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. 2020 Dec 1;98(11):skaa341. doi: 10.1093/jas/skaa341

Ractopamine-induced fiber type-specific gene expression in porcine skeletal muscles is independent of growth

Andrea M Gunawan 1, Con-Ning Yen 2, Brian T Richert 1, Allan P Schinckel 1, Alan L Grant 2, David E Gerrard 2,✉
PMCID: PMC7706407  PMID: 33259597

Abstract

Feeding ractopamine (RAC), a β-adrenergic agonist (BAA), to pigs increases type IIB muscle fiber type-specific protein and mRNA expression. However, increases in the abundance of these fast-twitch fiber types occur with other forms of muscle hypertrophy and thus BAA-induced changes in myosin heavy chain (MyHC) composition may simply be associated with increased muscle growth known to occur in response to BAA feeding. The objective of this study was to determine whether RAC feeding could change the MyHC gene expression in the absence of maximal muscle growth. Pigs were fed either an adequate diet that supported maximal muscle hypertrophy or a low nutrient diet that limited muscle growth. RAC was included in diets at 0 or 20 mg/kg for 1, 2, or 4 wk. Backfat depth was less (P < 0.05) in pigs fed the low nutrient diet compared with the adequate diet but was not affected by RAC. Loin eye area was greater (P < 0.05) in pigs fed an adequate diet plus RAC at 1 wk but did not differ among remaining pigs. At 2 and 4 wk, however, pigs fed the adequate diet had greater loin eye areas (P < 0.05) than pigs fed the low nutrient diet regardless of RAC feeding. Gene expression of the MyHC isoforms, I, IIA, IIX, and IIB, as well as glycogen synthase, citrate synthase, β 1-adrenergic receptor (AR), and β 2-AR were determined in longissimus dorsi (LD) and red (RST) and white (WST) portions of the semitendinosus muscles. MyHC type I gene expression was not altered by RAC or diet. Feeding RAC decreased (P < 0.01) MyHC type IIA gene expression in all muscles, but to a greater extent in WST and LD. MyHC type IIX gene expression was lower (P < 0.05) in WST and LD muscles in response to RAC but was not altered in RST muscles. RAC increased (P < 0.05) MyHC type IIB gene expression in all muscles, but to a greater extent in RST. β 1-AR gene expression was unaffected by RAC or diet, whereas the expression of the β 2-AR gene was decreased (P < 0.001) by RAC. No significant RAC * diet interactions were observed in gene expression in this study, indicating that RAC altered MyHC and β 2-AR gene expression in porcine skeletal muscles independent of growth.

Keywords: beta-adrenergic agonist, fiber type, myosin heavy chain, pig, ractopamine, skeletal muscle

Introduction

Four adult myosin heavy chain (MyHC) isoforms, slow type I and fast types IIA, IIX, and IIB, are expressed in mature pig longissimus dorsi (LD) muscle and reflect the fiber-type composition of muscle (Lefaucheur et al., 1998; Gunawan et al., 2007a). Muscle growth occurs as a result of hypertrophy of existing muscle fibers and not from increasing in muscle fiber number (Ashmore et al., 1972; Lefaucheur and Vigneron, 1986). The bulk of muscle hypertrophy occurs due to an increase in the size of glycolytic fibers that predominate in more superficially located muscles (Cassens and Cooper, 1971; Rosser et al., 1992) suggesting that increases in the proportion of fast-twitch fibers in the muscle lead to greater postnatal lean growth potential. This idea is supported by Karlsson et al. (1993) who reported that selection for increased muscle growth within domesticated pigs resulted in an increased proportion of IIB fibers. However, this does not eliminate the possibility that animals experiencing greater muscle growth postnatal simply develop more muscle with greater proportions of fast-contracting muscle fibers.

The addition of ractopamine (RAC), the commercially available β-adrenergic agonist (BAA) PAYLEAN (Elanco Animal Health, Greenfield, IN), into finishing pig diets increases muscle deposition (Mersmann et al., 1987; Merkel, 1988; Watkins et al., 1988; Warriss et al., 1990a, 1990b; Crome et al., 1996). When feeding RAC, the resulting muscle hypertrophy and improvement of carcass composition are greatly impacted by the nutrition and lysine level in the diet(Adeola et al., 1990; Mitchell et al., 1991; Xiao et al., 1999; Schinckel et al., 2003; Apple et al., 2004; Carr et al., 2005). Studies have shown that the effects of RAC are most effective with at least 16% crude protein (Jones et al., 1987) and 1% to 1.2% lysine (Herr et al., 2001). Any nutrient levels less than the recommended ranges will blunt the effect of RAC; therefore, we utilized different diets in this study to determine the effects of RAC with and without normal muscle growth. An adequate diet was formulated to meet the nutritional needs for optimal muscle growth, whereas the low nutrient diet was formulated to contain deficient levels of lysine and other essential amino acids required for normal muscle growth.

Consistent with the aforementioned hypothesis, skeletal muscle of pigs fed RAC becomes more glycolytic (Eisemann et al., 1987; Vestergaard et al., 1994) due to an increased presence of type IIB fibers (Oksbjerg et al., 1990, 1994; Aalhus et al., 1992; Depreux et al., 2002; Paulk et al., 2014). In particular, we have shown that type IIA and IIX MyHC expression reduced in pigs fed RAC, while the fastest MyHC isoform is upregulated within 12 h of BAA feeding (Gunawan et al., 2007b). Again, however, these effects of RAC on fiber type-specific gene expression are confounded with the fact that treated animals are experiencing exaggerated muscle growth. Therefore, the objective of this study was to determine if RAC differentially alters muscle fiber type-specific gene expression in porcine skeletal muscles during periods of suboptimal muscle growth.

Materials and Methods

Animals

This study was performed as a randomized complete block design with a 2 × 2 × 3 factorial arrangement. Two diets (adequate and low nutrient) and two levels of RAC-HCL (RAC; Paylean, Elanco Animal Health, Greenfield, IN; 0 or 20 mg/kg) were evaluated at three times (1, 2, or 4 wk). Diet compositions are presented in Table 1. Ninety-six crossbred pigs (Duroc × [York × Landrace]; barrows and gilts) weighed approximately 90 kg were blocked into four groups. Four pens were randomly assigned within each block to one of four dietary treatments: adequate, adequate plus 20 mg/kg RAC, low nutrient, or low nutrient plus 20 mg/kg RAC for a total of 16 pens. Each pen contained six pigs (three barrows and three gilts), and there were four pen replicates per treatment. Pigs were subjected to a 4-wk growth period in which RAC treatments of 1, 2, or 4 wk were supplemented at different start times. As such, all pigs were harvested on the same day after a 4-wk growth period, as previously described in Gunawan et al. (2007b). The Purdue Animal Care and Use Committee approved all procedures for the care and use of pigs.

Table 1.

Composition of adequate and low nutrient diets

Adequate Low Nutrient
Ingredient, %
 Corn 67.38 48.42
 Soybean meal (48% CP) 24.53 0.00
 Soybean hulls 0.00 19.00
 Dehydrated alfalfa (17% CP) 0.00 15.00
 Choice white grease 5.00 1.00
 Limestone 0.84 0.00
 Dicalcium phosphate 1.04 1.45
 Vitamin premix1 0.15 0.15
 Trace mineral premix2 0.10 0.10
 Phytase3 0.08 0.08
 Salt 0.25 0.25
 Corn gluten meal (60% CP) 0.00 14.50
 Lysine-HCL 0.30 0.00
 dl-Methionine 0.11 0.00
 l-Threonine 0.16 0.00
 l-Tryptophan 0.01 0.00
 Selenium premix4 0.05 0.05
Total 100 100
Calculated nutrients
 Metabolizable energy, Kcal/kg 3,531 2,894
 Net energy, Kcal/kg 2,691 2,046
 Crude protein (CP), % 17.49 17.50
 Total lysine, % 1.15 0.50
 SID lysine, % 1.00 0.35
 SID methionine, % 0.35 0.30
 SID methionine plus cysteine, % 0.60 0.53
 SID threonine, % 0.69 0.46
 SID tryptophan, % 0.19 0.09
 SID isoleucine, % 0.62 0.53
 SID valine, % 0.69 0.62
 Calcium, % 0.65 0.65
 Phosphorus, % 0.55 0.54
 Phytase-available phosphorus, % 0.35 0.39
 Fat, % 8.16 3.82
 Crude fiber, % 2.38 11.26
 Acid detergent fiber, % 3.24 13.60
 Neutral detergent fiber, % 8.15 21.31
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1Provided per kilogram of the diet: vitamin A, 3,969 IU; vitamin D3, 397 IU; vitamin E, 26.5 IU; vitamin K, 1.3 mg; riboflavin, 5.3 mg; pantothenic acid, 13.2 mg; niacin, 19.8 mg; B12, 23.2 mg.

2Provided available minerals per kilogram of the diet: iron, 97 mg; zinc, 97 mg; manganese, 12.0 mg; copper, 9.0 mg; iodine, 0.37 mg.

3Provided 0.3 ppm Se.

4Provided 480 FTU of phytase per kg of the diet (Phyzyme, Danisco Animal Nutrition/DuPont, St. Louis, MO).

The adequate diet was formulated to contain 17.5% crude protein (CP) and 1.00% standardized ileal digestible (SID) lysine, while all other amino acids were balanced on a modified NRC (1998) implied ratio basis. The adequate diet was designed to support optimal muscle growth. The low nutrient diet was formulated to provide 17.5% crude protein, 0.35% SID lysine, and approximately 80% of the level of other amino acids included in the adequate diet. Furthermore, the low nutrient diet contained 80% of the energy of a typical corn–soybean meal diet at ad libitum dietary intake. The fiber sources used in the low nutrient diet were selected due to their broad availability as a feed ingredient and readily fermentable neutral detergent fiber (NDF) content with minimal palatability issues for swine at these inclusion levels. Thus, the low nutrient diet was designed to maintain body weight and limit muscle growth during the finishing phase of the pigs. Diets and water were provided ad libitum. In order to calculate performance, pigs and feeders were weighted every 7 d during the study for average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency.

Meat quality data collection

Meat quality and carcass composition data were collected at 24 h postmortem. Loin eye area (LEA), fat thickness over the 10th rib, and subjective color, marbling (NPPC, 2000), and firmness scores (NPPC, 1991) were evaluated on the cut surface of the LD muscle between the 10th and 11th ribs (NPPC, 1991; 2000). Two 2.5-cm loin chops were collected caudal to the 11th rib to determine objective color scores and water-holding capacities using the drip loss method (Rasmussen and Stouffer, 1996). Objective color measurements were determined using a Hunter Lab 45°/0° D25-PC2Δ Colorimeter (Hunter and Associates Laboratory Inc., Reston, VA, USA) with a 64-mm diameter port size. Mean CIE-L* (lightness), a* (redness), and b* (yellowness) values were collected from three separate locations on the surface of each chop.

Muscle sampling

Pigs (average weight 100 ± 15 kg) were processed at the Purdue Meat Science Research laboratory according to normal industry procedures. Semitendinosus (ST) and LD muscles were removed immediately post-exsanguination (within 5 min). Furthermore, the ST muscle was visually categorized and separated into red (RST) and white semitendinosus (WST). Samples were frozen in liquid nitrogen and stored at −80 °C.

Total RNA preparation

Total RNA was extracted from porcine skeletal muscle using the single-step method (Chomczynski and Sacchi, 1987) with modifications from the study of Gunawan et al. (2007b). Briefly, 100 mg of skeletal muscle powdered in liquid nitrogen was placed into 3 mL of solution D containing 4 M guanidium isothiocyanate (Sigma, St. Louis, MO), 25 mM sodium citrate (pH 7), 0.5 % sarcosyl, and 0.1 M 2-mercaptoethanol, in a 50-mL conical tube held on ice. Tissue was homogenized using a Polytron Homogenizer (Brinkman Instruments, New York, NY) at speed 6, and then transferred to a fresh 15-mL conical tube. Next, 300 μL of 2 M sodium acetate (pH 4) was added and vortexed. Protein and lipids in the preparation were extracted by sequentially adding 3 mL phenol saturated with diethyl pyrocarbonate-treated water (pH 4.3) and 600 μL chloroform-isoamyl alcohol (40:1 v/v). After thorough vortexing, tubes were placed on ice for 15 min. Samples were centrifuged at 10,000 × g at 4 °C for 20 min to separate aqueous and organic phases. The aqueous phase (upper) was carefully transferred to a fresh 15-mL conical tube taking care to avoid disturbing the interphase. Precipitation of the RNA was facilitated by adding 3 mL isopropanol and holding samples at −20 °C for 1 h. Precipitated RNA was then collected by centrifugation at 10,000 × g at 4 °C for 30 min. Pellets were then redissolved in 0.9 mL solution D, and then 0.9 mL isopropanol was added and RNA was allowed to precipitate at −20 °C for 1 h. Precipitated RNA was then collected by centrifugation at 21,000 × g at 4 °C for 30 min. Isopropanol was discarded, and the pellet was washed twice with 1 mL 75% ethanol (v:v). Pellets were air-dried for 10 min and resuspended in 50 μL of TE-8 containing 1 M tris-HCl (pH 8), 0.2 M ethylenediaminetetraacetic acid (EDTA) (pH 8), and 0.1 M diethyl pyrocarbonate-treated water.

RNA quantification

Total RNA isolated from skeletal muscle was measured for RNA quality using the RiboGreen quantification kit (Molecular Probes, Eugene, OR). RNA was first treated with 4 U recombinant DNAse (Ambion, Indianapolis, IN) in 2 μL of a 10× DNase buffer containing 100 mM Tris-HCl, pH 7.5, 25 mM MgCl2, 5 mM CaCl2, and 3 μL nuclease-free water (Ambion, Indianapolis, IN) and digested for 30 min at 37 °C. Five microliters DNase Inactivation Reagent (Ambion, Indianapolis, IN), a resin that binds DNase, were added to the reaction and mixed by flicking the tubes. RNA was then loaded into 0.22-μm Spin-X Centrifuge Tube Filters (VWR International, West Chester, PA) and centrifuged at 10,000 × g for 1 min.

A 1-μL sample of purified RNA was diluted to 250 μL with 200 mM Tris-HCl and 20 mM EDTA, pH 7.5 (TE-buffer; Ambion, Indianapolis, IN), and 25 μL of diluted RNA was mixed with 75 μL TE-buffer and 100 μL RiboGreen reagent, diluted 2,000-fold in TE-buffer. Excitation was at 480 nm, and emission (520 nm) was measured using a GENios Pro Fluorometer (Tecan, Durham, NC).

cDNA synthesis

RNA was reverse transcribed to cDNA. First, cDNA master mix was prepared in 5 U Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), 0.5 U SUPERase-In (Ambion, Indianapolis, IN), 10 mM dithiothreitol (DTT) (Invitrogen, Carlsbad, CA), and 5X First-strand buffer (Invitrogen, Carlsbad, CA). Total RNA (0.5 μg) in 5 μL nuclease-free water was added to 100 ng/μL random hexamers and 100 μM of each dNTP. Samples were denatured at 65 °C for 5 min and then chilled to 4 °C on ice. Four microliters of cDNA master mix were added to denatured RNA and sequentially incubated at 25 °C for 10 min, 37 °C for 50 min, 70 °C for 10 min, and chilled to 4 °C on ice. Finally, cDNA samples were diluted with nuclease-free water and aliquoted so that gene quantification was based on 50 ng of total RNA per 5 μL.

Real-time PCR

Porcine-specific primer sequences for adult MyHC isoform types I, IIA, IIX, and IIB, glycogen synthase (GS), citrate synthase (CS), β 1- and β 2-adrenergic receptors (ARs), and β-actin have been described previously (Gunawan et al., 2007a). Quantification standards were composed of aliquots of PCR products in 10-fold serial dilutions ranging from 109 to 101 molecules. Standards were amplified in triplicate and used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each cDNA aliquot, amplified in duplicate, based on individual threshold cycle. PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA) according to optimized gene-specific protocols (Gunawan et al., 2007b). All genes of interest expression were normalized to β-actin and displayed as log starting abundance in the figures, as previously described in Gunawan et al. (2007b).

Statistical analysis

Data were analyzed as a 2 × 2 × 3 factorial arrangement with pen as the experimental unit. The main effects of this statistical model include diet (adequate and low nutrient), RAC (0 or 20 ppm), and time (1, 2, and 4 wk). There was no sex effect; therefore, the data were pooled together for the main effects. Meat quality and carcass composition data were analyzed using the general linear model (GLM) procedure of SAS. Gene expression data were analyzed using the GLM procedure of SAS with the log value of starting abundance for each gene as the independent variable. Gene expression data also included the main effect of muscle (WST, RST, and LD). Data were reported as least squares means, and an analysis of variance was used to determine the differences among those means. Student–Newman–Keuls procedure of SAS was used to separate the means when a significant F-test (P < 0.05) was observed.

Results

Performance

The effects of dietary RAC and diet on growth performance and pork quality over time are shown in Table 2. Moreover, the effects of RAC and diet on ADG, ADFI, and feed efficiency are reported by week in Table 3. Slaughter weight was not different at the end of 1 wk. However, at 2 and 4 wk, pigs fed RAC included in the adequate diet weighed more (P < 0.05) when harvested than those fed RAC in the low nutrient diet. Furthermore, the inclusion of RAC in the low nutrient diet numerically reduced the slaughter weight of pigs in comparison to those fed the low nutrient diet without RAC.

Table 2.

Effects of dietary RAC (R) and diet (D) on growth performance and pork quality1 over time (T)

Diet Adequate Low nutrient
RAC2 0 ppm 20 ppm 0 ppm 20 ppm
Time, wk 1 2 4 1 2 4 1 2 4 1 2 4
Slaughter weight, kg 96.19cd 98.94bcd 111.98ab 96.90bcd 104.75bc 118.79a 90.95d 96.33cd 105.43b 90.72d 91.32d 98.91bcd
ADG, kg/d 0.85bc 0.61cd 0.84bc 0.94ab 1.10a 0.92ab 0.09e 0.46d 0.51d 0.08e 0.10e 0.35d
ADFI, kg/d 1.92f 2.11ef 2.61bcd 2.07ef 2.41cde 2.75bc 1.39g 2.25def 2.96b 0.98g 1.98ef 3.54a
Gain:feed 0.46a 0.29abc 0.32ab 0.45a 0.46a 0.34ab 0.03d 0.21bcd 0.16bcd 0.01d 0.05d 0.08cd
Carcass weight, kg 68.6bcd 70.78bc 83.24a 68.85bcd 75.00 b 85.86a 63.51 d 66.30cd 73.32b 64.50cd 63.96 d 69.41bcd
Dressing, % 71.29bc 71.60bc 74.51 70.97 72.00bc 72.26b 69.86cd 68.80d 69.48cd 71.04bc 70.02cd 70.17cd
Backfat, cm 1.67b 1.937ab 2.06ab 1.79b 2.00ab 2.18a 1.21d 1.46bcd 1.73b 1.22d 1.42bcd 1.66bc
LEA, cm2 36.17de 40.93bc 46.49a 40.81bc 42.26b 49.19a 33.19e 36.73de 38.31cd 34.92de 38.71bcd 38.79bcd
pH, 24 h 5.64 5.55 5.57 5.67 5.52 5.59 5.69 5.56 5.63 5.62 5.58 5.64
L* 3 — 55.22 54.47 — 56.95 55.76 — 56.2 55.43 — 56.06 55.69
a* 3 — 8.72ab 9.32a — 7.14c 8.12b — 8.63ab 9.29a — 7.75bc 8.78ab
b* 3 — 13.5 13.93 — 13.23 13.22 — 13.85 13.72 — 13.06 13.84
Color4 2.81bc 3.21a 2.75bc 2.88abc 2.81bc 2.50c 2.81bc 3.13ab 2.56c 2.81bc 3.00ab 2.63bc
Marbling4 2.22a 1.58bc 1.13c 2.19ab 1.81ab 1.31bc 2.00ab 1.91ab 1.47b 1.63abc 1.88ab 1.41bc
Firmness4 2.16abc 1.69c 2.31ab 2.03bc 2.31ab 2.69a 2.06bc 2.16abc 2.41ab 2.41ab 2.47ab 2.63a
Drip loss, % 1.81cd 2.48cd 4.47a 1.81cd 2.64cd 4.10ab 1.80cd 2.22cd 2.89bc 1.53d 2.12cd 2.24cd
P-value
SEM D R T D × T R × T D × R D × R × T
Initial weight, kg 1.53 0.91 0.89 — — — 0.74 —
Slaughter weight, kg 1.91 <0.0001 0.89 <0.0001 0.26 0.99 0.03 0.38
ADG, kg/d 0.1 <0.0001 0.74 0.09 0.06 0.76 0.001 0.03
ADFI, kg/d 0.39 0.04 0.59 <0.0001 <0.0001 0.24 0.18 0.1
Gain:feed 0.07 <0.0001 0.79 0.84 0.09 0.91 0.09 0.32
Carcass weight, kg 1.44 <0.0001 0.83 <0.0001 0.05 0.89 0.15 0.49
Dressing, % 0.01 <0.0001 0.85 0.09 0.02 0.36 0.02 0.68
Backfat, cm 0.08 <0.0001 0.7 0.0003 0.87 0.96 0.47 0.96
LEA, cm2 1.46 <0.0001 0.01 <0.0001 0.02 0.68 0.38 0.66
pH, 24 h 0.05 0.26 0.8 0.01 0.74 0.9 0.59 0.53
L* 3 0.55 0.65 0.15 0.16 0.72 0.98 0.19 0.7
a* 3 0.26 0.27 0.0002 0.002 0.91 0.46 0.18 0.98
b* 3 0.21 0.47 0.05 0.2 0.78 0.57 0.71 0.11
Color4 0.14 0.95 0.18 0.0002 0.89 0.34 0.28 0.58
Marbling4 0.24 0.95 0.92 0.0004 0.13 0.61 0.29 0.98
Firmness4 0.2 0.18 0.01 0.02 0.59 0.46 1 0.36
Drip loss, % 0.45 0.004 0.43 <0.0001 0.03 0.67 0.61 0.99
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1Data are reported as least square means. Initial weights were 90.1, 90.4, 90.5, and 89.8 kg for adequate diets at 0 and 20 ppm and low nutrition diets at 0 and 20 ppm, respectively.

2RAC inclusion level in diets is indicated as 0 or 20 ppm.

3Measures are lightness (L*, greater value indicates a lighter color); redness (a*, greater value indicates a more red color); and yellowness (b*, greater value indicates a more yellow color).

4NPPC scale of 1 to 5 for color, marbling, and firmness, respectively: 1 = pale pinkish, traces, very soft; 5 = dark purplish red, abundant, very firm.

a-dMeans with different superscript letters differ by P < 0.05.

Table 3.

Effects of dietary RAC (R) and diet (D) on growth performance1

Diet Adequate Low nutrient P-value
RAC2 0 ppm 20 ppm 0 ppm 20 ppm SEM D R D × R
ADG, kg/day
 0 to 1 wk 0.85a 0.94a 0.09b 0.08b 0.16 0.0003 0.83 0.75
 0 to 2 wk 0.73b 1.02a 0.28c 0.09c 0.08 <0.0001 0.57 0.02
 0 to 4 wk 0.77b 0.99a 0.35c 0.17c 0.06 <0.0001 0.76 0.01
 1 to 2 wk 0.61b 1.1a 0.46c 0.1d 0.04 <0.0001 0.17 <0.0001
 1 to 4 wk 0.73b 1.01a 0.48c 0.22d 0.04 <0.0001 0.76 <0.0001
 2 to 4 wk 0.84a 0.92a 0.51b 0.35b 0.06 <0.0001 0.49 0.06
ADFI, kg/day
 0 to 1 wk 1.92a 2.07a 1.39b 0.98b 0.17 0.0005 0.46 0.13
 0 to 2 wk 2.02a 2.24a 1.82ab 1.48b 0.16 0.01 0.70 0.10
 0 to 4 wk 2.22 2.41 2.14 2.01 0.14 0.12 0.85 0.27
 1 to 2 wk 2.11 2.41 2.25 1.98 0.16 0.37 0.97 0.09
 1 to 4 wk 2.36 2.58 2.54 2.76 0.15 0.30 0.21 0.98
 2 to 4 wk 2.61c 2.75c 2.96b 3.54a 0.02 <0.0001 0.08 0.02
Gain:feed
 0 to 1 wk 0.46a 0.45a 0.03b 0.01b 0.12 0.004 0.91 0.93
 0 to 2 wk 0.37a 0.46a 0.12b 0.03b 0.06 0.0001 0.95 0.18
 0 to 4 wk 0.36a 0.42a 0.12b 0.02b 0.04 <0.0001 0.88 0.13
 1 to 2 wk 0.29b 0.46a 0.21c 0.05d 0.02 <0.0001 0.76 <0.0001
 1 to 4 wk 0.31b 0.40a 0.19c 0.07d 0.02 <0.0001 0.85 <0.0001
 2 to 4 wk 0.32a 0.34a 0.16b 0.08c 0.02 <0.0001 0.14 0.02
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1Data are reported as least square means.

2RAC inclusion level in diets is indicated as 0 or 20 ppm.

a-cMeans with different superscript letters differ by P < 0.05.

The average daily gain was greater (P < 0.001) in pigs fed the adequate diet at the end of 1 wk as compared with pigs fed the low nutrient diet, regardless of RAC inclusion. However, the inclusion of RAC in the two diets had differential (P < 0.01) effects on the gain at 2 and 4 wk. Dietary RAC increased (P < 0.01) ADG in pigs fed the adequate diet but decreased (P < 0.01) ADG in pigs fed the low nutrient diet.

Pigs fed the low nutrient diet consumed less (P < 0.05) feed than those fed the adequate diet during the first wk, but at the end of 2 wk, intake was the same among diets. Feed intake was greatest (P < 0.05) at week 4 in pigs fed the low nutrient diet with RAC included. Feed efficiency was greatest (P < 0.001) in pigs fed the adequate diet. Furthermore, pigs fed the low nutrient diet with RAC had the lowest overall (P < 0.05) feed efficiency. Feed efficiency of pigs fed the low nutrient diet improved (P < 0.05) to the level of those fed the adequate diet by the end of 2 wk, but was lower (P < 0.05) than pigs fed the adequate diet plus RAC. At 4 wk, feed efficiency decreased (P < 0.05) in pigs fed the adequate diet plus RAC to the level of pigs fed the low nutrient diet.

Carcass characteristics

Carcass weight was not different at 1 wk, but at 2 wk, pigs fed RAC in the adequate diet had heavier (P < 0.05) carcasses than pigs fed the low nutrient diet regardless of RAC presence. Feeding the adequate diet yielded heavier (P < 0.05) carcasses than the low nutrient diet, regardless of RAC inclusion by 4 wk. Dressing percentage was not different at 1 wk, but pigs fed adequate diet yielded a greater (P < 0.05) dressing percentage than pigs fed the low nutrient diet through 2 and 4 wk.

Pigs fed the low nutrient diet had less (P < 0.05) backfat depth than pigs fed the adequate diet over the entire course of the study. Furthermore, backfat was unaffected by RAC regardless of diet. Loin eye area was greatest (P < 0.05) in pigs fed the adequate diet plus RAC at 1 wk but was not different among the remaining treatments. LEA increased (P < 0.05) faster in RAC pigs fed the adequate diet compared with RAC pigs fed the low nutrient diet. Regardless of RAC inclusion, LEA was not different in pigs fed the adequate diet at either 2 or 4 wk; however, these pigs had greater LEA (P < 0.05) than pigs fed the low nutrient diet at 2 and 4 wk.

Gene expression

The expression of the β-actin gene did not vary between muscles or treatments (Figure 1). Type I MyHC gene expression had a significant (P < 0.05) time * muscle interaction (Figure 2). Transcript abundance decreased (P < 0.001) in WST at 4 wk but was unaffected at 1 and 2 wk. No additional effects of time on type I MyHC gene expression in RST or LD muscles were noted. A significant (P < 0.05) four-way interaction, however, was noted for MyHC type I gene expression (Supplementary Figure S1).

Figure 1.

Figure 1.

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Effect of muscle (RST, WST, and LD) on β-actin gene expression (log starting abundance) averaged across time and all dietary and RAC treatments (n = 96). There was no effect of time, diet, or RAC on gene expression. Means bearing different letters differ (P > 0.05).

Figure 2.

Figure 2.

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Effect of time (wk) and muscle (RST, WST, and LD) on type I MyHC gene expression (log starting abundance) across all dietary and RAC treatments (n = 32). Means bearing different letters differ (P < 0.05).

RAC decreased (RAC * muscle; P < 0.01) type IIA MyHC gene expression to a greater extent in WST and LD muscles than in RST muscle (Figure 3A). Muscle type IIA MyHC gene expression did not differ in pigs consuming the low nutrient diet (time * diet), whereas the expression increased (P < 0.01) at 2 and 4 in pigs fed the adequate diet (Figure 3B). RAC decreased (P < 0.0001) type IIA MyHC to the greatest extent at 1 wk in the adequate diet, whereas the largest decrease (P < 0.0001) in the low nutrient diet was not apparent until 2 wk (Figure 3C).

Figure 3.

Figure 3.

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Effect of RAC (0 or 20 ppm) and muscle (RST, WST, and LD) on type IIA MyHC gene expression (log starting abundance) across time and all dietary treatments (A; n = 48). Effect of time (wk) and diet (adequate or low nutrient) on type IIA MyHC gene expression (log starting abundance) across all muscles and RAC treatments (B; n = 48). Effect of time (wk), diet (adequate or low nutrient), and RAC administration (0 or 20 ppm) on type IIA MyHC gene expression (log starting abundance) across all muscles (C; n = 24). Means bearing different letters differ (P < 0.05).

RAC did not affect type IIX MyHC gene expression at 1 or 2 wk, but decreased (time * RAC; P < 0.05) expression at 4 wk (Figure 4A). Diet had no effect on type IIX MyHC gene expression in RST and LD muscles; however, the expression was greater (diet * muscle; P < 0.01) in WST muscles from pigs fed the adequate diet (Figure 4B). RAC decreased (RAC * muscle; P < 0.05) type IIX MyHC gene expression in WST and LD muscles but had no effect in RST (Figure 4C).

Figure 4.

Figure 4.

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Effect of time (wk) and RAC (0 or 20 ppm) on type IIX MyHC gene expression (log starting abundance) averaged over all muscles and dietary treatments (A; n = 48). Effect of diet (adequate, A; low nutrient, LN) and muscle (RST, WST, and LD) on type IIX MyHC gene expression (log starting abundance) across time and all RAC treatments (B; n = 48). Effect of RAC (0 or 20 ppm) and muscle (RST, WST, and LD) on type IIX MyHC gene expression (log starting abundance) across time and all dietary treatments (C; n = 48). Means bearing different letters differ (P < 0.05).

RAC increased (time * RAC; P < 0.05) type IIB MyHC gene expression at all time points compared with controls, with the largest increase at 1 wk (Figure 5A). Type IIB MyHC gene expression in WST and LD muscles was not affected by diet; however, type IIB MyHC gene expression was decreased (diet * muscle; P < 0.05) in RST muscles from pigs fed low nutrient diet compared with the adequate diet (Figure 5B). Furthermore, RAC increased (RAC * muscle; P < 0.01) type IIB MyHC gene expression in all muscles studied; however, the greatest (P < 0.0001) extent was in RST muscle (Figure 5C).

Figure 5.

Figure 5.

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Effect of time (wk) and RAC (0 or 20 ppm) on type IIB MyHC gene expression (log starting abundance) across all muscles and dietary treatments (A; n = 48). Effect of diet (adequate, A; low nutrient, LN) and muscle (RST, WST, and LD) on type IIB MyHC gene expression (log starting abundance) across time and all RAC treatments (B; n = 48). Effect of RAC (0 or 20 ppm) and muscle (RST, WST, and LD) on type IIB MyHC gene expression (log starting abundance) across time and all dietary treatments (C; n = 48). Means bearing different letters differ (P < 0.05).

Pigs fed the adequate diet had greater (P < 0.05) GS gene expression than those fed the low nutrient diet (Figure 6A). Furthermore, GS was differentially (P < 0.01) expressed in different skeletal muscles. Expression was the greatest in WST (P < 0.01) compared with LD muscle and lowest (P < 0.01) in RST muscle (Figure 6B). A time * RAC interaction indicated that GS gene expression varied (P < 0.05) over time with RAC administration (Figure 6C). Expression was greatest (P < 0.0001) in the muscles from pigs fed RAC 1 wk compared with controls. However, the expression in muscles from RAC pigs was not different from controls at 2 wk. Yet, RAC decreased (P < 0.01) GS gene expression at 4 wk compared with controls.

Figure 6.

Figure 6.

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Effect of diet (adequate or low nutrient) on GS gene expression (log starting abundance) across all muscles, time, and RAC treatments (A; n = 144). Effect of muscle (RST, WST, and LD) on GS gene expression (log starting abundance) across time and all dietary and RAC treatments (B; n = 96). Effect of time and RAC (0 or 20 ppm) on GS gene expression (log starting abundance) across all muscles and dietary treatments (C; n = 48). Means bearing different letters differ (P < 0.05).

CS gene expression was greatest (P < 0.05) in RST muscles compared with WST and LD muscles (Figure 7A), but RAC decreased (P < 0.05) CS gene expression in all muscles (Figure 7B). Additionally, CS gene expression decreased (P < 0.05) over time regardless of RAC (Figure 7C).

Figure 7.

Figure 7.

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Effect of muscle (RST, WST, and LD) on CS gene expression (log starting abundance) across time and all dietary and RAC treatments (A; n = 96). Effect of RAC (0 or 20 ppm) on CS gene expression (log starting abundance) across time, and all muscles and dietary treatments (B; n = 144). Effect of time (wk) on CS gene expression (log starting abundance) across all muscles, and dietary and RAC treatments (C; n = 96). Means bearing different letters differ (P < 0.05).

β 1-AR gene expression was not different in the porcine skeletal muscles studied and was unaffected by dietary treatments and RAC in this study (Figure 8). However, the expression of the β 2-AR gene was greatest (P < 0.01) in RST muscles compared with WST and LD muscles (Figure 9A). Additionally, RAC decreased (P < 0.001) β 2-AR gene expression equally in all muscles (Figure 9B).

Figure 8.

Figure 8.

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Effect of muscle (RST, WST, and LD) on β 1-AR gene expression (log starting abundance) across time and all dietary and RAC treatments (n = 96). There was no effect of diet, time, or RAC on gene expression. Means bearing different letters differ (P > 0.05).

Figure 9.

Figure 9.

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Effect of muscle (RST, WST, and LD) on β 2-AR gene expression (log starting abundance) across time and all dietary and RAC treatments (A; n = 96). Effect of RAC (0 or 20 ppm) on β 2-AR gene expression (log starting abundance) across time and all muscles and dietary treatments (B; n = 144). Means bearing different letters differ (P < 0.05).

Together, these data indicate that the growth-promoting effects of RAC are maximal in pigs fed an adequate diet and detrimental to pigs fed a low nutrient diet. Pigs fed the low nutrient diet grew the slowest, and the inclusion of RAC to these pigs further decreased feed and growth efficiency. Therefore, while RAC improves muscle growth in pigs fed the adequate diet, muscle growth is negatively affected by RAC in pigs fed the low nutrient diet. In conclusion, adding RAC to a diet deficient in lysine reduces muscle growth.

Discussion

RAC is fed to pigs during the finishing phase (Watkins et al., 1988; Aalhus et al., 1990; Depreux et al., 2002), a period of rapid muscle growth in pigs.

RAC-induced muscle hypertrophy is largely impacted by nutrition (Adeola et al., 1990; Mitchell et al., 1991; Xiao et al., 1999; Apple et al., 2004; Carr et al., 2005). The ability of RAC to improve carcass composition is affected by the lysine level in the diet (Schinckel et al., 2003). Greatest increases in dressing percentage and lean gain are observed after RAC administration in pigs fed lysine at a level required for maximal daily protein accretion (1.0% to 1.2% or 25g/d lysine) than pigs fed low-lysine diets (0.55% to 0.7% or 15g/d lysine; Herr et al., 2001). Therefore, reducing lysine to a deficient level may block RAC-induced muscle hypertrophy. Furthermore, when RAC was fed in a 16%, 20%, or 24% crude protein diet, average daily gain, dressing percentage, and loin eye area improved with increased dietary protein (Jones et al., 1987). Conversely, feeding cimaterol in a protein-restricted diet (14% crude protein) blunts the β-agonist effect on weight gain or protein or fat accretion (Mersmann et al., 1987). As a result, the manufacturer recommends greater crude protein requirements for pigs fed RAC to support additional protein deposition. Therefore, an adequate protein supply is required for the maximal effects of β-agonist on growth performance and body composition. Earlier, we reported that RAC differentially alters the MyHC gene expression in porcine skeletal muscles (Gunawan et al., 2007b). Unfortunately, untreated pigs were only sampled at the end of the study which occurred over a 4-wk period of growth, whereas treated pigs were fed RAC for 1, 2, or 4 wk before slaughter. Because all animals were growing in this study, we were unable to determine if increased gene expression was a direct result of RAC administration or simply a result of normal muscle growth. Furthermore, it is not known whether RAC induces differences in MyHC gene expression in the absence of muscle growth. Therefore, in the present study, two diets (adequate or low nutrient) were utilized to control muscle growth.

The well-documented effects of RAC on carcass composition and pork quality (Moody et al., 2000) were observed in this study in pigs fed the adequate diet, whereas low nutrient-fed pigs were negatively affected by RAC. After the administration of 20 ppm RAC, the average daily gain was increased at 2 and 4 wk in pigs fed the adequate diet, yet was decreased at 2 and 4 wk in the low nutrient diet, compared with controls. Feed efficiency was numerically increased by RAC in the adequate diet and numerically decreased by RAC in the low nutrient diet. Furthermore, the differential effects of RAC on growth performance in the two diets carried over into carcass measurements. Slaughter weight and hot carcass weight of pigs fed the adequate diet were numerically increased by RAC but were numerically decreased by RAC in pigs fed the low nutrient diet. Although RAC did not significantly increase all measures of growth in pigs fed the adequate diet, they are consistent with a previous report of increased slaughter weight and hot carcass weight in pigs fed 10 and 20 ppm RAC (Crome et al., 1996). RAC increased loin eye area at 1 wk in the adequate diet, but no differences were detected in the low nutrient diet, indicating that RAC-induced growth of lean muscle was blocked in this diet. Fat depth at the 10th rib was not improved by the inclusion of RAC in the adequate or low nutrient diet. This is consistent with a report that RAC, fed at dietary concentrations of 5, 10, or 20 ppm, does not reduce the 10th rib backfat depth (Stites et al., 1991). Contrasting these data, RAC improved 10th rib backfat thickness and loin eye area in pigs (Watkins et al., 1990; Crome et al., 1996); however, these studies utilized pigs with greater backfat than those in our study (2.69 or 3.15 vs. 1.7 cm). Pigs fed the low nutrient diet weighed less than those fed the adequate diet at all time points in our study, which demonstrates the desired change in growth we were looking for. Furthermore, diminished muscle size is a consequence of protein undernutrition (Garlick et al., 1975; Smith et al., 1982). Generally, feeding a protein-restricted diet decreases weight gain and lean tissue growth and increases fat accretion and feed intake (Bracher-Jakob and Blum, 1990). Although the low nutrient diet, deficient in lysine, was fed ad libitum, consumption was below that of the adequate fed pigs, a scenario often reported observed with acclimation to high protein-based diets (Adeola, 1990). Together, these data demonstrate the differential effects of RAC on body growth of pigs fed the adequate or low nutrient diet, suggesting that the adequate diet supported muscle hypertrophy and the low nutrient diet effectively limited muscle growth due to low lysine content and reduced feed intake.

Of the three muscles studied, we show that GS gene expression is greatest in glycolytic muscle, whereas CS gene expression is greatest in oxidative muscle. Furthermore, CS gene expression decreased over time in all pigs studied regardless of diet or RAC inclusion level, suggesting that normal muscle growth occurs in the absence of RAC administration and results in a decrease of CS gene expression. The major energy storage molecule in porcine skeletal muscle is glycogen, which is regulated by GS, the rate-limiting enzyme for synthesis. GS gene expression is decreased in pigs fed the low nutrient diet, which provided less lysine and amino acids to support protein synthesis, implying that available nutrients were utilized for growth and not stored as glycogen. These expression data suggest that as pigs grow, glycolytic metabolism may increase at the expense of oxidative metabolism in skeletal muscle. Type IIB fibers are the largest (Cassens and Cooper, 1971; Rosser et al., 1992), so as hypertrophy of existing muscle fibers increases muscle mass (Davies, 1972; Swatland, 1973), type IIB fibers account for the largest increase in cross-sectional area. Furthermore, muscle fibers in the glycolytic LD muscle are predominantly composed of type IIB MyHC transcripts (Lefaucheur et al., 1998), suggesting that glycolytic metabolism is associated with a high proportion of type IIB fibers. These data are consistent with our previous study (Gunawan et al., 2007a) and that of Lefaucheur and Vigneron (1986), suggesting that glycolytic metabolism corresponds with type IIB MyHC gene expression in porcine skeletal muscle and predominates over oxidative metabolism as animals mature. Therefore, altered gene expression in porcine skeletal muscles is associated with muscle growth and fiber type in the absence of RAC administration. Adding complexity to the relationship between the effect of RAC and normal muscle growth, protein turnover varies with fiber-type composition (Goldberg, 1967; Garlick et al., 1989). In particular, protein turnover is greatest in muscles with greater proportions of type I fibers (Dadoune et al., 1978; Millward et al., 1978; Bates and Millward, 1983). Associated with a 3-fold greater rate of protein synthesis (Goldberg, 1967), type I fibers have 5- to 6-fold more MyHC mRNA per microgram muscle tissue than type IIB fibers (Habets et al., 1999). Muscle hypertrophy, regardless of RAC administration, increases the abundance of type IIB fibers (Aalhus et al., 1992; Depreux et al., 2002); therefore, muscle growth is likely associated with increased efficiency of protein accretion as a result of reduced protein turnover in predominantly fast-twitch muscle. Although a clear association exists between increased muscle hypertrophy and the overall muscle fiber-type composition, a cause-and-effect relationship between β-agonist administration and muscle fiber-type composition has not been identified. Therefore, the objective of this study was to determine if an interaction exists between RAC-induced alterations in gene expression and muscle hypertrophy.

There was no effect of the low nutrient diet on type IIA MyHC gene expression at 1 wk; however, the expression was reduced at 2 and 4 wk. Pigs consuming the low nutrient diet showed decreased type IIX MyHC gene expression only in WST muscles, whereas type IIB MyHC gene expression was only decreased in RST muscles. These data indicate that the expression of type I MyHC, and initially type IIA, is spared in pigs fed the low nutrient diet at the expense of type IIX and IIB MyHC. Restriction of dietary protein intake affects the muscles of various myosin isoform compositions differently, especially during fetal and early postnatal growth (Santidrián et al., 1980; Dwyer and Stickland, 1992). Muscles in which fast-twitch myosin isoforms predominate lose mass and protein content faster during feed deprivation than those in which slow-twitch isoforms predominate (Brodsky et al., 2004). Furthermore, restricting dietary protein intake inhibits MyHC synthesis and results in a transition toward type I MyHC (Brodsky et al., 2004). However, a significant 4-way week * diet * RAC * muscle interaction observed in our study indicates that RAC decreases type I gene expression in WST muscle only in pigs fed the low nutrient diet. Therefore, it appears that RAC administration, in combination with feed restriction, decreases type I MyHC gene expression in a predominantly fast-twitch muscle. These data are inconsistent with a study by White et al. (2000), suggesting that restricted feed intake increases type I MyHC mRNA and protein in a predominantly fast-twitch muscle. Therefore, these data demonstrate the differential effect of RAC on MyHC gene expression in adequate and low nutrient diets.

The greatest reduction in type IIA MyHC gene expression in response to RAC was observed at 1 wk in pigs fed the adequate diet and 2 wk in pigs fed the low nutrient diet. After this decrease, the gene expression of type IIA MyHC began to approach that of controls in both diets. We have shown that RAC decreases type IIA MyHC gene expression at 1 wk but is not different from controls at 4 wk in LD muscle from pigs fed the adequate diet (Gunawan et al., 2007b). Therefore, the level of nutrition differentially affected RAC-induced gene expression of type IIA MyHC. Feed restriction induces atrophy of fast-twitch muscle fibers, increasing the proportion of IIA fibers at the expense of IIB fibers (Solomon et al., 1994), indicating that poor nutrition induced the upregulation of type IIA MyHC gene expression, which delayed RAC-induced downregulation of the gene.

All fast MyHC genes had significant RAC by muscle interactions, suggesting that RAC has muscle-specific effects. MyHC type IIA gene expression was reduced after RAC administration to a greater extent in LD and WST muscles than RST. These data are especially interesting because the expression of type IIA MyHC is normally less in LD and WST than in RST (Gunawan et al., 2007a). Similarly, RAC reduced type IIX MyHC gene expression in LD and WST muscles, whereas it had no effect in the slow-twitch RST muscle. This differential effect of RAC is noteworthy because these three muscles normally express type IIX MyHC at the same level (Gunawan et al., 2007a). In contrast to the effect on type IIA MyHC, RAC increased type IIB gene expression in all muscles studied with the greatest increase observed in RST, the muscle that normally expresses the least type IIB MyHC (Gunawan et al., 2007a). Although RAC increased the relative type IIB MyHC gene expression to the greatest extent in RST, the absolute level remained lower than that of LD and WST muscles before RAC administration, indicating that differential expression of MyHC isoforms in skeletal muscles is maintained after RAC administration. These data are consistent with those of other authors who show that β-agonists consistently increase weight and protein content to the greatest extent in muscles that are predominantly fast twitch (Deshaies et al., 1981; Beermann et al., 1986; Bohorov et al., 1987; Kim et al., 1987). Early data indicate that RAC increases the gene expression of muscle proteins, including α-actin, myosin light chain, and type II MyHC (Kim et al., 1987; Bergen et al., 1989; Smith et al., 1989; Helferich et al., 1990; Grant et al., 1993). These data indicate that increased skeletal muscle accretion in RAC-fed pigs is due to an increase in myofibrillar protein synthesis. However, using classical histochemical procedures, RAC was demonstrated to differentially affect muscle proteins such that the frequency of type IIA fibers decreased and IIB increased (Aalhus et al., 1992). Using a whole muscle ELISA-based assay, RAC was demonstrated to increase the abundance of type IIB MyHC and decrease type IIA and IIX (Depreux et al., 2002). Brown et al. (2016) also demonstrated that RAC incorporation shifts metabolism toward faster MyHC mRNA isoforms within 27 d. Mechanistically, β-agonist may cause a conversion of one muscle fiber type to another or simply cause hypertrophy in a subset of fast-twitch fibers, thereby altering MyHC profiles. Regardless, alteration of fiber-type composition in the muscle of pigs fed β-agonist creates a faster contracting, whiter muscle.

The gene expression of β 1-AR was not differentially expressed in porcine skeletal muscles nor was affected by RAC administration. However, the β 2-AR gene expression was greater in RST than WST and LD muscles. These data are supported by a study by Williams et al. (1984), indicating that β-AR density is greater in the membranes of slow-twitch muscles than fast twitch. Furthermore, in rats, clenbuterol stimulates hypertrophy of fast-twitch fibers in the predominantly slow-twitch soleus muscle, whereas type I fibers were unaffected. We observed a RAC-induced reduction in β 2-AR gene expression at all time points in pigs fed either diet, providing further evidence that the mechanism by which RAC affects MyHC gene expression may be related, in part, to differential regulation of β-ARs in different muscles and fiber types. RAC was originally thought to be a ligand that agonizes the β 1-AR (Smith et al., 1990; Moody et al., 2000), whereas clenbuterol and cimaterol are specific for the β 2 subtype (O’Donnell, 1976; Kim and Sainz, 1990). However, RAC has recently been shown to bind both porcine β 1- and β 2-ARs although β 2-ARs more effectively increase intracellular cAMP (Mills et al., 2003). We have reported a reduction in β 2-AR gene expression over time after RAC administration (Gunawan et al., 2007b); however, sample size and limitations of the real-time PCR assay may have led to increased error, which may have masked small differences in β 2-AR gene expression over time. An abundance of data suggests that muscle hypertrophy occurs as soon as 1 to 2 wk after β-agonist administration (Emery et al., 1984; Reeds et al., 1986). More recently, an anabolic effect of clenbuterol was demonstrated by 2 d in rats using body weight gain as an indicator of muscle growth (McElligott et al., 1989). Further research is needed to determine at what point the direct effect of RAC on the expression of genes in porcine skeletal muscle is detectable.

Although the RST muscle is less responsive to RAC-induced hypertrophy, the gene expression of type IIB MyHC is increased to the greatest extent in RST, compared with WST and LD muscles. RAC alters fiber-type composition of muscle to increase the abundance of fast-twitch isoforms (Gunawan et al., 2007b; Paulk et al., 2014; Almeida et al., 2015). Slow-twitch muscle possesses a greater number of fibers capable of transitioning to fast isoforms, whereas the majority of fibers in a fast-twitch muscle are already fast isoforms and may be unable to transition further in response to RAC administration. Therefore, slower muscles show greater fiber type shifting in response to β-agonist administration. These data are consistent with results from our study, suggesting that the muscle-specific response to RAC correlates to initial fiber type profile and not β-AR density. However, receptor sensitivity to long-term downregulation requires further investigation. RAC-induced growth responses are generally reduced over time of administration (Dunshea et al., 1993; Williams et al., 1994) and are associated with β-AR downregulation (Moody et al., 2000). However, we did not observe the downregulation of RAC-induced gene expression of β 1-AR or MyHC isoforms I, IIX, and IIB at 4 wk, indicating that the expression of these genes does not correlate with studies showing decreases in growth performance after long periods of RAC administration. Together, the chronic administration of RAC stimulates gene expression even though muscle growth promotion declines. Although our study focuses on the RAC effect on gene expression, further studies will be necessary to determine if MyHC gene expression will be translated to the protein level.

Several conclusions can be drawn from this research. Pigs fed the low nutrient diet showed that delayed RAC reduced type IIA MyHC gene expression, and type IIB MyHC gene expression decreased only in the RST muscles of pigs fed the low nutrient diet. These data suggest that poor nutrition increases gene expression of type IIA MyHC such that RAC-induced downregulation of type IIA MyHC is delayed. However, RAC-induced gene expression of type IIX and IIB MyHC isoforms was no different in pigs fed the adequate or low nutrient diet. Because pigs fed the low nutrient diet failed to grow as well as the adequate diet pigs, we can conclude that RAC alters gene expression in the absence of muscle hypertrophy, indicating that RAC has a direct effect on gene expression in porcine skeletal muscles.

Supplementary Material

skaa341_suppl_Supplementary_Figure_S1
Click here for additional data file. (285.3KB, docx)

Glossary

Abbreviations

AR

adrenergic receptor

BAA

β-adrenergic agonist

cAMP

cyclic adenosine monophosphate

cDNA

complementary deoxyribonucleic acid

CS

citrate synthase

dNTP

deoxynucleoside triphosphate

GS

glycogen synthase

LD

longissimus dorsi

LEA

loin eye area

mRNA

messenger ribonucleic acid

MyHC

myosin heavy chain

NDF

neutral detergent fiber

PCR

polymerase chain reaction

RAC

ractopamine

RST

red semitendinosus

SID

standardized ileal digestible

ST

semitendinosus

WST

white semitendinosus

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

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