Urea Recycling in Ruminants

Animals have a certain state of protein metabolism, varying from negative to positive protein balances. This balance level is influenced e.g. by the efficiency of nitrogen (N) utilization in animals. A simple strategy to increase the efficiency of N utilization is by reducing the N content in the feed converted to urea, for which a correlation of about r2=0.77 was found. However, this was mainly based on studies with mature or slow growing, small ruminants in which most of the absorbed N is converted to urea to maintain the N balance of the whole body close to zero (Lapierre and Lobley, 2001). More recent and extensive data show much weaker correlations between N intake and urea production for growing sheep (r2=0.33) and cattle (r2=0.58). Moreover, this strategy is not always realizable due to minimal absolute N requirements in animal feed, especially for growing animals.

In addition to N intake, the protein balance level is influenced by the efficiency of N recycling in animals, especially in ruminants. Nitrogen recycling takes place between blood and the digestive tract in the form of endogenous protein-N, secreted-N (e.g. enzymes in saliva) and urea-N (Reynolds and Kristensen, 2008).

In this chapter, the recycling of urea-N is explained. Amino acids and ammonia, which are absorbed from the digestive tract, are converted to urea in the liver. Urea (re)enters the digestive tract, mainly through the rumen wall, where it can be absorbed again or be (re)used for microbial protein synthesis and finally anabolic purposes.

Amino acids and ammonia are absorbed into the portal bloodstream and converted into urea in the liver (ureagenesis). Urea can reenter the rumen, where it can be absorbed (again) or be used for microbial protein synthesis.

Absorption of amino acids and ammonia

Urea is the mammalian end-product of the amino acid metabolism. In the rumen, proteins are degraded into amino acids and finally into ammonia (NH3) by means of rumen fermentation (Shingu et al., 2007). Then, absorption of both amino acids and NH3 through the rumen wall and entrance into the portal circulation to the liver can take place (figure 3.1). The NH3 absorption depends on the pH and the ratio of NH3 to NH4 in the rumen (Siddons et al., 1985).

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Ureagenesis

In the liver, detoxification of NH3 takes place, because urea is synthesized from the nitrogen (N) compound of both NH3 and amino acids (which appear in the portal circulation due to absorption from the intestine into the blood) (Obitsu and Taniguchi, 2009). The synthesis of urea, called ureagenesis, takes place by means of the urea or ornithine cycle. This cycle of biochemical reactions occurs in many animals that produce urea ((NH2)2CO) from ammonia (NH3), mainly in the liver and to a lesser extent in the kidney. The key compound is ornithine, which acts as a carrier on which the urea molecule is built up. At the end of the reaction sequence, urea is released by the hydrolysis of arginine, yielding ornithine to start the cycle again (Bender, 2008). Mitochondrial ammonia and cytosolic aspartate are precursors for the ornithine cycle (Van den Borne et al., 2006). The presence of arginine is needed to produce ornithine in the body, so higher levels of this amino acid should increase ornithine production. Furthermore, ornithine, citrulline and arginine (all components of the ornithine cycle) seem to stimulate urea synthesis, with a concurrent decrease in plasma ammonia.

Temporarily high ammonia fluxes seem to stimulate amino acid utilization for ureagenesis (Milano and Lobley, 2001). Urea is produced in the liver in greater amounts than which is eliminated in the urine. This is because urea from the liver is released to the blood circulation and then, next to excretion in the urine also is reabsorbed in the distal renal tubules, where it maintains an osmotic gradient for the reabsorption of water (Bender, 2008). Furthermore, urea from the blood can re-enter the digestive tract via saliva, secretions or directly across the rumen wall in the form of endogenous proteins or urea respectively (Lapierre and Lobley, 2001; Shingu et al., 2007; Obitsu and Taniguchi, 2009). Thus not all urea is secreted directly into the urine after entering the bloodstream.

Entry into digestive tract

Entry of urea into the digestive tract is, until certain concentrations (sheep: 6 mM (= 84 mg/L); cattle: 4 mM (= 56 mg/L) (Harmeyer and Martens, 1980; cattle: 80 mg/L (Kennedy and Milligan, 1978)) partly affected by plasma urea concentrations (Harmeyer and Martens, 1980). Above these concentrations, boundary layer effects with NH3 inhibit the urea entry into the digestive tract (Lapierre and Lobey, 2001). Urease activity is lower with increased NH3 concentrations and N intake (Marini et al., 2004). This inhibits the entry of urea into the digestive tract (Kennedy and Milligan, 1978). Thus high ammonia concentrations in the rumen result in a lower gut entry rate (Kennedy and Milligan, 1978; Bunting et al., 1989a).

Urea, which flows from the blood into the rumen and enters the digestive tract, is hydrolyzed by bacterial urease to carbon dioxide (CO2) and ammonia (NH3) (figure 3.1). NH3 can be either reabsorbed into the blood or be used as N source for microbial protein synthesis or microbial growth (Sarraseca et al., 1998; Shingu et al., 2007). This latter process may provide a mechanism for the salvage of urea-N into bacterial protein which can be digested and yields amino acids to the animal when they are absorbed in the lower parts of the digestive tract. Thus, urea nitrogen incorporated in microbial protein and possibly absorbed in the gut gets ‘a second chance’ for absorption and deposition/anabolic purposes. Therefore, urea recycling can be regarded as a mechanism with positive effects at the protein balance of ruminants.

Gut entry location and gut entry rate (GER)

The gut entry rate (GER) of urea is simply the amount of urea N recycled into the digestive tract. The amount of urea which entered the digestive tract that can be used for anabolic purposes depends e.g. on the gut entry location (Lapierre and Lobley, 2001). Urea appears to enter all parts of the digestive tract, including via saliva and pancreatic juice, but with different rates. The GER could be influenced by the concentration gradient of urea between the plasma and the fluids in the digestive tract (Harmeyer and Martens, 1980). The concentration gradient is again dependent on the activity of ureolytic bacteria and could therefore be influenced by diverse bacteria-influencing compounds in the feed. Also, the presence of carrier mediated, facilitative urea transport mechanisms have been reported in the ovine colon and rumen epithelia (Ritzhaupt et al., 1997). The carrier mediated, facilitative urea transporters in the ovine colon and rumen epithelia permit bi-directional flows (Ritzhaupt et al., 1997), and thus may the total gut entry rate (GER) be underestimated if urea molecules are reabsorbed without being metabolized (Lapierre and Lobley, 2001).

Post-stomach tissues can greatly influence the (GER) (up to 70%), but their contribution to potential anabolic salvage of N is not certain.

The majority of conversions of urea into anabolic compounds occur in the fore-stomach, mainly the rumen (Kennedy and Milligan, 1980). As summarized by Lapierre and Lobley (2001), in sheep, the part of the total gut urea entry (GER) transferred to the rumen varies from 27 to 60% (Kennedy and Milligan, 1978) and 27 to 54% (Siddons et al., 1985) depending on type of diet. This proportion seems to increase when animals get high levels of rumen-degradable energy in feed (Lapierre and Lobley, 2001; Theuer et al., 2002).

Also saliva contributes to the total urea entry into the rumen, depending on the type of diet ingested. E.g. this proportion varies extensively from 15 (Kennedy and Milligan, 1978) to almost 100% (Norton et al., 1978) in sheep. It has been found in growing beef steers that forage diets, e.g. alfalfa hay, result in higher proportions of saliva entering the gut (36% of GER) (Taniguchi et al., 1995) compared to high concentrate diets (17% of GER) (Guerino et al., 1991). Thus the fore-stomachs are important for the anabolic salvage of N, however, this depends on the type of feed ingested (and animal species).

Small intestine

Also the small intestine contributes to the anabolic salvage of N. It has been found in sheep that 37 and 48% of the total GER of urea entered the small intestine in case of grass silage and dried grass, respectively (Siddons et al., 1985). However, the quantities of anabolic N formed may by small, e.g. because ammonia production seems to exceed urea entry across the small intestine, although this depends on the type of feed ingested (Lapierre and Lobley, 2001).

Hindgut

Likely most microbial protein synthesized from urea that enters the hindgut is excreted. All the evidence so far would suggest that hindgut usage of urea involves only catabolic fates, at least in terms of amino acids supply to the animal (Lapierre and Lobley, 2001).

Fate of urea that enters the digestive tract

Urea that enters the gut by means of saliva or flowing through the gut wall can be used for anabolic purposes or is transformed into ammonia and returned to the liver (Lapierre and Lobley, 2001). Much of the NH3 in the GI tract is reabsorbed and used in the liver for the synthesis of glutamate and glutamine, and then a variety of other nitrogenous compounds (Bender, 2008).

Urea-N that entered the gut contributed for 33% of the rumen ammonia flux in sheep offered dried grass, while this percentage was lower in case of grass silage (Siddons et al., 1985). Lapierre and Lobley (2001), based on several references, summarized that sheep, dairy cows and growing steers have a efficient reuse of N because urea-N atoms can return to the gut on more than one occasion. This increases the overall probability of appropriation towards an anabolic fate. This multiple-recycling process can result in improvements of 22 to 49% of GER used for anabolic purposes in both cattle and sheep (Lapierre and Lobley, 2001). A substantial proportion of urea that enters the digestive tract is returned to the body as ammonia in both sheep (32 to 52%; Sarreseca et al., 1998) and cattle (26 to 41%; Archibeque et al., 2001). This means that a large proportion of net ammonia absorption across the PDV is due to recycled N, rather than arising directly from ingested N. These anabolic and catabolic fates of urea then explain why net appearances of amino acid-N and ammonia across the PDV can equal or exceed apparent digestible-N (Lapierre and Lobley, 2001). The net result of all these N transactions is that the apparent conversion of digestible N into net absorbed amino acid N can be high, with individual values of 27 to 279% calculated for both cattle and sheep. These ‘efficiencies’ are lower (24 to 58%) when other inputs are considered, mainly the urea-N inflow into the rumen. Apparent digestible N represents the net available N to the animal and thus the amino acid absorption cannot normally exceed this unless other N sources like amino acids obtained due to catabolism (released on a net basis during submaintenance intake) or urea recycling. N recycling via the digestive tract increases the opportunity for catabolism N to be reconverted to an anabolic product. This recycling can be considered analogous to the synthesis and breakdown of proteins within tissues, where the dynamic flow maintains metabolic fluidity with minimum loss (see figure …; Lapierre and Lobley, 2001).

SUMMARIZED UREA RECYCLING KINETICS

Thus, urea-N kinetics can, as an approximation, be considered as a mechanism, where hepatic synthesis is similar to digested N, with one-third lost via the kidneys into urine, while the remaining two-thirds is returned to the digestive tract. Half of this is then reconverted to anabolic N (mainly amino acids) that can be reabsorbed and used for productive purposes. Most of the remaining half of GER is reabsorbed as ammonia that is reconverted to urea and can be further re-partitioned between urinary loss and GER (see figure…). The process thus allows conversion of a catabolic products (urea-N) into anabolic forms, contains these for longer within the body, and provides the animal with increased opportunities to utilize products derived from dietary N (Lapierre and Lobley, 2001).

Figure… Urea recycling: values in circles represent the fraction of hepatic ureagenesis destined either for urinary output or to gut entry rate (GER); values in rectangles represent the fractions of gut entry rate lost in feces, returned as ammonia to the hepatic ornithine cycle or converted to anabolic products (mainly amino acid N). Thus, on average, 33% of hepatic urea-N flux is eliminated in urine while 67% enters the various sites of the digestive tract. Of this latter N, 10% is lost in feces, 40% is reabsorbed directly as ammonia, while the remaining 50% is reabsorbed as anabolic-N sources (mainly AA’s). Data are simplified means for steers, dairy cows and sheep (from Archibeque et al., 2001; Sarraseca et al., 1998; summarized by Lapierre and Lobley, 2001)

Efficiency of N utilization

In both cattle and sheep, the inefficient use of intake-N is associated with large ammonia absorption representing on average 0.46 and 0.47 of N available from the lumen of the gut (digestible N plus urea-N entry across the PDV) (Lapierre and Lobley, 2001). As mentioned earlier, one strategy is to reduce the amount of N directed towards ammonia absorption and hepatic ureagenesis, but the situation is more complex than that. The target of reduction of ammonia absorption has to be integrated in a wider context where this decrease would result 1) from a smaller degradation of dietary N into the rumen or 2) from an increased utilization of rumen ammonia for microbial protein synthesis. Lowered N degradation can result from diet manipulation. Lapierre and Lobley (2001) summarized from several studies that cattle fed concentrate-based diets had decreased ammonia absorption, both in absolute amounts and relative to digested N, compared with forage rations. Increased utilization of N for bacterial synthesis can also be influenced by dietary manipulation, particularly provision of additional energy. From several studies, it can be concluded that supplements of rumen fermentable energy sources increase the transfer of urea into the rumen, and therefore the capture of dietary N and GER into anabolic products, mainly amino acids. However, there appear to be upper limits to the overall efficiency of the process (Lapierre and Lobley, 2001). The limited data available suggest that a maximum of 50 to 60% of dietary N, or 70 to 90% of apparently digested N, will be converted into amino acids released into the portal vein. Energy sources may also improve utilization of dietary and urea-N by less direct means, e.g. by energy-sparing effects within the cells of the gut tissues rather than alteration of rumen fermentation (Lapierre and Lobley, 2001).

Intrarumen recycling

Recycling of N can also occur within the rumen, due to the presence of proteolytic bacterial and protozoa. These ‘graze’ and digest the rumen bacteria, increasing ammonia content and release within the rumen, and reducing microbial N outflow within the rumen because of increased recycling of bacteria (Lapierre and Lobley, 2001). Thus changes in the microbial population of the rumen can have substantial effect on anabolic N flow. Such modifications of the rumen microflora may contribute to the differences in N recycling and conversion to amino acids that occur between diets and animal species (Lapierre and Lobley, 2001).

Amino acid supply

In many circumstances, inefficiencies for conversion of feed N to animal protein may not be a feature of total amino acid supply, but rather depend more on the profile of absorbed amino acids. Hereby you can think of e.g. limiting essential amino acids.

In ‘short’ the definition of urea recycling is: the flow of urea from the blood into the digestive tract so that urea nitrogen salvage could happen.

Figure … Use of [15N15N] urea and isotopomer analysis of urinary [15N15N], [14N15N] and [14N14N] urea to quantify flows and fates of urea that enters the digestive tract. Part of the infused [15N15N]urea enters the digestive tract were it can be excreted in the faeces or is hydrolyzed to [15N]ammonia. This latter is either used by the microbial population to synthesize bacterial proteins ([15N]) or it is absorbed directly as [15N]ammonia. [15N]ammonia is removed by the liver were [15N14N]urea is formed. The ratio of [14N15N]:[14N14N]urea in the urine reflects the proportion of urea flux that is converted to ammonia in the digestive tract and returned directly to the hepatic ornithine cycle (Lapierre and Lobley, 2001).

The utilities of urea recycling

Both ruminants and non-ruminants, including omnivores, have a mechanism in which urea produced by the liver can enter the intestinal tract and where it is used for microbial protein production or urea production. However, the amount of urea recycled in ruminants is in much larger proportions compared to non-ruminants, which emphasizes the importance of urea recycling in ruminants (Lapierre and Lobley, 2001). Next to reducing feed costs (due to the lower dietary N contents required), there are three important reasons to obtain a good and efficient urea recycling in ruminants (Huntington and Archibeque, 1999):

• Maximization of the microbial functioning in the rumen;
• Optimization of the amino acid supply to the host ruminant – improvements of adaptation;
• Minimization of the negative effects of nitrogen excretion into the environment.

Maximization of microbial functioning

In ruminants, synthesis of urea by the liver can exceed apparent digestible N. This would result in a negative N balance (even at high intakes) if no salvage mechanism existed to recover some of this N (Lapierre and Lobley, 2001). Recycling of urea synthesized in the liver can provide a substantial contribution to available N for the gut. Lapierre and Lobley (2001) summarized that this can increase the digestible N inflow from 43 to 85% in growing steers, 50 to 60% in dairy cows and 86 to 130% in growing sheep. Moreover, in veal calves shifts the major origin of absorbable amino acids in the small intestine after weaning from milk protein to microbial protein (Obitsu and Taniguchi, 2009). With this, it is important to realize that a higher level of urea recycling results in a higher production of microbial protein. This protein source will be largely used for anabolic uses and performance which will result, on the long term, in improved production efficiency (Lapierre and Lobley, 2001). What urea-N recycling does is to increase N transfers through the body to convert more of the N into anabolic form and thus acts as a conservation mechanism. Therefore, the combined inflows of dietary N and urea GER can be considered analogous to protein turnover within the body, where the anabolic and catabolic processes of synthesis and degradation greatly exceed inputs (intake) and outputs (oxidation and gain). This is believed to provide an overall plasticity to allow rapid response to any challenges or changes in metabolic status.

Optimization of amino acid supply – adaptation

As a consequence of the salvage mechanism to recover some N, nitrogen and urea recycling in ruminants are important regarding the adaptation to different environmental (living) circumstances but mainly to nutritional conditions. Examples are periods of dietary protein deficiency or an asynchronous supply of carbohydrates and proteins (Reynolds and Kristensen, 2008). Ammonia and microbial protein produced in the gut and urea synthesized in the liver are major components in N-recycling transactions (Obitsu and Taniguchi, 2009). An increase in the total urea flux, caused by the return to the ornithine cycle from the gut entry, is considered to serve as a labile N pool in the whole body to permit metabolic plasticity under a variety of physiological (productive), environmental and nutritional conditions (Obitsu and Taniguchi, 2009; Lapierre and Lobley, 2001). Therefore, ruminant species have different characteristics of their urea recycling due to different living conditions varying from tropical conditions with poor quality feed to intensive systems in temperate/cold conditions with high quality feed. High ambient temperatures seem to increase urea production but reduce urea gut entry (Obitsu and Taniguchi, 2009).

Minimization of N excretion into the environment

Finally, a more efficient urea recycling in ruminants results in a less urea-N excretion in the urine. This is will minimize the negative effects of nitrogen excretion into the environment (Huntington and Archibeque, 1999).