Alpha-tocopherol acetate and alpha lipoic acid may mitigate the development of wooden breast myopathy in broilers at an early age.

1. The objective of this study was to identify the effects of the antioxidant alpha-tocopherol acetate (ATA) and alpha lipoic acid (ALA) which have anti-inflammatory effects on developmental onset, severity and the progression of wooden breast (WB) based on Pectoralis major (P. major) muscle morphology and expression of genes associated with WB during the first three weeks post-hatch.2. A total of 160 newly hatched Ross 708 broiler chicks were randomly assigned in a replicated trial to either a control group or three dietary treatments (ATA 160 mg/kg feed, ALA 500 mg/kg feed or in combination).3. Microscopic changes associated with WB began at one week of age in all groups. The ATA acetate and ALA fed in combination decreased WB severity at two weeks of age (P = 0.05) and ATA alone or in combination reduced severity at three weeks of age compared to the control group (P = 0.05). Expression of myogenic determination factor 1 and peroxisome proliferator-activated receptor gamma was reduced in all dietary treatments compared to the control at three weeks of age (P ≤ 0.05), which suggested reduced muscle degeneration and lipid deposition.4. ATA and ALA fed both independently and in combination had a positive effect on mitigating WB severity microscopically as early as two weeks of age.

Phosphorylation state of pyruvate dehydrogenase and metabolite levels in turkey skeletal muscle in normal and pale, soft, exudative meats.

1. Turkey production has increased dramatically as genetic selection has succeeded in increasing body weight and muscle yield to fulfil increasing consumer demand. However, producing fast-growing, heavily muscled birds is linked to increased heat stress susceptibility and can result in pale, soft, exudative (PSE) meat. Previous studies indicated that pyruvate dehydrogenase kinase 4 (PDK4) is significantly reduced in PSE samples, suggesting this as a candidate gene associated with the development of this problem.2. The objective of this study was to determine whether pre-market thermal challenge results in PSE meat as a result of differential expression of PDK4. Two genetic lines of turkeys were used in this study; the Randombred Control Line 2 (RBC2) and a commercial line. Turkeys were exposed to a pre-market thermal challenge of 12 h at 35°C followed by 12 h at 27°C for 5 d. Birds were slaughtered and processed according to industry standards. Pectoralis major samples were categorised as PSE or normal based on marinade uptake and cook loss indicators. In the first experiment, the relative expression of pyruvate dehydrogenase (PDH) and the phosphorylation state of PDH in normal and PSE turkey meat were analysed by western blotting. In the second experiment, the same samples were used to measure metabolite levels at 5 min post-mortem, comparing the normal to the PSE samples.3. The results of the first experiment showed that PSE samples had significantly lower total PDH (P = 0.029) compared to normal meat. However, there was no significant difference in the degree of phosphorylation of sites 1, 2 or 3. In the second experiment, there were no significant differences in glycogen, lactate, glycolytic potential or ATP when comparing PSE to control samples.4. These results suggested that a reduction in PDK4 expression alone does not explain the development of PSE meat.

Temperature effect on proliferation and differentiation of satellite cells from turkeys with different growth rates.

Poultry selected for growth have an inefficient thermoregulatory system and are more sensitive to temperature extremes. Satellite cells are precursors to skeletal muscle and mediate all posthatch muscle growth. Their physiological functions are affected by temperature. The objective of the current study was to determine how temperature affects satellite cells isolated from the pectoralis major (p. major) muscle (breast muscle) of turkeys selected for increased 16 wk body weight (F line) in comparison to a randombred control line (RBC2) from which the F line originated. Pectoralis major muscle satellite cells were thermally challenged by culturing between 33°C and 43°C to analyze the effects of cold and heat on proliferation and differentiation as compared to control temperature of 38°C. Expression levels of myogenic regulatory factors: myogenic differentiation factor 1 (MYOD1) and myogenin (MYOG) were quantified by quantitative polymerase chain reaction (qPCR). At all sampling times, proliferation increased at a linear rate across temperature in both the RBC2 and F lines. Differentiation also increased at a linear rate across temperature from 33 to 41°C at all sampling times in both the F and RBC2 lines. Satellite cells isolated from F line turkeys were more sensitive to both hot and cold temperatures as proliferation and differentiation increased to a greater extent across temperature (33 to 43°C) when compared with the RBC2 line. Expression of MYOD1 and MYOG increased as temperatures increased from 33 to 41°C at all sampling times in both the F and RBC2 lines. These results demonstrate that satellite cell function is sensitive to both cold and hot temperatures and p. major muscle satellite cells from F line turkeys are more sensitive to temperature extremes than RBC2 satellite cells.

Effect of the timing of posthatch feed restrictions on the deposition of fat during broiler breast muscle development.

The effect of the timing of posthatch feed restriction on adipose deposition and adipogenic gene expression in the broiler pectoralis major muscle was studied by applying a 20% feed restriction either the first or second week after hatch. Broiler chicks at hatch were divided into a full-fed (control) group and a 20% feed restriction group. The expression of adipogenic genes, peroxisome proliferator-activated receptor gamma (PPARγ), and CCAAT/enhancer-binding protein alpha (C/EBPα) were measured. The expression of both PPARγ and C/EBPα was affected by the wk 1 feed restriction with expression significantly increased during the first week posthatch. The deposition of fat within the pectoralis major muscle was affected by the timing of the feed restriction. Extensive fat depots were present by 27 d of age in the pectoralis major muscle of the wk 1 restricted group compared with the control. Fat deposition was eliminated when the 20% feed restriction occurred in wk 2. Taken together, these results demonstrate that the timing of early posthatch feed restrictions in chicks is critical in the deposition of fat in the pectoralis major muscle and expression of adipogenic genes.

Effect of the timing of posthatch feed restrictions on broiler breast muscle development and muscle transcriptional regulatory factor gene expression.

The effect of the timing of an immediate posthatch feed restriction on broiler pectoralis major muscle development was studied by applying a 20% feed restriction either the first or second week after hatch. Pectoralis major muscle morphological structure and the expression of the myogenic transcriptional regulatory factors, myogenic determination factor 1 (MyoD), myogenic regulatory factor 4 (MRF4), and myogenin, were measured. Broiler chicks at hatch were divided into a full-fed (control) group and a 20% feed restriction treatment administered either the first or second week posthatch. At the end of the feed restriction, the chicks were placed on a full feed ad libitum diet with no further restrictions. Muscle fiber diameter and fiber bundle size of the pectoralis major muscle were smaller in the wk 1 restricted group than the control group by 7 d of age. By 15 d of age through the duration of the study, d 43, both endomysial and perimysial connective tissue spacing were diminished in the wk 1 feed-restricted group. The expression of MyoD, MRF4, and myogenin was affected by the wk 1 feed restriction. The expression of MyoD and MRF4 was significantly increased during the first week posthatch. Both of the genes have been shown to be expressed during proliferation especially MyoD, which is required for muscle cell proliferation. In contrast, myogenin expression was significantly decreased. Myogenin expression is required for differentiation to occur. The morphological changes and gene expression changes observed with the wk 1 feed restriction were eliminated by moving the 20% feed restriction to wk 2, which is after the period of maximal myogenic satellite cell mitotic activity. Taken together, these results demonstrate that the timing of early posthatch feed restrictions to chicks is critical for the morphological development of the pectoralis major muscle and the expression of genes required for muscle satellite cell proliferation and differentiation.

The effect of nutritional status and muscle fiber type on myogenic satellite cell fate and apoptosis.

Satellite cells (SC) are multipotential stem cells that can be induced by nutrition to alter their cellular developmental fate, which may vary depending on their fiber type origin. The objective of the current study was to determine the effect of restricting protein synthesis on inducing adipogenic transdifferentiation and apoptosis of SC originating from fibers of the fast glycolytic pectoralis major (p. major) and fast oxidative and glycolytic biceps femoris (b. femoris) muscles of the chicken. The availability of the essential sulfur amino acids Met and Cys was restricted to regulate protein synthesis during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. Reductions in Met/Cys concentrations from the control level resulted in increased lipid staining and expression of the adipogenic marker genes peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation in the p. major SC. Although b. femoris SC had increased lipid staining at lower Met/Cys concentrations, there was no increase in expression of either adipogenic gene. For both muscle types, SC Met/Cys, concentration above the control increased the expression of peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation. As Met/Cys concentration was decreased during proliferation, a dose-dependent decline in all apoptotic cells occurred except for early apoptotic cells in the p. major, which had no treatment effect (P < 0.05). During differentiation, decreasing Met/Cys concentration caused an increase in early apoptotic cells in both fiber types and no effect on late apoptotic cells except for an increase in the p. major 7.5/24 mg/L of Met/Cys treatment. In general, the viability of the SC was unaffected by the Met/Cys concentration except during proliferation in the p. major 0/0 mg/L of Met/Cys treatment, which increased SC viability. These data demonstrate the effect of nutrition on SC transdifferentiation to an adipogenic lineage and apoptosis, and the effect of fiber type on this response in an in vitro context.

The effect of nutritional status on myogenic gene expression of satellite cells derived from different muscle types.

Satellite cells (SC) are a multipotential stem cell population responsible for facilitating posthatch muscle fiber hypertrophy. The proliferation and differentiation of SC is sensitive to nutritional regimen, and the SC response to nutrition varies depending upon their muscle type of origin. The objective of the current study was to determine the effect of altering protein synthesis on the expression of several key genes regulating SC activity and the effect of muscle type. Satellite cells isolated from the fast glycolytic pectoralis major and the fast oxidative and glycolytic biceps femoris were studied. These genes included the myogenic regulatory factors myogenic determination factor 1 (MyoD) and myogenin, the cell-membrane associated proteoglycans syndecan-4 and glypican-1, the extracellular matrix proteoglycan decorin, and the transcription factor paired box 7. Protein synthesis potential varied by the concentration of the sulfur amino acids Met and Cys during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3.0/9.6, 1.0/3.2, or 0/0 mg/L. A consistent pattern of gene expression emerged following Met/Cys manipulation as increasing reductions in mRNA expression for all genes were observed as Met/Cys concentration decreased, whereas increased Met/Cys concentration caused either no change or had a small negative effect on mRNA expression. Reduced paired box 7 expression would limit myogenic specification of SC, whereas decreased myogenic regulatory factor expression would affect subsequent myogenic development of the SC. Decreased levels of decorin affect SC response to growth factors like myostatin and transforming growth factor ß, and extracellular matrix organization. These data highlight the importance of nutrition on the expression of genes critical for satellite cell activation, proliferation and differentiation, and growth factor signal transduction.

Modulation of turkey myogenic satellite cell differentiation through the shedding of glypican-1.

Glypican-1 is a cell membrane heparan sulfate proteoglycan. It is composed of a core protein with covalently attached glycosaminoglycan, and N-linked glycosylated (N-glycosylated) chains, and is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. Glypican-1 plays a key role in the growth and development of muscle by regulating fibroblast growth factor 2 (FGF2). The GPI anchor of glypican-1 can be cleaved, resulting in glypican-1 being secreted or shed into the extracellular matrix environment. The objective of the current study was to investigate the role of glypican-1 shedding and the glycosaminoglycan and N-glycosylated chains in regulating the differentiation of turkey myogenic satellite cells. A glypican-1 construct without the GPI anchor was cloned into the mammalian expression vector pCMS-EGFP, and glypican-1 without the GPI anchor and glycosaminoglycan and N-glycosylated chains were also cloned. These constructs were co-transfected into turkey myogenic satellite cells with a small interference RNA targeting the GPI anchor of endogenous glypican-1. The soluble glypican-1 mutants were not detected in the satellite cells but in the cell medium, suggesting the secretion of the soluble glypican-1 mutants. Soluble glypican-1 increased satellite cell differentiation and enhanced myotube formation in the presence of exogenous FGF2. The increase in differentiation was supported by the elevated expression of myogenin. In conclusion, the shedding of glypican-1 from the satellite cell surface acts as a positive regulator of satellite cell differentiation and sequesters FGF2, permitting further differentiation.

Expression profiles for genes in the turkey major histocompatibility complex B-locus.

The major histocompatibility complex (MHC) is a highly polymorphic region of the genome essential to immune responses and animal health. In galliforms, the MHC is divided into 2 genetically unlinked regions (MHC-B and MHC-Y). Many MHC-B genes are involved in adaptive or innate immunity, yet others have nonimmune or unknown functions. The sequenced MHC-B region of the turkey (Meleagris gallopavo) contains 40 genes, the majority of which are predicted transcripts based on comparison with the chicken or quail, without direct evidence for expression. This study was designed to test for the presence of MHC-B gene transcripts in a panel of immune and nonimmune system tissues from domestic turkeys. This analysis provides the first locus-wide examination of MHC-B gene expression in any avian species. Most MHC-B genes were broadly expressed across tissues. Expression of all predicted genes was verified by reverse-transcription PCR, including B-butyrophilin 2 (BTN2), a predicted gene with no previous evidence for expression in any species. Previously undescribed splice variants were also detected and sequenced from 3 genes. Characterization of MHC-B expression patterns helps elucidate unknown gene functions and potential gene coregulation. Determining turkey MHC-B expression profiles increases our overall understanding of the avian MHC and provides a necessary resource for future research on the immunological response of these genes.

The effect of nutritional status on myogenic satellite cell proliferation and differentiation.

Early posthatch satellite cell (SC) mitotic activity is a critical component of muscle development and growth. Satellite cells are stem cells that can be induced by nutrition to follow other cellular developmental pathways. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation of SC, using variable concentrations of Met and Cys to modulate protein synthesis. Broiler pectoralis major SC were cultured and treated with 1 of 6 different Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. The effect of Met/Cys concentration on SC proliferation and differentiation was measured, and myonuclear accretion was measured by counting the number of nuclei per myotube during differentiation. The 30/96 mg/L Met/Cys treatment resulted in the highest rate of proliferation compared with all other treatments by 72 h of proliferation (P < 0.05). Differentiation was measured with Met/Cys treatments only during proliferation and the cultures receiving normal differentiation medium (R/N), normal proliferation medium and differentiation medium with variable Met/Cys (N/R), or both proliferation and differentiation receiving variable Met/Cys treatments (R/R). Differentiation responded in a dose-dependent manner to Met/Cys concentration under all 3 of these treatment regimens, with a degree of recovery in the R/N regimen cells following reinstatement of the control medium. Reductions in both proliferation and differentiation were more pronounced as Met/Cys concentrations were further reduced, whereas increased differentiation was observed under the increased Met/Cys concentration treatment when applied during differentiation in the N/R and R/R regimens. The number of nuclei per myotube was significantly decreased in the severely Met/Cys restricted treatments (P < 0.05). These data demonstrate the sensitivity of pectoralis major SC to nutritional availability and the importance of optimal nutrition during both proliferation and differentiation for maximizing SC activity, which will affect subsequent muscle mass accretion.

Differential gene expression between normal and pale, soft, and exudative turkey meat.

In response to high consumer demand, turkeys have been intensively selected for rapid growth rate and breast muscle mass and conformation. The success in breeding selection has coincided with an increasing incidence of pale, soft, and exudative (PSE) meat defect, especially in response to heat stress. We hypothesized that the underlying mechanism responsible for the development of PSE meat arises from differences in expression of several critical genes. The objective of this study was to determine differential gene expression between normal and PSE turkey meat using a 6K turkey skeletal muscle long oligonucleotide microarray. Breast meat samples were collected from Randombred Control Line 2 turkeys at 22 wk of age, and classified as normal or PSE primarily based on marinade uptake (high = normal, low = PSE). Total RNA was isolated from meat samples with the highest (normal, n = 6) and the lowest (PSE, n = 6) marinade uptake. Microarray data confirmation was conducted using quantitative real-time PCR. Selection of differentially expressed genes for pathway analysis was performed using a combination of fold change (FC) ranking (FC < -1.66, FC >1.66) and false discovery rate (<0.35) as criteria. The calcium signaling pathway was highlighted as the top canonical pathway associated with differential gene expression between normal and PSE turkey. Dramatic downregulation of fast-twitch myosin heavy chain coupled with upregulation of slow-twitch myosin and troponin C suggested a switch of skeletal muscle isoforms, which may alter muscle fiber arrangement and formation of actin-myosin complexes. Changes in expression of genes in the actin cytoskeleton signaling pathway also suggest altered structures of actin filaments that may affect cell motility as well as strength and flexibility of muscle cells. Substantial downregulation of pyruvate dehydrogenase kinase, isozyme 4 was observed in PSE samples, suggesting altered regulation of the aerobic metabolic pathway in the birds that developed PSE meat defect.

Differential expression of genes characterizing myofibre phenotype.

Skeletal muscle is composed of metabolically heterogeneous myofibres that exhibit high plasticity at both the morphological and transcriptional levels. The objective of this study was to employ microarray analysis to elucidate the differential gene expression between the tonic-'red' anterior latissimus dorsi (ALD) muscle, the phasic-'white' posterior latissimus dorsi (PLD) and 'mixed'-phenotype biceps femoris (BF) in 1-week-and 19-week-old male turkeys. A total of 170 differentially expressed genes were identified in the muscle samples analysed (P < 0.05). Gene GO analysis software was utilized to identify top gene networks and metabolic pathways involving differentially expressed genes. Quantitative real-time PCR for selected genes (BAT2D, CLU, EGFR and LEPROT) was utilized to validate the microarray data. The largest differences were observed between ALD and PLD muscles, in which 32 genes were over-expressed and 82 genes were under-expressed in ALD1-PLD1 comparison, and 70 genes were over-expressed and 70 under-expressed in ALD19-PLD19 comparison. The largest number of genes over-expressed in ALD muscles, as compared to other muscles, code for extracellular matrix proteins such as dystroglycan and collagen. The gene analysis revealed that phenotypically 'red' BF muscle has high expression of glycolytic genes usually associated with the 'white' muscle phenotype. Muscle-specific differences were observed in expression levels of genes coding for proteins involved in mRNA processing and translation regulation, proteosomal degradation, apoptosis and insulin resistance. The current findings may have large implications in muscle-type-related disorders and improvement of muscle quality in agricultural species.

Heparan sulfate proteoglycans, syndecan-4 and glypican-1, differentially regulate myogenic regulatory transcription factors and paired box 7 expression during turkey satellite cell myogenesis: implications for muscle growth.

The heparan sulfate proteoglycans have been shown to play essential roles in the proliferation and differentiation of myogenic satellite cells. Myogenic regulatory factors (MRF) and paired box 7 (Pax7) are essential transcription factors for satellite cell myogenesis. The objective of the current study was to investigate whether the expression of the MRF and Pax7 is, in part, regulated by the heparan sulfate proteoglycans, syndecan-4, and glypican-1, whose expression has been shown to differentially affect satellite cell proliferation and differentiation. To test this objective, small interfering RNA was used to knockdown the gene expression of glypican-1 and syndecan-4. The effect on the expression of MRF and Pax7 was measured at the mRNA level by real-time quantitative PCR. The knockdown of the glypican-1 gene decreased mRNA expression of MyoD, myogenin, MRF4, and Pax7 expression during proliferation and differentiation of turkey satellite cells; whereas knockdown of the syndecan-4 gene increased mRNA expression of MyoD and MRF4 expression during cell proliferation but not during differentiation and had no effect on myogenin and Pax7 expression. These data suggested that the precise expression of the MRF are dependent upon the appropriate expression of glypican-1 and syndecan-4 during satellite cell proliferation and differentiation, and Pax7 expression is influenced by glypican-1.

Versican, matrix Gla protein, and death-associated protein expression affect muscle satellite cell proliferation and differentiation.

Our previous transcriptional profiling study using a turkey skeletal muscle-specific oligonucleotide microarray revealed over 3,000 genes that were differentially expressed at 3 critical stages of muscle development: 18 d embryonic, 1 d posthatch, and 16 wk of age. The genes versican, matrix Gla protein (MGP), and death-associated protein (DAP) were selected to study for their potential effects on muscle satellite cell proliferation and differentiation, as their functions in other tissues are suggestive of possible key roles in the regulation of myogenesis and they are differentially expressed throughout muscle development in the turkey. Using small interfering RNA to knockdown the expression of these genes during proliferation and differentiation, each of the genes was found to differentially affect proliferation and differentiation. Versican and MGP predominantly affected proliferation with line effects, but later stages of differentiation were affected by the knockdown of versican and MGP. The underexpression of DAP inhibited myotube formation, which is a necessary stage in the development of muscle fibers. Without myotube development, muscle fiber formation will be inhibited or abolished. This is the first report that these genes with no previously documented functions with regard to muscle development play a critical role in muscle cell proliferation and differentiation.

Characterization of a 6K oligonucleotide turkey skeletal muscle microarray.

Consumer demand for lean, inexpensive meat products has driven the domestic turkey (Meleagris gallopavo) industry to unprecedented production; however, this has coincided with an increase in growth-induced myopathies and meat quality defects. With the aim of developing a new tool for the study of turkey growth and development at the muscle transcriptome level, a 6K oligonucleotide microarray was constructed, the Turkey Skeletal Muscle Long Oligo (TSKMLO) microarray. Skeletal muscle samples were collected at three critical stages in muscle development: 18-day embryo (hyperplasia), 1-day post-hatch (hypertrophy), and 16-week (market age) from two genetic lines of turkeys: RBC2, a line maintained without selection pressure, and F, a line selected from the RBC2 line for increased 16-week body weight. Oligonucleotides were designed from sequences obtained from skeletal muscle cDNA libraries from the three developmental stages. Several unique controls, including mismatch and distance controls and scrambled sequences, were designed for 30 genes. Quality control hybridizations were completed, confirming the validity and repeatability of the array. Control features were evaluated across two larger experiments comparing developmental stage within genetic line or genetic line within each developmental stage, totaling 70 arrays. Mismatch and scrambled sequences appeared to be useful controls of specific hybridization for most genes. In addition, quantitative real-time RT-PCR confirmed microarray results. This creation and assessment of the TSKMLO array provides a valuable community resource for the study of gene expression changes related to turkey muscle growth and development.

The influence of age on maternal inheritance of breast muscle morphology in turkeys.

It was apparent in previous studies at our institution using turkeys that measurements of muscle fibers and extracellular spacing were not adequate to explain what was observed in entire pectoralis major muscle sections. A rating system was developed in which muscle sections were rated from 1 (little extracellular matrix and indistinct muscle fibers) to 5 (large extracellular space and distinct muscle fibers). Maternal inheritance was observed at 16 wk of age but not at 8 or 20 wk of age. The purpose of the current study was to determine the effect of age on maternal inheritance. A line (F) selected long-term for increased 16-wk BW, its randombred control (RBC2), and reciprocal crosses between them were compared from 8 through 18 wk of age. Samples of pectoralis major muscle were obtained in a manner to avoid muscle contraction. After being fixed and cross-sectioned, the muscle samples were stained with hematoxylin and eosin and rated by 4 individuals. No significant difference among genetic groups was observed in scores at 8 wk of age. At 10 wk of age, the F line had lower scores than the other genetic groups. Maternal inheritance was suggested at 12 wk of age. The scores for RBC2 were higher than those for F, whereas the F x RBC2 cross did not differ from the pure RBC2 line score at this age. Although the RBC2 x F scores were higher than the pure F-line scores at 12 wk, they were lower than those of the F x RBC2 crosses. From 14 through 18 wk of age, the scores for the RBC2 line were higher than those for the F line and the maternal inheritance was absolute because the value for the individual crosses did not differ from that of the maternal parent. Based on the results, the type of mating used to produce commercial turkeys would have a major effect on breast muscle morphology from 12 through 18 wk of age.

Effect of glypican-1 covalently attached chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

Glypican-1 is a cell membrane heparan sulfate proteoglycan that is composed of a core protein and covalently attached glycosaminoglycan (GAG) chains and N-linked glycosylated (N-glycosylated) chains. The glypican-1 GAG chains are required for cell differentiation and responsiveness to fibroblast growth factor 2 (FGF2). The role of glypican-1 N-glycosylated chains in regulating cell activities has not been reported. The objective of the current study was to investigate the role of glypican-1 N-glycosylated chains and the interaction between N-glycosylated and GAG chains in turkey myogenic satellite cell proliferation, differentiation, and FGF2 responsiveness. The wild-type turkey glypican-1 and turkey glypican-1 with mutated GAG chain attachment sites were cloned into the pCMS-EGFP mammalian expression vector and were used as templates to generate glypican-1 N-glycosylated 1-chain and no-chain mutants with or without GAG chains by site-directed mutagenesis. The wild-type glypican-1 and all glypican-1 N-glycosylated 1-chain and no-chain mutants with or without GAG chains were transfected into turkey myogenic satellite cells. Cell proliferation, differentiation, and FGF2 responsiveness were measured. The overexpression of glypican-1 N-glycosylated 1-chain and no-chain mutants without GAG chains increased cell proliferation and differentiation compared with the wild-type glypican-1 but not the glypican-1 N-glycosylated mutants with GAG chains attached. Cells overexpressing glypican-1 N-glycosylated mutants with or without GAG chains increased cell responsiveness to FGF2 compared with wild-type glypican-1. These data suggest that glypican-1 N-glycosylated chains and GAG chains are critical in regulating turkey myogenic satellite cell proliferation, differentiation, and responsivness to FGF2.

Changes in satellite cell proliferation and differentiation during turkey muscle development.

Posthatch muscle growth is determined by the activation, differentiation, and fusion of satellite cells. Satellite cells composing an individual muscle are heterogeneous, which will differentially affect muscle growth. The proliferation and differentiation of turkey primary pectoralis major muscle cells were investigated in vitro at 1 d of age and at 4, 8, 16, 26, 35, 45, and 54 wk of age. The turkey was selected for these studies because turkey skeletal muscle fibroblasts do not grow in primary muscle cell cultures. Results from the proliferation analysis showed a decrease in proliferation by 8 wk of age. Differentiation into myotubes was significantly decreased by 4 wk of age and myotube diameter was decreased. The changes in muscle weight relative to total BW were measured for the anterior latissimus dorsi, biceps brachii, pectoralis major, sartorius, biceps femoris, and gastrocnemius muscles to compare the relative growth of different muscles. The age at which the muscles reached their maximum relative weight was muscle-dependent, with the biceps brachii plateauing the earliest at 4 wk and the sartorius the latest at 45 wk of age. These data suggested that changes in myogenic cells begin to occur early in muscle development and the activity of the satellite cells during these initial stages of posthatch growth is critical in overall muscle mass accumulation.

Effect of transforming growth factor-beta1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken.

During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-beta1 signal is carried by Smad proteins into the cell nucleus, inhibiting the expression of key myogenic regulatory factors including MyoD and myogenin. However, the molecular mechanism by which TGF-beta1 inhibits muscle cell proliferation and differentiation has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on in vivo skeletal muscle growth and development. A chicken line, Low Score Normal (LSN) with reduced muscling and upregulated TGF-beta1 expression, was used and compared to a normal chicken line. The injection of TGF-beta1 at embryonic day (ED) 3 significantly reduced the pectoralis major (p. major) muscle weight in the normal birds at 1 wk posthatch, whereas no significant difference was observed in the LSN birds. The difference between normal and LSN birds in response to TGF-beta1 is likely due to different levels of endogenous TGF-beta1 where the LSN birds have increased TGF-beta1 expression in their p. major muscle at both 17 ED and 6 wk posthatch. Smad3 expression was reduced by TGF-beta1 from 10 ED to 1 wk posthatch in normal p. major muscle. Unlike Smad3, Smad7 expression was not significantly affected by TGF-beta1 until posthatch in both normal and LSN p. major muscle. Expression of MyoD was reduced 35% by TGF-beta1 during embryonic development in normal p. major muscle, whereas LSN p. major muscle showed a delayed decrease at 1 d posthatch in MyoD expression in response to the TGF-beta1 treatment. Myogenin expression was reduced 29% by TGF-beta1 after hatch in normal p. major muscle. In LSN p. major muscle, TGF-beta1 treatment significantly decreased myogenin expression by 43% at 1 d posthatch and 32% at 1 wk posthatch. These data suggested that TGF-beta1 reduced p. major muscle growth by inhibiting MyoD and myogenin expression during both embryonic and posthatch development. Furthermore, TGF-beta1 also reduced the expression of the cell adhesion receptor beta1 integrin subunit during embryonic and posthatch muscle growth in normal and LSN chickens. Therefore, the reduction of beta1 integrin in response to TGF-beta1 is also associated with decreased posthatch muscle growth. The results from this study indicate that TGF-beta1 inhibits skeletal muscle growth by regulating MyoD and myogenin expression. These data also suggest that a beta1 integrin-mediated alternative pathway is likely involved in the TGF-beta1-induced reduction of muscle growth.

Effect of transforming growth factor-beta1 on decorin expression and muscle morphology during chicken embryonic and posthatch growth and development.

During skeletal muscle development, transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation, as well as a regulator of extracellular matrix (ECM) production. Decorin, a member of the small leucine-rich ECM proteoglycans, binds to TGF-beta1 and modulates TGF-beta1-dependent cell growth stimulation or inhibition. The expression of decorin can be regulated by TGF-beta1 during muscle proliferation and differentiation. How TGF-beta1 affects decorin and muscle growth, however, has not been well documented in vivo. The present study investigated the effect of TGF-beta1 on decorin expression and intracellular connective tissue development during skeletal muscle growth. Exogenous TGF-beta1 significantly decreased the number of myofibers in a given area at both 1 d and 6 wk posthatch. The TGF-beta1-treated muscle had a significant decrease in decorin mRNA expression at embryonic day (ED) 10, whereas protein amounts decreased at 17 ED and 1 d posthatch compared to the control muscle. Decorin was localized in both the endomysium and perimysium in the control pectoralis major muscle. Transforming growth factor-beta1 reduced decorin in both the endomysium and perimysium from 17 ED to 6 wk posthatch. Compared to the control muscle, the perimysium space in the pectoralis major muscle was dramatically decreased by TGF-beta1 during embryonic development through posthatch growth. Because decorin regulates collagen fibrillogenesis, a major component of the ECM, the reduction of decorin by TGF-beta1 treatment may cause the irregular formation of collagen fibrils, leading to the decrease in endomysium and perimysium space. The results from the current study suggest that the effect of TGF-beta1 on decorin expression and localization was likely associated with altered development of the perimysium and the regulation of muscle fiber development.