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.

Incubation temperature and time of hatch impact broiler muscle growth and morphology.

The adult myogenic population of stem cells, called satellite cells, initially develop in late-term embryos. Satellite cells are the only myogenic cell that repair damaged myofibers and increase post-hatch growth. The objective of the current study was to determine if incubation temperatures and time of hatch impact growth and pectoralis major (p. major) muscle morphology. Eggs were incubated at a constant 37.8°C; however, from d 14 to 18, the eggs were subject to 39.5°C for 0, 3, or 12 h per day. Chicks were divided into early, mid, or late hatch groups based upon the time they emerged from the shell. Growth and feed efficiency were measured throughout the 63-day trial, while meat quality and muscle morphology were evaluated at the time of processing. The chicks incubated at an increased temperature for 12 h per d had reduced (P < 0.01) body weights throughout the trial compared to the 3 h treatment and control. The early hatch broilers were heavier (P < 0.01) at 63 d compared to mid and late hatch broilers. Chicks from the 12 h incubation treatment had an increased (P = 0.01) gain to feed ratio compared to the control. Broilers from the 12 h incubation treatment had lower (P < 0.01) p. major weights compared to the 0 and 3 h treatments. Early hatch broilers had heavier p. major weights (P < 0.01) compared to mid and late hatch groups. The 12 h incubation treatment also reduced the number of broilers with moderate to severe myopathic attributes compared to the control. Similarly, there were fewer late hatch birds with fibrotic and necrotic p. major muscles compared to the early hatch group. Together, these data demonstrate that altering incubation temperature is a feasible management strategy to improve muscle morphology without negatively impacting meat quality parameters.

Influence of temperature and growth selection on turkey pectoralis major muscle satellite cell adipogenic gene expression and lipid accumulation.

P. MAJOR: Immature poults have an inefficient thermoregulatory system, and therefore extreme ambient temperatures can impact their internal body temperature. Satellite cells, the only posthatch myonuclei source, are multipotential stem cells and sensitive to temperature. Selection for faster-growing, high-yielding birds has altered satellite-cell properties. The objective of the current study was to determine how temperature affects adipogenic properties of satellite cells isolated from the pectoralis major ( ) muscle of Randombred Control line ( ) and F line turkeys selected only for increased 16-wk body weight from the RBC2 line. Satellite cells were cultured at 2°C incremental temperatures between 33 and 43°C and compared to cells cultured at the control temperature of 38°C to ascertain temperature effects on lipid accumulation and expression of adipogenic genes: CCAAT/enhancer-binding protein-ß ( ), peroxisome proliferator-activated receptor-γ ( ), and stearoyl-CoA desaturase ( ). During proliferation, the amount of quantifiable lipid in both F and RBC2 satellite cells increased at temperatures above 38°C ( P <  0.01) and decreased at temperatures below 38°C ( P < 0.01). Above 38°C, RBC2 satellite cells had more lipid ( P = 0.02) compared to the F line, whereas there were few differences between lines below 38°C. At 72 h of proliferation, expression of C/EBPß , PPARγ , and SCD decreased ( P ≤  0.02) as temperatures increased from 33 to 43°C in both cell lines. During differentiation expression of C/EBPß increased ( P <  0.01) as temperatures increased from 33 to 43°C in both cell lines. In F line satellite cells, PPARγ expression decreased ( P <  0.01) with increasing temperatures during differentiation, whereas there was no linear trend in RBC2 cells. During differentiation expression of SCD increased as temperatures increased ( P <  0.01) in RBC2 cells, and there was no linear trend within the F line. Results from the current study suggest that environmental temperature can affect p. major satellite cellular fate; however, selection for increased body weight had little impact on these cellular responses.

Regulation of turkey myogenic satellite cell migration by MicroRNAs miR-128 and miR-24.

Myogenic satellite cells are an adult stem cell responsible for all post-hatch muscle growth in poultry. As a stem cell population, satellite cells are highly heterogeneous, but the origin of this heterogeneity remains unclear. Heterogeneity is, in part, regulated by gene expression. One method of endogenous gene regulation that may contribute to heterogeneity is microRNAs (miRNAs). Two miRNAs previously shown to regulate poultry myogenic satellite cell proliferation and differentiation, miR-128 and miR-24, were studied to determine if they also affected satellite cell migration. Satellite cell migration is an essential step for both proliferation and differentiation. During proliferation, satellite cells will migrate and align to form new myofibers or donate their nuclei to existing myofibers leading to muscle fiber hypertrophy or regeneration. Transient transfection of miRNA specific mimics to each miRNA reduced migration of satellite cells following a cell culture scratch at 72 h of proliferation when the cultures were 90 to 100% confluent. However, only the migration in cells transfected with miR-24 mimics at 24 and 30 h following the scratch was significantly reduced (P ≤ 0.05) to around 70% of the distance migrated by controls. Alternately, transfection with inhibitors specific to miR-128 or miR-24 significantly (P ≤ 0.05) increased migration between 147 and 252% compared to their controls between 24 and 48 h following the scratch. These data demonstrate that miR-128 and miR-24 play a role in myogenic satellite cell migration, which will impact muscle development and growth.

Spatial influence on breast muscle morphological structure, myofiber size, and gene expression associated with the wooden breast myopathy in broilers.

The wooden breast (WB) myopathy is identified by the palpation of a rigid pectoralis major (p. major) muscle and is characterized as a fibrotic, necrotic p. major disorder in broilers. The objective of the current study was to determine spatial morphological and gene expression differences at 4 locations within WB affected muscle from different genetic lines. Morphology was evaluated in 2 broiler lines expressing the WB myopathy (Lines A and B) and a line without WB (Line C) at 3 ventral locations and one anterodorsal location in the p. major muscle. In WB affected muscle of Line A, fibrosis was greatest in the anterior locations of WB affected muscle. In Line B muscle, fibrosis was greatest in the anteroventral region and minimal in the anterodorsal or posterior regions. Average p. major myofiber diameter was 30% larger in Lines A and B compared to Line C. However, in Line A there were no differences between the percentage of large fibers (diameter >70 µm) in unaffected and WB affected muscles at any sampling region. The percentage of small fibers (diameter <10 µm), likely small regenerating fibers, and expression of myogenic determination factor 1 (MYOD1) and myogenin were increased in Line A WB affected muscle compared to unaffected muscle. In Line B, the percentage of small fibers and MYOD1 expression in WB affected muscle was not different from unaffected muscle. Connective tissue organization within WB affected muscle was also different in Lines A and B, which may be attributed to decorin, a proteoglycan that mediates collagen crosslinking, growth factor signaling, and cell growth. Decorin expression was increased at all locations within Line A. However, in Line B decorin was increased only in the fibrotic regions of the p. major. The compiled results provide evidence that the WB myopathy is not uniform throughout the entire p. major muscle and the anterior end of the p. major muscle was more affected by the condition.

Influence of chick hatch time and access to feed on broiler muscle development.

The effect of hatch time and the timing of access to feed on growth rate and breast muscle development was assessed in Ross 308 broiler chickens. Chicks were removed from the incubator upon hatching, and classified as early (EH), midterm (MH), or late (LH) hatchers, based on the duration of their incubation. Feed and water were available either immediately at hatch, or 24 h after the conclusion of the hatch period. Hatchling body weight was uniform regardless of hatch time. Subsequently, bodyweight was increased in EH compared to LH birds following immediate access to feed, until 7 d in female, and 14 d in male birds. Relative breast weight was increased until 28 d in birds with immediate access to feed, and also EH and MH birds regardless of access to feed. Pectoralis major muscle morphology and expression of the myogenic regulatory factors myogenic determination factor 1 (MYOD1) and myogenin, and the proteoglycans syndecan-4, glypican-1, and decorin were measured. Myogenin and glypican-1 stimulate satellite cell (SC) differentiation. Glypican-1 expression was unaffected by treatment. A late increase in myogenin expression was observed in MH birds with delayed access to feed, and all LH birds. Syndecan-4 and MYOD1, expressed in proliferating SC, and decorin, which stimulates satellite cell proliferation and differentiation, were variably upregulated in the first wk posthatch in the same birds. These data suggest SC were activated and proliferating, but had reduced differentiation in later hatching and feed deprived birds. Conversely, EH birds with immediate access to feed had maximal myofiber width at 7 d, while fiber width was increased in birds with immediate access to feed compared to those with delayed access to feed through 40 d of age. These results demonstrate that delaying chick access to feed for 24 h upon removal from the incubator will impair muscle growth. Additionally, hatch time influences muscle development, with accelerated muscle growth in EH and MH, compared to LH birds, irrespective of access to feed.

Influence of hatch time and access to feed on intramuscular adipose tissue deposition in broilers.

The effect of hatch time and subsequent access to feed on intramuscular adipose tissue deposition was studied in the pectoralis major muscle of male Ross 308 broiler chickens. Based on their hatch time chicks were classified as early (EH), midterm (MH), or late (LH) hatchers, with an average incubation duration of 497.7 h for EH, 508.8 h for MH, and 514.5 h for LH birds. Chicks were provided access to feed either immediately at hatch, or 24 h after the conclusion of the hatch window. Expression of the adipogenic regulatory genes peroxisome proliferator-activated receptor gamma (PPARγ), and stearoyl-CoA desaturase (SCD), were measured at the time of hatch, and zero, one, 4, 7, 28, and 40 d. Intramuscular adipocyte cell width and visualization of adipose tissue deposition was observed at 28 and 40 d. Expression of PPARγ was increased in the pectoralis major of LH birds at the time of hatch, zero, and one d. The expression of PPARγ at one and 7 d, and SCD at 7 d were increased in all birds that received delayed access to feed. At 28 d, adipocyte cell width was increased in LH birds with delayed access to feed, compared to EH and MH birds with delayed access to feed and LH birds with immediate access to feed. At 40 d, adipocyte cell width was increased in all birds that received delayed access to feed. Also at 40 d, there was a trend (P = 0.078) for more extensive intramuscular adipose tissue deposition in LH than EH birds, and in birds with delayed access to feed (P = 0.075). These data indicate delayed access to feed increases intramuscular adipose tissue deposition in the pectoralis major muscle, and suggest that hatch time influences this regulation.

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.

INVITED REVIEW: Evolution of meat animal growth research during the past 50 years: Adipose and muscle stem cells.

If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose- and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed.

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.

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.

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.

Deep transcriptome sequencing reveals differences in global gene expression between normal and pale, soft, and exudative turkey meat.

Previous studies from our laboratory suggested that differential expression of genes between normal and pale, soft, and exudative (PSE) turkey is associated with development of the PSE syndrome. However, a detailed understanding of molecular mechanisms responsible for the development of this meat defect remains unclear. The objective of this study was to extend and complement our previous work by using deep transcriptome RNA sequence analysis to compare the respective transcriptome profiles and identify molecular mechanisms responsible for the etiology of PSE turkey meat. Turkey breasts (n = 43) were previously classified as normal or PSE using marinade uptake as an indicator of quality (high = normal; low = PSE). Total RNA from breast muscle samples with the highest (n = 4) and lowest (n = 4) marinade uptake were isolated and sequenced using the Illumina GA(IIX) platform. The results indicated differential expression of 494 loci (false discovery rate < 0.05). Changes in gene expression were confirmed using quantitative real-time PCR. Pathway analysis of differentially expressed genes suggested abnormalities of calcium homeostasis and signaling pathways regulating actin cytoskeleton structure as well as carbohydrate metabolism and energy production in PSE samples. Dysregulation of postmortem glucose oxidation in PSE turkey was suggested by both dramatic downregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA, the most downregulated gene, and a decrease in the protein product (P = 0.0007) as determined by immunoblot analysis. These results support the hypothesis that differential expression of several genes and their protein products contribute to development of PSE turkey.

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 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.

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.

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.

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.