1. IMPORTANCE AND USE OF NONPROTEIN NITROGEN AND UREA

Brief historical summary

Urea was discovered in 1773 by Rouelle and its composition established by Prout in 1818. From the formula

it can be calculated that pure urea contains 47 percent of nitrogen compared to 16 percent for most proteins. The first synthesis of urea, in 1828, is credited to Wohler who evaporated an aqueous solution of ammonium cyanate to dryness. Around 1880 Weiske and associates in Germany proved that the addition of asparagine to a basal ration fed to sheep gave higher nitrogen retention. This led, in part, to the suggestion of Zuntz in 1891 that the rumen microflora were able to break down cellulose as a source of energy and convert NPN into true protein. Tappeiner in Germany in 1884 had reported that large quantities of volatile fatty acids, especially acetic, were produced as a result of the fermentation of carbohydrates within the rumen. In 1900, Kellner reported that two sheep retained an average of 0.6 gram nitrogen on a basal ration compared with 2.4 grams when either ammonium acetate or asparagine was added to the basal feed. During the period 1904 to 1925, Morgen found that urea could replace 30 to 50 percent of the protein in rations of cattle and sheep, and Voltz demonstrated that growth of lambs occurred on a diet of starch, alkali-washed straw, minerals and urea. Scarcity of vegetable proteins for feeds during the first world war stimulated research on urea synthesis and on its use in ruminant feeds in Germany; this work has been reviewed in detail by Krebs (1937). Not all studies gave favorable results. In summarizing the earlier research, Krebs concluded that there remained considerable doubt whether NPN compounds were converted to protein in amounts significant to ruminants. The apparent protein-sparing action of urea was suggested to result from the neutralization of organic acids in the rumen by ammonia. It is now clear that some of these failures resulted from the fact that the rations fed were often too high in true protein to demonstrate a value for the NPN source being tested. Research in the United Kingdom and the United States produced evidence supporting the view that urea could effectively replace part of the protein in rations for ruminants. Bartlett and Cotton (1938) in the United Kingdom reported that when urea supplemented the protein in a ration for young cattle, satisfactory growth resulted. Hart et al. (1939) found that urea or ammonium bicarbonate could replace some of the plant proteins for growing cattle and yield muscle tissue of normal protein content. At the same time, Work and Henke (1939) in Hawaii demonstrated the value of urea for dairy heifers. Studies with lactating cows (Archibald, 1943; Owen, Smith and Wright, 1943; Rupel, Bohstedt and Hart, 1943) also showed that urea could replace part of the vegetable protein sources with resulting satisfactory milk yield and composition. A number of nitrogen balance studies, as reviewed by Reid (1953), produced evidence that urea-nitrogen fed to ruminants was indeed retained in the body, and that the tissues of the growing animals were of normal composition. This demonstration by several groups of workers that young cattle gained body weight much more rapidly, over a substantial period of time, when urea was added to low-protein diets that otherwise would support little or no weight gain, was accepted as strong evidence that the urea was utilized for growth. Subsequently, digestion studies showed that urea supplements sometimes increased the digestibility of cellulose and crude fiber of low-protein rations. Balance studies gave evidence of increased nitrogen retention by animals that gained extra weight with supplemental urea. In vitro fermentation techniques and analyses of rumen ingesta were useful in showing that, as urea or ammonia decreased, true protein content increased in the fermentation medium. Finally chemical and microbiological analyses and the use of tracers removed all doubt that urea nitrogen was, in fact, converted in the rumen into amino acids and true protein which subsequently appeared as tissue- and milk-proteins.

A critical shortage of vegetable protein feeds for feeding livestock, during the war period of the early 1940s, stimulated widespread use of urea in the United States to replace protein sources in the practical feeding of cattle and sheep. Its initial usage was temporarily set back because of the death of a few cattle from urea toxicity, owing to improper mechanical mixing and the resulting excessive intake of urea. Not until the 1950s did urea become a generally accepted ingredient in cattle feeds following extensive research demonstrating its safety and usefulness in many kinds of rations.

Schoenemann and Kilian (1960) considered the research published in Germany after Krebs' (1937) review. They concluded that the prewar German work did not prove that considerable amounts of urea could be utilized because the methods used in the experiments were not good enough to establish proof. These authors fed N¹⁵ and showed that 8 percent of the dose was excreted in the urine. The milk of goats contained 15 percent of the N¹⁵ urea administered, clearly showing that urea was utilized, in agreement with earlier data (Watson et al., 1949). They tried administration of urea twice a day, every four hours and also continuously, through a rumen fistula, without finding any definite difference in growth rate of lambs.

Importance of urea as a feed ingredient

World industrial capacity for the production of urea was estimated to be 1.91 million metric capacity tons in 1959. This increased to 4.82 million by 1963 and was expected to reach 9 million metric tons by 1966 (Hodges, 1965). In 1959 three fourths of the productive capacity was in the United States and Japan and most of the rest in western Europe. By 1970 it is anticipated that more than 50 percent of all urea production capacity will be in countries other than the United States, Japan or western Europe.

Table 1. - Estimated production and importation of urea in the United States

Source: Hodges, 1965.
¹ Imports minus exports.

Most of the urea produced is used as fertilizer. Table 1 shows the total production of urea in the United States, the amount produced for feeding purposes and the net imports. There are no accurate data on the amount of urea used as feed for ruminants. In addition to that produced specifically for feed, some fertilizer-grade urea and some of the urea imported are used for feeding purposes. Hodges estimated that as much as 190,900 metric tons may have been used for feed in the United States in 1963 and suggests this figure could increase to 250,000 metric tons by 1970. In the United Kingdom the estimated use of urea as feed reached 1,600 metric tons in 1965. Figures are not available for other countries, but large quantities are used in the U.S.S.R. and eastern European countries.

Nonprotein nitrogen compounds used in ruminants feeds

Several NPN compounds have been studied as ingredients of livestock feeds. Some of these are shown in Table 2. Belasco (1954) assayed the availability of the nitrogen in most of these compounds, for growth of mixed rumen cultures, by using an in vitro fermentation procedure. All compounds proved useful sources of nitrogen except biuret, but none exceeded urea in availability. In addition to the compounds listed, asparagine was extensively studied by the early German scientists as an ingredient of livestock feed, and ammonium formate has been tested more recently with some success.

Table 2. - Nonprotein nitrogen sources for ruminants

¹ Nitrogen × 6.25. –
² Feed grade urea is diluted with varying amounts of materials to prevent lumps forming.
³ Includes cottonseed, soybean, linseed, coconut and similar meals from which the oil has been extracted.

Mechanism of urea utilization

When urea from feed sources enters the rumen, it is rapidly dissolved and hydrolyzed to ammonia by bacterial urease. The ammonia can then be utilized by the bacteria for synthesis of amino acids required for their growth. Amino groups are also split from amino acids and from intact proteins and used by bacteria in the same manner. Protein synthesis within the rumen by micro-organisms is very closely associated with the activity of these same organisms in breaking down cellulose and other carbohydrate materials and in the formation of organic acids as by-products of this fermentation process. The solubilities of natural proteins vary greatly and thus the rate at which they are hydrolyzed and utilized by bacteria differs appreciably. There is evidence, however, that a fairly high proportion of the more soluable proteins such as casein will be utilized by bacteria in about the same way as the ammonia from urea. For the less soluble proteins such as zein, the process of ammonia liberation is much less rapid and fairly large proportions of the protein may pass through the rumen to the abomasum without being broken down. When ammonia is produced too rapidly in the rumen or if the concentration becomes too high, appreciable amounts are absorbed directly into the bloodstream, reconverted to urea in the liver, excreted through the kidneys in the urine and thus lost to the animal. There is, however, always a small amount of urea in the bloodstream and other body fluids. This urea finds its way into the saliva and re-enters the rumen. Urea has been shown to pass into the rumen directly through the rumen wall from the circulating blood.

Schmidt-Nielsen et al. (1957) observed that camels had an unusual ability to conserve nitrogen under conditions of nutritional stress. On normal rations which supplied ample energy, protein and water, 40 percent of the urea filtered in the glomeruli of the kidneys was excreted in the urine, but on a low-protein ration only 1 to 2 percent of the urea was excreted, the rest being recycled into the rumen. The ability to recycle and utilize urea differs among various species of ruminants. Water intake and the nature of the ration also influence the response.

Houpt (1959) used an isolated rumen procedure, in anesthetized sheep, to demonstrate that urea was secreted from the bloodstream into the rumen in amounts about 15 times greater than by way of saliva. This secretion or recycling appears to occur under normal conditions. It has been proposed that this mechanism will supply nitrogen to preserve the rumen microbial population when the feed supply is limited or of very low nitrogen content.

Watson et al. (1949) fed urea labeled with N¹⁵ to sheep twice daily in gelatin capsules just before the regular feed of a low-protein basal ration. The sheep were slaughtered after being fed the labeled urea for 4 days, and their blood, liver and kidneys were examined for N¹⁵-containing protein. Since the protein separated from these tissues contained N¹⁵ in excess of the amount found in control animals, it was concluded that urea was utilized by ruminants for the formation of body protein.

By feeding to lambs purified diets in which urea was the only source of nitrogen (Loosli et al., 1949), it was demonstrated that the 10 amino acids essential for growth were all synthesized in the rumen. The lambs gained in body weight and produced wool of normal amino-acid composition. They remained in positive nitrogen balance over several months of the test.

Virtanen (1966) reported the production of more than 4,000 kilograms of milk in a year by cows fed diets in which urea and ammonium salts were the only nitrogen sources, along with purified carbohydrates as the energy source. The milk protein was normal in amount and composition as were also the water-soluble vitamins. These studies and many others leave no doubt of the effective conversion of urea nitrogen into tissue and milk proteins of ruminants.

A great many in vitro studies have been carried out in the past 20 years on the activity of rumen microflora and the utilization of NPN compounds. The literature relative to the role of specific rumen bacteria in the synthesis of protein has been reviewed by Doetsch and Robinson (1953). Some of the different major types of bacteria found in the rumen have been isolated and their growth requirements defined. While these studies have contributed greatly to the understanding of the reactions that occur in the process of digestion in ruminants, the results are not reviewed in this study because interest is here largely directed toward the practical use of urea in feeding livestock.

Biological value of microbial protein

Numerous experiments with cattle and sheep have led to the view that the quality of dietary protein is of relatively little importance because all nitrogen sources are largely converted to microbial protein in the rumen and the host animal is presented with protein of more or less standard quality regardless of its diet. Research has shown that the biological value of proteins is much less variable for ruminants than for nonruminants.

Johnson et al. (1942) found biological values of approximately 60 for lambs on a 12 percent protein diet regardless of the protein source, which agreed closely with the results of Harris and Mitchell (1941). Analyses of bacterial protein show lower values for methionine than are found in high-quality food proteins. Following this lead, Loosli and Harris (1945) observed that methionine supplements improved the performance of lambs on urea-containing diets, and Lofgreen, Loosli and Maynard (1947) found significant differences in the biological value of different proteins fed as 10 percent of the diet to lambs. When urea supplied 40 percent of the dietary protein, the biological value of the ration was 71, with urea + methionine the value was 74, while dried egg protein gave 80. These results and others all confirm the view that the rumen micro-organisms convert urea to protein of sufficiently high quality to support efficient production.

Microbial protein tends to have a biological value of 60 to 70. When mixed rations or specific proteins of higher biological value are fed, the bacteria tend to degrade its value. When low quality protein such as maize protein is fed, its value is improved by the rumen microflora. While there is a possibility that amino-acid supplementation of practical rations may prove beneficial in the future for the highest levels of milk production or maximal rates of gain with beef cattle, research results to date have not demonstrated the value of supplementation.