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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
Department of Human Nutrition, Food and Animal Sciences, University of Hawaii at Manoa, Honolulu 96822
1 Corresponding author: ykim{at}hawaii.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: myostatin • propeptide • broiler • antibody
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During developmental myogenesis, myostatin appears to suppress myoblast proliferation and differentiation by controlling cell cycle progression and the level of myogenic regulatory factors (Thomas et al., 2000; Taylor et al., 2001; Langley et al., 2002; Rios et al., 2002), explaining the muscle hypertrophy observed in animals with nonfunctional myostatin mutations. Myostatin also plays a role in postnatal skeletal muscle growth. Conditional myostatin knockout, exhibiting only a postnatal inactivation of myostatin, caused a dramatic increase in muscle mass (Grobet et al., 2003). Transgenic mice overexpressing myostatin propeptide, which is known to suppress the biological activity of myostatin, postnatally exhibited an increase in skeletal muscle mass mostly through hypertrophy of muscle fiber (Yang et al., 2001). Postnatal control of muscle mass by myostatin appears to be through the inhibition of satellite cell proliferation (McCroskery et al., 2003; McFarland et al., 2006), the activation of the ubiquitin proteolytic system in conjunction with the suppression of the IGF-I/PI(3)K/AKT hypertrophy pathway (McFarlane et al., 2006), or both. Many studies also have shown that there exists a negative correlation between the level of myostatin and muscle mass under various physiological or pathological conditions that induce muscle loss or gain in animal (Carlson et al., 1999; Kirk et al., 2000; Lalani et al., 2000; Mendler et al., 2000; Wehling et al., 2000; Armand et al., 2003; Yamaguchi et al., 2006) and human models (Gonzalez-Cadavid et al., 1998; Reardon et al., 2001; Welle et al., 2002). These findings together indicate that blocking myostatin activity during embryonic development or postnatal growth will be an effective strategy to improve skeletal muscle growth of animals.
Anti-myostatin antibodies have been tried as a means of modulating the activity of myostatin in laboratory animal models. Hypertrophy of muscle fiber resulted after the administration of antimyostatin antibodies to adult mice (Whittemore et al., 2003). In a dystrophic mouse model, antimyostatin antibody administration ameliorated the muscular dystrophic conditions with an increase in body mass, whole muscle cross-sectional area, and muscle fiber areas (Bogdanovich et al., 2002). Consistent with the above results, we have recently shown that in ovo administration of a monoclonal antimyostatin antibody that was raised against mature myostatin improved posthatch broiler growth and skeletal muscle mass (Kim et al., 2006), suggesting that skeletal muscle growth of meat-producing animals can be improved by blocking myostatin activity. Because polyclonal antibodies offer some advantage, such as cost of production, over monoclonal antibodies, in this study, we generated a polyclonal antibody against an unprocessed recombinant myostatin and examined the effect of in ovo administration of the antibody on posthatch broiler growth and skeletal muscle mass.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ampicillin, chloramphenicol, isopropyl-ß-D-thiogalac-topyranoside, lysozyme, Luria-Bertani agar, and Luria-Bertani broth were purchased from Fisher Scientific (Pittsburgh, PA). Anti-rabbit IgG conjugated to alkaline phosphate (AP), anti-goat IgG conjugated to AP, and bromo-chloro-indolyl phosphate and nitroblue tetrazolium liquid substrate were purchased from Sigma (St. Louis, MO). Recombinant mouse GDF-8, recombinant human bone morphogenetic protein-(BMP)2, BMP-3, and BMP-5, recombinant human TGF-ß3, and porcine TGB-ß1 and ß2 were purchased from R&D Systems (Minneapolis, MN). The PCR reaction mixture was purchased from Gibco BRL Inc. (Rockville, MD). Unless otherwise indicated, all other chemicals and reagents were purchased from Fisher Scientific or Sigma.
Construction of Myostatin Expression Vector and Expression of Recombinant Chicken Myostatin
The procedure for myostatin expression vector construction was the same as described in our previous study (Kim et al., 2006) except the PCR primer design. The primer combination was designed to yield a PCR product corresponding to bases 1 to 1,125 of the reported unprocessed chicken myostatin mRNA sequence (GeneBank Accession number, AF019621 [GenBank] ). The forward and reverse primers were 5'-ATGCAAAAGCTAGCAGTCTATG-3' and 5'-TCATGAGCACCCGCAACGATC-3', respectively. Myostatin expression was also the same as described previously (Kim et al., 2006). The expression of recombinant myostatin with molecular weight of 46 kDa (AVM46) was confirmed by SDS-PAGE analysis.
Isolation of Myostatin Inclusion Bodies and Purification of Recombinant Myostatin
Inclusion bodies containing unprocessed myostatin were prepared from cell pellets as described previously (Kim et al., 2006). Myostatin inclusion bodies were solubilized (3 mg protein/mL) in commercially available 50 mM CAPS buffer (pH 11) containing 0.3% N-laurylsarcosine and 1 mM dithiothreitol (DTT; Protein Refolding Kit, Novagen, WI). Inclusion body proteins were fractionated by SDS-PAGE under reducing conditions. The myostatin band was cut out and electro-eluted to prepare purified myostatin. The electro-eluted myostatin proteins were dialyzed first in 20 mM Tris buffer (pH 8.5) containing 1 mM DTT and second in the same buffer without DTT. Dialysis volume was 50 times the volume of sample, and each dialysis was done at 4°C with dialysis buffer change at 5 h intervals. The electro-eluted unprocessed myostatin was used as an antigen for polyclonal antibody production and as a coating antigen in ELISA. The purity of the antigen was verified by SDS-PAGE (Figure 1
).
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The Institutional Animal Care and Use Committee, University of Hawaii approved the protocol for the production of polyclonal antibodies using rabbits. New Zealand White female rabbits weighing approximately 2 kg were subcutaneously injected with antigen (1 mg) suspended 1:1 in Complete Freund’s adjuvant solution (1 mL). Three additional boosters were given 2 to 3 wk apart after primary immunization. Final bleedings were done 4 d after the last booster. Collected blood was placed at 4°C overnight and centrifuged at 10,000 x g for 10 min to collect sera.
Anti-myostatin antibodies were purified by Protein A affinity chromatography (Affi-gel protein A MAPS II kit, BioRad) following the manufacturer’s instruction. The purified IgG (antimyostatin antibody; pAb-AVM46) in each fraction were analyzed by SDS-PAGE under reducing and nonreducing conditions.
Enzyme-Linked Immunosorbent Assay
Enzyme-linked immunosorbent assay was used to examine antibody titers during the immunization. Coating antigen was the recombinant chicken myostatin that was purified as described earlier. Then 2.5 µg of recombinant myostatin proteins (50 µg/mL) were added to each well of a microELISA plate and incubated for 2 h at room temperature. The plate was washed with PBS (20 mM sodium phosphate, 15 mM NaCl, pH 7.4), and 100 µL of 1% BSA in PBS was added to each well, then incubated at room temperature for 1 h. The plate was washed with PBS, then 50 µL of various diluted antiserum was added to each well containing antigen for 1 h at room temperature. After incubation, the plate was washed 3 times with PBS-0.05% Tween-20, then 50 µL of antirabbit IgG AP conjugate in PBS-0.05% Tween-20 (1:10,000) were added and incubated at room temperature for 1 h. The plate was washed 3 times with PBS-0.05% Tween-20, and 50 µL of 4-nitrophenyl phosphate was added and incubated for 30 min in the dark at room temperature. The reaction was stopped by adding 25 µL of 3 N NaOH, and optical density was measured at 405 nm on a microplate reader.
SDS-PAGE Analysis
The SDS-PAGE was performed on mini gels by the method of Laemmli (1970) using 18% polyacrylamide gels in the presence of 0.1% SDS under reducing or nonreducing conditions. The gels were stained with Coomassie blue or subject to electophoretic transfer onto a nitrocellulose membrane for Western blot analysis.
Western Blot Analyses
Antibody binding specificity was examined using Western transfer and immunoblotting. Proteins fractionated by SDS-PAGE were electrophoretically transferred onto nitrocellulose membrane while immersed in Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS). After transfer, membranes were blocked with Tris buffered saline (125 mM NaCl, 25 mM Tris, pH 8.0) buffer containing 0.5% Tween 20 for 1 h at room temperature. Membranes were incubated for 1 h at room temperature in Tris buffered saline containing the polyclonal antimyostatin antibody (pAb-AVM46, 0.05 µg/mL). After washing, the membrane was reacted to AP conjugated anti-rabbit IgG (1:10,000 times dilution; Sigma, St. Louis, MO) for 1 h at room temperature. After washing, blots were developed using bromo-chloro-indolyl phosphate and nitroblue tetrazolium.
In Ovo Injection of pAb-AVM46
In experiment 1, 170 fertilized broiler eggs from a local hatchery were divided into 3 groups: no injection (Control, 37), injection into the albumen (Albumen, 66), and injection into the yolk (Yolk, 67). Egg weights were between 57 to 70 g with mean values and standard deviations being 63.2 ± 2.30 (Control), 63.2 ± 2.30 (Albumen), and 63.2 ± 2.29 (Yolk), respectively. In 1 preliminary study, no difference in BW was observed between the broilers from eggs injected with no-immune IgG (27 birds) into the albumen and the broilers from eggs with no injection (20 birds) during the 23-d growout period. Another preliminary study also demonstrated no difference in growth during the 5 wk growout period between the broilers from eggs injected with buffer alone in the albumen (22 birds) and the broilers from eggs with no injection (47 birds). Thus, we did not include a group with buffer alone or nonimmune IgG. On d 3 of incubation, eggs were injected with 35 µg of the pAb-AVM46 in 50 µL of PBS into the albumen or the yolk as described previously (Kim et al., 2006); then the eggs were placed into an egg incubator until hatching. Experiment 2 was designed to examine the dose effects of pAb-AVM46 injection into the yolk on posthatch broiler growth and skeletal muscle mass. One hundred sixty-two fertilized eggs were divided into 3 groups: no injection (Control, 33), 9 µg of pAb-AVM46 injection into the yolk (Yolk 1, 60), and 70 µg of pAb-AVM46 injection into the yolk (Yolk 2, 69). Egg weights were between 57 to 71 g with mean values and standard deviations being 63.3 ± 3.03 (Control), 63.4 ± 2.90 (Yolk 1), and 63.2 ± 2.96 (Yolk 2), respectively. On d 3 of incubation, eggs were injected with pAb-AVM46, then placed into an egg incubator until hatching.
Bird Care
Egg incubation and animal care were approved by the Institutional Animal Care and Use Committee. After hatching, chicks were placed to battery pens equipped with an electric heater. Initially, 3 pens were randomly allocated for each group. Because the size of the pen was not adjustable, the number of animals in each pen was adjusted as animals grew, resulting in gradual increase in number of pens for each group. Heater temperature was set to 35, 32, and 30°C in the first, second, and third week, respectively. After the third week, the heater was turned off, and the room temperature was maintained at 26.6°C in a thermostatically controlled building with a 12L:12D light cycle. During the first 3 wk, chicks were fed a commercial broiler starter diet containing 22% CP, 1% lysine, and 0.45% methionine, then they were fed a commercial grower diet containing 16.0% protein, 0.5% lysine, and 0.25% methionine. Water and diet were provided ad libitum. Weekly BW were recorded, and group feed consumption was measured daily. Birds were killed by CO2 inhalation after 3 h of fasting at 4 wk in experiment 1 and at 5 wk in experiment 2. After CO2 inhalation, the birds were weighed, decapitated at the first cervical vertebra, bled, scalded at 56°C for 30 s, defeathered, and dressed. The abdominal contents were eviscerated, and heart, liver, spleen, and abdominal fat were separated and weighed. At 24 h later, breast muscle was separated and weighed. Whole thighs and legs with bone in were also separated and weighed.
Statistical Analysis
The ANOVA was performed by GLM procedure using JMP software (SAS Institute, Cary, NC). The model included main effects of treatment, sex, and treatment x sex interaction. Tukey’s honestly significant difference test was used to compare the means in treatment groups when a significant effect was observed (P 
0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Western blot analysis, the polyclonal antimyostatin antibody showed high affinity to the myostatin propeptide, but almost no affinity to mature myostatin (GDF-8; Figure 2). When cross-reactivity of pAb-AVM46 with some members of the TGF-ß superfamily was examined in Western blot analysis, porcine TGF-ß1 and recombinant human BMP-2 showed an affinity to the pAb-AVM46, but porcine TGF-ß2, recombinant human TGF-ß3, BMP-3, and BMP-5 did not show affinity to the antibody (Figure 3). Very weak binding of the antibody to GDF-8 was observed (Figure 3).
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Table 1 summarizes the hatchability after in ovo administration of the pAb-AVM46 and number of birds killed at 4 wk in experiment 1. Consistent with previous studies that reported a decreased hatchability after in ovo injection of growth factors, amino acids, or IgG (Kocamis et al., 1998; Kocamis et al., 1999; Kim et al., 2006), hatchability was decreased by in ovo injection of the antibody. At 3 and 10 d posthatch, 4 chicks from each group were killed to collect blood for titer measurement. We could not detect titer values at 3 or 10 d posthatch in any of the treatment groups. Whereas no leg deformation was observed in the control birds, posthatch leg deformations that made the birds unable to stand properly were observed in the injection groups; thus 2 birds in the Albumen group and 3 birds in the Yolk group were culled.
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). Regardless of treatment, a significant sex effect was observed on BW, starting at 3 wk after hatch. When the feed/gain ratios were calculated for each treatment during the growout period, they were 1.47, 1.50, and 1.49 for the Control, Albumen, and Yolk groups, respectively. Because group feed consumption was measured, the data could not be statistically analyzed. Table 3 summarizes the effect of in ovo administration of the pAb-AVM46 on carcass weight, dressing percent, combined thigh and leg weight, breast muscle weight, and weight of some internal organs. Except for the combined thigh and leg weight, no significant treatment effect was observed on any of the parameters measured. The birds from eggs administered with the pAb-AVM46 into the yolk had 7.6% lower combined thigh and leg weight than that of control birds.
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summarizes hatchability, number of birds killed at 5 wk, and number of birds showing leg problems. Consistent with the results of experiment 1, some birds injected with the pAb-AVM46 showed posthatch leg deformation. Four and two birds from the Yolk 1 and 2 groups, respectively, were culled in the early growout period. Some leg deformation was also noticed at execution from each group (Table 4).
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). The feed/gain ratios for the Control, Albumen, and Yolk groups during the whole growout period were 1.55, 1.66, and 1.63, respectively. Consistent with experiment 1, birds from eggs injected with higher dose of the pAb-AVM46 had significantly lighter combined thigh and leg weight than control birds (Table 6). The difference was about 11.6%. The percent of the combined thigh and leg weight to BW of the Yolk 2 group was also significantly lower than that of the control group (20.95 vs. 23.08%). No difference was observed between the control birds and birds from eggs injected with a lower dose (9 µg).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Binding Characteristics of Polyclonal Antimyostatin Antibody
The polyclonal antimyostatin antibody (pAb-AVM46) that was produced against unprocessed myostatin showed high affinity to the myostatin propeptide with little affinity to the mature form of myostatin (Figure 2
), indicating that the majority of the polyclonal antibody populations recognize epitopes present in the propeptide region of unprocessed myostatin. The amino acid sequence of the active form of myostatin is well conserved, whereas the propeptide region shows sequence variability among mammalian species (McPherron et al., 1997). It, thus, is likely that the sequence variability present in the propeptide region of unprocessed myostatin induced the production of antibodies recognizing epitopes mostly in the propeptide region.
Because pAb-AVM46 showed strong affinity to the propeptide, we examined whether pAb-AVM46 could recognize the propeptide in skeletal muscle. In leg muscle, the antibodies showed affinity to 2 proteins with molecular weight 37 and 30 kDa. The location of the 37-kDa band matched the location of the propetide when we put propetide and skeletal muscle proteins side by side in Western blot analysis (Figure 4). It was, thus, tempting to suggest that in Western blot analysis, the pAb-AVM46 could recognize propeptide in skeletal muscles. Considering that unprocessed myostatin and mature myostatin were reportedly detectable in skeletal muscles using Western blot analysis (Sharma et al., 1999; Sato et al., 2006), it was expected that the pAb-AVM46 could also recognize the unprocessed myostatin. Because no band at the molecular weight of unprocessed myostatin (50 kDa) showed affinity to the pAb-AVM46 in our numerous Western blot analyses, it was questionable that the 37-kDa band detectable by the pAb-AVM46 in skeletal muscle was due to the presence of propeptide. In our Western blot analysis with skeletal muscles from cattle, pig, and mouse, the 37-kDa band was detectable by the pAb-AVM46 with almost equal band strength (Lee, 2003). Because the propeptide has sequence variability among species, unlike mature myostatin, the above result further raised a possibility that the 37-kDa band detectable by the pAb-AVM46 resulted from a nonspecific affinity rather than due to the presence of propeptide. Therefore, we did partial purification of the 37-kDa protein detectable by the pAb-AVM46 using a combination of ion-exchange and hydrophobic chromatography; then the SDS-PAGE fractionated 37-kDa band was subjected to N-terminal (10 amino acids) sequence analysis (data not shown). The N-terminal sequence was PHQYPALTPE, and a Blast search resulted in 90% match to human and mouse aldolase A (363 amino acids with 39.2 kDa). Because the adolase A sequence is well preserved among mammalian species and most abundant in skeletal muscle, it is highly probable that the 37-kDa band detectable by the pAb-AVM46 in skeletal muscle was aldolase A. There is the possibility of propeptide contamination in our partial purification of the 37-kDa protein detectable by the pAb-AVM46; thus further validation is necessary for this antibody to be used in measuring tissue propeptide concentration using ELISA or Western blot.
Effects of In Ovo Administration of pAb-AVM46 on Posthatch Broiler Growth and Muscle Mass
Myostatin, similar to many other TGF-ß family members, is produced as a prepropeptide composed of a N-terminal propeptide and a C-terminal mature form (McPherron et al., 1997). The mature form of myostatin is released upon removal of N-terminal propeptide by proteolysis at the conserved tetrabasic (RSRR) site (Lee and McPherron, 2001). Mature myostatin protein, like many other TGF-ß members, forms a homodimer that binds to ActRIIB receptors located on the muscle cells for its signal transduction process of regulating skeletal muscle mass (Lee and McPherron, 2001). The mature myostatin dimer remains biologically inactive by forming a latent complex with myostatin propeptide (Lee and McPherron, 2001; Thies et al., 2001; Zimmers et al., 2002) until its activation by releasing the propeptide through proteolysis via the BMP1/TLD family of metalloproteinases (Wolfman et al., 2003). In support of the myostatin inhibitory role of propeptide, overexpressing propeptide increased muscle mass in mice (Lee and McPherron, 2001; Yang et al., 2001).
Because the pAb-AVM46 raised against the unprocessed myostatin showed strong affinity to propeptide, we postulate that in ovo administration of this antibody might suppress posthatch skeletal muscle growth in broilers if binding of the antibody to propeptide suppresses latent complex formation of myostatin, thus enhancing mature myostatin biological activity. Our current results of decreased thigh and leg weight upon in ovo administration of the pAb-AVM46 support this postulation. The decrease in combined thigh and leg weight was dose dependent: 7.6% decrease with 35-µg administration and 11.6% decrease with 70-µg administration. Similar to our previous study in which the effect of in ovo administration of monoclonal antimyostatin antibody was investigated on posthatch skeletal muscle growth (Kim et al., 2006), the response of decreased skeletal muscle growth was only observed when the antibody was administered into the yolk not into the albumen area. Even though we verified that in ovo injection of nonimmune IgG into the albumen did not affect posthatch broiler growth, we did not examine the effect of in ovo injection of nonimmune IgG into the yolk on posthatch broiler growth. For this reason, a question can be raised whether the response of suppressed skeletal muscle growth observed in the Yolk group was truly due to the effect of the antibody. Previously, we observed that in ovo administration of a monoclonal antimyostatin antibody into the yolk improved posthatch skeletal muscle growth of broilers (Kim et al., 2006). Therefore, it is likely that the response of suppressed skeletal muscle growth observed in the Yolk group was due to the effect of the antibody.
Interestingly, we observed leg deformations from 6 to 28.6% in some broilers from eggs injected with the antibody. The leg deformation appeared to be mostly related to the inability of the knee joint to hold the leg straight. In some broilers, the leg deformation was severe and detectable within 2 wk posthatch, but in some broilers, the problem was mild with curved legs. The pAb-AVM46 showed strong cross-reactivity with BMP-2 and weak cross-reactivity with TGF-ß1. It is also well known that TGF-ß1 and BMP-2 regulate bone and cartilage formation (van den Berg et al., 2001; Ryoo et al., 2006). Taken together, it is only speculated that the leg deformation observed in broilers administered in ovo with the pAb-AVM46 was associated with the cross-reactivity of the antibody with BMP-2 and TGF-ß1. However, it should be noted that in our previous study (Kim et al., 2006), we did not observe any leg deformation after in ovo administration of a monoclonal antimyostatin antibody that showed cross-reactivity with BMP-2 but not with TGF-ß1. At the same time, leg deformation was also observed in the group injected with the antibody into the albumen, a group that did not show posthatch decreased muscle growth upon in ovo administration of the antibody. Therefore, to understand whether the cross-reactivity of the pAb-AVM46 caused the leg deformation, more focused studies are needed, including examination of the ability of the antibody to suppress the biological activity of BMP-2 and TGF-ß1, examination of the bone or cartilage development after targeted administration of the antibody to a specific area at a specific developmental period, or both. Unlike the pAb-AVM46 that was generated against the recombinant unprocessed myostatin, polyclonal antibodies generated against peptide fragments of the propeptide or myostatin would recognize much less epitopes, resulting in less cross-reactivities. Future studies, thus, probably need to consider using polyclonal antibodies generated against peptide fragments of myostatin or propetide in investigating the effect of antimyostatin antibodies on body and skeletal muscle growth.
In conclusion, the results of this study indicate that unprocessed full-length myostatin as an immunogen produces antibody populations having affinity mostly to the propeptide with little to the mature form. The decreased muscle weight observed in the broilers from eggs injected with the antibody in the yolk suggests that myostatin propeptide suppresses the biological activity of myostatin in broilers and that immunoneutralization of the propeptide decreases muscle growth. Thus, it appears that enhancing propeptide activity is a potential means to improve skeletal muscle growth in broilers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 13, 2007. Accepted for publication February 10, 2007.
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