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OCR for page 28
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
OCR for page 29
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
OCR for page 30
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
OCR for page 31
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
OCR for page 32
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
OCR for page 33
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
OCR for page 34
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
OCR for page 35
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
OCR for page 36
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~.
Representative terms from entire chapter:
amino acids
