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Nitrogen Metabolism in Tissues At the tissue level, protein nutrition of ruminants in- volves amino acid metabolism as in nonruminant spe- cies. Studies in cattle (Black et al., 1957; Downes, 1961) have shown that the same amino acids are essential in ruminants as in nonruminants since they are not synthe- sized in tissues in adequate amounts and must be ab- sorbed from the gastrointestinal tract (GIT). A primary difference between ruminant and nonruminant species is that protein quality is dependent upon the availabiity of amino acids leaving the rumen rather than that in the ingested diet. Ruminants undoubtedly require some op- timum ratio of amino acids for most efficient utilization of absorbed amino acids, but the understanding of tissue metabolism of amino acids in ruminants has not pro- gressed as much during the past 10 years as has the un- derstanding of protein metabolism within the GIT. A1- though there is interest and considerable speculation about amino acid requirements of ruminants (Hogans 1975; Bergen, 1979; Wolfrom et al., 1979), there is lim- ited information on amino acid requirements of rumi- nant species. The increase in nitrogen balance of sheep (Nimrick et al., 1970), cattle (Fenderson and Bergen, 1972; Richardson and Hatfield, 1978), wool growth (Reds et al., 1973), and lactation in cows (Clark, 1975b) following postruminal administration of certain amino acids suggests that amino acid requirements may be dif- ferent than the supply from the rumen and that the effi- ciency of nitrogen utilization in high-producing rumi- nants can be improved by manipulation of postruminal amino acid supply. It is recognized that there is a cellular requirement for all the amino acids incorporated into body proteins, but because the nonessential amino acids can be synthesized by certain tissues within the body if sufficient nonspe- cific nitrogen and carbon precursors are present, only the 10 dietary amino acids essential for the growing rat (EAA) will be considered here. These are leucine (Leu), 7 57 isoleucine (Ile), valine (Val), sulfur amino acids (S-AA, Met, Cys), phenylalanine and tyrosine (Phe-Tyr), threonine (Thr), tryptophan (Trp), lysine (Lys), argi- nine (Arg), and histidine (His). AMINO ACID METABOLISM A simplified diagram of amino acid metabolism is given in Figure 13. Free Amino Acid Pools Most of the amino acids in the body are bound by peptide bonds in proteins. A small portion of the amino acicis are free and equilibrate in pools. The major pools of free amino acids are in extracellular and intracellular tissue fluids and blood. The free EAA in the bloodstream arise from degrada- tion of tissue proteins and absorption from the GIT. Nearly all the absorption occurs in the mucosal cells of the small intestine as free amino acids or as di- and tri- peptides. Most of the peptides are hydrolyzed in the in- testinal mucosa to free amino acids before passage to the blood. A portion of the amino acids derived from pro- tein digestion in the intestine may be used for protein synthesis or oxidation by the cells of the intestine before they enter the vascular system. The absorbed amino ac- ids are transported by the blood through the portal vein to the liver before being carried to other tissues. Most of the transport is as free amino acids in plasma, but there is evidence of transport of amino acids to tissues as free amino acids in red blood cells and as peptides (McCor- mick and Webb, 1982~. There is some variation in ratio of free amino acids present in plasma and whole blood (Heitmann and Bergman, 1980) reflecting the ability of red blood cells to concentrate certain amino acids. The proportions of amino acids absorbed from the GIT are
58 Ruminant Nitrogen Usage nput a) Diet b) Synthesis Synthesis of Nonprotein Compounds Free Amino Acid Pool (s) .. 1 ~ - Tissue Proteins Proteins Lost from Body | ~Oxidation FIGURE 13 Simplified model showing flow of amino acids in mammalian metabolism. temporarily reflected in the free amino acid pools of plasma after feeding diets that result in large excesses or deficiencies of amino acids passing into the duodenum (Bergen, 1979~. Frequently, there is no postprandial rise in plasma amino acids in functional ruminants (Theurer et al., 1966; Fenderson and Bergen, 1972~. Between pe- riods of absorption or during fasting, the concentration of EAA increases, that of nonessential amino acid de- creases, and the ratios of free EAA more closely reflect the amino acids present in proteins of body tissues. The concentration of total free amino acids in tissues is 5 to 10 times higher than in plasma, indicating that cells accumulate amino acids against a concentration gradient. Uptake of free amino acids by cells is by active transport across cell membranes, but free amino acids are continuously leaving cells as well (Christensen, TABLE 16 Extraction of Amino Acids by Various Tissuesa 1982~. The distribution ratio between free amino acids in tissues and plasma varies widely for various amino acids due to differerlces in transport systems for different amino acids. When tissues are synthesizing protein, there is a net uptake of amino acids from the blood, but in times of inadequate dietary energy or protein intake there may be a net loss of free amino acids from tissues such as skeletal muscle (Ballard et al., 1976) . The extrac- tion of amino acids from the blood by tissues such as the mammary gland may not be in proportion to the ap- pearance of amino acids in proteins (Mepham, 1982~. The ratio of amino acids leaving skeletal muscle con- tains higher proportions of free glutamine and alanine and lower proportions of free branched chain amino ac- ids and glutamic acid than are present in muscle pro- teins. These differences reflect catabolism of certain amino acids within muscle and the role of alanine and glutamine as a means of transporting ammonia to the liver. The interorgan movement of amino acids and their metabolites may also be beneficial for more ade- quately meeting the nutritional needs of all body tissues. The concept of "protein reserves" is based upon degra- dation of protein to amino acids in certain tissues for transport to other tissues for utilization. It has been esti- mated that the "protein reserves" of the lactating cow can be as high as 27 percent of body protein. A summary of amino acid extraction by various or- gans of ruminants is provided in Table 16. Compared with other organs, the mammary gland most efficiently retains the EAA extracted from the blood. The liver re- moves high proportions of Met, Phe, and Tyr and low proportions of Cys, Vat, Leu, and Ile. In sheep there is high umbilical uptake of Vat, Leu, Ile, Phe, Lys, and Arg relative to the other EAA (Meter et al., 1981~. The extracted Lys and His were retained most efficiently, Cow, Mammary Sheep, Liver Sheep, Kidney Sheep, Fetus Calf, Hind Limb Gland Reference: McCormick Bickerstaffe Wolff et al. Bergman et al. Lemons et al.and Webb and Annison (1972) (1974) (1976)(1982) (1974) Leucine 2.8 2.0 9.88.4 42.2 Isoleucine 2.8 0.7 12.24.6 38.9 Valine 2.1 2.0 6.83.6 26.0 Phenylalanine 20.2 5 6 9.22.8 39.8 Tyrosine 16.0 4.4 6.14.8 41.8 Threonine 7.7 2.5 6.04.4 37.8 Lysine 6.7 5.3 8.916.8 58.5 Histidine 8.2 9.0 6.17.0 29.4 Cystine 5.8 9.3 -- 18.4 Methionine 15.7 12.7 -6.4 58.2 Arginine 7.7 11.1 17.71.5 53.0 a Extraction of free amino acids from plasma. Expressed as: (input-output) . input.
and Vat, Len, Ile, Phe, and Tyr least efficiently by the fetus. Leu and Lys are removed in higher proportion than other amino acids by tissues of the hind limb. These data are based upon plasma free amino acids, which may not be the only source of amino acids to tissues and the degree of extraction would be expected to vary di- rectly with degree of limitation of each amino acid. Mc- Cormick and Webb (1982) have reported that amino ac- ids are also extracted from plasma as proteins and peptides and from erythrocytes by the hind limb of calves. Reamination of keto acids also might be a source of amino acids for certain tissues. Detailed studies have not been conducted with tissues from ruminants, but evidence from rats indicates that labeled amino acids can be incorporated into newly syn- thesized protein of skeletal muscle and liver without complete mixing with the intracellular amino acid pool, supporting the concept of intracellular compartmental- ization of free amino acids. If free amino acids are com- partmentalized in cells, withdrawal of amino acids for synthesis and oxidation might not occur from a common pool. The concept of free amino acid pools is more complex than illustrated in Figure 13. There are many pools of free amino acids in the body that vary in size, ratio of amino acids, and efficiency of amino acid extraction from plasma. Although large quantities of amino acids pass through the free amino acid pools, there is limited storage of free amino acids in the body, and conse- quently the free amino acid pools do not represent a re- serve of amino acids for protein synthesis. Most of the amino acids are bound in proteins and excess amounts of amino acids are oxidized. For efficient utilization of die- tary nitrogen, the animal is therefore dependent upon a continuous supply of amino acids of the proper balance. Utilization of Amino Acids Removal from the free amino acid pools is mainly for synthesis of body proteins or oxidation. The use of amino acids for gluconeogenesis in the fed ruminant is contro- versial. Wolff et al. (1972) have suggested that between 11 and 30 percent of the glucose synthesized in fed sheep is derives] from amino acids. Bruckental et al. (1980), however, have suggested that amino acids contribute only 1 to 2 percent of the glucose need of the high-yield- ing cow where glucose and amino acids are both in short supply relative to demand. At any rate, the use of amino acids for gluconeogenesis is only competitive in meeting the protein requirement of the animal if the carbon skel- eton of the most limiting EAA is used or if it causes a shortage of precursors for synthesis of nonessential amino acids. Tamminga and Oldham (1980) estimated that no more than one-fourth of the amino acids used for Nitrogen Metabolism in Tissues 59 gluconeogenesis could be from EAA. It is conceivable that feeding excess protein to provide carbon from non- essential amino acids for gluconeogenesis might be ben- eficial at certain times. Limited amounts of some amino acids are used for synthesis of nonprotein compounds (e.g., creatine, nucleic acids, thyroxine) or excreted in the urine. Amino acid flux is toward protein synthesis since the Michaelis constants of the enzymes that deaminate amino acids are in the millimolar range, while the en- zymes initiating protein synthesis are in the micromolar range. Thereby, when amino acid concentrations are low, greater proportions are bound to synthetic than ox- idative enzymes. However, because one or more amino acids or other factors may limit protein synthesis and because free amino acids are transported from one tissue to another by the blood, extraction of amino acids by the liver results in continuous loss of amino acids by oxida- tion. Since smaller proportions of an amino acid pro- vided in excess are trapped by synthetic enzymes, excess amounts of the amino acid accumulate and plasma con- centrations increase. As plasma concentrations increase, the proportion shunted toward oxidation increases. Protein Synthesis Synthesis and degradation of body protein is continu- ous, but proteins in different tissues as well as various proteins within tissues turn over at different rates. In the very young ruminant the largest quantity of protein syn- thesized is in skeletal muscle (Combe et al., 1979), but increased growth of the GIT associated with consump- lion of dry feed results in an increased proportion of to- tal protein synthesis in the GIT. Of protein synthesis in sheep (Davis et al., 1981) and cattle (Lobley et al., 1980), 30 to 40 percent of the total synthesis occurs in the GIT, 10 to 20 percent in the skin, 15 to 20 percent in skeletal muscle, and 4 to 8 percent in the liver. The GIT and hide contain about 6 to 20 percent, respectively, of the total body protein but due to rapid turnover account for 30 to 40 percent ant] 10 to 20 percent, respectively, of total protein synthesized per clay. Skeletal muscle, at 40 percent of total body protein, accounts for at least 50 percent of nitrogen retained by a growing animal but only about 20 percent of daily protein synthesis. The fractional rate of protein synthesis is much faster in the GIT, liver, and hide than in skeletal muscle. After the period of rapid growth of the GIT in ruminants, the rel- ative growth rate of the GIT, hide, and liver is less than that of the empty body, but because of high turnover, most of the protein synthesis still occurs in these tissues rather than in skeletal muscle. As animals mature, the net gain in body protein ap- proaches zero, but large quantities of protein continue
60 Ruminant Nitrogen Usage to be synthesized due to continued turnover. Lobley et al. (1980) estimated protein synthesis of a mature cow was 1.9 to 3.1 kg per day with 1.0 to 2.1 kg per day occurring in noncarcass components. Large quantities of protein are synthesized in the mammary gland of lac- tating animals. A cow producing 30 kg of milk contain- ing 3 percent protein secretes 900 g of protein per day. Since there is little degradation of secreted proteins, syn- thesis probably is only slightly over 900 g per day. It is not known if lactation alters the fractional rate of pro- tein synthesis in other body tissues, but at a minimum, protein synthesis in the noncarcass part of the body must equal that of the mammary gland. The net amino acid requirement for milk protein synthesis, however, is much higher because the proteins are secreted and lost from the body. As tissue proteins turn over, a high pro- portion of the released amino acids can be reutilized, although efficiency may vary with relationships of pro- portions of EAA being released and those required for the protein being synthesized. Since hydroxyproline and 3-methylhistidine are not reutilized, their removal re- flects turnover rate. Turnover of proteins may account for a greater proportion of the total energy needs than the total amino acid needs of the body. Synthesis of Nonprotein Compounds Amino acids are used for the synthesis of a number of nonprotein compounds including creatine, glutathione, carnitine, melanin, dopamine, epinephrine, nore- pinephrine, thyroid hormones, histamine, carnosine, anserine, taurine, S-adenosylmethionine, nicotinic acid, serotonin, polyamines, y-aminobutyric acid, purines, pyrimidines, heme, hydroxylysine, and hy- droxyproline. The EAA involved include the sulfur amino acids, Arg, Lys, Phe-Tyr, Trp, and His. Only a few of these losses have been quantitated but in total probably account for less than 1 percent of absorbed amino acids. Excretion of creatinine is proportional to body weight and related to the phosphocreatine pool, predominantly in skeletal muscle. Estimates of daily creatinine-nitrogen excretion in cattle and sheep are 3.8 to 9.4 and 8.4 mg per kg body weight per day, respec- tively (Brody et al., 1934, McLaren et al., 1960~. Allan- toin-nitrogen, an end product of purine metabolism that is related to digestible organic matter intake and probably reflects absorbed and nonutilized purines from rumen microbes, has been estimated to be 14 ma/ kg feed organic matter per day in sheep fed chopped hay but only 0.7 mg/kg feed organic matter per day in sheep given soluble nutrients by intragastric infusion (Anto- niewicz and Pisulewski, 1982~. In cattle, the excretion of 3-methyl His in the urine is correlated with liveweight and estimated to be 0. 6 to 0. 7 mg/kg per day (Harris and Milne, 1981~. In sheep, the 3-methyl His arising from degradation of tissue proteins is not quantitatively ex- creted in the urine (Harris and Milne, 1980~. Excretion of free amino acids from the body in urine is a minor loss under most conditions. There seemed to be no net removal of EAA by the kidney of mature sheep fed at maintenance, fasted, or made acidotic (Bergman et al., 1974~. Amino Acid Oxidation The major irreversible loss of amino acids from the body is by oxidation. Oxidation of the EAA occurs al- most totally in the liver of ruminants. There is consider- able catabolism of the branched-chain amino acids in skeletal muscle and other extra-hepatic tissues of nonru- minant species, but this does not seem to be the case in ruminants (Coward and Buttery, 1982~. Amino acid ox- idation in the liver has not been critically studied in ru- minants under different nutritional and physiological conditions, but it is known that large portions of free amino acids are removed from blood by the liver (Wolff et al., 1972; Heitmann and Bergman, 1980~. In sheep fed at maintenance, nearly all the amino acids added by the portal-drained viscera seemed to be removed from blood plasma by the liver. With greatly reduced absorp- tion of amino acids from the GIT, such as during fast- ing, removal of amino acids by the liver was main- tained. The net escape of amino acids from the liver needs to be reinvestigated in light of erythrocytes and peptides as forms of amino acid transport. Increasing amino acid intake above requirement in- creases oxidation. Available evidence suggests that ex- cesses of EAA, due to high absorption from the GIT or by a relative excess due to a scarcity of one or more amino acids, are removed from the free amino acid pools by oxidation in the liver. Certain proteins represent a direct loss of amino acids from the body. Proteins in hair and scurf, wool, secreted proteins such as milk, proteins secreted or sloughed into the GIT that are not subsequently digested, and proteins retained in the conceptus represent protein losses from the body. Growth of hair and wool requires higher pro- portions of Val, Leu, Ile, Lys, and Thr and sulfur- containing amino acids as compared with whole body proteins. The amino acids found in higher proportions in milk proteins (Arg, Leu, Ile, and Val) also seem to be more extensively oxidized in the mammary gland as compared with Met, Phe, Tyr, and Trp (Oldham, 1981~. Nitrogen Excretion Waste nitrogen, principally as urea, arising from de- amination of amino acids or ammonia absorbed from the digestive tract, is excreted in the urine, some in milk,
Nitrogen Metabolism in Tissues 61 or back into the digestive tract. That nitrogen returned to the reticulo-rumen supplements the diet and contrib- utes to the amount of nitrogen available for microbial growth (Cocimano and Leng, 1967; Kennedy and Milli- gan, 1978; Kennedy et al., 1981, 1982~. The amount of urea-N recycled into the rumen appears c~epenctent on the animal and dietary conditions. Kennedy and Milligan (1980) related clearance of plasma urea to the concentration of rumen ammonia. Their regression developed for cattle fed hay and grain, or hay and sucrose was: Y - 59 - 0.41X ~ 0.00086X2; where Y = clearance of plasma urea in the rumen (mllh/kg BOO), and X = concentration of rumen ammonia (ma N/L). To calculate influx, it is then necessary to relate plasma urea concentration to either dietary IP or ruminal am- monia concentrations. More data are available that re- late plasma urea concentration to ruminal ammonia concentration. Kennedy and Milligan (1980) found a closer relationship between plasma urea concentration and ruminal ammonia concentration than between plasma urea and dietary crude protein. A linear regres- sion of plasma urea concentration on rumen ammonia concentration was developer] from data of Glenn et al. (1983~: Y = 79.0 + 14.5X, where Y = plasma urea-N (ma N/L), and X = ruminal ammonia-N (ma N/ 100 ml) . This relationship permits calculation of plasma urea concentration from ruminal ammonia concentration. Next, ruminal ammonia concentration is needed. This can be estimated from crude protein content and the total digestible nutrient (TDN) content of a diet (Roffler and Satter, 1975a) according to the following equation: Ruminal NH3-N (ma N/ 100 ml) - 38.73 - 3.04IP + 0.171IP2 - 0.49TDN + 0.0024 TDN2; R2= 0.92, where IP = dietary crude protein (percent), and TDN = total digestible nutrients (percent) = 1.02 digestible organic matter (DOM). From these relationships, the amount of urea-N recy- cled per kilogram body weight per day could be calcu- lated. Cattle in the study reported by Kennedy and Mil- ligan (1980) were consuming about 2.5 percent of their body weight daily as dry matter. For this level of intake, amounts of urea-N recycled for diets of various crude protein and DOM contents were calculated. Two re- gressions were determined: (1) Y = 0.1255 + 0.00426X- 0.003886X2; R2 = 0.94; where (2) where Y- X urea-N recycled (g N/daylkg BOO), and dietary IP (percent); Y = 121.7 - 12.01X + 0.3235X2; R2= 0.97; Y - urea-N recycled (percent of N intake), and X = dietary IP (percent). The latter regression is perhaps the most convenient for calculating the amount of urea-N recycled to the rumen. This regression indicates that a diet containing 4 percent IP will lead to urea-N recycled into the rumen equaling 86 percent of dietary N. indicating the significance of this activity in animals fed low-protein diets. For a diet containing 12 percent IP, this value drops to about 25 percent, and for a diet containing 20 percent IP, only 7 percent of the ingested nitrogen is recycled. Rapidly growing or heavily lactating animals may have lower plasma urea concentrations than the sheep used to develop these urea recycling equations. Tissue or milk synthesis may act as a nitrogen sink, reducing urea synthesis and plasma urea concentration. Highly pro- ductive animals might therefore be expected to recycle less urea into the rumen than less productive ruminants fed a comparable diet. Endogenous protein, from saliva ant] cells sloughed from rumen epithelium, is an additional source of nitro- gen for the rumen microbes, but quantitative informa- tion in this area is meager. Furthermore, availability of the nitrogen in keratinized rumen epithelial cells for ru- men microbes in unknown. The amount of nitrogen from endogenous protein recycled into the rumen may equal the amount of recycled urea found in highly pro- ductive animals. PROTEIN REQUIREMENTS The amino acid requirements of ruminants could be estimated by summing the net removal of free amino acids from the free amino acid pools (Figure 12~. Practi- cally, this is not possible because all losses have not been quantitaterl. Because of ease of analysis, the experimen- tal approach used most frequently has been to measure nitrogen rather than amino acid metabolism and con- vert nitrogen to crude protein (N x 6. 2S). Nitrogen bal
62 Ruminant Nitrogen Usage ance procedures have provided much of the knowledge currently available on protein requirements of animals. Since the body continues to lose nitrogen in the urine and feces, even when dietary intake of nitrogen is nil, these losses were considered to reflect a minimum nitro- gen metabolism required to support basic body func- tions and were termed endogenous (Mitchell, 1962~. This parallels energy metabolism with heat production continuing despite starvation. The adclitional nitrogen metabolism associated with dietary intake of protein has been termed exogenous. The net protein requirement is the sum of that for maintenance and that expected to be retained in tissues as growth, in the conceptus, woo} growth, or excreted in milk. The factorial equation to estimate net protein re- quirement (g/d) - (FPN + UPN + SPN) + (RPN + YEN ~ LPN). Requirements for absorbed protein (AP) are determined by assigning metabolic efficiencies for use of absorbed amino acids for various functions. Requirements for Maintenance Metabolic Fecal Protein (FPN). PAN is made up of the undigested fraction of endogenous proteins lost in the feces. Endogenous protein (nitrogen) enters all seg- ments of the GIT. It consists of enzymes, mucus, epithe- lial cellular debris, serum, lymph, bile, and urea. FPN is considered to represent endogenous proteins lost through the digestive tract as a result of feed intake. Es- timates of the quantity of FPN have been made by feed- ing animals protein-free diets and measuring nitrogen lost in the feces or by feeding diets containing different concentrations of protein and regressing digestible pro- tein against dietary protein to zero protein intake. The latter method usually results in a lower estimate of FPN. In nonruminant species fed low-fiber diets, FPN is re- lated to dry matter intake; however, in ruminants fed diets varying in fiber content it is more closely related to fecal dry matter. In cattle and sheep, FPN ranges from 6 to 8 percent of fecal dry matter. Swanson (1982) has esti- mated FPN, g/d = 0.068 x fecal dry matter. An alter- native estimate, if data on digestibility of the diet are not available, is FPN, g/d - 0.03 x ciry matter intake (g/ d). Baser] on this relationship and a DM digestibility of 0.66, FPN = 0.09 x indigestible dry matter (IDM). Mason and Fredericksen (1979) characterized nitrogen fractions in sheep feces anal found that much of the fecal nitrogen is microbial debris arising from undigested ru- men microbes and from microbial action in the large intestine and cecum. The quantity of nitrogen excreted in the feces increases and that excreted in the urine de- creases with increaser! passage of fermentable substrates to the large intestine (Mason et al., 1981~. FPN obvi- ously is of body origin when animals are fed nitrogen free diets, but when animals are fed protein, it is not known how much of the nitrogen captured by the mi- crobes in the lower GIT is of body origin and should be considered a true maintenance requirement rather than as a second excretory pathway for waste nitrogen arising from the inefficient use of absorbed nitrogen. Endogeno?~s Urinary Protein (UPN). UPN is the ni- trogen (protein equivalent) lost in the urine when ani- mals are fed nitrogen-free diets. After feeding nitrogen- free diets for 5 to 7 days, urinary nitrogen is excreted at a relatively constant level, irrespective of the diet fed. Creatine, urea, ammonia, allantoin, uric acid, hippuric acid, and small quantities of amino acids contribute to UPN. UPN is difficult to estimate in ruminants because there is some absorption of amino acids when they are fed nitrogen-free diets as a result of microbial growth originating from nitrogen recycled into the rumen. Swanson (1977) estimated UPN in cattle fed low-protein diets to be UPN, g/d = 2.75 x wt0 5. ARC (1980) esti- mate(1 UPN, g/d in cattle to be: 16.07 x in wt - 42.24. Forsheep,Swanson(1982)estimatedUPN,g/dc 1.125 wt0 55, and the ARC (1980) estimate for UPN is: 0.1468 x wt + 3.375. More recently 0rskov (1982) has mea- surec] loss of nitrogen in the urine of cattle and sheep nourished by intragastric infusion. When nitrogen-free infusates were given, urinary nitrogen losses were 300 to 400 mg N/wt0 75, which were considerably higher than nitrogen lost in the urine when ruminants are fed pro- tein-free diets and about triple the estimates above. Ani- mals maintained by intragastric infusion excrete very little nitrogen in the feces, and 0rskov and MacLeod (1982) suggested that metabolic fecal nitrogen mea- sured in feces of ruminants fed nitrogen-free diets is mainly endogenous nitrogen derived from breakdown of tissue protein but incorporated into microbial debris and excreted in the feces. We are recommending the equations of Swanson (1977, 1982~. Scum, Protein (SPN). SPN is protein lost from the surface of the body as hair, scurf, and secretions. The estimated loss in cattle is SPN, g/d - 0.2 x wt0 6 but is variable depending upon type of hair coat, weather, and ambient temperature. Requirements for Tissue Growth, Lactation, and Pregnancy Tissue Protein (RPN). RPN deposition has been esti- mated by determination of body composition of grow- ing animals. Many of these studies have been summa- rized elsewhere (ARC, 1980; Byers, 1982b; NRC, 1984~. Net protein gain is a multiple of weight gain and compo
Nitrogen Metabolism in Tissues sition of the gain, which are influenced by rate of gain, physiological maturity, previous nutrition, sex, and use of hormonal adjuvants. Three summaries have been made for purposes of estimating net protein require- ments of growing cattle by ARC (1980), Robelin and Daenicke (1980), and NRC (1984~. The equation of ARC (1980) to estimate the protein content of empty body gain (EBWG) of cattle of me- dium frame and gaining 0.6 kg EBWG/d is: k t i /k EBWG 0.8893e°8893tnEBW The correction factors for other types of cattle include a subtraction of 10 percent for small breeds, 10 percent for females, and 1.3 percent for each 0.1 kg/d more than 0.6 kg/d and an addition of 10 percent for large breeds, 10 percent for intact males and 1.3 percent for each 0.1 kg/d gain less than 0.6 kg/d to values calculated for me- dium steers gaining 0.6 kg/~. The equations of Robelin and Daenicke (1980) to esti- mate protein content in EBWG are: Lipid content of EBW (kg) - L Daily lipid deposition (kg/d) = ebO + bllnEBW + b2(1nEBW)~ - 1 = EBW (b1 + 2b21nEBW) EBWG Daily protein deposition (kg/d) = p FFM = fat free mess = EBW - L and = as al (EBWG - I) FFM`a~- i' an al ho be be Early maturing steers 0.1616 1.060 - 6.311 1.8110 0.0000 Early maturingbulls 0.1541 1.060 -1.680 0.0189 0.1609 Late maturing bulls 0.1541 1.060 -5.433 1.5352 0.0000 The equation of NRC (1984) for estimating protein content of shrunk live weight gain (LWG) is: Daily protein deposition (am) = p - LWG (268 - 29.4 x Meal energy per kg EBWG). The discussion and source of those conclusions are in NRC (1984~. For breeds with medium frame and implanted with hormonal adjuvants: Steers: Retained energy (Mcal/~) = 0.063S EBW0 7s x EBWGi 097 Heifers: Retained energy (Mcal/~) and = 0.0783 EBW0 75 x EBWGi ~9 EBWG = 0.956 (LWG) EBW = 0.891 (LOO) Modifications include: 1. Cattle without hormonal adjuvants contain 5 per- cent more energy per unit of gain. 2. Medium-frame bulls are equivalent to medium- frame steers of a 15 percent lighter weight. 3. Large-frame animals are equivalent to medium- frame animals of the same sex of a 15 percent lighter weight. A summary of the application of these three estimates for medium-frame steers of different weights and gain- ing 0. 5, 1. 0, and 1. ~ kg EBWG per day is given in Table 17. In the 250- to 400-kg weight range, all three methods resulted in similar estimates of net protein require- ments. The ARC approach resulted in low estimates for lighter weights and high estimates at the heavier weights. The NRC approach gave high estimates at lighter weights and very low estimates at heavier weights. It is not certain which equation is most representative of growth of cattle. For medium-frame beef cattle that are fattening, the NRC (1984) method may be most ap- propriate. Either ARC (1980) or Robelin and Daenicke (1980) is closer to the recommendations for dairy ani- mals approaching maturity without fattening (NRC, 1978~. The NRC (1984) equations and modifications have been chosen for use here. The ARC (1980) equations to estimate the protein content of empty body gain of sheep are: Males: kg protein/kg EBWG = 0 8995 e 0.8164 eO.81641nEBW Females: kg protein/kg EBWG = EBW 1 3032 The protein content of wool is estimated (ARC, 1980) to be protein, g/d = 3 ~ 0.1 x protein in g/d retained in other issues. A summary of the protein content of gain of sheep is given in Table 18. Lactating animals often lose weight in early lactation and gain during late lactation ant! the dry period. Com- position (g protein/kg EBW) of weight gain or loss of adult cattle has been estimates] to be 175 to 188 (Reic! and Robb, 1971) and 160 (NRC, 1978~. Protein content of empty body weight changes in adult ewes ranged from SO to 70 g protein/kg EBW in a study by Rattray et al. (1974~. Products of Conception (YPN). YEN include pro- tein gain in the fetus and growth of the uterus and re- lated tissues. Rattray et al. (1974) and Ferrell et al. (1976) have estimated the protein content of the mam- mary gland and the gravid uterus during pregnancy of sheep and cattle, respectively. Most protein deposition
64 Ruminant Nitrogen Usage TABLE 17 Estimated Net Protein Requirements for Growth of Cattle of Different Body Weights and Gaining at Different Rates Empty Body Weight, kg Gain lSO kg 200 kg 250 kg 300 kg by EBWG/d NRCa ARCb FC NRC ARC F NRC ARC F NRC ARC F . 0.5 .0 .5 101 81 93 197 151 186 290 212 279 (g protein per animal per day) 92 78 88 83 76 84 74 75 79 176 147 177 158 143 168 140 140 158 258 205 265 229 200 252 201 196 237 350 kg 400 kg 500 kg 600 kg NRC ARC F NRC ARC F NRC ARC F NRC ARC F 0.5 66 74 74 59 73 70 44 71 60 29 69 51 1.0 122 138 149 106 136 139 74 133 120 44 130 101 1.5 174 193 223 148 191 209 98 185 180 51 181 151 _ . . . a Estimates derived from NRC (1984~. bEstimates derived from ARC (1980~. CEstimates derived from Robelin and Daenicke (1980). in the mammary gland occurs during the last 30 days of pregnancy and is much less than that in the gravid uterus. Estimates of protein deposition in the fetus and uterus (kg/d) of cattle during 141 to 281 clays and sheep during days 63 to 147 from conception (ARC, 1980) are: Cattle: Protein (g/~) = (34 375) [e(~.s3s7 - 13. 120le ~ 0 00262X _ 0.00262X Sheep: Protein (g/d) A BAND. - , ~r~ ~_, ~ r {~ ~ `2A7C~ _ ~ ~ Amp-0-~601X _ 0.00~1X)] - (U.0~4) Let's A. -- ~ -five= where: X = days post conception. The daily gain of protein in the products of conception for cattle and sheep are summarized in Tables 19 and 20. Lactation (LPN). The protein in milk is a multiple of quantity and composition of milk. The LPN require- ment (g/d) can be estimated from: Milk N (g/kg) x 6.25 x milk yield (kg/d). Total nitrogen of milk includes a TABLE 18 Protein Retention in Gain of Growing Sheepa Empty Males and Castrates Females Body Weight Gain Woola (kg) (g/kg Gain) (gldlkg Gain) 10 160 20 148 30 142 40 138 50 135 19.0 17.8 17.2 16.8 16.5 Gain Woola (g/kg Gain) (g/d/kg Gain) 147 128 119 113 108 17.7 15.8 14.9 14.3 13.8 a Values for sheep above 10 kg empty body weight and non-Merino breeds. nonprotein component that is largely waste products of nitrogen metabolism and when known it may be more correct to use values for true protein content of milk rather than total N x 6.25. Representative values for true protein content of milk from cattle and sheep are given in Table 21. There is genetic variation in the pro- tein content of cow's milk and the value in Table 21 is more typical of the Friesian breed. There is a relation- ship between fat and protein content of milk (Overman et al., 1939), and for producers who usually know the fat content of milk, but not true protein, it would possi- ble to estimate protein content from fat content (NRC, 1978~. It is recognized that there is considerable variation in protein content of the products of animal production due to genetics, rate of production, and nutritional his TABLE 19 Protein Retention in Fetus and Gravid Uterus of Cattle at Different Stages of Gestation Age (Week from Conception) 20 22 24 26 28 30 32 34 36 38 Protein Gain (gld)a 13.7 18.3 24.2 31.6 40.8 52.2 66.1 82.8 102.8 126.6 a Corrected for uterus of nonpregnant cow.
Nitrogen Metabolism in Tissues 65 TABLE 20 Protein Retention in Fetus and Gravid Uterus of Sheep at Different Stages of Gestation Age (Week from Conception) Protein Gain (g/d) a 2.4 3.9 6.3 9.5 13.9 19.5 0 2 14 6 18 20 a Corrected for uterus of nonpregnant sheep. TABLE 21 Protein Content of Milk g Protein/kg Milka 30.0 47.9 Cattle Sheep a Corrected for nonprotein nitrogen content of milk (0.55 g N/kg for sheep and 0.30 g N/kg for cattle) . tory, as well as other factors. It is not the intent of this presentation to exhaustively review all of these variables for all classes of ruminants, but rather to present repre- sentative data that are needed to estimate protein re- quirements at the tissue level. Committees for each of the species will need to present more detailed data to more adequately predict protein requirements. Efficiency of Protein Utilization. The requirement for AP can be determined by correcting the sum of the net protein requirements for maintenance and produc- tion by the efficiency with which absorbed amino acids are transferred into product protein. The efficiency with which absorbed amino acids are used for produc- tion is difficult to determine, and there are few estimates for producing ruminants. Optimum values for effi- ciency of amino acid utilization are obtained when pro- tein is limiting procluction. In addition, there is varia- tion in utilization of different amino acids; the amino acid present in lowest amount relative to requirement is used most efficiently. If one amino acid is limiting, then the utilization of other amino acids will be reduced to some extent related to the deficiency of the limiting amino acid and the relative excess of the other amino acids. Excess amino acids resulting from overfeeding proteins or because of a limiting amino acid are rapidly removed from the body by oxidation and not stored. Data on efficiency of utilization of mixtures of amino acids that might be representative of absorption are very limited. One approach to estimate these values has been to calculate the biological value of absorbed nitrogen (NRC, 1978, 1984~. Estimated efficiencies for growing cattle range from 0.60 to 0.81 and 0.70 for lactating cows. A similar approach (ARC, 1980) has been to esti- mate efficiency from: (RPN + UPN)/(IP - FP). With diets limiting in nitrogen, the efficiency for nitrogen use in cattle and sheep is 0.75. It is important to evaluate any efficiency data in the context of the conditions (rela- tive to requirements) that they are gathered. The two major pathways of amino acid metabolism are protein synthesis or oxidation (Figure 13~. Efficiency of transfer of amino acids into product protein can then be calculated from: (Amino acids in product)/(Amino acids in product + Amino acids oxidized) or from: (Amino acid nitrogen in product)/(Amino acid nitrogen in product + Urea nitrogen formed from amino acids in metabolism). This method can be used to determine the efficiency of use of individually labeled amino acids. When the amino acid being studied is limiting produc- tion, it is used with high efficiency compared with other amino acids. In calves, utilization of methionine was 0.82 when methionine was limiting growth (Mashers and Miller, 1979~. Oldham (1981) and Oldham and A1- derman (1982) calculated efficiency of utilization of ab- sorbed amino acids from several studies using urea pro- duction to estimate amino acid oxidation and found the values to range from 0.6 to 0.8 for lactating ruminants and from 0.27 to 0.75 for growing ruminants when en- dogenous urinary nitrogen was included with product nitrogen. Storm et al. (1983) have reported a value of 0.66 for the efficiency of utilization of truly digested bacterial nitrogen for nitrogen retention in lambs. Based upon the fact that amino acid utilization is lower when protein is fed at or above requirement and amino acid balance usually will be less than maximum, it appears that efficiency of amino acid use should be 0.65 for lactating ruminants and 0.50 for growing rumi- nants. There is a need for additional research to derive more adequate estimates of efficiency of amino acid uti- lization, since these values have such a great impact on the calculated requirement for AP. Additional Roles of Amino Acids. In addition to serving as substrates for protein synthesis, there may be some requirement of amino acids for other needs in the body that under certain conditions might justify feeding additional protein. The role of amino acids in gluconeo- genesis has been briefly discussed. Under most practical feeding conditions, it does not appear necessary to feed protein to supply amino acids for synthesis of glucose. The relationships between amino acid metabolism and energy utilization may be economically important with certain ruminant production systems and should be fur- ther investigated. Possible roles of amino acids discussed by Oldham (1981) include effects of amino acids on feed consumption, digestion in the rumen, regulation of hor- mone secretion, and lipoprotein metabolism in the liver.
