Directory UMM :Data Elmu:jurnal:S:Small Ruminant Research:Vol35.Issue3.Jun2000:

Small Ruminant Research 35 (2000) 235±247

In¯uence of dietary intake and lasalocid on serum
hormones and metabolites and visceral organ growth
and morphology in wether lambs
K.C. Swansona, L.P. Reynoldsb, J.S. Catonb,*
a

b

Department of Animal Science, University of Kentucky, Lexington, KY 40546, USA
Department of Animal and Range Sciences, North Dakota State University, Fargo, ND 58105, USA
Accepted 30 June 1999

Abstract
Twenty-four black face, crossbred wether lambs (32.4  6.1 kg) were assigned to one of four treatments arranged in a 2  2
factorial. Individually penned wethers were fed a pelleted total mixed diet at low intake (LI; 60% of ad libitum) or high intake
(HI; 95% of ad libitum); diets contained either low lasalocid (LL; 0 mg per lamb daily) or high lasalocid (HL; 40 mg per lamb
daily). Measurements of serum hormones and metabolites were taken during two 3-day collection periods following a 14-day
adjustment to treatments. After 42±45 day on treatments, wethers were slaughtered and weight (wt) of liver and intestinal
segments were recorded and tissues subsampled. Serum insulin and glucose concentrations were increased (p < 0.10) in lambs

on HI compared with those on LI. Total ruminal VFA concentration (millimolar) was increased (p < 0.10) with greater for HI
vs LI. Compared with LI, HI had greater (p < 0.10) ®nal BW, eviscerated BW (EBW), and total visceral wt; colon fresh, dry,
and dry fat-free wt; cecum dry wt; liver fresh wt, dry wt and dry fat-free wt, liver RNA : DNA ratio, RNA content, and protein
content; and duodenum RNA content. Liver DNA concentration was decreased (p < 0.10) in HI vs LI. Neither labeling index
nor morphology of intestinal segments were in¯uenced (p > 0.10) by intake or lasalocid. Lasalocid had little in¯uence on
metabolic hormones or growth and development of visceral organs. These data indicate that high intake increased serum
insulin and glucose concentrations, liver wt and cell size. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Intake; Ionophore; Visceral growth; Insulin; Growth hormone

1. Introduction
In ruminants, it has been estimated that empty
visceral tissue accounts for 45±65% of total energy
expenditure while contributing to only 6±10% of
*

Corresponding author. ‡1-701-231-7653; fax: ‡1-701-2317590.
E-mail address: caton@plains.nodak.edu (J.S. Caton).

total body weight (wt) (Burrin et al., 1989; Reynolds
and Tyrrell, 1989). However, the role of dietary

factors in regulating visceral growth and development is poorly understood. Previous work in lambs
has shown that visceral organ mass generally
increases with increased intake (Rompala and
Hoagland, 1987; Burrin et al., 1990; Rompala et al.,
1991). However, little work has been done evaluating
the role of dietary factors in regulating visceral growth

0921-4488/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 1 - 4 4 8 8 ( 9 9 ) 0 0 0 9 2 - 9

236

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

and development at the cellular level, especially in
ruminants.
Ionophores are used extensively in ruminant livestock production. Generally, data suggest that ionophores change ruminal metabolism by altering the
ruminal micro¯ora to favor propionic acid production.
However, a complete understanding of the mode of
ionophore action remains elusive (Galyean and

Owens, 1991). Increases in whole animal energetic
ef®ciency resulting from ionophores is often greater
than can be explained by changes in ruminal fermentation (Harmon et al., 1993). When ionophores are fed,
the digestive tract of the animal is exposed to the
ionophore until it is either excreted or modi®ed so that
it is no longer biologically active (Spears, 1990).
Therefore, ionophores may have direct effects on
the intestines which could explain some of the
observed responses.
The objectives of this study were to determine the
effects of intake and lasalocid on ruminal fermentation, visceral organ weights, cell proliferation, intestinal morphology and metabolic hormones in growing
wether lambs fed concentrate diets.

2. Materials and methods
2.1. Animals, dietary treatments and sampling
periods
Twenty-four black face, crossbred wether lambs
(32.4  6.1 kg) were assigned to one of four treatments arranged in 2  2 factorial. Individually penned
wethers were fed once daily (07:00 h) a pelleted total
mixed diet (Table 1) at high intake (HI; 95% ad

libitum) or low intake (LI; 60% ad libitum). The diet
either did not contain supplemental lasalocid (LL) or
had lasalocid added (HL; 40 mg per lamb daily). Ad
libitum intakes were determined with pretrial measurements. Lambs were bedded on wood chips, and
water was freely available. Lambs were handled in a
manner consistent with institutional animal care and
use protocols.
Blood samples were collected into serum separator
tubes (Becton Dickinson Vacutainer Systems, Rutherford, NJ) via jugular venipuncture before feeding on
the ®rst 3 days of each collection period. Samples
were allowed to clot for a minimum of 30 min and

Table 1
Composition of diets fed to growing wether lambsa,b
Item

Percentage of diet (DM basis)

Alfalfa meal
Beet pulp

Corn
Barley
Soybean meal (44%)
Dry molasses
Vitamins and minerals

10.1
10.3
40.2
20.1
12.0
5.5
1.8

a
Lasalocid (Bovatec 68; Hoffman±LaRoche Nutley, NJ) was
incorporated into the diets for lambs on the high lasalocid
treatments; analysis from Hoffman±LaRoche revealed that lambs
on high lasalocid treatments received 39.6 mg/hd daily.
b

Chemical analysis revealed 5.8% ash, 16.0% CP, 22.5% NDF,
and 10.7% ADF.

then were centrifuged at 1560g for 30 min. Serum was
decanted and stored at ÿ208C until analyzed for
hormones and metabolites.
2.2. Ruminal fermentation
Samples of whole ruminal contents were taken
immediately following slaughter. Ruminal samples
were analyzed for pH with a portable pH meter (Model
SA230, Orion, Cambridge, MA) ®tted with a combination electrode. Ruminal samples were strained
through four layers of cheesecloth, and the ¯uid
portion was acidi®ed with 7.2 N H2SO4 at the rate
of 1 ml of acid/100 ml of ruminal ¯uid. Samples were
stored frozen (ÿ208C).
Thawed ruminal samples were centrifuged at
10,000g for 10 min and the ¯uid portion analyzed
for ammonia concentrations by the colorimetric procedure of Broderick and Kang (1980). Ruminal ¯uid
was also analyzed for VFA concentrations using gas
chromatography with 2-ethylbutyric acid as the internal standard (Caton et al., 1993).

2.3. Serum hormones and metabolites
Blood serum was analyzed for growth hormone
(GH) using radioimmunoassay (RIA) procedures as
we have previously described for cattle (Reynolds
et al., 1990). The GH assay utilized NIDDK-oGH-I4 (biopotency ˆ 1.5 IU/mg) as the radioiodination
preparation, USDA-bGH-B-1 (biopotency ˆ 1.4 IU/

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

mg) as the reference standard, NIDDK-oGH-2 as the
primary antiserum, and sheep-anti-rabbit gamma globulin as the secondary antiserum.
To 12  75 mm2 polypropylene tubes, 500 ml of
serum was diluted with 100 ml of PBS (0.01 m phosphate, 0.14 m NaCl, pH 7.3) plus 1% (wt/vol) BSA
(RIA Grade; Sigma, St. Louis, MO). After this dilution
step, 200 ml of primary antiserum (1 : 20,000 in PBS)
was added, and the tubes were incubated for 24 hours
at 48C. Iodinated GH (100 ml) was then added, and
tubes were incubated for an additional 72 hours at 48C.
After this 72 hours incubation, 100 ml of secondary
antiserum was added and tubes were incubated for

another 72 hours at 48C. Finally, 3 ml of cold PBS was
added, tubes were centrifuged at 1500 g for 30 min,
the liquid was decanted, and tubes were counted on a
Beckman Gamma 5500 scintillation counter (Beckman Instruments, Arlington Heights, Illinois).
All samples were run in a single assay and intraassay variation was determined by assaying replicates
(n ˆ 6) of a pool of lamb plasma in the same assay.
Resulting mean SD concentration of growth hormone in the lamb plasma pool was 1.02  0.09 ng/ml
(c.v. ˆ 21.9%). To validate further the growth hormone assay, the pooled lamb plasma was assayed at
volumes of 200, 300, 400, 500, 600, and 700 ml, which
yielded an inhibition curve that was parallel to that of
the reference standard.
Blood serum was analyzed for insulin concentrations using a commercially available RIA kit (Coat-ACount, Diagnostic Products Corporation, Los
Angeles, CA) and procedures similar to those reported
previously by Reynolds et al. (1985, 1990). A glucose
oxidase kit (glucose, procedure no. 510, Sigma Diagnostics, St. Louis, MO) was used to determine serum
glucose concentrations. Serum urea nitrogen was
measured using a urea nitrogen kit (procedure no.
640, Sigma Chemical). We have previously reported
similar procedures for measuring glucose and urea
nitrogen in serum of cattle (Reynolds et al., 1985,

1990).
2.4. Slaughter procedures
Lambs were on treatment for 42±45 days and were
slaughtered by exsangination, after stunning via captive bolt, over a 4-day interval immediately following
the second collection period. Final live BW was

237

determined and, after slaughter and evisceration, visceral organs were obtained. Eviscerated body weight
(EBW, BW without viscera) was determined after
removal of visceral organs. Total visceral weight
(including ®ll) was calculated by subtracting EBW
from ®nal BW. Portions of small intestine segments
(duodenum, jejunum, ileum, cecum, and colon) were
obtained by using the following anatomical landmarks
(Widdowson et al., 1976; Jin et al., 1994): duodenum,
pyloric±duodenal junction to duodenal±jejunal junction (cranial third of small intestine): jejunum, duodenal±jejunal junction to jejunal±ileal junction
(middle third of small intestine): and ileum, jejunal±ileal junction to ileal±cecal junction (caudal third
of small intestine). In addition, the cecum was separated from the colon, and the colon was obtained up to
the rectal±anal junction (Widdowson et al., 1976; Jin

et al., 1994).
2.5. Tissue sampling procedures
To obtain intestinal tissue samples for analysis, the
mesentery was removed and small intestinal segments
were obtained as described above. For jejunum, the
mid point was located and a 10 cm section was
obtained. For duodenum, ileum, and colon 10 cm
sections were taken 5 cm posterior to the pancreatic
duct, 40 cm anterior to the ileal±cecal junction, and
40 cm posterior to the ileal±cecal junction, respectively. For cecum, a 10 g sample was taken midway
between the tip and the ileal±cecal junction on the side
distant from the cecal vein.
Intestinal samples were weighed and 1 cm3 samples
for liver and 1 cm wide cross-sections of each intestinal segment were immediately ®xed in 10% neutral
buffered formalin, Bouin's, or in Carnoy's solution for
48, 24, and 6 hours respectively, and transferred to
70% ethanol until they were embedded in paraf®n
(Reynolds and Redmer, 1992). Histological sections
(5 mm) of paraf®n-embedded intestinal segments were
mounted onto glass slides using standard histological

techniques, as described previously (Reynolds and
Redmer, 1992).
Additional 5 g samples of liver and each intestinal
segment, were frozen in liquid nitrogen and stored at
ÿ708C until analyzed for DNA, RNA and protein
concentrations (Reynolds et al., 1985, 1990; Reynolds
and Redmer, 1992).

238

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

After intestinal (duodenum, jejunum, ileum, cecum
and colon) tissue samples were obtained, the remainder of each segment was gently stripped by hand of
digesta and then fresh empty weights were determined. Liver weights were also recorded. Weights
of the tissue samples obtained for histological and
biochemical analyses were included in the fresh
weight for each tissue. Tissue samples were lyophilized to determine dry tissue weights. Dry fat-free
tissue weights were determined by the Soxhlet fat
extraction method (AOAC, 1990).
2.6. Tissue DNA, RNA and protein concentrations
and contents
To evaluate cellular growth of liver and intestinal
tissue segments, 0.5 g of each sample was homogenized in ®ve volumes of TNE buffer (0.05 M Tris,
2.0 M NaCl, 0.002 M EDTA, pH 7.4) using a polytron
(Brinkman, Westbury, NY). Diphenylamine and orcinol procedures were used to determine DNA and RNA
concentrations, respectively (Reynolds and Redmer,
1992; Jin et al., 1993a). Standards were DNA Type I
from calf thymus and RNA Type IV from calf liver
(Sigma Chemical, St. Louis, MO). Concentrations of
protein in tissue homogenates were determined by the
method of Bradford (1976) with bovine serum albumin (Fraction V, Sigma Chemical) as the standard.
Tissue DNA, RNA and protein contents were calculated by multiplying tissue concentrations by fresh
tissue weights (Reynolds and Redmer, 1992; Jin et al.,
1993a). Concentrations and contents of DNA were
used as an index of tissue hyperplasia, and ratios of
RNA : DNA and protein : DNA were used as indexes
of tissue hypertrophy (Baserga, 1985; Reynolds et al.,
1985, 1990).
2.7. Relative rate of cell proliferation (PCNA
labeling) in situ
To estimate the relative rate of cell proliferation,
histological detection of proliferating cell nuclear
antigen (PCNA) an endogenous nuclear protein that
is not only critical for DNA synthesis and cell replication but also is present in increasing abundance during
the S phase of the cell cycle was used (Zheng et al.,
1994; Fricke et al., 1997). Presence of PCNA in
speci®c nuclei in formalin-®xed intestinal sections

was detected immunohistochemically with a speci®c
monocloned primary antibody (mouse anti-PCNA
monoclonal; Boehringer Mannheim, Indianapolis,
IN) and a biotyinylated secondary antibody (horse
anti-mouse IgG; Vector Lab., Burlingame, CA) in
combination with avidin : biotinylated peroxidase
complex (ABC) reagents (Vectastain, Vector Lab.),
and 3,3'-diaminobenzidine as the substrate (Zheng
et al., 1994; Fricke et al., 1997). The tissue sections
were incubated with the PCNA antibody (1 : 2000 in
PBS containing 0.3% Triton X-100 (Mallingkrodt,
Paris, KY), and 1.5% normal horse serum (Vector
Lab.)) for 1 h at room temperature. Mouse IgG ascites
¯uid (ICN Biochemicals, Costa Mesa, CA) was used
on control slides in place of the anti-PCNA primary
antibody.
A computerized image analysis system (Roche
Image Analysis Systems, Elon College, NC) was used
to evaluate PCNA labeling (Jin et al., 1994; Fricke
et al., 1997). The total area of PCNA-labeled crypt cell
nuclei was determined for 10 randomly chosen ®elds
(26,376 mm2) per tissue for each lamb. Area of individual crypt cell nuclei was determined for each
intestinal segment by measuring the diameter of 10
nuclei in two dimensions per tissue per wether and
using the formula for the area of an ellipse
(A ˆ r1r2). The number of PCNA-labeled crypt cell
nuclei per unit area was then calculated for each
intestinal segment by dividing the total PCNA-labeled
area by the average individual nuclear area.
2.8. Intestinal morphometry
Bouin's-®xed intestinal sections were stained with
PAS (1% periodic acid and Schiff's reagent) and
Harris' hematoxylin to visualize tissue morphometry
(Reynolds and Redmer, 1992). The computerized
image analysis system was used to determine morphometry of intestinal segments, including villus
length, villus width and crypt depth (Jin et al.,
1993b, 1994). Ten villi and their associated length,
width and crypt depth were measured for each intestinal segment from each wether.
2.9. Statistical analysis
Collection of samples was not done for one lamb in
period 1 and another lamb in period 2 because these

239

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

animals refused to eat. Both lambs were in the low
intake-low lasalocid treatment. The lamb removed
from sampling period 2 was also removed from
slaughter and tissue collection.
Data were analyzed by GLM procedures of SAS,
1988. Period data were subjected to split±plot analysis
(Gill and Hafs, 1971). The model for serum hormones
and metabolites included effects of intake, lasalocid,
intake by lasalocid, animal within intake by lasalocid,
period, period by intake, period by lasalocid and
period by intake by lasalocid. Animal within intake
by lasalocid was used as the error term to test for
intake and lasalocid effects. When signi®cant F tests
were observed (p < 0.10) means were separated by the
least signi®cant difference (LSD) method.
A two factor factorial design was used for ®nal BW,
EBW, total visceral weight and fermentation data and
included effects of intake, lasalocid and intake by
lasalocid. Liver and intestinal (duodenum, jejunum,
ileum, cecum and colon) tissue weights and RNA,
DNA and protein contents; and liver RNA, DNA and
protein concentrations were also analyzed in this manner. When signi®cant F tests were observed (p < 0.10)
for the intake by lasalocid interaction, simple effect
means were separated by the method of LSD.
Intestinal tissue data that did not depend on individual tissue weight were subjected to split±plot analysis (Gill and Hafs, 1971). The model for intestinal
RNA, DNA and protein concentrations, morphometry
and PCNA labeling included effects of intake, lasalocid, intake by lasalocid, animal within intake by
lasalocid, tissue, tissue by intake, tissue by lasalocid
and tissue by intake by lasalocid. Animal within intake
by lasalocid was used as the error term. When intake
by lasalocid interactions were not signi®cant
(p> 0.10), main effect means were reported.

3. Results and discussion
3.1. Serum hormones and metabolites
No intakelasalocid interactions (p < 0.10)
were observed for serum insulin, glucose or urea
nitrogen concentrations. Serum insulin and glucose
concentrations were greater (p < 0.02) for HI
compared with the LI groups (Table 2). Brockman
and Laarveld (1986), Del®no et al. (1988), McFadden
et al. (1990) and Lapierre et al. (1992) also found
increases in blood insulin concentrations due to
increased intake in ruminants and Reynolds et al.
(1992) showed an increase in serum glucose concentrations as well. Serum urea nitrogen was not
affected (p > 0.10) by intake level which agrees
with results of McFadden et al. (1990). Concentrations
of serum insulin, glucose and urea nitrogen were
not affected (p > 0.10) by lasalocid, which is
consistent with results of other investigations
(Paterson et al., 1983; Shetaewi and Ross, 1991;
Quigley et al., 1992).
There was an intakelasalocid interaction
(p < 0.10) in serum growth hormone concentration.
Growth hormone concentration, with a standard error
of 0.93, was greatest for HILL (3.80 ng/ml) and least
for HIHL (1.80 ng/ml) with LILL (2.18 ng/ml) and
LIHL (3.74 ng/ml) treatments intermediate (p < 0.10).
Our GH data in HI, and not LI, lambs agrees with the
work of Duff et al. (1994) who suggested that lasalocid
could decrease serum GH concentrations in cattle fed
concentrate diets. Interestingly, in our study, GH in LI
lambs responded differently to lasalocid than HI
lambs, resulting in the interaction. Reasons for this
are unclear; but, may be related to an increased
energetic ef®ciency resulting from lasalocid in HI

Table 2
In¯uence of intake and lasalocid on serum insulin, urea N and glucose in growing wether lambs
Item

Insulin (mU/ml)
Glucose (mg/dl)
Urea N (mg/dl)
a

Intakea

Lasalocidb
c

LI

HI

P

LL

HL

P

8.36
79.4
20.4

29.9
98.4
18.6

0.02
0.01
0.17

19.8
91.8
18.5

18.5
86.0
20.5

0.78
0.25
0.13

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake or lasalocid.
b

SE
c

3.45
3.56
0.92

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K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

Table 3
In¯uence of intake and lasalocid on ruminal pH, ammonia, total VFA concentrations, and VFA proportions in growing wether lambs
Intakea

Lasalocidb
c

SE
c

Item

LI

HI

P

LL

HL

P

Ruminal pH
Ammonia (mg/dl)
Total VFA (mM)
Acetate : propionate

6.77
20.2
41.9
1.85

5.82
17.3
98.7
1.57

0.01
0.59
0.01
0.05

6.41
17.8
64.9
1.78

6.17
19.6
75.7
1.64

0.25
0.73
0.50
0.32

0.15
3.75
11.45
0.10

Mol/100 mol
Acetate
Propionate
Butyrate

48.5
26.7
8.5

44.8
29.5
11.3

0.10
0.19
0.20

48.5
27.7
9.0

44.8
28.4
10.8

0.1
0.8
0.4

1.5
1.5
1.5

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake or lasalocid.
b

lambs and (or) an uncoupling of hormonal mechanisms in LI lambs.
3.2. Ruminal fermentation
Total ruminal VFA concentrations were greater
(p < 0.01) for HI vs LI (Table 3). In contrast, ruminal
pH and acetate : propionate ratio were less (p < 0.05)
in lambs on HI compared with those on LI. There also
was a tendency (p ˆ 0.10) for the molar proportion of
acetate to decrease with increased intake. Hart and
Glimp (1991) reported no differences in ruminal
fermentation values with differing levels of intake
in lambs fed a 90% concentrate diet. However, in
their case, the lowest restricted level of dietary intake
was 85% of ad libitum intake, which may not have
been low enough to elicit a response.
Although we found no differences in ruminal
fermentation due to lasalocid supplementation,
molar proportion of acetate tended (p ˆ 0.10) to
be less with than without lasalocid. According to
Bergen and Bates (1984), the most consistently
observed effect on ruminal fermentation due to
ionophore supplementation is the increased proportion of propionic acid with a concomitant decline
in the molar proportion of acetate and butyrate. It is
unclear why these effects of lasalocid on ruminal
fermentation were not observed in the present study.
In addition, we found no effect (p > 0.10) of intake or
lasalocid levels on ruminal ammonia concentration
(Table 3).

3.3. Body and visceral tissue weights
Final BW, eviscerated BW, (EBW) and total visceral weight (wt) were increased (p < 0.10) in the HI
compared with the LI groups (Table 4). The total
visceral wt : BW ratio also tended (p ˆ 0.10) to
decrease with increased intake. However, lasalocid
did not in¯uence (p > 0.10) ®nal lamb BW, eviscerated BW, total visceral wt, or the total visceral
wt : BW ratio (Table 4).
Visceral tissue weight (wt) data are presented in
Table 5. For duodenum, no effects (p > 0.10) of intake
or lasalocid were observed. In jejunum, the ratio of dry
wt : fresh wt was increased (p < 0.03) in HI compared
with LI, but the other tissue wt measurements were not
affected (p > 0.10) by intake; however, in jejunum,
fresh wt : EBW tended (p ˆ 0.10) to decrease with
increased intake. For ileum, dry wt and dry wt : fresh
wt ratio were greater (p < 0.07) in lambs receiving HI
compared with LI treatments but the other tissue wt
measurements did not differ (p > 0.10). Cecal fresh
wt, dry wt, and dry fat-free wt were increased
(p < 0.02) in high intake compared with LI groups,
but the other tissue wt measurements were not in¯uenced (p > 0.10) by intake (Table 5). For colon, fresh
wt, dry wt and dry free-fat wt were increased
(p < 0.09) in HI compared with LI groups, but other
tissue weight measurements were not in¯uenced
(p > 0.10) by intake.
However, for fresh wt, dry wt and dry fat-free wt of
ileum and fresh wt and dry fatfree wt of cecum, the

241

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247
Table 4
In¯uence of intake and lasalocid on ®nal BW and total visceral weights in growing wether lambs
Item

BW (kg)
Eviscerated BW (kg)
Total visceral wt (kg)d
Total visceral wt: eviscerated BW

Intakea

Lasalocidb
c

SE
c

LI

HI

P

LL

HL

P

38.1
25.2
13.0
0.52

47.0
31.9
15.1
0.48

0.01
0.01
0.02
0.10

42.8
28.7
14.1
0.50

42.3
28.4
13.9
0.49

0.87
0.90
0.84
0.64

1.83
1.35
0.17
0.062

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake or lasalocid.
d
Includes digesta ®ll.
b

intakelasalocid interaction was signi®cant (p < 0.10;
Table 6). Fresh and dry fatfree wt of ileum were
highest in the HILL treatment and lowest in the LILL
and HILL treatments with the LILL treatment intermediate (p < 0.10). Dry wt of ileum was highest
(p < 0.10) in the HILL treatment, with no differences
(p > 0.10) among the other three treatments. Cecum
fresh wt was highest in the HILL treatment and lowest
in the LILL treatment, with the LIHL and HIHL
treatments intermediate (p < 0.10). Cecum dry fat-free
wt was highest in the HILL and HIHL treatments and
lowest in the LILL treatment, with the LIHL treatment
intermediate (p < 0.10).
Consistent with our observations, other investigators have shown that fresh weights of small and large
intestine generally increase in response to increased
intake (Rompala and Hoagland, 1987; Burrin et al.,
1990; Rompala et al., 1991; Fluharty and McClure,
1995). However, our data suggest that individual
segments of the large intestine are in¯uenced by intake
to a greater extent than individual segments of the
small intestine. Interestingly, lasalocid had no in¯uence (p > 0.38) on visceral tissue weights (Table 5),
except in ileal and cecal tissue (Table 6). In these two
tissues it appears that lasalocid will reduce fresh wt in
lambs fed 95% ad libitum. Biological signi®cance of
this response is likely minimal in light of other data
presented in this manuscript indicating that lasalocid
has little effect on visceral organ mass (Table 5) or
cellular proliferation in the intestine (Table 9).
For liver, fresh weight, dry weight and dry fat-free
weight, and the ratios of fresh weight : EBW, dry
weight : EBW and dry fat-free weight : EBW were
increased (p < 0.08) in HI compared with LI treatments (Table 5). The ratio of dry wt : fresh wt, for

liver, was not in¯uenced (p > 0.10) by intake. Consistent with our observations, other investigators have
also shown increases in liver fresh weights in ruminants with increased intake (Murray et al., 1977;
Rompala and Hoagland, 1987; Burrin et al., 1990;
Rompala et al., 1991; Lobley et al., 1994; Fluharty and
McClure, 1995). Lasalocid did not in¯uence
(p > 0.10) liver tissue wt measurements. This is in
agreement with Fluharty et al. (1996) who reported
that supplementation of lasalocid did not affect fresh
liver, small intestine or large intestine wt in lambs.
3.4. Tissue DNA, RNA and protein
Intestinal concentrations (across duodenum, jejunum, ileum cecum and colon) of RNA, DNA and
protein and ratios of RNA : DNA and protein : DNA
were not in¯uenced (p > 0.10) by intake (Table 7).
These data indicate that number of cells per gram of
tissue was not affected by treatments. In addition, cell
size as re¯ected by RNA : DNA and protein:DNA
ratios was not affected by intake or lasalocid (Table
7). Lobley et al. (1994) showed no differences in RNA
and protein concentrations in segments of the small
and large intestine due to increased intake in lambs.
Burrin et al. (1992), however, reported an increase in
duodenal RNA concentration and decreased duodenal
and jejunal DNA concentrations due to increased
intake in sheep. Burrin et al. (1990) also reported
an increase in the protein : DNA ratio of the duodenum and jejunum in response to increased intake in
sheep.
Concentrations of liver RNA and protein and the
ratio of protein : DNA were not in¯uenced (p > 0.10)
by intake (Table 8). In contrast, liver DNA concentra-

Table 5
In¯uence of intake and lasalocid on visceral tissue weights in growing wether lambs
Item

Intakea

Lasalocidb

SE

c

c

LL

HL

P

LI

HI

Duodenum
Fresh wt (g)
Dry wt (g)
Dry fat-free wt (g)
Fresh wt : EBW (g/kg)
Dry wt : EBW (g/kg)
Dry fat-free wt : EBW (g/kg)
Dry wt : fresh wt

313
57.4
48.2
12.7
2.33
1.96
0.183

340
64.5
52.4
10.9
2.05
1.67
0.190

0.30
0.12
0.29
0.18
0.24
0.17
0.19

330
61.1
50.6
11.9
2.19
1.82
0.185

323
60.7
50.1
11.7
2.19
1.82
0.188

0.77
0.93
0.91
0.89
0.99
0.99
0.55

17.9
3.16
2.77
0.95
0.14
0.14
0.003

Jejunum
Fresh wt (g)
Dry wt (g)
Dry fat-free wt (g)
Fresh wt : EBW (g/kg)
Dry wt : EBW (g/kg)
Dry fat-free wt : EBW (g/kg)
Dry wt : fresh wt

299
55.1
44.9
12.17
2.24
1.82
0.184

307
61.1
47.2
9.87
1.95
1.52
0.201

0.77
0.21
0.57
0.10
0.24
0.15
0.03

308
58.3
47.2
11.2
2.11
1.71
0.189

298
57.8
44.8
10.8
2.08
1.63
0.196

0.67
0.91
0.56
0.78
0.89
0.70
0.38

18.0
3.40
2.91
0.95
0.17
0.15
0.005

Ileum
Fresh wt (g)
Dry wt (g)
Dry fat-free wt (g)
Fresh wt : EBW (g/kg)
Dry wt : EBW (g/kg)
Dry fat-free wt : EBW (g/kg)
Dry wt : fresh wt

364
68.3
52.7
14.7
2.74
2.12
0.188

394
79.6
57.1
12.6
2.52
1.82
0.203

0.27
0.04
0.27
0.13
0.30
0.11
0.7

388
75.3
55.9
13.9
2.67
1.99
0.193

370
72.6
53.9
13.4
2.59
1.94
0.197

0.52
0.62
0.62
0.67
0.72
0.78
0.71

19.3
3.80
2.81
0.93
0.15
0.13
0.006

Cecum
Fresh wt (g)
Dry wt (g)
Dry fat-free wt (g)
Fresh wt : EBW (g/kg)
Dry wt : EBW (g/kg)
Dry fat-free wt : EBW (g/kg)
Dry wt : fresh wt

49.9
8.35
6.49
2.01
0.34
0.26
0.167

59.8
10.49
7.89
1.91
0.33
0.25
0.175

0.02
0.01
0.02
0.54
0.87
0.64
0.36

54.7
9.19
7.15
1.94
0.32
0.25
0.168

55.0
9.66
7.23
1.99
0.34
0.26
0.175

0.94
0.54
0.89
0.78
0.46
0.71
0.38

2.77
0.55
0.41
0.12
0.02
0.02
0.006

Colon
Fresh wt (g)
Dry wt (g)
Dry fat-free
Fresh wt : EBW (g/kg)
Dry wt : EBW (g/kg)
Dry fat-free wt : EBW (g/kg)
Dry wt : fresh wt

399
121
40.8
16.1
4.88
1.64
0.302

476
156
47.2
15.2
4.89
1.51
0.328

0.04
0.03
0.09
0.55
0.98
0.37
0.24

433
134
43.7
15.4
4.73
1.55
0.308

443
143
44.4
15.9
5.03
1.59
0.321

0.78
0.53
0.85
0.76
0.56
0.74
0.54

25.6
10.8
2.55
1.16
0.36
0.10
0.015

Liver
Fresh wt (g)
Dry wt (g)
Dry fat-free wt (g)
Fresh wt : EBW (g/kg)
Dry wt : EBW (g/kg)
Dry fat-free wt : EBW (g/kg)
Dry wt : fresh wt

658
201
171
26.5
8.10
6.89
0.31

969
295
242
30.3
9.22
7.58
0.30

0.01
0.01
0.01
0.01
0.02
0.08
0.62

795
245
204
27.7
8.52
7.137
0.31

832
252
209
29.1
8.80
0.34
0.30

0.55
0.73
0.75
0.33
0.54
0.57
0.17

43.6
13.9
11.3
1.01
0.32
0.27
0.01

a

P

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake or lasalocid.
b

243

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247
Table 6
In¯uence of intake and lasalocid on ileum and cecum weights in growing wether lambs (simple effect means)a
Item

LI

Ileum
Fresh (g)
Dry (g)
Dry fat-free (g)
Cecum
Fresh (g)
Dry fat-free (g)

HI

LL

HL

LL

HL

348c
63.8c
49.1c

380c,d
72.2c
56.2c,d

428d
86.8d
62.6d

361c
72.4c
51.6c

46.1c
5.89c

53.8c,d
7.09c,d

63.3c
8.41d

56.2d,e
7.37d

SE

Pb

28.53
5.62
4.15

0.08
0.04
0.03

4.10
0.60

0.07
0.06

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% ad libitum); LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid
(40 mg/hd daily).
b
P equals observed signi®cance level for intakelasalocid interaction.
c
Means within a row that do not have a common superscript differ (p < 0.10).
d
Means within a row that do not have a common superscript differ (p < 0.10).
e
Means within a row that do not have a common superscript differ (p < 0.10).

tion decreased (p < 0.06), and ratio of RNA : DNA
increased (p < 0.02), in HI compared with LI groups
(Table 7). These data indicate that cell number, per
gram of tissue is decreased and cell size is increased in
the liver of lambs fed at high levels compared to
restricted lambs. Lobley et al. (1994) reported no
differences in liver RNA and protein concentrations
due to increased intake in lambs. Conversely, Burrin

et al. (1992) reported increases in the protein concentration and the ratio of protein : DNA in livers of sheep
in response to increased intake. Lasalocid did not
in¯uence (p > 0.10) liver RNA, DNA or protein concentrations or the ratio of protein : DNA. The ratio of
RNA : DNA; however, was increased (p < 04) in the
liver of lambs on the HL compared with the LL
groups, indicating a possible increase in liver cell size

Table 7
In¯uence of intake and lasalocid on intestinal and liver RNA, DNA, and protein concentrations (mg/g of tissue) and RNA : DNA and
protein : DNA ratios in growing wether lambs
Item

Intakea
LI

Lasalocidb
HI

c

P

LL

SE
HL

c

P

Intestine
RNA (mg/g)
DNA (mg/g)
Protein (mg/g)
RNA : DNA
Protein : DNA

3.43
6.23
58.7
0.61
10.7

3.50
5.92
59.6
0.69
12.0

0.68
0.58
0.98
0.16
0.23

3.40
6.21
59.4
0.62
11.0

3.53
5.94
58.9
0.69
11.6

0.52
0.56
0.65
0.20
0.50

0.13
0.67
2.56
0.05
0.79

Liver
RNA (mg/g)
DNA (mg/g)
Protein (mg/g)
RNA : DNA
Protein : DNA

4.51
3.38
167
1.36
51.6

4.41
2.79
155
1.62
57.5

0.71
0.06
0.24
0.02
0.29

4.28
3.21
162
1.38
53.0

4.64
2.97
161
1.61
56.2

0.20
0.44
0.94
0.04
0.56

0.20
0.22
7.15
0.07
3.90

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake and lasalocid.
b

244

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

Table 8
In¯uence of intake and lasalocid on visceral tissue RNA, DNA, and protein contents (mg) in growing wether lambs
Item

Intakea
LI

Lasalocidb
c

HI

P

LL

SE
HL

c

P

Duodenum
RNA
DNA
Protein

1.22
2.15
20.4

1.54
2.35
24.3

0.06
0.55
0.10

1.32
2.28
22.1

1.44
2.22
22.7

0.46
0.84
0.81

0.11
0.23
1.66

Jejunum
RNA
DNA
Protein

1.05
1.93
18.1

1.16
2.02
18.8

0.30
0.71
0.73

1.10
2.05
19.6

1.11
1.91
17.3

0.94
0.58
0.30

0.07
0.17
1.49

Ileum
RNA
DNA
Protein

1.23
3.03
20.8

1.28
3.44
22.3

0.72
0.33
0.57

1.24
3.41
22.0

1.26
3.07
21.1

0.86
0.43
0.70

0.09
0.31
2.99

Cecum
RNAd
DNA
Protein

0.16
0.25
2.72

0.18
0.23
3.31

0.22
0.63
0.14

0.18
0.27
3.03

0.16
0.21
3.01

0.19
0.06
0.97

0.01
0.02
0.28

Colon
RNA
DNA
Protein

1.21
1.66
22.4

1.35
1.63
26.0

0.35
0.92
0.38

1.27
1.64
24.6

1.29
1.64
23.8

0.90
0.99
0.86

0.11
0.21
2.87

Liver
RNA
DNA
Protein

2.98
2.24
110

4.30
2.70
151

0.01
0.13
0.01

3.40
2.53
126

3.87
2.41
134

0.25
0.70
0.55

0.29
0.21
9.26

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake and lasalocid.
d
Intakelasalocid interaction (p < 0.10).
b

in response to lasalocid. This data, in conjunction with
ileal wt changes in response to lasalocid (Table 6)
indicate that lasalocid may have slight, but measurable
effects on visceral organ mass and metabolism. However, these slight changes do not explain reported
effects of lasalocid on animal performance and feed
ef®ciency.
For jejunum, ileum, cecum and colon, intake did not
affect (p > 0.10) RNA, DNA or protein contents
(Table 8). For duodenum; however, the contents of
RNA was increased (p < 0.06) in lambs on the HI
compared with LI treatment. In addition, protein
content of the duodenum tended (p ˆ 0.10) to increase
in HI lambs whereas the DNA content was not in¯uenced (p > 0.10) by intake. Liver RNA and protein

contents also were increased (p < 0.01) in HI compared with LI lambs (Table 8). Liver DNA content,
however, tended (p ˆ 0.13) to increase with increased
intake. These data indicate that liver protein synthesis
and deposition were increased in lambs on HI. Lasalocid had no effects on RNA, DNA and protein contents of duodenum, jejunum, ileum, colon or liver.
However, for the cecum contents of DNA were
decreased (p < 0.06) in HL compared with LL treatments. Protein content in cecum was not affected
(p > 0.10) by lasalocid. Analysis of RNA content in
cecum showed an intakelasalocid interaction
(p < 0.10) wherein cecal RNA content was increased
(p < 0.10) in the HILL (0.21 mg) treatment compared
with the LILL (0.16 mg), LIHL (0.17 mg) and HIHL

245

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

Table 9
In¯uence of intake and lasalocid on proliferating cell nuclear antigen (PCNA) labeling in sections of intestinal tissues in growing wether lambs
Intakea

Item

2 d

PCNA-labeled area (mm )
Area of individual nuclei (mm2)
Number of PCNA-labeled nucleid

Lasalocidb
c

SE
c

LI

HI

P

LL

HL

P

1261
29.6
43.9

1460
29.9
50.4

0.17
0.75
0.30

1284
29.9
44.1

1437
29.6
50.1

0.35
0.82
0.38

98.7
1.06
4.13

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake and lasalocid.
d
Ten ®eld (26,376 mm2 each) were measured per tissue per lamb.
b

Table 10
In¯uence of intake and lasalocid on intestinal morphology in growing wether lambs
Item (mm)

Crypt depth
Villus length
Villus width

Intakea

Lasalocidb

SE

c

LL

HL

P

372
525
113

380
523
110

0.51
0.99
0.29

LI

HI

P

364
514
110

388
533
112

0.11
0.14
0.52

c

10.6
9.14
1.94

a

LI ˆ low intake (60% of ad libitum); HI ˆ high intake (95% of ad libitum).
LL ˆ low lasalocid (0 mg/hd daily); HL ˆ high lasalocid (40 mg/hd daily).
c
P equals observed signi®cance level for main effects of intake and lasalocid.
b

(0.15 mg) treatments. The intakelasalocid interaction for RNA content in cecum may simply re¯ect the
interaction observed for cecal tissue weight since the
RNA content is calculated by multiplying the tissue
RNA concentration and fresh tissue weight.
3.5. Cellular proliferation and morphometry
Proliferating cell nuclear antigen was immunolocalized in histological sections of intestinal segments
to determine if intake or lasalocid affected the rate of
intestinal cell proliferation. However, total area of
PCNA labeling per ®eld (26,376 mm2) was not
affected (p > 0.10) by intake or lasalocid (Table 9).
Area of individual crypt cell nuclei and number of
PCNA-labeled nuclei per ®eld (26,376 mm2) also were
not affected (p > 0.10) by intake or lasalocid. These
data indicate that relative rate of intestinal cell proliferation was not affected by intake or lasalocid.
There were also no differences (p > 0.10) in crypt
depth, villus length and villus width due to intake
or lasalocid (Table 10). However, there were tendencies for crypt depth (p ˆ 0.11) and villus length

(p ˆ 0.14) to be increased in HI compared with LI
treatments.
These data, in conjunction with other recent reports
(Swanson et al., 1999) indicate that changes in intestinal cellular proliferation and morphology can not
explain alterations in ef®ciency and production associated with changes in intake or lasalocid supplementation.

4. Conclusions
As expected intake level in¯uenced ruminal fermentation, serum hormones and metabolites and visceral mass. Production and ef®ciency changes usually
observed with changes in intake do not appear to be
explained by changes in intestinal cellular proliferation. Increases in growth hormone in response to
lasalocid were intake dependent. Changes in visceral
mass and growth were minimal in response to lasalocid and cannot explain changes in production and
ef®ciency often reported from ionophore supplementation.

246

K.C. Swanson et al. / Small Ruminant Research 35 (2000) 235±247

Acknowledgements
The authors thank the National Hormone and Pituitary Program (NIDDK and University of Maryland
School of Medicine) and also the USDA Animal
Hormone Program for the gift of reagents for the
growth hormone RIA. In addition, the authors would
like to express their appreciation to Tim Johnson and
Terry Skunberg for animal care, Ruth Weis, Marsha
Kapphahn, Jim Kirsch, and Kim Kraft for laboratory
assistance and Julie Berg for clerical assistance.

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