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Nitrogen Metabolism in the Large Intestine Postruminal fermentation primarily in the cecum and large intestine of ruminant animals received little atten- tion until the advent of intestinal cannulation. As little as 4 percent of the total organic matter digestion occurs in the cecum plus large intestine with low intakes of for- age diets for sheep (Ulyatt et al., 197Sa), but with cattle fed at a high level of intake, up to 37 percent of the total energy digestion can occur past the terminal ileum (Zinn and Owens, 1981b). Digestion in the cecum and large intestine can compensate for incomplete digestion in the rumen where residence time, ammonia supply, or pH may limit extent of digestion. This shift in site of fermentation from the rumen to the large intestine can alter energy and amino acid availability for the animal, microbial yield, as well as fecal N loss. Loss of N in feces is typically the greatest source of N loss to a ruminant animal and must be considered in protein metabolism models. Physiology and digestion in the large intestine have been reviewed recently by Ulyatt et al. (1975a), Hoover (1978), Stevens et al. (1980), Wrong et al. (1981), and 0rskov (1982~. Nitrogen enters the cecum plus large intestine from the ileum and by diffusion through the intestinal wall. Input from the ileum consists of undigested feed protein (IUP), indigestible feed protein (IIP), undigested bacte- rial protein (BCP), plus endogenous N secreted or sloughed from the earlier sections of the intestinal tract (FPN) . Amounts of free amino acids or peptides entering the large intestine are insignificant (Clarke et al., 1966) . Basecl on nucleic acid concentrations at various seg- ments of the small intestine, Ben-Ghedalia (1982) sug- gested that some bacteria may grow in the last half of the small intestine and contribute to the N supply at the end of the ileum. Ileal N (undigested IUP, undigested BCP, and IIP) has been reported to include 45 to 60 percent amino N. 3 to 4 percent nucleic acid N. from 1 to 13 percent ammonia N. and up to 15 percent urea N 53 (Clarke et al., 1966; Coelho da Silva et al., 1972a; Van't Klooster, 1972~. The remaining 8 to 40 percent of the total N is presumably hexosamine and mucus glycopro- tein. Urea is present in ileal contents at concentrations from SO to lOO percent of that in blood. This is derived from diffusion into the small intestine (Hecker, 1971) and suggests that ureolytic bacteria are not prevalent in the small intestine. Urea is rapidly hydrolyzes] on entry into the cecum plus large intestine. From 14 to 37 per- cent of the total urea turnover in sheep has been attrib- uted to urea hydrolysis in the cecum and large intestine (Hecker, 1971; Hogan, 1973; Nolan et al., 1976~. To- gether with degradation of N compounds from undi- gested feed, bacterial and endogenous sources, hyclroly- sis of urea that diffuses into the large intestine from the blood stream helps maintain ammonia-N concentra- tions in the cecum and large intestine between 6 and 27 mM in sheep, although levels below 4 mM have been reported with ruminant animals fed diets containing higher amounts of grain (Williams, i965; Hecker, 1971; Kern et al., 1974~. Sampling methods and sample han- dling will alter estimates of ammonia concentration of intestinal and fecal matter (Wrong et al., 1981~. Under most feeding conditions more N enters the large intestine from the ileum than leaves as fecal pro- tein (UP) leading to a net absorption of 0.5 to 2 g daily in sheep (Clarke et al., 1966; Hecker, 1971; 0rskov et al., 1971b; Coelho da Silva, 1972a; Thornton et al., 1970) and O to 5 g in cattle (Van's Klooster and Boekholt, 1972, Zinn and Owens, 1982~. Nevertheless, the amount of nitrogen passing to the terminal ileum per day is highly correlated with the supply excreted in feces (Zinn and Owens, 1982~. Nitrogen absorption from the cecum and large intestine into the blood stream or through diffu- sion to other organs is enhanced by the high large intesti- nal pH (7 to 9) with roughage rations and is thought to
54 Ruminant Nitrogen Usage be primarily ammonia. Ammonia can be utilized by bacteria in the large intestine for BCP synthesis, be pas- sively absorbed into the portal blood system, or passed with FP. Diffusion of ammonia is primarily on the non- ionized form. Evidence from nonruminants suggests that pH dictates the fate of ammonia, with more ammo- nia in feces having a lower pH (Down et al., 1972~. In- creased availability of energy in the large intestine, achieved through infusion of starch, glucose, or sucrose, will increase FP and decrease urinary protein (UP) ex- cretion (Thornton et al., 1970; 0rskov et al., 1971b; Mason et al., 1977~. Part of this change is due to an in- crease in BCP in feces (Mason et al., 1977), and a part of the increase is in the soluble N fraction, probably associ- ated with a decreased fecal pH. Such a shift from N ex- cretion as UP to FP invalidates certain traditional indi- ces of protein value for ruminants, namely apparent digestibility, the concept of biological value, ant] possi- bly metabolic fecal N (FPN). Although generally more N enters the large intestine from the ileum than exits as FP, the magnitude of transfer of nitrogen may depend on diet, intake level, animal species, and other factors. With in vitro preparations, active uptake of amino acids by the colon has been demonstrate`] (Scharrer, 1978~. Yet, transport of amino acids to the serosa re- mains unproven. Several types of reasoning have been used to suggest that amino acids are absorbed from the large intestine. With horses, feeding of urea or infusion of protein into the cecum can increase N retention (Slade et al., 1970; Reitnour and Salsbury, 1972~. Disappear- ance of i4C amino acids (Hoover and Heitman, 1975) or i5N microbial protein (Slade et al., 1971) from the ce- cum also could reflect amino acid absorption. However, similar results could occur when microbial digestion in the cecum yields ammonia and volatile fatty acids to be absorbed and used by tissues for synthesis of nonessential amino acids. The low concentrations of free amino acids in the cecum and large intestine might be interpreted to suggest that sufficient quantities of amino acids are not available in the free form for absorption. Low concen- trations of amino acids in the large intestine reflect the rapid uptake and catabolism of amino acids by intesti- nal microbes. Wrong et al. (1981) concluder] that amino acid absorption from the large intestine, except in the newborn animal, is quantitatively insignificant. Never- theless, absorbed ammonia becomes available for ami- nation reactions in tissues and urea synthesis for recy- cling. From 4 to 37 percent of the total tract DOM digest- ibility by ruminants occurs in the cecum plus large intes- tine. High concentrations of volatile fatty acids and branched chain fatty acids reflect fermentation and pro- teolysis (Hecker, 1971; Kern et al., 1974~. With high concentrate rations, lactate production (Kern et al., 1974) may lower pH or ammonia may become limiting (Williams, 1965) . Since infusions of glucose, starch, and gelatin all increase the amount of fecal N as well as the amounts of bacterial components (DAP, RNA) excreted in feces of sheep (Mason et al., 1977), available energy is thought to be the factor limiting BCP synthesis in the large intestine of sheep under most dietary conditions. Fecal excretion (UP) has been related to (1) intake of nitrogen and (2) either dry matter intake or fecal dry matter output in attempts to estimate (a) true digestibil- ity of fed protein (IP) and (b) the amount of FEN lost by animals to feces. Regression of apparent digestibility of N against N concentration of the diet gives a slope that represents true digestibility of IF. True digestibility val- ues from a number of trials and summaries are listed in Table 15. True digestibility estimates range from 85 to 95 per- cent of feed N (Table 15~. These are for the total diges- tive tract, not for specific N component from the small TABLE 15 Estimates of True Digestibility and Metabolic Fecal Nitrogen Source Schneider, 1947 Halter and Reid, 1959 Halter and Reid, 1959 Anderson and Lamb, 1967 Harris et al., 1972 Harris et al., 1972 Harris et al., 1972 Harris et al., 1972 Stallcup et al., 1975 Boekholt, 1976 NRC, 1976 Swanson, 1977 Mason and Fredericksen, 1979 Dror and Tagari, 1980 Preston, 1982 Waldo and Glenn, 1982 Calculated from Morrison, 1959 Green roughages, N = 65 Dry roughages, N = 75 Silages, N = 25 Concentrates, N = 29 All feeds, N = 197 FEN True N (g/kg of Digestibility 1:)M Intake) Species Diet 91 92.9 88.3 8S.4 86.6 85.0 908 91.8 90.2 83.3 87.7 89.8 92.0 84.0 90.3 86.1 89.0 87.0 82.8 95.0 93.6 35 31 21 31 21 38 40 36 33 26 29 30 29a 14a 34 29 38 30 27 38 35 Ovine All Ovine All Bovine Forage Bovine Forage Bovine Forage Bovine Conc. Bovine All Bovine Mixed Bovine Mixed Bovine All Ovine Ovine Forage and mixed All Bovine Mixed Bovine Mixed Mixed Mixed Mixed Mixed Mixed Forage Forage Forage Conc. All
Nitrogen Metabolism in the Large Intestine 55 intestine as in Table 14. Values are surprisingly constant considering the wide variations in digestible energy con- tent and protein sources used in various diets. Fraction- ation of feces (Mason, 1969) led to the conclusion that true N digestibilities with sheep feel various diets ranged from 73 to 96 percent. FPN or nondietary fecal N. is the inevitable loss asso- ciated with production of feces. For nonruminant ani- mals F:PN has been attributed primarily to erosion of the intestinal lining since increased dietary fiber increases F"PN (Mukherjee and Kehar, 1949) and feeding of a pu- rified completely digested diet reduces fecal output, and thereby FPN to zero. FPN for nonruminants usually is correlated more closely with fecal output (IOM intake) than DM intake and thereby may be a result of micro- bial fermentation in the large intestine. With rumi- nants, part or all of MEN has been regarded as microbial N either synthesized in the large intestine or indigestible BCP passed through from the rumen (Mason and Fre- dericksen, 1979~. When all nutrients are provided to ru- minants through infusion of purified, absorbable nutri- ents, the quantity of feces produced and the amount of FP decline (0rskov and MacLeod, 1982~. Fermentation in the rumen will reduce the amount of potentially di- gestible material available for fermentation in the large intestine. The amount of BCP synthesized in the rumen or large intestine is normally related to supply of DOM and, at least in the rumen, it should be negatively re- lated to dietary fiber level. Efficiency of microbial growth (BCPFOM), however, is usually higher with di- ets containing more ADF. Hence, BCP and BCPFOM may change in opposite directions as dietary roughage level is altered. AcIding an inert fiber to a diet for calves can alter the relationship of FP to IOM intake (Stro- zinski and Chandler, 1972), yet FPN appears to be cor- related more closely with indigestible organic matter (IOM) output than DM intake (Swanson, 1982~. If F:PN in ruminants is a combination of (1) microbial residues from the (a) rumen or (b) cecum and large intestine plus (2) indigestible eroded or secreted protein from the ~li- gestive tract as suggested in the PDI system of protein evaluation (Walclo and Glenn, I-982), several factors would be needed to estimate its magnitude. These in- clude: (l) site, (2) extent of organic matter digestion, and (3) the amount of indigestible residue pushed through the digestive tract. Chemical subdivision of FPN into microbial versus nonmicrobial fractions does not define the origin of the N. Origin is critical in models of protein metabolism. FP, which originates from intes- tinal tissue, whether or not it is subsequently incorpo- rated into BCP, must be charged against tissue reserves of essential and nonessential amino acids. In contrast, protein synthesized from NPN in the digestive tract is appropriately charged against nonspecific N reserves, such as plasma urea. No discount for biological value applies to the latter fraction. FPN has been estimated by several procedures. These include (1) the intercept of the plot of apparently digestible protein against dietary protein level, (2) direct measurement with diets having 100 percent true protein digestibility or labelled isotopes as discussed by Strozinski and Chandler (1972), and (3 by enteral infusions of digestible nutrients (0rskov and MacLeod, 1982~. FPN values, being the intercept of the regression of fecal N or estimated by detergent proce- dures are also listed in Table 15. Estimates range from 21 to 38 g protein per kilogram DM intake. This value has been subdivided into portions for roughage and con- centrate portions of the diet by Dror and Tagari (1980) and has been attributed by some workers to the type of diet fed (Institut National de la Recherche Agro- nomique, 1978~. 0rskov and MacLeod (1982) main- tained steers and cows with intragastric infusions of digestible nutrients and measured FP and UP losses. In- fusions reduced FP but elevated UP loss compared to feeding of N-free diets. This led the authors to conclude that FPN is a result of microbial fermentation some- where in the digestive tract, and without microbial ac- tivity, FPN approaches zero. Yet, UP loss increased in magnitude similar to the decrease in FPN, suggesting that turnover of protein of the intestine results in irre- versible loss of N to the system by either one route or another. Differentiation between the two may not be feasible although the combination of FPN and UPN may be more constant. Whether infusions decreased turn- over of protein of the digestive tract has not been deter- mined. Indigestible fiber present in the intestine may absorb secretions and abrade more cells from the lumen of the intestine and thereby increase FP. Based on the more general equations of Harris et al. (1972), digestible protein = (0.84 to 0.92) IPDM - (0.021 to 0.04) (r2 > 0.90), one can calculate total FP. Combining terms, subtracting from IF, and multiplying by DM intake reveals that fecal protein (FP) = (8 to 16) IF (in kg) + (21 to 40) DM intake (in kg). Here, FPN is calculated as a function of DM intake and ranged from 21 to 40 g protein per kilogram dry matter intake. Using the assumption that all protein has a true di- gestibility of 90 percent, Swanson (1977) calculated FPN based on an extensive literature review. He con- cluded that FPN was 25 to 40 g protein per kilogram dry matter intake or 61.5 g N per kilogram IDM excreted. For calculation by the current system, FPN was as- sumed to equal 30 g protein per kilogram DM intake. With an assumed `dietary DM digestibility of 67 percent, PEN was calculated to be 90 g protein per kilogram IOM (30/0.33~. Although this means of estimating FPN is ap- pealing, results may be misleading. Depending on the foodstuff category chosen, FPN estimated by regression
56 Ruminant Nitrogen Usage can vary by 70 percent (Harris et al., 1972~. This could reflect experimental error or could suggest that FPN is not a constant proportion of feed intake or fecal output. Secondly, dry matter intake ant] protein intake are cor- relatecl in most studies, so FPN and true protein digest- ibility cannot be estimated independently. As illustrated in Table 15, the FPN estimate increases as the estimate of true digestibility of protein increases. Thirdly, true digestibility of protein calculated by regression across feedstuffs generally exceeds values measured with iso- topically labeled feed proteins. In conclusion, mathe- matical separation of fecal nitrogen into that from dietary versus endogenous origin by regression appears variable and without a biological basis. However, some method to subdivide FP into indigestible IF and FPN and make practical accounting of this nitrogen fraction, which totals from 20 to 68 percent of the total nitrogen loss by animals, as described in early studies by Blaxter and Mitchell (1948), is necessary to displace the concept of protein digestibility and generate requirement values in the newer systems of protein metabolism of ruminant animals. Fecal N consists of 45 to 65 percent amino nitrogen, 5 percent nucleic acid nitrogen, and 3 percent ammonia nitrogen (Coelho da Silva, 1972a; Van't Klooster and Boekholt, 1972; Hogan, 1973~. The residual nitrogen consists of partially degraded nucleic acids, bacterial cell walls, and glycoprotein, as well as nitrogen bound to fiber components. Separation by sonication and mod- ified fiber solubility procedures (Mason, 1969) has sug- gested that 7 to 28 percent of feces is undigested dietary N. 16 to 59 percent is water-soluble N. and 38 to 74 per- cent is bacterial plus endogenous debris N (Mason, 1969; Mason and Fredericksen, 1979; Plouzek and Trenkle, 1982~. The latter fraction can be subdivided, and, ac- cording to concentrations of diamino-pimelic acid and ribonucleic acicl, is presumably largely bacterial debris, especially bacterial cell walls (0rskov et al., 1971b; Ma- son et al., 19774. In conflict with this general concept that bacterial debris comprises a large fraction of the fecal N. some isotope studies with bacterial cell walls indicate that cell walls are readily rligestec] in the rumi- nant's small intestine (Hogenraad and Hird, 1970; Bird, 1972), and nucleic acid nitrogen concentrations in feces are generally below 5 percent of fecal nitrogen (Coelho da Silva et al., 1972a) . These studies would indicate that the amount of intact bacteria in feces is small. Although subdividing FP is useful to determine true digestibility of protein, compositional analysis of feces does not reveal the point of origin of F P. Liberated, non- utilized N from endogenous secretions of the intestines can be absorbed and excretes] in urine or recycled. The amount of N in the BCP fraction of feces could originate from endogenous essential amino acids or from urea cy- cled to the digestive tract. If energy available to the mi- crobes of the large intestine is the factor that limits mi- crobial protein synthesis, then N in the bacterial plus endogenous debris fraction of feces is not a suitable indi- cator of endogenous protein loss. Nevertheless, some es- timate of the total amount of F1< must be calculated to be included in calculations of the total N economy of the ruminant animal. Indigestible BCP synthesized during ruminal fermentation, should logically be charged against nonspecific or N available in the rumen (RAP), but this fraction is not necessarily determined by extrap- olation across protein intakes, since with a protein- deficient diet, this fraction may be reduced. It appears more logical to use an intercept estimate of FPN that is not fundamentally based than to underestimate the to- tal N required to replace inevitable losses. Nitrogen bound to acid-cletergent fiber (one index of IIP) comprises from 1 to 75 percent of feed N and has been used to predict apparent N digestibility of heat- damaged forage (Goering et al., 1972, Thomas et al., 1982~. Recovery of feed acid-detergent fiber-N in feces, however, differs with feedstuffs and generally ranges from 39 to 90 percent (Goering et al., 1972; Zinn and Owens, 1982~. Insolubility in an acid pepsin solution (PIN) has also been employed as an index of indigestibil- ity (IIP) for nonruminants (AOAC, 1980) and rumi- nants (Goering et al., 1972~. Indiscriminate binding of protein or ammonia N to fiber fractions with heating or in the intestinal tract can reduce N availability drasti- cally. Several implications of the complexity of fermenta- tion in the large intestine are apparent. Protein metabo- lism schemes must ultimately charge excreted nitrogen against its origin. The quantity of FP to be charged di- rectly against IP due to incligestibility should be to the amount of IF that is truly IIP. Indicators of IIP have been used to predict indigestibility of heat-damaged feeds (Goering et al., 1972, Thomas et al., 1982), but their usefulness for feeds not damaged by heat remains to be determined. The capacity to recover and recycle nitrogen from the cecum and large intestine gives the ruminant animal a means to alter the efficiency of nitrogen utilization when demands are altered. This means that biological value of N can increase as recycling increases. The mag- nitude of this adjustment with various feeding condi- tions must be determined before N utilization in the large intestine and biological value of metabolizable protein can be properly assessed and calculated.
