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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal Sciences, University of Florida, Gainesville 32611, and and
North Florida Research and Education Center, Marianna 32446
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: beta-agonist • cull cow • muscle fiber • ractopamine • satellite cell
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Many studies exist detailing the positive effects of RAC on swine performance and carcass composition (Dunshea et al., 1993, 1998; Sainz et al., 1993). At the cellular level in pigs, beta-agonists increase the size of type IIB muscle fibers. Cross-sectional area and fiber diameters were larger in pigs fed RAC (Aalhus et al., 1992) and an increase in the relative amount of myosin IIB was apparent (Depreux et al., 2002). A second effective means of altering feed efficiency and carcass composition is the use of steroid implants. In growing steers, treatment with Revalor-S (trenbolone acetate + estradiol; TBA) resulted in an increase in type IIB fibers without a change in the size or number of type I muscle cells (Fritsche et al., 2000).
Satellite cells, or muscle stem cells, are responsible for postnatal muscle fiber growth and repair (Mauro, 1961; Schultz et al., 1978). This typically quiescent population of cells lies adjacent to the muscle fiber under the basal lamina and is identified in vivo by their expression of Pax7. Pax7 is a member of the paired-box family of transcriptional mediators and is implicated in the establishment of the satellite cell lineage (Seale et al., 2000). Mice devoid of Pax7 exhibit severe muscle size and functional deficits that are due to an absence of satellite cells (Mansouri et al., 1996, Seale et al., 2000). The animals die within 3 to 4 wk of age. Pax7 does not alter proliferation rates but does inhibit satellite cell differentiation and apoptosis (Olguin and Olwin, 2004; Relaix et al., 2006; Zammit et al., 2006).
Due to limited information regarding aged bovine muscle fiber size, satellite cell numbers, and growth capabilities, we examined LM fiber morphometrics and myonuclei numbers in cull cows fed RAC and TBA.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cows were fed in 4 pens, with implant status and dietary treatment as the main effects. All diets were fed ad libitum in self-feeders. The BCS of all cows on arrival was uniform (4.3 ± 0.03; Carter et al., 2006). One-half of the cows in each pen were implanted with Revalor-IS (80 mg of trenbolone acetate plus 16 mg of estradiol; Intervet, Millsboro, DE), whereas the remainder received no implant. The basal diet was fed to one-half of the cows (2 pens) for the duration of the 92 d on feed. The remaining one-half (2 pens) were fed the same basal diet from d 0 to 55. On d 56, a pelleted supplement containing RAC was added to the basal diet, delivered to the empty self-feeders, and fed ad libitum for the remaining 35 d on feed.
The basal diet consisted of (DM basis) soybean hulls (21.1%), citrus pulp (19.7%), cracked corn (14.4%), wheat middlings (14.2%), cottonseed hulls (12.7%), cottonseed meal (7.0%), liquid molasses (7.0%), vitamins and minerals (included sodium bicarbonate; 2.1%), tallow (1.3%), and urea (0.4%). The diet provided 87.6% DM, 14% crude protein (DM basis), and 79.5% TDN and was formulated to meet the nutrient requirements of a nonpregnant, nonlactating, beef cow predicted to gain 2.06 kg/d. The pelleted, premixed supplement (type B premix) consisted of wheat middlings (97.6%) and ractopamine HCl (2.4%; Optaflexx 45, Elanco Animal Health, Greenfield, IN) and was formulated to provide approximately 15 mg/kg when combined at the proper rate in our basal diet, depending on DMI (projected to be approximately 13.6 kg·head–1·d–1).
This study was designed to mimic standard housing and management practices in a feedyard. Individual animal intake was not monitored, and it is acknowledged that the implanted cows may have had a greater intake. Thus, the projected range of RAC was between 15 and 16.5 ppm. The type B premix with RAC was randomly sampled and analyzed for the concentration of the experimental compound before blending and feeding to ensure accurate delivery of the formula at the prescribed rate. Analytical results indicated that the B premix contained on average 2.15 g/kg of RAC (as-fed basis) and would adequately provide the targeted level of RAC.
The basal diet also included an ionophore [Rumensin 80 (monensin, granulated), Elanco Animal Health] formulated at the rate of 22 mg/kg of feed. Feed samples were collected randomly over the feeding period and analyzed for monensin concentration, which averaged 22.22 mg/kg.
Slaughtering and Sample Collection
On d 92, cows were slaughtered under USDA inspection in a commercial slaughter facility located in Center Hill, FL. Preslaughter BW was 501.96 ± 6.90 kg for light cows and 522.15 ± 7.09 kg for the heavy cows. After a 48-h chill period and carcass data collection, 10 wholesale ribs were randomly selected from each treatment group (n = 40). The 6th-rib steak from each wholesale rib section was removed. Two 1 x 1 x 1-cm portions of the 6th-rib LM were suspended in OCT tissue freezing medium (Fisher Scientific, Hampton, NH), frozen by submersion in supercooled isopentane, and stored at –80°C.
Immunohistochemistry
Three serial cryosections (12 µm), one for each fiber isoform, were collected on frost-resistant slides (Fisher Scientific) for each LM sample. Two sets of serial cryosections were collected for each animal, and the protocol of Watson et al. (2003) was followed with modifications. Nonspecific antigen sites were blocked with 5% horse serum in PBS for 20 min at room temperature. Cryosections were incubated for 60 min at room temperature with the primary antibodies. Antibodies and dilutions were: 
-dystrophin (Abcam, Cambridge, MA), 1:500; undiluted, supernatant, myosin heavy chain type 1 (BAD.5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City); myosin heavy chain type 2A (SC.71, Developmental Studies Hybridoma Bank); myosin heavy chain type 2A (SC.71, Developmental Studies Hybridoma Bank); myosin heavy chain type 2B (BF.F3, Developmental Studies Hybridoma Bank); 1:5 diluted, supernatant Pax7 (Developmental Studies Hybridoma Bank). The myosin heavy chain antibodies were directed toward bovine skeletal muscle myosin (Schiaffino et al., 1989).
After extensive washing with PBS, tissues were incubated for 40 min with goat anti-mouse Alexa Flour 568 (1:500; Invitrogen, San Diego, CA) for -dystrophin, or goat anti-mouse biotin (1:100; Vector Laboratories, Burlingame, CA), followed by steptavidin Alexa Flour 488 (1:500; Invitrogen) for Pax7 and myosin heavy chain isoform detection. After Pax7 immunostaining, Hoechst 33245 (1 µg/mL in PBS) was used to identify total nuclei. Fiber-associated nuclei (FAN) were visualized with propidium iodide (1 µg/mL; Invitrogen).
After a final PBS wash, the cover slips were mounted on the slides, and immunostaining was evaluated using an Eclipse TE 2000-U stage microscope (Nikon, Lewisville, TX) equipped with an X-Cite 120 epifluorescence illumination system (EXFO, Mississauga, Ontario, Canada). Images were captured at 100x magnification using a DXM 1200F digital camera (Nikon) and analyzed for individual muscle fiber area and diameter and total number of FAN per field using the NIS-Elements computer system (Nikon). For each set of serial cryosections, 4 images from the same area of each cryosection were collected for each myosin heavy chain isoform (Figure 1). Fibers that were reactive with the antibody to the specific myosin heavy chain isoform were counted, and fiber area was defined as the region constrained by -dystrophin immunostaining. Diameter was measured by the computer system, rotating every 90° around the fiber and taking a diameter measurement, and subsequently averaging the measurements. For each animal, a minimum of 475 fibers was measured and used for analysis. Fiber-associated nuclei was defined as propidium iodide-stained cells contained within an -dystrophin boundary. Nuclei that were identified with Hoechst dye and that were Pax7-positive were counted as being a satellite cell.
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).
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; Cranwell et al., 1996a; Schnell et al., 1997). Fiber frequencies were tabulated and compared by 
2 analysis using PROC FREQ (SAS Inst. Inc., Cary, NC). Treatment group frequencies within a fiber type were compared with one another by a 2-sample t-test for proportions. Data for fiber area and diameter were sorted and analyzed by individual fiber type, whereas FAN and Pax7 nuclei were not sorted. Data were analyzed with PROC MIXED of SAS, where implant status, dietary treatment, and their interaction were the fixed effects. Random effects included BW replicate, truckload, and animal within treatment. Each combination of BW replicate and truckload were grouped and used in the random statement. Pairwise comparisons between the least squares means of the factor levels were computed by using the PDIFF option of the LSMEANS statement. The UNIVARIATE procedure of SAS was used to generate histograms and to analyze the distributions of fiber diameter and area within each treatment group for each fiber type. |
RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Muscle fiber diameters were measured following colocalization of myosin and dystrophin. There was a significant (P < 0.01) RAC x TBA interaction for type I fibers. The main effects of TBA and RAC increased (P < 0.05) the diameter and computed cross-sectional area (CSA) of type I fibers (Table 1). Feeding RAC to TBA implanted cull cows did not further increase the LM fiber diameter (P = 0.88) or CSA (P = 0.77). No change (P > 0.05) in the CSA or diameters of type II fibers occurred in response to TBA, RAC, or TBA + RAC. No differences (P > 0.05) in the percentage of type I and type II fibers were observed across the control, RAC, and TBA treatments (Table 1). There was an increase (P < 0.001) in the percentage of type II fibers in the LM of cull cows treated with RAC/TBA.
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). Curve of best fit for type II muscle fiber histograms do not differ (P = 0.18) between the groups (Figure 4).
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). Satellite cell number, as measured by antiPax7 at 48 h postmortem, did not change (P > 0.89) in response to any treatment (Table 2).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). A 2-fold increase in the numbers of type IIB fibers, at the expense of the type IIA fibers, was recorded in RAC-supplemented hogs (Depreux et al., 2002). In the current study, neither type IIB nor type IIX muscle fibers were detected by immunocytochemistry. The population of type IIA muscle fibers remained constant at approximately 68% of the total fibers in cattle fed RAC or implanted with TBA. A greater percentage of type IIA fibers at the expense of type I fibers was observed in TBA implanted cows fed RAC. This result is intriguing in light of our inability to detect a change in the proportion of type II fibers in cows fed RAC only. In cattle and mice, it is well established that beta agonists increase the percentage of type IIA fibers at the expense of type I fibers (Vestergaard et al., 1994; Rajab et al., 2000; Bricout et al., 2004). By contrast, anabolic agents have no effect on the distribution of type I or type IIA fibers (Ono et al., 1996; Fritsche et al., 2000). The underlying mechanism of action of RAC/TBA that elicits a change in myosin isoforms remains unknown and warrants further investigation.
The type IIA antibody used in this study reportedly cross reacts with myosin IIX and IIB in cattle (Duris et al., 2000) and type IIX fibers in swine (Depreux et al., 2000). Based on the assumption of that all type II fibers are immunoreactive, 60% the LM fibers in cull cows are classified as fast, a value in agreement with prior publications. Using enzymatic techniques, Brandstetter et al. (1998) identified the percentage of type I, type IIA, and type IIB fibers as 25, 25, and 50%, respectively, in the LM. Fritsche et al. (2000) reported a similar percentage with 20 to 30% of fibers classified as oxidative and approximately 55% of the total fibers present as fast glycolytic fibers. In a similar manner, the inability to detect type IIB fibers may be a limitation of the immunodetection method. Watson et al. (2003) failed to detect type IIB fibers in harbor seals using the same antibody. Duris et al. (2000) reported this antibody demonstrates a low specificity for type II myosin heavy chain in bovine tissues and indicated that antimyosin IIB (N3.36) is a suitable alternative. However, monoclonal N3.36 failed to detect type IIB fibers in the LM of cull cows fed RAC. Our inability to detect myosin type IIB fibers agrees with Tanabe et al. (1998) and Toniolo et al. (2005), who were unable to establish the presence of myosin IIB in bovine LM by semiquantitative reverse transcription-PCR. Chikuni et al. (2004) also reported a lack of myosin IIB mRNA as measured by real-time PCR, and hypothesized the absence of this isoform may explain the differences between beef and pork. Thus, the inability of RAC to shift myosin expression to the fastest isoform may be unique to cattle. Alternatively, fiber type shifts in response to RAC may occur only in muscle that normally expresses limited amounts of myosin IIB.
Quartile analysis of all type II fibers demonstrates no apparent difference in the percentage of fibers present with a larger diameter in RAC cull cows by comparison with control suggesting that the response of IIA fibers was minimal. Indeed, control animals appear to have the greatest percentage of large diameter fibers that may represent type IIB. The unresponsive nature of type II fibers of cull cows to RAC in the current study may reflect the muscle environment. Finishing hogs fed normal or supraphysiological levels of RAC contained an equivalent amount of myosin IIA in the LM as control animals. By contrast, the semintendinosus muscle contained less myosin IIA in response to RAC (Depreux et al., 2002). Alternatively, the resident type II population may be refractile to growth enhancing agents due to the advanced age of the animal. The differential response of bovine and porcine type II muscle fibers to the beta agonist is intriguing and warrants further investigation.
A substantial increase in the size of type I fibers was evident in cows treated with RAC or TBA. Administration of TBA to cull cows increased LM area and carcass fat-free lean (Cranwell et al., 1996b). In a similar manner, implantation of feedlot steers with TBA increased type I and IIA CSA in the LM (Hughes et al., 1998). Thus, the larger diameter type I fibers in cull cows receiving TBA reflects previous reports. Conversely, an increase in LM type I CSA by RAC supplementation is novel. No change in the size of type I fibers is apparent in finishing hogs supplemented with RAC (Aalhus et al., 1992). The LM type I fibers from lambs fed cimaterol showed no change to a modest 15% increase in fiber CSA (Beermann et al., 1987; Kim et al., 1987). The 30% increase in type I CSA found in RAC-supplemented cows suggests that this population of cells is 2 to 3 times more responsive in cattle than other species. Further support for species differences is reflected by a 35% larger type I CSA in bulls fed cimaterol (Vestergaard et al., 1994). However, cull cows are unique in that their type II fiber population is completely refractile to RAC induced hypertrophy, whereas cimaterol stimulated hypertrophy in bulls (Vestergaard et al., 1994). The mechanism behind the differential response in cattle may be a reflection of the age of the animal. During aging in humans, the numbers of type IIA/X muscle fibers and size are reduced (Lee et al., 2006; Verdijk et al., 2006). Type I fiber CSA remains unchanged largely, but the percentage of type I fibers are increased. In addition, the ability of the muscle to respond to hypertrophic events is altered in extreme age (for review see Carmeli et al., 2002). Advanced age in rodents, birds, and humans demonstrates an impaired ability to increase in size that is associated with reduced type II mucle fiber numbers (Carson et al., 1995; Blough and Linderman, 2000; Short et al., 2005; Lee et al., 2006). The percentage of type I and II fibers is established by 24 mo of age in cattle, irrespective of breed, and the LM is composed predominantly of type I and IIB (Wegner et al., 2000; Kirchofer, et al., 2002). In the event that type II fibers are limited in their protein synthetic capacity and are intolerant to hypertrophic stimuli, the nutrients supplied by diet coupled with the partitioning agents lead to an excess of available substrate for type I fiber growth.
One of the primary reasons for supplementing livestock with RAC or TBA is to improve carcass value. Schroeder et al. (2005a, b) reported an increase in ribeye area and fat-free lean in steers and heifers fed RAC. Therefore, based on the findings above we predicted that cull cows receiving 15 ppm RAC daily for 35 d would possess a larger REA. However, no improvement in carcass characteristics, including REA, due to RAC or TBA supplementation was detected (Carter et al., 2006). This may be a reflection of an unresponsive type II fiber population. Approximately 30% of the total number of fibers was present as type I. Based on the 30% increase in type I size found in young bulls (Vestergaard et al., 1994), we would predict a minimal 9% increase in REA. This small change may require more animals to reach statistical significance.
Postnatal skeletal muscle growth is accomplished through the satellite cell population. These normally quiescent muscle stem cells become mitotically active, proliferate, and fuse into existing muscle fibers (for review see Collins, 2006). The number of satellite cells declines with age, and the activation potential of these cells is reduced in older individuals (Collins and Partridge, 2005). In aged rats and elderly humans, satellite cells represent 1 to 2% of the total myonuclei (Gallegly et al., 2004; Sajko et al., 2004; Brack et al., 2005). The number of Pax7 immunopositive satellite cells in cull cows represents approximately 1% of the total number of myonuclei, in close agreement with rodent data. Satellite cells isolated from TBA implanted steers exit the dormant state sooner than their contemporaries, suggesting that the anabolic steroid affects self-renewal and subsequent proliferation. In addition, these cells fused into larger muscle fibers in vitro (Johnson et al., 1998). The enhanced myogenic capabilities may account for the larger fiber sizes found in TBA-implanted cull cows. Alternatively, TBA increased circulating levels of IGF-I and autocrine synthesis of the growth factor (Thompson et al., 1989; White et al., 2003; Kamanga-Sollo et al., 2004). It is possible that the increased type I fiber diameters and calculated CSA are a product of elevated IGF-I activities.
Satellite cells proliferate, differentiate, and fuse with the muscle fibers to provide FAN for increased contractile gene expression and maintenance of the myonuclear domain (Aberle et al., 2001). The lack of an increase of FAN found in all the supplemented cows indicates that the 2 growth promotants may act through a similar pathway of altered protein synthesis, degradation rates, or both. It was reported that RAC stimulates protein synthesis without an apparent effect on satellite cell cycle kinetics or fusion (Shappell et al., 2000). Beta-agonists may also augment nutrient supply to the muscle cell by increasing blood flow. Muscle accretion is supported and possibly bolstered by the enhanced delivery of substrates and energy needed for protein synthesis (Mersmann, 1998). In several swine and cattle studies, the mechanism of muscle growth due to ractopamine supplementation was attributed to enhanced protein synthesis (Smith et al., 1987; Dunshea et al., 1993, 1998; Williams et al., 1994). Beermann et al. (1987) and Kim et al. (1987) reported that DNA concentration per gram of protein was less in beta-agonist-supplemented lambs, and both groups concluded that muscle growth was due to a reduction in protein degradation and independent of satellite cell activity. A similar observation was reported for rats (Maltin et al., 1986) and lambs (Bohorov et al., 1987) fed clenbuterol. The discrepancies between increased protein synthesis or reduced degradation rates may be a reflection of the beta-agonist fed and the beta-receptor isoform activated.
Conclusion
Supplementing cull cows with RAC or TBA alone or the combination increased LM fiber CSA and diameter for type I fibers, although had no effect on type II fibers. There were no fiber type shifts between the different myosin heavy chain isoforms due to the presence of TBA or RAC. Fiber-associated or satellite cell numbers were not affected by the RAC or TBA treatments. The lack of effect on FAN and constant satellite cell numbers suggests that any hypertrophy occurred due to changes in protein synthesis, degradation rates, or both. It is hypothesized that because protein synthesis is limited in older animals, this prevented the response to TBA and RAC normally seen in younger animals.
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Footnotes |
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2 Corresponding author: sjohnson{at}animal.ufl.edu
Received for publication September 12, 2006. Accepted for publication April 17, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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