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Effects of Dietary Clenbuterol on Metabolism of the Hindquarters

in Steers

Article  in  Journal of Animal Science · March 1988

DOI: 10.2527/jas1988.662342x · Source: PubMed

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 Effects of Dietary Clenbuterol on Metabolism of the
Hindquarters in Steers
J. H. Eisemann, G. B. Huntington and C. L. Ferrell

J ANIM SCI 1988, 66:342-353.

The online version of this article, along with updated information and
services, is located on the World Wide Web at:
http://jas.fass.org/content/66/2/342

www.asas.org

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 EFFECTS OF D I E T A R Y C L E N B U T E R O L ON M E T A B O L I S M
OF THE H I N D Q U A R T E R S IN STEERS 1

J. H. E i s e m a n n , G. B. H u n t i n g t o n a n d C. L. Ferrell 2'3

U S D A - A R S , R o m a n L. H r u s k a U.S. M e a t A n i m a l R e s e a r c h C e n t e r
Clay Center, NE 6 8 9 3 3
and
Beltsville A g r i c u l t u r a l R e s e a r c h C e n t e r
Beltsville, MD 2 0 7 0 5

ABSTRACT
The objective of this study was to measure acute (d 1) and chronic (d 9) effects of dietary
clenbuterol on heart rate, blood flow, oxygen uptake, and net uptake/release of metabolites in the
hindquarters of growing steers. The design was a single reversal with two 9-d periods of control or 8
mg clenbuterol/d with 5 d between periods. Within 2 h of initial consumption of 2 mg clenbuterol
(d 1), heart rate and blood flow doubled and arterial plasma concentrations of glucose, L-lactate
and nonesterified fatty acid (NEFA) increased, whereas cx-NH2 N and NH 3 concentrations decreased,
demonstrating an acute response. Uptake of oxygen increased and net uptake of cz-NH2 N de-
creased. Net release of both L-lactate and NEFA increased. On d 9, there were no acute responses
to clenbuterol consumption; however, heart rate, blood flow, and NEFA concentration remained
chronically elevated, and plasma concentrations of acetate and propionate decreased compared
with control feeding. Net uptake of c*-NH2 N, oxygen and release of L-lactate by the hindquarters
chronically increased on d 9 of clenbuterol feeding. Changes in both blood flow and arteriovenous
(AV) concentration difference contributed to changes in uptake/release. The chronic metabolic
changes and oxygen uptake were consistent with increased N retention in the hindquarters through
increased protein synthesis, decreased use of acetate and increased reliance on NEFA for cellular
energy. In conclusion, the data show that the perturbation of homeostatic regulation by dietary
clenbuterol on d 1 evolved to establishment of homeorhetic regulation by d 9 that is consistent
with increased skeletal protein accretion in growing steers.
(Key Words: Cattle, Blood Flow, Metabolism, Regulation.)

Introduction
C h a n g e s in m e t a b o l i c r e g u l a t i o n a n d n u t r i e n t
p a r t i t i o n i n g d u r i n g g r o w t h are s u b t l e a n d t h e r e -
f o r e are d i f f i c u l t t o m e a s u r e or assess. O n e w a y
to study the regulation of nutrient partitioning
is t o p e r t u r b t h e p h y s i o l o g y o f t h e g r o w i n g ani-
1Mention of a trade name, proprietary product, or
specific equipment does not constitute a guarantee or
warranty by the USDA and does not imply approval
to the exclusion of other products that may be suitable.
2The authors thank B. Larsen, L. Laaker, and K.
Sorensen for feeding and care of the steers; M. Buschow
and R. Jaeger for assistance at surgery; C. Felber, D.
Robertson, J. Whitt, and E. Zetina for technical sup-
port; and J. Rosch for typing the manuscript.
3The authors with to thank Boehringer lngelheim
Animal Health, Inc., for providing the clenbuterol used
in this study.
Received April 1, 1987.
Accepted August 13, 1987.
m a l in such a w a y t h a t a c c r e t i o n of m u s c l e or
a d i p o s e tissue is s u b s t a n t i a l l y c h a n g e d . T h e r e
are a n u m b e r of a g e n t s c a p a b l e o f e f f e c t i n g
such changes, t h e m o s t d r a m a t i c o f w h i c h are
c o m p o u n d s k n o w n collectively as /3-adrenergic
agonists. T w o o f t h e s e c o m p o u n d s , c l e n b u t e r o l
a n d c i m a t e r o l , are orally active a n d are effective
in m a n y species i n c l u d i n g pigs, s h e e p a n d c a t t l e
( B a k e r et al., 1 9 8 4 ; D a l r y m p l e et al., 1 9 8 4 ;
R i c k s et al., 1 9 8 4 ; H a n r a h a n et al., 1986). A t
levels t h a t did n o t i n h i b i t gain, c l e n b u t e r o l feed-
ing increased p r o t e i n c o n t e n t of t h e 9 t h to 1 1 t h
rib s e c t i o n b y 13% a n d d e c r e a s e d fat c o n t e n t
b y 20% in cattle (Ricks et al., 1 9 8 4 ) . Even m o r e
d r a m a t i c carcass c h a n g e s were o b s e r v e d in cattle
in r e s p o n s e to c i m a t e r o l ( H a n r a h a n et al., 1 9 8 6 ) .
O f i n t e r e s t are t h e u n d e r l y i n g m e t a b o l i c
changes a n d c o n t r o l s t h a t m u s t b e a l t e r e d to
b r i n g a b o u t t h e carcass changes previously cited.
T o d a t e , t h e m e c h a n i s m s r e s p o n s i b l e for a l t e r i n g
n u t r i e n t p a r t i t i o n i n g in r e s p o n s e to/3-adrenergic
agonists are n o t well d e f i n e d . T h e o b j e c t i v e s o f

342 J. Anim. Sci. 1988. 66:342-353

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 CLENBUTEROL AND HINDQUARTERS METABOLISM IN STEERS
TABLE 1. COMPOSITION OF FEED
Ingredient % of dry mattera
Cracked corn 65.00
Ground alfalfa hay 12.25
Soybean meal 15.00
Molasses 5.00
Urea .96
CaCO3 1.25
NaCI .49
Trace mineral premix b .05
Vitamin A, Dc +
acrude protein = 18.6%; 2.96 Mcal ME/kg dry mat-
ter.
bproviding in mg/kg DM: Fe, 50; Mn, 40; Zn, 30;
Cu, 8; I, .5; and Co, .1.
Cproviding in IU/kg DM: A, 1762 and D, 176.
our study were to compare the acute and chron-
ic effects of the metabolic regulator, clenbu-
terol, on blood flow, heart rate and metabolism
in the hindquarters of growing steers.
Materials and Methods
Animals and Diets. Four Hereford steers, 11
mo of age, were gentled and adapted to a diet
(table 1) formulated to meet protein require-
ments for 1.0 kg daily weight gain (NRC, 1984)
at restricted intake beginning 1 mo before sur-
gery. They were housed in individual stalls dur-
ing January and February 1986 in an enclosed
barn that had skylights to provide a natural
daily light pattern.
Feed was withheld for 48 h and water for 24
h before surgical insertion of indwelling cathe-
ters and placement of an ultrasonic blood flow
probe 4. The steers were anesthetized with sodi-
um thiamylal (1.26 g/100 kg body weight [BW] )
in sterile .15 M NaC1 and maintained under
anesthesia with 1 to 4% halothane in 02. With
the steer in right lateral recumbency, an ultra-
sonic flow probe 16 mm in diameter was placed
around the abdominal aorta distal to the renal
vessels through a side incision on the left flank
perpendicular to the spine. The incision was ex-
tended toward the patella to access the caudal
branch of the circumflex iliac vessels. Teflon
catheters (id, 1.27 mm; od, 2.29 ram) were in-
4Transonic Systems, Inc., Ithaca, NY.
Downloaded from jas.fass.org by guest on July 10, 2011
343
serted 25 to 40 cm into the circumflex iliac
artery and vein until the catheter tips were
located by palpation of the vessels distal (1 to 4
cm) to the probe body in the aorta and in a
similar position in the inferior vena cava, respec-
tively. The incision on the left side was then
closed and the steer was moved to left lateral
recumbency. A similar incision was made on
the right side for insertion of a second set of
catheters into the right circumflex iliac vessels
and palpation of catheter tips in the abdominal
aorta and vena cava. The probe cable and cathe-
ters were exteriorized on the Iumbar shelf and
stored in a gauze patch. The steers were given
injections of antibiotic (penicillin G procaine in
dihydrostreptomycin solution) beginning the
day before and continuing for 3 d after surgery.
Following surgery, and between sampling days,
catheters were filled with a .15 M NaC1 solution
containing 200 units heparin/ml, 1% (v/v) ben-
zyl alcohol, and 1% (v/v) penicillin G procaine
in dihydrostreptomycin sulfate solution. On
sampling days, catheters were flushed between
samplings and filled with .15/14 NaC1 containing
25 units heparin/ml.
Following surgery, the steers were moved to
individual stalls in a completely enclosed room
having a light:dark cycle of 16L:8D. Room
temperature was 18 C. The diet was fed to
steers at 195 kcal metabolizable energy 9 (kg
BW"Ts 9 day) - 1 in four equal aliquots at 6-h
intervals beginning at 0700. Steers consumed all
their feed prior to and on each sampling day.
The experiment began 2 wk after surgery. Aver-
age body weight was 241 + 5 kg.
Design. The experimental design was a bal-
anced, single reversal including a 9-d control
period and a 9-d period of clenbuterot feeding
(8 mg/d divided equally across feedings) with a
5-d interim period. Nitrogen retention returned
to control values 1 d after withdrawal of cima-
terol (40 /ag/kg BW) in lambs nourished by
intragastric infusion (Beermann et al., 1986b).
Meals with clenbuterol were prepared daily for
each steer to assure daily presentation of 8 nag/
steer. Meals fed during sampling were prepared
separately to assure that each meal contained 2
mg clenbuterol.
Blood samples were collected on d 1 and 9
of each period. Blood (25 ml) was withdrawn
simultaneously from the abdominal aorta (A)
and inferior vena cava (V) at 30-min intervals
beginning at 0700 for one feeding cycle (6 h,
13 samples/steer). Blood samples were collected
in heparinized syringes and immediately placed
344 EISEMANN ET AL.
on ice. Aliquots of whole blood were removed
for later analyses, packed cell volume (PCV)
was determined, and the samples were centri-
fuged at 1500 • g for 20 min at 4 C to obtain
plasma. Plasma was aliquoted and stored at - 2 0
C.
Total oxygen was determined on individual
heparinized whole blood samples, sealed in 1-ml
syringes until analyzed with a Lex-O2-Con K
oxygen analyzer s . Equal aliquots of blood were
pooled within arterial or venous source and steer
on sampling days for determination of volatile
fatty acids (VFA) by the method of Reynolds
et al. (1986). Similarly, for analysis of D-3-hy-
droxybutyrate, 1 ml of whole blood from each
sample was combined with .1 ml of 6N per-
chloric acid and mixed. After completion of
sample collection, the acidified whole blood
was centrifuged at 12,000 x g for 30 min at 4
C. The supernatant fraction was neutralized
with 6N KOH and centrifuged at 1500 x g for
10 min at 4 C. The supernatant fraction from
the second centrifugation was stored frozen
until analyzed by the method of Williamson
and Mellanby (1974).
Plasma concentration of glucose was mea-
sured by a glucose oxidase method 6 modified
to use 3,3r-dimethoxybenzide-dihydrochlOride
as the coupling agent, c~-amino N (a-NH2 N,
leucine equivalent) by the method of Broderick
and Kang (1980) modified to include sample
dialysis, urea by a diacetylmonoxine method 7
and ammonia by a hypochlorite method s on
individual samples b y automated procedures.
Plasma concentration of L-lactate was deter-
mined with a membrane-immobilized enzyme
system involving lactate oxidase 9. Plasma con-
centration of nonesterified fatty acids (NEFA,
palmitate equivalent) were analyzed on a pooled
sample using an enzymatic assay 1~ The kit
instructions were modified by diluting reagents
1 + 1 with deionized water, adding one-half the
volume of reagents specified and incubating for
20 min. Recoveries were 94.1% (SE = .4%).
Analysis of individual samples indicated no
trend toward increased NEFA concentrations
throughout sampling during control or clenbu-
terol periods (data not shown), indicating that
s Lexington Instruments, Waltham, MA.
#Technicon Industrial Method No. 566-79T, Tech-
nicon Industrial Systems, Tarrytown, NY.
7Technicon Industrial Method No. 339-01.
e Technicon Industrial Method No. 337--74T.
Downloaded from jas.fass.org by guest on July 10, 2011
use of heparin to flush catheters was not alter-
ing NEFA concentrations.
A transit-time ultrasonic blood-flow meter a
(Model T101) was used to measure blood flow
and heart rate at the time of each blood sample.
Integrated average blood flow rates monitored
at 10-s intervals were averaged for 5 min, en-
compassing the blood sampling interval, to ob-
tain a single value for blood-flow rate at each
blood sampling interval.
Calculations. Plasma flow was calculated
using the following equation:
Plasma flow (L/min) =
(1 - (PCV/100)).Blood flow (L/rain).
Net uptake or release of nutrients was calcu-
lated as the product of arteriovenous (AV)
concentration difference and whole blood (or
plasma) flow.
For variables analyzed on individual samples,
mean values were calculated for each steer and
sample day. For each variable, the mean values
from each steer during the control and treated
periods were used to calculate a paired t-sta-
tistic for d 1, to evaluate acute response to
clenbuterol. The variation among animal means
was used to test the effect of treatment. The
same procedure was used with d-9 data to
evaluate chronic response to clenbuterol.
Resu Its
Steers consumed all feed offered during
measurements. On d 1 of clenbuterol feeding,
steers consumed the 0700 feeding in approxi-
mately 15 min. Following the initial feeding,
steers consumed little feed for the remainder of
the 24-h period. Steers gradually resumed
consumption over the next 4 d. Average daily
intake was 3.87 + .09 kg dry matter (712 g
crude protein and 11.46 Mcal metabolizable
energy)/steer. Over the 23-d experimental
period, steers gained 20 + 2 kg body weight.
Initial consumption of clenbuterol caused a
rapid doubling of heart rate (figure la). Heart
rate decreased slightly from this peak but
remained elevated and relatively constant
throughout the duration of sampling on d 1.
There was some fluctuation in heart rate after
control feeding (figure la), but no dramatic
changes. Blood flow showed a response simi-
lar to that of heart rate (figure la). Mean values
of both heart rate and blood flow were increased
(P<.01) with clenbuterol compared with
 CLENBUTEROL AND HINDQUARTERS METABOLISM IN STEERS 345

D0y I Doy 9
(figure 5a). Arterial c o n c e n t r a t i o n o f urea was
180
B n o t a f f e c t e d (figure 3a). Mean arterial c o n c e n -
i60
3E t r a t i o n s o f glucose ( P < . 0 2 ) , L-lactate ( P < . 0 1 )
~ i40
i
/ and N E F A ( P < . 0 1 ) w e r e higher, c o n c e n t r a t i o n s
<~ 120
I o.S~'-o""o-o-or O-o-O-o.o o f t~-NH2 N ( P < . 0 5 ) and NH 3 (P<.O1) w e r e
I00
lad l o w e r and c o n c e n t r a t i o n s o f O2, acetate,
~ a0 propionate, D-fl-hydroxybutyrate and urea
~ 60 w e r e n o t altered o n d 1 o f c l e n b u t e r o l com-
<
~ 40 p a r e d w i t h c o n t r o l f e e d i n g (table 2). On d 9,
' ' ' I I I t h e r e w e r e no a c u t e changes in arterial c o n c e n -
t r a t i o n o f any o f t h e m e t a b o l i t e s m e a s u r e d
i 16 (figure 2b t o 5b) following c l e n b u t e r o l feeding.
14 F u r t h e r , t h e r e were no c h r o n i c r e s p o n s e s t o
J
12 ,o.-O..o-O'~176 ~ c l e n b u t e r o l in m e a n arterial c o n c e n t r a t i o n s o f
iO
/ o-~a-o-o- o-o.o_o.o.o-o
e ,~
o
0

m 4
1700 0900 1100 1300
'
0700
' '
0900
' I
IlO0
I
13100
6.6 f ~
T I M E OF D A Y ( h ) T I M E OF D A Y ( h ) 6.4 / '~
E 6,2 ~' ~ ~Q
Figure 1. Heart rate and blood flow to the hind-
o 6.0 b.Cl
quarters in steers at each sampling time. Sampling
J
began at 0700 and continued for 6 h at 30-min inter- 5.8
vals. The arrow indicates time of feeding. A: Samples 5.1B

taken on d 1 of control ( e - - e ) or clenbuterol (o--o)

5.4
treatment. Average SEM for control and clenbuterol
, , , , , , ,
treatments, respectively, was: heart rate (heats/min) 5.21 I I ' ' ~ ' '

3.2 and 4.4; blood flow (liters/min) 1.00 and 1.12. B:

Samples taken on d 9 of control ( e - - e ) or clenbuterol
+
~ 3.C
(o--o) treatment. Average SEM for control and clenbu-
terol treatment, respectively, was: heart rate (beats/
rain) 5 and 4; blood flow (liters/rain) .82 and 1.02. 0
Z
2.0

b..o.~..o,.o-O
< 1.0
w
c o n t r o l feeding o n d 1 (table 2). Packed cell
I I I i i I I
v o l u m e was also increased ( P < . 0 5 ) o n d 1 o f
clenbuterol compared with control feeding 20
+ +
r
(table 2). By d 9 o f c l e n b u t e r o l feeding, a c u t e
changes in heart rate and blood flow were no _= 16
1
=0 I
longer observed, following consumption of E
clenbuterol at the beginning of the sampling IZ

period. However, both heart rate and blood I0

flow remained c h r o n i c a l l y elevated (P<.01) 8

after 9 d of clenbuterol compared with control 60700

I .
0900
. , ,
liO0
, +
1300
9

0700
, ,

0900
, ,

Im~
, ,

1300
feeding (figure l b and table 2). T I M E OF DAY ( h ) T I M E OF D A Y ( h )
O n d 1, c l e n b u t e r o l f e e d i n g caused a transi-
Figure 2. Arterial 02 concentration, arteriovenous
ent increase in arterial concentration of O2 (fig- concentration difference and 02 uptake in steers at
ure 2a), a progressive increase in arterial concen- each sampling time. Sampling began at 0700 and con-
tration o f glucose (figure 3a) and L-lactate tinued for 6 h at 30-min intervals. The arrow indicates
(figure 4a) and a progressive decrease in arterial time of feeding. A: Samples taken on d 1 of control
( e - - e ) or clenbuterol (o--o) treatment. Average SEM
c o n c e n t r a t i o n o f NH 3 (figure 3a) and a-NH2 N for control and clenbuterol treatments, respectively,
was: Arterial 02 (raM) .20 and .21; arteriovenous 02
(mM) .23 and .23; 02 uptake (mmol/min) 1.07 and
2.01. B: Samples taken on d 9 of control ( e - - e ) or
clenbuterol (o--o) treatment. Average SEM for control
9yellow Springs Instrument Co., Inc., Yellow and clenbuterol treatments, respectively, was: Arterial
Springs, OH. 02 (raM) .16 and .20; arteriovenous 02 (mM) .17 and
l~ Chemicals USA, Dallas, TX. .23; 02 uptake (mmol/min) 1.86 and 2.25.

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34-6 EISEMANN ET AL.

8
. . z~ ~ z ~

~o

~z .u
b.,

~ ~ ~ . . . .
Z

z~
Om <
-6
~z
ooo ZZZZ . . . . .
<

Z
0
m~
ea~

eL

Om
~Z

[...,

Z .~ o,
.. B.-~
e~

gE'~

Downloaded from jas.fass.org by guest on July 10, 2011

 CLENBUTEROL AND HINDQUARTERS METABOLISM IN STEERS 347

A B hindquarters (table 3; figure 4a). In addition,

extraction ratios for O2 and/3-hydroxybutyrate
. o . . ~ --o''o
10 ~. decreased (P<.10) on d 1 of clenbuterol com-
8
pared with control feeding. On d 9, clenbuterol
feeding caused both decreased AV concentra-
tion difference and extraction ratio for O2,
acetate and /3-hydroxybutyrate compared with
' ' ' ' i I I . . . . I I I
control feeding. The AV concentration differ-
ences of L-lactate remained negative with
chronic clenbuterol compared with control
z tc
feeding (table 3).
4( On d 1, net uptake of O2 increased (figure
w
2a; table 4) and net uptake of 0t-NH2 N de-
creased (figure 5a, table 4) whereas net release
of both L-lactate (figure 4a; table 4) and NEFA
I t , , I I i . . . . i | *

r A ,o
18
~E .3

5t H
tl
z 4F ,o,"~

.I
,= :t/
. . . . . I
o'oo'o~o' ,~o',r o,oo o,0o ,,oo ,~o
TIME OF DAY(h) TIME OF DAY(h)
1
Figure 3. Arterial plasma glucose, urea-N and o
NH 3-N concentrations in steers at each sampling time.
Sampling began at 0700 and continued for 6 h at 30-
min intervals. The arrow indicates time of feeding. A:
Samples taken on d 1 of control ( e - - e ) or clenbuterol
(o . . . . o) treatment. Average SEM for control and clen-
buterol treatments, respectively, was: Glucose (mM)
.25 and .89; urea-N (mM) 1.0 and 1.3; NH~ (raM)
.019 and .006. B: Samples taken on d 9 of control
+!
t
( o - - e ) or clenbuterol (o--o) treatment. Average SEM
for control and clenbuterol treatments, respectively,
was: Glucose (mM) .25 and .22; urea N (mAd) 1.0 and
1.5 ; NH 3 (mM) .023 and .014.
!!'
-4

J I 9 . . . . . . J . i i

02, /3-hydroxybutyrate, carbohydrate, or nitro-

0 7 •
0900 ,(X) 1300 0700 0900 I100 1300

TIME OF DAY(h) TIME OF DAY(h)

genous compounds compared with control
feeding (table 2). In contrast, concentrations of Figure 4. Arterial plasma lactate concentration,
arteriovenous concentration differences and lactate
NEFA were chronically elevated (P<.01) and uptake/release in steers at each sampling time. Sam-
concentrations of acetate (P<.02) and propi- piing began at 0700 and continued for 6 h at 30-min
onate (P<.07) were decreased by d 9 of clenbu- intervals. The arrow indicates time of feeding. A: Sam-
terol compared with control feeding. ples taken on d 1 of control ( o - - o ) or clenbuterol
(o---o) treatme0t. Average SEM for control and clen-
Arteriovenous (AV) concentration differ-
buterol treatments, respectively, was: L-lactate (raM)
ences for acetate (P<.05), propionate (P<.05) .07 and .23; arteriovenous lactate (raM) .03 and .04;
and (~-NH2 N (P<.01) were decreased, as was lactate uptake/release (mmol/min) .12 and .33. B:
extraction ratio, following initial clenbuterol Samples taken on d 9 of control ( o - - o ) or clen-
consumption (table 3; figure 5a). The AV buterol (o--o) treatment. Average SEM for control
and clenbuterol treatments, respectively, was: L-lac-
concentration difference was more negative for tate (mAd) .04 and .05; arteriovenous lactate (mM) .04
L-lactate (P<.01) and NEFA (P<.05), indicat- and .02; lactate uptake/release (mmol/min) .16 and
ing increased metabolite release from the .14.

Downloaded from jas.fass.org by guest on July 10, 2011

348 EISEMANN ET AL.

sss~... ~g{~. ~.~z~~176

. zz~

m~

Z
0
F-
<

I
<
r~ -1

z
d
[..,
< . . . . . ~ o ~
,4
Z

Z
0 8

#.

<
8~
-4
0 I

g "~ X

,4 4 9 9 9 ~ "~ ~ 44
I
II

..~ ~ II
o ~, o

Downloaded from jas.fass.org by guest on July 10, 2011

 CLENBUTEROL A N D H I N D Q U A R T E R S METABOLISM IN STEERS 349

A .B
-~.0

1.8
e
Z 1.6

1,4

III I.Z t \ t
E-,
Z
~ .. .

~t
.1 <
I i I i . . . . I I I
[.-,
Z .1~

T
U .011

| .. ~ q~ .

.04 Z
1
5_< .oz ,d
mr U
0

!l I
- .02 . . . . I i I Z
<
A

-~
ei
0700 OgO0 II~ ' 1300 0700 OlO0 IlO0 I~O0 e
o
T I M E OF D A Y ( h i T I M E OF D A Y ( I I )
Z
Figure 5. Arterial plasma ~-amino nitrogen (c~-NH2 ~ q ~ ~. .
N), arteriovenous concentration difference and c~-NH2
N uptake in steers at each sampling time, Sampling
began at 0700 and continued for 6 h at 30-min inter-
vals. The arrow indicates time of feeding. A: Samples
taken on d 1 of control ( o - - e ) or ctenbuterol (o--o)
treatment. Average SEM for control and clenbuterol
treatments, respectively, was: ~-NH~ N (mM) .20 and
.04; arteriovenous a-NH~ N (raM) .03 and .02; ~-NH~
N uptake (mmol/min) .12 and .12. B: Samples taken
on d 9 of control ( o - - ~ ) or clenbuterol (o--o) treat- I
ment. Average SEM for control and clenbuterol treat-
ments, respectively, was: ~-NHz N (raM) .21 and .12;
arteriovenous e~-NH~ N (raM) .03 and .02; ce-NH2 N
uptake (mmol/min) .13 and .11. <
X "x~

I
increased w i t h c l e n b u t e r o l c o m p a r e d w i t h con-
t r o l feeding. By d 9, c l e n b u t e r o l f e e d i n g caused
a s u s t a i n e d increase in u p t a k e o f 0 2 (figure 2 b ;
t a b l e 4), increased u p t a k e of a-NH2 N (figure e~

5b; t a b l e 4) a n d increased n e t release o f L-lac-

t a t e (figure 4 b ; t a b l e 4) c o m p a r e d w i t h c o n t r o l 4-
feeding. N e t u p t a k e o f o t h e r m e t a b o l i t e s was
n o t altered ( P > . 1 0 ) b y a c u t e (d 1) or c h r o n i c
(d 9) f e e d i n g of c l e n b u t e r o l .
"~ o ~.o. u ... =t z
Dis cussio n
The rapidity of the cardiovascular responses
to initial c l e n b u t e r o l feeding (figure l a ) p r o b -

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350 EISEMANN ET AL.
ably indicates that the drug is absorbed in the
proximal sections of the gastrointestinal tract.
Clenbuterol has been described mainly as a
132-adrenergic agonist because of effects on
bronchodilation; however, the marked tachy-
cardia caused by administration of the com-
pound to steers suggests fll-adrenergic activity,
as defined by Lands et al. (1967). By d 9,
chronic tachycardia in response to clenbuterol
feeding was still present (figure lb). Williams et
al. (1986) reported tachycardia in young bull
calves fed clenbuterol at 20 lag/kg BW; however,
adaptation, indicated by a return o f heart rate
to pretreatment values, occurred b y d 3 of
treatment. Acute tachycardia in response to
clenbuterol (Herbert et al., 1985) or cimaterol
(Beermann et al., 1986a) was observed in sheep.
An acute increase in blood flow to the hind-
quarters in response to cimaterol was observed
also in sheep (Beermann et al., 1986a). Increased
PCV in response to clenbuterol on d 1 (table 2)
may reflect splenic contraction, generally
described as an a-adrenergic mediated event
(Ignarro and Titus, 1968).
Isoproterenol, a nonselective 13-adrenergic
agonist, acutely increased both heart rate and
stroke volume to increase cardiac output in the
guinea pig (Van de Walle and Martin, 1985).
Total peripheral resistance was decreased. In
response to isoproterenol, absolute blood flow
to the carcass and portal-drained visceral (PDV)
tissues was increased, as was the proportion of
cardiac output distributed to the carcass of the
guinea pigs, suggesting a selective vasodilation
in response to a /3-adrenergic agonist, in addi-
tion to increased overall flow. We have observed
increased blood flow to both the hindquarters
and PDV in steers following initial ingestion of
2 mg clenbuterol, and the increase in blood flow
to the hindquarters was greater than that to the
PDV (unpublished observations). Administra-
tion of clenbuterol to horses acutely increased
cardiac output by 44%, whereas cardiac output
was increased only 15% after 8 d of clenbuterol
treatment (16/ag/kg BW; Claussen, 1981). These
cited studies support the concept that the ini-
tial increase in blood flow to the hindquarters
was caused by increased cardiac output, as well
as selective vasodilation in response to clenbu-
terol. Further, these studies suggest that the
large (47%) chronic increase in blood flow to
the hindquarters on d 9 of clenbuterol feed-
ing (table 2) partly reflects a change in distri-
bution of cardiac output. The increased heart
rate on d 9 o f clenbuterol feeding would in-
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crease cardiac output 33% if stroke volume
were unchanged. The change in distribution of
cardiac output may be due to stimulation of
arterial fl-adrenergic receptors located in skele-
tal muscle (Smith and Hamlin, 1977) and(or)
may be a result of increased tissue metabolism.
The acute changes in blood concentration of
metabolites (figures l a to 5a) reflect the extent
of metabolic perturbation induced by clenbuter-
ol. Of the metabolites exhibiting changes in
concentration, those associated with carbo-
hydrate and lipid metabolism (glucose, L-lac-
tate, /~-hydroxybutyrate and N E F A ) increased,
whereas those related to N metabolism (0~-NH2
N, NH3) decreased. Combined information on
changes in concentration and uptake or release
elucidates whether entry or exit from the plas-
ma pool is altered to produce nonsteady-state
conditions. Our data suggest that increased con-
centration of glucose reflects increased entry
through an increase in liver gluconeogenesis
and(or) glycogenolysis. Similarly, increased
entry of lactate through an increase in peri-
pheral glycolysis and of N E F A through in-
creased lipolysis are consistent with observed
changes. Thornton et al. (1985) demonstrated a
direct acute effect of clenbuterol added to in
vitro incubations of sheep adipose tissue to
increase lipid mobilization. The acute changes
in carbohydrate and lipid metabolites were
similar to ~ and 13-adrenergic-mediated metabo-
lic effects previously reported in sheep (Bassett,
1970).
Decreased concentration of~-NH2 N coupled
with decreased peripheral uptake on d 1 indi-
cate a decrease in entry rate, most likely through
decreased protein degradation. Short-term
studies using skeletal muscle from rats demon-
strated a/3-adrenergic-mediated decrease in pro-
tein degradation (Li and Jefferson, 1977) and
amino acid release (Garber et al., 1976). The
time course of changes in a-NH~ N concentra-
tion suggesting decreased protein degradation is
consistent with the observed increase in N reten-
tion on d 1 of clenbuterol (Herbert et al., 1985)
or cimaterot (Beerman et al., 1986b) infusion in
lambs nourished by intragastric infusion. Epine-
phrine (a and j3-adrenergic agonist) had no effect
on in vitro protein synthesis in diaphragm mus-
cle from rats (Nutting, 1982).
Chronic effects of clenbuterol on metabolite
concentrations contrasted with acute effects
observed on d 1. Lack of acute metabolic
changes on d 9 demonstrates adaptation to the
drug and a change in tissue sensitivity to clen-
 CLENBUTEROL AND HINDQUARTERS METABOLISM IN STEERS
buterol. The change in sensitivity could be re-
lated to receptor of post-receptor mediated
events. Concentrations of V F A decreased and
concentrations of N E F A increased chronically,
whereas concentration of variables associated
with carbohydrate and N metabolism were un-
changed. Previous studies demonstrated a posi-
tive correlation between whole body irreversi-
ble loss or oxidation and b l o o d concentrations
of acetate (Annison et al., 1967; Pethick et al.,
1981) or NEFA (Lear and Ford, 1966; Pethick
et al., 1983; Eisemann et ai., 1986); however,
this association did not hold for leucine (Else-
mann et al., 1986). Decreased entry of acetate
from the portal-drained viscera or decreased
endogenous hepatic production may be re-
sponsible for decreased entry. Decreased AV
concentration difference (table 3) and a trend
toward decreased acetate uptake in the hind-
quarters (table 4) supports decreased acetate
use (exit) for oxidation and(or) lipogenesis on
a whole-body basis. Chronically elevated arterial
N E F A concentrations suggest continued lipid
mobilization and oxidation with chronic clen-
buterol treatment. N E F A could also be directly
oxidized, from hydrolysis of triacylglycerol in
muscle tissues, without entering the plasma
pool. Consistent with the tendency toward de-
creased acetate uptake, there was a trend for
increased lipid mobilization from the hind-
quarters; however, other body depots may
make a larger contribution to total lipid mobili-
zation.
For O~ and metabolites monitored, mean
AV differences and extraction ratios changed in
similar directions with both acute and chronic
clenbuterot feeding (table 3). Increased use of
02 in the hindquarters (table 4), indicating in-
creased oxidative metabolism, could result from
increased catabolism of N E F A or glucose. If
glucose is metabolized to lactate, rather than
completely oxidized to H 2 0 and COs, this
would not be accompanied by an increase in 02
consumption in the hindquarters because ATP
production would be at the substrate level
rather than coupled to electron transport. The
large release of lactate on d 1 of clenbuterol
feeding and continued release on d 9 (table 4)
11Calculated stoichiometrieally based on: 1 glucose
+ 6 0 z ~ 6 CO2 + 6 HzO. Each mole lactate release is
equivalent to .5 mole glucose.
12Calculated stoichiometrically based on: 1 3-
hydroxybutyric acid + 4.5 0 2 ~ 4 CO 2 + 4 HzO.
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3 51
suggests increased glycolysis with clenbuterol
feeding. Mobilization and increased oxidation
of NEFA inhibits glucose oxidation at pyruvate
dehydrogenase, with consequent increased
release of lactate (Newsholme and Leech,
1983). The fact that glucose uptake was not
altered on d 9 of clenbuterol feeding, but that
lactate release was (table 4), exemplifies this
metabolic shift. On the average, glucose uptake
corrected for lactate release 11 accounted for
39.8 and 29.1%, maximum, of 02 uptake for
control and clenbuterol treatments, respec-
tively, on d 9. The potential contribution of
f3-hydroxybutyrate to O212 use was constant at
19.8 and 21.9%, respectively. Because acetate is
used for both oxidation and lipid synthesis,
potential contribution to 02 consumption was
not calculated.
By d 9 of treatment, hindquarters uptake of
a-NHR N was increased, suggesting an increase
in protein deposition in response to chronic
clenbuterol treatment. Chronic administration
of clenbuterol to rats resulted in either an in-
crease (Emery et al., 1984) or no change (Reeds
et al., 1986) in fractional synthesis rate of mus-
cle protein. Also, the fractional synthesis rate
of muscle protein was not altered in sheep with
chronic clenbuterol administration (Bohorov et
al., 1987). Emery et al. (1984) administered 2
mg-(kg BW~ - 1 subcutaneously, whereas
Reeds et al. (1986) fed clenbuterol at approxi-
mately 160 to 5 6 0 / l g . ( k g BW-d) - 1 . Bohorov
et al. (1987) included clenbuterol in the feed to
effect a dose of 17 m g . ( h d . d ) - 1 for sheep from
27 to 40 kg BW. Dose or method of administra-
tion may contribute to the variation in observed
effects.
Oxygen consumption by the hindquarters
was increased 3.06 mmol/min (4.41 mol/d) by
chronic clenbuterol treatment. Assuming tight-
ly coupled mitochondria (i.e., 1 mol O2 con-
sumed equals 6 mol ATP formed), increased 02
consumed is equivalent to the formation of a
maximum of 26.5 tool of ATP. The increment
in a-NH 2 N uptake was .216 mol/d or 148.5 g
crude protein (assuming 16% N and average
molecular weight 110 g/mol amino acid). If 5
mol ATP are the minimum required/peptide
b o n d formed (equivalent to 1 tool amino acid
or 110 g protein; Millward and Garlick, 1976),
then 6.75 tool of ATP or 25.6% of the increased
ATP would be needed if the 148.5 g increment
in crude protein resulted from protein synthesis.
Increased NEFA oxidation could provide the
needed ATP. Increased substrate cycling (News-
352 EISEMANN ET AL.
h o l m e and Crabtree, 1976), o t h e r energy-
requiring processes or a decrease in A T P pro-
d u c t i o n / m o l 02 consumed m a y c o n t r i b u t e to
t h e additional increase in 02 c o n s u m p t i o n .
Comparing our data for steers during c o n t r o l
feeding with data f r o m a group of heavier (345
kg) Hereford • Angus steers~(Prior et al., 1984),
m e t a b o l i t e concentrations, A V differences, ex-
t r a c t i o n ratios and net uptake/release were simi-
lar for glucose, acetate and p r o p i o n a t e but not
for lactate. We observed either a net release of
lactate f r o m the hindquarters or no net m e t a b o -
lism (net u p t a k e not different f r o m 0), whereas
Prior et al. (1984) observed net u p t a k e of lac-
tate. Bell et al. (1975) observed a net release of
lactate f r o m the hind limb of u n t r e a t e d y o u n g
steers as well. Because lactate is a precursor for
lipid synthesis in bovine adipose tissue (Prior,
1978; Whitehurst et al., 1978), net or unidirec-
tional m e t a b o l i s m of lactate in the hindquarters
m a y vary with the phase o f growth.
Data on A V c o n c e n t r a t i o n differences of
acetate, propionate, and ~-NH2 N (table 3)
d e m o n s t r a t e the critical need to measure b l o o d
flow as well as A V c o n c e n t r a t i o n b e f o r e making
m e t a b o l i c differences. Decreased A V difference
on d 9 is offset by increased blood flow, thus
there is no net effect (P>.10) on u p t a k e of
either V F A (table 4), although there are trends.
In contrast, there is b o t h decreased A V concen-
tration difference and uptake of ~-NH2 N on d
1 despite the increased b l o o d flow to t h e
hindquarters.
A c u t e and chronic m e t a b o l i c responses to
clenbuterol feeding illustrates two types of
metabolic control, homeostasis and h o m e o -
rhesis (Bauman and Currie, 1980; Bauman et
al., 1982). The steers showed an a c u t e general
catabolic response to a t3-adrenergic agonist by
releasing stored nutrients and decreasing
peripheral anabolic processes (table 4). Changes
in c o n c e n t r a t i o n s of m e t a b o l i t e s d e m o n s t r a t e
the extent to which steady state was altered
and show the need for rapid interplay of h o m e -
ostatic c o m p e n s a t o r y mechanisms. Chronic
a d a p t a t i o n is indicated by lack o f acute re-
sponse in any variable measured to clenbuterol
ingestion by d 9, as well as chronic changes in
c o n c e n t r a t i o n only of m e t a b o l i t e s whose con-
centration is related to irreversible loss (e.g.,
acetate and N E F A ) and indicates that the steers
had reached a new steady state or were moving
along a m o r e tightly regulated c o n t i n u u m . De-
creased 0~-NH2 N uptake by the hindquarters on
d 1 and increased ~-NH2 N u p t a k e on d 9, with
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no change in a ~-NH2 N c o n c e n t r a t i o n on d 9
(i.e., no relationship b e t w e e n c o n c e n t r a t i o n and
hindquarters uptake), illustrates changing me-
tabolic control and the i m p o r t a n c e of chronic,
h o m e o r h e t i c regulation.
Our data detail m e t a b o l i c changes that con-
tribute to altered n u t r i e n t partitioning, increased
muscle and decreased fat accretion previously
observed in response to clenbuterol feeding.
A c u t e and chronic changes illustrate h o m e o -
static and b o m e o r h e t i c regulatory mechanisms,
respectively, for m e t a b o l i c control. Perhaps the
data on a-NH2 N m e t a b o l i s m best illustrate t h e
critical need for chronic evaluation of regula-
t o r y agents to understand regulatory mecha-
nisms involved in c o o r d i n a t i o n of nutrient par-
titioning during growth.
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