Urea Metabolism and Recycling in Ruminants

Daniel Getahun1, Tewodros Alemneh2*, Dawit Akeberegn3, Mebrate Getabalew1 and Derebie Zewdie1 1Department of Animal Science, College of Agricultural and Natural Resource Science, Debre Berhan University, Ethiopia 2Woreta City Office of Agriculture and Environmental Protection, South Gondar Zone, Amhara Regional State, Ethiopia 3Debre Berhan City Municipality Office, Meat Inspection & Hygiene, Semen Shewa Zone, Amhara Regional State, Ethiopia


Introduction
Urea and ammonia, in addition to amino acids (AA), peptides and microbial crude proteins (MCP), play an important role in nitrogen digestion and metabolism in ruminants. Also, urea could be used to replace a portion of dietary protein in ruminants [1].
Thereafter, some studies were conducted on the use of NPN in ruminant diets. During the 1970s and 1980s, multiple studies were conducted on the utilization of urea as a replacement for protein in ruminant diets, especially its effect on dry matter intake [2], rumen fermentation [1,3] milk yield and reproduction-related parameters [4,5]. Ruminants can utilize non protein nitrogen as a dietary protein source via their rumen microorganisms [6].
In most mammalian species, a large amount of endogenous urea-N is excreted via the urine. However, ruminants have evolved a mechanism that allows constant recycling of urea-N to the gastrointestinal tract (GIT), particularly to the rumen, where urea-N can be used as a source of N for microbial protein, which is the major contributor to the metabolizable protein supply to the small intestine. Urea-N recycling to the GIT and its utilization for anabolic use is influenced by several dietary and ruminal factors.
Major dietary factors which regulate the proportion of hepatic urea output returning to the GIT and its subsequent fate are: dietary N concentration and N intake [7,8]; total dry matter intake [9]; feed processing [10][11][12]; oscillating dietary N levels [13] and amount as well as frequency of feeding dietary N that is degraded in the rumen [14,15]. In juxtaposition with dietary factors, ruminal factors such as ruminal NH3-N concentration, ruminal bacterial urease activity, ruminally-fermentable carbohydrate (RFC), ruminal concentrations of volatile fatty acids (VFA) and CO 2 , and ruminal P H also play a significant role in trans-epithelial movement of blood urea-N into

Ammonia Production and Ammonia Toxicity
Usually, ruminants are much less efficient than no ruminants in utilizing high quality dietary proteins. A dominant feature of N Digestion and metabolism is microbial conversion of some dietary protein to NH 3 . In ruminants, ammonia is generated by amino acids obtained from the diet, from catabolism of glutamine by enterocytes, or from peripheral tissues such as skeletal muscle. In mammals, at least 20 metabolic reactions generate ammonia, with the glutaminase, glutamate dehydrogenase and purine nucleo-tide cycle pathways producing most of the ammonia [16]. Ammonia generation in the gut results from two main processes: one is microbial degradation of nitrogenous compounds within the gut lumen; the other is microbial hydrolysis of urea passing across the gut wall from the blood and intestinal fluids [3].
The primary source of NH 3 within the rumen is dietary protein, except for ruminants consuming diets very low in protein. The UIP and indigestible intake protein (IIP) usually pass to the duodenum without affecting NH 3 production in the rumen. Degradable dietary NPN can be converted rapidly and quantitatively to NH 3 dissolved nucleic acids in the rumen are also degraded extensively by rapid action of bacterial peptidases and deaminases to produce NH 3 [17] Much of the daily ammonia load is derived from the catabolism of amino acid obtained from dietary proteins. In animals with high protein intake, 10% to 15% of the protein is delaminated and used for energy, with resultant ammonia production [18]. Urea recycling is significantly related to NH 3 production and absorption in the gastrointestinal tract (GIT) of ruminants. All NH3 absorbed from the rumen epithelium, small intestinal mucosa, and large intestinal mucosa travels via the portal vein to the liver; body tissue NH 3 also en-ters the liver. Liver metabolism has a central role in the integration of body N metabolism. Ammonia in the liver is detoxified by conversion to urea, urea can then is recycled directly into the rumen, small intestine, or large intestine; it can enter the rumen in saliva, be excreted by the kidney, or be secreted in milk or sweat [19].

Metabolism of Urea in the Liver
Under normal physiological and nutritional conditions, NH 3 absorbed into the portal vein is efficiently extracted by the liver and detoxified by conversion to urea or glutamine. Over a wide range of portal NH3 concentrations and on a variety of diets, the liver is able to extract 70 to 95% of portal NH3. As a result, hepatic NH 3 removal is on average slightly higher (4%) than portal absorption [17]. The structure and function of the liver attests to the importance of removing potentially toxic NH 3 from blood of

Rumen Ureolytic Bacteria
Rumen ureolytic bacteria play an important role in dietary urea hydrolysis, for they produce ureases that catalyze the breakdown of urea to ammonia (NH 3 ) and carbon dioxide [21,22]. In the rumen, the ammonia can be assimilated by many rumen bacteria for the synthesis of microbial proteins [22]. However, the efficiency of urea N utilization in ruminants is low and this is attributed to the rapid hydrolysis of urea to NH3, which occurs at a higher rate than NH 3 utilization by rumen bacteria [23]. Due to the difficulty in cultivating rumen bacteria, only a small number of bacteria have been isolated [24]. The lack of sufficient understanding of the ruminal microbiome is one of the major knowledge gaps that hinder effective enhancement of rumen functions [25]. In addition, limited information about rumen urea-degrading bacteria makes regulation of the urea hydrolysis rate by targeting predominant ureolytic bacteria difficult.

Urea Transport
Urea transport Across the Rumen Epithelium: Urea produced in the liver is transferred across the rumen wall from the blood and then it is hydrolyzed to ammonia by resident bacteria [26]. As is already known, urea transport across the ruminant wall is mediated via urea transporters in the epithelium membrane [27]. These transporters allow the passage of urea across cell membranes, down a concentration gradient [28]. Facilitative urea transporters are derived from the UT-A and UT-B genes [29]. UT-B mRNA or protein expressions have been characterized in the rumen epithelium [30]. In the study of Coyle et al. [31], UT-B transporters were identified to be specifically localized to certain regions of tissue in the bovine gastrointestinal tract. Hepatic urea-N synthesis has two fates i.e., it is either excreted in the urine or is recycled back to the GIT via salivary secretions or by the direct transfer across the epithelial tissues of the digestive tract [32]. All mammalian species have the mechanism of urea-N recycling to the GIT. However, in ruminants the amount of urea-N recycled to the GIT (as a proportion of total hepatic urea-N output) varies between 29 to 99%, which is much greater compared to non-ruminants (15 to 39%) [33]. Animals utilize the released urea-N in a number of ways. In sheep, 30-50% of the urea that enters the digestive tract is returned to the host as ammonia, whereas this value is 25-40% for cattle [26].  However, the relative contributions of these routes can vary enormously depending on a complex interaction of factors, including the composition of diet ingested. For example, in cattle fed a concentrate diet, saliva secretion accounted for 17% of the total gut entry of urea, whereas in cattle fed a forage diet this value increased to 36% [26]. In high and rapidly growing ruminants, urea-N recycling to the GIT is so important that it can increase the N availability to the GIT from 43 to 130% [26]. Total urea synthesis in the liver can be as high as 33 to 99% of N intake. Of that total endogenous hepatic urea-N production in the liver, 1 to 71% of urea-N is excreted in the urine and about 29 to 99% enters the GIT. In the GIT 16 to 70% of urea-N (as a proportion that enters the GIT) is utilized for anabolic purposes and 3 to 21% is lost in feces. Unutilized urea-N (i.e., NH3-N) is returned to the ornithine cycle (17 to 80% of urea-N that enters GIT) for urea synthesis. The data depicted in this figure are obtained from urea-N kinetic measurements obtained from intra-jugular infusion of 15N15N-urea [36].

Urea Entry into the Rumen:
Available data from literature [10,34] shows that between 27 to 60% (combined salivary contributions and transfer across the rumen wall) of the GIT entry is to the rumen. The quantity of urea-N transfer to different sections of the GIT is regulated by characteristics of the ruminant diet.
Huntington [11] demonstrated that in steers fed high concentrate diets, up to 95% of urea-N (as a proportion of urea-N entry to the GIT) enters the rumen, as compared to 62.5% in steers fed high forage diet. Urea-N can enter the rumen via direct transfer of blood urea-N across the ruminal wall or via salivary secretions. Salivary urea-N entry to the rumen calculated as difference between total splanchnic flux and urinary excretions rate as a percent of total hepatic urea-N production represented 72% in steers fed high forage diets as compared to 21% in those fed high concentrate diet [11]. High roughage diets stimulate rumination, thus increasing the flow of salivary secretions to the rumen. Reports from other studies also show that salivary flow of urea-N into the rumen as a percent of total urea-N entry to the GIT was 36% in forage-fed [17] and 16% in concentrate-fed [37] ruminants. Recently, Ludden et al. [38] showed that UT-B proteins are present in the parotid gland in sheep and may be involved in the facilitated carrier-mediated transfer of urea-N into the saliva.

Urea Entry into the Small and Large Intestine:
In ruminants, up to 70% of the total portal-drained viscera flux of urea can enter post-stomach (small intestine) compartments [26] of which up to 90% of total portal-drained viscera flux of urea is to the mesentericdrained viscera in animals fed high fiber diets [11] as compared to only 19% in animals fed high concentrate diets [32]. However, most of the urea-N that enters post-stomach compartments is returned back to the ornithine cycle as NH3 for re-synthesis of urea [26]. Small amounts of urea-N are recycled to the hind gut (cecum and colon) and, even though bacteria residing in the hind gut utilize recycled urea-N for protein synthesis, because there are no mechanisms for digestion and absorption of microbial protein formed in the hind gut, it is eventually lost in the feces [33].

Factors Affecting Urea Recycling
All factors that influence the production, absorption, and transfer of NH 3 and urea will affect urea recycling in ruminants.
Kennedy and Milligan [10] reported that urea transfer to the rumen was inversely related to the rumen NH 3 concentration and suggested that the NH3 concentration was a factor regulating urea entry into the rumen. There was a marked reduction of urea transfer to the rumen when the ruminal NH 3 concentration was elevated by continuous NH 3 infusion into it. Tracer studies have indicated that a supplemental energy source such as grain, starch, or dried beet pulp, significantly increased endogenous urea degradation in the gastrointestinal tract. It is possible that the rumen was the site of increased degradation because Kennedy and Milligan, [10] reported that dietary sucrose greatly enhanced the rate of transfer of urea to the rumen. On the other hand, urea transfer from blood to the GIT might not be controlled by the urea concentration in plasma alone. In sheep and cattle, the upper limits of the blood urea concentration above which urea transfer was no longer linearly related to plasma urea concentrations were 6.0 mm and 4.0 mm, respectively. Elevation of plasma urea above these concentrations did not further increase rumen NH 3 . Norton et al. [39] found that transfer of urea into the post-ruminal tract is correlated with both plasma urea concentration and its production rate.

Inhibition: Increasing intraruminal ammonia concentration
decreases the urea flux across the rumen wall [40]. According to Remond et al. [41], ammonia absorption may be responsible for reducing urea flux. The effect of ammonia on urease activity is long term [42] rather than short term, and the mechanism by which ammonia regulates the trans-epithelial flux of urea during shortterm variations is not yet known.

Impact of Animal Factors on Urea Recycling: Production of
urea is largely a substrate-driven process. Thus, animal productivity can impact urea recycling by impacting how much N is available for urea synthesis. When cattle use more N for productive purposes (i.e., growth or lactation), less N are available for urea synthesis and therefore less urea are recycled to the gut. Bailey [43] compared urea recycling in forage-fed steers weighing 208 and 391 kg; the larger steers were physiologically more mature and deposited less N in tissue proteins than the younger steers. The more mature cattle had greater urea synthesis and greater urea recycling than the younger cattle that deposited more tissue protein. Thus, body protein utilization impacts urea recycling.

Urease Inhibitors in Ruminants:
Urea hydrolysis to ammonia in the rumen is very rapid, which can override its utilization by the ruminal microorganisms and lead to ammonia toxicity and wastage of nitrogen of feeds. Therefore, slowing down the urea hydrolysis may reduce ammonia loss and improve urea utilization.
Coated urea or slow release urea products as protein supplements could constantly supply ammonia to ruminal microorganisms for their growth without the potential toxicity associated with feedgrade urea [44], which may also improve nutrient utilization for low-quality forages and reduce plasma ammonia concentrations [45]. Another strategy, which has been explored for many years to decrease the urease activity in the rumen, is the use of urease inhibitors (Table 1). A number of urease inhibitors such as acetohydroxamic acid (AHA), phosphoric phenyl ester diamide (PPD), N-(n-butyl) thiophosphoric triamide (NBPT), boric acid, bismuth compounds and hydroquinone decrease ureolytic activity.
However, some of these compounds pose potential risks to animal and human health, thus precluding their use in production. These inhibitors usually work very well when tested in vitro [46][47][48][49].  [47] Vaccination, jack bean urease Calves a) Increased growth rate and feed efficiency [48] Conclusion Urea is one of the major non-protein nitrogen feeds for ruminants and the optimal utilization of urea in feed can alleviate to some extent the cost of dietary protein. Urea is hydrolyzed quickly by ureolytic bacteria in the rumen. The ability of the liver to detoxify NH3 to urea appears to be similar in ruminant and nonruminant species, the principal difference being that the production of NH 3 by foregut fermentation in ruminants is extremely variable and dependent on feed sources, whilst in non-ruminants, NH 3 is produced in the hindgut and, therefore, absorption into the portal vein is affected far less by diurnal feed cycles. Despite the high rates of uptake from the gut, which result from rapid fermentation of soluble N in forage diets, the ruminant liver is extremely adept at detoxifying NH 3 to urea. However, there is evidence to suggest that NH 3 detoxification to urea imposes a metabolic 'cost' in terms of amino acid deamination. This could explain observations of poor N retention in forage-fed ruminants, although further specific metabolic studies are required to identify mechanism which could explain this interaction of NH 3 with amino acid metabolism.
Also urea that is produced in the rumen is circulating in to gastro intestinal tract of ruminants.