Eukaryotic inhibitors or activators elicit responses to chemosensory compounds by ruminal isotrichid and entodiniomorphid protozoa.

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the degradable fraction of rumen particulates. These results extend findings from aerobic ciliate models to explain how rumen protozoa have adapted physiology for their specific ecological niches.

Muscle fiber characteristics of pectoralis major muscle as related to muscle mass in different Japanese quail lines.

The objectives of this study were to investigate the muscle fiber characteristics of the pectoralis major muscle, and its relation to growth performance in the random bred control (RBC) and heavy weight (HW) Japanese quail lines at 42 days of age. The HW line had greater body (232.0 v. 100.2 g, P < 0.001) and pectoralis major muscle (19.0 v. 6.2 g, P < 0.001) weights than the RBC line. Color differences were observed between the superficial and deep regions of the pectoralis major muscle, with the superficial region showing a higher value of lightness than the deep region of the RBC or HW lines (P < 0.001). The percentage of the superficial region in the pectoralis major muscle was higher in the HW line compared with the RBC line (46.2% v. 38.0%, P = 0.017). There were no significant differences in the total fiber number in the superficial and deep regions between the two quail lines (P = 0.718). The HW quail line showed a larger mean fiber cross-sectional area (CSA; 375.5 v. 176.6 µm², P < 0.001) and type IIA fiber CSA (243.7 v. 131.9 µm², P < 0.001) than the RBC quail line. The HW line also had greater CSA percentage (60.2% v. 34.2%, P < 0.001) and number percentage (41.6% v. 14.2%, P < 0.001) of type IIB fibers, although there were no significant differences in type IIB fiber CSA between the RBC and HW lines (P = 0.219). Therefore, greater body and muscle weights of the HW line are caused by differences in muscle fiber characteristics, especially the proportion of type IIB fiber and the CSA of type IIA fiber, compared with the RBC line. The results of this study suggest that muscle fiber hypertrophy has more impact on body and muscle weights of the different quail lines than muscle fiber hyperplasia.

Comparative growth performance in different Japanese quail lines: the effect of muscle DNA content and fiber morphology.

The aim of this study was to investigate the DNA content and morphological characteristics of muscle fibers, and their relation to the growth performance in random bred control (RBC) and heavy weight (HW) Japanese quail lines. The 2 lines were of similar embryo size at 6 and 8 d of incubation; however, HW quail were significantly larger than their counterparts after 10 d of incubation (P < 0.05). The hatch weight of the HW quail line was approximately 1.3-fold higher than the RBC quail line (P < 0.001). After 15 d posthatch, the BW and pectoralis major muscle weight (PMW) exhibited remarkable differences between the 2 quail lines. The RBC line showed a faster rate of increase in PMW (2.7- vs. 2.1-fold) and total DNA mass (2.2- vs. 1.6-fold) between 0 and 4 d posthatch. The HW line exhibited a greater rate of the PMW (33.0- vs. 12.9-fold) and total DNA mass (10.3- vs. 4.0-fold) between 4 and 15 d posthatch than the RBC line. Moreover, the greatest increase in total DNA mass occurred between 0 and 8 d posthatch for the RBC line and 4 to 15 d posthatch for the HW line. These differences in the DNA content indicate a difference in the hypertrophic potential of muscle fibers between the 2 quail lines. The cross-sectional area of muscle fibers was 1.3-fold greater in the HW line compared with the RBC line at 8 d posthatch (158.5 vs. 97.11 µm(2), P < 0.001), and this difference increased with age (over 2.1-fold greater in the HW line). Thus, the most important time windows affecting ultimate body and muscle weights in the RBC and HW quail lines are between 0 to 8 d and 4 to 15 d posthatch, respectively. Rapid muscle growth rate and a greater muscle mass in the HW quail line may be partially due to the hypertrophic potential of muscle fibers, which is characterized by larger fiber size.

Temporal myosin heavy chain isoform expression transitions faster in broiler chickens compared with Single Comb White Leghorns.

Myosin heavy chain (MyHC), one of the major components in the contractile machinery of skeletal muscle fibers, is found in several isoforms during myogenesis. During chicken development, embryonic, neonatal, and adult MyHC isoforms are expressed. Broiler chickens have been selected for fast and large muscle growth, whereas Single Comb White Leghorn (SCWL) chickens have been selected for egg laying capabilities. This has led to an obvious difference in muscle growth and development with broilers being much larger than SCWL. The objective of this study was to determine if differences in muscle growth and development of SCWL and broilers are associated with differences in temporal expression of MyHC isoforms in skeletal muscle between the 2 breeds. Pectoralis major muscle (PM) was collected from SCWL and broilers at embryonic d 15, 17, and 19 and 1, 5, 11, 20, 27, and 33 d posthatch with n = 3 samples per time point and breed. Western blotting using 3 monoclonal antibodies (EB165, 2E9, and AB8) was performed to compare the expression patterns of embryonic/adult, neonatal, and adult isoforms of MyHC, respectively, for all time points in both SCWL and broiler chickens. Both broiler and SCWL chickens began expressing the neonatal MyHC isoform on d 5; however, SCWL chickens expressed the neonatal isoform much longer than broilers. The SCWL chickens had sustained expression of the neonatal MyHC isoform through d 27, whereas in broiler chickens the neonatal isoform was not expressed at d 20. Pectoralis major tissue from broiler chickens expressed the adult MyHC isoform as early as d 20, whereas the SCWL chickens began expressing the adult isoform later. The rate of transition to neonatal and adult MyHC isoforms in broilers and Leghorns is consistent with the faster maturation and growth of broilers relative to Leghorns. This relationship between faster growth of the PM and the rate of transition of MyHC isoforms within the fast skeletal muscle of the PM may indicate a selection marker for improvement of broiler PM growth.

The ontogeny of delta-like protein 1 messenger ribonucleic acid expression during muscle development and regeneration: comparison of broiler and Leghorn chickens.

Delta-like protein 1 (DLK1) has been implicated in the muscle hypertrophy observed in DLK1 transgenic mice, callipyge sheep, and mouse paternal uniparental disomy 12 and human paternal uniparental disomy 14 syndromes. The current study was aimed to determine chicken DLK1 (gDLK1) mRNA expression during primary muscle cell differentiation and during muscle regeneration after cold injury and to compare gDLK1 mRNA expression during skeletal muscle development in layers and broilers. In chicken primary muscle cell culture, gDLK1 mRNA expression was significantly increased from 12 to 48 h (P < or = 0.05) when the nascent myotubes were actively formed at d 2 to 3. Myogenin, a late myogenic marker gene, mRNA expression peaked at 36 to 48 h. Myogenic differentiation 1 (MyoD) and paired box gene 7 (Pax7), early myogenic marker genes, mRNA expression gradually decreased during myogenic differentiation. During muscle regeneration, the expression of MyoD and Pax7 peaked at d 2 (P < or = 0.05), and myogenin mRNA expression peaked at d 4 (P < or = 0.05). The induction of gDLK1 gene appeared between d 7 to 10 postinjury (P < or = 0.05) when myotubes were actively formed as also demonstrated in histological sections. The expression of gDLK1 was slowly downregulated to the control levels at d 14 when the damaged muscle appeared nearly healed. These data suggest that gDLK1 may be involved in the late myogenic stages of primary muscle cell differentiation and muscle regeneration. The gDLK1 mRNA in the muscle tissues was very abundant at embryonic ages but decreased after hatching in both broiler and layer chickens. Compared with layers, broiler muscle at embryonic d 13 had a 3-fold greater expression of DLK1 (P < or = 0.01). In addition, the gDLK1 mRNA expression at d 1, 11, and 33 post-hatch was significantly higher in broilers than layers (P < or = 0.05). Therefore, the relatively greater expression of the gDLK1 gene in muscles of broilers compared with layers suggests that gDLK1 may serve as a new selection marker for high muscle growth in chickens. These findings may provide new insight into chicken muscle development and regeneration.

Technical note: a gene delivery system in the embryonic cells of avian species using a human adenoviral vector.

Adenovirus (Ad) has been used in vivo and in vitro as a vector to carry a foreign gene for efficient gene delivery into various cell types and tissues of animals. The aim of the current study was to evaluate the Ad delivery system in primary avian cells. Primary cells isolated from the embryonic pectoralis major muscles of the chicken and quail were cultured and incubated with human recombinant Ad serotype 5 (Ad5) containing sequences encoding either the green fluorescence protein (GFP) gene alone, as a tracking marker, or both GFP and murine 3-hydroxyisobutyryl-CoA hydrolase (mHIBCH) as a target gene. The fluorescent GFP images showed the successful delivery of a target gene using Ad5 in the primary avian cultured cells. In addition, immunostaining of the myosin heavy chain (MyHC) in these cells indicated that a large population of the cells was myogenic. Colocalization of GFP-positive cells with MyHC staining was mostly found in MyHC-negative cells, indicating successful delivery of Ad5 into a large population of mononucleated cells. Furthermore, the current fluorescence study detected the dual expression of GFP and mHIBCH protein in GFP-positive cells. Finally, Western blot analysis confirmed that the Ad-mediated expression of mHIBCH protein was specific in primary cultures of avian myogenic cells and that the mHIBCH protein expression was continued for 15 d after infection in chicken primary cells. These data demonstrate that Ad5 is a feasible tool to express foreign genes in primary cultured cells of avian species, providing a new approach to study the function of genes of interest in muscle development and metabolism.

Turkey breast meat functionality differences among turkeys selected for body weight and/or breast yield.

Functional meat characteristics were studied in three turkey lines, (1) RBC2: representing 1960s commercial turkeys, (2) F-line: a line selected for body weight (BW) and (3) C-line: a fast growing commercial line with enhanced breast muscle yield. The RBC2s Warner-Bratzler shear force values for the Pectoralis major (PM) were lower than the F- and C-lines' values (P<0.05). The WHC of the breast muscle from the C-line was lower compared with the RBC2 line (P<0.05), with the F-line being intermediate between, though not different from either the RBC2 or C-lines. A trend was observed, as the thermally induced meat gels from the RBC2 line PM had the highest storage modulus (G'), the F-line was intermediate and the C-line had the lowest storage modulus (P=0.09). These results suggest that selection for increased growth and breast muscle yield may be associated with decreased meat functionality in modern commercial turkeys.

Myofibrillar 1-D fingerprints and myosin heavy chain MS analyses of beef loin at 36 h postmortem correlate with tenderness at 7 days.

This study investigated the potential for relating changes in electrophoretic protein patterns derived from the longissimus dorsi of beef cattle 36 h postmortem with tenderness at 7 days. We report finding a significant correlation (R(2)=0.82) between electrophoretic l. dorsi myofibrillar fingerprints at 36 h postmortem and tenderness at 7 days, as determined by Warner-Bratzler shear analysis. In addition, we have used mass spectrometric analyses to identify fragments of bovine myosin heavy chain that are significantly correlated with tenderness. Furthermore, this method offers the potential to increase our understanding of the fundamental cellular mechanisms underlying the proteolytic breakdown of muscle proteins during the aging process.

Ventricular myosin heavy chain isoform expression is altered in vitro in low score normal chickens.

The low score normal (LSN) chicken exhibits a genetic muscle weakness and altered in vitro myogenesis compared to the normal White Leghorn chicken. The ventricular myosin heavy chain isoform has been reported to be the initial muscle-specific contractile protein expressed during myogenesis. The goals of this study were to determine whether altered myogenesis of the LSN satellite cells in culture was accompanied by delayed ventricular myosin heavy chain expression and to further characterize the altered myogenic events exhibited by the LSN chicken. Immunocytochemical and ELISA analyses were employed to document the temporal expression of the ventricular myosin heavy chain during LSN chicken myogenesis. Satellite cells derived from the LSN chicken pectoralis major exhibited lower (P