Ruminants as a source of environmental pollutants

Ruminant production is beset by two major problems resulting from inefficiencies in ruminal fermentation: nitrogen and methane emission. It has been estimated that globally ruminal fermentation produces 86 million tonnes of methane a year⁽¹⁾. Anaerobic decomposition of manure from ruminants is estimated to release a further 8·5 million tonnes of methane a year⁽¹⁾. Methane is produced by methanogenic archaea during the final stages of the anaerobic fermentation of plant biomass in the rumen, a process which involves the integrated activities of a variety of different microbial taxa⁽Reference McAllister, Okine and Mathison^12, Reference Moss, Jouany and Newbold¹³⁾. Plant carbohydrates including starch, cell-wall polymers and water-soluble carbohydrate are hydrolysed to simple sugars and undergo microbial fermentation via pyruvate, resulting in the production of volatile fatty acids (principally acetate, propionate and butyrate), CO₂ and hydrogen as end-products. The latter two products are the primary substrates for ruminal methanogenic archaea. Although methane generation is considered to be an undesirable process, resulting in ruminal-based inefficiencies in the use of plant biomass, it provides an important hydrogen sink for the rumen ecosystem. The accumulation of hydrogen produced during microbial fermentation can be toxic to certain ruminal micro-organisms unless it is removed⁽Reference Martin, Morgavi and Doreau¹⁴⁾. Methane production can be calculated from the stoichiometry of the main volatile fatty acids formed during fermentation; acetate and butyrate production results in methane production as they are hydrogen-producing reactions, while propionate formation is a hydrogen-consuming reaction resulting in lowered methane production⁽Reference Moss, Jouany and Newbold¹³⁾. Animal-based approaches to methane production are discussed in Scollan et al.⁽Reference Martin, Morgavi and Doreau³⁾, but plant-based approaches are also appropriate as the ruminant diet has been shown to alter the molar proportions of volatile fatty acids, and consequently can reduce ruminal methane production and methane emission⁽Reference Moss, Jouany and Newbold^13–Reference Edwards, Kim, Scollan, Andrews and Andrews¹⁷⁾. Initially, efforts focused on dietary changes involved either adjustment of a major dietary component(s) or the use of supplements to manipulate ruminal fermentation patterns (and subsequently volatile fatty acids production) through the perturbation of microbial populations which contribute to and/or produce ruminal methane. These methods include the application of yeast (Saccharomyces cerevisiae), Aspergillus oryzae extracts, ionophores (i.e. monensin), organic acids (i.e. fumarate, malate), defaunation and using starch-enriched concentrate feeding systems⁽Reference Boadi, Benchaar and Chiquette¹⁶⁾. The potential of plant extracts containing essential oils to manipulate rumen function in order to improve nutrient use efficiency and environmental burden has been demonstrated⁽Reference Boadi, Benchaar and Chiquette^16, Reference Hart, Yáñez-Ruiz and Duval¹⁸⁾ and the provision of linseed fatty acid as a dietary supplement can also depress ruminal methanogenesis⁽Reference Martin, Rouel and Jouany¹⁹⁾. Future advances could involve the identification of other plants' secondary and primary products which can advantageously perturb rumen function. For example, at the whole forage level, an in vitro fermentation study has shown that perennial ryegrass cultivars differing in nutrient composition (for example, water-soluble carbohydrate) could be exploited to mitigate enteric methane emission in a pasture-based production system⁽Reference Lovett, McGilloway and Bortolozzo²⁰⁾.

Ruminants are also responsible for the direct deposition of N in urine and faeces as urea and ammonia. While in low input systems this does fulfil a role in supplying organic fertiliser, the resource is poorly captured. Soil micro-organisms catalyse conversion to nitrites (by Nitrosomonas spp.) and then to nitrates (by Nitrobacter spp.). Where nitrate run-off from land enters water sources, it is extremely toxic to aquatic life, and to infants if it enters the source of drinking water supply⁽Reference Subbarao, Ito and Sahrawat⁷⁾. Although poorly understood, the deposition of N onto land contributes to the soil-borne capacity for N₂O generation by supplying substrate, in addition to the direct emission of N₂O in manure⁽Reference Subbarao, Ito and Sahrawat⁷⁾. A solution to this problem would simply be to decrease stocking density, but this does not address the need to maintain production. A forage-specific effect on the production of atmospheric pollutants has been identified, with the application of slurries from sheep fed kale silage resulting in more N₂O production than after application of slurry from sheep fed lucerne or ryegrass silage⁽Reference Cardenas, Chadwick and Scholefield²¹⁾. Interestingly, although leguminous crops fix N (so do not require additional N fertiliser) leguminous pastures can also give rise to N leaching, thus permitting N₂O production⁽Reference Subbarao, Ito and Sahrawat⁷⁾. Realistic targets for environmental improvement include an increased use of precision agricultural techniques for fertiliser application and increasing N use efficiency (the ability of plants to take up and sequester N) of crop plants⁽Reference Subbarao, Ito and Sahrawat⁷⁾. A further opportunity is to exploit the natural presence of N-fixing endophytic bacteria on the roots of non-leguminous crop plants, including grasses⁽Reference Ferreira, Fernandes and Döbereiner^22–Reference Cocking²⁴⁾, the success of which will enable decreased requirement to add N fertilisers to land for forage production. These bacteria are apparently able to colonise (at low level compared with numbers present in the soil) not just the root surface, but also the apoplastic spaces (possibly xylem elements) to overcome oxygen toxicity of the nitrogenase enzyme⁽Reference Baldani, Caruso and Goi^23, Reference Cocking²⁴⁾. The potential of this has been demonstrated by yield improvements in wheat inoculated with Azospirillum ⁽Reference Boddey, Baldani and Baldani²⁵⁾, although this may be mediated by hormonal effects rather than by the modest increase in N fixation⁽Reference Andrews, Raven, Hodge, Andrews and Andrews²⁶⁾. An alternative strategy to address N pollution is to move to a more intensive livestock production system which removes waste to a point source⁽¹⁾, but does not fully address the problem of minimising N waste in the first place.

The rumen is a complex ecosystem which essentially functions in the conversion of plant material to microbial protein, driven by the fermentation of plant biomass by a community of bacteria, anaerobic fungi, ciliate protozoa and methanogenic archaea⁽Reference Hobson and Stewart²⁷⁾. The diversity of these microbial taxa is only now becoming truly acknowledged, due to the application of cultivation-independent techniques⁽Reference Edwards, McEwan and Travis^28–Reference Tuckwell, Nicholson and McSweeney³²⁾. However, it is with this complex microbial population that the source of ruminal inefficiencies in feed use lies. Forage supplies the nutrients required for microbial proliferation. However, it is believed that there is an imbalance in the relative delivery of protein and carbohydrate which favours the abundance of protein breakdown products during a period of celluloysis⁽Reference MacRae and Ulyatt^33, Reference MacRae, Theodorou, Abberton, Amdrews, Skøt L and Theodorou³⁴⁾. The hyper-ammonia-producing bacteria are key contributors to an excessive production of ammonia in the rumen. Hyper-ammonia-producing bacteria can derive energy from amino acids by deamination with a consequential generation of ammonia, which can be assimilated by the rumen bacteria and used for microbial protein synthesis. However, when carbohydrate is limiting, microbial growth rates are slow meaning that an excess of ammonia is produced relative to what can be re-assimilated by the microbial population to generate microbial protein. The excess ammonia cannot be used by the animal, and enters the blood stream for processing into urea before it is removed in the form of excreta. This extremely poor nutrient use efficiency underlies the low retention of dietary N by ruminants, which is in the order of just 20–30%⁽Reference MacRae and Ulyatt^33, Reference Dewhurst, Mitton and Offer³⁵⁾. Therefore an important target is to find a strategy that can improve N use efficiency in ruminant production systems.

Plant-based solutions

It is undisputed, and supported by an abundance of literature, that different forage species have different intrinsic qualities in terms of digestibility. Some of these qualities are easily understood, such as increasing protein content of the crop and transfer into ruminant product, albeit with associated N outputs. However, the mechanisms of action of other plant components are less understood. Many plant species contain a diverse collection of secondary products including condensed cyanogenic glycosides, glucosinolates, alkaloids, tannins, phenolics and saponins⁽Reference Bennett and Wallsgrove^36, Reference Kliebenstein³⁷⁾. Although originally believed to be undesirable by-products of primary metabolism, the roles of these secondary products are now thought to be mainly defensive (excluding interactions with the specialist feeders), for example, to prevent attack by insect herbivores⁽Reference Bennett and Wallsgrove^36–Reference Metlen, Aschehoug and Callaway³⁸⁾. Indeed, many of these chemicals can be induced by developmental stage, damage, pathogenesis and environment⁽Reference Bennett and Wallsgrove^36, Reference Metlen, Aschehoug and Callaway^38, Reference Bidart-Bouzat and Imeh-Nathaniel³⁹⁾. Furthermore, the presence of secondary metabolites in forage also has implications for large herbivores via the perturbation of rumen function. One example is the non-protein amino acid mimosine which is found in the tropical legume Leucaena leucocephala. Although mimosine is toxic, it has been found that ruminants can graze Leucaena following an adaptive change in the rumen microflora favouring proliferation of Synergistes jonesii which facilitates detoxification of the mimosine derivative⁽Reference Allison, Mayberry and McSweeney^40–Reference Wallace⁴²⁾. In terms of potential beneficial effects on ruminant digestion, two groups of compounds have attracted most attention; the condensed tannins (proanthocyanidins) and phenolics. Commonly used tanniferous forage species include the trefoils (Lotus species), sainfoin (Onobrychis viciifolia) and chicory (Cichorium intybus). Tannins have been shown to have a beneficial effect on ruminant production and parasite load when included in ruminant diets, typically at less than 6% DM⁽Reference Min, Barry and Attwood^43, Reference Hoste, Jackson and Athanasiadou⁴⁴⁾. However, tannins can also have negative effects on feed utilisation by decreasing digestibility and microbial growth in the rumen, especially when dietary protein is low⁽Reference Niederhorn and Baumont⁴⁵⁾. It is thought that the inclusion of tannins in feed improves nutrient use efficiency in the rumen by complexing with protein making it unavailable for degradation, thus increasing the pool of ‘bypass protein’ which is degraded post-ruminally in the abomasum⁽Reference Reed⁴⁶⁾. An alternative explanation is that the protein protective effect is mediated via the formation of tannin-protease complexes⁽Reference Aerts, Barry and McNabb⁴⁷⁾, thereby limiting degradative potential⁽Reference McMahon, McAllister and Berg^48, Reference Theodorou, Barahona, Kingston-Smith and Brooker⁴⁹⁾. Despite the complexity of the biosynthetic pathways involved in tannin synthesis, molecular approaches have shown that it is possible to alter the tannin content of forage⁽Reference Robbins, Paolocci and Hughes^50–Reference Dixon, Xie and Sharma⁵²⁾. Much remains to be understood about the fundamentals of the regulation of tannin biosynthesis in planta, especially regulation by myb and myc genes⁽Reference Marles, Ray and Gruber⁵³⁾. Some plant species, including the forage and model legume Medicago sativa, contain saponins. Saponins are amphipathic glycosides which can also complex with protein and, although the results are variable, forage saponins have been linked with decreased methane production (reviewed in Niederhorn and Baumont⁽Reference Niederhorn and Baumont⁴⁵⁾). Likewise, some plant species (e.g. Trifolium pratense, red clover) contain the enzyme polyphenol oxidase (PPO) which catalyses the oxidative complexing of plant phenolics with cellular proteins⁽Reference Parveen, Threadgill and Moorby⁵⁴⁾, thus decreasing the availability of forage protein for degradation⁽Reference Lee, Olmos Colmenero Jde and Winters^55, Reference Theodorou, Kingston-Smith and Winters⁵⁶⁾. Genetic control of PPO is complex, involving at least six genes⁽Reference Winters, Heywood and Farrar⁵⁷⁾ and mutants with lower than normal PPO activities have been identified in red clover⁽Reference Lee, Tweed and Minchin⁵⁸⁾. The protein protective benefits of PPO-containing forage is largely exploited in silage, where there is an adequate window in time for this oxygen-dependent enzyme to be catalytically active⁽Reference Lee, Olmos Colmenero Jde and Winters^55, Reference Lee, Tweed and Minchin⁵⁸⁾. However it is estimated that mastication by grazing cattle takes approximately 3 min (at approximately 55–60 chews per bolus and a chew rate of 15–18 per minute⁽Reference Abrahamse, Dijkstra and Vlaeminck⁵⁹⁾); so given that PPO is active within 5 min post-damage there is the potential for exploitation of this trait in the protection of protein before the down bolus is swallowed into the anaerobic rumen environment⁽Reference Lee, Tweed and Minchin⁵⁸⁾.

Recent research has indicated that plants can affect nutrient use efficiency in more subtle ways than merely on a compositional basis (reviewed in Kingston-Smith et al.⁽Reference Kingston-Smith, Davies and Edwards⁶⁰⁾). In a grazing situation, the ruminant ingests living plant material, which invokes endogenous stress responses upon exposure to the primary environmental stresses of the rumen: anoxia, elevated temperature and invasion by micro-organisms. These conditions are found in the field, for example during flooding, in high summer and during attack by pathogenic micro-organisms (e.g. mildew, rust etc). As plants cannot move away from adverse conditions they have evolved mechanisms to enable them to withstand periods of environmental stress, but these efforts are of limited duration in the rumen as the plant cells will eventually die⁽Reference Kingston-Smith, Bollard and Armstead⁶¹⁾. However, how the plant cells die and how they use their endogenous constituents to prevent death has an impact on nutrient availability for the ruminal micro-organisms. For instance, it has been demonstrated that exposing forage species to the ruminal conditions of heat and anoxia in the absence of a microbial inoculum is sufficient to promote rapid degradation of plant protein by the plant's own proteases⁽Reference Kingston-Smith, Bollard and Armstead^61–Reference Kingston-Smith, Merry and Leemans⁶³⁾. Stress-induced proteolysis in plants is a phenomenon that has been widely reported for biotic and abiotic stresses⁽Reference Simova-Stoilova, Demirevska and Petrova^64–Reference Howarth and Ougham⁶⁷⁾. Finding a way to inhibit this stress-induced proteolysis would therefore produce immediate benefits in terms of decreasing the relative availability of protein breakdown products in recently ingested forage⁽Reference Kingston-Smith and Thomas⁶⁸⁾. This is not a trivial concern, because proteolysis is highly regulated at transcriptional and post-translational levels. In addition, plant cells are highly compartmented which means that proteases could be present at relatively low abundance on a whole cell basis, but could be present at high concentrations within an organelle. Hence, low abundance proteases may have an apparently disproportionately large effect if co-localised with their protein substrate prior to or as a result of stress-induced activation. For example, the majority of protease activity is in vacuoles⁽Reference Feller and Dalling⁶⁹⁾, which can account for up to 70 % of the cell volume in mesophyll cells, but about 70% of the soluble protein of the plant cell is located in the chloroplasts, accounting for about 25% of the mesophyll cell volume⁽Reference Winter, Robinson and Heldt⁷⁰⁾. To date, there is little evidence that forage variety improvement comes about on the basis of selection for total protease activity in the standing crop⁽Reference Kingston-Smith, Bollard and Shaw^71, Reference Pichard, Tesser and Vives⁷²⁾. Research is required to identify control points in proteolysis as the plant cells die, and how amenable these are for manipulation for forage improvement.

It is clear that to fully understand rumen function consideration must be paid to all components of the ecosystem and how they interact, particularly the plant-microbe ‘interactome’. Microbial colonisation of newly ingested plant material by a complex microbial community is a pre-requisite for ruminal degradation of plant biomass⁽Reference McAllister, Bae and Jones^73–Reference Koike, Yoshitani and Kobayashi⁷⁵⁾. The dynamics of this process are poorly understood, although recent work has demonstrated that this process is similar to other microbial colonisation events in nature in that the biofilm phenotype prevails⁽Reference Mayorga, Huws and Kim⁷⁶⁾. Biofilms are defined as attached microbes which are enveloped in exopolymeric substances⁽Reference Costerton, Cheng and Geesey⁷⁷⁾. This ‘slimy matrix’ encases the colonising microbes, offering them protection from predation and concentrating their plant degradative enzymes (Fig. 1).

Fig. 1.

Scanning electron microscopy image of a biofilm community on a perennial ryegrass leaf following 2 h of incubation under ruminal conditions. Scale: 1 μm.

The initial attachment of ruminal bacteria and anaerobic fungi to plant material occurs within minutes of its ingestion⁽Reference Koike, Yoshitani and Kobayashi^75, Reference Edwards, Huws and Kim^78, Reference Edwards, Kingston-Smith and Jimenez⁷⁹⁾. Ciliate protozoa also rapidly colonise ingested plant material⁽Reference Mayorga, Huws and Kim⁸⁰⁾, although it is currently unclear whether this reflects attachment per se. Following initial colonisation events, the production of extracellular polymeric substances by the colonising bacteria ensues. The production of exopolymeric substances is quick and reaches its maxima early on in the colonisation process, although the exact timing of this is dependent on the plant material. For example, under ruminal conditions exopolymeric substances production on perennial ryegrass leaf and stem material is maximal after 1 h and 4 h of incubation respectively⁽Reference Mayorga, Huws and Kim⁷⁶⁾. This observation is not really surprising considering the heterogeneity of the plant as a microbial substrate⁽Reference Kingston-Smith, Bollard and Thomas⁸¹⁾.

As well as spatial differences associated with differences in plant structure, the organisation of plant metabolites within these structures also has an impact on the ability of microbes to utilise plant nutrients. The mosaic distribution of condensed tannin containing plant cells within Lotus corniculatus leaf tissue, for example, results in the preferential microbial degradation of non-tanniferous areas of leaf tissue (Fig. 2). As well as the structural heterogeneity of plant material, it appears that the nature of the plant may also influence the timing of the changes in the population composition of colonising microbes. Differences in the populations of rumen bacteria colonising Lotus corniculatus over time have been reported, with a change in the population composition evident after 8 h of ruminal incubation⁽Reference Edwards, Kim, Scollan, Andrews and Andrews¹⁷⁾. In contrast, with perennial ryegrass the temporal difference in the colonising bacterial population composition occurred after just 2 h of ruminal incubation⁽Reference Mayorga, Huws and Kim⁷⁶⁾. The mechanism underlying this temporal change in the bacterial populations colonising fresh plant material is currently unclear; however, this appears to be correlated with a decrease in exopolymeric substances quantity, and as such quorum sensing may play a role. With due consideration of how plants and microbes interact in the rumen, it will be possible to understand the mechanistic basis for many of the feed-specific effects identified by decades of research. Not only will this be of academic interest, but it will also allow a refinement of screening parameters, plus provide the potential for enhanced throughput by utilisation of molecular markers, to derive improved forage more rapidly.

Fig. 2.

Rumen microbial degradation of Lotus corniculatus. Leaf discs (5 mm) of L. corniculatus were anaerobically incubated in 10% (v/v) rumen fluid at 39°C for 24 h prior to being stained for the presence of condensed tannins (purple) with 4-dimethylaminocinnamaldehyde. Representative light microscopy images of leaf discs before (A) and after 24 h of incubation (B) show the preferential degradation of non-tanniferous areas of the leaf tissue (C).

Opportunities for selective breeding for impact mitigation

Selective breeding of forage has enabled significant improvements in livestock productivity to be made. Considerable progress has been made with respect to the yield, persistency and tolerance to abiotic and biotic stresses of the grasses and clovers used in temperate agriculture. In recent years, considerations of quality and of reducing deleterious environmental impacts associated with livestock production have been emphasised alongside these agronomic traits. Recently, improved grass varieties containing elevated contents of water soluble carbohydrates (sucrose, glucose, fructose and fructan) with an approximate average annual water soluble carbohydrate content of 24% (e.g. AberDart) have proved commercially successful. In research trials, feeding ruminants with grasses containing increased carbohydrate have resulted in significant improvements in the incorporation of N into milk and meat when used as grazed and conserved feed⁽Reference Miller, Moorby and Davies⁸²⁾. This effect could be due to a more favourable provision of protein breakdown products and readily fermentable carbohydrate than in control grass varieties with lower carbohydrate content. Of course, an improved retention of N inputs means decreased N outputs to the benefit of the local and wider environment. Future opportunities for improvement could arise from capitalising on the observations of species-specific differences in endogenous rates of induced proteolysis⁽Reference Kingston-Smith and Thomas^68, Reference Shaw⁸³⁾.

The results described above, together with efforts to understand the rumen interactome in terms of the microbial competition and the ecological niches required by methanogens, in addition to how their proliferation can be minimised, enable the identification of key traits required for the development of the next generation of improved forages⁽Reference Edwards, Kim, Scollan, Andrews and Andrews¹⁷⁾. The ability to incorporate new selection criteria in plant breeding programmes is greatly facilitated by the integration of molecular marker-based approaches into germplasm development, together with genomics and modelling⁽Reference Abberton, Marshall and Humphreys⁸⁴⁾. In grasses and white clover, interspecific hybridisation and introgression of genes from related species has an important role to play. Modern breeding programmes will increasingly be based on high throughput phenotyping techniques operating in parallel with next generation sequencing to ensure that enhanced understanding of gene function can be rapidly incorporated into variety development.