Rumen Ecosystem Research Articles

Metagenomics and its Application in Rumen Ecosystem: Potential Biotechnological Prospects

Genetic and biological diversity of microorganism s is an important area of scientific research. Considering the importance of ruminants in livestock strategies their ability to converts locally available feedstuff to animal products should be improved. Recent advances in the molecular biology and genomics now offer new opportunities to conduct a more holistic examination of the structure and function of rumen communities. To understand the complex microbial communities function and how microbes interact within their niches represents a major challenge for rumen microbiologists today. Metagenomics has the potential for providing insight into the functional dimensions of the rumen genomic database and will help to achieve a major goal of rumen microbiology; the complexities of microbial communities function and interaction among these microbes. This review summarizes the molecular methods of culture-indep endent insight 'metagenomics' and their recent application to studies of the rumen ecosystem for enhancing the livestock productivity.

Dietary phytochemicals as rumen modifiers: a review of the effects on microbial populations

In the recent years, the exploration of bioactive phytochemicals as natural feed additives has been of great interest among nutritionists and rumen microbiologists to modify the rumen fermentation favorably such as defaunation, inhibition of methanogenesis, improvement in protein metabolism, and increasing conjugated linoleic acid content in ruminant derived foods. Many phytochemicals such as saponins, essential oils, tannins and flavonoids from a wide range of plants have been identified, which have potential values for rumen manipulation and enhancing animal productivity as alternatives to chemical feed additives. However, their effectiveness in ruminant production has not been proved to be consistent and conclusive. This review discusses the effects of phytochemicals such as saponins, tannins and essential oils on the rumen microbial populations, i.e., bacteria, protozoa, fungi and archaea with highlighting molecular diversity of microbial community in the rumen. There are contrasting reports of the effects of these phytoadditives on the rumen fermentation and rumen microbes probably depending upon the interactions among the chemical structures and levels of phytochemicals used, nutrient composition of diets and microbial components in the rumen. The study of chemical structure-activity relationships is required to exploit the phytochemicals for obtaining target responses without adversely affecting beneficial microbial populations. A greater understanding of the modulatory effects of phytochemicals on the rumen microbial populations together with fermentation will allow a better management of the rumen ecosystem and a practical application of this feed additive technology in livestock production.

The effect of pineapple waste (Ananas comosus (L). Merr) subtitution on mixed basal diet of Elephant grass and calliandra on rumen ecosystem of sheep

The aim of this study was to evaluate the effect of pineapple waste substitution to mixed basal diet of Elephant grass and calliandra on rumen ecosystem. Pineapple waste was substituted to basal of Elephant grass and calliandra leaves (3:2) at the level of 0% (RA); 10% (RB); 20% (RC); 30% (RD); 40% (RE) and 50% (RF). In this experiment 24 Indonesian local male sheep (9-10 months old, 15.3 kg average body weight) were used, and were divided into 6 groups of dietary treatment (4 sheep each). Every group was offered one of the experimental diets (RA to RF) in a Completely Randomized Design. Pineapple waste was offered gradually for one month until the level of 50% (RF) was reached. The animals were adapted to experimental diets for about 14 days prior to the data collection period. Rumen fluids from each animal was taken (5 hours after morning feeding) for pH, ammonia concentration; bacteria and protozoa population analysis. The results showed that substitution of pineapple waste up to 30% had no effect on pH, but when the level was increased up to 40 and 50%, the pH (P 0.05). Increasing pineapple waste given increased population of amyllolytic bacteria but decreased the population of cellulolytic bacteria. On the Elephant grass and calliandra basal diet with the proportion of 3 : 2, the best substitution of pineapple waste was up to 20%. Key words : Pineapple Waste, Rumen, Bacteria, Protozoa, Ammonia

Using microbial fatty acids to improve understanding of the contribution of solid associated bacteria to microbial mass in the rumen

This study sought to distinguish liquid-(LAB) and detached (SAB 1) and undetached (SAB 2) solid-associated bacteria through their fatty acid (FA) and purine base (PB) profiles. Fatty acids and PB were also evaluated as internal microbial markers for estimating microbial biomass associated with rumen particles. Four merino rams fitted with rumen cannulae and fed dehydrated alfalfa pellets provided rumen contents. In 3 consecutive weeks, rumen contents were collected and samples of LAB and SAB 1, total rumen content (TRC), washed rumen particles (WRP) and rumen particles after SAB 1 extraction (ERP) were obtained and analysed for PB and FA. The SAB 2 biomass composition was estimated from the non-NDF organic matter (OM) remaining in ERP. The concentration of total SAB biomass in particles was estimated using both PB and odd and branched-chain fatty acids (OBCFA). Concentrations of PB and OBCFA were highly correlated among the different rumen fractions. Marked differences between LAB and SAB populations occurred with LAB having higher PB content, lower FA content and a higher proportion (g/100 g fatty acids) of OBCFA than did SAB. The chemical composition of SAB 1 and SAB 2 was similar, except for the 15% higher crude protein content of the latter. The concentration of OBCFA (mg/g microbial OM) did not differ between bacterial fractions. The PB/OBCFA ratio (mg/mg) was higher in LAB (2.08) than in SAB (0.94). The ratio between branched-chain and odd-linear-chain FA was higher in LAB (2.26) than in SAB (1.46). Extraction of PB and OBCFA from WRP with our SAB detachment procedure was 61% and 31%, respectively. Estimated SAB 1 and total SAB biomass (mg OM/g WRP) were 158 and 266, and 47 and 164, respectively, using PB and OBCFA as microbial markers. This study suggests that the OBCFA have potential as internal microbial markers in rumen ecosystem studies.

Molecular characterization of ruminal bacterial diversity in vitro

PCR analysis is a sensitive and specific tool to detect and monitor microorganisms in complex environmental samples. The amplification of 16S ribosomal DNA sequences followed by gel electrophoresis under denaturing gradient (DGGE) has been a powerful technique to genetically evaluate microbial ecosystems. Changes in rumen microbial populations were investigated in vitro using a basal diet with different lipid sources. PCRs were performed with two different sets of primers in order to amplify 16S rRNA sequences, and the amplified fragments were submitted to DGGE analysis. The findings presented in this study show that distinct microbial communities were present in each treatment. The presence of soybean oil seems to maximize growth of bacterial population, whereas fish oil appears to reduce growth. We demonstrated the successful application of molecular ecological techniques to analyze the structure and composition of bacterial communities in rumen ecosystems.

Advances in microbial ecosystem concepts and their consequences for ruminant agriculture

Microbial transformations in the rumen ecosystem have a major impact on our ability to meet the challenge of reducing the environmental footprint of ruminant livestock agriculture, as well as enhancing product quality. Current understanding of the rumen microbial ecosystem is limited, and affects our ability to manipulate rumen output. The view of ruminal fermentation as the sum of activities of the dominant rumen microbiota is no longer adequate, with a more holistic approach required. This paper reviews rumen functionality in the context of the microbiota of the rumen ecosystem, addressing ruminal fermentation as the product of an ecosystem while highlighting the consequences of this for ruminant agriculture. Microbial diversity in the rumen ecosystem enhances the resistance of the network of metabolic pathways present, as well as increasing the potential number of new pathways available. The resulting stability of rumen function is further promoted by the existence of rumen microbiota within biofilms. These protected, structured communities offer potential advantages, but very little is currently known about how ruminal microorganisms interact on feed-surfaces and how these communities develop. The temporal and spatial development of biofilms is strongly linked to the availability of dietary nutrients, the dynamics of which must also be given consideration, particularly in fresh-forage-based production systems. Nutrient dynamics, however, impact not only on pathway inputs but also the turnover and output of the whole ecosystem. Knowledge of the optimal balance of metabolic processes and the corresponding microbial taxa required to provide a stable, balanced ecosystem will enable a more holistic understanding of the rumen. Future studies should aim to identify key ecosystem processes and components within the rumen, including microbial taxa, metabolites and plant-based traits amenable to breeding-based modification. As well as gaining valuable insights into the biology of the rumen ecosystem, this will deliver realistic and appropriate novel targets for beneficial manipulation of rumen function.

Redirecting rumen fermentation to reduce methanogenesis

Methane production in ruminants has received global attention in relation to its contribution to the greenhouse gas effect and global warming. In the last two decades, research programs in Europe, Oceania and North America have explored a variety of approaches to redirecting reducing equivalents towards other reductive substrates as a means of decreasing methane production in ruminants. Some approaches such as vaccination, biocontrols (bacteriophage, bacteriocins) and chemical inhibitors directly target methanogens. Other approaches, such as defaunation, diet manipulations including various plant extracts or organic acids, and promotion of acetogenic populations, seek to lower the supply of metabolic hydrogen to methanogens. The microbial ecology of the rumen ecosystem is exceedingly complex and the ability of this system to efficiently convert complex carbohydrates to fermentable sugars is in part due to the effective disposal of H2 through reduction of CO2 to methane by methanogens. Although methane production can be inhibited for short periods, the ecology of the system is such that it frequently reverts back to initial levels of methane production though a variety of adaptive mechanisms. Hydrogen flow in the rumen can be modelled stoichiometrically, but accounting for H2 by direct measurement of reduced substrates often does not concur with the predictions of stoichiometric models. Clearly, substantial gaps remain in our knowledge of the intricacies of hydrogen flow within the ruminal ecosystem. Further characterisation of the fundamental microbial biochemistry of hydrogen generation and methane production in the rumen may provide insight for development of effective strategies for reducing methane emissions from ruminants.

Application of Biotechnology to Improve Post-Ingestion Forage Quality in the Rumen

Several methods are currently employed to manipulate rumen fermentation to enhance postingestion nutritive value of fibrous forages through use of biotechnology including inoculants of native and recombinant rumen microorganisms, natural adaptation and microbial feed enzymes. In this essay, progress and problems related to manipulation of rumen ecosystem through inoculation of natural and genetically modified rumen microorganisms are discussed. Also advancements in exogenous fibrolytic enzymes to improve digestibility of fibrous diets is briefly reviewed.

Dose–response effects of essential oils on in vitro fermentation activity of the rumen microbial population

Numerous bioactive compounds in plant-derived products are secondary toxic metabolites produced by plants as a mechanism of defence against herbivores and invading microbes. Among them, essential oils (EO), which are known for their antimicrobial effects have been proposed as modulators of rumen fermentation. However, there is little information on their dose–response effects on the rumen ecosystem. Using five natural EO from Thymus vulgaris, Origanum vulgare, thymol chemo-type of O. vulgare, Cinnamomum verum, and Anethum graveolens, and three pure constituents thymol, carvacrol, and cinnamaldehyde, we examined the in vitro response of the rumen microbial ecosystem evaluated through production curves of volatile fatty acids (VFA), ammonia, and gas. Three types of EO molecules were discriminated, being carvone (a terpene), thymol, and carvacrol (phenolic EO originated from the terpene pathway), and cinnamaldehyde (a phenolic EO originating from the phenyl-propane pathway). The carvone-based EO produced a linear non-threshold profile that negatively affected end-products of fermentation. A threshold profile, characterized by a virtual stop of fermentation when doses were higher than the threshold level, occurred for thymol/carvacrol-based EO. The effect of cinnamaldehyde-based EO was a negative sigmoid profile characterized by a reduction of protein degradation at low additive concentration, without changes in VFA production, and a negative effect on all fermentation variables at higher concentrations. Loss of the methanogenesis was a feature of the transition point in this profile. Results show that EO induces at least three types of dose–response in the rumen ecosystem. Knowing the type of response, as well as the effective dose, could help determine which compound would be most appropriate for a particular production objective, such as decreasing protein degradation. Results also highlight the toxic nature of EO upon rumen microbes, and that there is a narrow dose window for the successful adoption of this technology.

The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review

This paper analyses the research progress in the use of molecular techniques based on ribosomal RNA and DNA (rRNA/rDNA) for rumen microbial ecosystem since first literature by Stahl et al. (1988). Because rumen microbial populations could be under-estimated by adopting the traditional techniques such as roll-tube technique or most-probable-number estimates, modern molecular techniques based on 16S/18S rRNA/rDNA can be used to more accurately provide molecular characterization, microbe populations and classification scheme than traditional methods. Phylogenetic-group-specific probes can be used to hybridize samples for detecting and quantifying of rumen microbes. But, competitive-PCR and real-time PCR can more sensitively quantify rumen microbes than hybridization. Molecular fingerprinting techniques including both denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and restriction fragment length polymorphisms (RFLP) can used to explore diversity of bacteria, protozoa and fungi in the rumen ecosystem. By constructing clone libraries of 16S/18S rRNA/rDNA of rumen microbes, more new microbes can be discovered and identified. For fungi, internal transcribed spacers (ITS) of fungi are better than 18S rRNA/rDNA for discriminating operational taxonomic units. In conclusion, 16S/18S rRNA/rDNA procedures have been used with success in rumen microbes and are quickly gaining acceptance for studying rumen microbial ecosystem, and will become useful methods for rumen ecology research. However, molecular techniques based on 16S/18S rRNA/rDNA don't preclude classical and traditional microbiological techniques. It should used together to acquire accurate and satisfactory results.

Effects of whole crop canola silage on Holstein dairy cow performance in early lactation

It is clear that providing sufficient fiber in dairy cattle diets has most positive effects on improvement of rumen ecosystem and feed intake efficiency. In semiarid countries such as Iran providing forage requirement of cattle is limited. So it is better to use plants with low water requirement, high growth rate and high forage yield per hectare. Canola (Brassica napus L.) recently has received consideration as a forage source for livestock (Lancaster et al., 1990). Canola is reasonably widely adapted and performs well in many areas of Iran. In addition to its low water requirements and high growth rate, its cultivation season is well matched with environmental condition in Iran. The main objective of this study was to evaluate effect of replacing maize silage with canola silage (as dry matter %) on Holstein dairy cow performance.

Effects and modes of action of live yeasts in the rumen

AbstractLive yeasts (Saccharomyces cerevisiae) are more and more widely used as feed additives for ruminants. They are considered as allochtonous microorganisms in the rumen environment, however, distributed daily to dairy cows or beef cattle they can survive in the digestive tract and interact with autochtonous microbial populations. The positive effects of yeast cells have been mainly demonstrated on growth and activity of fibre-degrading bacteria and fungi, on stabilisation of rumen pH and prevention of lactate accumulation, on ruminal microbial colonization and on the set up of fermentative processes during the pre-weaning period. Modes of action of yeast probiotics depend on their viability and stability in the rumen ecosystem. Up to now, the main modes of action identified are the supply of growth factors to rumen microorganisms, oxygen scavenging inducing more favourable conditions for the anaerobic communities, and nutritional competition with autochtonous ruminal species.

Changes in rumen ecosystem and feed dry matter degradability of buffalo which received rumen content of cattle through cross inoculation

The research was done to identify changes in rumen ecosystem of buffalo which received rumen content of cattle. As much as three head of fistulated male buffaloes (live weight of 450-550 kg) and three fistulated female cattle (live weight 250-380 kg) were used. This experiment was done three stage as follows: pre-inoculation, inoculation and post-inoculation. In Pre-inoculation, the sample of rumen content was taken two hours before morning feeding and directly observed for pH rumen liquor, ammonia nitrogen (NH 3 -N) and total volatile fatty acid (VFA). During the last three days of the first week of pre and post inoculation, the in situ dry matter digestibility was conducted. The samples were incubated for 0, 3, 6, 9, 12, 24, 48, and 72 hours in the rumen of the experimental animals. Napier grass, leucaena, and wheat pollard were incubated separately in the nylon bags to determine DM digestibility. The cross inoculation (total of rumen content) was done less than 10 minutes. In post-inoculation, the sample of rumen content was taken at two hours before morning feeding as long as two days of two weeks after inoculation. Changes in rumen ecosystem (pH, NH 3 -N and total VFA) were tested by using Completely Randomized Design. Result showed that the transfer of rumen content from cattle to buffalo significantly reduced the pH level in the rumen of buffalo. However, it had no significant effect on the ammonia concentration in the rumen of buffalo and showed significantly decreased of bacteria but it was not affect on the total count of protozoa and fungi. The transfer of rumen content from cattle to buffalo significantly decreased the value of insoluble and potential digestible fraction of leucaena. The DM effective degradability of Napier grass was significantly increase (P<0.05) within outflow rate 0.02 and 0.04 h -1 after cross inoculation. In wheat pollard, a significantly decrease (P<0.05) was occurred when outflow rate was observed at 0.04, 0.06 and 0.08 h -1 Key Words: Rumen Ecosystem, Cross Inoculation, Degradability

Bacterial Mechanisms to Overcome Inhibitory Effects of Dietary Tannins

High concentrations of tannins in fodder plants inhibit gastrointestinal bacteria and reduce ruminant performance. Increasing the proportion of tannin-resistant bacteria in the rumen protects ruminants from anti-nutritional effects. The reason for the protective effect is unclear, but could be elucidated if the mechanism(s) by which tannins inhibit bacteria and the mechanisms of tannin resistance were understood. A review of the literature indicates that the ability of tannins to complex with polymers and minerals is the basis of the inhibitory effect on gastrointestinal bacteria. Mechanisms by which bacteria can overcome inhibition include tannin modification/degradation, dissociation of tannin-substrate complexes, tannin inactivation by high-affinity binders, and membrane modification/repair and metal ion sequestration. Understanding the mechanism of action of tannins and the mechanism(s) bacteria use to overcome the inhibitory effects will allow better management of the rumen ecosystem to reduce the anti-nutritional effects of tannin-rich fodder plants and thereby improve ruminant production.

The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce group-specific fingerprints

Identification of microbial community members in complex environmental samples is time consuming and repetitive. Here, ribosomal sequences and hidden Markov models are used in a novel approach to rapidly assign fungi to their presumptive genera. The ITS1 and ITS2 fragments from a range of axenic, anaerobic gut fungal cultures, including several type strains, were isolated and the RNA secondary structures predicted for these sequences were used to generate a fingerprinting program. The methodology was then tested and the algorithms improved using a collection of environmentally derived sequences, providing a rapid indicator of the fungal diversity and numbers of novel sequence groups within the environmental sample from which they were derived. While the methodology was developed to assist in investigations involving the rumen ecosystem, it has potential generic application in studying diversity and population dynamics in other microbial ecosystems.

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.

Ecology of uncultivated Oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches.

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Population dynamics of rumen microbes using modern techniques in rumen enhanced solid incubation

The microbial ecology of the rumen is very complex. Different species of bacteria, protozoa, and fungi are involved in digestion of plant material in ruminants. In spite of complicated interrelationships among the various groups of microorganisms in the rumen ecosystem, Bacteria and Archaea are believed to play a major role because of their numerical predominance and metabolic diversity. In this work we are presenting the results for microbial population dynamics of rumen microbes during two-stage anaerobic digestion of grass. The reactors were inoculated with fresh rumen content. Fluorescent in situ hybridization, confocal laser scanning microscopy and epifluorescence microscopy were employed for microbial investigation. It was observed that Bacteria dominated in the hydrolytic reactor (1st stage) whereas Archaea were predominant in the methanogenic reactor (2nd stage). The stability of the methanogenic reactor was result of the dominance of Methanosaeta species (mainly the filamentous type).

II. Quantitative aspects of phosphorus absorption in ruminants.

Phosphorus absorption in ruminants was analysed from a database described in a previous article. For common values of ingested phosphorus (2.5-5.0 g x kg(-1) of DM), 0.73 of dietary phosphorus is absorbed. The remaining variability is probably due to phosphorus quality. Phosphorus absorbed from silage, cereal, cereal by-products and hay differs greatly. The current true absorption coefficient used to calculate daily phosphorus supply is a constant value in the current systems and often it underestimates the true absorption resulting in an excess of phosphorus being supplied in the diets. Adjusting the true absorption coefficient values requires better characterisation of the phosphorus supplied by each feedstuff. Dietary influences (phytate phosphorus, crude fibre, etc.) were investigated but trials assessing the ration effect on phosphorus absorption are lacking. Since rumen microbes have specific phosphorus requirements, particularly for cell-wall degradation, the feedstuffphosphorus availability for the rumen ecosystem is discussed.

Rumen Manipulation to Improve Animal Productivity

Anaerobic rumen microorganisms mainly bacteria, protozoa and fungi degrade ligno-cellulosic feeds consumed by the ruminants. The ruminants in developing countries are predominantly maintained on low grade roughage and grazing on degraded range land resulting in their poor nutrient utilization and productivity. Hence, manipulation of rumen fermentation was tried during last two decades to optimize ruminal fermentation for improving nutrient utilization and productivity of the animals. Modification of rumen microbial composition and their activity was attempted by using chemical additives those selectively effect rumen microbes, introduction of naturally occurring or genetically modified foreign microbes into the rumen and genetically manipulation of existing microbes in the rumen ecosystem. Accordingly, rumen protozoa were eliminated by defaunation for reducing ruminal methane production and increasing protein outflow in the intestine, resulting in improve growth and feed conversion efficiency of the animals. Further, Interspecies trans-inoculation of rumen microbes was also successfully used for annulment of dietary toxic factor. Additionally, probiotics of bacterial and yeast origin have been used in animal feeding to stabilize rumen fermentation, reduced incidence of diarrhoea and thus improving growth and feed conversion efficiency of young stalk. It is envisaged that genetic manipulation of rumen microorganisms has enormous research potential in developing countries. In view of feed resource availability more emphasis has to be given for manipulating rumen fermentation to increase cellulolytic activity for efficient utilization of low grade roughage.