approved by regulatory authorities for use in Brazil and Chile and has received a favorable opinion from the scientific panel of the European Food Safety Authority, with approvals in various jurisdictions expected in the near future. As a substantial body of research on 3-nitrooxypropanol is now available, this review offers a timely analysis of the opportunities and challenges of using 3-nitrooxypropanol to mitigate enteric CH4 emissions in ruminant livestock.Methane (CH4) from enteric fermentation accounts for 3 to 5% of global anthropogenic greenhouse gas emissions, which contribute to climate change. Cost-effective strategies are needed to reduce feed energy losses as enteric CH4 while improving ruminant production efficiency. Mitigation strategies need to be environmentally friendly, easily adopted by producers and accepted by consumers. However, few sustainable CH4 mitigation approaches are available. Recent studies show that the chemically synthesized CH4 inhibitor 3-nitrooxypropanol is one of the most effective approaches for enteric CH4 abatement. 3-nitrooxypropanol specifically targets the methyl-coenzyme M reductase and inhibits the final catalytic step in methanogenesis in rumen archaea. Providing 3-nitrooxypropanol to dairy and beef cattle in research studies has consistently decreased enteric CH4 production by 30% on average, with reductions as high as 82% in some cases. Efficacy is positively related to 3-NOP dose and negatively affected by neutral detergent fiber concentration of the diet, with greater responses in dairy compared with beef cattle when compared at the same dose. This review collates the current literature on 3-nitrooxypropanol and examines the overall findings of meta-analyses and individual studies to provide a synthesis of science-based information on the use of 3-nitrooxypropanol for CH4 abatement. The intent is to help guide commercial adoption at the farm level in the future. There is a significant body of peer-reviewed scientific literature to indicate that 3-nitrooxypropanol is effective and safe when incorporated into total mixed rations, but further research is required to fully understand the long-term effects and the interactions with other CH4 mitigating compounds.

Betaine Modulates Rumen Archaeal Community and Functioning during Heat and Osmotic Stress Conditions In Vitro.

Rumen archaea play an important role in scavenging ruminal hydrogen (H2) and thus facilitate rumen fermentation. They require optimum temperature and osmolality for their growth and metabolism; however, a number of external factors may put archaea under heat and osmotic stress. Betaine is an osmolyte, molecular chaperone, and antioxidant; therefore, it bears potential to combat against these stressors. In this in vitro study, three betaine levels, namely, 0 (control), 51 (low), and 286 (high) ppm, were used. Each of these was subjected to two temperatures (39.5 and 42°C) and two osmolality conditions (295 and 420 mOsmol kg−1) with n = 6 per treatment. Sequencing analyses of the solid phase (which use solid materials containing primarily fibrous materials of low-density feed particles) and the liquid phase (rumen fermenter liquid) using 16S rRNA revealed that more than 99.8% of the ruminal archaea in fermenters belong to the phylum Euryarchaeota. At the genus level, Methanobrevibacter was the most prevalent in both phases, and Methanosaeta was only detected in the liquid phase. The genera Methanobrevibacter and Methanobacterium both showed a positive correlation with methane (CH4) formation in the liquid and solid phases, respectively (P < 0.05). Heat stress increased the relative abundance of genus Methanimicrococcus at the expense of candidate archaeal genus Vadin CA11 (P < 0.05). In the solid phase, osmotic stress significantly reduced the Shannon and Simpson indices of diversity, and relative abundance was higher for Methanobrevibacter at the expense of Methanimicrococcus. In the liquid phase, osmotic stress increased not only the abundance-based coverage estimator (ACE) and singles parameters of diversity but also the relative abundances of Methanosphaera and Methanobacterium. The overall decrease in all gas parameters and estimated metabolic hydrogen ([2H]) utilization was observed during osmotic stress conditions (P < 0.05). Betaine enhanced the diversity of solid phase archaea as indicated by the increase in ACE and singles during heat stress, and only a high dose improved all diversity parameters in the liquid phase during osmotic stress (P < 0.05). Thus, betaine alleviates the effects of heat stress and osmotic stress on the archaea community.

Survey of rumen microbiota of domestic grazing yak during different growth stages revealed novel maturation patterns of four key microbial groups and their dynamic interactions

BackgroundThe development and maturation of rumen microbiota across the lifetime of grazing yaks remain unexplored due to the varied lifestyles and feed types of yaks as well as the challenges of obtaining samples. In addition, the interactions among four different rumen microbial groups (bacteria, archaea, fungi and protozoa) in the rumen of yak are not well defined. In this study, the rumen microbiota of full-grazing yaks aged 7 days to 12 years old was assessed to determine the maturation patterns of these four microbial groups and the dynamic interactions among them during different growth stages.ResultsThe rumen microbial groups (bacteria, archaea, protozoa and fungi) varied through the growth of yaks from neonatal (7 days) to adult (12 years), and the bacterial and archaeal groups were more sensitive to changes in growth stages compared to the two eukaryotic microbial groups. The age-discriminatory taxa within each microbial group were identified with the random forest model. Among them, Olsenella (bacteria), Group 10 sp., belonging to the family Methanomassiliicoccaceae (archaea), Orpinomyces (fungi), and Dasytricha (protozoa) contributed the most to discriminating the age of the rumen microbiota. Moreover, we found that the rumen archaea reached full maturation at 5 approximately years of age, and the other microbial groups matured between 5 and 8 years of age. The intra-interactions patterns and keystone species within each microbial group were identified by network analysis, and the inter-interactions among the four microbial groups changed with growth stage. Regarding the inter-interactions among the four microbial groups, taxa from bacteria and protozoa, including Christensenellaceae R-7 group, Prevotella 1, Trichostomatia, Ruminococcaceae UCG-014 and Lachnospiraceae, were the keystone species in the network based on betweenness centrality scores.ConclusionsThis study depicted a comprehensive view of rumen microbiota changes in different growth stages of grazing yaks. The results revealed the unique microbiota maturation trajectory and the intra- and inter-interactions among bacteria, archaea, fungi and protozoa in the rumen of grazing yaks across the lifetime of yaks. The information obtained in this study is vital for the future development of strategies to manipulate rumen microbiota in grazing yaks for better growth and performance in the harsh Qinghai-Tibetan Plateau ecosystem.

The structure of microbial populations in Nelore GIT reveals inter-dependency of methanogens in feces and rumen

BackgroundThe success of different species of ruminants in the colonization of a diverse range of environments is due to their ability to digest and absorb nutrients from cellulose, a complex polysaccharide found in leaves and grass. Ruminants rely on a complex and diverse microbial community, or microbiota, in a unique compartment known as the rumen to break down this polysaccharide. Changes in microbial populations of the rumen can affect the host’s development, health, and productivity. However, accessing the rumen is stressful for the animal. Therefore, the development and use of alternative sampling methods are needed if this technique is to be routinely used in cattle breeding. To this end, we tested if the fecal microbiome could be used as a proxy for the rumen microbiome due to its accessibility. We investigated the taxonomic composition, diversity and inter-relations of two different GIT compartments, rumen and feces, of 26 Nelore (Bos indicus) bulls, using Next Generation Sequencing (NGS) metabarcoding of bacteria, archaea and ciliate protozoa.ResultsWe identified 4265 Amplicon Sequence Variants (ASVs) from bacteria, 571 from archaea, and 107 from protozoa, of which 143 (96 bacteria and 47 archaea) were found common between both microbiomes. The most prominent bacterial phyla identified were Bacteroidetes (41.48%) and Firmicutes (56.86%) in the ruminal and fecal microbiomes, respectively, with Prevotella and Ruminococcaceae UCG-005 the most relatively abundant genera identified in each microbiome. The most abundant archaeal phylum identified was Euryarchaeota, of which Methanobrevibacter gottschalkii, a methanogen, was the prevalent archaeal species identified in both microbiomes. Protozoa were found exclusively identified in the rumen with Bozasella/Triplumaria being the most frequent genus identified. Co-occurrence among ruminal and fecal ASVs reinforces the relationship of microorganisms within a biological niche. Furthermore, the co-occurrence of shared archaeal ASVs between microbiomes indicates a dependency of the predominant fecal methanogen population on the rumen population.ConclusionsCo-occurring microorganisms were identified within the rumen and fecal microbiomes, which revealed a strong association and inter-dependency between bacterial, archaeal and protozoan populations of the same microbiome. The archaeal ASVs identified as co-occurring between GIT compartments corresponded to the methanogenic genera Methanobrevibacter and Methanosphaera and represented 26.34% of the overall archaeal sequencesdiversity in the rumen and 42.73% in feces. Considering that these archaeal ASVs corresponded to a significant part of the overall diversity of both microbiomes, which is much higher if one includes the interactions of these co-occurring with other rumen archaea ASVs, we suggest that fecal methanogens could be used as a proxy of ruminal methanogens.

Comparison of rumen archaeal diversity in adult and elderly yaks (Bos grunniens) using 16S rRNA gene high-throughput sequencing

This study was conducted to investigate the phylogenetic diversity of archaea in the rumen of adult and elderly yaks. Six domesticated female yaks, 3 adult yaks ((5.3±0.6) years old), and 3 elderly yaks ((10.7±0.6) years old), were used for the rumen contents collection. Illumina MiSeq high-throughput sequencing technology was applied to examine the archaeal composition of rumen contents. A total of 92 901 high-quality archaeal sequences were analyzed, and these were assigned to 2 033 operational taxonomic units (OTUs). Among these, 974 OTUs were unique to adult yaks while 846 OTUs were unique to elderly yaks; 213 OTUs were shared by both groups. At the phylum level, more than 99% of the obtained OTUs belonged to the Euryarchaeota phylum. At the genus level, the archaea could be divided into 7 archaeal genera. The 7 genera (i.e., Methanobrevibacter, Methanobacterium, Methanosphaera, Thermogymnomonas, Methanomicrobiu, Methanimicrococcus and the unclassified genus) were shared by all yaks, and their total abundance accounted for 99% of the rumen archaea. The most abundant archaea in elderly and adult yaks were Methanobrevibacter and Thermogymnomonas, respectively. The abundance of Methanobacteria (class), Methanobacteriales (order), Methanobacteriaceae (family), and Methanobrevibacter (genus) in elderly yaks was significantly higher than in adult yaks. In contrast, the abundance of Thermogymnomonas in elderly yaks was 34% lower than in adult yaks, though the difference was not statistically significant. The difference in abundance of other archaea was not significant between the two groups. These results suggested that the structure of archaea in the rumen of yaks changed with age. This is the first study to compare the phylogenetic differences of rumen archaeal structure and composition using the yak model.

Effects of plants containing secondary metabolites as feed additives on rumen metabolites and methanogen diversity of buffaloes

Four fistulated adult Murrah buffaloes were fed on a basal diet consisting of wheat straw and concentrate mixture in a 4 × 4 Latin square design to study the effects of feeding plants containing secondary metabolites on rumen metabolites and methanogen diversity. The four groups were Control (no additive), Mix-1 (ajwain oil and lemon grass oil in a 1 : 1 ratio @ 0.05% of dry matter intake), Mix-2 (garlic and soapnut in a 2 : 1 ratio @ 2% of dry matter intake) and Mix-3 (garlic, soapnut, harad and ajwain in a 2 : 1 : 1 : 1 ratio @ 1% of dry matter intake). In each phase of 30 days’ duration, after 19 days of feeding, rumen liquor was sampled for two consecutive days at 0, 2, 4, 6 and 8 h post-feeding, whereas rumen content was sampled at 0 h feeding. The pH of the rumen liquor was recorded at every collection and then the rumen liquor of every collection was pooled day-wise and animal-wise. These pooled samples were used for estimation of rumen metabolites like ammonia, lactic acid and volatile fatty acids. Microscopic counting of protozoa was done in both 0 h and pooled samples of rumen liquor. Rumen contents collected from different locations of rumen were processed for enzyme estimation. The rumen contents were squeezed and the liquid portion was used for DNA isolation, which was further processed to determine methanogen diversity. Daily intake of feed was similar (P &gt; 0.05) in all the four groups. The ammonia-N concentration and ciliate protozoa population were reduced significantly in the treatment groups supplemented with additives. Rumen pH, lactic acid, volatile fatty acids and enzyme activities were not affected (P &gt; 0.05) by feeding of any of these additives. Methanogenic diversity comparison was made between the Control and Mix-1 group. The basic local alignment search tool (BLAST) analysis of the 133 (44 from the Control group and 89 from the Mix-1 group) sequences showed similarity of the sequences of rumen archaea by up to 97% to the known sequences of rumen methanogens. The sequences with minimum length of 750 bp were selected for phylogenetic analysis. Per cent identity of these sequences with that of the available nearest neighbour as calculated by MEGA 5.03 software showed identity of the clones in the range of 88–97%. The clones were similar with Methanobrevibacter smithii ATCC 35061, uncultured Methanobrevibacter sp. clone MEME95 and M. ruminantium M1. Overall, feeding of any of these feed additives to fistulated buffaloes did not affect feed intake, rumen pH, or rumen metabolites except ammonia and enzyme profile. Methanogen diversity showed the possibility of Methanobrevibacter as the major methanogen in buffalo rumen liquor.

Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows

The evaluation of greenhouse gas mitigation strategies requires the quantitative assessment of individual methane production. Because methane measurement in respiration chambers is highly accurate, but also comprises various disadvantages such as limited capacity and high costs, the establishment of an indicator for estimating methane production of individual ruminants would provide an alternative to direct methane measurement. Methyl-coenzyme M reductase is involved in methanogenesis and the subunit α of methyl-coenzyme M reductase is encoded by the mcrA gene of rumen archaea. We therefore examined the relationship between methane emissions of Holstein dairy cows measured in respiration chambers with 2 different diets (high- and medium-concentrate diet) and the mcrA DNA and mcrA cDNA abundance determined from corresponding rumen fluid samples. Whole-body methane production per kilogram of dry matter intake and mcrA DNA normalized to the abundance of the rrs gene coding for 16S rRNA correlated significantly when using qmcrA primers. Use of qmcrA primers also revealed linear correlation between mcrA DNA copy number and methane yield. Regression analyses based on normalized mcrA cDNA abundances revealed no significant linear correlation with methane production per kilogram of dry matter intake. Furthermore, the correlations between normalized mcrA DNA abundance and the rumen fluid concentration of acetic and isobutyric acid were positive, whereas the correlations with propionic and lactic acid were negative. These data suggest that the mcrA DNA approach based on qmcrA primers could potentially be a molecular proxy for methane yield after further refinement.

SPECIAL TOPICS — Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options1

The goal of this review was to analyze published data related to mitigation of enteric methane (CH4) emissions from ruminant animals to document the most effective and sustainable strategies. Increasing forage digestibility and digestible forage intake was one of the major recommended CH4 mitigation practices. Although responses vary, CH4 emissions can be reduced when corn silage replaces grass silage in the diet. Feeding legume silages could also lower CH4 emissions compared to grass silage due to their lower fiber concentration. Dietary lipids can be effective in reducing CH4 emissions, but their applicability will depend on effects on feed intake, fiber digestibility, production, and milk composition. Inclusion of concentrate feeds in the diet of ruminants will likely decrease CH4 emission intensity (Ei; CH4 per unit animal product), particularly when inclusion is above 40% of dietary dry matter and rumen function is not impaired. Supplementation of diets containing medium to poor quality forages with small amounts of concentrate feed will typically decrease CH4 Ei. Nitrates show promise as CH4 mitigation agents, but more studies are needed to fully understand their impact on whole-farm greenhouse gas emissions, animal productivity, and animal health. Through their effect on feed efficiency and rumen stoichiometry, ionophores are likely to have a moderate CH4 mitigating effect in ruminants fed high-grain or mixed grain-forage diets. Tannins may also reduce CH4 emissions although in some situations intake and milk production may be compromised. Some direct-fed microbials, such as yeast-based products, might have a moderate CH4-mitigating effect through increasing animal productivity and feed efficiency, but the effect is likely to be inconsistent. Vaccines against rumen archaea may offer mitigation opportunities in the future although the extent of CH4 reduction is likely to be small and adaptation by ruminal microbes and persistence of the effect is unknown. Overall, improving forage quality and the overall efficiency of dietary nutrient use is an effective way of decreasing CH4 Ei. Several feed supplements have a potential to reduce CH4 emission from ruminants although their long-term effect has not been well established and some are toxic or may not be economically feasible.

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.

Structure of the Archaeal Community of the Rumen

Members of the domain Archaea contribute about 0.3 to 3.3% of the microbial small subunit (16S and 18S) rRNA in the rumen (22, 39, 60). Archaea have a range of different metabolisms and are found in many habitats (6), but those known to exist in the rumen are strictly anaerobic methanogens. Yanagita et al. (59) observed that 2.8 to 4.0% of ruminal microorganisms displayed autofluorescence characteristic of F420, a methanogen cofactor, able to be seen under UV illumination during microscopy. Taken together with the small subunit rRNA abundance data, this suggests that a large part of the archaeal population is made up of methanogens. Most species of methanogens can grow using H2 and often formate as their energy sources and use the electrons derived from H2 (or formate) to reduce CO2 to CH4. Some species can grow with methyl groups, oxidizing some to CO2 to produce electrons that are used to reduce further methyl groups to methane. A few species can grow with acetate, effectively dissimilating acetate to CH4 and CO2. However, acetate is not metabolized to CH4 to any significant extent in the rumen (13). This is probably because the rate of passage of rumen contents through the rumen is greater than the growth rate of acetate-utilizing methanogens (53). In a normally functioning rumen, proteins and polymeric carbohydrates, which usually make up the largest part of the incoming feed, are fermented by a mixed microbial community to volatile fatty acids (VFAs), NH4+, CO2, and H2. The hydrogen is metabolized by the methanogens. The VFAs are taken up by the animal across the rumen wall and serve as major carbon and energy sources for the ruminant. A part of the VFAs, undigested feed components, and microbial cells leave the rumen and enter the rest of the animal's digestive tract. The central role of H2 in the rumen fermentation (12) means that, although methanogenic archaea make up only a small part of the rumen microbial biomass, they play an important role in rumen function and animal nutrition. Efficient H2 removal leads to a nutritionally more favorable pattern of VFA formation and to an increased rate of fermentation by eliminating the inhibitory effect of H2 on the microbial fermentation (26, 53). The rumen can be simplistically described as an open system with discontinuous solid (feed) and liquid (saliva and drinking water) inputs and multiple fractions that have different turnover rates (53). The methanogens in the rumen are found free in the rumen fluid, attached to particulate material and rumen protozoa, associated as endosymbionts within rumen protozoa, and attached to the rumen epithelium. The methanogens associated with these different fractions can be expected to have different growth rates since they will be removed from the rumen at different rates. In addition, the animal itself and the feed also influence the rate of passage of digesta through the rumen system (25). These different habitats may allow niche division among the methanogens and may explain some of the observed phylogenetic diversity of rumen archaea.

Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence

Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.

Molecular diversity of rumen methanogens from sheep in Western Australia.

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobrevibacter: Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.

Methane-suppressing effect of myristic acid in sheep as affected by dietary calcium and forage proportion

The efficiency of myristic acid (14 : 0) as a feed additive to suppress CH4 emissions of ruminants was evaluated under different dietary conditions. Six sheep were subjected to a 6 x 6 Latin square arrangement. A supplement of non-esterified 14 : 0 (50 g/kg DM) was added to two basal diets differing in their forage:concentrate values (1 : 1.5 and 1 : 0.5), which were adjusted to dietary Ca contents of 4.2 and 9.0 g/kg DM, respectively. Comparisons were made with the unsupplemented basal diets (4.2 g Ca/kg DM). The 14 : 0 supplementation decreased (P<0.001) total tract CH4 release depending on basal diet type (interaction, P<0.001) and dietary Ca level (P<0.05, post hoc test). In the concentrate-based diet, 14 : 0 suppressed CH4 emission by 58 and 47 % with 4.2 and 9.0 g Ca/kg DM, respectively. The 14 : 0 effect was lower (22 %) in the forage-based diet and became insignificant with additional Ca. Myristic acid inhibited (P<0.05) rumen archaea without significantly altering proportions of individual methanogen orders. Ciliate protozoa concentration was decreased (P<0.05, post hoc test) by 14 : 0 only in combination with 9.0 g Ca/kg DM. Rumen fluid NH3 concentration and acetate:propionate were decreased (P<0.05) and water consumption was lower (P<0.01) with 14 : 0. The use of 14 : 0 had no clear effects on total tract organic matter and fibre digestion; this further illustrates that the suppressed methanogenesis resulted from direct effects against methanogens. The present study demonstrated that 14 : 0 is a potent CH4 inhibitor but, to be effective in CH4 mitigation feeding strategies, interactions with other diet ingredients have to be considered.

Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens

Molecular diversity of rumen archaea was analyzed by PCR amplification and sequencing of two 16S rRNA clone libraries prepared from the bovine rumen fluid using two different archaea-specific primer sets. The first library of 19 clones which was generated with primers D30 and D33, produced essentially two groups of sequences, one affiliated with Methanomicrobium mobile (21% of clones) and the other – with the uncultured archaeal sequences from anaerobic digester, which are distantly associated with Thermoplasma (79% of clones). The second library of 25 clones, which was generated with primers 0025e Forward and 1492 Reverse, produced a higher degree of diversity: in addition to the previous two groups, with the M. mobile- (56%) and Thermoplasma-associated sequences (20%), four clones (16%) were identified as Methanobrevibacter spp. The remaining two sequences were associated with unidentified archaeal sequences from the rumen and swine waste. Phylogenetic placement of eight almost complete 16S rRNA sequences revealed the existence of a novel cluster of the rumen Euryarchaeota, which is not affiliated with the known methanogenic archaea.