How can cows derive nutrients from cellulose

However, Methanosarcina have rarely been isolated from the rumen Beijer, ; Rowe et al. Although members of the domain Archaea do possess the nitrate reductase gene Cabello et al. The genome of Mbb. Scheme of hydrogenotrophic and methylotrophic methanogenesis.

Adapted from Thauer et al. MF, methanofuran; MPT, tetrahydromethanopterin. How do the archaea respond to nitrate and products of its reduction such as nitrite? Community analysis of ruminal digesta from cattle or other ruminants receiving nitrate has so far been restricted to fairly broad characterization by ribosomal intergenic spacer analysis Lin et al.

Both of the latter take advantage of developments in rapid, accurate DNA sequencing. Metagenomic analysis enables the full gene profile that separates these phenotypes to be elucidated Wallace et al. Clearly this is a major area for development in our quest to understand the effects of nitrate on methanogenesis.

Available evidence from qPCR of archaeal 16S rRNA gene abundances suggests that archaeal abundance declined almost fold in goats receiving nitrate Asanuma et al. The bacterial community of the rumen is very much more complex than that of the archaea. Although several thousand bacterial species may be present Fouts et al.

Despite the large number of species, the predominant bacteria form only a narrow subset of the domain Bacteria, comprising mainly Bacteroidetes and Firmicutes, with smaller numbers of Proteobacteria and other phyla Kim et al.

Bacteria do not carry out methanogenesis, but are involved in the degradation of plant materials, which provides the substrates for methanogenesis by archaea, principally hydrogen.

The main hydrogen producers are considered to be Firmicutes, particularly Ruminococcus sp. Stewart et al. Although the vast majority of the bacterial community are strict anaerobes, many possess electron transport chains Russell and Wallace, that can potentially be linked to nitrate reductase activity. Bacteria are generally considered to be primarily responsible for the reduction of nitrate and nitrite in the mixed ruminal community of adapted animals Leng, ; Lin et al.

Allison and Reddy made presumptive identification of Selenomonas sp. Large Gram-positive rods formed another predominant group, but their identity is uncertain. Iwamoto et al. They also confirmed that Selenomonas ruminantium was active in nitrate and nitrite reduction, but only ssp. All three species reduced nitrate in pure culture Iwamoto et al. The species mainly responsible for nitrate reduction changed with diet in the goat study of Asanuma et al.

Numbers of W. Asanuma et al. The concentration of intracellular nitrate reductase-mRNA was higher when S. Transcription of the nitrate reductase gene was also suggested to be enhanced in response to a deficiency of energy and electron supply. Mannheimia succiniciproducens , V. Nitrate supplementation increased the percentage of C. Yoshii et al. Other studies in which increased numbers of nitrate-reducing bacteria or nitrate reductase activity were observed did not identify the specific bacterial species Lin et al.

Thus, most evidence points to a very significant role for S. Clearly, information on the ruminal bacteria that reduce nitrate and nitrite is very sparse and indeed is largely based on textbook properties Stewart et al. Given the large number of species, including those that have not yet been cultivated Kenters et al. This is a major gap in our knowledge that impacts upon our understanding of how nitrate might be used to mitigate methane emissions.

The other major issue is the species of ruminal bacteria that might be sensitive to the toxic effects of nitrate. Marais et al. As with the archaea, community analysis of ruminal digesta from cattle or other ruminants receiving nitrate has so far been restricted to fairly broad characterization by ribosomal intergenic spacer analysis Lin et al.

Cellulolysis by bacteria, in particular, is absolutely fundamental to optimally productive rumen fermentation. Therefore, it is important that we understand how the cellulolytic population responds to dietary nitrate, particularly as some of the key species seem to be sensitive to nitrate and its more reduced intermediates.

Once again, this is a major gap in our understanding of nitrate as a feed additive to lower methane emissions. Over ciliate species have been described from various ruminants Williams and Coleman, They can be divided into two orders in the class Trichostomatida, Vestibuliferida, and Entodiniomorphida Small and Lynn, Similar species inhabit the digestive tract of various vertebrates, and almost all the members of Entodiniomorphida inhabit the rumen or large intestine of large herbivorous mammals.

Metabolically, the protozoa are rather similar to bacteria in the substrates used and products formed Williams and Coleman, However, they differ in that they possess a cytoplasmic organelle, the hydrogenosome, which has evolved from mitochondria Embley et al.

As its name implies, the hydrogenosome forms hydrogen, and it contains electron transport carriers that might conceivably relay electrons during nitrate reduction. The ciliate protozoa, because they produce abundant amounts of hydrogen, form a central component of substrate supply for methanogenesis. This is reflected in the intimate association between ciliates and archaea.

Archaea can be seen to colonize the outer surface of protozoa Vogels et al. How important are protozoa in nitrate metabolism in the mixed ruminal community?

Rumen protozoa were reported to accelerate nitrate reduction when co-cultured with bacteria Yoshida et al. The protozoal fraction had greater ability for nitrate and nitrite reduction than the bacterial fraction, and inhibition of methane production by nitrate was greatest in the protozoal fraction.

Similar results were obtained by Lin et al. Both these studies confirm that protozoa play an integral part in nitrate metabolism, and indeed may be vital for the safe use of nitrate because of their activity in reducing nitrite. Furthermore, there may be a symbiotic relationship between protozoa and associated bacteria, whereby both reduce nitrate and the protozoa mainly reduce nitrite Lin et al. How does the ciliate protozoal community respond to nitrate and its reduction products?

Several papers suggest a negative effect. Sar et al. In contrast, van Zijderveld et al. Given that protozoa may have a crucial role in the safe use of nitrate as a feed additive, we need to know much more about protozoal metabolism of nitrate and nitrite and their response to dietary nitrate.

The other main category of eukaryotic microorganism in the rumen is the anaerobic fungi. Perhaps 20 different species are present, all belonging to the phylum Neocallimastigomycota Orpin and Joblin, ; Gruninger et al. Indeed variations in ITS regions of ribosomal RNA genes rather than the hypervariable regions within the genes themselves are used to distinguish the different taxa Fliegerova et al. Their main function is plant fiber breakdown, indeed they are the only rumen cellulolytic species that physically as well as enzymically degrade plant fiber Ho et al.

Thus they are particularly valuable to animals consuming poorer quality forages Gordon and Phillips, The fungi, as do the protozoa, possess hydrogenosomes, and they are significant producers of hydrogen Marvin-Sikkema et al. Lin et al. The main concern if fungi are generally suppressed by nitrate would be reductions in fiber digestion, particularly of the more recalcitrant plant cell walls. When Jones observed decreased methane production in response to nitrate, the effect was interpreted as a possible consequence of nitrate raising the redox potential, E h , of the medium; however, most subsequent studies have considered nitrate to be an alternative hydrogen sink to methane production Figure 3.

Methane is produced in the rumen predominantly by the hydrogenotrophic route, whereby hydrogen and carbon dioxide are the principal substrates Hungate, Several compounds or their metabolites that could act as alternative hydrogen sinks to methane have been identified, including sulfate and propionate precursors like fumarate and acrylate Newbold et al. In contrast, Iwamoto et al. The addition of nitrate is intended to provide an alternative hydrogen sink, in other words a competition for available hydrogen.

The alternative hydrogen sink hypothesis is used most often to explain how nitrate lowers methane production in anaerobic ecosystems. There may be other mechanisms involved as well, however.

N-oxide intermediates, such as nitrite and nitrous oxide, may suppress methanogenesis directly Ungerfeld, As methanogens do not contain menaquinone or appreciable amounts of b- or c- cytochromes, and obtain energy exclusively by electron transport-linked phosphorylation Thauer et al.

Other possible impacts may arise from the toxicity of nitrate or its products to ruminal microorganisms, altering their metabolism, particularly hydrogen production. If the sensitive species are hydrogen producers, methane production may be decreased by the lower supply of hydrogen. In support of such a mechanism, Marais et al. However, other, non-hydrogen producing bacteria were equally sensitive to nitrite.

Clarification of the relative sensitivity of different microbial species to the products of nitrate reduction and their ability to adapt their metabolism would greatly enhance our understanding of the mechanism of action of nitrate in inhibiting ruminal methanogenesis. Although hydrogen and carbon dioxide are the predominant substrates for methanogenesis, others are present too.

The relatively recently described group of Thermoplasmata Poulsen et al. Trimethylamine is formed from trimethylglycine and choline.

The former is particularly abundant in beet pulp, which was a component of the diet used by Poulsen et al. It would be instructive to determine if nitrate or its reduction products influence methanogenesis from methylamines. If the main mode of action is disruption of the redox status of key cellular electron carriers, presumably methanogenesis from methylamines would be affected as for the hydrogenotrophic methanogens.

Thus, although the hydrogen sink is usually considered to be the mechanism whereby nitrate inhibits ruminal methanogenesis, future studies to develop nitrate as a feed additive must take account of the relative importance of each of the other potential mechanisms, in order to be sure that the perceived mechanism, a possible target for manipulation, is correct. The reason for finding out what is the real mechanism is quite simple. Four moles of hydrogen are consumed to produce one mole of methane.

At best, assuming completion of the assimilatory pathway, four moles of hydrogen will be used to convert nitrate to ammonia. This reduction might be consistent with either mechanism. More studies need to be done to determine if lower amounts of nitrate may be adequate to have a significant inhibitory effect on methanogenesis. The thermodynamically favorable reduction of nitrate preferentially directs hydrogen away from methanogenesis, but could also draw hydrogen away from other processes such as propionogenesis van Zijderveld et al.

In terms of the formation of the main fermentation products, the VFAs are key nutrients for the host animal. Farra and Satter observed a shift in the VFA profile from propionate to acetate when diets high in nitrate were fed to dairy cows. The butyrate concentration was also significantly lowered. The same phenomenon has been observed in many other studies Lee and Beauchemin, , although butyrate concentration increased when nitrate was added to the diet in the beef study of Troy et al.

The explanation for this change in propionate is that nitrate is an alternative electron acceptor to endogenous fumarate in many propionate-producing bacteria see below. Thus, nitrite is formed rather than succinate, which would then be decarboxylated to propionate, and the balance of VFA moves away from propionate. Following absorption, propionate is the only VFA that is glucogenic, so a lower molar propionate production rate would generally be considered to be detrimental to nutrition Leng et al.

The rumen evolved as an organ whereby the passage through the gut of plant fiber is retarded, enabling fibrolytic microorganisms more time to degrade cellulose, hemicellulose, and pectin polymers. Fiber breakdown is key to the efficient utilization of forage feedstuffs.

However, a major survey concluded that, if adaptation was conducted carefully when nitrate was introduced, no detrimental effects on feed intake or weight gain would occur Lee and Beauchemin, In monitoring animal health by measuring blood methemoglobin, mean values are normally reported.

However, the key indicator is not the mean but the extreme response. The death of one or two animals is a much greater loss than a depression in average performance. Extreme responders do exist even when animals are gradually adapted to feeding nitrate.

Cockrum et al. Similarly, when Duthie et al. Duthie et al. Individual animals also vary in the extent of methane reduction when fed nitrate; Troy et al. Understanding individual animal responses to nitrate, including both animal and microbial components, is necessary to improve safety. It is increasingly clear there is a complex interaction between the rumen microbiome, diet, and host animal and that host genotype influences the rumen microbiome King et al.

For methemoglobin, specific animal factors that influence concentrations include rates of feed consumption, nitrite absorption from the rumen, re-oxidation of nitrite to nitrate within animal tissues, oxidation of methemoglobin to hemoglobin and recycling of nitrate to the rumen. As indicated in Figure 4 , information is sparse on many of these factors and to improve safety when feeding nitrate, the critical factors require identification.

For example, although nitrate can be recycled to the rumen Leng, , it is not known if nitrate is concentrated into saliva from plasma as in humans Cockburn et al. Careful adaptation of ruminants to nitrate-containing diets may not only allow the rumen microbiome to adapt but also the host animal. Godwin et al. As inorganic phosphate also increases erythrocyte methemoglobin reductase activity, ensuring adequate dietary phosphorus in nitrate-fed animals may improve clearance of blood methemoglobin.

The animal factor most amenable to manipulation is rate of feed intake. Conditions which encourage rapid feed consumption such as restricted vs. Practical feeding strategies should avoid situations that encourage rapid feed consumption. Documented processes are show as solid arrows whilst those inferred are shown as broken arrows.

The type of diet offered may influence rates of rumen nitrate and nitrite reduction. Tillman et al. As nitrate reductase is a molybdenum pterin cofactor-containing enzyme Magalon et al. In agreement, blood methemoglobin Troy et al. Because of the greater bulk of high forage diets, slower feeding rates on these diets compared to high concentrate diets, together with higher rumen pH, may be important in avoiding methemoglobin accumulation.

Gradual introduction of nitrate to the diet is intended to decrease nitrite accumulation through enhancement of the kinetics of nitrite reduction to ammonia and hence toxic methemoglobin concentrations. Typically, dietary nitrate is increased step-wise over time. However, the total length of time to adapt animals to nitrate varies widely as does time on each step: 7—21 day total adaptation time and 2—7 day on each step van Zijderveld et al.

The optimal strategy based on comparison of responses does not seem to have been investigated. There is a need to establish minimum conditions for successful adaptation to nitrate-containing diets. During adaptation, the rates of nitrate and nitrite reduction Alaboudi and Jones, ; Lin et al.

However, the rate of nitrite reduction remains less than that of nitrate reduction, thus still favoring nitrite accumulation. The reason for successful adaptation may be that increased rates of nitrate and nitrite reduction increase net conversion of nitrate to less toxic compounds such as ammonia. To minimize risk to the animal, the rate of nitrite reduction should be greater than that of nitrate.

Increased nitrite reduction may be achieved nutritionally, by manipulating the rumen microbiome or by introducing specific novel microorganisms. Electron donors, such as formate and lactate, when included together with nitrate in vitro , increased nitrite reduction and reduced methane production to a greater extent than for nitrate alone Iwamoto et al.

However, the fermentation of diets including cellulose, hemicellulose, and starch which are normally fed to productive livestock will produce lactate and formate as intermediates or end-products of fermentation. In vivo , therefore, any benefits in increased nitrate reduction resulting from increased lactate and formate supply by alternative nutritional strategies are likely to be limited. Adding sulfate to nitrate-containing diets might lower nitrite concentrations, as sulfate-reducing bacteria, specifically Desulfovibrio species, are able to reduce nitrite in vitro but not nitrate Mitchell et al.

Methane production was also decreased both in vitro Patra and Yu, ; Wu et al. Methemoglobin was not detected when sulfate was added to a nitrate-containing diet van Zijderveld et al. In addition, high dietary sulfur intakes can be toxic inducing polioencephalomalacia and therefore caution must be exercised in the use of dietary sulfate Drewnoski et al.

Introducing microorganisms containing nitrite reductase into the rumen may increase nitrite reduction. However wild type Escherichia coli but not a genetically modified strain with enhanced nitrite reductase activity lowered rumen nitrite and blood methemoglobin concentrations Sar et al.

Sakthivel et al. In general, a lack of information about the members of the rumen microbiome responsible for nitrate and nitrite reduction and how the microbiome changes when nitrate is included in the diet restricts attempts to manipulate the microbiome to enhance nitrate and nitrite reduction.

For example, protozoa may be responsible for a substantial proportion of nitrate reduction in the rumen Lin et al. However, this study used ruminal fluid from animals not adapted to nitrate. Recently, Asanuma et al. It will be vital to analyze the ruminal microbiome in detail in order to understand the microbiological basis for different responses. Although the energetics of reducing nitrate to ammonia via the dissimilatory pathway are more favorable than converting hydrogen and carbon dioxide to methane, in vivo decreases in methane emissions are less than would be expected when stoichiometrically 1 mol 62 g of nitrate, fully reduced to ammonia in the rumen, should lower methane formation by 1 mol 16 g.

Several explanations are possible for lower than expected decreases in methane emissions. Lee et al. Second, nitrite may be metabolized to end-products other than ammonia such as nitrogen and nitrous oxide gasses. Since the environmental objective of feeding nitrate is to lower GHG emissions, then production of nitrous oxide, which has a global warming potential more than 10 times greater than methane, is not desirable.

Clearly nitrous oxide emissions must be accounted for when assessing the benefits of feeding nitrate. Thirdly, feeding nitrate might, by lowering feedback inhibition, increase total hydrogen production and thus the effect of nitrate on methane production would be less than predicted. Research should be directed to maximizing decreases in methane emissions for a given intake of nitrate. It is also important to ensure maximum conversion of nitrate to ammonia, the primary substrate for microbial protein synthesis so that nitrate can replace dietary rumen degradable protein sources analogous to the use of urea.

Nitrate should not be added to diets already adequate in rumen-degradable nitrogen supply as excretion of excess nitrogen can lead to increased nitrous oxide production from soil after manure application. In many experiments, nitrate intakes have been balanced by inclusion of urea in control diets Lee and Beauchemin, ; animal performance on the nitrate and urea-containing control diets have been similar.

However, there is little evidence concerning animal performance on nitrate-containing diets compared with control diets where nitrate replaces protein rather than urea.

There is a need for experimental designs which include a negative control treatment for dietary rumen degradable protein so that nitrate and urea supplementation can be compared. If conditions for feeding nitrate which achieve both maximum conversion of nitrate to ammonia and lowering of methane production limit the amount of nitrate that can be fed, then an alternative approach is to use nitrate in combination with other strategies known to lower methane emissions.

Combining strategies for lowering methane emissions with different mechanisms has scarcely been investigated. Addition of nitrate and fumarate did not affect intake, nutrient utilization, microbial protein supply, and blood profile Pal et al. Patra and Yu in an in vitro study, found that combining inhibitors of methane production with complementary mechanisms at low doses could be more effective and practical in mitigating methane emissions from ruminants without impairing feed digestion.

Combination of saponins and nitrate may be such a practical strategy. When sulfate and nitrate were fed to sheep van Zijderveld et al. Of course, there is a need for long-term performance experiments with large numbers of animals to better assess persistency of single- or combination-strategy approaches to methane mitigation on feed intake, performance, meat, and milk characteristics.

The possible consequences of a successful outcome to current ruminant methane research have prompted much discussion and some experimental and data analysis. They participate in vital pathways such as fatty acid synthesis and gluconeogenesis. Primarily rumen is hosted by various microorganisms that assist the host organism in the digestion of complex polysaccharides of the dairy cow.

The infants derive basal flora from the environment, feed consumed, partners, etc. The first floor of the rumen of neonates is colonized by enterococcus and streptococcus species which transform the gut environment to anaerobic This helps in the recruitment of strict anaerobes in the gut to maintain the anaerobic ambience.

Facultative anaerobes and aerobes are present in very less quantities, approximately fold lesser in comparison with anaerobic organisms. The digestive capability of the dairy cow is directly proportional to the existing rumen microflora activity. Most of the organisms present in the rumen are non-culturable, whereas the culturable biota were studied in all aspects 34 , The constitution of gut microbiota varies with the host, indicating a solid environmental-driven specificity of the host.

The microbial composition of the feces in twins was more similar than in siblings This implies the involvement of host genetics in deciding the individual gut microflora. Individuals also vary in fungal and archaeal compositions. The choice and development of gut microflora hence is a collaborative play of host genetics as well as environment.

It is an ardent fact that the physiology of the individual has a strong relationship with gut microbial development.

Apart from this, microflora differs from section to section in gut regions. Strict segregation of microflora between digestive and epithelium starts in the early stages of the life of a calf.

The methanogenic composition also differs down the gastrointestinal tract. In neonatal calves the phylum Bacteroides is predominant, whereas in adult animals the phyla Prevotella and Bacteroidetes are abundant. Studies indicate that the microflora of day-old calves has Prevotella Methanogens and cellulolytic members were observed at 3—4 days of age, and this population is similar to that of matured mammals. Cellulolytic flora is present in 1-day individuals, indicating their importance in the ruminant system.

Surprisingly, the rumen microflora of day-old calves harbors more profuse yet ephemeral microorganisms in comparison with adult organisms. Metagenomic studies indicate that archaea 0. Bacteria occupies the major portion of gut microflora, and their presence is crucial for the health of the dairy cow. They aid in the fermentation and degradation of plant polymers by the secretion of various enzymes 37 , The communal interaction of various bacteria enables the breakdown of the ingested fiber.

The identification of these bacteria and their unique functionality has become the focal point of many studies. With the advancement of next generation sequencing technology, microbiological techniques, culture free approaches, and genetic engineering it has become easier to step forward in studying the role of commensal flora in host metabolism. Gene sequencing helped to identify and classify bacteria based on 16 s rRNA and physicochemical properties.

The predominant microflora in the rumen are Proteobacteria, Bacteroidetes , and Firmicutes. The coordinated metabolism of the microflora in which the metabolic product of one organism acts as a substrate for the other allows the sequential digestion of plant polymers 39 , The bacterial consortium is highly complex, and hence most of the bacteria are uncultured. The flora which are dominant and have a specific role in the metabolism are covered here so far.

The cell wall of a plant is comprised of a hemicellulose matrix with embedded cellulose fibers in it. The initial degradation of this matrix is carried out by a particular taxon of bacteria that secretes cellulolytic enzymes 24 , In general, bacteria contribute to most of the xylanase and endoglucanase activities in the rumen. The first order cellulolytic bacteria includes Ruminococcus flavefaciens, Ruminococcus albus , and Fibrobacter succinogenes. Also, Butyrivibrio fibrisolvens is present in a lesser extent in comparison with the above said organisms.

Apart from these, other uncultured bacteria can also act upon the substrate to degrade cellulose fibers. Some organisms like Cellulosilyticum ruminicola H1 , from the rumen of Yak, also have the capability to produce lignocellulolytic enzymes. On the other hand, coculturing of some organisms implicated negative interaction and decreased enzyme efficiency This inhibition is found to be an effect of the bacteriocins secreted as a part of the defense mechanism and competition for the substrate For instance, R.

Non-cellulolytic bacteria also secrete bacteriocins and are supposed to be tough competitors for different substrates in a rumen environment 45 , The end products of cellulolytic bacterial interaction act as substrates for different microflora that start further degradation of such compounds. Other important polymers, such as starch, are hydrolyzed by Selenomonas ruminantium, Succinomonas amylolytica, Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminobacter amylophilus , and Prevotella species, whereas pectin is degraded by Lachnospira multiparus and Succinovibro dextrinosolvens.

Besides, the constitution of bacteria changes with the type of feed consumed by the host 5 , 47 , Animal feeding differs in various places.

High fiber feed is rich in cellulose whereas high grain feed is packed with starchy material. This influences the type of bacteria required to digest the material consumed and has a strong impact on microflora constitution in the gut environment 49 , Sugar and starch fermenters constitute most of the rumen bacteria. Maximum energy is extracted from the plant polysaccharides as the end products of bacterial fermentation serve as substrate to many other organisms.

Megasphaera elsdenii acts upon lactate end product of bacterial fermentation and Veillonella alcalescens utilizes succinate, acetate, and hydrogen 51 , Recent metagenomic studies on gut microflora of various mammalian species revealed that in ruminant and herbivore microflora the anabolic pathways for the synthesis of amino acids AAs are more prevalent in comparison to carnivores.

This is because the diet of a carnivore would be rich in protein, and therefore the constitution of gut microbiota is chosen to be more proteolytic. In the point of herbivores, the diet is fiber rich, and carbohydrate is the core source of energy Hence in the microbiota of rumen, the AA synthesis pathways are commonly seen.

Indeed, a certain cellulolytic activity some organisms also exhibit potent proteolytic activity, such as B. This activity is considered to be crucial as the rumen environment has lesser protein and ammonia that act as nitrogen sources for AA and protein synthesis Other classes of bacteria include sulfate-reducing bacteria that assist in the reduction of sulfate to H 2 S.

In addition, it has to be noted that the rumen microbiota is fine-tuned depending upon the dietary changes to assist degradation and fermentation of various complex compounds. They also have communal relations with each other and with the host to ensure their survival as well as maximum energy production.

They also play a role in supplying VFAs and proteins to the host organism. Disturbances in concentrations of microbiota sometimes have a heavy impact on the host system and may lead to diseases. Different types of bacteria are listed in Table 2. Major archaea members of rumen microbiota are listed in Table 3. Metagenomic studies and 16 s rRNA sequencing analyses revealed the presence of archaea in the rumen environment.

Studies revealed that about 3. Most of the ruminal methane is produced via the reduction of CO 2 rather than dissimilating acetate. The process of CO 2 reduction requires electrons which come from various sources, including methylamine, methanol, formate, and hydrogen produced as metabolic intermediates 72 , Archaea are clustered under Euryarchaeota and are classified as Methanomicrobiales, Methanosarcinales, Methanococcales, Methanobacteriales , and Methanopyrales.

Most of the ruminant methanogens fall under one of the three categories identified. Apart from this, another set of uncultured ruminal archaea were categorized under rumen cluster C RCC. A study on the ruminal archaea community of red deer, cattle, and sheep disclosed the fact that their composition is maintained throughout different species.

They are more conserved when compared to the bacterial members. The dominant archaea species stood same in all the rumens. Species belonging to Methanobrevibacter is found to be dominant in rumen. About Methane production by various archaea is mediated by cytochrome in few methanogens, whereas alternative complexes mediate this process in some methanogens.

The genus Methanosarcinales comprises of methanogens and has the capability to grow on a wide range of substrates. Hydrogen concentration in the environment plays a crucial role in the production of methane. Cytochrome-based methanogens have higher growth yields when compared with non-cytochromic methanogens.

Non-cytochromic methanogens need lesser hydrogen concentration to produce methane whereas cytochromic methanogens need about fold higher concentrations of hydrogen for the optimal growth. This is the reason for the presence of non-cytochromic methanogens in higher concentrations in the rumen.

Hydrogen utilization by methanogens is crucial as it decreases the pressure, allowing the conversion of endergonic metabolic reactions to exergonic reactions. This makes bacterial fermentation energetically favorable Hydrogen consumption by methanogens stands as a good example of the symbiotic relationship between methanogenic and cellulolytic bacteria, wherein the hydrogen produced by the latter is consumed by the former for its survival.

Coculturing of rumen methanogens and ruminal fungus has a heavy influence on cellulolytic and fermentation activities. Hydrogen transfer among methanogens and other microflora in rumen is best described by coculturing methanogens with protozoa. Even though archaea and bacteria fall prey to protozoa, methanogens get habituated inside and help in the generation of energy by consuming the hydrogen produced during the metabolism 74 — Hydrogen consumption by methanogens forms the root of symbiosis with other microbiota in the rumen for maximal energy production 77 — The commensal interactions of methanogens with protozoa and other rumen microbiota facilitate the complete degradation of complex plant polymers.

The methane production is directly related to the amount of fodder and hemicellulose degradation 80 — The commensal interaction of methanogens with other microbiota in the rumen enhances energy production to a maximum extent.

But the gas production has a hinderance effect on the overall energy harvested from the ingested feed. Protozoa are unicellular organisms bound by pellicle or cuticle in the rumen. They are the simplest forms of eukaryotes found in the universe Table 3. Most of the protozoa are parasitic as they feed on microorganisms, organic matter, and cell debris. Ciliates are more prevalent in ruminant gut in comparison with several flagellate species. Ciliates are subcategorized into Vestibuliferida and Entodiniomorphida with 25 genera.

Protozoa in the rumen have specialized functions tuned to survive in a rumen environment 83 , Most of the protozoa are anaerobic, but very few species are supposed to sequester oxygen. Oxygen sequestration from the environment is advantageous to the host as it maintains the anaerobic ambience of the reticulorumen.

This also helps in the survival of strict anaerobes and promotes the digestive degradation. Various complex carbohydrates viz. Degradation of fats, proteins, and carbohydrates is facilitated by direct engulfing The lignocellulosic digestion capacity by protozoa is presumed to be the result of lateral gene transfer from the bacteria they engulf Protozoa prey on selective species of bacteria, and the reason for feeding on particular bacteria is not clearly understood 87 — Ciliates play a crucial role in fermentation and plant fiber degradation.

The products obtained as a result of protozoan fermentation are found similar to that of bacteria. In contrast to bacteria, protozoa divide at a much slower rate 15—24 h. To overcome the washing out of protozoa before division, they tend to reside in the lower layers of the rumen. Many methanogens reside on the protozoan surface for H 2. Hydrogen gas is produced is used for the reduction of CO 2 to methane.

Protozoa are capable of engulfing and store more starch at once, which decreases acid production by lowering pH Protozoa holotrich produces pectin esterase, invertase, amylase, and polygalactouronase to degrade plant sugars and fibers. Protozoa also produce cellulolytic and hemicellulolytic bacteria in lower quantities compared with that of entodiniomorphids. Ciliates in the rumen secrete proteolytic enzymes, resulting in the production of AAs and ammonia.

The type of engulfed microbiota decides the nitrogen metabolism of the protozoa. Generation of nitrogenous compounds in turn influences the recycling of nitrogen. Rumen ciliates also influence ammonia as well as VFA production. The symbiosis of protozoa and rumen bacteria were investigated and showed that the presence of rumen protozoa effected the bacterial composition in rumen.

Absence of protozoa has a positive effect on the growth of cellulolytic and hemicellulolytic bacteria. No proper effect of protozoa on methane production is observed. Variations in the composition of digested material in both omasum and abomasum are observed in defaunated and faunated animals. It is an ardent fact that protozoa influence many processes in the metabolism of host 92 — Rumen is a repository of anaerobic fungi with an explicit capacity of lignocellulose degradation. They are deliberate members of plant fiber degradation.

Fungi also exhibit proteolytic activity. In the fungal structure, polycentric or monocentric thallus is observed, and the zoospores are polyflagellate or uniflagellate. Asexual life cycle of anaerobic fungi is mostly observed Most of the fungi are not present alone in the rumen of the animals but are vividly present along the digestive tract.

Fungal species were also isolated from the feces and saliva of the dairy cow. Domestic animals host Chytridiomycetes for assisting their digestion. But in the case of high grain diets, fungal population decreases.

Enzymes secreted by fungal cultures degrade lignin, hemicellulose, starch, and cellulose In addition, fungi are strict anaerobes, and hence carbohydrate fermentation is the sole source of energy production.

Fungi are devoid of cytochromes and mitochondria that are coplayers of oxidative phosphorylation. Despite that, they contain Hygrogenosomes that facilitate the generation of energy. Hydrogenosomes are mitochondrial derivatives that occurred during evolution, and they are not only confined to fungal genera. Various anaerobic eukaryotes and trichomonads are also found to contain this organelle. They also provide room for ATP production and pyruvate conversion.

Commensal interplay of fungi and bacteria is a well-studied concept. In vitro studies were carried out to understand the degradative dynamics of fungi when cocultured with cellulolytic bacteria. Cellulose degradation capacity of the fungi increases manifold with Megasphera elsdenii, Selenomonas ruminantium , and Viellonella alcalescens.

Xylan consumption is increased by coculturing Neocallimastix frontalis with cellulolytic bacteria like Selenomonas ruminantium, Prevotella ruminicola , and Succinivibrio dextrinosolvens Table 3. On the other hand, coculturing with Streptococcus brevis or Lachnospira sp. These bacteria release a polypeptide into the broth that has detrimental effects on cellulolytic activity of the fungus.

The fungal activity in the degradation of cellulosic materials is considered minimum than that of bacteria. This might be due to their larger doubling time, inhibition by bacteria, competition for substrates, and decreased retention. Nevertheless, they exhibit remarkable activity in the degradation of lignocellulosic material, as the rhizoids pervade the cell wall of plants and make it easily accessible by the rest of the rumen microbiota Bacteriophages are obligate parasites and play a crucial role in rumen microbiota.

Bacteriophages infect bacteria and lyse them after their replication Table 3. Through lysis, the overall bacterial population is maintained in the host digestive environment. Bacterial lysis releases bacterial proteins that act as precursors of AA synthesis Bacteriophages are found to vary with the organism, i. This may be used by the researchers to destroy a particular genus of microbes from the rumen environment.

Very little information is known about the bacteriophages infecting protozoans, methanogens, and archaea. It was identified that siphophages are capable of infecting methanogenic bacteria. The knowledge about the enzymatic profile and genetic makeup of rumen phages is limited and yet to be explored to manipulate the rumen environment Disturbances in the homogeneity of gastrointestinal microflora have severe effects on the digestive system and various organs. This disharmony in the communal relationship also causes various metabolic disorders, including bloat, ruminal acidosis, hypoglycemia, diarrhea, ulcers in gastrointestinal GI tract, and retivuloperitonitis Figure 1.

The rumen tympany, also called as bloat, is associated with a condition in which excess gas is accumulated in the rumen. This is observed in animals fed with higher quantities of grains or forages 99 , which can be categorized into free gas and frothy bloat. Esophagus obstructions external particles cloths and fruit material, etc.

Frothy bloat is the result of feed ingestion, which continuously produces froth that cannot be easily expelled from the stomach. Testing with a stomach tube helps in figuring the type of bloat. If the causative agents are physical obstructions, they have to be removed manually to ensure the gas expulsion. Frothy bloat contains both hydrophobic and hydrophilic properties. The foam is the result of partial digestion of polymeric compounds including, lipopolysaccharides, fatty acids, glycans, and glycolipids.

Presence of these partially digested compounds increases rumen viscosity and hinders gas removal. Gaseous distension exerts pressure on the nearby organs causing edema, pain, organ failure, and death. Several practices that are employed to treat free bloat and frothy bloat include using a stomach tube to remove gas and partially digested feed, anti-foaming agent administration, and the placement of fistula or cannula Apart from physical factors, the microbiota in the rumen also contribute to the development of gas.

Gas is generated as a result of methanogenic bacterial action upon various substrates. This methane, hydrogen, and CO 2 gases produced in excess when left unattended by downstream flora results in the accumulation of gas in the stomach. The hydrogen gas produced as a part of methanogen metabolism also has to be addressed. It is a well-known fact that the rumen environment is highly anaerobic.

But excess CO 2 can cause subtle changes in the rumen. It is nevertheless necessary to attend to the excess production of these gases to maintain ruminal microbial harmony. Hence to maintain the environment, probiotics can be used to replenish the rumen flora. Treated and high fiber feed also helps in relieving the stress caused by methanogenic bacteria Ruminal acidosis is caused by the consumption of more fermentable carbohydrate-rich feed material than grainy feeds , Molasses, sugar beets, potatoes, and cereal grains result in acidosis.

Fermentation of such compounds result in higher amounts of lactic acid production and hence pH of rumen is drastically reduced , Due to this, many gram-negative bacteria are destroyed releasing endotoxin into the rumen.

All these results in low pH, accumulation of fluid, disturbance of microbiota, and partial digestion. Low pH and acid production have destructive effects on the inner epithelium of the stomach causing ulcers as well as mucosal inflammation.

Drastic fall in pH also inhibits the cellulolytic bacteria but enhances propionate-producing bacteria in the rumen. Rumen microbiota alteration leads to improper metabolism which can cause liver dysfunction, lung-related diseases, and can also lead to death — Hypoglycemia is a disorder observed when the rate of glucose uptake is very less in comparison to the rate of utilization , Vitamin B 12 plays a key role in the synthesis of glucose from propionate, and its deficiency is also related to the occurrence of hypoglycemia.

In new-born calves and lambs in a cold environment, hypoglycemia leads to death. For this reason, an organism depends primarily on dietary carbohydrates for glucose rather than synthesis. Deficiency in glucose supply caused hypoglycemia in all the animals. On the other hand, hypoglycemia is also seen in animals whose diet is rich in inhibitors of fatty acid beta oxidation in the kidney and liver.

Required amounts of AAs, fatty acids, ambience, and vitamins have to be provided for treating hypoglycemia — The bolus moves back down the esophagus and into the rumen. Saliva produced during this process acts as a buffer, ensuring that the rumen pH remains fairly stable. The rumen has a capacity of approximately 40 gallons and is the primary site of digestion and absorption of nutrients. It is covered with finger-like papillae which increase the surface area and allow for greater absorptive capacity.

The rumen is home to a diverse microbial community made up of many species of bacteria, protozoa, and fungi. The relationship between the microorganisms and the cow is symbiotic, meaning it is beneficial to both. The wet, warm, anaerobic environment is a place that microorganisms can thrive, and the cow derives energy and protein to meet its nutrient requirements.

Microorganisms secrete enzymes with break down large pieces of fibrous material, specifically cellulose, and create volatile fatty acids or VFAs. Volatile fatty acids acetate, propionate, and butyrate are absorbed as an energy source for the cow.