Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition (1976)

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2 Mechanism of NPN Utilization in the Ruminant It has been almost a century since Weiske et al. (1879) reported that ruminants could convert NPN to protein. During the following 60 years, this subject was intensively researched by German nutritionists. Krebs (1937) reviewed their research and summarized the status of the field at that time. Studies on this subject in the United States began in Wisconsin. Hart et al. (1939) reported that either urea or ammonium carbonate was used by growing dairy heifers. They also found that dietary soluble carbo- hydrates increased NPN utilization. This was the forerunner of a series of experiments that had as a common goal the study of the metabolic aspects of NPN utilization by ruminants. Another landmark in NPN re- search was conducted by Loosli et al. (1949), who demonstrated that urea could serve as the sole dietary nitrogen source for lambs. Using the purified diet approach, they found that the 10 amino acids that are dietary essentials for the laboratory rat were synthesized within the rumen. Lambs fed these diets grew and remained in positive nitrogen balance during the test. Results of similar studies have yielded infor- mation on the mechanism of NPN utilization and have provided the facts for establishing the guidelines for the use of NPN in practical ruminant rations. Urea was approved in the United States as a feed ingredient in ruminant diets in 1940 by the Association of American Feed Control Officials.

4 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition AMMONIA AS A COMMON DENOMINATOR It is important to indicate that NPN compounds are normal constituents in the biological fluids of ruminants, even when NPN is absent from the diet. Also, natural feedstuffs that are fed to ruminants contain a variable amount of NPN. Thus, the ruminant continually uses NPN as a normal dietary and metabolic constituent. . Ammonia is the common denominator in the utilization of NPN by ruminants (Hungate, 1966). If the rumen microorganisms cannot de- grade the compound in question to yield free ammonia, it is useless as a nitrogen source to the microorganisms. When urea is the substrate, the following steps appear to be involved in its complete utilization: Microbial. Urease .' 2 2. Carbohydrates f Volatile Fatty Acids (VF A) + Keto Acids , j Microbial Enzymes 3. NH3 + Keto Acids ^ - Amino Acids 4. Amino Acids ' Microbial Protein c »*. u- i n * . Animal Enzymes in the „ 5. Microbial Protein -re - * — TT - ,. T , — r= - •- Free Abomasum and Small Intestines Amino Acids 6. Free amino acids are absorbed from the small intestine and used by the host animal. Similar schemes would be appropriate for other NPN sources if en- zymic action is needed for hydrolysis. However, different enzymes may be involved for each NPN compound. Bloomfield et al. (1960) reported that step number one usually proceeds at a faster rate than step number two. This is especially true if the lignocellulose complex of poor-quality forages is the primary carbohydrate source in the diet. In this case the keto acids necessary for amino acid synthesis are limiting; thus there may be a considerable loss of ammonia through the ruminal wall, re- sulting in poor utilization of dietary nitrogen. If the rate of urea intake

Mechanism of NPN Utilization in the Ruminant 5 is reduced in such diets to conform to the rate of cellulose hydrolysis, the efficiency of nitrogen utilization can be improved (Campling et al., 1962). The kinetics of urea metabolism are presently receiving much study, and the basic experiments of Nolan and Leng (1972) are typical of these studies. Biuret metabolism in the ruminant is also an active field of study (Tiwari et al., 1973a,b). It is beyond the scope of this review, however, to cover the kinetics of NPN utilization. AMMONIA PRODUCTION IN THE RUMEN Most ruminal bacteria prefer ammonia to amino nitrogen for the syn- thesis of microbial protein (Bryant, 1963; Hungate, 1966). Ammonia can arise from the degradation of dietary protein, microbial protein, and NPN compounds. In addition, urea is recycled to the rumen via saliva and through the wall of the rumen. A majority of dietary protein is hydrolyzed to peptides and amino acids by microbial enzymes. The free amino acids and peptides may be incorporated into microbial pro- tein or deaminated with the production of ammonia and volatile fatty acids (VFA), which may be absorbed from the rumen or used by mi- crobes as carbon skeletons for amino acid synthesis. The level of di- etary protein and its solubility greatly influence ammonia production, which in turn affects the utilization of dietary NPN compounds. This important aspect has been discussed by Church (1969). The optimum concentration of ruminal ammonia required for maxi- mum cell yield has not been established. This is understandable, per- haps, because the concentration depends upon such factors as level of feeding, solubility of dietary protein, availability of carbohydrates and minerals to the microbes, frequency of feeding, etc. Recent in vitro research by Satter and Slyter (1974) indicated that the tungstic acid precipitable nitrogen was 90 percent of maximum when NH3 concen- tration was 1-2 mg NH3-N/100 ml fluid. Increasing the ammonia con- centration to 8 mg increased the output some. In vivo results by (Slyter et al. (1973) indicate that nitrogen retention of steers was improved by maintaining ruminal ammonia concentrations above these values. E. L. Miller (1973) also studied this problem in vivo and reported that the greatest microbial flow from the rumen was achieved with rumen am- monia concentration of approximately 28 mg NH3-N/100 ml fluid. Perhaps the increased performance with the higher ammonia concentra- tions in vivo than in vitro can partially be explained by the possible beneficial effects of ammonia outside the rumen, i.e., synthesis of non- essential amino acids in the liver, etc. This area is receiving considerable research interest at the present time.

6 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition As indicated earlier, NPN must first be converted to ammonia; the reaction is mediated by microbial enzymes. In the case of urea, the hydrolytic enzyme is urease, which is produced by many species of ruminal bacteria (Slyter et al, 1968). Urea is rapidly hydrolyzed under most ruminal conditions. In fact, one danger of high levels of urea feeding is excess ammonia production, which may lead to ammonia toxicity. Results of studies on methods of inhibiting urease activity (Tillman and Sidhu, 1969) have not yielded useful information for practical application. Most ammonium salts, such as ammonium sulfate, ammonium phosphate, and ammonium salts of volatile fatty acids are ionized in the rumen and do not have to undergo enzyme hydrolysis. In contrast to urea, both biuret and uric acid are hydrolyzed by in- duced enzymes (an enzyme that only appears after the substrate is present), biuretase and uricase, respectively. A long period (3 weeks) is needed for the induction of peak biuretase activity, while uricase (Oltjen et al, 1968) is induced at a faster rate. Because of biuret's slow rate of hydrolysis and lack of toxicity, even when used at high dietary levels, some workers feel that it might become the NPN source of choice when it is fed to supplement the diets of ruminants grazing or fed low-quality roughages. AMMONIA METABOLISM BY RUMEN MICROORGANISMS A wide variety of NPN compounds will support growth of ruminal bac- teria in vitro (Belasco, 1954; Henderickx, 1967). Allison (1969) re- viewed the biosynthesis of amino acids by rumen bacteria. In general, amination and transamination reactions appear to be responsible for the major part of ammonia assimilation by the microflora. Glutamic dehydrogenase (Hoshino et al, 1966) plays a key role in the initial fixation of ammonia to a carbon skeleton, and glutamate-oxaloacetate and glutamate-pyruvic transaminases are important in the transfer of ammonia to other carbon skeletons, which are present in rumen fluid. Other dehydrogenase and transaminase enzyme systems also play a part in ammonia assimilation by rumen bacteria (Chalupa, 1972). Rumen microflora can use NPN for protein synthesis if the necessary carbon skeletons are present or if these can be synthesized fast enough from dietary carbohydrate or alternate carbon sources. The most im- portant single fermentation characteristic is the amount of fermentable energy available in the diet for microbial growth (protein synthesis) above that needed for maintaining equilibrium in the rumen between the feed protein degraded and the microbial protein resynthesized.

Mechanism of NPN Utilization in the Ruminant 7 Burroughs et al. (1971d,e, 1974a) proposed a system for the evaluation of feeds based on estimated urea fermentation potential (UFP). The UFP was estimated on the basis of the fermentable energy of a given feed and the amount of the feed or diet protein degraded in the ru- men. This system recognizes that microbial protein synthesis is pri- marily dependent upon energy availability and that the conversion of rumen degraded dietary protein into microbial protein represents an energy cost. Primary sources of carbon fragments that arise from carbohydrate fermentation are CO2 and VFA's. However, there are specific require- ments for isobutyrate, indole-3-acetate, isovalerate, 2-methylbutyrate, and phenylacetate to provide for the synthesis of the specific amino acids (Allison, 1969). There are potential sources of keto acids in rumen fluid, but the branched-chain VFA'S arise mainly from the deamination of branched-chain amino acids provided by dietary protein. It is significant that the feeding of protein-free diets causes a depression in the concen- tration of these acids (0rskov and Oltjen, 1967; Oltjen, 1969; Chalupa et al., 1970), with isovalerate and isobutyrate being greatly influenced. However, evidence regarding a possible need to supplement high-urea diets with a combination of branched-chain VFA is not clear-cut; some researchers obtained increased responses (Hemsley and Moir, 1963; Cline et al., 1966; Hume, 1970), while others have received little re- sponse (Oltjen et al, 1971). Including branched-chain VFA in the diet did not change the rumen protozoa numbers, cellulolytic bacterial numbers, nor the microbial amino acid composition (Oltjen et al., 1971). This aspect of NPN utilization needs further study, especially when high-forage or high-NPN-containing diets are fed. The high levels of the enzyme activities present in the rumen would indicate that insufficient carbon skeletons could limit ammonia assimi- lation. This observation is in agreement with the calculations of Balch (1967) showing that the level and type of dietary carbohydrate has a large influence on the efficiency of NPN utilization. Although many predominant species of rumen bacteria have been studied in pure culture, the microflora are complex and much remains to be learned about microbial interrelationships. This makes it difficult to generalize about requirements for certain nutrients, especially the B vitamins. It appears that specific B vitamins are needed by certain strains of ruminal bacteria; and, if they are not present at adequate con- centrations in the rumen fluid, growth of some bacterial strains may cease. It is known that rumen bacteria produce as well as use certain B vitamins (Bruggemann and Giesecke, 1967). Apart from their impor- tance in the metabolism of the host animal, B vitamins may play an

8 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition important part in regulating microbial interrelationships in the rumen. It has been reported that dietary urea appears to stimulate B-vitamin synthesis in the rumen (Teeri and Colovos, 1963; Briggs et al., 1964). Hunt et al. (1954) found that rumen microbes, which use urea and decompose cellulose, have a requirement for sulfur that can be met by sulfate; sulfate is reduced to sulfide prior to being incorporated into the amino acids, cystine, and methionine. Bruggemann et al. (1962) found that there is an increase in the number of sulfate-reducing bac- teria in rumen fluid when protein is replaced by urea. Several workers have reported that the N:S ratio in NPN-containing rations for sheep should be about 10:1 for the most effective utilization of these com- pounds. In cattle the ratio should be 12-15:1. The species difference is probably due to the requirement of the sheep for more sulfur- containing amino acids for wool production. There is no evidence to indicate that the sulfur needs of the ruminal microbes from sheep are different than those of cattle. Minerals are required by rumen bacteria and the host animal, but substitution of NPN for dietary protein does not increase requirements for these beyond those stated in present feeding standards. Since NPN and grain are combined in a mixture to replace protein supplements in isonitrogenous diets, it must be remembered that this combination of NPN plus carbohydrate source may contain lower levels of essential minerals than does the replaced protein supplement. Therefore, the use of NPN compounds in ruminant diets makes it quite important for nu- tritionists to carefully consider the amounts and balance of all nutrients in diets containing NPN. QUANTITY OF MICROBIAL PROTEIN SYNTHESIZED IN THE RUMEN Many workers have conducted research that points to the abilities and limitations of ruminal bacteria to synthesize microbial protein. Hungate (1966) has summarized these factors. Synthesis of microbial protein from dietary nitrogen depends upon the amount and the nature of dietary constituents as well as the amount of high-energy materials, primarily ATP, that can be derived from these. Since the rumen is anaerobic and the major substrate is carbohydrate, the amount of di- gestible carbohydrate represents the level of energy-yielding materials. Also, oxygen is needed for oxidation of carbohydrate and energy pro- duction; thus, efficiency of protein synthesis is much lower in anaerobic than aerobic systems. Hungate (1966) suggested that a cell yield of 10-20 percent in anaerobic systems would be expected. Purser (1970), using Hungate's data, estimated a yield of 18.3 g of digestible protein

Mechanism of NPN Utilization in the Ruminant 9 for each digestible megacalorie in ruminants. Using the value proposed by Purser (1970) and the National Research Council (NRC) (1971b) requirements for protein in dairy cows, Chalupa (1972) calculated that a dairy cow entirely dependent upon the microbial protein synthesized in the rumen for milk protein synthesis would produce only 10 kg of milk per day. This value is close to the quantity of milk actually pro- duced by cows fed protein-free urea containing purified diets (Virtanen, 1966). The cow actually produces considerably more than 10 kg of milk daily, leading Chalupa (1972) to suggest several possible explana- tions: (1) that the NRC requirements for digestible protein are too high, (2) that greater than anticipated yields of ATP are obtained, (3) that at least 50 percent of the dietary protein was not degraded in the rumen for the synthesis of microbial protein, and (4) that the combination of microbial protein and dietary protein, which presumably bypassed the rumen (50 percent) was just sufficient to meet the digestible protein re- quirement as set by the NRC. A number of recent studies (Hogan and Weston, 1970; Lindsay and Hogan, 1972;0rskovera/., 1972; Bucholtz and Bergen, 1973;Thomas, 1973) indicate that microbial yields considerably greater than 18.3 g of digestible protein per digestible megacalorie occur in the rumen. Data from these and other reports indicate that the cell yield in the rumen may range from 10 to 30 g of digestible protein for each digestible megacalorie. It is not surprising that a range is indicated because of such factors as nutritional adequacy of the diet, availability of cofactors, spe- cific microbial population in the rumen, turnover rate, lysis, etc. It is important to clearly define the cell yield to more precisely determine the usefulness of NPN in ruminant diets. It has been known for many years that some dietary protein, depend- ing upon its solubility and upon rumen conditions, does bypass ruminal degradation. If dietary protein is replaced with NPN, less protein is avail- able for ruminal bypass. Therefore, the replacement of too much dietary protein with NPN compounds beyond certain limits could lead to pro- tein deficiencies in high-producing animals. Ruminants fed urea-contain- ing purified diets presented 10-30 percent less protein to the abomasum (Tucker and Fontenot, 1970) than those fed isolated soy protein con- taining purified diets. It is possible that future research on protein nutrition in ruminants will reveal methods of feeding NPN that meet the nitrogen requirements of the bacteria and enable the animal to obtain all of the microbial pro- tein possible by this means. In addition, the animal may be fed dietary protein that has been treated with formaldehyde, tannins (Peter et al, 1971;Driedgerand Hatfield, 1972; Nimrick etal., 1972), or other ma-

10 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition terials to effectively reduce or prevent microbial degradation in the rumen. By the proper selection of the treated dietary proteins and/or "protected" amino acid supplements, the quantity and quality of pro- tein presented to the abomasum could support optimum production in the ruminant with a minimum of preformed dietary protein. QUALITY OF MICROBIAL PROTEIN SYNTHESIZED IN THE RUMEN Chalupa (1972) summarized much of the available information on bac- terial vs. protozoal protein found in rumen fluid and reported the fol- lowing averages, respectively: crude protein content, 55 vs. 38 percent; true digestibilities, 66 vs. 88 percent; biological value, 78 vs. 77 percent; and net protein utilization, 55 vs. 67 percent. He points out that the lower than usual crude protein percentages reported were probably due to contamination of the samples with digesta found in rumen fluid. Chalupa (1972) also constructed aminograms on bacterial and proto- zoal proteins using available data in the literature. Higher quantities of leucine, phenylalanine, lysine, and tyrosine were found in protozoal protein, indicating that it has superior nutritional value, which is borne out by results of feeding trials with laboratory nonruminants. Allison (1969) found that the amino acid pattern in the bacterial cell wall was different from that of cellular constituents. Using the data of Hoogen- raad and Hird (1970), Chalupa (1972) plotted aminograms of cell wall and non-cell wall constituents and reported that cell walls contained lower levels of all amino acids. The cell walls of bacteria account for about 15 percent of the dry weight of these microbes; however, they are more resistant to the proteolytic enzymes. Thus, their presence reduces the digestibility of the bacterial protein. Therefore, the net utilization of bacterial protein, which contains a greater proportion of cell walls, is less than that of protozoal protein. These results indi- cate that it would be desirable to alter the ratio of bacteria to protozoa by increasing numbers of the latter. Abou Akkada and El-Shazly (1965) reported a significantly greater nitrogen retention by faunated sheep compared to defaunated sheep. Experimental results are not always in agreement: High-roughage diets promote a protozoal biomass nearly as great as that of bacterial, while high-grain diets reduce or completely eliminate protozoa. Yet the biological value of rumen fluid dry matter from high-grain diets was higher than that of hay-fed steers (Little et al, 1965), suggesting that other factors were involved. Smith et al. (1969) reported that 80 percent of the nitrogen found in ruminal bacteria was in protein and 20 percent was in nucleic acid. Nucleic acid in rumen fluid is primarily of microbial origin. When nu-

Mechanism of NPN Utilization in the Ruminant 11 cleic acids were added to rumen fluid in vitro, both RNA and DNA were degraded rapidly (Smith and McAllan, 1970). There was no degra- dation in the abomasum. Consequently, ruminants present a high and continuous supply of nucleic acids to the small intestine. Digestibility of nucleic acids in the small intestine is high (Smith and McAllan, 1970). Condon (1971) has studied the metabolic fate of nucleic acids reaching the small intestine of sheep. His results are as follows: 1. Lost to the host animal-63 percent a. Undigested and lost in feces-20 percent b. Excreted as purine derivatives in urine-43 percent 2. Possible value to the host animal-37 percent a. Contribution to ammonia pool from degradation of purine bases-11 percent b. Contribution to ammonia pool from degradation of pyrimidine bases 10 percent c. Contribution to ammonia pool from degradation of aminoiso- butyrate and beta-alanine -16 percent. Nucleic nitrogen is of limited value to the animal, thus a high production of nucleic acid in the microbial protein increases the loss of nitrogen. Replacement of dietary protein with NPN compounds increases the pro- portion of microbial protein and increases the nucleic acid content of protein presented to the abomasum and the small intestine. Protein requirements of ruminants, as is true of all animals, must be evaluated in terms of the amounts of amino acids absorbed from the intestinal tract in relation to those needed for productive purposes. Amino acids found in the abomasum and the small intestine have their origin in microbial protein, dietary protein that escaped rumen degrada- tion, and endogenous secretions. The amount of degradation of dietary proteins is greatly dependent upon solubility of the protein in rumen fluid. Chalupa (1972) has considered the solubilities of various protein sources relative to rumen bypass and the factors affecting solubilities. The solubility of dietary protein is important in the utilization of am- monia released from NPN compounds, because certain proteins may be degraded to ammonia in the rumen as quickly as the NPN compound itself, while others are less soluble. The optimum condition would be to formulate diets using the less-soluble dietary proteins with the more- rapid, ammonia-releasing compounds in order not to have an excess of ruminal ammonia. It would be desirable to decrease ruminal proteolysis and/or deamination, thereby forcing the microbes to use ammonia from NPN sources. Also, it requires energy to synthesize amino acids. Wohlt

12 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition et al. (1973) have classified many proteins according to their solubility. Heat treatment reduces solubility of oil meal proteins, thereby increas- ing their nutritive values for ruminants (Sherrod and Tillman, 1962, 1964). The problems of the elucidation of the total amino acid requirements of ruminants for productive purposes and the determination of whether specific diets cause amino acid deficiencies or imbalances are complex; the main complication concerns the varying degrees of microbial altera- tion of dietary proteins in the reticulorumen. These problems have been studied by different methods: Some have examined plasma amino acid profiles of ruminants receiving postruminal administrations of a single amino acid or mixtures of amino acids, while others have measured the performance of ruminants as well as monitored the plasma profiles. Treatment of dietary proteins to reduce or inhibit microbial degradation in the rumen has also been tested by others. Postruminal administration of specific amino acids and protein of high biological value offers promise of increasing the production of rumi- nants (Reis and Schinckel, 1964). This subject has been reviewed by L. F. Nelson (1970). In sheep, the first limiting amino acid is methionine, and an excellent response was obtained (Schelling and Hatfield, 1968) when supplemental methionine was placed in the abomasum. Nimrick et al. (1970a,b,c) found that, after the requirement for methionine was met, lysine and threonine became limiting. Cattle fed urea-containing purified diets had lowered plasma concentrations of valin&, isoleucine, leucine, and phenylalanine, but increased levels of serine and glycine (Oltjen and Putnam, 1966;Oltjen, 1969) compared to cattle fed iso- lated soybean protein. Also, less dietary nitrogen was retained when the cattle were fed urea-containing diets. When combinations of valine, isoleucine, leucine, and phenylalanine were infused into the abomasum of steers, the utilization of the urea-containing diet was improved, almost equaling the performance of steers fed the soy protein-contain- ing diet (Oltjen et al, 1970). These workers also found that the infusion of glycine and serine depressed the utilization of the soy protein diet, demonstrating the importance of amino acid balance in ruminants. In general, dietary supplements of amino acids in ruminant diets have not given consistent responses, regardless of the response criteria used. It must be emphasized that the urea-containing purified diets em- ployed by the University of Illinois and USDA (Beltsville, Md.) re- searchers contained NPN as the sole nitrogen source making the protein in the abomasum solely of microbial and endogenous origin. This is a much different condition from that found with practical diets in which urea furnishes a smaller percentage of the total dietary nitrogen. However,

Mechanism of NPN Utilization in the Ruminant 13 even when practical diets containing some urea were fed to cattle, plasma levels of valine, isoleucine, leucine, and lysine were lower (Little et al, 1969) than those found in cattle fed diets containing only natural protein. Because gains were also lower in urea-fed steers, the importance of amino acid supplementation at the lower gut when urea is fed at high levels is emphasized. If further research identifies the amino acids needed by ruminants under specific conditions, development of prod- ucts for incorporation in the diet to bypass degradation in the rumen appears feasible. This is presently an exciting field for research, but the necessary practical guidelines must await results of future experi- ments. AMMONIA METABOLISM IN THE HOST ANIMAL Ammonia is absorbed from the reticulorumen as well as the omasum, small intestine, and cecum. The reticulorumen is considered to be the largest absorption area. The liver, as well as the mucosal cells of the reticulorumen, can use ammonia. The mucosal cells contain transami- nases, and glutamine synthesis has been reported here (Hoshino et al, 1966). In an extensive study of the mucosal cellular structure, Chalupa et al., 1970) found that transaminase activities of mucosal cells were lower per unit of cellular materials than in liver cells. The large mass of mucosal cells (almost 1 percent of the ruminant's body weight) would indicate that these cells also play a role in the overall metabolism of dietary nitrogen by this animal. AMMONIA TOXICITY Ammonia is a weak base with a pKa of 8.8 at 40° C; therefore, there is a close relationship between ruminal fluid pH and the ratio of ammonia to ammonium ions. The lipid layer of the rumen mucosa is permeable to ammonia, allowing rapid absorption. Also, the alkaline buffering capacity of rumen fluid is not great in comparison with its ability to buffer acids. Because of poor management or improper formulation, the feeding of high levels of dietary urea may result in a rapid accumu- lation of ammonia in rumen fluid. This is accompanied by a rise in rumen fluid pH with rapid absorption of ammonia across the rumen wall. When the rate of ammonia absorption exceeds the capacity of the liver to convert it to urea, ammonia accumulates in the blood and toxicity may result. Lewis et al. (1957) reported that changes in the rumen ammonia con-

14 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition centration of sheep fed various diets were paralleled by changes in portal blood ammonia levels. Peripheral ammonia did not increase until the ruminal levels exceeded 60 millimoles/liter. Toxic symptoms were ap- parent when the concentration in the peripheral blood exceeded 0.6-0.9 millimoles/liter. Acute ammonia toxicity symptoms in the ruminant ap- pear to be progressive as follows: The animal becomes nervous and un- easy, salivates excessively, and demonstrates muscular tremors; these symptoms are followed by incoordination, respiratory difficulty, and frequent urination and defecation; the front legs begin to stiffen, and the animal becomes prostrate; violent struggling, bellowing, and termi- nal tetanic spasms are found in most animals; the jugular pulse is marked, and bloating is common; death occurs within 0.5-2.5 h after the initial symptoms are observed. It appears that rumen fluid NH3-N levels of 80 mg/100 ml will cause toxicity and can be used as a diagnostic tool. Blood NH3 -N levels causing toxicity are difficult to determine, and it is suggested that levels of 1 mg/100 ml will cause toxicity in cattle. There are usually no char- acteristic lesions found on necropsy examination; however, congestion, hemorrhages, and pulmonary edema are common. Predisposing factors to urea toxicity in cattle appear to be (1) lack of an adequate adaptation period to urea-containing diets, suggesting that it is important to start feeding urea at low levels and increase gradually over a period of several days, especially if high levels of urea are fed; (2) fasting prior to urea consumption; (3) the feeding of urea in diets composed primarily of poor-quality roughages; (4) the feeding of diets that promote a high pH in ruminal fluid; and (5) low water intake. Er- rors in formulation and improper mixing of urea with other diet in- gredients are probably the major factors causing urea toxicity in the feeding of ruminants. An effective treatment for urea toxicity for cattle, if applied before tetanic spasms occur, is to immediately administer 20-40 liters of cold water orally. Cold water will lower ruminal fluid temperature and thereby reduce ureolysis. It will also dilute the concentration of am- monia and reduce its rate of absorption from the rumen. Four liters of either dilute acetic acid or vinegar given with cold water is more effec- tive than cold water alone. Acetic acid will neutralize the toxic effects of free ammonia. Also, Coombe et al. (1960) and Hogan (1961) have reported that ammonia is absorbed through the ruminal wall at a much faster rate at a high ruminal pH than at a low ruminal pH. There is concern among animal production specialists regarding the possibility that ammonia toxicity in some members of the cow herd will increase the incidence of abortions in the surviving cows. It is felt

Mechanism of NPN Utilization in the Ruminant 15 that high levels of blood ammonia might be toxic to the fetus, even though the cow survives. Oklahoma researchers (Word et al., 1969) induced severe toxicity symptoms in pregnant cows and then averted death by acetic acid treatment. They found no abortions in any of the cows, even though toxicity symptoms were well advanced in all cows be- fore acetic acid was administered, indicating that the fetus is resistant to high levels of blood ammonia. Also, subsequent reproductive perfor- mance was not affected in cows subjected to this condition. There ap- pears to be little likelihood of urea toxicity in ruminants if proper levels are fed and judicious management practices are used. SUMMARY AND CONCLUSIONS NPN compounds are widely used as dietary nitrogen sources for rumi- nants, but are of little, if any, value in nonruminant diets. For this reason, the following points apply almost entirely to ruminants: 1. Ammonia is the common denominator in the utilization of NPN compounds by ruminants. Microorganisms living in the rumen produce enzymes that hydrolyze the dietary NPN source to yield ammonia. Also, there is simultaneous enzymatic hydrolysis of complex dietary carbohydrates to produce carbon skeletons, which combine with am- monia to form amino acids. These amino acids are used for the syn- thesis of microbial protein, which is later digested, absorbed, and utilized by the animal for productive purposes. Most rumen bacteria prefer ammonia nitrogen to that supplied by peptides and amino acids, suggesting that some dietary NPN in rumi- nant diets is desirable for maintaining a viable rumen microflora. 2. The quantity of protein synthesized in the rumen by micro- organisms depends upon the nature and amounts of dietary con- stituents, a point that will be discussed in much detail in later sections of this report. However, if all dietary factors are present in the rumen and in optimum proportions, it appears that yields ranging from 10- 30 g of digestible protein for each digestible megacalorie can be ex- pected. In purified-diet studies in which urea furnished all of the dietary nitrogen, thereby causing the animal to be entirely dependent upon microbial protein for supplying its requirements for growth or milk production, it was found that rates of growth and production were 65 percent of optimum. These results indicate that the rate of protein synthesis might be too slow, the quality of the microorganism protein might be poor, or both.

16 Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition 3. The quality of a bacterial protein is lower than that of protozoal protein; however, attempts to shift ruminal ratios of bacteria to proto- zoa have not always improved results. Microbial protein contains about 20 percent nucleic acid, which is not utilized efficiently by the animal for protein synthesis. Also, the high proportion of cell walls in the mi- crobes reduces protein quality. When cattle are fed diets in which urea is the only nitrogen source and compared to control animals receiving natural protein, they have slower rates of growth and lower blood levels of isoleucine, leucine, phenylalanine, and valine, but increased blood levels of glycine and serine. 4. Major research is needed on the means of feeding sufficient NPN to maintain a viable microbial population that in turn would supply the major portion of the protein needed by the host animal. Accompanying research is needed to determine how to protect high-quality dietary pro- tein from ruminal degradation. The development of such methods would make it possible to efficiently use NPN sources in diets of high- performance animals. 5. Prevention of urea toxicity is a management function; and, if proper management is exercised, the incidence of ammonia toxicity is extremely low. Toxicity symptoms were described, and treatment methods were suggested. Research results indicate that high levels of rumen and blood ammonia do not increase the incidence of abortions in cow herds. Furthermore, subsequent reproductive performances of cows surviving high levels of rumen and blood ammonia were not affected.