Download Excess amino acid supply improves methionine and leucine

Published December 8, 2014
Excess amino acid supply improves methionine and leucine
utilization by growing steers1
M. S. Awawdeh,* E. C. Titgemeyer,*2 G. F. Schroeder,* and D. P. Gnad†
*Department of Animal Sciences and Industry and †Department of Clinical Sciences,
Kansas State University, Manhattan 66506-1600
ABSTRACT: In 2 experiments, 6 ruminally cannulated Holstein steers (205 ± 23 and 161 ± 14 kg initial
BW in Exp. 1 and 2, respectively) housed in metabolism
crates were used in 6 × 6 Latin squares to study the
effects of excess AA supply on Met (Exp. 1) and Leu
(Exp. 2) use. All steers received a diet based on soybean
hulls (DMI = 2.66 and 2.45 kg/d in Exp. 1 and 2, respectively); ruminal infusions of 200 g of acetate/d, 200 g
of propionate/d, and 50 g of butyrate/d, as well as abomasal infusion of 300 g of glucose/d to provide energy
without increasing the microbial protein supply; and
abomasal infusions of a mixture of all essential AA
except Met (Exp. 1) or Leu (Exp. 2). Periods were 6 d,
with 2-d adaptations and 4 d to collect N balance data.
All treatments were abomasally infused. In Exp. 1,
treatments were arranged as a 2 × 3 factorial, with 2
amounts of L-Met (0 or 4 g/d) and 3 AA supplements
(no additional AA, control; 100 g/d of nonessential AA
+ 100 g/d of essential AA, NEAA + EAA; and 200 g/d
of essential AA, EAA). Supplemental Met increased (P
< 0.01) retained N and decreased (P < 0.01) urinary N
and urinary urea N. Retained N increased (P < 0.01)
with NEAA + EAA only when 4 g/d of Met was provided,
but it increased (P < 0.01) with EAA with or without
supplemental Met. Both AA treatments increased (P <
0.01) plasma urea and serum insulin. Plasma glucose
decreased (P = 0.03) with supplemental Met. In Exp.
2, treatments were arranged as a 2 × 3 factorial with
2 amounts of L-Leu (0 or 4 g/d) and 3 AA supplements
(control, NEAA + EAA, and EAA). Supplemental Leu
increased (P < 0.01) retained N and decreased (P < 0.01)
urinary N and urinary urea N. Both AA treatments
increased (P < 0.01) retained N, and they also increased
(P < 0.01) urinary N, urinary urea N, and plasma urea.
Serum insulin increased (P = 0.06) with supplemental
Leu and tended (P = 0.10) to increase with both AA
treatments. Supplementation with excess AA improved
Met and Leu use for protein deposition by growing
cattle.
Key words: amino acid, cattle, growth, leucine, methionine, utilization
2006 American Society of Animal Science. All rights reserved.
INTRODUCTION
In vivo (Lobley et al., 1995) and in vitro (Mutsvangwa
et al., 1996, 1999) studies suggested that metabolic ammonia (NH3) loading might lead to inefficient use of
dietary AA by metabolically consuming AA to provide
α-amino N to support ureagenesis. We recently observed no negative effects of NH3 loading on protein
deposition when Met, Leu, or His limited cattle performance (Awawdeh et al., 2004, 2005; McCuistion et al.,
2004). Excess dietary N had different effects on protein
1
Contribution No. 06-39-J from the Kansas Agric. Exp. Stn., Manhattan. This research was supported by NRI Competitive Grants
Program/CSREES/USDA, Award No. 2003-35206-12837.
2
Corresponding author: [email protected]
Received September 28, 2005.
Accepted February 9, 2006.
J. Anim. Sci. 2006. 84:1801–1810
doi:10.2527/jas.2005-557
deposition, depending on the N source. For example,
supplementation with essential AA had no significant
effects on His use, but supplementation with a mixture
of essential and nonessential AA improved His use in
cattle (McCuistion et al., 2004).
We have demonstrated that Met and Leu are used
by growing cattle with efficiencies less than the NRC
(1996) values (Awawdeh et al., 2004, 2005), and that
efficiency of Leu use depends on the animal’s nutritional status (Awawdeh et al., 2005). Our long-term
objective is to evaluate use efficiencies of individual AA
under different nutritional conditions. We have measured the use efficiency for Met, Leu, and His under
NH3 loading (Awawdeh et al., 2004, 2005; McCuistion
et al., 2004), for Met with supplemental energy
(Schroeder et al., 2006a), and for His with excess AA
supply (McCuistion et al., 2004).
Because Met, Leu, and His are metabolized differently throughout the body, excess AA could have differ-
1801
1802
Awawdeh et al.
Table 2. Ruminal and abomasal infusates (Exp. 1 and 2)
Table 1. Diet composition (Exp. 1 and 2)
Item
% of DM
Item
Ingredient
Pelleted soybean hulls
Wheat straw
Cane molasses
Dicalcium phosphate
Sodium bicarbonate
Calcium carbonate
Urea
Magnesium oxide
Trace mineralized salt1
Vitamin premix2
Sulfur
Bovatec-683
83.4
7.4
3.7
2.0
1.2
1.0
0.3
0.4
0.2
0.2
0.1
+
Nutrient
OM
N
89.6
2.1
1
Composition (g/kg, minimum guarantee): NaCl (960 to 990), Mn
(≥ 2.4), Fe (≥ 2.4), Mg (≥ 0.5), Cu (≥ 0.32), Zn (≥ 0.32), I (≥ 0.07), and
Co (≥ 0.04).
2
Provided 4,410 IU of vitamin A, 2,205 IU of vitamin D, and 45
IU of vitamin E per kilogram of diet DM.
3
Supplied 33 mg of lasalocid per kilogram of diet DM.
ent effects on their use by growing cattle. For example,
the first committed step in Met catabolism is cystathionine synthesis, a process that competes with methylation for homocysteine; His catabolism is regulated in
part by supply of AA other than His; Leu is catabolized
throughout the body, rather than principally in the
liver, and the initial step is transamination. Our objective was to examine effects of excess AA on the wholebody protein deposition when Met or Leu was the most
limiting AA.
MATERIALS AND METHODS
The Kansas State University Institutional Animal
Care and Use Committee approved all procedures involving animals in this study.
In 2 experiments, 6 ruminally cannulated Holstein
steers (205 ± 23 and 161 ± 14 kg initial BW in Exp. 1
and 2, respectively) fitted with ruminal and abomasal
infusion lines were used in 6 × 6 Latin squares to study
the effects of excess AA on Met (Exp. 1) and Leu (Exp.
2) use. Steers were housed in individual metabolism
crates in a temperature-controlled room (21°C) under
continuous lighting.
Before initiation of each experiment, steers were
adapted to the basal diet (Table 1) for 2 wk and to
ruminal and abomasal infusions for 5 d. All steers had
free access to water and received the same basal diet
in equal proportions at 12-h intervals, with DMI averaging 2.66 and 2.45 kg/d in Exp. 1 and 2, respectively.
The basal diet, which was characterized for metabolizable AA supply by Campbell et al. (1997), was formulated to provide adequate ruminally degraded protein,
but small amounts of metabolizable AA. All steers received continuous ruminal infusions of 200 g of acetate/
Control
NEAA
+ EAA
0 or 4
10
20
0 or 4
10
20
10
10
10
5
10
10
100
50
—
—
—
—
300
60
30
34
0 or 4
15
35
0 or 4
15
40
20
25
20
10
20
20
130
60
15
25
10
10
300
90
42
17
Abomasal infusate
L-Met
(Exp. 1)
(Exp. 2)
L-Leu (Exp. 1)
(Exp. 2)
1
L-HisⴢHClⴢH2O (74.0%)
2
L-LysⴢHCl (feed grade; 78.8%)
3
L-Thr (feed grade; 98%)
L-Phe
L-Arg
4
L-Trp (feed grade; 98%)
L-Ile
L-Val
L-Glu
Glycine
L-Ala
L-Asp
L-Pro
L-Ser
Dextrose
6 M HCl
NaOH
NaCl
EAA
g/d
0 or 4
20
50
0 or 4
20
60
30
40
30
15
30
30
100
50
—
—
—
—
300
120
54
—
mg/d
Folic acid
PyridoxineⴢHCl
Cyanocobalamin
10
10
0.1
10
10
0.1
g/d
Ruminal infusate
Acetic acid
Propionic acid
Butyric acid
10
10
0.1
200
200
50
200
200
50
200
200
50
1
Provided 7.4, 11.1, and 14.8 g of His/d for control, nonessential
AA + essential AA (NEAA + EAA), and EAA, respectively.
2
Provided 15.8, 31.5, and 47.3 g of Lys/d for control, NEAA + EAA,
and EAA, respectively.
3
Provided 9.8, 19.6, and 29.4 g of Thr/d for control, NEAA + EAA,
and EAA, respectively.
4
Provided 4.9, 9.8, and 14.7 g of Trp/d for control, NEAA + EAA,
and EAA, respectively.
d, 200 g of propionate/d, and 50 g of butyrate/d as well
as abomasal infusions of 300 g of glucose/d to supply
additional energy without increasing the microbial protein supply. To ensure that the most limiting AA for N
retention was Met in Exp. 1 and Leu in Exp. 2, all
steers received continuous abomasal infusions of an
AA mixture (Table 2; control group), as described by
Greenwood and Titgemeyer (2000), that supplied
nonessential and all essential AA, except for Met in
Exp. 1 and Leu in Exp. 2.
In Exp. 1, treatments (Table 2) were arranged as a
2 × 3 factorial, with 2 amounts of L-Met (0 or 4 g/d) and
3 AA supplements (Table 2), including no additional
AA (control), 100 g/d of nonessential AA + 100 g/d of
essential AA (NEAA + EAA), and 200 g/d of essential
AA (EAA). All treatments were delivered to the abomasum via continuous infusion. To ensure that the steers
were able to respond to supplemental Met, the 4 g/d of
supplemental Met was less than the steers’ require-
Amino acid utilization by cattle
ments for maximal N retention under our experimental
conditions (Campbell et al., 1997). The EAA and NEAA
+ EAA treatments were estimated to increase the metabolizable AA supply above that of the control group
by approximately 40%. The 2 groups of AA (NEAA +
EAA and EAA) were selected to examine if the effects
of excess AA supply were dependent on AA type (essential vs. nonessential) or simply on the amount of N
provided.
Each experimental period lasted 6 d, with 2 d for
adaptation to treatment and 4 d for total fecal and
urinary collections. Short adaptation periods are adequate because cattle rapidly adapt to changes in nutrients supplied postruminally (Moloney et al., 1998), and
2-d adaptations have been validated for our experimental model (Schroeder et al., 2006b).
Abomasal infusate for the control treatment was prepared by dissolving the branched-chain AA (L-Val, LLeu, and L-Ile) in 1 kg of water containing 60 g of 6 M
HCl. Once the branched-chain AA were dissolved, the
remaining AA, except L-Glu, were added to the mixture.
Glutamate was dissolved separately in 500 g of water
containing 30 g of NaOH. After all AA were dissolved,
the 2 solutions of AA were mixed together, 300 g of
glucose was added, and water was added to bring the
total weight of the daily infusate to 4 kg.
The EAA and NEAA + EAA treatments were prepared by replacing 2 kg of water from the control infusate with solutions containing AA. For EAA treatment,
branched-chain AA were dissolved in 1.7 kg of water
containing 60 g of 6 M HCl. After the branched-chain
AA were dissolved, the remaining AA (L-Arg, L-His, LLys, L-Phe, L-Thr, and L-Trp) were added to the mixture,
and water was added to bring the final weight to 2 kg.
The NEAA + EAA treatment was prepared by replacing
2 kg of water from the control infusate with 1 kg of the
EAA solution and 1 kg of the NEAA solution. The NEAA
solution was prepared by dissolving L-Glu and L-Asp
with 800 g of water containing 11 g of NaOH. Once
those dissolved, the remaining AA (Gly, L-Ala, L-Pro,
and L-Ser) were added to the solution, and water was
added to bring the final weight to 2 kg.
The AA treatments were balanced for Na and Cl (from
HCl and NaOH used to prepare the AA solutions) by
adding 34 g of NaCl to the control, 17 g of NaCl to NEAA
+ EAA, and 24 g of NaOH to EAA. PyridoxineⴢHCl (10
mg), folic acid (10 mg), and cyanocobalamin (100 ␮g)
were added to the abomasal infusate because steers
maintained under our experimental conditions were deficient in one or more of these vitamins (Lambert et al.,
2004). Methionine was dissolved separately in water
and added to the mixture according to treatment (0 or
4 g).
Ruminal infusates for each steer were prepared by
mixing 200 g of acetic acid/d, 200 g of propionic acid/d,
50 g of butyric acid/d, and 3.55 kg of water/d. Infusion
lines of flexible polyvinylchloride tubing (2.4-mm i.d.)
were placed in the rumen and abomasum through the
ruminal cannula. A perforated vial was attached to the
1803
end of the ruminal infusion lines to avoid direct infusion
of VFA onto the ruminal wall. Rubber flanges (8-cm
diam.) were attached to the end of the abomasal infusion lines to ensure that they remained in the abomasum. Solutions were continuously infused into the rumen and abomasum by using a peristaltic pump.
Representative samples of the basal diet for each
period were collected daily and stored (−20°C) for later
analysis. Orts, if any, were collected on d 2 through 5,
composited, and stored (−20°C) for later analysis. Feces
and urine for each steer were collected from d 3 through
6 of each period and were weighed to determine output.
Urine was collected in buckets containing 300 mL of 6
M HCl to prevent NH3 loss. Representative samples of
feces (10%) and urine (1%) were saved, composited by
period, and stored (−20°C) for later analysis. Three observations were not obtained in Exp. 1 due to failures
in the collection of excreta. Six observations were not
obtained in the original design in Exp. 2 as a result of
failure to collect excreta. Thus, in Exp. 2, the Latin
square was modified by adding an additional period
(observations were collected from 5 steers), with a treatment distribution to compensate for the missing data,
to increase the final number of observations to 35.
Samples of the diet, orts, and feces were analyzed for
DM (105°C in a forced-air oven for 24 h) and OM (weight
loss upon ashing at 450°C for 8 h) to calculate digestibilities. Composite samples of the diet, orts, wet feces, and
urine were analyzed for N by using a Leco FP 2000
Nitrogen Analyzer (Leco Corporation, St. Joseph, MI)
to calculate N retention.
Jugular blood samples were collected 4 h after the
morning feeding on the last day of each period. Blood
was collected into vacuum tubes (Becton Dickinson,
Franklin Lakes, NJ) containing sodium heparin, immediately chilled on ice, and centrifuged for 20 min at
1,000 × g to obtain plasma. Blood also was collected
into vacuum tubes without additives, allowed to clot
for 30 min at room temperature, and centrifuged for 20
min at 1,000 × g to obtain serum. Samples were stored
(−20°C) for later analysis of plasma glucose, urea, and
AA, and of serum insulin and IGF-I.
Plasma glucose concentrations were measured according to methods of Gochman and Schmitz (1972).
Plasma and urinary urea were measured according to
the method of Marsh et al. (1965), and urinary and
ruminal fluid NH3 concentrations were measured by
the method of Broderick and Kang (1980). Plasma AA
were measured by gas chromatography with a commercial kit (EZ:faast, Phenomenex, Torrance, CA).
Insulin was measured with an insulin RIA kit (intraassay CV of 2.0% and sensitivity of 0.024 ng/mL;
DSL-1600, Diagnostic Systems Laboratories, Webster,
TX), and IGF-I was measured with an active IGF-I
coated-tube, 2-site immunoradiometric assay kit (intraassay CV of 1.0% and sensitivity of 5.0 ng/mL; DSL5600, Diagnostic Systems Laboratories).
Data were analyzed statistically as a Latin square
by using the MIXED procedure of SAS, Release 8.1
1804
Awawdeh et al.
Table 3. Effect of supplemental Met and AA on nitrogen balance and diet digestibility of growing steers (Exp. 1)1
P value
No Met
Item
n
4 g of Met/d
Control
NEAA
+ EAA
EAA
Control
NEAA
+ EAA
EAA
SEM2
Met
Control
vs. AA
EAA vs.
EAA +
NEAA
Met ×
(Control
vs. AA)
Met ×
(EAA vs.
EAA +
NEAA)
6
4
5
6
6
6
Nitrogen, g/d
Dietary
Infused
Total intake
Fecal
Urinary
Urea
Ammonia
Retained
57.9
34.6
92.5
20.2
49.9
37.0
1.7
22.3
56.9
61.1
118.0
19.4
77.9
60.9
3.9
21.6
55.6
63.2
118.8
18.6
70.8
56.8
6.3
29.8
56.9
35.0
91.9
19.1
45.3
33.4
1.5
27.5
56.0
61.5
117.6
19.4
60.6
47.2
4.0
37.5
57.2
63.5
120.8
18.6
65.7
53.2
2.0
36.4
1.3
—
1.3
1.0
2.3
2.1
1.2
2.6
0.91
0.19
0.97
0.35
0.18
0.67
0.66
<0.01
<0.01
0.10
<0.01
<0.01
0.49
<0.01
<0.01
0.01
<0.01
0.03
0.48
0.66
0.64
0.83
0.17
0.35
0.51
0.09
0.14
0.31
0.15
0.18
0.97
0.02
0.02
0.06
0.08
Diet digestibility, %
DM
OM
75.2
78.0
75.6
77.9
75.5
77.7
75.0
77.0
75.2
77.9
75.1
77.5
1.1
1.2
0.74
0.70
0.80
0.81
0.94
0.81
0.88
0.68
0.99
0.98
1
NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA.
For n = 6.
2
(SAS Inst. Inc., Cary, NC). The model contained the
effects of Met, AA, Met × AA, and period. Steer was
included as a random variable. Treatment means were
computed using the LSMEANS option. Orthogonal contrasts were used to evaluate the effects of AA treatments and their interactions with Met: 1) control vs.
average of EAA and NEAA + EAA, and 2) EAA vs.
NEAA + EAA. Pairwise t-tests among least squares
means were used to derive P values for comparisons
that could not be easily described by the contrasts.
In Exp. 2, experimental housing, periods, diet, basal
infusions, sample collection, laboratory analyses, and
statistical analysis were the same as in Exp. 1, except
for Leu being the most limiting AA instead of Met.
Treatments were arranged as a 2 × 3 factorial, with 2
amounts of L-Leu (0 or 4 g/d) and 3 AA supplements
(control, NEAA + EAA, and EAA). The AA treatments
were the same as in Exp. 1, except for Met replacing
Leu (Table 2). On the basis of data from Awawdeh
et al. (2005), 4 g/d of Leu was less than the steers’
requirements under our experimental conditions.
RESULTS AND DISCUSSION
Experiment 1
Diet digestibilities and N retention data in response
to supplemental Met and AA are presented in Table 3.
Diet DM and OM digestibilities were not altered (P
≥ 0.68) by treatments and averaged 75.3 and 77.7%,
respectively. Fecal N was not affected (P ≥ 0.48) by
treatments.
Supplementation with 4 g of Met/d increased (P <
0.01) retained N from 24.6 to 33.8 g/d, as a result of
decreased (P < 0.01) urinary N excretion, indicating
that Met was indeed the limiting AA in our experiment.
Similar responses to Met have been consistently ob-
served with our experimental model (Campbell et al.,
1996, 1997; Awawdeh et al., 2004). Also, supplemental
Met decreased (P < 0.01) urinary urea N excretion. The
decreases in urinary N and urinary urea N, and the
increases in retained N in response to supplemental
Met were or tended to be greater for the NEAA + EAA
treatment than for the control or the EAA treatment
[Met × (EAA vs. NEAA + EAA) interaction; P = 0.02
for urinary N, P = 0.08 for N retention]. Both AA treatments led to increases (P < 0.01) in urinary urea N
that were closely proportional to the increases in total
urinary N. Both AA treatments increased (P = 0.01)
urinary NH3-N. The NEAA + EAA treatment increased
(P < 0.01) urinary N less with 4 g of supplemental Met/
d than with no supplemental Met (increases of 15.3 vs.
28.0 g/d, respectively). The EAA treatment increased
urinary N the same amount with or without supplemental Met (increases of 20.9 and 20.4 g/d, respectively).
The increases in N intake in response to the EAA treatment exceeded the increases in urinary N, resulting in
N retention being increased 8.2 g/d by the EAA
treatment.
The increases in retained N in response to the EAA
and NEAA + EAA treatments imply that both AA treatments improved the efficiency of Met use because they
increased N retention with the same amount of supplemental Met. This response could be attributed to the
energy supplied by the AA treatments (Schroeder et
al., 2006a). Our observations that increased AA supply
improved, or had no negative impact on, protein deposition when Met limited N retention is in agreement with
the finding of McCuistion et al. (2004) that supplying
excess AA had either no negative effect or a positive
effect on protein deposition when His limited steers’
performance.
Although it could be argued that our AA treatments
improved N retention by supplying a colimiting AA
1805
Amino acid utilization by cattle
Table 4. Effect of supplemental Met and AA on serum hormone and plasma metabolite concentrations of growing
steers (Exp. 1)1
P value
No Met
Item
n
4 g of Met/d
Control
NEAA
+ EAA
EAA
6
5
5
Serum
Insulin
IGF-I
Met ×
(EAA vs.
NEAA +
EAA)
SEM2
Met
Control
vs. AA
1.02
174
0.2
21
0.75
0.45
<0.01
0.99
0.18
0.72
0.54
0.40
0.73
0.07
4.2
5.0
4.0
5.0
0.2
0.1
<0.01
0.03
<0.01
0.93
0.69
0.60
0.56
0.21
0.44
0.48
6.3
185
29
9.5
5.1
132
254
540
43
121
187
107
95
59
67
158
107
39
29
272
8.0
165
27
7.8
5.4
139
221
484
50
162
260
151
102
91
49
152
152
47
34
379
1.3
11.7
1.5
0.5
0.6
7.2
18.7
44.3
6.7
8.3
13.8
11.3
5.5
5.5
2.8
12.5
15.7
4.0
3.1
15.2
<0.01
0.04
0.08
0.54
0.06
0.02
0.71
<0.01
0.22
0.65
0.05
0.56
0.35
0.19
<0.01
<0.01
0.57
0.41
0.87
<0.01
0.01
<0.01
0.94
0.48
0.88
0.06
0.97
0.16
0.97
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.38
0.08
0.48
0.24
0.86
0.84
0.17
<0.01
0.21
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.14
<0.01
<0.01
<0.01
<0.01
0.49
0.56
0.83
0.06
0.86
0.42
0.05
0.04
0.28
0.19
0.99
0.01
<0.01
0.14
0.78
<0.01
0.09
0.17
0.02
0.82
0.69
0.72
0.15
0.04
0.77
0.23
<0.01
0.16
0.79
0.64
0.88
0.96
0.03
0.52
0.56
0.32
0.38
0.21
0.04
0.48
Control
NEAA
+ EAA
EAA
6
6
6
0.68
176
0.88
198
2.9
5.1
10.8
157
28
7.5
5.4
142
205
533
41
75
122
75
61
38
48
117
71
29
19
163
ng/L
0.62
180
0.95
141
1.16
186
3.5
5.2
4.8
5.3
4.6
5.2
6.5
167
30
8.4
4.5
134
253
640
56
88
143
97
77
41
57
175
89
38
25
206
4.0
200
28
7.6
4.5
123
179
822
44
114
209
90
72
53
74
335
74
33
21
302
4.7
187
32
8.1
4.4
113
265
665
55
163
279
135
100
78
59
307
143
52
36
426
Plasma
Urea
Glucose
Met ×
(Control
vs. AA)
EAA vs.
NEAA +
EAA
mM
␮M
Met
Ala
Asn
Asp
α-Aminobutyric acid
Glu
Gln
Gly
His
Ile
Leu
Lys
Ornithine
Phe
Pro
Ser
Thr
Trp
Tyr
Val
1
NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA.
For n = 6.
2
other than Met, N balance responses to supplemental
Met have been demonstrated to be linear up to 6 g/d
(Campbell et al., 1997) and 10 g/d (Lambert et al., 2002).
Also, the lack of the increase in retained N in response
to the NEAA + EAA treatment when no supplemental
Met was provided supports a conclusion that the responses to the AA treatments were not a result of supply
of a colimiting AA.
If deposited protein equals retained N × 6.25 and
protein of tissue gain contains 2.0% Met (Ainslie et al.,
1993), the calculated incremental efficiencies of use of
the 4 g of supplemental Met/d were 16, 50, and 21% for
steers receiving the control, NEAA + EAA, and EAA
treatments, respectively. The 50% value for the NEAA
+ EAA treatment seems to be an anomaly resulting
from the lack of response to this treatment in the absence of supplemental Met. Our efficiency of use of supplemental Met for the control animals (16%) was comparable to that previously reported in growing steers
(Campbell et al., 1996; Awawdeh et al., 2004; Schroeder
et al., 2006a) but was much less than the 66% predicted
by the NRC (1996) equations.
Effects of supplemental Met and AA on blood metabolites and hormones are presented in Table 4. Serum
IGF-I concentration was not affected by treatments.
Serum insulin and plasma urea concentrations increased (P < 0.01) in response to both AA treatments.
Supplemental Met decreased (P ≤ 0.03) plasma concentrations of urea and glucose.
Supplemental Met increased (P < 0.01) its plasma
concentration as a result of increased supply, but the
magnitude of increase was small enough to suggest that
4 g of Met/d did not exceed the steers’ requirement
(Campbell et al., 1997; Lambert et al., 2002). Supplementation with Met decreased (P ≤ 0.05) plasma concentrations of Ala, Pro, Leu, and Val. Also, supplemental Met decreased (P < 0.01) plasma concentrations of
Gly and Ser, but these decreases in response to Met
were greater when steers received the AA treatments
than when they received the control treatment [Met ×
(control vs. AA) interaction; P ≤ 0.04], likely because the
greater concentrations achieved when AA treatments
were infused allowed for greater responses to supplemental Met. Similar responses to supplemental Met
1806
Awawdeh et al.
were observed for Leu, Ser, and Val in growing steers
(Titgemeyer and Merchen, 1990; Campbell et al., 1997;
Awawdeh et al., 2004). The decreases in Ala, Gly, Leu,
Pro, Ser, and Val might be due to increased uptake and
use of these AA for protein deposition as Met, the first
limiting AA, became available in greater amounts. In
part, the ability of supplemental Met to reduce plasma
Ser concentrations also could be attributed to the use
of Ser during of Met transsulfuration to produce cystathionine. However, the much greater concentrations of
Ser in steers receiving no supplemental Met might also
suggest that the elevated Ser concentrations are reflective of metabolic disturbances during Met deficiency
and that, as expected, supplementation with the AA
treatments exacerbated the Met deficiency. When Met
supply was not limiting, excess AA did not substantially
affect the plasma concentration of Ser (Lambert, 2001).
Supplemental Met increased (P = 0.02) plasma concentrations of Glu. The lessened demands for N removal
when supplemental Met was provided, as indicated by
greater N retention, might have resulted in less amidation of Glu to Gln, although Gln concentrations were
not consistently decreased in response to supplemental
Met. Methionine tended (P = 0.06) to increase plasma αaminobutyric acid, likely produced from α-ketobutyric
acid (Costa et al., 1985), which is a product of cystathionine cleavage.
Plasma concentrations of Ala, Pro, and Ser increased
(P < 0.01) in response to the NEAA + EAA treatment,
likely as a result of increased supply. Also, some EAA
(Ile, Leu, Phe, and Val) increased (P < 0.01) in response
to the NEAA + EAA treatment, likely due to increased
supply of these AA. Plasma concentration of ornithine
increased in response to the NEAA + EAA treatment
when supplemental Met was provided, but there was
no effect when no supplemental Met was provided [Met
× (control vs. AA) interaction; P < 0.01].
The EAA treatment increased plasma concentrations
of Ile, Leu, Lys, Phe, Thr, Trp, Tyr, and Val above those
observed with the NEAA + EAA treatment [(EAA vs.
NEAA + EAA); P < 0.01], likely due to greater amounts
infused. Also, plasma concentration of ornithine increased with the EAA treatment, and the increase was
greater when supplemental Met was provided [Met ×
(control vs. AA) interaction; P < 0.01].
Although Ala and Ser were not included in the EAA
treatment, the EAA treatment increased plasma concentration of these AA; however, the responses to the
EAA treatment tended to be less (P ≤ 0.14) than that
observed for the NEAA + EAA treatment. Most of the
N resulting from catabolism of infused EAA should be
removed from the system as urea. Because Ala acts as
an interorgan transporter of N, the increase in plasma
concentration of Ala in response to the EAA treatment
might be a result of increased interorgan N transport.
Both AA treatments decreased (P = 0.01) plasma concentrations of Met and tended (P = 0.06) to decrease
Glu concentrations. The decreases in plasma Met concentrations in response to AA treatments could be ex-
plained by an increased Met uptake and subsequent
use in protein deposition, as indicated by increased retained N in response to these AA treatments. The decreases in plasma Glu concentrations in response to AA
treatments might be a result of increased urea synthesis to remove excess N infused. The AA treatments
provided AA beyond the AA requirements for protein
deposition, as indicated by increased urinary urea (Table 3) and plasma urea concentrations (Table 4). Thus,
excess AA must be removed from the system, mainly
via ureagenesis. Glutamate N can be utilized in ureagenesis via transamination to aspartate (Parker et al.,
1995), which will contribute 1 of the 2 N used in urea
synthesis. The decrease in Glu concentration in response to AA treatments also might be due to increased
amidation to Gln.
Experiment 2
Diet digestibilities and N retention data in response
to supplemental Leu and AA are presented in Table 5.
Diet DM and OM digestibilities were not altered (P
≥ 0.13) by treatments and averaged 74.5 and 77.0%,
respectively. Total N intake increased (P < 0.01) with
AA treatments, as a result of additional N infused. Fecal
N was not affected (P > 0.12) by treatments.
There were no interactions (P ≥ 0.32) between supplemental Leu and AA treatments for urinary and retained
N. Supplementation with 4 g of Leu/d increased (P <
0.01) retained N from 30.6 to 34.6 g/d, as a result of
decreasing (P < 0.01) urinary N from 54.9 to 51.1 g/d.
This was an expected response because Leu was purposefully designed to be the limiting AA (Löest et al.,
2001; Awawdeh et al., 2004). Urinary urea N decreased
(P < 0.01) from 45.3 to 40.1 g/d, but urinary NH3-N
increased (P = 0.04) from 2.2 to 3.2 g/d in response to
supplemental Leu. Although the increase in urinary
NH3-N resulting from supplementation with Leu was
significant, the low amounts of urinary NH3-N here, as
well as in Exp. 1, indicate that our steers were not
experiencing metabolic acidosis, which would have increased urinary NH3 excretion more dramatically (Vagnoni and Oetzel, 1998).
Both AA treatments increased (P < 0.01) urinary excretion of N and urea N. However, the increases in
urinary N in response to AA treatments (increase of
22.0 and 23.8 g/d) were less than the increases in intake
N (26.4 g/d), resulting in increases (P = 0.01) in retained
N in response to both AA treatments. Also, the increases
in urinary N were greater (P = 0.05) in response to the
EAA treatment than to the NEAA + EAA treatment,
resulting in a greater (P = 0.13) retained N for the
NEAA + EAA treatment than for the EAA treatment
(increase of 4.3 vs. 2.2 g/d). Urinary NH3-N increased
(P = 0.03) in response to both AA treatments. The increases in retained N in response to AA treatments
might have resulted from supplying additional energy.
Because retained N was increased at the same amounts
of Leu, both AA treatments improved the efficiency of
1807
Amino acid utilization by cattle
Table 5. Effect of supplemental Leu and AA on N balance and diet digestibility of growing steers (Exp. 2)1
P value
No Leu
Item
n
4 g of Leu/d
Control
NEAA
+ EAA
EAA
Control
NEAA
+ EAA
EAA
SEM2
Leu
Control
vs. AA
EAA vs.
EAA +
NEAA
Leu ×
(Control
vs. AA)
Leu ×
(EAA vs.
EAA +
NEAA)
7
5
6
5
6
6
Nitrogen, g/d
Dietary
Infused
Total intake
Fecal
Urinary
Urea
Ammonia
Retained
53.4
33.0
86.5
18.2
40.2
30.5
1.9
28.0
53.7
58.4
112.2
18.3
60.9
51.5
1.8
33.0
53.2
59.3
112.6
18.5
63.5
53.7
2.9
30.7
53.6
33.5
87.1
19.0
35.3
25.4
2.1
32.7
55.3
58.9
114.2
19.4
58.4
47.0
3.8
36.3
54.1
59.8
113.9
19.8
59.5
47.7
3.8
34.6
1.1
—
1.1
0.9
0.9
1.7
0.6
1.4
0.32
0.50
0.43
0.56
0.73
0.14
0.12
<0.01
<0.01
0.04
<0.01
<0.01
0.56
<0.01
<0.01
0.03
<0.01
0.93
0.69
0.05
0.28
0.32
0.13
0.56
0.75
0.32
0.95
0.25
0.66
0.73
0.95
0.42
0.58
0.38
0.81
Diet digestibility, %
DM
OM
74.1
76.6
74.8
77.5
74.7
77.2
75.2
77.7
75.1
77.5
73.1
75.6
1.0
1.1
0.96
0.76
0.70
0.70
0.17
0.13
0.25
0.17
0.22
0.27
1
NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA.
For n = 6.
2
Leu use. Our observation that increased AA supply
improved protein deposition when Leu limited N retention is in agreement with our findings in Exp. 1, in
which Met was limiting, and with those of McCuistion
et al. (2004), in which His was limiting. We expected
that excess branched-chain AA (Val and Ile) might increase Leu catabolism by increasing branched-chain
α-keto acid dehydrogenase (Block, 1989). In contrast,
supplying a complete mixture of AA that contained Ile
and Val in amounts greater than the steers’ requirements improved protein deposition in Exp. 2.
Based on previous work from our laboratory (Awawdeh et al., 2005), 4 g of Leu/d was selected to be less
than the animals’ requirements to ensure that steers
were able to respond to supplemental Leu. If retained
N was completely deposited as tissue protein (retained
N × 6.25) and if tissue protein gain contains 6.7% Leu
(Ainslie et al., 1993), the calculated incremental efficiencies of use of the 4 g of supplemental Leu/d averaged
41%, without large differences among the AA treatments (49, 34, and 41% for steers receiving the control,
NEAA + EAA, and EAA treatments, respectively). The
average value (41%) for the incremental efficiency of
supplemental Leu use agreed with our previously reported values (Awawdeh et al., 2005) but was less than
the 69% value predicted by the NRC (1996). The 41%
value is greater than that for Met in Exp. 1 (16% for
the control group), however, indicating that different
AA might have different efficiency values.
Effects of supplemental Leu and AA on blood metabolites and hormones are presented in Table 6. Serum
IGF-I concentrations were not affected (P ≥ 0.19) by
any treatment. Supplemental Leu decreased (P ≤ 0.03)
plasma concentrations of urea and tended to increase
(P = 0.06) serum insulin concentrations. Both AA treatments increased (P < 0.01) plasma urea concentrations,
decreased (P < 0.01) plasma glucose concentrations, and
tended (P = 0.10) to increase serum insulin concentrations. The insulin response to the AA treatments tended
to be greater (P = 0.12) when 4 g of supplemental
Leu/d was provided. Decreased glucose concentrations
in response to AA treatments could reflect increased
use of glucose in support of increased tissue deposition
or responses to alterations in insulin.
Plasma Leu concentrations increased (P < 0.01) in
response to supplemental Leu, likely due to increased
supply; the observed increase tended (P = 0.14) to be
greater with AA treatments, predominantly due to
lower concentrations achieved when AA treatments
were infused, which allowed for greater responses to
supplemental Leu. The magnitude of increase in
plasma Leu in response to supplemental Leu was small
enough to suggest that 4 g of Leu/d was less than the
steers’ requirements. Supplementation with Leu decreased (P ≤ 0.02) plasma concentrations of Asn, Ile,
Thr, Tyr, and Val, and tended (P ≤ 0.17) to decrease
plasma Asp, Gly, Met, ornithine, and Ser concentrations, in large part resulting from increased uptake
and use in protein deposition as Leu, the limiting AA,
became more available. Plasma concentrations of Ile,
Tyr, and Val in response to infused Leu were previously
reported in growing steers (Greenwood and Titgemeyer,
2000; Löest et al., 2001; Awawdeh et al., 2005).
The NEAA + EAA treatment increased (P < 0.01)
plasma concentrations of Ala, Pro, and Ser, likely as a
result of increased supply. Also, plasma concentrations
of a number of EAA (Ile, Lys, Met, Phe, Thr, Trp, Tyr,
and Val) increased (P ≤ 0.02) in response to the NEAA
+ EAA treatment, likely due to increased supply. The
EAA treatment increased (P ≤ 0.02) plasma concentrations of Ile, Lys, Met, Phe, Thr, Trp, Tyr, and Val,
compared with those of the NEAA + EAA treatment,
likely due to greater amounts being infused.
1808
Awawdeh et al.
Table 6. Effect of supplemental Leu and AA on serum hormone and plasma metabolite concentrations of growing
steers (Exp. 2)1
P value
No Leu
Item
4 g of Leu/d
Control
NEAA
+ EAA
EAA
7
5
6
n
Serum
Insulin
IGF-I
Leu ×
(Control
vs. AA)
Leu ×
(EAA vs.
NEAA
+ EAA)
SEM2
Leu
Control
vs. AA
EAA vs.
NEAA
+ EAA
0.99
260
0.1
54.2
0.06
0.67
0.10
0.44
0.87
0.19
0.12
0.43
0.91
0.30
3.6
5.44
0.3
0.1
0.03
0.35
<0.01
<0.01
0.07
0.76
0.90
0.32
0.14
0.20
3.8
24.9
2.1
0.72
1.1
6.8
17.7
39.0
4.0
13.2
12.9
3.2
5.2
4.4
3.1
11.0
9.5
2.6
2.1
26.6
<0.01
0.70
<0.01
0.09
0.29
0.91
0.27
0.08
0.44
<0.01
0.21
0.17
0.14
0.23
0.24
0.13
0.02
0.51
0.02
<0.01
0.05
0.04
0.89
<0.01
<0.01
0.92
0.32
0.29
0.05
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.28
<0.01
0.20
0.69
0.41
0.76
0.57
<0.01
0.83
<0.01
0.01
<0.01
0.02
<0.01
<0.01
<0.01
<0.01
0.02
<0.01
<0.01
0.14
0.81
0.40
0.41
0.56
0.79
0.35
0.10
0.05
0.21
0.54
0.27
0.40
0.94
0.22
0.87
0.91
0.16
0.45
0.32
0.39
0.99
0.42
0.10
0.74
0.64
0.51
0.85
0.18
0.14
0.20
0.91
0.41
0.73
0.88
0.12
0.51
0.30
0.22
0.36
Control
NEAA
+ EAA
EAA
5
6
6
0.65
262
0.99
269
4.1
5.32
ng/L
0.68
280
0.67
274
0.70
203
2.9
5.65
4.1
5.31
4.1
5.11
Plasma
mM
Urea
Glucose
2.6
5.60
␮M
Leu
Ala
Asn
Asp
α-Aminobutyric acid
Glu
Gln
Gly
His
Ile
Lys
Met
Ornithine
Phe
Pro
Ser
Thr
Trp
Tyr
Val
31
213
33
7.4
11
85
258
450
42
130
85
25
54
41
52
65
69
41
20
273
23
267
34
10.3
14
85
241
511
40
253
114
32
77
62
68
105
113
47
25
529
22
213
31
8.9
14
90
223
349
44
300
124
40
84
78
47
80
137
51
30
672
37
203
28
7.1
9
87
227
414
33
103
71
25
45
37
46
54
55
35
15
207
38
263
29
7.9
13
86
227
478
46
194
99
28
71
56
67
106
104
45
20
422
33
211
29
8.8
14
85
228
307
41
266
129
36
84
75
46
50
119
55
30
592
1
NEAA + EAA = 100 g/d nonessential + 100 g/d essential AA; EAA = 200 g/d essential AA.
For n = 6.
2
Both AA treatments decreased (P = 0.05) plasma concentrations of Leu, likely due to increased uptake and
use of Leu in protein deposition, as indicated by increased N retention in response to AA treatments. Also,
both AA treatments increased (P < 0.01) plasma concentrations of Asp and α-aminobutyric acid. The increases
in plasma concentrations of α-aminobutyric acid in response to AA treatments might be a result of increased
Met supply, inasmuch as α-aminobutyric acid production can result from Met transsulfuration. Plasma His
concentrations were increased with AA treatments
when supplemental Leu was provided but not without
it [Leu × (control vs. AA) interaction; P = 0.05].
General Discussion
We examined the effects of excess AA on the wholebody protein deposition under conditions in which either Met or Leu was limiting. To achieve that, the diet
was formulated to provide deficient amounts of digestible AA, and then supplements containing all essential
AA, except Met in Exp. 1 or Leu in Exp. 2, were pro-
vided. If the AA under study were not limiting, negative
effects of excess N, if any, on AA use might not lead to
any changes in performance because an excess supply
of the AA under study could allow for optimal performance, even in the face of reduced efficiency of use. For
example, Hagemeier et al. (1983) demonstrated that
supply of excess Arg had no negative effect on swine
performance when Lys supply was adequate, but when
Lys supply was limiting, excess Arg decreased performance. Similarly, excess Leu decreased retained N in
pigs when Ile supply was limiting, but did not affect
retained N when Ile was not limiting (Langer and Fuller, 2000).
We expected that excess AA might decrease retained
N as a result of increased catabolism of the limiting AA
to support ureagenesis or as a result of an AA imbalance
or both. Various mixtures of excess AA have led to AA
imbalances and, subsequently, to decreased performance of chicks (Park and Austic, 2000), rats and mice
(Sauberlich, 1956; Peng, 1979), and pigs (Southern and
Baker, 1982; Hagemeier et al., 1983; Edmonds and
Baker, 1987). Supplying excess Met (0.177 g of Met/kg
Amino acid utilization by cattle
of BW) decreased retained N in Holstein calves (Abe et
al., 1999). In Exp. 2, the greatest amount of Met (EAA
treatment) was roughly two-thirds of that provided by
Abe et al. (1999; 0.12 vs. 0.177 g/kg of BW). Also, supplying excess Lys (0.43 g/kg of BW) did not decrease retained N in cattle (Abe et al., 2001). In our experiments,
supplemental Lys was provided at 0.23 g/kg of BW in
Exp. 1 and 0.29 g/kg of BW in Exp. 2 for the EAA
treatment. The discrepancy between our findings and
those of studies that observed negative effects of supplying excess AA (leading to AA imbalance) can possibly
be attributed to species differences, to the supply of
single AA vs. mixtures containing all EAA (except the
limiting AA under investigation), or to the amount of
excess AA provided.
In our study, excess AA improved protein deposition
when Leu supply limited steers’ performance (Exp. 2).
When Met supply limited steers’ performance (Exp. 1),
EAA increased protein deposition, regardless of supplemental Met, but excess NEAA + EAA increased protein
deposition only when 4 g of supplemental Met/d was
provided. When His supply was very limited (no additional His was provided), only a mixture of NEAA and
EAA, but not EAA alone, increased protein deposition
(McCuistion et al., 2004). It is clear that the effects on
protein deposition of supplying excess AA are dependent upon the AA being studied and on the source of
excess AA.
We demonstrated that excess AA improved the efficiency of use of Met and Leu because retained N increased at the same amounts of the limiting AA. The
efficiency of use of Met from the basal diet in Exp. 1
was 105% for the control and increased to an average
of 121% for the NEAA + EAA and EAA treatments.
The basal Met supply was 2.7 g/d provided by the diet
(Campbell et al., 1997), and the amounts of Met deposited in the body were calculated assuming that deposited protein equaled retained N × 6.25 and that protein
of tissue gain contained 2.0% Met (Ainslie et al., 1993).
Similarly, additional AA included in the AA treatments
in Exp. 2 increased the efficiency of use of the basal
dietary Leu from 81% for control to an average of 92%
for the NEAA + EAA and EAA treatments. The basal
Leu supply was 14.4 g/d provided by the diet (Campbell
et al., 1997), and the amounts of Leu deposited in the
body were calculated assuming that deposited protein
equaled retained N × 6.25 and that protein of tissue
gain contained 6.7% Leu (Ainslie et al., 1993). For both
Met and Leu, the efficiencies for use of the basal AA
supply are likely overestimated due to the routine overestimation of protein deposition by the N balance technique. The improvements in efficiency in response to
the NEAA + EAA and EAA treatments could be explained as a result of excess AA being used as a source of
energy. Gerrits et al. (1996) demonstrated that protein
deposition was improved in preruminant calves by supplying a protein-free energy source when the protein
supply was limiting, indicating that supplemental energy improves the use efficiency of dietary protein for
1809
tissue deposition. Increasing the energy supply linearly
increased retained N in growing steers limited by Met,
resulting in improved use efficiency of Met (Schroeder
et al., 2006a). The efficiency of use of the limiting AA
may be improved because energy supply determines
the protein turnover rate in ruminants by decreasing
protein degradation and, subsequently, increasing protein deposition (Asplund, 1994). In our study, additional
AA N included in the AA treatments was used for protein deposition with poor efficiencies of 21% (Exp. 1)
and 11% (Exp. 2), suggesting that they were not used
predominantly as a source of limiting AA. When all
supplemental energy provided to pigs was from protein,
the efficiency of protein use was 17% (Fuller and Crofts,
1977), similar to our values.
It is also possible that the net effects observed in
response to the AA treatments were a balance of benefits resulting from the energy supplied and of detriments from AA imbalances or increases in AA catabolism. If both positive and negative effects were present,
it is clear that the positive effects outweighed the negative ones. Because we did not provide energy to the
control treatment in amounts equal to the AA treatments, it is impossible to separate these responses.
The effects of supplemental Met, Leu, and AA on
plasma glucose and serum insulin levels are dependent
on the AA being studied and on the protein (AA) status
of the animals. For example, Met decreased plasma
glucose but had no effect on serum insulin levels (Exp.
1). On the other hand, Leu increased serum insulin but
did not affect plasma glucose levels (Exp. 2). Although
the same AA mixtures, except for the limiting AA, were
used in both experiments, excess AA had different effects on glucose and insulin. Excess AA increased serum
insulin in both experiments, but decreased plasma glucose only when Leu was limiting (Exp. 2).
Different AA have different use efficiency values, and
the efficiency values, at least for Leu and Met, are less
than those predicted by the NRC (1996). Our estimated
efficiencies of use for Leu and Met raise doubts about
the validity of using the single equation for all AA developed by Ainslie et al. (1993) and adopted by the NRC
(1996).
We previously studied the effects of ruminal NH3
loading on protein deposition in growing steers limited
by Met, Leu, or His supply (Awawdeh et al., 2004, 2005;
McCuistion et al., 2004). From the current study and
that of McCuistion et al. (2004), excess N from AA does
not negatively affect protein deposition by cattle, at
least when His, Leu, or Met is limiting. This finding
contrasts the suggestions of in vitro (Mutsvangwa et
al., 1996, 1999) and in vivo (Lobley et al., 1995) studies
that excess N might lead to inefficient use of dietary
AA as a result of metabolically consuming AA to provide
α-amino N to support ureagenesis. Excesses of AA supply do not seem to penalize the use of Met and Leu by
growing steers.
1810
Awawdeh et al.
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