The use of direct-fed microbials for mitigation of ruminant methane emissions: a review

Concerns about the environmental effect and the economic burden of methane (CH₄) emissions from ruminants are driving the search for ways to mitigate rumen methanogenesis. The use of direct-fed microbials (DFM) is one possible option to decrease CH₄ emission from ruminants. Direct-fed microbials are already used in ruminants mainly to increase productivity and to improve health, and are readily accepted by producers and consumers alike. However, studies on the use of DFM as rumen CH₄ mitigants are scarce. A few studies using Saccharomyces cerevisiae have shown a CH₄-decreasing effect but, to date, there has not been a systematic exploration of DFM as modulators of rumen methanogenesis. In this review, we explored biochemical pathways competing with methanogenesis that, potentially, could be modulated by the use of DFM. Pathways involving the redirection of H₂ away from methanogenesis and pathways producing less H₂ during feed fermentation are the preferred options. Propionate formation is an example of the latter option that in addition to decrease CH₄ formation increases the retention of energy from the diet. Homoacetogenesis is a pathway using H₂ to produce acetate, however up to now no acetogen has been shown to efficiently compete with methanogens in the rumen. Nitrate and sulphate reduction are pathways competing with methanogenesis, but the availability of these substances in the rumen is limited. Although there were studies using nitrate and sulphate as chemical additives, use of DFM for improving these processes and decrease the accumulation of toxic metabolites needs to be explored more. There are some other pathways such as methanotrophy and capnophily or modes of action such as inhibition of methanogens that theoretically could be provided by DFM and affect methanogenesis. We conclude that DFM is a promising alternative for rumen methane mitigation that should be further explored for their practical usage.     

Methane production and diurnal variation measured in dairy cows and predicted from fermentation pattern and nutrient or carbon flow

Many feeding trials have been conducted to quantify enteric methane (CH₄) production in ruminants. Although a relationship between diet composition, rumen fermentation and CH₄ production is generally accepted, the efforts to quantify this relationship within the same experiment remain scarce. In the present study, a data set was compiled from the results of three intensive respiration chamber trials with lactating rumen and intestinal fistulated Holstein cows, including measurements of rumen and intestinal digestion, rumen fermentation parameters and CH₄ production. Two approaches were used to calculate CH₄ from observations: (1) a rumen organic matter (OM) balance was derived from OM intake and duodenal organic matter flow (DOM) distinguishing various nutrients and (2) a rumen carbon balance was derived from carbon intake and duodenal carbon flow (DCARB). Duodenal flow was corrected for endogenous matter, and contribution of fermentation in the large intestine was accounted for. Hydrogen (H₂) arising from fermentation was calculated using the fermentation pattern measured in rumen fluid. CH₄ was calculated from H₂ production corrected for H₂ use with biohydrogenation of fatty acids. The DOM model overestimated CH₄/kg dry matter intake (DMI) by 6.1% (R²=0.36) and the DCARB model underestimated CH₄/kg DMI by 0.4% (R²=0.43). A stepwise regression of the difference between measured and calculated daily CH₄ production was conducted to examine explanations for the deviance. Dietary carbohydrate composition and rumen carbohydrate digestion were the main sources of inaccuracies for both models. Furthermore, differences were related to rumen ammonia concentration with the DOM model and to rumen pH and dietary fat with the DCARB model. Adding these parameters to the models and performing a multiple regression against observed daily CH₄ production resulted in R² of 0.66 and 0.72 for DOM and DCARB models, respectively. The diurnal pattern of CH₄ production followed that of rumen volatile fatty acid (VFA) concentration and the CH₄ to CO₂ production ratio, but was inverse to rumen pH and the rumen hydrogen balance calculated from 4×(acetate+butyrate)/2×(propionate+valerate). In conclusion, the amount of feed fermented was the most important factor determining variations in CH₄ production between animals, diets and during the day. Interactions between feed components, VFA absorption rates and variation between animals seemed to be factors that were complicating the accurate prediction of CH₄. Using a ruminal carbon balance appeared to predict CH₄ production just as well as calculations based on rumen digestion of individual nutrients.     

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.     

Comparison of the effects of lanthanum,cerium and praseodymium on rumen fermentation,nutrient digestion and plasma biochemical parameters in beef cattle

The objectives of the trial were to compare the effects of supplementing rare earth elements (REE) lanthanum (La), cerium (Ce) and praseodymium (Pr) on rumen fermentation, nutrient digestion, methane (CH₄) production, nitrogen (N) balance and plasma biochemical parameters in beef cattle. Four Simmental male cattle, aged 12 months, with initial average liveweight of 333 ± 9 kg and fitted with rumen cannulas, were fed with a basal ration composed of concentrate mixture and maize silage. Animals received a basal ration without adding REE (Control) or three treatments, i.e. supplementing LaCl₃, CeCl₃ or PrCl₃ at 204 mg/kg DM to the basal ration, respectively, which were allocated in a 4 × 4 Latin square design. Each experimental period lasted 15 d, consisting of 12 d for pre-treatment and three subsequent days for sampling. Results showed that all tested levels of REE tended to increase neutral detergent fibre digestibility (p = 0.064) and tended to decrease rumen CH₄ production (p = 0.056). Supplementing LaCl₃ and CeCl₃ decreased total N excretion and urinary N excretion, increased N retention (p < 0.05), tended to increase total urinary purine derivatives (PD) (p = 0.053) and microbial N flow (p = 0.095), whereas supplementing PrCl₃ did not affect N retention, urinary PD and microbial N flow. No differences were found in the effects of nutrient digestibility, CH₄ production and plasma biochemical parameters among LaCl₃, CeCl₃ and PrCl₃. Further trials using graded levels of LaCl₃, CeCl₃ and PrCl₃ in a wide range are needed to obtain more pronounced results for comparing effects of La, Ce and Pr on rumen fermentation and nutrient digestion in beef cattle.     

Fruit and fibre: the nutritional value of figs for a small tropical ruminant,the blue duiker (Cephalophus monticola)

Tropical forests throughout the world are home to a guild of small ruminants that consume fruit as a substantial portion of their diet. Because the rumen is relatively inefficient at digesting nonstructural carbohydrates and only slowly digests cellulose, the feeding adaptations of frugivorous ruminants are enigmatic. We examined the nutritional value of wild figs to blue duikers, one of the smallest and most frugivorous ruminants, through chemical analyses and a series of digestion trials with six species of wild African figs. These figs were high in fat, protein, cell wall, lignin and Ca : P ratios, low in sugar and starch, and high in unextractable, fibre‐bound tannins when compared with many other fruits. The fibre‐bound tannins and protein caused protein digestibility and nitrogen balance to be consistently low or negative. The high fibre content of the figs allowed duikers to only digest 30–60% of energy contained in the figs. However, duikers were able to consume enough digestible energy to maintain body mass during 4‐day trials. Therefore, a ruminant digestive system is beneficial to mammals eating high fibre, high‐tannin tropical fruit like figs, especially if the mammal is small enough to harvest a sufficient amount to meet its daily energy requirements and has adaptations for reducing the effects of tannins on protein digestibility.     

The functional significance of the browser-grazer dichotomy in African ruminants

The allometric relationships for the fermentation rate of dry matter, the total energy concentration of volatile fatty acids (VFAs), the energy supplied from VFA production and the mass of the digesta contents within the rumen or caecum and proximal colon (hindgut) were used to test whether the digestive strategies of grazing and browsing African ruminants differ. The wet and dry mass of the contents of the rumen and hindgut were allometrically related to body mass (BM). These relationships did not differ between browsing and grazing ruminants. The fermentation rates in the rumen were strongly allometric and the intercepts of the relationships did not differ between browsers and grazers. The fermentation rates in the hindgut were not allometrically related to BM and did not differ between ruminants with different feeding habits. Likewise, the total energy concentration of the VFAs in the rumen and hindgut showed no allometric scaling and did not differ between browsing and grazing ruminants. The energy supplied by VFA production in both the rumen and hindgut of African ruminants scaled at around 0.8 with BM. Only in the case of the energy supplied by VFAs in the rumen were there significantly different intercepts for browsing and grazing ruminants. The energy supplied by VFA production in the rumen was inadequate to meet the energy requirements for maintenance of browsers and small grazers. The retention time of digesta in the alimentary tract was positively related to BM although there was no difference in the allometric relationships for grazers and browsers. The results of these analyses suggest that, after controlling for the effects of body mass, there is little difference in digestive strategy between African ruminants with different morphological adaptations of the gut.     

Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein

Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH₄) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH₄ emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH₄ emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats.     

Comparative nutrient extraction from forages by grazing bovids and equids: a test of the nutritional model of equid/bovid competition and coexistence

Summary Ruminants are unevenly distributed across the range of body sizes observed in herbivorous mammals; among extant East African species they predominate, in numbers and species richness, in the medium body sizes (10–600 kg). The small and the large species are all hind-gut fermenters. Some medium-sized hind-gut fermenters, equid perissodactyls, coexist with the grazing ruminants, principally bovid artiodactyls, in grassland ecosystems. These patterns have been explained by two complementary models based on differences between the digestive physiology of ruminants and hind-gut fermenters. The Demment and Van Soest (1985) model accounts for the absence of ruminants among the small and large species, while the Bell/Janis/Foose model accounts both for the predominance of ruminants, and their co-existence with equids among the medium-sized species (Bell 1971; Janis 1976; Foose 1982). The latter model assumes that the rumen is competitively superior to the hind-gut system on medium quality forages, and that hind-gut fermenters persist because of their ability to eat more, and thus to extract more nutrients per day from high fibre, low quality forages. Data presented here demonstrate that compared to similarly sized grazing ruminants (bovids), hind-gut fermenters (equids) have higher rates of food intake which more than compensate for their lesser ability to digest plant material. As a consequence equids extract more nutrients per day than bovids not only from low quality foods, but from the whole range of forages eaten by animals of this size. Neither of the current nutritional models, nor refinements of them satisfactorily explain the preponderance of the bovids among medium-sized ungulates; alternative hypotheses are presented.     

The determination of digesta particle size in some herbivores

Methods for determining and describing digesta and faecal particle size were studied. Higher mean particle size values were demonstrated for wet sieving as compared to dry sieving. Equines had larger faecal particle size than ruminants and rabbits. Particle size decreased with body weight in rumen-reticulum and facese for the ruminants. The fibre particle size was the same in rabbit hard and soft faeces and similar to sheep and goat faeces.The excretion pattern of Cr-mordanted fibre in sheep was shown to be influenced by the fraction that was labelled in the rumen. Retention time in the rumen and whole tract increased with increasing particle size.In rabbits, <75 μ or > 300 μ Cr-mordanted fibre particles gave similar marker retention times.     

A causal analysis of diet composition in free ranging cattle in reed-dominated vegetation

Summary Theoretical and observed diets of free-ranging yearling cattle in an area of reed-dominated vegetation were related to constraints inherent to ruminants (rumination time as reflected in the zone of acceptable crude fibre content of the ration; rumen fill) and in more general terms, to requirements for minerals (Na, Ca, Mg, P), for protein and for metabolisable energy in order to balance the growth rate achieved by experimental animals. Theoretical diets in spring were mainly limited by feeding time; theoretical diets in summer and autumn were mainly limited by rumen capacity. The high proteinto-energy ratio in the forage species caused protein intake to exceed the animals' demands. Mineral intake was in excess of requirements except for sodium. Yearling cattle in our study area maximized their energy intake, with feeding time, rumen capacity and crude fibre content of the forage as important constraints.     

Effect of fumarate reducing bacteria on in vitro rumen fermentation,methane mitigation and microbial diversity

The metabolic pathways involved in hydrogen (H₂) production, utilization and the activity of methanogens are the important factors that should be considered in controlling methane (CH₄) emissions by ruminants. H₂ as one of the major substrate for CH₄ production is therefore should be controlled. One of the strategies on reducing CH₄ is through the use of hydrogenotrophic microorganisms such as fumarate reducing bacteria. This study determined the effect of fumarate reducing bacteria, Mitsuokella jalaludinii, supplementation on in vitro rumen fermentation, CH4 production, diversity and quantity. M. jalaludinii significantly reduced CH₄ at 48 and 72 h of incubation and significantly increased succinate at 24 h. Although not significantly different, propionate was found to be highest in treatment containing M. jalaludinii at 12 and 48 h of incubation. These results suggest that supplementation of fumarate reducing bacteria to ruminal fermentation reduces CH₄ production and quantity, increases succinate and changes the rumen microbial diversity.     

Methanogens and Methanogenesis in the Rumens and Ceca of Lambs Fed Two Different High-Grain-Content Diets

The amount and nature of dietary starch are known to influence the extent and site of feed digestion in ruminants. However, how starch degradability may affect methanogenesis and methanogens along the ruminant''s digestive tract is poorly understood. This study examined the diversity and metabolic activity of methanogens in the rumen and cecum of lambs receiving wheat or corn high-grain-content diets. Methane production in vivo and ex situ was also monitored. In vivo daily methane emissions (CH₄ g/day) were 36% (P < 0.05) lower in corn-fed lambs than in wheat-fed lambs. Ex situ methane production (μmol/h) was 4-fold higher for ruminal contents than for cecal contents (P < 0.01), while methanogens were 10-fold higher in the rumen than in the cecum (mcrA copy numbers; P < 0.01). Clone library analysis indicated that Methanobrevibacter was the dominant genus in both sites. Diet induced changes at the species level, as the Methanobrevibacter millerae-M. gottschalkii-M. smithii clade represented 78% of the sequences from the rumen of wheat-fed lambs and just about 52% of the sequences from the rumen of the corn-fed lambs. Diet did not affect mcrA expression in the rumen. In the cecum, however, expression was 4-fold and 2-fold lower than in the rumen for wheat- and corn-fed lambs, respectively. Though we had no direct evidence for compensation of reduced rumen methane production with higher cecum methanogenesis, the ecology of methanogens in the cecum should be better considered.     

Biology of gut anaerobic fungi

The obligately anaerobic nature of the gut indigenous fungi distinguishes them from other fungi. They are distributed widely in large herbivores, both in the foregut of ruminant-like animals and in the hindgut of hindgut fermenters. Comparative studies indicate that a capacious organ of fermentative digestion is required for their development. These fungi have been assigned to the Neocallimasticaceae, within the chytridiomycete order Spizellomycetales. The anaerobic fungi of domestic ruminants have been studied most extensively. Plant material entering the rumen is rapidly colonized by zoospores that attach and develop into thalli. The anaerobic rumen fungi have been shown to produce active cellulases and xylanases and specifically colonise and grow on plant vascular tissues. Large populations of anaerobic fungi colonise plant fragment in the rumens of cattle and sheep on high-fibre diets. The fungi actively ferment cellulose which results in formation of a mixture of products including acetate, lactate, ethanol, formate, succinate, CO2 and H2. The properties of the anaerobic fungi together with the extent of their populations on plant fragments in animals on high-fibre diets indicates a significant role for the fungi in fibre digestion.     

丁酸调控幼龄反刍动物瘤胃上皮发育研究进展

瘤胃是反刍动物营养物质消化吸收和代谢的重要器官,其发育状态直接影响反刍动物生产性能和健康。初生犊牛和羔羊,瘤胃功能尚未发育完全,不能够充分消化和吸收固体饲料。因此,在幼龄时期,通过营养调控手段促进反刍动物的瘤胃发育对维持动物健康及提高生产性能具有重要意义。丁酸是瘤胃微生物降解植物性饲料的主要产物,也是瘤胃上皮及宿主的重要能量来源。丁酸调控幼龄反刍动物瘤胃上皮发育是一个历久弥新的话题。主要介绍了幼龄反刍动物瘤胃上皮形态及功能的发育以及丁酸调控幼龄反刍动物瘤胃上皮发育的研究进展。     

The intraruminal papillation gradient in wild ruminants of different feeding types: Implications for rumen physiology

Browsing and grazing ruminants are thought to differ in the degree their rumen contents are stratified—which may be due to different characteristics of their respective forages, to particular adaptations of the animals, or both. However, this stratification is difficult to measure in live animals. The papillation of the rumen has been suggested as an anatomical proxy for stratification—with even papillation indicating homogenous contents, and uneven papillation (with few and small dorsal and ventral papillae, and prominent papillae in the atrium ruminis) stratified contents. Using the surface enlargement factor (SEF, indicating how basal mucosa surface is increased by papillae) of over 55 ruminant species, we demonstrate that differences between the SEF_dorsal or SEF_ventral and the SEF_atrium are significantly related to the percentage of grass in the natural diet. The more a species is adapted to grass, the more distinct this difference, with extreme grazers having unpapillated dorsal and ventral mucosa. The relative SEF_dorsal as anatomical proxy for stratification, and the difference in particle and fluid retention in the rumen as physiological proxy for stratification, are highly correlated in species (n = 9) for which both kind of data are available. The results support the concept that the stratification of rumen contents varies among ruminants, with more homogenous contents in the more browsing and more stratified contents in the more grazing species. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Effect of feeding dehydrated and ensiled tanniferous sainfoin (Onobrychis viciifolia) on nitrogen and mineral digestion and metabolism of lambs

The effects of tanniferous sainfoin on digestion and metabolism have been investigated in 12 lambs in an incomplete cross-over design (n = 6). Effects of condensed tannins (CT) were evaluated by comparing dehydrated and ensiled sainfoin treated with and without polyethylene glycol (PEG). Dehydrated and ensiled grass-clover mixtures served as controls. The lambs were fed the treatment diets, including a mineral supplement, for 21 d. During the last 7 d excreta, rumen fluid and blood were sampled. The CT of sainfoin decreased rumen fluid ammonia concentration (p < 0.001) and increased the plasma concentration mainly of essential amino acids (p < 0.001). Body retention of phosphorus, calcium and magnesium was lower with sainfoin compared to PEG-treated sainfoin (p < 0.05). Sainfoin without PEG resulted in lower digestibilities of organic matter and neutral detergent fibre than sainfoin with PEG and the grass-clover mixture (p < 0.001). Ensiling of sainfoin led to the lowest N-retention. In conclusion, the reduction in ruminal ammonia and urine-N losses by sainfoin CT did not improve N-retention.     

Relationship between efficiency of nitrogen utilization and isotopic nitrogen fractionation in dairy cows: contribution of digestion v. metabolism?

Animal tissues are naturally ¹⁵N enriched relative to their diet and the extent of this difference (Δ¹⁵N_animal-diet) has been correlated to the efficiency of N assimilation in different species. The rationale is that transamination and deamination enzymes, involved in amino acid metabolism are likely to preferentially convert amino groups containing ¹⁴N over ¹⁵N. However, in ruminants the contribution of rumen bacterial metabolism relative to animal tissues metabolism to naturally enrich animal proteins in terms of ¹⁵N has been not assessed yet. The objective of this study was to assess the impact of rumen and digestion processes on the relationship between Δ¹⁵N_animal-diet and efficiency of N utilization for milk protein yield (milk N efficiency (MNE); milk N yield/N intake) as well as the relationship between the ¹⁵N natural abundance of rumen bacteria and the efficiency of N use at the rumen level. Solid- and liquid-associated rumen bacteria, duodenal digesta, feces and plasma proteins were obtained (n=16) from four lactating Holstein cows fed four different diets formulated at two metabolizable protein supplies (80% v. 110% of protein requirements) crossed by two different dietary energy source (diets rich in starch v. fiber). We measured the isotopic N fractionation between animal and diet (Δ¹⁵N_animal-diet) in these different body pools. The Δ¹⁵N_animal-diet was negatively correlated with MNE when measured in solid-associated rumen bacteria, duodenal digesta, feces and plasma proteins, with the strongest correlation found for the latter. However, our results showed a very weak ¹⁵N enrichment of duodenal digesta (Δ¹⁵N_{duodenal digesta-diet} mean value=0.42) compared with that observed in plasma proteins (Δ¹⁵N_{plasma protein-diet} mean value=2.41). These data support the idea that most of the isotopic N fractionation observed in ruminant proteins (Δ¹⁵N_{plasma protein-diet}) has a metabolic origin with very little direct impact of the overall digestion process on the existing relationship between Δ¹⁵N_{plasma protein-diet} and MNE. The ¹⁵N natural abundance of rumen bacteria was not related to either rumen N efficiency (microbial N/available N) or digestive N efficiency (metabolizable protein supply/CP intake), but showing a modest positive correlation with rumen ammonia concentration. When using diets not exceeding recommended protein levels, the contribution of rumen bacteria and digestion to the isotopic N fractionation between animal proteins and diet is low. In our conditions, most of the isotopic N fractionation (Δ¹⁵N_{plasma protein-diet}) could have a metabolic origin, but more studies are warranted to confirm this point with different diets and approaches.     

Effects and modes of action of live yeasts in the rumen

Live 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. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.