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