Review: Ruminal microbiome and microbial metabolome: effects of diet and ruminant host.

The rumen contains a great diversity of prokaryotic and eukaryotic microorganisms that allow the ruminant to utilize ligno-cellulose material and to convert non-protein nitrogen into microbial protein to obtain energy and amino acids. However, rumen fermentation also has potential deleterious consequences associated with the emissions of greenhouse gases, excessive nitrogen excreted in manure and may also adversely influence the nutritional value of ruminant products. While several strategies for optimizing the energy and nitrogen use by ruminants have been suggested, a better understanding of the key microorganisms involved and their activities is essential to manipulate rumen processes successfully. Diet is the most obvious factor influencing the rumen microbiome and fermentation. Among dietary interventions, the ban of antimicrobial growth promoters in animal production systems has led to an increasing interest in the use of plant extracts to manipulate the rumen. Plant extracts (e.g. saponins, polyphenol compounds, essential oils) have shown potential to decrease methane emissions and improve the efficiency of nitrogen utilization; however, there are limitations such as inconsistency, transient and adverse effects for their use as feed additives for ruminants. It has been proved that the host animal may also influence the rumen microbial population both as a heritable trait and through the effect of early-life nutrition on microbial population structure and function in adult ruminants. Recent developments have allowed phylogenetic information to be upscaled to metabolic information; however, research effort on cultivation of microorganisms for an in-depth study and characterization is needed. The introduction and integration of metagenomic, transcriptomic, proteomic and metabolomic techniques is offering the greatest potential of reaching a truly systems-level understanding of the rumen; studies have been focused on the prokaryotic population and a broader approach needs to be considered.

Dynamic role of single-celled fungi in ruminal microbial ecology and activities.

In ruminants, high fermentation capacity is necessary to develop more efficient ruminant production systems. Greater level of production depends on the ability of the microbial ecosystem to convert organic matter into precursors of milk and meat. This has led to increased interest by animal nutritionists, biochemists and microbiologists in evaluating different strategies to manipulate the rumen biota to improve animal performance, production efficiency and animal health. One of such strategies is the use of natural feed additives such as single-celled fungi yeast. The main objectives of using yeasts as natural additives in ruminant diets include; (i) to prevent rumen microflora disorders, (ii) to improve and sustain higher production of milk and meat, (iii) to reduce rumen acidosis and bloat which adversely affect animal health and performance, (iv) to decrease the risk of ruminant-associated human pathogens and (v) to reduce the excretion of nitrogenous-based compounds, carbon dioxide and methane. Yeast, a natural feed additive, has the potential to enhance feed degradation by increasing the concentration of volatile fatty acids during fermentation processes. In addition, microbial growth in the rumen is enhanced in the presence of yeast leading to the delivery of a greater amount of microbial protein to the duodenum and high nitrogen retention. Single-celled fungi yeast has demonstrated its ability to increase fibre digestibility and lower faecal output of organic matter due to improved digestion of organic matter, which subsequently improves animal productivity. Yeast also has the ability to alter the fermentation process in the rumen in a way that reduces methane formation. Furthermore, yeast inclusion in ruminant diets has been reported to decrease toxins absorption such as mycotoxins and promote epithelial cell integrity. This review article provides information on the impact of single-celled fungi yeast as a feed supplement on ruminal microbiota and its function to improve the health and productive longevity of ruminants.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

Aerobic cultivation of anaerobic rumen protozoa, Entodinium caudatum and Epidinium caudatum.

Rumen protozoa, primarily ciliates, are one of the important groups of strictly anaerobic microbes living in the rumen. Despite their ubiquitous occurrence in the rumen and significant contribution to host animals, it is still poorly understood why they live only in the rumen and similar environment. Because rumen protozoa require strict anaerobic conditions to sustain their viability and grow, only a few laboratories equipped with protozoology expertise and anaerobic facilities can grow rumen protozoa in laboratory. Also for the same reason, only a few species have been grown and maintained as laboratory cultures for research. Prompted by a recent study, we hypothesized that anaerobic rumen protozoa could also be cultivated aerobically if antioxidants were included in the media. Indeed, our experiments showed that the cultures of both Entodinium caudatum and Epidinium caudatum, two major rumen protozoal species, could be cultured successfully in aerobic media supplemented with ascorbic acid, glutathione and α-ketoglutarate as antioxidants. Anaerobic fermentation was maintained through the fermentation characteristics and microbial populations were altered to some extent under aerobic conditions. The antioxidants also enhanced the revival of cryopreserved stock cultures of both rumen protozoal species. The results of this study may facilitate and promote future research in which rumen protozoa need to be cultured in laboratory.

Probiotics: From Isolation to Application.

Probiotics have become highly recognized as supplements for humans and animals because of their beneficial effects on health and well-being. The present review aims to provide an overview of different steps through which microbial strains become applicable probiotics in food and/or feed industries. Isolation of potential probiotic strains is the first step. Lactic acid bacteria are the most frequently used microorganisms as probiotics, which can be isolated from human, animal, plant, and environment. The next steps are identification of the isolates and characterization of them based on the main selection criteria for any potential probiotic microorganism, including resistance to gastric acidity and bile salt, adherence to mucus and/or intestinal epithelial cells and cell lines, and antimicrobial and antagonism activity against potentially pathogenic microbes. There are additional probiotic properties that may be considered for selection of probiotic strains with specific effects, such as cholesterol reduction ability, antioxidant activity, or cytotoxic effect against cancer cells. However, a potential probiotic does not need to fulfill all such selection criteria. As the last step, safety status of probiotics for humans is verified by taxonomy clarification, in vitro and in vivo tests, human trials, and genome sequencing.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Rumen microbiota and dietary fat: a mutual shaping.

Although fat content in usual ruminant diets is very low, fat supplements can be given to farm ruminants to modulate rumen activity or the fatty acid (FA) profile of meat and milk. Unsaturated FAs, which are dominant in common fat sources for ruminants, have negative effects on microbial growth, especially protozoa and fibrolytic bacteria. In turn, the rumen microbiota detoxifies unsaturated FAs (UFAs) through a biohydrogenation (BH) process, transforming dietary UFAs with cis geometrical double-bonds into mainly trans UFAs and, finally, into saturated FAs. Culture studies have provided a large amount of data regarding bacterial species and strains that are affected by UFAs or involved in lipolysis or BH, with a major focus on the Butyrivibrio genus. More recent data using molecular approaches to rumen microbiota extend and challenge these data, but further research will be necessary to improve our understanding of fat and rumen microbiota interactions.

DISEASE COMPLEXITY IN A DECLINING ALASKAN MUSKOX (OVIBOS MOSCHATUS) POPULATION.

The muskox ( Ovibos moschatus ) population inhabiting the eastern North Slope (ENS) of Alaska, US declined dramatically during 1999-2006, whereas populations in western Alaska (WA) were stable or increasing. To understand morbidity and mortality factors contributing to the decline, Alaska Department of Fish and Game conducted pathologic investigations of carcasses from 2005 until 2008. Additionally, archived sera from both ENS and WA muskoxen collected during 1984-92, before the documented beginning of the ENS decline; sera collected during 2000, near the beginning of the decline; and contemporary sera (from live capture-release, adult females) collected during 2006, 2007, and 2008 were analyzed to determine whether prevalence of antibody to potential pathogens differed in the two areas or changed over time. The pathogens investigated were those that were believed could cause lameness or poor reproduction or adversely affect general health. Furthermore, trace mineral levels, hemograms, and gastrointestinal parasites were evaluated in live adult females captured 2006-08. Pathologic investigations identified several comorbid conditions, including predation, polyarthritis caused by or consistent with Chlamydophila spp. infection, hoof lesions, copper deficiency, contagious ecthyma, verminous pneumonia, hepatic lipidosis suggestive of negative energy balance, and bacterial bronchopneumonia due to Trueperella pyogenes and Bibersteinia trehalosi . Pathogens suspected to be newly introduced in the ENS muskox population on the basis of serologic detection include bovine viral diarrhea, respiratory syncytial virus, Chlamydophila spp., Brucella spp., Coxiella burnetii , and Leptospira spp., whereas parainfluenza virus-3 antibody prevalence has increased in the WA population. Although multiple disease syndromes were identified that contributed to mortality and, in combination, likely limited the ENS muskox population, further holistic investigations of disease agents, trace mineral status, and nutritional factors in conjunction with intensive demographic and environmental analyses would provide a better understanding of factors that influence Alaskan muskox populations.

Medicinal herbs as a potential strategy to decrease methane production by rumen microbiota: a systematic evaluation with a focus on Perilla frutescens seed extract.

Mitigation of the methane (CH4) emission from ruminants is needed to decrease the environmental impact of ruminant animal production. Different plant materials and chemicals have been tested, but few are both effective and practical. Medicinal herbs contain biological compounds and antimicrobials that may be effective in lowering the CH4 production. However, few studies have systematically evaluated medicinal herbs for their effect on CH4 production or on the rumen microbiota. In this study, extracts from 100 medicinal herbs were assessed for their ability to decrease CH4 production by rumen microbiota in vitro. The extracts of 12 herbs effectively lowered the CH4 production, with the extract of Perilla frutescens seeds being the most effective. The major components of P. frutescens seed extract were identified, and the effects of the extract on the fermentation characteristics and populations of rumen methanogens, fungi, protozoa, and select bacteria were also assessed. The decreased CH4 production induced by the P. frutescens seed extract was accompanied by an increased abundance of Ruminobacter, Selenomonas, Succinivibrio, Shuttleworthis, Pseudobutyrivbrio, Anaerovibrio, and Roseomonas and a decreased abundance of Methanobrevibacter millerae. The abundance of Pedobacter, Anaeroplasma, Paludibacter, Ruminococcus, and unclassified Lachnospiraceae was positively correlated with the CH4 production, with no effects on volatile fatty acids. This study suggests that medicinal herbs may be used to mitigate the CH4 emission from ruminants.

Effects of plants containing secondary compounds and plant oils on rumen fermentation and ecology.

A number of experiments have been conducted to investigate effects of tropical plants containing condensed tannins and/or saponins present in tropical plants and some plant oils on rumen fermentation and ecology in ruminants. Based on both in vitro and in vivo trials, the results revealed important effects on rumen microorganisms and fermentation including methane production. Incorporation and/or supplementation of these plants containing secondary metabolites have potential for improving rumen ecology and subsequently productivity in ruminants.

The effect and mode of action of saponins on the microbial populations and fermentation in the rumen and ruminant production.

The growing public concerns over chemical residues in animal-derived foods and threats of antibiotic-resistant bacteria have renewed interest in exploring safer alternatives to chemical feed additives in ruminant livestock. Various bioactive phytochemicals including saponins appear to be potential 'natural' alternatives to 'chemical' additives in modulating rumen fermentation favourably and animal performance. Saponins are a diverse group of glycosides present in many families of plants. The primary effect of saponins in the rumen appears to be to inhibit the protozoa (defaunation), which might increase the efficiency of microbial protein synthesis and protein flow to the duodenum. Furthermore, saponins may decrease methane production via defaunation and/or directly by decreasing the activities (i.e. rate of methanogenesis or expression of methane-producing genes) and numbers of methanogens. Saponins may also selectively affect specific rumen bacteria and fungi, which may alter the rumen metabolism beneficially or adversely. The ammonia-adsorption and modulation of digesta passage in the rumen by saponins have also been implicated in altering rumen metabolism, but their physiological responses are likely to be negligible compared with microbiological effects. The effects of saponins on rumen fermentation have not been found to be consistent. These discrepancies appear to be related to the chemical structure and dosage of saponins, diet composition, microbial community and adaptation of microbiota to saponins. There is need for systematic research based on chemical structures of saponins, nutrient composition of diets and their effects on rumen microbial ecosystem to obtain consistent results. The present paper reviews and discusses the effects and mode of action of saponins on microbial community and fermentation in the rumen, and ruminant performance.

Microbial fatty acid conversion within the rumen and the subsequent utilization of these fatty acids to improve the healthfulness of ruminant food products.

Consumers are aware of foods containing microcomponents that may have positive effects on health maintenance and disease prevention. In ruminant milk, meat, and milk products; these functional food components include eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), 9c11t-conjugated linoleic acid, and vaccenic acid (11t-18:1). Modifying ruminal microbial metabolism of fatty acid in rumen through animal diet formulation is an effective way to enhance these functional fatty acids in ruminant-derived food products. However, it requires an understanding of the interrelationship between supply of lipid through the diet and rumen fermentation. Lipids in ruminant diets undergo extensive hydrolysis and biohydrogenation in the rumen. Apparent transfer efficiency of eicosapentaenoic acid and docosahexaenoic acid from feed to milk is very low (1.9 to 3.3%), which is, to a large extent, related to their extensive biohydrogenation in the rumen. Therefore, feeding a rumen-protected supplement containing eicosapentaenoic acid and docosahexaenoic acid, can be used to bypass the rumen. Ruminant-derived foods also contain different types of conjugated linoleic acid isomers, which are intermediates of rumen biohydrogenation of linoleic acid (9c12c-18:2). The predominant isomer of conjugated linoleic acid is 9c11t, which has numerous health benefits in animal models. The concentration of conjugated linoleic acid in ruminant-derived food products can be significantly enhanced through animal diet modification. We conclude that most current functional food products from ruminants have potential for their health-supporting properties, and for this market to succeed, an evidence-based approach should be developed in humans.

Meta-analysis of the influence of Saccharomyces cerevisiae supplementation on ruminal parameters and milk production of ruminants.

The effects of yeast supplementation on intake, production, and rumen fermentation characteristics have been widely studied, but results are inconsistent between different studies. A quantitative meta-analysis was applied to 110 papers, 157 experiments, and 376 treatments dealing with yeast supplementation in ruminants. The objective was first to highlight the major quantitative effects of live yeast supplementation on intake, rumen fermentation, and milk production, and second, to identify major differences in experimental conditions between studies that can affect the response to treatment. Some of these experimental conditions are referred to as interfering factors. Yeast supplementation increased rumen pH (+0.03 on average) and rumen volatile fatty acid concentration (+2.17 mM on average), tended to decrease rumen lactic acid concentration (-0.9 mM on average), and had no influence on acetate-to-propionate ratio. Total-tract organic matter digestibility was also increased by yeast supplementation (+0.8% on average). Yeast supplementation increased dry matter intake (DMI; +0.44 g/kg of body weight; BW), milk yield (+1.2 g/kg of BW), and tended to increase milk fat content (+0.05%), but had no influence on milk protein content. Dose effects of yeast supplementation, expressed as log(10) [1+(cfu per 100 kg of BW)], globally confirmed the qualitative effects observed in the first analysis. The positive effect of yeast supplementation on rumen pH increased with the percentage of concentrate in the diet and with the DMI level. It was negatively correlated with the level of dietary neutral detergent fiber (NDF). The positive effect of yeast supplementation on rumen volatile fatty acid concentration increased with DMI and crude protein levels. The positive effect of yeast supplementation on organic matter digestibility increased with the percentage of concentrate and NDF in the diet. The negative effect of yeast supplementation on lactic acid concentration tended to decrease when the DMI level and the percentage of concentrate in the diet increased. The effects of interfering factors were globally similar when either dose effect or qualitative effect of yeast was taken into account. Although rumen fermentation efficiency per se was not measured, these results suggest an improvement in rumen fermentation by yeast supplementation. This effect could, however, be modulated by several different factors such as DMI, percentage of concentrate or NDF in the diet, or species.

[Determination of the inhibitory effect of a biologically active preparation on the reproduction of micro-organisms from the intestines of ruminants]. / Stanovenie inhibicného úcinku biologicky aktívneho prípravku na rozmnozovanie niektorých mikroorganizmov z intestina prezúvavcov.

A procedure for the production of biologically active substances from pine and spruce needles was worked out within the research project Utilization of Tree Verdure. The product was referred to as a biologically active preparation. The fields of its possible uses include agriculture (animal production-feeding), cosmetics (production of pastes, soaps, etc.), pharmaceutical industry (after additional conditioning the product can be used for manufacturing medicinal drugs for the control of inflammatory diseases and the like). The biologically active preparation, i. e. the pine needle extract, was studied as to its effect on some of the most numerous microorganisms in the intestines of ruminants. As found, this preparation has a partial inhibitive action on the growth and reproduction of microorganisms. The biologically active preparation is an extract isolated from the needles by means of 0.3% sodium hydroxide. Besides extractive substances it also contains the residues of organellae of cellular nature (which got into the extract through the filter during the process of preparation) and a small amount of fibre (0.33%). The chemical composition of the biologically active preparation is varied: until now about 220 substances, many of which are biologically active, have been found to be present in the product.