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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal Sciences and Industry, Kansas State University, Manhattan, 66506;
and
Intervet Inc., Millsboro, DE 19966; and
and
Cactus Research Ltd., Amarillo, TX 79116
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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0.05). Greater days on feed decreased (P < 0.05) ADG, G:F, DMI, and the number of yield grade 1 and 2 carcasses. Also, greater days on feed increased (P < 0.05) HCW, dressing percentage, and the number of prime and choice carcasses, as well as the number of yield grade 4 and 5 carcasses. Increasing days on feed decreased (P < 0.05) the abundance of ß1-AR and ß3-AR mRNA and increased (P < 0.05) the abundance of ß2-AR mRNA in skeletal muscle samples obtained at slaughter. Ractopamine had no effect (P > 0.10) on the abundance of ß1-AR or ß3-AR mRNA, but tended (P = 0.09) to increase ß2-AR mRNA. Additional time-course studies with primary muscle cell cultures revealed that advancing time in culture increased (P < 0.001) ß2-AR mRNA but had no effect (P > 0.10) on ß1-AR or ß3-AR mRNA. We conclude that days on feed and RAC are affecting ß-AR mRNA levels, which could, in turn, impact the biological response to RAC feeding in yearling steers.
Key Words: ß-adrenergic receptor • ractopamine-hydrogen chloride • skeletal muscle tissue • steer
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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), and RAC has been reported to preferentially bind to the ß1-AR (Moody et al., 2000). Moreover, the ß2-AR is the most abundant receptor subtype in bovine skeletal muscle and adipose tissue (Sillence and Matthews, 1994).
Prior studies with feeding of RAC to yearling beef steers demonstrated increased ADG, improved G:F, increased carcass weight gain, and limited effects on USDA yield grade and quality grade (Johnson, 2004; Laudert et al., 2004). Moody et al. (2000) found that external factors such as diet, dose, treatment length, age, BW, sex, and genetics impacted the biological response of an animal to ß-AA. Species, muscle type, and expression of specific ß-AR genes are also impacted to a different magnitude, depending on the ß-AA type (Bridge et al., 1998). Greife et al. (1989) and Vestergaard et al. (1994) demonstrated that ß-AA administration resulted in greater ADG responses in animals of heavier initial BW compared with animals of lighter initial BW. Others have pointed out that physiological maturity may account for differences in the ability of ß-AA to repartition nutrients (Schiavetta et al., 1990). No studies have determined if the response to RAC in yearling steers is affected by the length of the feeding period.
The objectives of our study were to evaluate the effects of feeding RAC to yearling steers slaughtered at 3 different days on feed on the following: 1) feedlot performance, 2) carcass characteristics, and 3) the abundance of ß-AR mRNA in skeletal muscle tissue. Additionally, a subsequent time-course study with bovine muscle cell cultures was conducted to measure the changes in the abundance of ß-AR mRNA in bovine muscle cell cultures.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). English x Continental steers (n = 2,252; 314 kg of initial BW) were fed at Cactus Research Ltd. This study was a randomized complete block design, and treatments were arranged in a 3 x 2 factorial design. Steers were blocked by arrival date, BW, and origin; randomly assigned to treatments within block; and allotted to 24 pens with 91 to 97 steers per pen. Pen served as the experimental unit for all of the analysis.
Pens of steers were assigned to slaughter dates of 150, 171, or 192 d. Within each slaughter date, steers either received RAC (200 mg/steer daily) for the final 28 d, or did not receive RAC (control). Steers were implanted with Revalor-IS (80 mg of trenbolone acetate and 16 mg of estradiol-17ß) at feedlot arrival and were reimplanted 75 d later with Revalor-S (120 mg of trenbolone acetate and 24 mg of estradiol-17ß). Steers were fed a steam-flaked corn-based diet. Diet composition is provided in Table 1. At the completion of the study, steers were weighed, and a 4% pencil shrink was applied to determine final weight. Steers were transported 113 km to a commercial slaughter facility (Tyson Fresh Meats Inc., Amarillo, TX). Carcass characteristics, including USDA yield and quality grades, were obtained 24 h after slaughter.
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The concentration of RNA was determined by absorbance at 260 nm. Electrophoresis of total RNA through a 1% agarose-formaldehyde gel followed by ethidium bromide staining to allow visualization of 28S and 18S ribosomal RNA (rRNA) was used to assess the integrity of RNA. Samples were then treated with DNase to remove any contaminating genomic DNA using a commercially available kit (DNA-free, Ambion, Austin TX). Total RNA (1 µg) was then reverse-transcribed to produce the first-strand complementary DNA (cDNA) using TaqMan Reverse Transcription Reagents and MultiScribe Reverse Transcriptase (Applied Biosystems, Foster City, CA) following the protocol recommended by the manufacturer. Random hexamers were used as primers in cDNA synthesis.
Real-Time Quantitative PCR
Real-time quantitative PCR was used to determine the quantity of ß1-AR, ß2-AR, and ß3-AR mRNA relative to the quantity of 18S rRNA in total RNA isolated from semimembranosus muscle of the steers. Measurement of the relative quantity of cDNA was carried out using TaqMan Universal PCR Master Mix (Applied Biosystems), 900 nM of the appropriate forward and reverse primers, 200 nM of the appropriate TaqMan detection probe, and 1 µL of the cDNA mixture. Bovine primers and probes for ß1, ß2, and ß3 receptors are presented in Table 2. Commercially available eukaryotic 18S rRNA primers and probes were used as an endogenous control (Applied Biosystems; GenBank, X03205). Assays were performed in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using thermal cycling parameters recommended by the manufacturer (50 cycles of 15 s at 95°C and 1 min at 60°C). Relative expression of mRNA for ß1, ß2, and ß3 receptors were normalized to 18S mRNA endogenous control and expressed in arbitrary units.
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; Johnson et al., 1998). All steers were fed RAC (200 mg/steer daily) during the final 28 d of the feeding period. Steers were anesthetized with sodium pentobarbital and then exsanguinated. Using sterile techniques, approximately 500 g of the semimembranosus muscle was dissected and transported to the cell culture laboratory. Subsequent procedures were conducted in a sterile field under a tissue culture hood. After removal of connective tissue, the muscle was passed through a sterile meat grinder. The ground muscle was incubated with 0.1% pronase in Earle’s Balanced Salt Solution for 1 h at 37°C with frequent mixing (Invitrogen, Frederick, MD). After incubation, the mixture was centrifuged at 1,500 x g for 4 min, the pellet was suspended in PBS (140 mM NaCl, 1 mM KH2PO4, 3 mM KCl, and 8 mM Na2HPO4), and the suspension was centrifuged at 500 x g for 10 min. The resulting supernatant was centrifuged at 1,500 x g for 10 min to pellet the mononucleated cells. The PBS wash and differential centrifugation were repeated twice. The resultant mononucleated cell preparation was suspended in cold (4°C) Dulbecco’s modified Eagle medium (DMEM; Invitrogen) that contained 10% fetal bovine serum (FBS; Invitrogen) and 10% (vol/vol) dimethyl-sulfoxide and were then frozen (Sigma, St. Louis, MO). Cells were stored in liquid N for future studies.
Primary cultures of satellite cells were plated on tissue culture plates (9.62 cm2/well) that were precoated with reduced growth factor-Matrigel (Becton Dickinson Labware, Franklin Lakes, NJ) diluted 1:9 (vol/vol) with DMEM. Cells were plated on 10% FBS-DMEM at a density of 0.22 g of original tissue weight/cm2 and incubated at 37°C, 5% CO2, in a water-saturated environment. At 24 h, the cells were rinsed with DMEM, and fresh 10% FBS-DMEM was added. At 96, 120, and 144 h postplating, total RNA was isolated from the cells using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA). Methods for establishing RNA concentration, cDNA synthesis, and quantity of mRNA were as previously described.
Statistical Analysis
Data for performance, carcass characteristics, and skeletal muscle gene expression were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). Data were arranged as a 3 x 2 factorial in a randomized complete block, with pen serving as the experimental unit for all feedlot and carcass characteristic analyses. The model contained the effects of RAC, days on feed, and RAC x days on feed.
Cell culture data were analyzed using the MIXED procedure of SAS. The model statement contained the effects of hours in culture, implant, and hours in culture x implant. Treatment means were computed using the LSMEANS option. The LSD procedure was used to separate means when the respective F-tests were significant (P < 0.05).
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). There was no overall change in DMI in response to RAC during the entire feeding period, but during the last 28 d, feed intake increased (P < 0.05) by 3.5% in RAC steers compared with controls (data not shown). Similar results were obtained by administering alternate ß-AA. When finishing Friesian steers were fed the ß-AA, L-644,969, a 30% linear increase in feed efficiency was observed as dose increased (Moloney et al., 1990). Conversely, in Hereford steers administered clenbuterol, there was no increase in feed efficiency (Ricks et al., 1984).
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). Similar decreases in ADG and G:F were demonstrated by Van Koevering et al. (1995), because steers were on feed for longer periods of time. In our study, we noted no interaction for RAC x days on feed, indicating the inclusion of RAC in the diet for 28 d before slaughter resulted in improved performance responses regardless of length of feeding period. Laudert et al. (2004) also reported that RAC inclusion increased ADG and gain efficiency, but had no effect on daily feed intake.
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). Feeding RAC increased (P = 0.08) ribeye area in cattle by 1.74 cm2, but had no impact on USDA quality and yield grades (Table 3). When finishing steers were fed clenbuterol, ribeye area was increased 11 to 16%, there was a 36 to 42% reduction in 12th-rib fat depth, and KPH percentage was less in clenbuterol steers compared with controls (Ricks et al., 1984). Likewise, in finishing heifers administered clenbuterol, researchers documented an 18% increase in ribeye area and a decrease in 12th-rib fat depth thickness and KPH percentage (Miller et al., 1988). In a trial in which clenbuterol was administered for 50 d to younger steers, researchers noted a 28% increase in ribeye area with no change in 12th-rib fat depth or KPH percentage (Schiavetta et al., 1990). Schiavetta et al. (1990) attributed the larger increase in ribeye area to the age of cattle at time of clenbuterol administration, suggesting that clenbuterol has greater effects on enhancing protein deposition and reducing protein degradation during the normal stages of rapid protein accretion that typically occur earlier in the feeding period. As well, the lack of effect of clenbuterol on fat thickness and perirenal fat in their study may be attributed to age of administration; the older cattle mentioned in previous studies had reductions in both 12th-rib fat depth and KPH percentage (Schiavetta et al., 1990). The varying results that have been noted with clenbuterol administration in cattle suggest that response may be impacted by physiological maturity.
Greater time on feed increased (P < 0.01) HCW, dressing percentage, and ribeye area; improved marbling score; and worsened USDA yield grade (Table 4). May et al. (1992) and Van Koevering et al. (1995) showed comparable effects of days on feed on HCW, ribeye area, marbling score, and yield grade.
Effect of RAC on Semimembranosus Muscle ß1-, ß2-, and ß3-AR mRNA Concentrations
Ractopamine feeding had no effect on the abundance of ß1-AR or ß3-AR mRNA (data not shown). In pigs administered RAC, Spurlock et al. (1994) reported no decrease in ß-AR density in pig LM. Likewise, Smith (1989) reported no significant decline in ß-AR density in skeletal muscle of pigs administered RAC.
Contradictory to our study and the aforementioned data, Walker et al. (In press) reported decreases in both ß1-AR and ß2-AR mRNA expression (P = 0.02) in LM tissue from Holstein steers administered RAC (200 mg/ d) for 28 d. It is likely that RAC may be eliciting a response through both the ß1-AR and ß2-AR due to the decrease in mRNA abundance of these receptors. Similar findings were noted by Rothwell et al. (1987) in that clenbuterol administration in rats significantly reduced ß2-AR density in skeletal muscle. In vitro findings from Hausdorff et al. (1990) explain that functional ß-AR was lost through chronic administration of high doses of ß-AA, which in turn downregulated ß-AR mRNA abundance; this would explain the decrease noted by Walker et al. (In press). To further demonstrate this phenomenon, Kim et al. (1992) reported a decrease in overall ß-AR density in skeletal muscle from rats administered cimaterol, and Spurlock et al. (1994) noted a significant decrease in overall ß-AR density in adipose tissue as a result of RAC treatment. Our data do not support previous findings, because the ß1-AR was not affected in our study.
In the current study, RAC had a tendency to increase (P = 0.09) the abundance of ß2-AR mRNA (Figure 1). We have also observed this in semimembranosus muscle from heifers administered RAC (200 mg/d) for 28 d (E. K. Sissom, C. D. Reinhardt, B. J. Johnson, Kansas State University, Manhattan; J. P. Hutcheson, W. T. Nichols, D. A. Yates, Intervet Inc., Millsboro, DE; R. S. Swingle, Cactus Research Ltd., Amarillo, TX; unpublished data). There is evidence that endogenous catecholamines can regulate ß2-AR density. Sillence et al. (1995) showed that ß2-AR density can be upregulated by ß-antagonist or agonist treatment in rats. Furthermore, Mills (2002) proposed that ß-AR subtypes located on the cell surface respond differently to various ligands. In addition, RAC may induce production of ß-AR protein on the muscle cell surface to overcome any downregulation that may occur as a result of ligand binding to its receptor and subsequent initiation of the signaling cascade. In the case of the increase in abundance of ß2-AR mRNA that we saw, RAC may be binding weakly to this receptor; this action may trigger synthesis of new ß2-AR protein that may explain the increase in ß2-AR mRNA that we observed as a result of RAC administration.
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). Conversely, ß2-AR mRNA abundance increased (P < 0.05) as days on feed increased (Figure 2).
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indicated that the anabolic responses of ß-AA are achieved independent of the ability of the ß-AA to bind to its receptor. Yet, Bridge et al. (1998) reported that the density of binding sites of ß-AR in bovine skeletal muscle cells from animals of different ages increased from 90 d fetal to 120 d fetal, and increased from 120 d fetal to adult. Possibly 1 of the reasons we still saw a positive performance response was due to the fact that the receptors had a stronger binding affinity for the ligand, even though the mRNA levels for the receptors were lower as cattle aged.
Another possible reason why we saw an increase in performance, yet a decrease in the abundance of ß1-AR mRNA over time, may be explained by observations from Greife et al. (1989). These researchers reported that when clenbuterol was administered to rats of different initial BW, heavy weight rats had greater weight gains as a result of clenbuterol administration than light-weight rats. In addition, Greife et al. (1989) reported clenbuterol significantly reduced perirenal fat. Greife et al. (1989) hypothesized that heavier rats had a greater response to clenbuterol as a result of an increase in protein deposition from energy that was released through the mobilization of body fat stores caused by clenbuterol. Similar results were noted when scrutinizing the relationship between ß-AA and age by Vestergaard et al. (1994). These researchers investigated the effects of cimaterol administration to young bulls of different initial weights and slaughter ages. Vestergaard et al. (1994) noted cimaterol increased daily gains by 10, 25, and 22% in light, middle, and heavy weight groups, respectively. As well, carcass weights were 18, 28, and 27 kg greater as a result of cimaterol treatment across the 3 weight groups, respectively. Cimaterol treatment decreased carcass fat percentage by 1.29, 1.31, and 1.44% across the 3 weight groups, respectively. In our study, days on feed increased KPH percentage, and RAC increased HCW and had a tendency to increase ribeye area, indicating that, although there were fewer ß1-AR as cattle were on feed for longer periods, it may be possible that the positive performance responses stemmed from more energy released from a larger fat store.
Muscle Cell Culture ß1-, ß2-, and ß3-AR mRNA Concentrations
Time in culture had no effect on the abundance of ß1-or ß3-AR mRNA (data not shown), but time in culture did have an effect (P < 0.001) on the abundance of ß2-AR mRNA (Figure 3). As cells were in culture beyond 120 h, the concentration of ß2-AR mRNA increased (P < 0.001) 40% from those cells isolated at 120 h to cells isolated at 144 h. The increase in ß2-AR mRNA over time that we observed in vitro agree with the in vivo findings previously mentioned.
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Footnotes |
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2 Corresponding author: bjohnson{at}ksu.edu
Received for publication August 15, 2006. Accepted for publication September 19, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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