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Degradation of Dietary Crude Protein in the Reticulo-Rumen INTRODUCTION Intake protein (IP) that passes to the omasum is often called"bypass" or "undegraded" protein (UIP) to differ- entiate it from protein synthesized by microbes (BCP) in the rumen and from endogenous secretions. These terms can be confusing and overlapping. The IP that passes to the omasum consists of two fractions. These are: (1) pro- tein that resists microbial attack in the rumen; and (2) protein that evades attack in the rumen and passes to the omasum without thoroughly mixing with ruminal con- tents. Protein flushed out of the rumen at feeding time and passing through the esophageal groove would fall into this category. The term "undegraded" protein is most suited to the first fraction, while "bypass" would be more suited to the second fraction. Measurements in vitro usually attempt to quantitate "undegraded pro- tein," while in vivo measurements include both frac- tions. The BCP synthesized in the rumen, UIP, and en- dogenous protein together total the amount of protein entering the omasum. Rumen microorganisms cause major transformations of dietary nitrogenous compounds. Most forms of non- protein nitrogen are converted almost quantitatively to ammonia. True protein is degraded to a variable extent to peptides and amino acids in the rumen. Peptides and amino acids are utilized for synthesis of BCP, or are fur- ther hydrolyzed and deaminated, producing ammonia as the major ens] product, which contains N. Although rumen microbes may supply 60 to 80 per- cent of the amino acids (protein) absorbed from the in- testine (AP), much interest has been focused on the amount of UIP. Medium- to high-producing ruminants rely on some IP escaping degradation in the rumen since the quantity of BCP is inadequate to support high rates of growth, wool production, or milk production. The 28 proportion of UIP must increase as production levels in- crease, using feeds and technology of the present. The supply of UIP can be a limiting factor at high levels of animal performance. This was illustrated in the work of Hogan and Weston (1967), which stimulated much re- search in N utilization by ruminants in the following decade. Insights gained during the last decade form the basis for much of the following discussion. MECHANISM OF PROTEIN DE GRADATION This topic has been reviewed by Tamminga (1979). Therefore, discussion will be limited to an overview of some of the major features. The IP entering the reticulo- rumen may be degraded by both bacteria and protozoa. Degradation involves basically two steps: (1) hydrolysis of the peptide bond (proteolysis) to produce peptides and amino acids; and (2) deamination and degradation of amino acids. Russell et al. (1983) suggest that the hy- drolysis of peptides to amino acids is the rate-limiting step. Free amino acid concentrations in ruminal ingesta are normally extremely low (Annison et al., 1959, Lewis, 1962), suggesting that proteolysis is normally the rate-limiting step in protein degradation. This view is supported by Nugent and Mangan (1978, 1981~. The proteolytic enzymes appear to be associated pri- marily with the bacterial cell wall with a small amount of cell-free activity probably resulting from cell lysis (A1- lison, 1970). An example is the protease produced by the rumen anaerobe Bacteroides amplophilus. This prote- ase is present on the outer cell surface ancl hydrolyzes protein extracellularly (Blackburn, 1968; Blackburn and Hullah, 1974~. Proteolytic enzymes are associated with many rumen bacteria, and proteolytic activity of rumen microorganisms is not greatly altered by diet
Degradation of Dietary Crude Protein in the Reticulo-Rumen 29 (Blackburn and Hobson, 1962; Allison, 1970~. As dis- cusssed later, diet can have an effect on protein degrada- tion in the rumen, perhaps indirectly through altering pH and bacterial numbers or types. Protease activity appears to be "trypsin-like" in na- ture. Craig and Broderick (1984) observed that when casein was incubated in vitro with rumen microorgan isms, losses of lysine and arginine were disproportion- ately large. Stern and Satter (1982) reported similar results with in vivo studies. Craig (1981) observed that the artificial trypsin substrate benzoylarginine ethyl es ter inhibited in vitro casein degradation, but synthetic substrates for chymotrypsin had little effect. These results imply that bacterial proteases may be trypsin- like in activity, preferentially exposing lysine and argi- nine residues to further degradation by microbial exo- peptidases and deaminases. This suggests that use of a trypsin inhibitor may reduce ruminal protein break- down and improve utilization of feed protein. Following proteolysis, liberated peptides or amino acids may leave the reticulo-rumen, be utilized for mi- crobial growth, or be degraded to ammonia and fatty acids. Amino acids are rapidly degraded in the rumen, and therefore only small quantities of free amino acids would be available for absorption or passage from the reticulo-rumen. The half-life of eight essential amino acids incubated with strained rumen fluid was 2 h or less (Chalupa, 19764. MEASURING PROTEIN DEGRADATION Measuring protein degradation by rumen microbes is a difficult task. There can be wide variation in protein degradation within and among feedstuffs, as well as sig- nificant differences among animals with regard to ru- men environment and retention time of feed in the reticulo-rumen. There are many sources of analytical error, the most important of which is distinguishing be- tween BCP and UIP. Considerable caution must be ex- ercised in applying the results of a single experiment, and replication of experiments or studies is necessary to help identify contributing variables. No single tech- nique or experimental design is fully adequate at the present time. Despite the difficulties of making in vivo measure- ments of protein degradation, in vivo measurements are essential' for they serve as the standard against which al1 chemical or in vitro methods for estimating protein deg- radation must be evaluated. Chemical or in vitro meth- ods for estimating protein degradation are important for screening or monitoring purposes, but they must be validated in vivo and must not serve as the only estimate of protein degradation. In Vivo Methods In vivo measurements are usually performed with surgically prepared animals equipped with cannulae in the rumen and abomasum or small intestine. Determination of digesta flow with a reentrant can- nula may be accomplished with total collection of the ingesta, or more commonly by use of an indigestible di- gesta marker and collection of spot samples (Zinn et al., 1980~. When using animals prepared with T-type can- nulae, spot samples are taken and flow rate of digesta is calculated by reference to digesta markers. Although in vivo measurements of protein flow to the intestine must be the primary source of information about protein deg- radation in the rumen, it must be recognized that mea- surement of digesta flow to the duodenum is subject to considerable error. Digesta markers currently used are not ideal markers and do not always reflect the solid or liquid phase that they are intended to represent. The use of digesta markers to measure flow to the small intestine has been reviewed (Faichney, 1975, 1980; Warner, 1981~. The amount of UIP can be estimated as the difference between IP and the sum of endogenous and BCP enter- ing the abomasum or smal1 intestine. Procedures for es- timating BCP are available, utilizing microbial markers such as nucleic acids, diaminopimelic acid (1)APA), aminoethylenephosphonic acid (AEP3, or one of the ra- dioisotopes, 35S, 32p, or ~5N (Clark, 1977~. Estimates of BCP based upon digesta or microbial marker techniques are subject to errors inherent in those techniques. In practice, some investigators use microbial markers present in bacteria only and therefore clo not include protozoa! protein in their estimates. Protozoal protein can be important under certain feeding conditions (Harrison and McAllan, 1980~. Estimates of endogenous protein are variable and difficult to obtain. Conse- quently, enclogenous protein is often ignored, leading to an overestimate of UIP when difference techniques are used. The extent of this error probably is not large. Another approach to estimate the amount of UIP is available. This method is based on the increase in flow of protein to the small intestine in response to incremen- tal additions of IP (Stern and Satter, 1982~. Unfortu- nately, this technique is useful only with feeds having a relatively high protein content. It assumes that protein content in the ration does not influence the measure- ment in question (Zinn et al., 1980~. In Situ Method for Estimating Protein Degradability While the use of cannulated animals can provide esti- mates of protein degradation in the rumen, in vivo esti
30 Ruminant Nitrogen Usage mates are labor intensive and time consuming. Alterna- tive techniques that can provide rapid, yet reasonable estimates of protein degradation for a wicle variety of feedstuffs are desirable. Unfortunately, alternative techniques tester] to date have one or more major limita- tions. One of the more promising approaches is the da- cron or nylon bag technique. Mehrez and 0rskov (1977) suggested that this in situ technique is suitable for deter- mining degradation of protein. The simplest applica- tion of the in situ technique for estimating protein deg- radation is to suspend the bag in the rumen for an arbitrary period of time, thus giving a relative estimate of protein degradation. Alternatively, the extent of pro- tein degradation can be determined at the moment when a predetermined percentage of the truly digestible organic matter has disappeared from the dacron bag, thus simulating the extent of digestion in the rumen of normally fed animals (0rskov and Mehrez, 1977~. Un- fortunately, ruminal retention time and ruminal or- ganic matter digestion vary among diets, intake levels, and many other conditions. Several methods have been used to combine in situ N disappearance and ruminal dilution rate information (0rskov and McDonald, 1979; Mathers and Miller, 1981; McDonald, 1981; Stern and Satter, 1982~. The first three methods are similar in approach and use rate constants for both nitrogen disappearance and passage rate. The procedure applied by Mathers and Miller in- volves the following: Fraction of protein degraded = A + kaBB/(kaB + kpB), where the terms in the equation are as previously described. It may be inappropriate to apply a single rate con- stant to the degradation of that portion of protein re- maining in the bag after the soluble protein has disap- peared. Several rate constants are probably involved with most feecistuffs, depending upon the number and amount of each type of protein present. Rate constants for digestion of N usually have more influence on pro- tein degradation than rate constants for passage from the rumen (0rskov and McDonald, 1979; Mathers and Miller, 1981~. The following example illustrates this point. Many protein supplements, ant] most feedstuffs, will have a ruminal passage rate (kpB) within the range of 0.03 to 0.07 h-i (Ganev et al., 1979; Hartnell and Satter, 1979; Lindberg, 1982; Stern and Satter, 1982~. Using an arbitrary value of .1 for LAB and 0.3 for A, pro- tein degradation would decrease from 0.84 to 0.71 as the rate constant (kpB) for passage of undigested residue from the rumen increased from 0.03 to 0.07 h- I. This is a rather modest change in degradation as a result of a large change in rumen retention time. The value used for LAB will determine, of course, how much influence kpB will have on protein degradation. Manipulations that increase kpB, such as increased feed intake, will have their greatest effect when LAB iS small (Mashers and Miller, 1981~. Stern and Satter (19823 have described a more empiri- cal approach for combining in situ N disappearance and ruminal dilution rate. Protein degradation is obtained by summing the product of protein remaining in the ru- men Determined in a rate of passage study) and the fractional disappearance of N from the dacron bag at 1O different time intervals. The approach is analogous to the method of Castle (1956) for calculating mean reten- tion time of digesta in the gastrointestinal tract. The ap- proach avoids reliance on a single rate constant for de- scribing the rate of protein degradation. The in vitro bag technique is subject to variables that can influence the estimate of protein degradation. Pore size of the cloth can influence the rate and extent of N disappearance from the bag. Entry of feed particles and colonization of bag contents by rumen bacteria can lead to an underestimate of protein degradation, and varia- lion in the washing technique can lead to error. A1- though the in situ bag technique is an imperfect and em- pirical approach, it incorporates animal and microbial factors helpful in quantitating protein degradation in the rumen. Protein Solubility as a Means of Estimating Protein Degradation Soluble proteins tend to be more rapidly or com- pletely degraded (Hendrickx and Martin, 1963~. Unfor- tunately, some segments of the feed industry have as- sumed that soluble protein is degraded in the rumen and insoluble protein is not. Early reports of animal work, often quoted to relate protein solubility with protein degradation in the rumen, reveal no basis for equating soluble protein with degradable protein and insoluble protein with undegradable protein, except for extreme examples such as zein and casein (McDonald, l9S2; Chalmers et al., l9S4; el-Shaz~y, 1958; Tagari et al., 1962; Little et al., 1963; Whitelaw and Preston, 1963; Tagari, 1969~. Soluble proteins are generally more vulnerable to proteolysis than insoluble proteins. Accessibility of pro- teins to proteases is much greater if the protein is in solu- tion. It seems likely, however, that some feed protein can be hydrolyzed directly from the solid state without an intervening soluble stage, similar to the digestion of cellulose. Relatively insoluble proteins such as zein can be extensively degraded in the rumen, given adequate time. It may be that much of the protein hydrolysis is occurring on the surface of the feed particle. Large differences exist between soluble proteins in the
Degradation of Dietary Crude Protein in the Reticulo-Rumen 31 rate at which they are hydrolyzed. Nugent and Mangan (1978) studier] the degradation of casein, fraction I leaf protein, and bovine serum albumin in vitro using sheep rumen fluid. All three proteins were buffer soluble but differed greatly in the rate at which they were hydro- lyzed (casein > fraction I leaf protein > bovine serum albumin). Treatment of bovine serum albumin with dithiothreitol, which breaks some of the disulfide bonds cross-linking the protein, caused a substantial increase in its rate of rumen proteolysis. It was concluded that differences in the rates of microbial hydrolysis of these proteins were caused by structural and not solubility differences. Mahadevan et al. (1980) further examined this question by incubating soluble and insoluble pro- teins with partially purified protease from Bacteroides Anglophiles, one of the principal proteolytic organisms in the rumen. Their results showed that serum albumin and ribonuclease A, both of which are buffer soluble, were relatively resistant to hydrolysis, and that buffer- soluble proteins from soybean meal, rapeseed meal, and casein were hydrolyzed at different rates. Interestingly, buffer-soluble and -insoluble proteins of soybean meal were hydrolyzed at almost identical rates. Mahadevan et al. (1980) concluded that buffer solubility of a protein is not an indication of susceptibility to hydrolysis by ru- men bacterial protease. A somewhat different perspective relating protein solubility and susceptibility to proteolysis has been dis- cussed by Pichard and Van Soest (1977~. They con- cluded from protein solubility and proteolysis studies that there are four general categories of N in ruminant feeds. Fraction A is a water-soluble NPN fraction con- taining primarily nitrate, ammonia, amines, and free amino acids. Insoluble fractions include a rapidly de- gradable protein fraction B1, a more slowly degradable protein fraction B2, and an unavailable fraction C. Application of this approach to partitioning of N in silages might have potential. Fermentation of forages increases the amount of N in fraction A and may in- crease the amount in fraction C if the forage has under- gone heat damage. Whether a combination of solubility and protease information can be used to predict in vivo protein degradation of forages remains to be demon- strated. Since some proteins are soluble in water, it would appear that fraction A for some feedstuffs would contain true protein in addition to nonprotein nitrogen. Stern and Satter (1982) evaluate<] the relationship be- tween N solubility in Burroughs mineral buffer (Bur- roughs et al., 1950), N disappearance from dacron bags, and in vivo measurements of degraded intake protein (DIP) for 34 total mixed diets containing various dietary N sources. They found that N solubility was highly cor- related with N disappearance from bags in the rumen for short exposure times, but as exposure time increased the correlation between these procedures progressively decreased, to be expected due to the dynamics of degra- dation. Correlation between in viva crude protein deg- radation and N disappearance from dacron bags be- came significant at 12 h of rumen exposure and increased to 0.68 at 24 h. The correlation between N solubility and degradation of protein in vivo was only 0.26, indicating that solubility may be a poor predictor of protein degradation, when the dynamics of ruminal passage are not taken into account. Solubility of a pro- tein varies with the solvent used (Crooker et al., 1978), and care is required in interpretation of some experi- mental results. Measurements of protein solubility were described by Wohlt et al. (1973) and Waldo and Goering (1979~. Information on solubility of proteins in buffers is pres- ently being used in France for estimating rumen protein degradation in a protein evaluation scheme known as the PDI system (Verite et al., 1979) . Protein solubility in mineral buffer and, in some cases, ammonia production in vitro (Verite and Demarquilly, 1978, Verite and Sau- vant, 1981) are related to published information on flow of N from the rumen in sheep and cattle fitted with oma- sal, abomasal, or intestinal cannulae. They concluded that, on average, all of the soluble dietary crude protein and 35 percent of the insoluble dietary crude protein were degraded in the rumen. The hazard in using such a constant is discussed above. Several feed companies in the United States are pres- ently using information on protein solubility in mineral buffers to formulate dairy rations. One group of compa- nies formulates diets to provide not less than 15 percent and not more than 25 percent of the total dietary protein as soluble protein, and has claimed that such formula- tions increase milk yields (Braund et al., 1978~. In Vitro Ammonia Production for Estimating Protein Degradation A common approach for estimating protein degrada- tion involves incubation of the test protein with rumen fluid and measurement of subsequent ammonia produc- tion. The chief advantage of this method is its simplic- ity, provided a source of ruminal ingesta is available. The method has several disadvantages, however, that limit its usefulness. Microbial growth and ammonia up- take occur simultaneously with protein degraclation and ammonia release. This frustrates quantitative measure- ments by making it difficult to equalize microbial growth across a variety of feedstuffs. Broderick (1978) has attempted to overcome this problem by inhibiting deamination and uptake of amino acids by the mi- crobes, thus enabling a direct measure of proteolysis. Another problem is that incubation conditions (sub
32 Ruminant Nitrogen Usage strafe, end products, pH) in a batch culture change with time, so rates of both ammonia production and uptake change. In vitro ammonia production has been the principal method used for obtaining estimates of protein degrada- tion for the ARC system of protein evaluation (Roy et al., 19773 and has been used in France with the PD] system mentioned earlier. Verite et al. (1979) comment that in vitro incubation with rumen digesta is probably superior to solubility for estimating protein degrada- tion, but that the procedure is not suitable for routine analysis. They feel that there is satisfactory agreement between the two methods for most feedstuffs, but that protein solubility gives lower estimates of degradation for cereals, soybean meals, and sugar beet pulps and higher estimates for horse beans and peas. With these feedstuffs, in vitro ammonia production estimates, rather than solubility estimates, were used, and these were termed "corrected solubility" values in their feed- stuff tables. It appears that when in vitro ammonia pro- duction and protein solubility give similar estimates of protein degradation for a class of feeds, solubility infor- mation is used. When agreement is not good, in vitro ammonia production is used. In summary, estimation of ruminal protein degrada- tion in the rumen is a complex problem. A primary diffi- culty, in vivo, in situ, and in vitro, is to distinguish be- tween microbial and dietary protein. Secondly, all of the laboratory or in vitro procedures for estimating in vivo protein degradation have one or more major flaws that can invalidate the estimates. It therefore appears necessary to continue with the tedious and costly in vivo experiments with cannulated animals to determine pro- tein degradation of the major feedstuffs. These determi- nations are also subject to error, and considerable repli- cation is advised. Equally important with the in vivo studies are the feeding variables (cited earlier) that can influence protein degradation in the rumen. Protein solubility, or in vitro methods, will continue to be the source of protein degradation estimates for the minor feedstuffs for the near future, even though these "short-cut" procedures are potentially misleading. On the other hand, protein solubility or in vitro methods can be used to monitor changes within a foodstuff or to screen similar feedstuffs. For example, Beever et al. (1976) found a high negative correlation (r2 = 0.96) be- tween soluble N in perennial ryegrass, determined as the N soluble after incubation with 0.01 percent pepsin in 0.1 N HC1 for 16 h, and the quantity of total nitrogen entering the small intestine. The solubility and, in this case, the degradation of the ryegrass protein were al- tered by drying at different temperatures and by for- maldehyde treatment. It is reasonable to expect solubil- ity or the in vitro methods to predict differences in protein degradation more accurately when applied to a group of similar feedstuffs that when used across a di- verse group of feedstuffs that differ in physical and chemical properties. EXTENT OF PROTEIN DEGRADATION IN THE RUMEN Both ruminant nutritionists and livestock producers seek more quantitative information on the extent of pro- tein degradation in the rumen. Three of the metaboliz- able protein systems that have been proposed to replace crude or digestible protein for ruminants are dependent upon protein degradation values (Burroughs et al., 1975a; Roy et al., 1977; Verite et al., 1979~. Table 6 and Appendix Table 2 contain a list of feedstuffs and esti- mates of the percentage of crude protein that escapes destruction in the reticulo-rumen. All of these estimates were obtained with sheep and/or cattle at various feed intakes and having abomasal or duodenal cannulae. It is clear from these tables that (1) estimates of the amount of protein escaping degradation in the reticulo-rumen are extremely variable, and (2) in vivo information is deficient or nonexistent for many feedstuffs of major di- etary importance. Part of the variation in degradation estimates is due to analytical error associated with the in vivo measurements and part to variation in feedstuffs, the diets used and amounts fed, the experimental ani- mals employed, and method of feeding and physical na- lure of the diet. The values for protein degradation in Table 6 must be used with caution. In some instances the values reported in Table 6 do not agree with other in vivo information on protein degradation where the feedstuff in question was part of a mixed diet, but where degradation of individual protein sources was not re- ported. Most evidence suggests that the small grains, such as barley and oats, have protein that is more clegrarlable than the protein in corn. Soybean meal protein is a rela- tively degradable protein. In vivo information on whole cottonseeds and cottonseed meal is very limited, but cot- tonseed meal prepared by the expeller process may be more resistant than that prepared by the solvent process (Broderick ant! Craig, 1980; Goetsch and Owens, 1985~. Many by-product feeds appear relatively resistant to ruminal degradation. Brewers grains, distillers grains, corn gluten meal, fish meal, blood meal, and meat and bone meal are more resistant than most feed grains and oil meals. Up to 50 percent or more of the protein in these feedstuffs escapes degradation. The protein in most forages is quite susceptible to deg- radation. The in vivo estimates of protein degradation in forages are variable, reflecting not only the difficulty
Degradation of Dietary Crude Protein in the Reticulo-Rumen 33 TABLE 6 Tentative Estimates of Undegraded Protein for Common Feedstuffs When Total Dry Matter Intake Is in Excess of 2 Percent of Body Weight Feed Number of Measurements Mean Fraction of Undegraded Protein Standard Deviation Feedgrains Barley Corn Sorghum grain Oil meals Cottonseed meal (solvent) Cottonseed meal (Prepress) Cottonseed meal (screw press) Linseed meal Peanut meal Rapeseed meal Soybean meal Sunflower meal By-product feeds Blood meal Brewers dried 2 3 8 6 2 2 1 2 1 10 2 1 grains 5 Corn gluten meal Distillers dried grains Fish meal Meat meal 1 Meat and bone meal 3 2 2 Forages Alfalfa hay 4 Alfalfa (dehydrated) Bromegrass hay Corn silage Timothy hay 3 2 1 2 0.21 0.60 0.52 0.41 0.36 0.50 0.44 0.30 0.23 0.28 0.24 0.82 0.53 0.55 0.07 0.06 0.15 0.12 0.02 0.07 0.08 0.14 0.05 0.14 0.06 0.62 0.07 0.80 0.12 0.76 0.60 0.28 0.62 0.32 0.27 0.42 0.11 0.08 0.04 0.12 0.11 in measuring degradation of low-protein foodstuffs but also the wide variation in forage protein content due to harvesting and method of preservation. Since forages provide the bulk of protein in many ruminant rations, more quantitative information on degraclation of forage protein is needed. It has been assumed in studies with protein degrada- tion that the individual amino acids are equally suscep- tible to degradation. There may be preferential hydro- lysis of some amino acids from the pepticle or protein molecule. Secondly, free amino acids may differ in their rates of degradation. Chalupa (1976) addressed the lat- ter question and noted that amino acids differ markedly in their rates of degradation by rumen microbes. Argi- nine and threonine were rapidly degraded. Lysine, phenylalanine, leucine, and isoleucine formed an inter- mediate group, while valine and methionine were least rapicily degraded. Nevertheless, all free amino acids were rapidly catabolized. Stern et al. (1983b) measured the relative loss of individual amino acids from protein in intestinally cannulated lactating dairy cows receiving incremental amounts of corn gluten meal. The six most degraded amino acids in descending order were lysine, isoleucine, histidine, arginine, valine, and leucine. The basic amino acids appear more extensively degraded than acidic amino acids (Stern and Satter, 1982~. This is different from the studies with free amino acids (Cha- lupa, 1976~. Besides knowing the extent of ruminal protein degra- dation for foodstuffs, it is important to know the relative value of protein sources for supporting animal produc- tion. With this in mind, Klopfenstein (1980, used the slope-ratio technique (Hegsted and Chang, 1965) for evaluating protein sources for growing ruminants. This approach should reflect not only on the amount of pro- tein that escapes ruminal clegradation, but also the qual- ity and availability of protein that escapes the reticulo- rumen. Klopfenstein (1980) calculated a "protein efficiency value" for nine different feeclstuffs, with soy- bean meal being assignee] an efficiency value of lOO per- cent. All other feeds were evaluated relative to soybean meal, depending on growth in beef cattle. The protein efficiency values for blood meal (ring dried), blood meal (conventional), corn gluten meal, brewers grains, dehy- drated alfalfa, meat meal, distillers grains and distillers grains (with solubles) were 250, 200, l9O, 190, 185, 173, and 137 percent, respectively. Although in vivo mea- surements of protein degradation were not made, these animal growth data support the concept that the protein supplements tested were more resistant to ruminal deg- radation than soybean meal. Lactating ewes have been used to evaluate protein sources of differing extents of degradation, and the correlation coefficient between milk yield and degradation of the protein supplement was - 0.89 (Gonzalez et al., 1979~. Similar information with lactating dairy cows is needed. The ultimate test of any nutrient or feedstuff is how well it supports animal production. Obviously protein degradation information is important, but care must be exercised in relying too heavily on data that are not quantitative and tell only part of the story. Animal re- sponse data are essential in the final evaluation of pro- tein supplements. FACTORS INFLUENCING PROTEIN DEGRADATION IN THE FORESTOMACH The extent to which protein is degraded in the rumen will depend upon microbial proteolytic activity in the rumen, microbial access to the protein, and rumen turn- over. Differences in the proteolytic potential of rumen digesta under a variety of feeding conditions have been
34 Ruminant Nitrogen Usage small. Microbial access to the protein seems to be the most important factor influencing protein degradation in the rumen. Tertiary Structure of the Protein The three-dimensional structure of protein is impor- tant in determining whether the protein will be de- graded or not. For example, ovalbumin is slowly de- graded because it is a cyclic protein having no terminal amino or carboxyl groups (Mangan, 1972~. Proteins with extensive cross-linking, such as clisulfide bonds, are less accessible to proteolytic enzymes and are relatively resistant to degradation (Nugent anti Mangan, 1978~. Examples of such proteins are hair and feathers. Pro- teins treated with formaldehyde have methylene cross- linking and are normally degraded to a lesser extent (Ferguson et al., 1967~. These and other features of pro- tein structure dictate the vulnerability of protein to hy- drolysis in the rumen. Proteins in feeds are composed primarily of four types: albumins, globulins, prolamines, and glutelins. Albumins and globulins are usually more soluble than prolamines and glutelins (Sniffer, 1974~. This is unfor- tunate because albumins and globulins usually have higher biological values than prolamines and glutelins. Rumen Factors Retention time of feed protein in the rumen can influ- ence protein degradation. Proteins retained for a short time are degraded to a lesser extent than those with a longer retention time. Ruminal retention time of dietary ingredients varies among animals (Belch and Cam- pling, 1965), among species (Church, 1969), and among diet ingredients (Hartnell and Satter, 1979~. Retention time is influenced by particle size of the feed (Belch and Campling, 1965) and by the quantity of feed eaten (Belch and Campling, 1965; Zinn et al., 1981; Lind- berg, 1982~. A comprehensive review of factors influ- encing digesta passage through the gut is available (Warner, 1981~. The amount of UIP in lactating cows eating either 8.2 or 12.9 kg of dry matter daily was 29 and 45 percent, respectively (Tamminga et al., 1979~. High-producing ruminants consuming large quantities of feed are likely to have a larger percentage of UIP than animals consuming low or moderate amounts of feed. The effect of level of intake on retention time of feed particles, however, is sometimes quite small (Hartnell and Satter, 1979; Varga and Prigge, 1982), and the im- pact on protein degradation may often be minor (Miller, 1973) or without effect (McAllan and Smith, 1983~. A summary of studies showing the relationship be tween level of feed intake and retention time in the ru- men reveals that intake has a large effect on ruminal retention time when daily intake is less than approxi- mately 2 percent of body weight. The decrease in rumi- nal retention time associated with increased feed intake is much diminished when feed consumption is in excess of approximately 2 percent of body weight (Prange, 1981~. A somewhat similar observation by Alwash and Thomas (1971) indicates! that mean retention time of forage particles was related to the log of feed intake. In conclusion, increased feed intake can reduce protein de- struction in the rumen, but the influence of feed intake on residence time in the rumen and therefore on protein degradation is diminished as intake is increased. Level of feed intake may have some effect on protein degrada- tion aside from influencing residence time. For exam- ple, lower rumen pH, which usually accompanies higher levels of feed intake, may reduce bacterial activ- ity and proteolytic activity. As pointed out earlier, vari- ation between protein sources in rate of protein degra- dation is greater than variation in retention time in the rumen and would therefore have more impact on extent of total proteolysis in the rumen. Increasing the dilution rate of rumen fluid has been demonstrated to increase the flow of protein from the rumen of sheep (Harrison et al., 1975) and steers (Cole et al., 1976b; Prigge et al., 1978~. Part of this increase is probably due to a net increase in BCP (Harrison et al., 197S; Harrison and McAllan, 1980) and part due to an increase in the amount of UIP (Hemsley, 19754. Rumen fluid dilution rates have been increased by feeding or by ruminal infusion of artificial saliva, sodium bicarbon- ate, or sodium chloride. Environmental temperatures can influence the resi- dence time of feed in the rumen. Kennedy et al. (1976) demonstrated that sheep in a cold environment had an increased rate of digests passage. This increased BCP and the amount of UIP. In a more recent study, Ken- nedy et al. (1982) found that the percentage of UIP in the rumen increased from 20 to 24 percent for alfalfa hay and from 40 to 49 percent for bromegrass hay when sheep were exposed to cold temperatures. No effect of temperature on extent of protein degradation of a barley-canola seed meal diet was observed. The turn- over time (h) of ~03Ru-phenanthroline in the rumen for the alfalfa, bromegrass, and barley-canola meal diets at warm and cold temperatures were: 18.4, 12.3; 15.6, 15.3; and38.9, 32.3. Feeding of monensin has been shown to reduce di- etary protein degradation in vitro (Whetstone et al., 1981) and in the rumen (Poos et al., 1979b; Isichei and Bergen, 1980j. Although the amount of information is limited, it appears that UIP can be increased by approx
Degradation of Dietary Crude Protein in the Reticulo-Rumen 35 imately one-third with monensin feeding. However, monensin may inhibit BCP synthesis (Chalupa, 1980b), resulting in little or no net increase in total protein sup- ply to the intestine. Rumen pH could affect protein degradation by alter- ing microbial activity and by changing protein solubil- ity. Rumen pH is normally between S.5 and 7.0, and proteins with an isoelectric point in this range would have altered solubility and possibly altered protein de- gradability. Also, fiber may limit microbial access to plant protein, and reduced fiber digestion at a lower pH might be involved as well (Ganev et al., 1979~. Proteolysis and deamination are affected by pH, but experimental results are conflicting. As reviewed by Tamminga (1979), the bulk of evidence suggests that the optimum pH for both proteolysis and deamination is be- tween 6 and 7. There are reports of lower pH optima for ruminal proteases and deaminases, but since activity of both will be largely dependent upon total bacterial numbers, rumen pH in a range between 6 and 7 should be compatible with maximum microbial activity. Un- der most feeding situations, pH in the rumen is in a range where extensive breakdown of dietary protein can occur. Little is known about the effect of ammonia concen- tration on proteolysis or deamir~ation. Since the main pathway of ammonia fixation by rumen bacteria may differ according to the prevailing concentration of am- monia (Erfle et al., 1977), it might be suggested that catabolic processes in rumen bacteria are influenced by ammonia concentration. For example, ammonia, through end procluct inhibition, might alter the rate of protein hydrolysis. Nikolic and Filipovic (1981), how- ever, were not able to demonstrate an effect of ammonia concentration on the degradation rate of corn protein. Very low ammonia concentrations would affect total proteolytic activity to the extent that ammonia might limit total microbial growth (Poos et al., 1979a). Feed Processing and Storage Many feecistuffs are exposed to heat during processing or storage. By-product feeds are frequently produced by an aqueous extraction process and are often dried for marketing. This exposure to heat can make the protein more resistant to degradation (Ferguson, 1975~. Ensiled feeds may experience elevated temperatures for a sus- tained period of time, resulting in more resistant protein (Merchen and Satter, 1983b). Feed processing methods such as pelleting, extrusion, and steam rolling and flaking may generate enough heat to alter feed protein. In terms of optimum protection of protein, however, it is likely that more heat is required than most commercial processing methods wil1 provide. Moisture level, quantity of soluble carbohydrate present in the feedstuff, maximum temperature, and time- temperature relationships are some of many factors that will influence the effects of feedstuff exposure to heat (Goering and Waldo, 1978~. Heat treatment of feeds to reduce protein degradation in the rumen has potential (Beever and Thomson, 1981), and quantitative infor- mation is needed. Protection produced by heating can be counter- balanced by decreases in total tract digestibility and bio- logical value. The Maillard reaction between sugar al- dehyde groups and the free amino groups of protein is responsible for much of the heat darnage to protein when reducing sugars are present. However, proteins can be damaged by reactions other than Maillard type. Condensation reactions make essential amino acids nu- tritior~ally unavailable (Ferguson, 1975~. Beever et al. (1981) noted that pelleting a mixture of Italian ryegrass and timothy, which had been dried at high temperature, reduced degradation of dietary pro- tein from 69 to 47 percent. The effect of pelleting in this experiment may be due to heat or to an influence on retention time of the forage in the rumen. Pelleting demonstrates how changing the physical form of a feed- stuff can influence protein degradation. Ensiling of feeds can convert large portions of true protein into NPN (Bergen et al., 1974; Goering and Waldo, 1974~. This may lower the amount of protein potentially available for passage from the rumen. For- mation of NPN is particularly evident with silages of high moisture content (Merchen and Satter, 1983b). However, other factors (including hydration rate) may ~nfluence these events. Chemical treatment of feedstuffs has been used to provide partial protection against breakdown in the ru- men. Feeding trials with formaldehyde-treated casein appeared very promising (Ferguson et al., 1967), and extensive experiments with formaldehyde treatment of forage have been conducted. Presently, formaldehyde- treatec] feeds are used in Europe. Although treatment of commercial protein supplements with formaldehyde has been disappointing, a combination of formic acid- formaldehyde has been used to assist preservation of direct-cut forages (Waldo, 1977a). This process is also employed in Europe. Tannins have been used to protect protein from deg- radation in the rumen. Driedger and Hatfield (1972) re- ported that addition of 10 percent tannin to soybean meal fed to lambs increased rate and efficiency of gain and nitrogen retention and decreased in vitro deamina- tion by 90 percent. The high level of tannin used in those experiments would appear not to be practical. Isopro
36 Ruminant Nitrogen Usage panol, propanol, and ethanol have been used to increase the resistance of protein in soybean meal to degradation by rumen microbes (Van der Aar et al., 1982~. Inhibi- tors of amino acid deamination in the rumen have been tested (Chalupa and Scott, 1976~. There is great potential for protecting feed protein from excessive destruction and loss in the rumen. One of the major advantages of feeding protected protein would be greater opportunity for utilization of NPN for BCP synthesis in the rumen and the economy inherent with NPN use. A balance is needed, however, in the amount of UIP and the amount of dietary nitrogen made available for BCP synthesis. Much remains to be learned about practical ways to alter protein degrada- tion in the rumen. For more complete summaries of ex- perimental work, the reader is referred to Chalupa (1975a), Clark (1975a), Ferguson (197S), Huber and Rung (1981), and Owens and Bergen (1983~.
