Evidence of vascular endothelial dysfunction in Wooden Breast disorder in chickens: Insights through gene expression analysis, ultra-structural evaluation and supervised machine learning methods.

Several gene expression studies have been previously conducted to characterize molecular basis of Wooden Breast myopathy in commercial broiler chickens. These studies have generally used a limited sample size and relied on a binary disease outcome (unaffected or affected by Wooden Breast), which are appropriate for an initial investigation. However, to identify biomarkers of disease severity and development, it is necessary to use a large number of samples with a varying degree of disease severity. Therefore, in this study, we assayed a relatively large number of samples (n = 96) harvested from the pectoralis major muscle of unaffected (U), partially affected (P) and markedly affected (A) chickens. Gene expression analysis was conducted using the nCounter MAX Analysis System and data were analyzed using four different supervised machine-learning methods, including support vector machines (SVM), random forests (RF), elastic net logistic regression (ENET) and Lasso logistic regression (LASSO). The SVM method achieved the highest prediction accuracy for both three-class (U, P and A) and two-class (U and P+A) classifications with 94% prediction accuracy for two-class classification and 85% for three-class classification. The results also identified biomarkers of Wooden Breast severity and development. Additionally, gene expression analysis and ultrastructural evaluations provided evidence of vascular endothelial cell dysfunction in the early pathogenesis of Wooden Breast.

Seasonal muscle ultrastructure plasticity and resistance of muscle structural changes during temperature increases in resident black-capped chickadees and rock pigeons.

Resident birds in temperate zones respond to seasonally fluctuating temperatures by adjusting their physiology, such as changes in basal metabolic rate or peak metabolic rate during cold exposure, or altering their organ sizes, so as to match the thermogenic requirements of their current environment. Climate change is predicted to cause increases in the frequency of heat and cold wave events, which could increase the likelihood that birds will face an environmental mismatch. Here, we examined seasonality and the effects of acute and chronic heat shock to 33°C and subsequent recovery from heat shock on the ultrastructure of the superficial pectoralis muscle fiber diameter, myonuclear domain (MND) and capillary density in two temperate bird species of differing body mass, the black-capped chickadee (Poecile atricapillus) and the rock pigeon (Columba livia). We found that muscle fiber ultrastructure did not change with heat treatment. However, in black-capped chickadees, there was a significant increase in fiber diameter in spring phenotype birds compared with summer phenotype birds. In rock pigeons, we saw no differences in fiber diameter across seasons. Capillary density did not change as a function of fiber diameter in black-capped chickadees, but did change seasonally, as did MND. Across seasons, as fiber diameter decreased, capillary density increased in the pectoralis muscle of rock pigeons. For both species in this study, we found that as fiber diameter increased, so did MND. Our findings imply that these two temperate birds employ different muscular growth strategies that may be metabolically beneficial to each.

Pectoral Vessel Density and Early Ultrastructural Changes in Broiler Chicken Wooden Breast Myopathy.

In wooden breast myopathy (WBM) of broiler chickens, the pectoralis major muscles show abnormally hard consistency and microscopical myodegeneration of unknown aetiology. To date, previous studies have focused primarily on chronic WBM and ultrastructural descriptions of early WBM are lacking. The aim of this study was to elucidate the pathogenesis of WBM by light microscopical morphometry of vessel density and the ultrastructural description of early WBM changes with transmission electron microscopy. The pectoral vessel density was compared between unaffected chickens (n = 14) and two areas of focal WBM in affected chickens (n = 14). The transverse myofibre area per vessel was highest in the unaffected area of muscle from cases of focal WBM, significantly higher (P = 0.01) than in macroscopically unaffected tissue, indicating that relatively decreased blood supply may trigger the development of WBM. The ultrastructural study included unaffected chickens (n = 3), two areas of focal WBM from affected chickens (n = 3) and areas of diffuse WBM from affected chickens (n = 3). The morphologically least affected myofibres within the WBM lesion areas in light microscopy exhibited ultrastructural changes of increased sarcoplasmic reticulum diameter and mitochondrial hyperplasia. Such changes originate typically from osmotic imbalance, for which the most likely aetiologies in WBM include tissue hypoxia or myodegeneration of the surrounding myofibres. The findings suggest that a relative reduction of blood supply in the major pectoral muscle occurs in the early phase of WBM, which may be linked to the ultrastructural changes of osmotic imbalance.

The Effect of the Wooden Breast Myopathy on Sarcomere Structure and Organization.

The wooden breast (WB) has been classically identified by the phenotypic presence of a wood-like pectoralis major (p. major) muscle. The WB-affected p. major muscle is characterized by necrotic muscle fibers and the replacement of muscle with connective tissue, water, and fat. The objective of the current study was to determine the effect of the WB myopathy on sarcomere organization by transmission electron microscopy. Sarcomere structure and organization were examined in two broiler lines with a high incidence of WB (Lines A and B) and another broiler line without WB (Line C). Affected muscle had an increase in smaller myofibers with diameters of 20 µm or less. Sarcomere organization decreased with fiber diameter in both Lines A and B. The structure and organization of sarcomeres in Line C were similar to WB-unaffected muscle in Lines A and B. Taken together, these data demonstrate that the WB myopathy detrimentally affects sarcomere organization in a broiler line-specific manner. Disorganization of sarcomere structure will affect the function of the p. major muscle as well as meat quality.

The effects of electrical stunning methods on broiler meat quality: effect on stress, glycolysis, water distribution, and myofibrillar ultrastructures.

This study was designed to compare the effects of different stunning systems on the meat quality of broilers. This was done by investigating meat water-holding capacity, meat color, muscle glycogen, and lactate concentrations, as well as blood parameters, low-field nuclear magnetic resonance (NMR) transverse relaxation, and myofibrillar ultrastructures. A total of 160 broilers were divided into 4 treatment groups: a low-voltage stunning (LS) with a constant voltage of 15 V at 750 Hz for 10 s; a midvoltage stunning (MS) with a constant voltage of 50 V at 50 Hz for 10 s; a high-voltage stunning (HS) with a constant voltage of 100 V at 50 Hz for 5 s; and a control group with no stunning (NS). Blood samples were collected immediately after cutting the neck. Pectoralis major muscles were removed from the carcass after chilling and placed in ice. Breast muscle pH, meat color, glycogen, and lactate contents were determined at both 2 and 24 h postmortem. Drip loss, cooking loss, pressing loss, cooked breast meat shear values, low-field NMR, and ultrastructures of myofibrils were determined 24 h postmortem. The NS and MS treatments significantly increased (P < 0.05) blood plasma corticosterone, initial rate of glycolysis, and drip loss, and significantly reduced (P < 0.05) initial muscle pH and shear force values when compared with LS and HS. The results of low-field NMR reflect that NS and MS significantly decreased (P < 0.05) NMR transverse relaxation population 1 (T21) and increased (P < 0.05) NMR transverse relaxation population 2 (T22). The myofibrils of NS and MS samples showed significantly (P < 0.05) longer sarcomere length when compared with the LS and HS samples. The meat color, cooking loss, pressing loss, and final pH were not affected by the stunning methods. This study indicates that NS and MS treatments reduce meat water-holding capacity and decrease meat shear force when compared with LS and HS.

Collagen content and architecture of the pectoralis muscle in male chicks and broilers reared under various nutritional conditions.

Varying chicken growth rates were induced with different nutritional regimes, and the collagen content and architecture of M. pectoralis (PT) were compared among 21-day-old chicks and broilers at 80 or 95 days of age. The percentage of muscle weight to live weight was higher in rapid growing chicks (8.4%) than slow growing chicks (6.3%). The 80-day-old broilers engaged in compensatory growth after the early slow growth period producing PT muscle at 11% of live weight. The 80- and 95-day-old chicks with restricted late growth after an early rapid growth period showed PT weight at 8% and 9% of live weight, respectively. Collagen content of the PT muscle markedly decreased from the chicks to the broilers. The collagen concentration was higher in the late-growth restricted broilers (1.67-1.88 mg/g) than the compensatory growth broilers (1.01-1.10 mg/g). Collagen concentration did not differ between the rapid and slow growing chicks (2.72 and 2.94 mg/g). Scanning electron micrographs showed thick and thin perimysia, and honeycomb endomysia. In the perimysia, a stack layer of collagen platelets and a reticular layer of collagen fiber cords were distinguished and collagen baskets of adipocytes were observed. The perimysial collagen fibers became thicker during growth of the chicks to broilers. However, in the late-growth restricted broilers, the perimysial collagen fibers seemed to have retarded development compared with the compensatory growth birds. The PT muscle of chickens develops optimally when body growth is enhanced. The PT muscle of the compensatory growth broilers had improved collagen architecture regardless of the marked decrease in collagen content.

Alterations in tissue aerobic capacity may play a role in premigratory fattening in shorebirds.

Migratory shorebirds show regulated seasonal increases in body mass (BM) even in captivity, consisting primarily, but not exclusively, of fat. We examined whether captive red knot (Calidris canutus) exhibited seasonal alterations in mitochondrial volume (liver, pectoral muscle) and/or succinate dehydrogenase (SDH) activity (liver, pectoral muscle, heart, small intestine) during three distinct life-cycle stages: stable BM, spring peak in BM, and as BM rapidly declined after the spring peak. Mitochondrial volume in liver and pectoral muscle and SDH activity in liver and heart did not alter with life-cycle stage. However, red knot undergoing premigratory fattening exhibited significantly lower pectoral muscle SDH activity in concert with significantly elevated activity in the small intestine compared with the other two time-points, suggesting that tissue metabolic rate alters with life-cycle stage. The increased intestinal SDH activity may indicate an elevation in energy assimilation at a time when intestine hypertrophy occurs, thus maximizing BM increase prior to putative migration. The concomitant decrease in pectoral muscle activity may act to reduce overall metabolic rate, or at least help counter the elevation in intestinal mass-specific metabolic rate. Both tissues hypertrophy prior to migration in wild red knot, but hypertrophy of the intestine precedes that of pectoral muscle. Indeed, it appears that the intestinal mass undergoes atrophy by the time pectoral muscle hypertrophy occurs in wild red knot. Thus, physiological adjustments in tissue metabolism may be an important factor in the life-history strategies of migrating shorebirds.

Comparison of chicken genotypes: myofiber number in pectoralis muscle and myostatin ontogeny.

This study was performed to evaluate breast muscle development in chicken genotypes divergently selected for muscularity. In the first experiment, 2 commercial broiler lines (a high breast yield, HBY, and a normal breast yield broiler strain-cross, NBY) and a Leghorn line were grown up to 35 d to evaluate BW, breast weight, and breast yield. At 7 and 21 d of age, pectoralis muscle was used to estimate myofiber density (MFD, number of myofibers per mm2) and total apparent myofiber number (MFN). In the second experiment, the ontogeny of myostatin was determined from broiler- and Leghorn-type chick embryos, at embryonic days 1 to 20 (E1 to E20), using reverse transcription (RT)-PCR. As expected, the Leghorn line had lower BW, breast weight, and breast yield than broiler lines. The HBY line showed higher breast yield at all ages evaluated, but lower BW at 21 and 35 d than the NBY line. The Leghorn line had 45% higher MFD than broilers, which indicates an increased cross-sectional area of the myofibers in broiler lines. No MFD difference was observed between the broiler strains (P > 0.05). The myofiber number of broilers was more than twice that of Leghorns and HBY had 10% higher MFN than the NBY line. Myofiber number was correlated to BW (r = 0.58), breast weight (r = 0.58), and breast yield (r = 0.69). Conversely, MFD showed negative correlation with BW, breast weight, and breast yield (r = -0.85, -0.83, and -0.88, respectively). No effect of genotype or interaction between genotype and embryonic age was observed for myostatin expression. This study showed that broilers have higher MFN in the breast muscles than Leghorn-type chickens, and that high breast yield of broiler strains may be due to increased MFN. Higher muscularity of broilers, as compared with Leghorns, was not attributed to lower expression of myostatin during embryonic development.

Inheritance of breast muscle morphology in turkeys at sixteen weeks of age.

The inheritance of morphology of the pectoralis major muscle in turkeys at 16 wk of age was studied in a randombred control line (RBC2), a subline (F) of RBC2 selected long term for increased 16 wk BW only, and F1 and F2 crosses of the F and RBC2 lines. Samples of pectoralis major muscle were obtained from 10 males and 10 females of each genetic group in a manner to avoid muscle contraction. After being fixed and cross sectioned, the muscle samples were stained with hematoxylin and eosin to view muscle morphology. The stained sections were analyzed for muscle fiber width, number of fibers in a 136-microm2 area, and extracellular matrix perimysial (PW) and endomysial (EW) width in areas of sections in which accurate measurements could be made. Because muscle damage was evident in some sections and, therefore, morphological measurements might not have provided a complete overview of muscle morphology, sections of the F2 crosses were subjectively rated by 4 people. The ratings ranged from 1 (little extracellular matrix and indistinct muscle fibers) to 5 (large extracellular space and distinct muscle fibers). Ratings of 2 to 4 were intermediate to these extremes. Creatine kinase concentrations of blood samples taken immediately prior to collecting muscle tissue were obtained and correlated with muscle section ratings within genetic group and sex. The F and RBC2 lines differed in PW and EW but not in individual fiber measurements. In the F1 generation, heterosis was -10.4% (P < or = 0.01), 19.7% (P < or = 0.05), -25.2% (P < or = 0.01), and -34.3% (P < or = 0.01), respectively, for fiber width, number of muscle fibers, PW, and EW. The F2 crosses differed only in EW based on measurements of sections in which accurate measurements could be made. However, based on subjective ratings of the muscle sections, possible maternal inheritance was suggested, as the orthogonal contrast was significant (P < or = 0.01) for crosses with F dams as F1 parents vs. those with RBC2 dams as F1 parents, confirming a previous study. The correlation coefficient between creatine kinase concentration and muscle section ratings was -0.282 (P < or = 0.01) after adjustment for line and sex effects.

Developmental states of the collagen content, distribution and architecture in the pectoralis, iliotibialis lateralis and puboischiofemoralis muscles of male Red Cornish x New Hampshire and normal broilers.

1. Developmental states of the collagen content, distribution and architecture in the pectoralis (PT), iliotibialis lateralis (ITL) and puboischiofemoralis (PIF) muscles of male Red Cornish x New Hampshire (RN, 80 d, body weight 2.9 kg) and normal (3.1 kg) broilers were evaluated. 2. In PT muscle the total amount of collagen was significantly greater in RN broilers (3.33 mg/g) than in normal ones (1.71 mg/g). This higher collagen content in RN broilers was based mainly on the closer mesh sizes of endomysial honeycomb. The collagen structures in the perimysia also differed between broiler types, when more collagen fibres were observed in RN broilers. 3. ITL muscle contained total collagen of 4.10 to 5.00 mg/g. Types I and III collagens were distributed on the perimysia at higher percentages in RN broilers (31.6%, 37.2%) than normal (15.6%, 30.8%), respectively. The thick bands of tough collagen fibres characteristic of ITL muscle perimysium in cockerels had not yet developed in these broilers. 4. Total collagen was 4.63 to 6.29 mg/g in PIF material with fascia. In PIF muscle the perimysial collagen fibres had not yet attained their full growth but consisted of densely packed fibrils. PIF muscle was characterised by the earlier maturing collagen structure. 5. These results show that a perimysial collagen structure in broilers is still in an undeveloped state. It is supposed that tenderness of broiler meat is attributed mainly to characteristics of the collagen distribution, in which the majority of types I and III collagens is distributed on the closer mesh of endomysial honeycomb.

Possible maternal inheritance of breast muscle morphology in turkeys at sixteen weeks of age.

During the study of inheritance of breast muscle morphology of turkeys during embryonic development and posthatch in two experiments, interesting results were obtained at 16 wk of age. In experiment 1, an experimental line (F) selected long term for increased 16-wk BW was crossed reciprocally with a commercial sire line (B). Samples of pectoralis major muscle were obtained from three males and three females of each genetic group. The F line was reciprocally crossed with a randombred control line (RBC2) in experiment 2, and p. major muscle samples were taken from 10 males and 10 females of each genetic group. The RBC2 line was the base population for the F line. The muscle samples were obtained in a manner to avoid contraction. After fixing, the muscle samples were stained with hematoxylin and eosin to view muscle morphology. Distinct morphological types were observed in the muscle samples for the B, F, and RBC2 lines. The pectoralis major muscle from the B line was, in general, characterized by large fibers with a well-defined extracellular matrix (perimysial and endomysial spaces). Similar to the B line, representative samples of the RBC2 line had a well-defined extracellular matrix but muscle fiber size was not as large as that of the B line. Representative samples of the F line were characterized by a greatly reduced extracellular matrix with the muscle fibers not well defined. For the reciprocal crosses in both experiments, representative samples indicated the offspring had breast muscle morphology similar to that of the female parent, suggesting maternal inheritance. There was no sex effect on breast muscle morphology in either experiment. In order to study the consistency of the results, muscle morphology of representative sections of muscle were subjectively rated by four individuals. The ratings ranged from 1 (little extracellar matrix and indistinct muscle fibers) to 5 (large extracellular space and distinct muscle fibers). Ratings of 2 to 4 were intermediate to these extremes. In experiment 1, ratings of the pure B line and the F sire x B dam cross were similar and higher than that of the pure F line and B sire x F dam cross, which did not differ in average rating. The results for experiment 2 were similar to those for experiment 1 in that the average ratings of the reciprocal crosses were similar to that of the female parental line. The results from the ratings support maternal inheritance of breast muscle morphology.

Is ribosomal capacity a potential metabolic marker of muscle development? Measurement by muscular biopsy.

Metabolic markers of muscle metabolism could help geneticists and nutritionists predict the breast meat development of chickens. The aim of the current study was to test the ribosomal capacity (CS), a potential metabolic marker, and to evaluate a simple biopsy method on the pectoralis major muscle. Ribosomal capacity was measured in three commercial meat chicken genotypes differing in their growth rate. Fast-, medium-, and slow-growing male chickens were fed using three commercial dietary programs of increasing energy and protein concentration (nine treatments). Biopsy was performed at 4 wk of age on the p. major 12 chickens per treatment. Fast-, medium-, and slow-growing chickens were slaughtered at market weight, i.e., 6, 8, and 12 wk of age, respectively, and breast meat (right and left p. major + minor) was dissected. A significant reduction in BW at slaughter (-1 to -3%) and breast meat yield (-4%) occurred only in biopsied medium-growing chickens but not in the other two types. Slow-growing chickens had a significantly lower CS (8.75 microg/mg) than the two other chicken types (9.40 and 9.46 microg/mg for fast- and medium-growing chickens, respectively). No significant dietary effect or interaction of dietary treatment with genotype was measured. The CS was not significantly correlated to breast meat development. Under conditions of the present experiment, CS may not be a relevant marker of subsequent breast meat development at 4 wk of age. The biopsy technique can easily be applied to other markers.

Breast muscle development in commercial broiler chickens.

Genetic and gender-related variations in breast muscle yield of broiler chickens may be attributed to differences in number and size of muscle cells (myofibers). In this study, male and female broilers from eight commercial strain crosses (SC) were compared for body and breast muscle weight with adjustment of the Gompertz function. Additionally, breast fillet dimensions (length, width, and depth) and myofiber density (myofiber number/area; MFD) were assessed. Live weight and breast muscle development was determined to 56 d of age at weekly intervals. MFD was assessed at 8 d of age. As expected, SC differed in BW, breast weight and yield, and breast fillet dimensions and had variations in growth curves. Maximal growth rate for breast weight was reached approximately 4 d after that of BW. Males and females showed different growth curves, with males having slower growth rate maturity parameter and reaching the maximal growth rate later than females for BW and breast weight. Breast depth was the breast measure with highest positive correlation to breast yield. SC differences could not be explained by MFD, but males had higher MFD density than females. The possible relationship of the MFD observations to total myofiber number is discussed.

Comparison of the histological and histochemical properties of skeletal muscles between carbon dioxide and electrically stunned chickens.

1. Histological and histochemical profiles of Musculus pectoralis (PT, type IIB fibres), M. iliotibialis lateralis (ITL, types IIA + IIB fibres) and M. puboischiofemoralis pars medialis (PIF, type I fibres) were compared in carbon dioxide (37%, 70 s) and electrically (14 V, 5 s) stunned male chickens. 2. Muscle materials were taken at 0, 4 and 24 h from carcases dressed and cooled with ice-water mixture for 30 min. Glycogen and fat contents, and adenosine triphosphatase and reduced nicotinamide adenine dinucleotide dehydrogenase activities of fibres were measured. 3. In PT muscle at 0 h, gas stunned chickens showed many fibres with high glycogen content but those electrically stunned contained few such fibres. Fibres from gas stunned birds had lost almost all their glycogen after 24 h of cold storage. 4. In the ITL muscle of gas stunned chickens at 0 h residual glycogen was observed in type IIB fibres. In contrast, in the electrically stunned birds it was in type IIA, showing the different effects of the stunning methods. During cold storage, glycogen disappeared earlier in type IIB than IIA fibres. 5. In PIF muscle with fibres of low glycogen content, the gas stunned chickens maintained a good fibre structure for 4 h or more, but the electrically stunned had already lost intact fibre structure at 4 h. 6. These results indicated that the carbon dioxide stunning was a better method for chicken welfare and meat quality than electrical stunning.

Identification of lipids as the main component of skeletal muscle Z-discs.

The existence of lipids in Z-discs of the vertebrate skeletal muscle was suggested by the results of microscopy using rhodamine 6G reagent, a fluorescent probe. After removal of lipids by treatment of myofibrils with 0.5% Trition X-100, intact configurations of Z-filaments composed of alpha-actinin were clearly visible under an electron microscope. These findings indicated that lipids were amorphous-matrix materials of the Z-disc. Lipids were proved to be the main component of Z-discs by their extraction from I-Z-I brushes with a mixture of methanol and chloroform and by analysing them. The total amount of lipids in Z-discs exceeded that of alpha-actinin and varied from 2.4 to 7.1 g per 100 g of myofibrillar proteins depending on the phenotype of skeletal muscle. The sum of the amounts of lipids and alpha-actinin was 4.5 g per 100 g of myofibrillar proteins in chicken pectoralis profundus muscle (fast type), while it was 10.1 g in chicken soleus muscle (slow type). Lipid classes were phospholipids, triacylglycerols, cholesterol and free fatty acids. Since the lipids extracted from I-Z-I brushes were hardly contaminated with those from the sarcoplasmic reticulum and mitochondria, their amounts and classes were considered to be characteristic of Z-discs. The lipids probably cement neighbouring Z-filaments electrostatically, reinforce the Z-disc structure, and play an important role in the force transmission of skeletal muscle myofibrils.

Ultrastructural changes and glutathione depletion in the skeletal muscle induced by protein malnutrition.

Ultrastructural changes and glutathione level were investigated in the pectoralis muscle of rats fed a low-protein diet. Electron microscopy demonstrated the ultrastructural changes of occasional myofibrils affected with protein deficiency that were characterized with the streaming and/or disruption of the Z-line and disintegration of sarcomeric striation. In the affected sarcomeres, sarcomere length was often elongated and fragmented thick filaments were present together with dense amorphous materials flowing from the damaged Z-line. Glutathione level of muscle in the low-protein diet group (5% casein) was reduced to about one-third of that in the control diet group (20% casein). The study suggests that depletion of glutathione by protein malnutrition is responsible for inducing myofibrillar damage through the excess leaking of Ca2+ into the cytosol.

The ultrastructure of skeletal and smooth muscle in experimental protein malnutrition in rats fed a low protein diet.

Light microscopy of the pectoralis muscle of rats on a low protein diet did not show such morphological alterations as atrophy, degeneration, or sarcoplasmic edema, but electron microscopy occasionally demonstrated ultrastructural changes only in the sarcomeres of myofibrils. In the affected sarcomeres, the Z-line was disrupted and often showed a jagged structure. The Z-substance with electron opacity was frequently present flowing along the long axis of myofibrils, here referred to as the streaming of Z-lines. In addition, regular striations formed by the reciprocal arrangement of thick and thin filaments disappeared from the affected sarcomeres, though these filaments were still discernible. Two or more consecutive sarcomeres in a single myofibril were occasionally involved in these changes. A further two or more neighboring sarcomeres at the same level of myofibrils were affected transversely by these structural alterations. On the other hand, the ultrastructure of the intestinal smooth muscle was not affected by protein deficiency. The study suggests that the ultrastructural damage induced by a low protein diet is attributed to the activation of endogenous protease by the excess leaking of Ca2+ into the cytosol as a result of lipid peroxidation of cell membrane by raised free radicals, owing to the depletion of glutathione production by protein deficiency. It also suggests that the smooth muscle cells differ in their susceptibility to protein deficiency from the skeletal muscle cells.

Fiber capillarization and ultrastructure of pigeon pectoralis muscle after cold acclimation.

We investigated the effect of 2 months of exposure to cold conditions (0-5 C) on capillarization and on fiber size, distribution and ultrastructure in the pectoralis muscle of nine pigeons (Columbia livia; mean body mass 700 31 g) and compared the results with measurements from four control birds (mean mass 715 42 g) kept at normal ambient temperature (22-23 C) for the same period. Superficial and deep portions of the muscles, taken from the central area of the right or left pectoralis major muscle, were perfusion-fixed in situ, processed for electron microscopy and analyzed by morphometry. Aerobic fibers represented the vast majority of fibers (93 1 %, mean s.e.m.) in all samples. After cold-acclimation, fiber sectional area was reduced and capillary density increased proportionally. There was no change in the degree of orientation (anisotropy) of capillaries, capillary-to-fiber ratio or fiber type distribution compared with controls. The volume density of mitochondria and lipid droplets in aerobic fibers and capillary diameter increased in response to cold, while the linear relationship between capillary length per fiber volume and fiber mitochondrial volume density remained unchanged. Capillary surface area, intrafiber lipid deposition and fiber mitochondrial volume density were all correlated in cold-acclimated pigeons. The results indicate a close match between the aerobic capacity of the highly aerobic fibers of the pectoralis muscle and their vascularization to meet the increased energetic demand of shivering.

Tissue-specific effects of hypothyroidism on postnatal muscle development in the barnacle goose.

The hypothesis that tissue-specific levels of thyroid hormones may be required for normal locomotor muscle development was investigated in the barnacle goose Branta leucopsis. Hypothyroidism was induced in goslings by treatment with methimazole from either 3 days or 2 weeks of age, and birds were killed at 7 weeks of age. The masses of the pectoralis, iliofibularis, semimembranosus and cardiac ventricle muscles were measured, and samples from these tissues were analysed for the mass-specific activity of the mitochondrial enzyme citrate synthase (CS). An ultrastructural electron micrograph analysis of the pectoralis was also carried out. No significant differences were found between the two hypothyroid groups except for the effect on the relative mass of the iliofibularis muscle. Developmental responses to hypothyroidism were found to be tissue-specific. Hypothyroidism resulted in a significantly lower relative cardiac ventricle mass (by 17 %) and CS activity of the leg muscles (by 34 %), while absolute leg muscle mass was not affected. The relative mass of the pectoralis was significantly lower (by 57 %) in hypothyroid birds and showed a significant, uniformly lower CS activity (by 60-83 %) as a result of a lower mitochondrial fractional volume. Haematocrit and capillary-to-fibre ratio in the pectoralis were also significantly lower in hypothyroid birds, and skeletal growth and plumage development were affected.

Characteristics of the pectoralis superficialis and semimembranosus of broiler strain chickens, bantam chickens, and the reciprocal crosses.

At hatch, chicks from bantam sire x broiler dam (BaBr) matings were as heavy as those from broiler sire x broiler dam (BrBr) matings. Both were heavier than pure bantam chicks (BaBa) or chicks from broiler strain sires and bantam dams (BrBa), but weights of the latter two genotypes were identical. The Pectoralis superficialis and Semimembranosus of pure bantam chicks weighed less than those of pure broiler chicks throughout the studies. Muscle weights in the BaBr chicks were markedly heavier than those of genetically similar BrBa chicks at hatch. Genetic influences were quickly expressed and weights of muscles in these two groups had converged by 14 d of age. Myofiber cross-sectional areas at 1 d of age were not clearly associated with genotype or maternal environment, but clear patterns had begun to develop by 4 d of age. Myofiber area of both muscles was greater in BrBr than BaBa chicks by this age and after. Myofiber areas in the reciprocal crosses converged toward each other and away from the pure line chicks. By 11 d, the area of Pectoralis myofibers of BrBr chicks was greatest, those of the reciprocal crosses was intermediate and equal to each other, and that of BaBa chicks was least. A similar trend was apparent in Semimembranosus fibers by 11 d but the differences did not become significant until 21 d of age. Significant differences in myofiber number of the Semimembranosus were present (BrBr > BaBr > BrBa > BaBa). Thus, differences in muscle mass were the result of differences in both fiber number and fiber size.