In¯uence of dietary intake and lasalocid on serum
hormones and metabolites and visceral organ growth
and morphology in wether lambs
K.C. Swanson
a, L.P. Reynolds
b, J.S. Caton
b,* aDepartment of Animal Science, University of Kentucky, Lexington, KY 40546, USA bDepartment of Animal and Range Sciences, North Dakota State University, Fargo, ND 58105, USAAccepted 30 June 1999
Abstract
Twenty-four black face, crossbred wether lambs (32.46.1 kg) were assigned to one of four treatments arranged in a 22 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
total body weight (wt) (Burrin et al., 1989; Reynolds and Tyrrell, 1989). However, the role of dietary factors in regulating visceral growth and develop-ment 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 *Corresponding author. 1-701-231-7653; fax:
1-701-231-7590.
E-mail address: caton@plains.nodak.edu (J.S. Caton).
and development at the cellular level, especially in ruminants.
Ionophores are used extensively in ruminant live-stock production. Generally, data suggest that iono-phores 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 fermen-tation (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 fermenta-tion, visceral organ weights, cell proliferafermenta-tion, intest-inal 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 (32.46.1 kg) were assigned to one of four treat-ments arranged in 22 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 mea-surements. 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, Ruther-ford, 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
then were centrifuged at 1560gfor 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 combi-nation 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 pro-cedure of Broderick and Kang (1980). Ruminal ¯uid was also analyzed for VFA concentrations using gas chromatography with 2-ethylbutyric acid as the inter-nal 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-I-4 (biopotency1.5 IU/mg) as the radioiodination preparation, USDA-bGH-B-1 (biopotency1.4 IU/
Table 1
Composition of diets fed to growing wether lambsa,b
Item Percentage of diet (DM basis) Alfalfa meal 10.1
aLasalocid (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.
bChemical analysis revealed 5.8% ash, 16.0% CP, 22.5% NDF,
and 10.7% ADF.
mg) as the reference standard, NIDDK-oGH-2 as the primary antiserum, and sheep-anti-rabbit gamma glo-bulin as the secondary antiserum.
To 1275 mm2 polypropylene tubes, 500ml of serum was diluted with 100ml of PBS (0.01 m phos-phate, 0.14 m NaCl, pH 7.3) plus 1% (wt/vol) BSA (RIA Grade; Sigma, St. Louis, MO). After this dilution step, 200ml of primary antiserum (1 : 20,000 in PBS) was added, and the tubes were incubated for 24 hours at 48C. Iodinated GH (100ml) was then added, and tubes were incubated for an additional 72 hours at 48C. After this 72 hours incubation, 100ml 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 1500g for 30 min, the liquid was decanted, and tubes were counted on a Beckman Gamma 5500 scintillation counter (Beck-man Instruments, Arlington Heights, Illinois).
All samples were run in a single assay and intra-assay variation was determined by intra-assaying replicates (n6) of a pool of lamb plasma in the same assay. Resulting mean SD concentration of growth hor-mone in the lamb plasma pool was 1.020.09 ng/ml (c.v.21.9%). To validate further the growth hor-mone assay, the pooled lamb plasma was assayed at volumes of 200, 300, 400, 500, 600, and 700ml, which yielded an inhibition curve that was parallel to that of the reference standard.
Blood serum was analyzed for insulin concentra-tions using a commercially available RIA kit (Coat-A-Count, 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 Diag-nostics, 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 cap-tive bolt, over a 4-day interval immediately following the second collection period. Final live BW was
determined and, after slaughter and evisceration, visc-eral 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 junc-tion (cranial third of small intestine): jejunum, duo-denal±jejunal junction to jejunal±ileal junction (middle third of small intestine): and ileum, jeju-nal±ileal junction to ileal±cecal junction (caudal third of small intestine). In addition, the cecum was sepa-rated 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, respec-tively. 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 cm3samples for liver and 1 cm wide cross-sections of each intest-inal 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 (5mm) 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
After intestinal (duodenum, jejunum, ileum, cecum and colon) tissue samples were obtained, the remain-der of each segment was gently stripped by hand of digesta and then fresh empty weights were deter-mined. 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 lyophi-lized 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 homoge-nized 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 orci-nol 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 albu-min (Fraction V, Sigma Chemical) as the standard. Tissue DNA, RNA and protein contents were calcu-lated 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 replica-tion 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 (26,376mm2) per tissue for each lamb. Area of indi-vidual 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 (Ar1r2). 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 mor-phometry 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 intes-tinal 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
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 man-ner. 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 indi-vidual tissue weight were subjected to split±plot ana-lysis (Gill and Hafs, 1971). The model for intestinal RNA, DNA and protein concentrations, morphometry and PCNA labeling included effects of intake, lasa-locid, intake by lasalasa-locid, 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 con-centrations 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 Intakea Lasalocidb SE
LI HI Pc LL HL Pc
Insulin (mU/ml) 8.36 29.9 0.02 19.8 18.5 0.78 3.45
Glucose (mg/dl) 79.4 98.4 0.01 91.8 86.0 0.25 3.56 Urea N (mg/dl) 20.4 18.6 0.17 18.5 20.5 0.13 0.92
lambs and (or) an uncoupling of hormonal mechan-isms 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 (p0.10) 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 (p0.10) 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 pro-portion 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 visc-eral 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 (p0.10) to decrease with increased intake. However, lasalocid did not in¯uence (p> 0.10) ®nal lamb BW, eviscer-ated 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 (p0.10) 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¯u-enced (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
Table 3
In¯uence of intake and lasalocid on ruminal pH, ammonia, total VFA concentrations, and VFA proportions in growing wether lambs
Intakea Lasalocidb SE
Item LI HI Pc LL HL Pc
Ruminal pH 6.77 5.82 0.01 6.41 6.17 0.25 0.15 Ammonia (mg/dl) 20.2 17.3 0.59 17.8 19.6 0.73 3.75 Total VFA (mM) 41.9 98.7 0.01 64.9 75.7 0.50 11.45 Acetate : propionate 1.85 1.57 0.05 1.78 1.64 0.32 0.10 Mol/100 mol
Acetate 48.5 44.8 0.10 48.5 44.8 0.1 1.5
Propionate 26.7 29.5 0.19 27.7 28.4 0.8 1.5
Butyrate 8.5 11.3 0.20 9.0 10.8 0.4 1.5
aLIlow intake (60% of ad libitum); HIhigh intake (95% of ad libitum). bLLlow lasalocid (0 mg/hd daily); HLhigh lasalocid (40 mg/hd daily). cPequals observed signi®cance level for main effects of intake or lasalocid.
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 inter-mediate (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 investiga-tors 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¯u-ence (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 treat-ments (Table 5). The ratio of dry wt : fresh wt, for
liver, was not in¯uenced (p> 0.10) by intake. Con-sistent with our observations, other investigators have also shown increases in liver fresh weights in rumi-nants 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, jeju-num, 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 duode-num and jejuduode-num 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 4
In¯uence of intake and lasalocid on ®nal BW and total visceral weights in growing wether lambs
Item Intakea Lasalocidb SE
LI HI Pc LL HL Pc
BW (kg) 38.1 47.0 0.01 42.8 42.3 0.87 1.83 Eviscerated BW (kg) 25.2 31.9 0.01 28.7 28.4 0.90 1.35 Total visceral wt (kg)d 13.0 15.1 0.02 14.1 13.9 0.84 0.17
Total visceral wt: eviscerated BW 0.52 0.48 0.10 0.50 0.49 0.64 0.062
Table 5
In¯uence of intake and lasalocid on visceral tissue weights in growing wether lambs
Item Intakea Lasalocidb SE
LI HI Pc LL HL Pc
Duodenum
Fresh wt (g) 313 340 0.30 330 323 0.77 17.9 Dry wt (g) 57.4 64.5 0.12 61.1 60.7 0.93 3.16 Dry fat-free wt (g) 48.2 52.4 0.29 50.6 50.1 0.91 2.77 Fresh wt : EBW (g/kg) 12.7 10.9 0.18 11.9 11.7 0.89 0.95 Dry wt : EBW (g/kg) 2.33 2.05 0.24 2.19 2.19 0.99 0.14 Dry fat-free wt : EBW (g/kg) 1.96 1.67 0.17 1.82 1.82 0.99 0.14 Dry wt : fresh wt 0.183 0.190 0.19 0.185 0.188 0.55 0.003 Jejunum
Fresh wt (g) 299 307 0.77 308 298 0.67 18.0 Dry wt (g) 55.1 61.1 0.21 58.3 57.8 0.91 3.40 Dry fat-free wt (g) 44.9 47.2 0.57 47.2 44.8 0.56 2.91 Fresh wt : EBW (g/kg) 12.17 9.87 0.10 11.2 10.8 0.78 0.95 Dry wt : EBW (g/kg) 2.24 1.95 0.24 2.11 2.08 0.89 0.17 Dry fat-free wt : EBW (g/kg) 1.82 1.52 0.15 1.71 1.63 0.70 0.15 Dry wt : fresh wt 0.184 0.201 0.03 0.189 0.196 0.38 0.005 Ileum
Fresh wt (g) 364 394 0.27 388 370 0.52 19.3 Dry wt (g) 68.3 79.6 0.04 75.3 72.6 0.62 3.80 Dry fat-free wt (g) 52.7 57.1 0.27 55.9 53.9 0.62 2.81 Fresh wt : EBW (g/kg) 14.7 12.6 0.13 13.9 13.4 0.67 0.93 Dry wt : EBW (g/kg) 2.74 2.52 0.30 2.67 2.59 0.72 0.15 Dry fat-free wt : EBW (g/kg) 2.12 1.82 0.11 1.99 1.94 0.78 0.13 Dry wt : fresh wt 0.188 0.203 0.7 0.193 0.197 0.71 0.006 Cecum
Fresh wt (g) 49.9 59.8 0.02 54.7 55.0 0.94 2.77 Dry wt (g) 8.35 10.49 0.01 9.19 9.66 0.54 0.55 Dry fat-free wt (g) 6.49 7.89 0.02 7.15 7.23 0.89 0.41 Fresh wt : EBW (g/kg) 2.01 1.91 0.54 1.94 1.99 0.78 0.12 Dry wt : EBW (g/kg) 0.34 0.33 0.87 0.32 0.34 0.46 0.02 Dry fat-free wt : EBW (g/kg) 0.26 0.25 0.64 0.25 0.26 0.71 0.02 Dry wt : fresh wt 0.167 0.175 0.36 0.168 0.175 0.38 0.006 Colon
Fresh wt (g) 399 476 0.04 433 443 0.78 25.6
Dry wt (g) 121 156 0.03 134 143 0.53 10.8
Dry fat-free 40.8 47.2 0.09 43.7 44.4 0.85 2.55 Fresh wt : EBW (g/kg) 16.1 15.2 0.55 15.4 15.9 0.76 1.16 Dry wt : EBW (g/kg) 4.88 4.89 0.98 4.73 5.03 0.56 0.36 Dry fat-free wt : EBW (g/kg) 1.64 1.51 0.37 1.55 1.59 0.74 0.10 Dry wt : fresh wt 0.302 0.328 0.24 0.308 0.321 0.54 0.015 Liver
Fresh wt (g) 658 969 0.01 795 832 0.55 43.6
Dry wt (g) 201 295 0.01 245 252 0.73 13.9
Dry fat-free wt (g) 171 242 0.01 204 209 0.75 11.3 Fresh wt : EBW (g/kg) 26.5 30.3 0.01 27.7 29.1 0.33 1.01 Dry wt : EBW (g/kg) 8.10 9.22 0.02 8.52 8.80 0.54 0.32 Dry fat-free wt : EBW (g/kg) 6.89 7.58 0.08 7.137 0.34 0.57 0.27 Dry wt : fresh wt 0.31 0.30 0.62 0.31 0.30 0.17 0.01
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 concen-tration 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 con-centrations 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 6
In¯uence of intake and lasalocid on ileum and cecum weights in growing wether lambs (simple effect means)a
Item LI HI SE Pb
LL HL LL HL
Ileum
Fresh (g) 348c 380c,d 428d 361c 28.53 0.08
Dry (g) 63.8c 72.2c 86.8d 72.4c 5.62 0.04
Dry fat-free (g) 49.1c 56.2c,d 62.6d 51.6c 4.15 0.03
Cecum
Fresh (g) 46.1c 53.8c,d 63.3c 56.2d,e 4.10 0.07 Dry fat-free (g) 5.89c 7.09c,d 8.41d 7.37d 0.60 0.06
aLIlow intake (60% of ad libitum); HIhigh intake (95% ad libitum); LLlow lasalocid (0 mg/hd daily); HLhigh lasalocid
(40 mg/hd daily).
bPequals observed signi®cance level for intakelasalocid interaction. cMeans within a row that do not have a common superscript differ (p< 0.10). dMeans within a row that do not have a common superscript differ (p< 0.10). eMeans within a row that do not have a common superscript differ (p< 0.10).
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 Lasalocidb SE
LI HI Pc LL HL Pc
Intestine
RNA (mg/g) 3.43 3.50 0.68 3.40 3.53 0.52 0.13 DNA (mg/g) 6.23 5.92 0.58 6.21 5.94 0.56 0.67 Protein (mg/g) 58.7 59.6 0.98 59.4 58.9 0.65 2.56 RNA : DNA 0.61 0.69 0.16 0.62 0.69 0.20 0.05 Protein : DNA 10.7 12.0 0.23 11.0 11.6 0.50 0.79 Liver
RNA (mg/g) 4.51 4.41 0.71 4.28 4.64 0.20 0.20 DNA (mg/g) 3.38 2.79 0.06 3.21 2.97 0.44 0.22 Protein (mg/g) 167 155 0.24 162 161 0.94 7.15 RNA : DNA 1.36 1.62 0.02 1.38 1.61 0.04 0.07 Protein : DNA 51.6 57.5 0.29 53.0 56.2 0.56 3.90
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. How-ever, 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 (p0.10) to increase in HI lambs whereas the DNA content was not in¯u-enced (p> 0.10) by intake. Liver RNA and protein
contents also were increased (p< 0.01) in HI com-pared with LI lambs (Table 8). Liver DNA content, however, tended (p0.13) to increase with increased intake. These data indicate that liver protein synthesis and deposition were increased in lambs on HI. Lasa-locid had no effects on RNA, DNA and protein con-tents of duodenum, jejunum, ileum, colon or liver. However, for the cecum contents of DNA were decreased (p< 0.06) in HL compared with LL treat-ments. 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
Table 8
In¯uence of intake and lasalocid on visceral tissue RNA, DNA, and protein contents (mg) in growing wether lambs
Item Intakea Lasalocidb SE
LI HI Pc LL HL Pc
Duodenum
RNA 1.22 1.54 0.06 1.32 1.44 0.46 0.11
DNA 2.15 2.35 0.55 2.28 2.22 0.84 0.23
Protein 20.4 24.3 0.10 22.1 22.7 0.81 1.66
Jejunum
RNA 1.05 1.16 0.30 1.10 1.11 0.94 0.07
DNA 1.93 2.02 0.71 2.05 1.91 0.58 0.17
Protein 18.1 18.8 0.73 19.6 17.3 0.30 1.49
Ileum
RNA 1.23 1.28 0.72 1.24 1.26 0.86 0.09
DNA 3.03 3.44 0.33 3.41 3.07 0.43 0.31
Protein 20.8 22.3 0.57 22.0 21.1 0.70 2.99
Cecum
RNAd 0.16 0.18 0.22 0.18 0.16 0.19 0.01
DNA 0.25 0.23 0.63 0.27 0.21 0.06 0.02
Protein 2.72 3.31 0.14 3.03 3.01 0.97 0.28
Colon
RNA 1.21 1.35 0.35 1.27 1.29 0.90 0.11
DNA 1.66 1.63 0.92 1.64 1.64 0.99 0.21
Protein 22.4 26.0 0.38 24.6 23.8 0.86 2.87
Liver
RNA 2.98 4.30 0.01 3.40 3.87 0.25 0.29
DNA 2.24 2.70 0.13 2.53 2.41 0.70 0.21
Protein 110 151 0.01 126 134 0.55 9.26
aLIlow intake (60% of ad libitum); HIhigh intake (95% of ad libitum). bLLlow lasalocid (0 mg/hd daily); HLhigh lasalocid (40 mg/hd daily). cPequals observed signi®cance level for main effects of intake and lasalocid. dIntakelasalocid interaction (p< 0.10).
(0.15 mg) treatments. The intakelasalocid interac-tion 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 immunolo-calized 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 (26,376mm2) 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 (26,376mm2) also were not affected (p> 0.10) by intake or lasalocid. These data indicate that relative rate of intestinal cell pro-liferation 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 tenden-cies for crypt depth (p0.11) and villus length
(p0.14) 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 intest-inal cellular proliferation and morphology can not explain alterations in ef®ciency and production asso-ciated with changes in intake or lasalocid supplemen-tation.
4. Conclusions
As expected intake level in¯uenced ruminal fer-mentation, serum hormones and metabolites and visc-eral mass. Production and ef®ciency changes usually observed with changes in intake do not appear to be explained by changes in intestinal cellular prolifera-tion. Increases in growth hormone in response to lasalocid were intake dependent. Changes in visceral mass and growth were minimal in response to lasa-locid and cannot explain changes in production and ef®ciency often reported from ionophore supplemen-tation.
Table 9
In¯uence of intake and lasalocid on proliferating cell nuclear antigen (PCNA) labeling in sections of intestinal tissues in growing wether lambs
Item Intakea Lasalocidb SE
LI HI Pc LL HL Pc
PCNA-labeled area (mm2)d 1261 1460 0.17 1284 1437 0.35 98.7
Area of individual nuclei (mm2) 29.6 29.9 0.75 29.9 29.6 0.82 1.06
Number of PCNA-labeled nucleid 43.9 50.4 0.30 44.1 50.1 0.38 4.13 aLIlow intake (60% of ad libitum); HIhigh intake (95% of ad libitum).
bLLlow lasalocid (0 mg/hd daily); HLhigh lasalocid (40 mg/hd daily). cPequals observed signi®cance level for main effects of intake and lasalocid. dTen ®eld (26,376
mm2each) were measured per tissue per lamb.
Table 10
In¯uence of intake and lasalocid on intestinal morphology in growing wether lambs
Item (mm) Intakea Lasalocidb SE
LI HI Pc LL HL Pc
Crypt depth 364 388 0.11 372 380 0.51 10.6
Villus length 514 533 0.14 525 523 0.99 9.14 Villus width 110 112 0.52 113 110 0.29 1.94
Acknowledgements
The authors thank the National Hormone and Pitui-tary 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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