Ruminal Microbial Protein Synthesis

Ruminal Microbial Protein Synthesis

Ruminants are distinguished from the rest of the animals by the morpho-physiological adaptation of the upper part of their stomach. This peculiarity allows them to turn roughages and low-quality proteins, even non-protein nitrogen (NPN), into quality nutrients for themselves such as the microbial protein and the volatile fatty acids (Dewhurst et al. 2000). It is known that the acquisition of protein sources demands large amounts of money to guarantee the feeding in any productive system. The use of protein concentrates increases the production costs and increases the risks and dependencies of the system. However, the microbial protein in rumen provides more than a half of the amino acids absorbed by the ruminants and it may be between 70 and 100 % of the nitrogen (N) available in the lower parts of the digestive tract of animals fed roughages with low protein content (Ørskov 1992). The studies on ruminant nutrition are addressed to formulate diets that encourage microbial protein production in rumen by reducing the supplements with non-degradable protein sources. From the ecological standpoint, they increase the carbon fixation into the microbial biomass and reduce the carbon losses in the form of carbon dioxide and methane (Blümmel et al. 1997).

Nitrogen Metabolism in Ruminal Microbial Protein Synthesis

The rumen is an important evolutionary advantage because it allows the animal to consume roughages and NPN. Nevertheless, from the point of view of the use of true protein in the diet, it is inefficient (Wu and Papas 1997). In dairy cows fed concentrates with high protein content, the efficiency in the conversion of N from the feed in milk N ranges from 18 to 32 % (Dewhurst et al. 2000).
Whether in the protein molecular or in the non-protein forms, the N reaches the rumen in small amounts through the diet and the saliva by means of the rumen lining. The nitrogenous complexes from the diet include proteins of various molecular weights and tertiary structure, peptides, amino acids, amides, ammonia salts, nitrates, nitrites, ammonia, and urea (Ruiz and Ayala 1987). There are numerous reviews on the N metabolism in rumen (Ørskov 1992, Stern et al. 1994, Dewhurst et al. 2000, Bach et al. 2005, and Nolan and Dobos 2005). They all agree in splitting the study of the N metabolism into three basic aspects: catabolic processes, anabolic processes, and factors affecting the N metabolism in rumen.

Rumen Degradation on Diet Protein

The rumen microbial ecosystem is made up mainly by species of bacteria, fungi, and protozoa, which are strictly anaerobic, in amounts between 109 and 1011 cfu/mL. The species diversity and their relative proportions within the microbial community depend primarily on the diet consumed by the animals (Febel and Fekete 1996). The literature states that the microbial population in rumen has high proteolytic capacity (Ørskov 1992, Bach et al. 2005, and Nolan and Dobos 2005), but no particular species is responsible for this capacity or determines it (Ruiz and Ayala 1987). Nevertheless, it is known that Bacteroides ruminicola and Peptostreptococcus sp. have high proteolytic capacity (Nolan and Dobos 2005).

The protein degradation in rumen depends on the conjunction of three catabolic processes: proteolysis, peptidolysis, and deamination. The bacteria proteases are endo- and exo-peptidases enzymes, bound to the cells, but located in the cell surface to have higher possibilities of interaction with the substrates. These exo-enzymes do not seem to be subject to the metabolic control. Nevertheless, due to their localization, any factor affecting the number or metabolic activity of the microbes will affect the proteolytic activity (Ruiz and Ayala 1987). The synergic action of different types of proteases produces peptides and amino acids (Wallace et al. 1997). The speed and rate of the degradation of the proteins depend on the proteolytic activity of the rumen microbiota, the type of diet protein (Bach et al. 2005), as well as on the rate of rumen outflow and the presence of enzymatic inhibitors (Ruiz and Ayala 1987), and anti-nutrients (McSweeney et al. 2001 and Min et al. 2003).

Protozoa play an important role in protein degradation because they engulf large feed particles and rumen bacteria (Van Soest 1994). Besides, they release considerable amounts of soluble protein to the rumen environment due to their capacity of degrading the insoluble protein from the engulfed feed and to the fact that they cannot use the ammonia N (Dijkstra 1994). The peptides and the amino acids produced and contained in those forms in the feed are carried to the inner part of the microbial cells. The peptides may still be degraded to amino acids by the action of the peptidases enzymes and release amino acids from their structure. Finally, the amino acids may be deaminated and produce volatile fatty acids, CO2 and ammonia. Also, they may be used again during the synthesis of microbial protein (Nolan and Dobos 2005).

The destination of the absorbed amino acids and peptides depend on the energy availability in rumen. If there is enough energy available, the microbes will use it for the protein synthesis. On the contrary, the priority will be in the catabolic pathways producing ATP to fulfill the energy demands for the rest of the metabolic functions of the microbe (Ørskov 1992).

The most important factors affecting the microbial degradation of the proteins in the diet are the type of protein, the interactions with other nutrients, mainly the energy complexes and predominant microbial population, which depend upon the type of ration, the passage rate, and the rumen pH (Bach et al. 2005).

Microbial Protein Synthesis

The bacteria, the protozoa and the fungi forming the ecosystem have different requirements of nutrients and metabolism (Bach et al. 2005). They all ferment the feed constituents (polysaccharides, sugars, proteins) to generate the ATP molecules required to keep their homeostasis and guarantee their growth. This process comprises the synthesis of monomers (such as the synthesis de novo of amino acids) and their polymerization (such as the elongation of the polypeptide chains) (Nolan and Dobos 2005). Rumen microbes are able of synthesizing de novo the ten amino acids essential for the tissues of the mammals (Nolan and Dobos 2005), as well as of obtaining this via most of the amino acids requirements (Ruiz and Ayala 1987).

The synthesis of these amino acids is carried out from ammonia and simple carbonated skeletons, produced during the feed degradation. Thus, ruminants subsist and have modest levels of production when they only have NPN (urea, ammonia) as N source in the diet (Virtanen 1966). Ammonia is the central intermediary in the N degradation and assimilation in rumen. The levels of ammonia in rumen range from 0 to 130 mg of N/100 mL, according to the N source and the postprandial time. According to Nolan and Dobos (2005), the concentration of this complex may exceed these figures, after the animals ingest fresh pastures.

The optimum concentration for the microbial protein synthesis is between 5.6 and 10.0 mg of NH3 /100 mL of rumen liquor when the energy availability does not limit the rumen ecosystem (Van Soest 1994). The possibility of using the ammonia allows the rumen microbes recycling large amounts of urea from the intermediary metabolism of the animal, as N source for the synthesis of microbial protein, when enough energy amounts are available. Other nitrogenous complexes may also be recycled through the saliva or the rumen lining such as the purine metabolites and the mucoproteins. This evolutionary adaptation of the ruminants reduces effectively the minimum N required and increases the time of survival of undernourished animals (Nolan and Dobos 2005).

When the ammonia amount is large due to the extensive degradation of the proteins, the surplus of ammonia is absorbed through the lining of the tract. Later, it is turned into urea in the liver to reduce the circulation of this complex by the organism because it is toxic to the animal. The urea produced may be recycled to the rumen to be used by part of the microbes or it is excreted in the urine of the animal with the consequent N loss (Ørskov 1992, Bach et al. 2005 and Nolan and Dobos 2005). This process, known as urea cycle, is the result of the adaptation of the ruminants to the inefficient use of the proteins in the rumen to prevent the toxicity of the ammonia molecules and use the N released afterwards. Thus, the energy availability in the rumen permits its incorporation to the microbial protein. The synthesis of the urea molecules in the liver demands energy, thus, it is an expensive process and affects negatively the animal production because a part of the energy available for maintenance or beef or milk production should be taken to compensate the situation created by an excess of ammonia in blood.

The rumen bacteria may also incorporate directly amino acids and peptides from the diet (Wallace et al. 1999). The diminished concentration of the free amino acids in the rumen indicates that they are used readily, although the rise in the first hours after the feeding suggests that the proteolysis occurs at higher rate than the use of amino acids. Around 30 % of the N from the diet degraded in the rumen is incorporated to the microbial protein in the form of peptides and amino acids (Ruiz and Ayala 1987).

Factor affecting the production of Microbial Protein Synthesis

In laboratory conditions, the requirements for the optimum microbial growth are reduced to a viable inoculum, an energy source, and nutrients providing the essential materials for the growth, as well as the absence of growth-inhibitor substances. An adequate physical and chemical medium should be added to these requirements (Pirt 1975).

However, it is obvious that in the rumen these conditions are not fulfilled because there are other factors affecting the microbial growth and the protein synthesis. In controlled conditions, a homogeneous mixed culture may be obtained, but this is not possible in the rumen due to the semi-continuous supply of feeds from the heterogeneous sources and the saliva (Dewhurst et al. 2000).

The sources of carbohydrates and proteins, the levels of voluntary intake, the feeding frequency, and the fodder/concentrate ratio in the diet are among the factors affecting the microbial protein synthesis (Febel and Fekete 1996). Also, there are the synchronization of the rumen functions, the fodder quality (Dewhurst et al. 2000), the rumen recycling of the microbes (Ørskov 1992), and the antinutritional factors of the plants (McSweeney et al. 2001 and Min et al. 2003).

The most important factor limiting the microbial protein synthesis in the rumen is the energy released in the rumen during the fermentation of carbohydrates to organic acids (Febel and Fekete 1996). The sources of carbohydrates are classified into two groups: those rich in non-structural carbohydrates (sugars, starches), and those rich in structural carbohydrates (pectins, cellulose, hemicellulose) (Patton 1994).

The characteristics of the source of carbohydrates affect the rate of microbial synthesis. The lower rates of microbial growth are produced when using cellulose as only energy source, but the degradation of the structural carbohydrates depends also on the amounts of lignin in the feed (Hespell 1988). The synthesis of microbial protein is increased sometimes by the inclusion of moderate quantities of carbohydrates readily fermentable in the diet (Dewhurst et al. 2000) because there is increase in the availability of substrates and the growth rate of the bacteria associated with the liquid phase of the digestion. By increasing the particle size of the fibrous fraction in diets with large amounts of energetic concentrates, there is increase in the efficiency of the microbial synthesis due to the improvement in the rumen conditions by the enhancement of the processes of rumination and salivation. Likewise, there is increment in the enhancement of the organic matter digestibility (Yang et al. 2002 and Yang and Beauchemin 2005).

The inclusion of starch in the diet of ruminants may affect in several forms the ruminant microbes, and the forecast of the final effect is not simple. The starches may have negative effects on the microbial synthesis in the rumen because their fermentation diminishes the rumen pH, affects the fiber degradation, increases the energy losses in the microbes, and declines the synthesis de novo of the amino acids (Russell and Wallace 1997).

Not all the energy sources have the same effect on the microbial protein synthesis. It has been proved that the soluble sugars (saccharose, lactose and fructose) increase even more the microbial protein synthesis in the rumen rather than when using supplements of cereals rich in starch (Chamberlain et al. 1993). Cone et al. (1989) proved that the starches of oat and barley are degraded from 2 to 2.8 times faster than those of corn; thus, the energy availability for the microbial synthesis and the negative effect of these sources on the rumen conditions of fermentation varied considerably between one source and the other.

Effect of the Protein Sources

The effect of the protein source on the microbial protein synthesis is complex, and it should be studied with careful attention. Dewhurst et al. (1999) proved that the effect of the protein supplementation on the microbial protein synthesis depends on the type of energy supplied in the concentrates.

The structure of the diet proteins defines their degradation in the rumen and its actual contribution to the N available for the microbes. The rate and extent of the diet protein degradation depends on the previously described factors. The ammonia is the main N source of the rumen microbes, but the diet availability of amino acids, peptides, and both together increases the growth of cellulolytic and amylolytic bacteria (Kernick 1991). This may be due to their direct incorporation to the microbial protein or to the fact that they increase the availability of carbonated skeletons that may be used as energy source or in the synthesis de novo of microbial amino acids (Van Soest 1994). There is no knowledge on the optimum concentration of peptides needed for maximizing the microbial protein synthesis in rumen (NRC 1996).

It is estimated that the bacteria need 1.2 g of N/kg of organic matter fermented in the rumen, but this value should not be applied to all types of rations (Bach et al. 2005). In general, it is believed that the rumen microbes do not have absolute requirements of amino acids, although Atasoglu et al. (2004) proved that some amino acids limit the microbial growth because the rumen microbes have difficulties for synthesizing them de novo (phenylalanine, leucine, isoleucine, lysine). The research of Atasoglu et al. (2004) suggests that the high-aminoacid-content diets may increase the growth of rumen microbes (Bach et al. 2005). However, it should be considered that the spread in the use of amino acids in rumen varies according to the source (Taghizadeh et al. 2005).

Effect of the level of Voluntary Intake

By increasing the intake, the energy costs of maintenance of the microbes are reduced because the time of permanence in rumen is also diminished. Besides, by increasing the intake, the particle flow is increased in the rumen. Thus, the number of bacteria adhered to the feed coming out of the rumen and passing to the abomasums and the duodenum is increased, the rumen recycle of the microbial protein is reduced (Van Soest 1994), and the microbial yield is increased.

However, some diets with high content of concentrates, rich in starch, provoke high intakes and tend to reduce the efficiency in microbial synthesis. This is due probably to the energy expenditure by keeping the homeostasis and the cell pH, when the rumen pH decreases.
By increasing the voluntary intake of the animals, the microbial N outflow from the rumen is increased (Webster et al. 2003); but there is not always a correlation between the voluntary intake and the microbial protein production (Firkins et al. 1984). Singh et al. (2007) found that the supply of microbial protein to the duodenum was increased linearly by increasing the voluntary intake. However, when expressing the microbial yield, according to the intake of digestible organic matter, there was not effect on the production of microbial protein.

Effect of the Synchronization of the Rumen Function and the Microbial Protein recycle in Rumen

The concept of synchronization in rumen is important to understand the ratio between the energy and the protein supplied to the rumen microbes. The microbial protein synthesis may be maximized, if the fermentable energy availability and that of N not degraded by the microbes in rumen are synchronized (Ørskov 1992). When the rate of protein degradation exceeds that of the carbohydrates, large amounts of N are lost in the form of ammonia. However, when the rate of degradation of the carbohydrates exceeds that of the protein, the microbial protein production may be impaired (Nocek and Russell 1988). The fermentation of the carbohydrates has little influence on the protein degradation rate by the extracellular proteases (Febel and Fekete 1996).

Nevertheless, the rumen degradation of the carbohydra-tes provides the energy in the form of ATP required by the microbes for their metabolism. Therefore, it defines if the peptides and amino acids produced during the extracellular degradation of the diet proteins are incorporated to the catabolic or anabolic pathways of the microbial metabolism. It is possible to change the synchronic effect of the diets, whether by changes in the ingredients and in the frequency of offering certain energetic or proteinic components of the ration or by the combination of some of these forms. It is not possible to define if the increase in the microbial protein synthesis is due to the inclusion of different ingredients, if it is the result of the energyprotein synchronization, or if it is a factor associated with the manipulation of the ingredients (level and type) (Dewhurst et al. 2000).

It is unknown the optimum relationship between the non-structural carbohydrates and the ammonia N. Hoover and Stokes (1991) suggested that in a continuous culture fermentor, in conditions of controlled pH, the maximum microbial growth is obtained with a ratio of non-structural carbohydrates/rumen degradable proteins of 2/1. Although this ratio was not attained in practical conditions, the importance of supplying adequate amounts of degradable N was indeed proved, when the energy is not a limiting factor (Bach et al. 2005).

There are contradictions in the literature on the effect of the synchronization of the supply of the protein and carbohydrate sources. In vivo studies have indicated positive responses by synchronizing the better availability of N and energy (Matras et al. 1991), whereas other in vitro studies did not report satisfactory effects (Newbold and Rust 1992). The concept of synchronization of the supply of protein and energy has a solid theoretical foundation, but the mixture of rumen microbes makes up a complex ecosystem. In it, the nutrients supply may be synchronized for some populations, and not, for some others. Also, the rumen recycle of microbes may contribute to stabilize the microbial growth when the N supply is not wellsynchronized (Bach et al. 2005).

The rumen recycle of the microbial protein reduces the microbial biomass yield per unit of fermented carbohydrates. The main responsible of this process is the protozoa. They consume large amounts of bacteria that are their main protein source (Bach et al. 2005) and engulf large amounts of fungus zoospores, which were used as additional source of proteins for their metabolic processes (Galindo et al. 1995).

Effect of the antinutritional factors

The most studied antinutritional factors are tannins, although there are many other secondary metabolites from plants that may affect the microbial populations in rumen (Marrero et al. 2002). That is the case of saponins, cyanogenic compounds and non-protein amino acids, lectins, alkaloids, and oxalic acid (Kumar and D’Mello 1995), as well as flavonoids and steroids (Galindo et al. 2000b). Tannins affect the microbial protein synthesis in different forms (Molan et al. 2001 and Makkar 2003), whether directly by their action on the rumen microbes (Marrero et al. 2002) or by their interaction with the nutrients they depend upon for energy and N supply (McSweeney et al. 2001 and Hedqvist 2004). Their effects on the rumen ecosystem are complex and they are according to the degree of tolerance of the forming species (McSweeney et al. 2001).

Due to their bacteriostatic and bactericide action (Makkar 1993), these compounds affect the growth rate and the microbial synthesis of proteins (Min et al. 2002), although the mechanisms are less known than their reactions with the diet proteins (Min et al. 2003). The microbial biomass production is increased by inactivating the condensed tannins (Bento et al. 2005 and Alipour and Rouzbehan 2007) because there is increase in the availability and the degree of synchronization in the nutrients fermentation (Makkar 2003). The antimicrobial action of the tannins is also manifested, to a larger extent, with protozoa, which decrease significantly due to the presence of tannins in the diet, although it is not clear if it is a direct inhibiting effect on the protozoa or from the negative action of these compounds on the bacteria populations (Wang et al. 1996).

It seems that the rumen fungi are in general more resistant to tannins and larger concentrations are needed to inhibit their activity in the rumen (Lowry et al. 1996). The condensed tannins react with the proteins and form tannin-protein complexes through hydrogen bonds, hydrofobe interactions, ionic bonds and covalent links (Schofield et al. 2001). These bonds may affect the enzymes secreted by the rumen bacteria and inhibit the fermentation of carbohydrates and proteins (Molan et al. 2001). Besides, tannins affect the rumen proteolysis because they associated with the soluble proteins of the diet protecting them from the microbial action.

Nevertheless, it is stated that the inclusion of tannins in the diet has a positive effect on the animal because it increases the quantity of diet protein non-degradable in rumen, without affecting significantly the outflow of microbial protein to the duodenum (McSweeney et al. 2001). This could be due to the bactericide action of these complexes is compensated by the action also exerted on the protozoa (Galindo et al. 2000a), which reduces the rumen recycle of bacteria biomass and compensates the reductions in the bacteria production. In recent years, researchers have been motivated by the study on the potential uses of saponins as complexes to manipulate rumen fermentation. Hess et al. (2003) demonstrated that the supplementation with fruits of Sapindus saponaria, rich in saponins, improved the outflow of microbial protein to the duodenum and the efficiency of the rumen fermentation. This outcome coincided with the reports from other authors when using Yuca schidigera as saponin source (Wang et al. 2000 and Santoso et al. 2004). This increase in the outflow of microbial proteins may be related to the inhibiting effect of the saponins on the protozoa population (Galindo et al. 2000a and Hu et al. 2005), which reduces the rumen recycle of bacteria N. Hu et al. (2005) observed that by increasing the content of saponins, the in vitro rumen fermentation pattern of a mixture of meal of forage and corn was affected by having increase in gas production and microbial protein, and decrease in the number of protozoa and in the methane concentration.

Makkar (2005) incubated in vitro 0.6 mg/mL of saponins obtained from different plant sources and, according to the saponin source, the gas production and the microbial protein source were affected. Recent studies show that many secondary metabolites from plants have potential to modify favorably the rumen fermentation, when used in relatively low concentrations. In adequate doses, the efficiency in microbial synthesis and the microbial yield are increased by including saponins (Hess et al. 2003) or condensed tannins (Puchala et al. 2005).

Importance of the microbial protein on the ruminant nutrition

The amino acids produced by rumen microbes are available for the host animal in the small intestine when the microbial protein flows with the digested material toward the lower parts of the gastrointestinal tract (Nolan and Dobos 2005). The metabolizable protein of the rumen microbes has a stable composition, similar to the nondegradable protein of the pastures and a variable amino acid profile, but generally adequate (Storm and Ørskov 1983).

The net digestibility of the amino acids in the small intestine is of around 85 %, with the exception of the diaminopimelic acid, which has a very low digestibility. The absorbed microbial amino acids are used by the animal in around 80 % (Storm et al. 1983).
The rumen microbes seem to supply protein for maintenance, slow growth, and initial pregnancy, but not for the fast growth, the final pregnancy of the start of the lactation (Ruiz and Ayala 1987). However, Stern et al. (1994) reported the importance of maximizing the efficiency of microbial synthesis to support the high levels of milk production.

In tropical productive systems, where roughages are the basis of the diet, the microbial protein may supply 100 % of the protein available for the ruminant (Ørskov 1992). The knowledge on the amount of microbial protein synthesized in these systems will permit to make a more efficient use of the pastures and forages as basic feed for the ruminants in this region, as well as of the supplements (foliage from shrubs and trees, harvest wastes, multi-nutrient blocks, and concentrates, etc). Thus, it influences positively the economic and environmental competitiveness of the cattle production (Posada et al. 2005).

Conclusion

The rumen microbes are able of incorporating their amino acids and peptides to the diet and of using the ammonia to synthesize de novo their amino acids such as the ten amino acids essential for the tissues of the mammals. The synthesis of microbial protein depends upon different factors such as the sources of carbohydrates and proteins, the level of voluntary intake, the synchronization of the rumen functions, the rumen recycle of microbes, and the antinutrients of the plants consumed. The microbial protein has a relevant role in ruminants fed diets with high-fiber content and low level of N. It is sometimes the only protein source for the animal. The understanding of the mechanisms involved in the diet N use and in the synthesis of microbial protein in rumen, the factors determining it, and the perfection of the methods of estimation and development of accurate predictions will permit to use more efficiently the feeding resources in the tropics.