16
Influence of dietary carnitine in growing sheep fed diets containing non-protein nitrogen A.M. Chapa a , J.M. Fernandez a,* , T.W. White a , L.D. Bunting b , L.R. Gentry a , J.C. Lovejoy c , K.Q. Owen d a Department of Animal Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803-4210, USA b Department of Dairy Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803-4210, USA c Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 70808-4124, USA d Lonza, Inc., Fair Lawn, NJ 07410, USA Accepted 2 October 2000 Abstract The influence of supplemental L-carnitine was investigated in growing sheep fed rations containing non-protein nitrogen (NPN). The experiment was conducted as a randomized block design with a 2 2 factorial arrangement of treatments. Lambs (77.4 kg BW, n 24) were fed a total mixed ration (12.1–13.6% CP) with two levels of L-carnitine (0 or 250 ppm) and two levels of NPN (urea contributing 0 or 50% of total dietary N) for a 50-day period. Jugular blood samples were collected at 0, 1, and 3 h post-feeding, and ruminal fluid samples were collected at 1 h post-feeding, during days 1, 8, 29, and 50 of the experiment. Average daily gain (121 versus 214 g) was lower (P < 0:0001) in lambs fed the NPN diets. Lambs consuming diets containing NPN had higher (P < 0:0001) ruminal fluid pH (6.6 versus 5.9), ruminal ammonia N (4.8 versus 2.8 mmol/l), and plasma ammonia N (177.1 versus 49.5 mmol/l) than lambs not fed NPN. Additionally, lambs fed the NPN diets had lower plasma urea N (14.5 versus 17.5 mmol/l; P < 0:003) and thyroxine (T 4 ) concentrations (65.8 versus 78.4 ng/ml; P < 0:02), and lower T 4 :triiodothyronine (T 3 ) ratio (37.9 versus 43.9; P < 0:02). Plasma glucose concentrations were higher (P < 0:05) in lambs fed L-carnitine (3.83 versus 3.70 mmol/l). Two oral urea load tests (OULT 1 and OULT 2) were conducted during the 50-day trial. Urea solutions (0.835 g/kg 0.75 BW) were administered as oral drenches. During the OULT 1 (day 10), plasma ammonia N and glucose concentrations were highest (P < 0:0001) in the lambs fed NPN with L-carnitine compared with lambs fed control, L-carnitine, and NPN diets. During the OULT 2 (day 50), plasma ammonia N was highest (P < 0:0001) in the NPN and NPN with L-carnitine groups compared with the control and L-carnitine groups. Plasma glucose was lowest (P < 0:04) in the NPN with L-carnitine group compared with the NPN and L-carnitine groups, but did not differ (P > 0:10) from the control group. Plasma urea N levels in both OULT 1 and OULT 2 were lower (P < 0:0001) in the NPN and NPN with L-carnitine groups compared with the control and L-carnitine groups. In the present experiment, production and plasma criteria were affected by NPN incorporation in the diets. Production criteria were not affected by inclusion of L-carnitine in the diet, however, L-carnitine reduced experimentally induced hyperammonemia by day 50 of the trial. # 2001 Elsevier Science B.V. All rights reserved. Keywords: L-Carnitine; Ammonia; Non-protein nitrogen; Insulin sensitivity; Sheep Small Ruminant Research 40 (2001) 13–28 * Corresponding author. Tel.:1-225-578-3442; fax: 1-225-388-3279. E-mail address: [email protected] (J.M. Fernandez). 0921-4488/01/$ – see front matter # 2001 Elsevier Science B.V. All rights reserved. PII:S0921-4488(00)00218-2

Influence of dietary carnitine in growing sheep fed diets containing non-protein nitrogen

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In¯uence of dietary carnitine in growing sheep feddiets containing non-protein nitrogen

A.M. Chapaa, J.M. Fernandeza,*, T.W. Whitea, L.D. Buntingb,L.R. Gentrya, J.C. Lovejoyc, K.Q. Owend

aDepartment of Animal Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803-4210, USAbDepartment of Dairy Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803-4210, USA

cPennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA 70808-4124, USAdLonza, Inc., Fair Lawn, NJ 07410, USA

Accepted 2 October 2000

Abstract

The in¯uence of supplemental L-carnitine was investigated in growing sheep fed rations containing non-protein nitrogen

(NPN). The experiment was conducted as a randomized block design with a 2� 2 factorial arrangement of treatments. Lambs

(77.4 kg BW, n � 24) were fed a total mixed ration (12.1±13.6% CP) with two levels of L-carnitine (0 or 250 ppm) and two

levels of NPN (urea contributing 0 or 50% of total dietary N) for a 50-day period. Jugular blood samples were collected at 0, 1,

and 3 h post-feeding, and ruminal ¯uid samples were collected at 1 h post-feeding, during days 1, 8, 29, and 50 of the

experiment. Average daily gain (121 versus 214 g) was lower (P < 0:0001) in lambs fed the NPN diets. Lambs consuming

diets containing NPN had higher (P < 0:0001) ruminal ¯uid pH (6.6 versus 5.9), ruminal ammonia N (4.8 versus 2.8 mmol/l),

and plasma ammonia N (177.1 versus 49.5 mmol/l) than lambs not fed NPN. Additionally, lambs fed the NPN diets had lower

plasma urea N (14.5 versus 17.5 mmol/l; P < 0:003) and thyroxine (T4) concentrations (65.8 versus 78.4 ng/ml; P < 0:02),

and lower T4:triiodothyronine (T3) ratio (37.9 versus 43.9; P < 0:02). Plasma glucose concentrations were higher (P < 0:05)

in lambs fed L-carnitine (3.83 versus 3.70 mmol/l). Two oral urea load tests (OULT 1 and OULT 2) were conducted during the

50-day trial. Urea solutions (0.835 g/kg0.75 BW) were administered as oral drenches. During the OULT 1 (day 10), plasma

ammonia N and glucose concentrations were highest (P < 0:0001) in the lambs fed NPN with L-carnitine compared with

lambs fed control, L-carnitine, and NPN diets. During the OULT 2 (day 50), plasma ammonia N was highest (P < 0:0001) in

the NPN and NPN with L-carnitine groups compared with the control and L-carnitine groups. Plasma glucose was lowest

(P < 0:04) in the NPN with L-carnitine group compared with the NPN and L-carnitine groups, but did not differ (P > 0:10)

from the control group. Plasma urea N levels in both OULT 1 and OULT 2 were lower (P < 0:0001) in the NPN and NPN with

L-carnitine groups compared with the control and L-carnitine groups. In the present experiment, production and plasma criteria

were affected by NPN incorporation in the diets. Production criteria were not affected by inclusion of L-carnitine in the diet,

however, L-carnitine reduced experimentally induced hyperammonemia by day 50 of the trial. # 2001 Elsevier Science B.V.

All rights reserved.

Keywords: L-Carnitine; Ammonia; Non-protein nitrogen; Insulin sensitivity; Sheep

Small Ruminant Research 40 (2001) 13±28

* Corresponding author. Tel.:�1-225-578-3442; fax: �1-225-388-3279.

E-mail address: [email protected] (J.M. Fernandez).

0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 1 - 4 4 8 8 ( 0 0 ) 0 0 2 1 8 - 2

1. Introduction

Carnitine (û-hydroxy-g-[trimethylammonio]buty-

rate), a naturally occurring quaternary amine com-

pound, is necessary for the transportation of long chain

fatty acids across the inner mitochondrial membrane

(Stryer, 1988; DeVivo and Tein, 1990). Although,

carnitine is not considered to be an essential nutrient

for mammals, studies in swine (Owen et al., 1996) and

cattle (LaCount et al., 1995, 1996a,b) have shown that

the addition of L-carnitine to diets may prove bene-

®cial for production.

Responses in ruminants to L-carnitine administra-

tion have been variable. Supplemental L-carnitine,

administered directly into either the rumen or aboma-

sum, was effective in increasing plasma, liver, and

milk concentrations of L-carnitine, however, L-carni-

tine had little effect on milk yield and composition

(LaCount et al., 1995). Yavuz et al. (1997) reported

that supplemental L-carnitine reduced plasma urea

nitrogen (N) concentrations in Holstein calves fed

diets containing 50% broiler litter. Supplemental L-

carnitine also reduced plasma urea N concentrations

and improved ADG in grazing beef calves (White

et al., 1998a). A study using heifers (Hill et al., 1995)

showed that dietary L-carnitine reduced marbling

score, but little to no consistent effects have been

found in steers (Hill et al., 1994).

In sheep, intravenous (Chapa et al., 1998) but not

oral (Morris et al., 1998) administration of L-carnitine

solutions reduced plasma ammonia N concentrations

during induced urea toxicosis. L-carnitine has also

been shown to protect mice against known lethal

amounts of ammonium acetate (O'Connor et al.,

1984, 1986; Costell et al., 1987; Matsuoka and Igisu,

1993). Studies using Channel cat®sh have shown that

L-carnitine increases the tolerance to environmental

ammonia (Burtle and Newton, 1991).

The mechanism by which L-carnitine alleviates

hyperammonemia is still under investigation. Never-

theless, the neurological symptoms observed during

acute hyperammonemia have been attributed to

derangements in glucose metabolism (Edjtehadi

et al., 1978; Fernandez, et al., 1988, 1990; Haliburton

and Morgan, 1989), and L-carnitine has been shown to

alter glutamatergic neurotransmissions (MinÄana et al.,

1997) and glucose metabolism (Chapa et al., 1998;

Peluso et al., 2000) during hyperammonemia.

The objective of this experiment was to determine

the in¯uence of supplemental L-carnitine on growth

and metabolic criteria of growing lambs fed a diet high

in NPN (constituting 50% of total dietary N).

2. Materials and methods

2.1. Animal care and diets

Twenty-four spring-born (born February±March

1997) Suffolk lambs (14 wethers and 10 ewe lambs)

were used in a feeding trial (June±August 1997) to

investigate the effects of supplemental L-carnitine on

growth and metabolic criteria of growing lambs fed a

diet high in NPN (constituting 50% of total dietary N).

The lambs, obtained from the Louisiana Agricultural

Experiment Station Sheep Unit, were blocked by sex,

strati®ed by weight, and randomly assigned to pens.

Prior to initiation of the experiment, the lambs were

dewormed and hooves properly trimmed. Lambs were

housed in individual indoor pens with a concrete ¯oor.

The concentrate-based experimental diet was for-

mulated to meet the recommended nutrient require-

ments of growing lambs (NRC, 1985; Table 1). The

dietary treatments included two levels of dietary NPN

as urea (0 and 50% of total dietary N; 12.1±13.6% CP

on a DM basis) and supplemented with two concen-

trations of L-carnitine (0 or 250 ppm) as Carniking

(50% L-carnitine, Lot # 101031; Lonza, Inc., Fair

Lawn, NJ), resulting in a randomized block design

with a 2� 2 factorial arrangement of treatments.

Lambs were individually fed and slowly adapted

(over 21 days) to the diets containing NPN as urea.

The L-carnitine was added to the appropriate diets

on day 1 of the collection period, to ensure that

any adaptation by the rumen micro-organisms to L-

carnitine would occur during the collection period.

During the 50-day collection period, lambs were

individually fed (0730) amounts to ensure 15% orts,

and had ad libitum access to the diets and water.

Individual intakes were recorded daily, and the orts

were removed prior to each feeding.

2.2. Blood and ruminal ¯uid sampling

On days 1, 8, 29, and 50 of the collection period,

following 16 h of feed deprivation, lambs were

14 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

weighed and blood was collected via jugular veni-

puncture into 7 ml evacuated tubes containing potas-

sium oxalate and sodium ¯uoride (Monoject Blood

Collection Tubes; Sherwood Medical, St. Louis, MO)

at 0, 1, and 3 h post-feeding. Ruminal ¯uid samples

(15 ml) were collected via stomach tube at 1 h post-

feeding and immediately placed in bottles containing

500 ml of saturated mercuric chloride as the preserva-

tive. Blood samples were placed in an ice bath,

transported to the laboratory, centrifuged (48C) at

1600� g for 15 min, and the plasma was harvested

and stored at ÿ208C until analyzed. Samples for

plasma ammonia N analysis were immediately depro-

teinized and analyzed. Plasma samples also were

analyzed for urea N, glucose, non-esteri®ed fatty acid

(NEFA), albumin, and insulin. Additionally, plasma

from the 0 h of each sampling day was analyzed for

total carnitine, triiodothyronine (T3), thyroxine (T4),

and cortisol.

2.3. Oral urea load test

An oral urea load test was conducted on days 10

(OULT 1) and 50 (OULT 2) of the collection period.

Following a 16 h fast, lambs were ®tted with a sterile

indwelling jugular vein catheter (Quik-Cath1, 14G

5.1 cm; Baxter Healthcare Corp., Deer®eld, IL), teth-

ered, and allowed 1 h to rest. A freshly prepared urea

solution was administered as an oral urea drench

(0.835 g/kg0.75 BW prepared with distilled water as

a 25% w/v solution) using a stomach tube at the

30 min blood sample. Blood samples were collected

via the jugular vein catheter into 4 ml tubes containing

potassium oxalate and sodium ¯uoride (Monoject

Blood Collection Tubes) at 0, 15, 30, 45, 60, 75,

90, 105, 120, 135, 150, 165, 180, 195, 210, 225,

240, 270, 300, 330, 360, 390, and 420 min. All plasma

samples from the OULT 1 and OULT 2 were analyzed

for ammonia N, urea N, glucose, and insulin

Table 1

Composition of experimental diets

Item Dietsa

Control NPN Carn NPN � Carn

Feed composition (as fed basis)

Corn 30.63 52.32 29.63 51.32

Soybean meal (49% CP) 18.60 0.14 18.60 0.14

Cottonseed hulls 43.74 37.50 43.74 37.50

Urea ± 2.68 ± 2.68

Oystershell flour 0.59 0.48 0.59 0.48

Trace mineral saltb 0.32 0.33 0.32 0.33

Vitamin premixc 0.10 0.10 0.10 0.10

Monocalcium phosphate 1.01 1.45 1.01 1.45

Carnitine premixd ± ± 1.00 1.00

Bovatece 5.00 5.00 5.00 5.00

Nutrient composition (% of DM)

DM 95.0 95.6 94.6 95.4

CP 12.1 13.6 12.3 13.1

a Experimental diets were Control � urea contributing 0% of the total dietary N and 0 g L-carnitine; NPN � urea contributing 50% of the

total dietary N and 0 g of L-carnitine; Carn � urea contributing 0% of the total dietary N and 250 ppm of L-carnitine; NPN� Carn � urea

contributing 50% of the total dietary N and 250 ppm of L-carnitine.b Trace mineral salt provided the following per kilogram of diet: sodium chloride, 4.4 g; manganese, 14 mg; iron, 8.5 mg; copper, 2.3 mg;

iodine, 0.23 mg; cobalt, 0.24 mg.c Provided the following per kilogram of diet: ribo¯avin, 2.6 mg; pantothenic acid, 10 mg; niacin, 18 mg; vitamin B12, 12 mg; biotin,

88 mg; choline, 176 mg; menadione, 1.7 mg; folic acid, 0.7 mg; thiamine, 0.9 mg; pyridoxine, 0.9 mg; ascorbic acid, 22 mg; vitamin A, 2205

IU; vitamin D, 661 IU; vitamin E, 18 IU.d Carnitine premix consisted of 250 ppm of L-carnitine (as Carniking, contains 50% L-carnitine; Lonza, Inc., Fair Lawn, NJ).e Bovatec (15% mixture) was added at a rate of 136 g/907 kg of diet.

A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28 15

concentrations. Additionally, plasma samples col-

lected at the 0 min were analyzed for ammonia N,

urea N, albumin, glucose, NEFA, and insulin. Plasma

samples for ammonia N analysis were deproteinized

and analyzed within 24 h of collection.

Ruminal ¯uid samples were collected via stomach

tube immediately prior to the oral urea drench (at the

30 min sample) and immediately placed in bottles

containing saturated mercuric chloride as the preser-

vative.

2.4. Intravenous glucose tolerance test and insulin

challenge

On day 11 of the collection period, a combined

intravenous glucose tolerance and insulin challenge

test (frequently sampled intravenous glucose tolerance

test; FSIGT) was conducted following the procedure

of Bergman (1997) as evaluated for sheep by Majorie

et al. (1997). Brie¯y, non-fasted lambs were ®tted with

a sterile indwelling jugular vein catheter (Quik-

Cath1, 14G 5.1 cm), tethered, and allowed 1 h to

rest. A bolus dose of a glucose solution (300 mg

glucose/kg BW; 50% w/v glucose±saline solution)

was administered via the jugular catheter after the

collection of the 1 min sample. After the collection of

the 19 min sample, a bolus dose of insulin-0.2% BSA±

saline solution (0.03 units of crystalline ovine insulin/

kg BW; Sigma Chemical Co., St. Louis, MO) was

administered via the jugular catheter. Blood samples

(3 ml) were collected via the jugular catheter into 4 ml

tubes containing potassium oxalate and sodium ¯uor-

ide (Monoject Blood Collection Tubes) atÿ10,ÿ1, 2,

4, 8, 14, 19, 22, 30, 40, 50, 60, 70, 80, 90, 120, 150, and

180 min. All plasma samples from the FSIGT were

analyzed for glucose and insulin. In addition, plasma

samples collected at ÿ10 min were analyzed for

ammonia N, urea N, albumin, glucose, NEFA, and

insulin. Samples for ammonia N analysis were imme-

diately deproteinized and analyzed.

2.5. Chemical analyses

Plasma ammonia N, urea N, and albumin contents

were determined using spectrophotometric methods

following the procedures outlined by Laborde et al.

(1995). Plasma glucose (Method No. 315; Sigma

Chemical Co.) and NEFA (NEFA-C Kit, ACS-ACOD

Method; Wako Chemicals USA, Inc., Richmond, VA)

were analyzed spectrophotometrically using commer-

cially available kits. Additionally, plasma from the 0 h

of each sampling day was analyzed for total carnitine

(Minkler and Hoppel, 1993). All samples subjected to

metabolite analyses were assayed in duplicate, and

measurements resulting in a 5% error or higher were

reanalyzed.

Plasma hormone measurements were determined

using established radioimmunoassay procedures.

Plasma insulin concentrations were determined using

a double-antibody radioimmunoassay procedure

(Kitchalong et al., 1995). The mean interassay CV

for the insulin radioimmunoassay was 14%. Plasma T3

(ICN Kit # 07-292102), T4 (ICN Kit # 07-290102),

and cortisol (ICN Kit # 07-221102) were determined

using commercially available coated-tube radioimmu-

noassay kits (ICN Biomedicals, Costa Mesa, CA) as

reported by Kitchalong et al. (1995). The mean intra-

assay CV for T3, T4, and cortisol were 5.4, 10, and 2%,

respectively. All samples subjected to hormone ana-

lyses were assayed in duplicate, and measurements

resulting in a 7% error or higher were reanalyzed.

Ruminal ¯uid pH was determined on fresh samples

immediately after sampling, and samples were stored

at ÿ208C until analysis for ruminal ammonia N con-

tent (Fernandez et al., 1997). Ruminal ¯uid samples

were assayed in duplicate, and measurements result-

ing in a 7% error were reanalyzed.

2.6. Calculations and statistical analyses

Production data, and plasma metabolite and hor-

mone measurements obtained from blood samples

collected during days 1, 8, 29, and 50 of the collection

period and during both OULT were analyzed using the

MIXED procedure of SAS (1992). Data were analyzed

as a randomized complete block design with sex as

block with individual lamb as the experimental unit.

The treatment structure was a 2� 2 factorial arrange-

ment with the two factors including two levels of urea

and two levels of L-carnitine. The main effects tested

were sex, level of L-carnitine, level of urea, time, and

their interaction. Differences among least-square

treatment means were separated using the Bonferroni

method (Steel and Torrie, 1980).

The Minimal Model is a computer-based modeling

procedure used to determine glucose effectiveness

16 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

(SG), a measure of non-insulin-dependent glucose

clearance, and insulin sensitivity (SI), a measure of

insulin-dependent glucose clearance (Bergman,

1997). Plasma glucose and insulin data points from

the FSIGT were used to calculate the SG and the SI for

each lamb. The calculated SG and SI were then ana-

lyzed using the MIXED procedure of SAS. Data were

analyzed as a randomized complete block design with

sex as block with individual lamb as the experimental

unit. The treatment structure was a 2� 2 factorial

arrangement with the two factors including two levels

of urea and two levels of L-carnitine. The main effects

tested were sex, level of L-carnitine, level of urea, and

their interaction. Differences among least-square

treatment means were separated using the Bonferroni

method (Steel and Torrie, 1980).

3. Results

3.1. Feed intake and weight response

There were no differences in total feed intake

caused by treatment (P > 0:10; Table 2). Although,

total feed intake was not different between treatments,

intakes tended to be higher in lambs fed NPN with L-

carnitine (P � 0:11) from days 1 to 7. Intake increased

over the experimental period (P < 0:0001; not

shown). Average daily gain (ADG) was lower

(P < 0:0001) in lambs on the NPN and NPN � L-

carnitine diets (Table 2). Body weight increased

throughout the collection period (time effect;

P < 0:0001). Body weights exhibited a NPN � day

interaction (P < 0:0001) in which lambs consuming

diets without NPN gained more weight when com-

pared to the lambs consuming diets with NPN (12.1

versus 7.1 kg, respectively). However, there was no

differences in ®nal weight caused by treatment

(P > 0:10; Table 2).

3.2. Responses in daily plasma and ruminal ¯uid

response

Following 16 h of feed deprivation, jugular blood

samples were obtained at 0, 1, and 3 h post-feeding

during days 1, 8, 29, and 50 of the collection period;

ruminal ¯uid samples were obtained at 1 h post-

feeding during the same days. Lambs fed the NPN diets

had higher (P < 0:0001) ruminal ¯uid pH (Table 3)

and higher (P < 0:0001) ruminal ammonia N (Table 3)

compared with lambs fed the non-NPN diets.

Plasma total carnitine was 75% higher (P < 0:002)

in the lambs fed L-carnitine than in the lambs not fed L-

carnitine (Fig. 1A). Lambs fed the L-carnitine diets

had higher concentrations of plasma total carnitine

during days 8, 29, and 50, compared with lambs not

fed the L-carnitine diets (L-carnitine� day effect,

P < 0:04; Fig. 1B).

Over all days, plasma ammonia N was higher

(P < 0:0001) in lambs fed the NPN diets than in

the lambs fed diets without NPN (Table 4). Plasma

ammonia N concentrations increased 1 h post-feeding

in lambs fed the NPN diets, but decreased in lambs fed

the non-NPN diets (NPN� hour effect, P < 0:0001;

Table 3). There was no effect of L-carnitine (P > 0:10)

nor was there an NPN � L-carnitine interaction

(P > 0:10) for plasma ammonia N. Over all days,

plasma urea N was lower (P < 0:003) in the lambs fed

Table 2

Effect of NPN and L-carnitine on initial BW, ®nal BW, and ADGa

Item Dietsb SEM

Control NPN Carn NPN � Carn

Initial BW (kg) 26.8 28.6 26.8 27.7 2.2

Final BW (kg) 39.3 34.0 38.5 36.5 0.3

ADG (g) 221.0 x 124.5 y 207.0 x 118.3 y 8.4

Feed intake (kg/day) 2.9 2.5 2.6 2.6 0.9

a Within a row means lacking a common letter (x, y) differ, P < 0:001.b Experimental diets were: Control � urea contributing 0% of the total dietary N and 0 ppm L-carnitine; NPN � urea contributing 50% of

the total dietary N and 0 ppm of L-carnitine; Carn � urea contributing 0% of the total dietary N and 250 ppm of L-carnitine;

NPN� Carn � urea contributing 50% of the total dietary N and 250 ppm of L-carnitine.

A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28 17

Table 3

Effect of NPN on plasma ammonia N, glucose, urea N, NEFA, and insulin concentrations, and ruminal pH and ammonia N levels, relative to

feedinga

Metabolite Hourb Dietsc,d SEM

Without NPN With NPN

Ammonia N (mmol/l) 0 59.3 f 74.6 f 8.9

1 49.8 xf 259.0 yg 9.5

3 39.2 f 194.9 h 8.2

Urea N (mmol/l) 0 9.41 xf 5.83 yf 0.2

1 9.16 xf 6.97 yg 0.2

3 7.70 xg 8.91 yh 0.2

Glucose (mmol/l) 0 3.79 f 3.79 f 0.03

1 3.74 f 3.64 g 0.04

3 3.99 xg 3.70 yfg 0.03

NEFA (mEq/l) 0 143.4 f 115.4 f 7.5

1 102.5 xg 149.6 yg 8.1

3 82.0 gh 111.5 f 7.0

Insulin (mU/ml) 0 35.3 f 43.1 f 8.0

1 86.2 xg 46.4 yf 8.0

3 103.4 xgh 59.6 yf 7.8

Ruminal fluid pH 5.9 x 6.6 y 0.02

Ruminal ammonia N (mmol/l) 28.0 x 48.1 y 3.1

a Experimental diets were without NPN � urea contributing 0% of the total dietary N, with and without supplemental L-carnitine

(250 ppm); or with NPN � urea contributing 50% of the total dietary N with and without supplemental L-carnitine (250 ppm). Overall LS

means for samples collected on days 1, 8, 29, and 50 of the collection period.b Time (h) relative to feeding following 16 h of feed deprivation.c Within a column, means lacking a common letter (f, g, h) differ, P < 0:01.d Within a row, means lacking a common letter (x, y) differ, P < 0:0001.

Fig. 1. Effect of (A) L-carnitine on plasma total carnitine and (B) L-carnitine and day on plasma total carnitine. Experimental diets contained 0

(No Carn) or 250 ppm of L-carnitine (Carn). There was an overall L-carnitine effect (P < 0:002; (A)) and an L-carnitine� day effect

(P < 0:04; (B)). The pooled SEM was 6.5 and 7.0 nmol/ml, for (A) and (B), respectively.

18 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

the NPN diets, compared with lambs fed the non-NPN

diets (Table 4). Plasma urea N concentrations

increased at 1 and 3 h post-feeding in the NPN-fed

lambs, while lambs fed the non-NPN diets exhibited a

decrease in urea N during the same period

(NPN� hour effect, P < 0:0001; Table 3).

Plasma glucose concentrations were higher

(P < 0:05) in the lambs fed the L-carnitine diets than

in the lambs not receiving supplemental L-carnitine

across all sampling days (Table 4). Lambs fed L-

carnitine had higher plasma glucose levels at day

50 than did lambs not fed L-carnitine (L-

carnitine� day effect, P < 0:06; Table 4). In addition,

lambs fed the NPN diets had lower plasma glucose at

3 h post-feeding than lambs fed the non-NPN diets

(NPN� hour effect, P < 0:0001; Table 3). Lambs fed

the NPN diets exhibited an increase in plasma NEFA

levels 1 h post-feeding, but lambs fed the non-NPN

diets showed a decrease during the same period

(NPN� hour effect, P < 0:0001; Table 3).

Plasma albumin was not affected by NPN or L-

carnitine (P > 0:10; data not shown). Lambs fed the

NPN diets had lower (P < 0:02) plasma T4 concen-

trations (Table 5). Plasma T3 was not affected

(P > 0:10) by either NPN or L-carnitine, however,

the T4:T3 ratio was lower (P < 0:02) in lambs fed

diets containing NPN (Table 5). Ewe lambs fed the

NPN diet had higher plasma cortisol than the ewe

lambs fed the non-NPN diets (10.1 versus 8.2 ng/ml),

whereas wethers fed the NPN diets had lower plasma

cortisol values than wether lambs fed the non-NPN

diets (7.5 versus 11.2 ng/ml) resulting in a NPN � sex

interaction (P < 0:01; data not shown).

Plasma insulin was higher in the lambs fed diets

containing NPN at 1 and 3 h post-feeding compared

with lambs fed the non-NPN diets (NPN� hour

effect, P < 0:002; Table 3). There was an L-

carnitine� day interaction (P < 0:06; Table 5) for

plasma insulin. Lambs on the NPN diets had the

lowest insulin concentrations on day 29 (NPN effect,

P < 0:04; Table 5).

3.3. Oral urea load test results

Samples drawn at the 0 min sample of the OULT 1

showed an effect of NPN. Ruminal ¯uid ammonia N

(P < 0:005) and plasma urea N (P < 0:0008; Table 6)

were lower, and plasma ammonia N (P < 0:05;

Table 4

Effect of NPN and L-carnitine on plasma metabolites concentrationsa

Metabolite Day Control Dietsb SEM

NPN Carn NPN � Carn

Ammonia N (mmol/l) 1c 67.8 211.5 58.7 202.5 19.4

8c 36.6 140.8 40.6 164.0 18.2

29c 52.9 191.4 49.8 171.3 24.0

50c 44.0 181.8 45.9 153.9 11.0

Avg.c 50.3 181.4 48.7 172.9 12.9

Urea N (mmol/l) 1c 9.93 7.56 8.90 7.94 0.5

8c 8.53 6.89 8.30 7.38 0.6

29c 8.77 6.60 8.25 6.30 0.5

50 8.36 7.49 8.25 8.06 0.6

Avg.c 8.90 7.13 8.62 7.43 0.4

Glucose (mmol/l) 1 4.15 4.01 4.17 4.05 0.10

8 3.70 3.54 3.83 3.68 0.05

29 3.68 3.57 3.84 3.64 0.10

50d 3.42 3.46 3.81 3.72 0.10

Avg.d 3.74 3.65 3.91 3.77 0.07

a Daily LS means for samples collected on days 1, 8, 29, and 50 of the collection period.b Experimental diets were without NPN � urea contributing 0% of the total dietary N, with and without supplemental L-carnitine

(250 ppm); or with NPN � urea contributing 50% of the total dietary N with and without supplemental L-carnitine (250 ppm).c NPN effect, P < 0:0001.d

L-carnitine effect, P < 0:0001.

A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28 19

Table 6) was higher, in the lambs fed the NPN diets.

Lambs fed the L-carnitine diets had higher plasma

glucose concentrations (P < 0:04; Table 6) than lambs

fed diets free of supplemental L-carnitine. Ruminal

¯uid pH was not affected by NPN at 0 min (P > 0:10;

Table 6).

During the OULT 1, plasma ammonia N was higher

in lambs fed the NPN diets (P < 0:0001; Fig. 2A).

Ewe lambs had higher plasma ammonia N levels in the

control, L-carnitine, and NPN with L-carnitine diets

compared with wethers, however, ewe lambs consum-

ing the NPN treatment had lower plasma ammonia N

compared with the wethers, resulting in an NPN � L-

carnitine� sex interaction (P < 0:04). During the

OULT 1, plasma urea N was lower in lambs fed the

NPN diets (P < 0:0001; Fig. 2B), and plasma glucose

concentrations were higher in L-carnitine-

supplemented lambs (P < 0:0001; Fig. 3A) as well

as in wether lambs compared with ewe lambs (sex

effect, P < 0:0001). Plasma insulin was lowest in the

NPN-fed lambs compared with lambs consuming the

non-NPN diets (P < 0:0001; Fig. 3B).

Samples drawn at 0 min of the OULT 2 showed an

effect of NPN. Concentrations of ruminal ¯uid ammo-

nia N (P < 0:0001), plasma urea N (P < 0:0001), and

plasma insulin (P < 0:009) were lower, and plasma

ammonia N was higher (P < 0:0002) in lambs consum-

ingtheNPNdiets(Table6).Atthe0 minsample,ruminal

¯uid pH was not affected by NPN (P > 0:10; Table 6).

During the OULT 2, plasma ammonia N was lower

in the lambs fed the L-carnitine and the NPN with L-

carnitine diets than in the lambs fed the NPN diet

(P < 0:02; Fig. 4A). Plasma urea N was lowest in

lambs fed diets containing NPN (P < 0:0001; Fig. 4B)

compared with the lambs fed diets not containing

supplemental NPN. Plasma glucose was lowest in

the lambs fed the NPN diets (P < 0:05; Fig. 5A),

additionally, lambs fed the NPN with L-carnitine diet

had the lowest plasma glucose values (NPN� L-

carnitine effect, P < 0:0003). Plasma insulin

Table 5

Effect of NPN and L-carnitine on plasma hormones concentrations in relation to feedinga

Metabolite Day Control Dietsb SEM

NPN Carn NPN � Carn

T4 (ng/ml) 1 71.24 56.03 65.03 67.41 2.73

8 72.39 58.82 70.94 67.92 2.94

29 79.25 56.62 68.58 64.31 3.42

50 99.45 76.54 102.55 82.74 6.41

Avg.b 80.58 62.00 76.78 70.60 2.36

T3 (ng/ml) 1 1.87 1.67 1.76 1.84 0.04

8 1.84 1.62 1.70 1.81 0.04

29 1.76 1.62 1.74 1.71 0.06

50 1.90 1.75 1.77 1.91 0.06

Avg. 1.84 1.67 1.74 1.82 0.03

T4:T3 ratio 1 38.33 33.56 37.92 36.76 1.44

8 39.40 36.43 38.78 37.73 1.62

29 46.32 35.13 39.92 37.48 2.08

50 51.83 44.20 57.66 42.88 3.34

Avg.c 43.97 37.33 43.57 38.71 2.00

Insulin (mU/ml) 1 96.1 52.2 56.5 53.1 25.0

8 78.8 43.1 75.7 54.9 17.0

29c 117.1 48.0 58.6 43.9 18.6

50 28.7 44.8 48.5 49.2 18.2

Avg. 80.2 47.0 59.8 50.3 14.2

a Daily LS means for samples collected on days 1, 8, 29, and 50 of the collection period.b Experimental diets were without NPN � urea contributing 0% of the total dietary N, with and without supplemental L-carnitine

(250 ppm); or with NPN � urea contributing 50% of the total dietary N with and without supplemental L-carnitine (250 ppm).c NPN effect, P < 0:04.

20 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

concentrations were highest (P < 0:007) in lambs fed

L-carnitine and lowest (P < 0:0007) in lambs fed NPN

(Fig. 5B). Plasma glucose was highest in the lambs fed

the L-carnitine diets but lowest in lambs fed the NPN

with L-carnitine diet (NPN� L-carnitine effect,

P < 0:01).

3.4. Results of the minimal model FSIGT

The SG value, or glucose effectiveness, was not

affected by NPN or L-carnitine (overall mean �0:0258� 0:007 minÿ1; P > 0:10). The SI value, or

insulin sensitivity, was not affected by L-carnitine

(overall mean � 0:337� 0:15� 10ÿ4 minÿ1 mUÿ1

mlÿ1; P > 0:10), however, lambs fed the NPN-con-

taining diets tended to have a higher SI value compared

with lambs fed the non-NPN diets (0:456�0:10� 10ÿ4 minÿ1 mUÿ1 mlÿ1 versus 0:217� 0:11

�10ÿ4 minÿ1 mUÿ1 mlÿ1; P � 0:13).

Samples drawn at the ÿ10 min of the FSIGT

showed no effect of NPN or L-carnitine on plasma

albumin concentrations (overall mean � 34:3� 1:5 g/

l; P > 0:10). Plasma NEFA (83.0 versus 133.9 meq./l;

P < 0:01) and urea N (15.7 versus 18.7 mmol/l;

P < 0:008) values were lower in lambs fed the diets

containing NPN.

4. Discussion

Production and metabolic responses to supplemen-

tal L-carnitine have been variable in ruminants. In the

present experiment, total feed intake and ADG ratio

were not affected by the addition of L-carnitine. Hill

et al. (1994) reported no effect of supplemental L-

carnitine on ADG, DMI, and gain:feed ratio in feedlot

steers. These ®ndings are in contrast to those of White

et al. (1998a) who found an improvement in ADG

when L-carnitine was supplemented to grazing wean-

ing calves, and DeRouen et al. (1998) who found that

weaned beef calves fed broiler litter-corn diets plus L-

carnitine had higher DMI and tended to have higher

Table 6

Baseline values of ruminal and plasma criteria in samples collected prior (0 min sample) to an oral urea load test during days 10 (OULT 1) and

50 (OULT 2) of the trial

Item Dietsa SEM

Control NPN Carn NPN � Carn

OULT 1

Ruminal ammonia N (mmol/l)b 5.85 11.27 5.68 10.98 2.1

Ruminal pH 6.6 6.7 6.6 6.7 0.04

Plasma ammonia (mmol/l)c 64.0 68.4 59.4 75.4 2.4

Plasma urea N (mmol/l)d 10.95 7.04 10.22 7.35 0.4

Plasma glucose (mmol/l)e 3.52 3.52 3.81 3.86 0.07

Plasma insulin (mU/ml) 30.5 21.9 30.2 21.6 4.3

OULT 2

Ruminal ammonia N (mmol/l)b 6.61 11.63 5.78 10.05 2.5

Ruminal pH 6.4 6.4 6.2 6.3 0.05

Plasma ammonia (mmol/l)c 76.4 108.2 71.3 116.0 3.8

Plasma urea N (mmol/l)d 11.42 6.32 10.29 6.76 0.4

Plasma glucose (mmol/l)e 3.48 3.79 3.93 3.36 0.07

Plasma insulin (mU/ml)f 63.3 36.5 90.9 48.3 5.7

a Experimental diets were: Control � urea contributing 0% of the total dietary N and 0 ppm L-carnitine; NPN � urea contributing 50% of

the total dietary N and 0 ppm of L-carnitine; Carn � urea contributing 0% of the total dietary N and 250 ppm of L-carnitine;

NPN� Carn � urea contributing 50% of the total dietary N and 250 ppm of L-carnitine.b NPN effect, P < 0:005.c NPN effect, P < 0:05.d NPN effect, P < 0:001.e

L-carnitine effect, P < 0:05.f NPN effect, P < 0:009.

A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28 21

ADG. From days 1 through 7 on the collection period,

lambs fed the NPN with L-carnitine diet tended to have

a higher feed intake compared with the other treat-

ments. Other studies reported decreases in ADG in

weanling calves supplemented with L-carnitine and

various protein sources (White et al., 1998b) and

decreases in DMI and gain:feed ratio in Holstein

calves fed broiler litter and L-carnitine (Yavuz et al.,

1997).

When NPN constitutes a large percentage of the

total dietary N in ruminant diets, reductions in growth,

ADG, N retention, feed ef®ciency, and milk produc-

tion were exhibited (Chalupa, 1972; NRC, 1976; Kertz

et al., 1982; Fernandez et al., 1997). Researchers have

proposed that the decreased performance associated

with the feeding of NPN may be caused by derange-

ments in intermediary metabolism (Chalupa, 1972;

Spires and Clark, 1979; Emmanuel et al., 1982; Fer-

nandez et al., 1988, 1997). In the present experiment,

two of the treatments contained NPN which consti-

tuted 50% of the total dietary N, resulting in a decrease

in ADG.

In addition to affecting growth parameters, ruminal

¯uid pH and ammonia N concentrations were higher in

lambs fed the NPN-containing diets. Ruminants fed

diets containing NPN generally experience increases in

ruminal pH and ammonia N concentrations (Chalupa,

1972; Kertz et al., 1982; Fernandez et al., 1997).

Feeding NPN as urea has been shown to increase rumen

pH because of the rapid production of ammonia through

urea hydrolysis, which contributes to the alkalinity of

the rumen ¯uid through formation of the ammonium

(NH4�) ion (Kertz et al., 1982; Visek, 1984).

In general, rapid hydrolysis of dietary urea within

the rumen via the action of the microbial enzyme

urease results in a concomitant increase in ruminal

Fig. 2. Changes in plasma (A) ammonia N and (B) urea N concentrations in sheep following an oral urea load test (OULT 1) on day 10. Urea

solution 0.835 g/kg0.75 was administered via a stomach tube at 30 min. Experimental diets were Control � urea contributing 0% of the total

dietary N and 0 ppm L-carnitine; NPN � urea contributing 50% of the total dietary N and 0 ppm of L-carnitine; Carn � urea contributing 0%

of the total dietary N and 250 ppm of L-carnitine; NPN/Carn � urea contributing 50% of the total dietary N and 250 ppm of L-carnitine.

Plasma ammonia N (2A) was in¯uenced by NPN (P < 0:0001) and NPN� time (P < 0:0001). Plasma urea N (2B) was in¯uenced by NPN

(P < 0:0001) and NPN� L-carnitine (P < 0:0002). The pooled SE for plasma ammonia N and urea N were 7.6 mmol/l and 0.15 mmol/l,

respectively.

22 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

¯uid pH and ammonia N concentration, which, in turn,

results in an increase in circulating ammonia N (Cha-

lupa, 1972; NRC, 1976; Kertz et al., 1982). Excess

ruminal ¯uid ammonia N in the presence of elevated

pH favors the formation of the free, non-ionized form

(NH3 versus NH4�), and it is this form of ammonia

that is rapidly absorbed across the rumen epithelium

into the portal and lymphatic circulation, resulting in

sub-clinical and clinical forms of hyperammonemia

(Spires and Clark, 1979; Visek, 1984; Fernandez et al.,

1990, 1997). In the present experiment, plasma ammo-

nia N concentrations were highest in lambs fed the

NPN-containing diets. This indicates that the higher

ruminal ¯uid pH and ammonia N concentrations

resulted in the elevated plasma ammonia N levels

observed in these sheep (average 177 mmol/l). We

considered these lambs to be experiencing subclinical

hyperammonemia (Fernandez et al., 1988). In con-

trast, lambs fed the non-NPN diets did not exhibit an

increase in plasma ammonia N 1 h post-feeding; this

could be due to the preformed, natural protein source

(soybean meal) that was used as the protein source

(NRC, 1976; Visek, 1984).

Plasma total carnitine concentrations increased

in the lambs fed the diets containing supplemental

L-carnitine. This indicates that dietary L-carnitine

was being absorbed by the lambs. Since, the concen-

trations of total carnitine increased throughout the

collection period, this suggests that the rumen

micro-organisms showed minimal adaptation to the

supplemented L-carnitine. This is in contrast to

LaCount et al. (1996a) who showed that L-carnitine

degradation was higher in ruminal ¯uid from cows that

had adapted to dietary L-carnitine supplementation for

2 weeks.

Supplementation of L-carnitine did not affect rum-

inal ammonia N or plasma ammonia N concentrations

in this study. This agrees with Morris et al. (1998) who

found that oral administration (1 g/day) of an L-carni-

tine solution in mature ewes for 10 days did not affect

Fig. 3. Changes in plasma (A) glucose and (B) insulin concentrations in sheep following an oral urea load test on day 10 (OULT 1). Urea

solution (0.835 g/kg0.75) was administered via a stomach tube at 30 min. Experimental diets were Control � urea contributing 0% of the total

dietary N and 0 ppm L-carnitine; NPN � urea contributing 50% of the total dietary N and 0 ppm of L-carnitine; Carn � urea contributing 0%

of the total dietary N and 250 ppm of L-carnitine; NPN/Carn � urea contributing 50% of the total dietary N and 250 ppm of L-carnitine.

Plasma glucose (3A) was affected by L-carnitine (P < 0:0001), whereas plasma insulin (3B) was affected by NPN (P < 0:0001). The pooled

SE for plasma glucose and insulin were 0.03 mmol/l and 1.7 mU/ml, respectively.

A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28 23

baseline ruminal ¯uid ammonia N or plasma ammonia

N concentrations. Similar observations were reported

in cows (LaCount et al., 1995, 1996a).

The ruminal and plasma ammonia N response to

supplemental L-carnitine has been variable. Yavuz

et al. (1997) reported that Holstein calves fed diets

containing broiler litter and L-carnitine had higher

ruminal ammonia N compared with calves fed a

similar diet containing broiler litter but void of sup-

plemental L-carnitine. In contrast, White et al. (1997)

showed a decrease in ruminal ammonia N in grazing

calves fed a molasses±urea based liquid supplement

containing L-carnitine. Moreover, plasma ammonia N

levels were not affected by L-carnitine in several

feeding trials conducted with calves (Yavuz et al.,

1997; DeRouen et al., 1998; White et al., 1998a).

Previous work in our laboratory has shown that

intravenous L-carnitine administration in sheep, prior

to an OULT, reduces the characteristic rise in plasma

ammonia N (Chapa et al., 1998). L-carnitine has been

shown to protect mice (Costell et al., 1987) and

Channel cat®sh (Burtle and Newton, 1991) against

elevated and even lethal levels of ammonia. In the

experiments noted above, the presence of L-carnitine

reduced circulating ammonia N levels in response to

environmentally and experimentally induced hyper-

ammonemia, however, this has not always been the

case (Morris et al., 1998). It is apparent that the

in¯uence of L-carnitine on circulating ammonia N

levels may be dependent on route and level of admin-

istration, the adaptation of the rumen microbial popu-

lation to L-carnitine, or even to the severity of the

hyperammonemia experienced. For instance, O'Con-

nor et al. (1986) reported that intraperitoneal injec-

tions of L-carnitine in mice offered the most protection

against experimentally induced acute hyperammone-

mia, followed by intravenous and intramuscular

administration.

Fig. 4. Changes in plasma (A) ammonia N and (B) urea N concentrations in sheep following an oral urea load test on day 50 (OULT 2). Urea

solution (0.835 g/kg0.75) was administered via a stomach tube at 30 min. Experimental diets were Control � urea contributing 0% of the total

dietary N and 0 ppm L-carnitine; NPN � urea contributing 50% of the total dietary N and 0 ppm of L-carnitine; Carn � urea contributing 0%

of the total dietary N and 250 ppm of L-carnitine; NPN/Carn � urea contributing 50% of the total dietary N and 250 ppm of L-carnitine.

Plasma ammonia N (4A) was affected by NPN (P < 0:0001), L-carnitine (P < 0:05), and an NPN� time effect (P < 0:0001). Plasma urea N

(4B) was affected by NPN (P < 0:0001) and NPN� L-carnitine (P < 0:0001). The pooled SE for plasma ammonia N and urea N were

7.7 mmol/l and 0.2 mmol/l, respectively.

24 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

In the present experiment, total carnitine concen-

trations almost doubled from baseline concentrations

by day 7 in the lambs fed supplemental L-carnitine,

however, this level of plasma carnitine was not suf®-

cient to reduce the hyperammonemia resulting from

the OULT1 (day 10). Nevertheless, an ammonia-

lowering effect by L-carnitine was noted by day 50

of the trial, but only during experimentally induced

hyperammonemia. During the OULT 2, plasma

ammonia N concentrations were lower in the lambs

fed diets containing L-carnitine and NPN with L-

carnitine. This could be attributed to an increase in

plasma total carnitine, since by day 50 of the trial,

plasma total carnitine concentrations had nearly

tripled from baseline levels.

Because ammonia is converted to urea in the liver,

the expected response to high plasma ammonia levels

is elevated plasma urea levels (Fernandez et al., 1990).

Plasma urea N levels in the lambs fed NPN were lower

in this study while L-carnitine had no effect on plasma

urea N. One explanation may be that lambs in this

study were fed at a level to meet the NRC (1985)

protein requirements for growing lambs and were not

fed excess dietary N. The elevated plasma ammonia N

levels may be explained by the high N solubility

associated with the dietary urea, whereas the lower

levels of plasma urea N may be explained by the

feeding of adequate (but not excess) dietary N in

accordance to NRC (1985) requirements (Preston

et al., 1965). Another possibility for the lower plasma

urea N levels is that the urea may have been excreted.

Sheep and cattle fed high levels of dietary NPN and

urea tend to have lower N retention because they

excrete greater amounts of N in the urine (Chalupa,

1972).

In the present experiment, supplemental L-carnitine

had no effect on plasma urea N concentrations. Some

studies have shown decreases in plasma urea N levels

with L-carnitine, suggesting a metabolic effect by the

supplement (Yavuz et al., 1997; White et al., 1997,

Fig. 5. Changes in plasma (A) glucose and (B) insulin concentrations in sheep following an oral urea load test on day 50 (OULT 2). Urea

solution (0.835 g/kg0.75) was administered via a stomach tube at 30 min. Experimental diets were Control � urea contributing 0% of the total

dietary N and 0 ppm L-carnitine; NPN � urea contributing 50% of the total dietary N and 0 ppm of L-carnitine; Carn � urea contributing 0%

of the total dietary N and 250 ppm of L-carnitine; NPN/Carn � urea contributing 50% of the total dietary N and 250 ppm of L-carnitine.

Plasma glucose (5A) was affected NPN (P < 0:05) and NPN� L-carnitine effect (P < 0:0003). Plasma insulin was in¯uenced by NPN

(P < 0:0007) and L-carnitine (P < 0:007). The pooled SE for plasma glucose and insulin were 0.05 mmol/l, and 2.8 mU/ml, respectively.

A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28 25

1998b), whereas other studies have not been able to

demonstrate an effect of L-carnitine on plasma urea N

(DeRouen et al., 1998; Morris et al., 1998).

Supplemental L-carnitine increased plasma glucose

concentrations in the present experiment. However,

the response of plasma glucose to L-carnitine has also

been variable. Chapa et al. (1998) reported that intra-

venous administration of L-carnitine (6.36 and

12.72 mmol/kg0.75 BW) increased plasma glucose in

sheep, however, this effect was lost when accompa-

nied by an OULT. Other studies have shown that

plasma glucose concentration was either decreased

(White et al., 1997), increased (Er¯e et al., 1971), or

was not affected (LaCount et al., 1995; Yavuz et al.,

1997; DeRouen et al., 1998; Morris et al., 1998) by L-

carnitine supplementation.

In this study, there was no effect of NPN on plasma

glucose, which suggests that the lambs on the NPN

treatments were not experiencing clinical hyperam-

monemia, since one of the characteristics of this acute

form of hyperammonemia is hyperglycemia (Bartley

et al., 1976; Emmanuel et al., 1982; Fernandez et al.,

1988, 1990). Nevertheless, plasma insulin was lower

in the lambs fed the NPN diets at 1 and 3 h post

feeding. This lends support that lambs fed the NPN-

containing diets were experiencing a subclinical

ammonia toxicity, since hypoinsulinemia has been

associated with clinical and subclinical hyperammo-

nemia (Emmanuel et al., 1982; Fernandez et al.,

1988).

The lower plasma insulin concentrations exhibited

by the NPN-fed lambs during both OULT could be in

response to subclinical hyperammonemia. Experi-

mentally induced hyperammonemia is associated with

hypoinsulinemia (Emmanuel et al., 1982; Fernandez

et al., 1988). Lambs fed diets containing supplemental

L-carnitine had higher plasma glucose concentrations

during both OULT 1 and 2. These results are in

contrast to Chapa et al. (1998) and Morris et al.

(1998), who found no effect of L-carnitine on plasma

glucose levels during an OULT.

It is not surprising that lambs fed NPN had

increased plasma NEFA levels 1 h post-feeding, since

it is known that a lower insulin, and more speci®cally,

a lower insulin:glucagon ratio, promotes lipolysis and

hepatic ketogenesis in ruminants (Brockman and

Laarveld, 1986). Nevertheless, insulin concentration

was not affected in lactating does fed diets containing

up to 50% of the total N (Fernandez et al., 1997). This

may be explained by the fact that homeorhetic

mechanisms supporting lactation differ from those

supporting growth, resulting in differences in NEFA

entry rate and lipid metabolism (Pethick and Dunshea,

1993).

Since hypoinsulinemia and hyperglycemia had

been shown to be characteristic of clinical forms of

induced hyperammonemia (Bartley et al., 1976;

Emmanuel et al., 1982; Fernandez et al., 1988), an

FSIGT was conducted early during the study to deter-

mine glucose effectiveness (SG), a measure of non-

insulin-dependent glucose clearance, and insulin

sensitivity (SI), a measure of insulin-dependent

glucose clearance (Bergman, 1997). In the present

experiment, there were no differences noted in SG or SI

due to L-carnitine or NPN. These results suggest that

L-carnitine and NPN did not in¯uence glucose clear-

ance as induced by glucose per se, and that L-carnitine

did not affect insulin-dependent glucose clearance

(Bergman, 1997; Majorie et al., 1997). However,

lambs fed diets with NPN tended to have a higher

SI, which suggests that these lambs were more insulin

sensitive compared with lambs fed diets without added

NPN (Majorie et al., 1997). If the NPN-fed lambs were

more insulin sensitive compared with their non-NPN-

fed counterparts, then plasma insulin levels should be

lower in the NPN-fed lambs because insulin would be

more effective. This could explain the hypoinsuline-

mia, however, if insulin is more effective, then the

hyperglycemic condition shouldn't occur. This is not

the case in clinical hyperammonemia, since both

conditions occur (Bartley et al., 1976; Emmanuel

et al., 1982; Fernandez et al., 1988). It should be

noted that most of the studies that showed hypoinsu-

linemia and hyperglycemia in light of hyperammone-

mia were short-term infusions. In this study, the lambs

on the NPN diets had lower plasma insulin concen-

trations but did not exhibit hyperglycemia. One pos-

sibility is that the lambs adapted to the NPN, since it

was fed for an extended period (Bartley et al., 1976;

Emmanuel et al., 1982; Fernandez et al., 1988).

The thyroid hormones, T3 and T4, are associated

with regulation of metabolic rate and modulation of

other growth- and metabolism-related hormones

(Brockman and Laarveld, 1986). Plasma T4 concen-

tration and the T4:T3 ratio were lower in lambs fed

NPN. Sex affected plasma T4 values with ewe lambs

26 A.M. Chapa et al. / Small Ruminant Research 40 (2001) 13±28

showing higher levels of this hormone compared with

wethers. Sex and NPN affected plasma cortisol con-

centrations, ewe lambs fed NPN had higher plasma

cortisol and wethers fed NPN had lower plasma

cortisol. Fernandez et al. (1997) found no effect of

NPN on cortisol concentrations in lactating goats.

5. Conclusion

Non-protein nitrogen incorporation in the diets of

growing Suffolk lambs reduced ADG and had pro-

found effects on critical blood metabolite and hor-

mone levels. Plasma total carnitine increased with

supplemental L-carnitine. However, the addition of

L-carnitine to the diets did not affect the lambs' growth

and had minimal effects on ruminal and blood char-

acteristics. Although plasma ammonia N levels were

not affected by L-carnitine early in the feeding trial

(day 10), L-carnitine did lower plasma ammonia N

later in the feeding trial (day 50) during induced

hyperammonemia. This suggests that supplementation

of L-carnitine with NPN may prevent or reduce the

development of hyperammonemia in ruminants.

Nevertheless, many questions still persist on the

micronutrient's mode of action and the level of L-

carnitine supplementation that would afford an ade-

quate and consistent level of protection to ammonia

toxicity under practical production conditions.

Acknowledgements

This experiment was supported, in part, by grants

from Lonza, Inc., Fair Lawn, New Jersey, and the

Louisiana Educational Quality Support Fund

(LEQSF-(1992-93)-ENH-TR-01 and LEQSF-

(1992-94)-RD-02), in co-operation with the Louisiana

Agricultural Experiment Station (Project LAB02936).

The publication has been approved by the Director of

the Louisiana Agricultural Experiment Station as

publication No. 00-11-0177. The authors wish to

express their appreciation to G. Trey Harding and

the staff at the LAES Central Station Sheep Unit

for their management and care of the sheep, as well

as their assistance during the collection period, and to

Dr. David C. Blouin for his assistance with the sta-

tistical analyses.

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