Evaluation of Capillary and Myofiber Density in the Pectoralis Major Muscles of Rapidly Growing, High-Yield Broiler Chickens During Increased Heat Stress Author(s): K. S. Joiner, G. A. Hamlin, R. J. Lien, and S. F. Bilgili Source: Avian Diseases, 58(3):377-382. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10733-112513-Reg.1 URL: http://www.bioone.org/doi/full/10.1637/10733-112513-Reg.1
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AVIAN DISEASES 58:377–382, 2014
Evaluation of Capillary and Myofiber Density in the Pectoralis Major Muscles of Rapidly Growing, High-Yield Broiler Chickens During Increased Heat Stress K. S. Joiner,AC G. A. Hamlin,A R. J. Lien,B and S. F. BilgiliB A B
Department of Pathobiology, College of Veterinary Medicine, 166 Greene Hall, Auburn University, AL Department of Poultry Science, College of Agriculture, 201 Poultry Science Building, Auburn University, AL Received 4 December 2013; Accepted 5 March 2014; Published ahead of print 6 March 2014
SUMMARY. Skeletal muscle development proceeds from early embryogenesis through marketing age in broiler chickens. While myofiber formation is essentially complete at hatching, myofiber hypertrophy can increase after hatch by assimilation of satellite cell nuclei into myofibers. As the diameter of the myofibers increases, capillary density peripheral to the myofiber is marginalized, limiting oxygen supply and subsequent diffusion into the myofiber, inducing microischemia. The superficial and deep pectoralis muscles constitute 25% of the total body weight in a market-age bird; thus compromise of those muscle groups can have profound economic impact on broiler production. We hypothesized that marginal capillary support relative to the hypertrophic myofibers increases the incidence of microischemia, especially in contemporary high-yield broilers under stressing conditions such as high environmental temperatures. We evaluated the following parameters in four different broiler strains at 39 and 53 days of age when reared under thermoneutral (20 to 25 C) versus hot (30 to 35 C) environmental conditions: capillary density, myofiber density and diameter, and degree of myodegeneration. Our data demonstrate that myofiber diameter significantly increased with age (P $ 0.0001), while the absolute numbers of capillaries, blood vessels, and myofibers visible in five 4003 microscopic fields decreased (P $ 0.0001). This is concomitant with marginalization of vascular support in rapidly growing myofibers. The myofiber diameter was significantly lower with hot environmental temperatures (P $ 0.001); therefore, the absolute number of myofibers visible in five 4003 microscopic fields was significantly higher. The incidence and subjective degree of myodegeneration characterized by loss of cross-striations, myocyte hyperrefractility, sarcoplasmic vacuolation, and nuclear pyknosis or loss also increased in hot conditions. Differences among strains were not observed. RESUMEN. Evaluacio´n de la densidad de capilares y miofibras en el mu´sculo pectoral superficial en pollos de engorde de ra´pido crecimiento y de alto rendimiento bajo condiciones aumentadas de estre´s por calor. El desarrollo del mu´sculo esquele´tico comprende desde de la embrioge´nesis temprana hasta la edad de la comercializacio´n en los pollos de engorde. Mientras que la formacio´n de las miofibras esta´ pra´cticamente concluida al momento de la eclosio´n, se puede incrementar la hipertrofia miofibrilar despue´s de la eclosio´n debido a la asimilacio´n de los nu´cleos de las ce´lulas sate´lite dentro de las miofibras. Como el dia´metro de las miofibras aumenta, la densidad capilar perife´rica a la miofibras se concentra en los ma´rgenes, lo que limita el suministro de oxı´geno y su difusio´n posterior a las miofibras, lo que induce microisquemia. Los mu´sculos pectorales superficiales y profundos constituyen el 25% del peso corporal total en un ave a la edad de mercado, por lo que el dan˜o de esos mu´sculos puede tener un profundo impacto econo´mico en la produccio´n de pollos de engorde. Se establecio´ la hipo´tesis de que el aporte capilar que esta marginalizado relacionado con las miofibras hipertro´ficas aumenta la incidencia de microisquemia, especialmente en pollos de engorde de alto rendimiento que se encuentran bajo condiciones de estre´s, tales como altas temperaturas ambientales. Se evaluaron diferentes para´metros en cuatro lı´neas diferentes de pollo de engorde a los 39 y 53 dı´as de edad que fueron criados en condiciones de termoneutralidad (20 a 25 C) y en condiciones de calor ambiental (30 a 35 C), los para´metros incluyeron: densidad capilar, dia´metro y densidad miofibrilar y el grado de miodegeneration. Los datos demuestran que el dia´metro de la miofibra aumento´ significativamente con la edad (P $ 0.0001), mientras que el nu´mero absoluto de capilares, vasos sanguı´neos y fibras musculares visibles en cinco campos microsco´picos de 400 3 disminuyeron (P $ 0.0001). Esto fue concomitante con la marginacio´n del soporte vascular en las miofibrillas de ra´pido crecimiento. El dia´metro de la miofibra fue significativamente menor en temperaturas ambientales calientes (P $ 0.001), por lo tanto, el nu´mero absoluto de fibras musculares visibles en cinco campos microsco´picos de 400 3fue significativamente mayor. La incidencia y el grado subjetivo de miodegeneration caracterizada por la pe´rdida de estrı´as cruzadas, hiperrefractibilidad de miocitos, vacuolizacio´n sarcopla´smica y picnosis o pe´rdida de nu´cleos tambie´n aumentaron en condiciones de calor. No se observaron diferencias entre las cepas. Key words: broiler chicken, capillary density, histomorphometrics, ischemia, myodegeneration, skeletal muscle
With the advent of industrial-scale agriculture during the early 20th century, intensive selection to improve broiler (chicken meat) production occurred and continues to occur at an astounding rate. Expansion of the broiler industry is driven by ever-increasing global consumer demand for sustainable, relatively inexpensive, and healthy protein sources. Vertical integration, advances in live production C
Corresponding author. E-mail: [email protected] The authors declared that they had no conflicts of interest with respect to their authorship or the publication of this article. This research was funded by the U.S.-Israel Binational Agricultural Research and Development Fund.
(breeding, nutrition, environmental control, and health programs), processing technologies (speed and automation), product diversification, and global trade have provided a market advantage for poultry as compared to other meat sources. According to the USDA Foreign Animal Service data on world markets and trade, approximately 81 million metric tons of broiler meat was produced worldwide in 2011, with a projected increase of 2.7% for FY2012 (27). Genetic selection for enhanced breast muscle development plays an important role in the high productivity and sustained economic efficiency of broiler chickens. Breast muscle (pectoralis major) development proceeds from early embryogenesis through marketing age. While myofiber formation is
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essentially complete at hatching, myofiber hypertrophy proceeds following hatch by the assimilation of satellite cell nuclei into the radially expanding myofiber (20). Satellite cells rapidly proliferate during the early growth phase; however, upon withdrawal from the cell cycle and differentiation, the number of satellite cells diminishes to less than 5% of total myofiber nuclei and toward the end of the growth phase become largely quiescent (1,14). Several growth factors including members of the fibroblast growth factor family, insulinlike growth factor I and II, and hepatocyte growth factor are able to stimulate or inhibit satellite cell proliferation and differentiation (13). Recently investigators have shown that nutritional or environmental treatments (heat conditioning or monochromatic green-light illumination) during late-term embryogenesis or first days posthatch increase breast muscle growth and weight at marketing age (9,10,11,12,13). Collectively, the increase in breast muscle growth is due to changes at the cellular and molecular levels, leading to increased satellite cell proliferation and differentiation with subsequent hyperplasia in the late term embryo (23). This is further enhanced by increased hypertrophy in the neonatal chick (13). A comparison between contemporary high-yield broiler chickens and a 1950s heritage line demonstrated that a market weight of approximately 2.3 kg is attained in 5 wk in the modern high-yield broiler, whereas it requires 16 wk in the heritage broiler line (26). Likewise, breast muscle mass increases at a rate of 6.1 g/day in the high-yield broiler as compared to 1.6 g/day in heritage line broilers. While the overall metabolism of the modern broiler has evolved, resulting in birds that are highly efficient at converting feed to body mass and increased muscle yield, so too have undesirable traits arisen, presumably due to increased stress from rapid growth rates (8). Rapid growth has been associated with sudden death due to cardiovascular failure, ascites, reduced adaptive immune function, poor reproductive performance, and reduction in bone strength due to alterations in calcium and phosphorous metabolism (4,15, 21,22,24,25). Enhanced breast muscle development is a major factor in genetic selection of contemporary broiler lines, and unfortunately the detrimental effects of rapid growth are often exacerbated in the pectoralis muscle groups. Increased radial growth of myofibers impairs the gas exchange capacity due to margination of the supporting capillaries and resultant increased diffusion distance of oxygen and various metabolites across the myofiber diameter (16). Functional ischemia is grossly evident at processing, as affected muscles are frequently pale and flaccid and may be discolored red or yellow-green due to associated hemorrhage or inflammatory infiltrates, respectively. This invariably diminishes carcass quality, resulting in economic loss. Vascular compromise with insufficient oxygen supply to muscles is exacerbated during periods of increased internal and/or external heat stress. Increased sarcoplasmic protein deposition during radial myofiber hypertrophy elevates oxygen demand and increases internal metabolic heat production. The heat load on rapidly growing birds is further intensified as expanding geographical spread of hot climatic conditions and energy costs for environmental cooling systems continue to increase. The effects of both internal metabolic heat production and high environmental temperatures synergistically contribute to the increasing prevalence of myofiber damage and less than optimal breast meat yield and quality. The present study was designed to evaluate the capillary and myofiber density in the pectoralis major muscles of standard yield and rapidly growing highyield broiler chickens reared in either thermoneutral or hot environmental conditions using histomorphometric assessment.
MATERIALS AND METHODS Chickens. One hundred forty-four, 1-day-old broiler chicks were obtained from two separate commercial poultry breeding companies, 1 and 2 (coded nomenclature to protect proprietary rights). Chicks were obtained from similarly aged breeder flocks so that chick weights and maternal immunity effects were comparable. Thirty-six standard yield broiler chickens were selected from each of the commercial poultry breeding companies 1 and 2 and designated strain A and strain B, respectively. Standard yield strains of broilers have been bred for several decades with the main emphasis placed on rapid growth and secondary emphasis placed on breast meat yield and other traits related to productive and reproductive performance. Thirty-six high-yield broiler chickens were also selected from each of the commercial breeding companies 1 and 2 and correspondingly designated strain C and strain D. High-yield strains have been developed during the past decade of intensive genetic selection pressure for high breast meat yield. Chickens were individually identified by wing banding and were reared in 12, 3.0 m 3 3.7 m, environmentally controlled rooms, each divided into two pens and bedded with pine shavings. The chickens were fed a standard commercial ration with ad libitum access to water. Experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal use guidelines. Auburn University is an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited institution. Study design. The experimental strategy was to evaluate capillary density and myofiber dynamics in each genetic strain A–D, subjected to two types of heat load: internal heat production due to metabolism and environmental temperature effects. Metabolic heat production is exacerbated in broilers reared to heavy body weight (about 3.5 kg) as compared to standard body weight (about 2.5 kg). Environmental heat load increases when broilers are reared in high ambient temperatures (30 to 35 C) versus thermoneutral ambient temperatures (20 to 25 C). Total heat stress in rapidly growing broilers is often a combination of both. Therefore, chickens from each strain A–D were reared to standard or heavy body weights in either high temperatures (30 to 35 C) or under thermoneutral (20 to 25 C) conditions with a 4 3 2 factorial arrangement of strains and temperature treatments. Seventy-two birds (18 from each strain A–D) were placed in 12 thermoneutral pens, and 72 birds (18 from each strain A–D) were placed in 12 high-temperature pens with a stocking density of six birds per pen. Temperatures in the thermoneutral treatment group were maintained at 29.4 to 32.2 C during week 1, 26.6 to 29.4 C during week 2, 23.8 to 26.6 C during week 3, and 20 to 25 C thereafter. Temperatures in the hot treatment group were maintained at 30 to 35 C throughout the trial. Nine birds from each strain A–D in the thermoneutral treatment group (n 5 36), and nine birds from each strain A–D in the hot treatment group (n 5 36) were humanely euthanatized and necropsied at 39 days of age and at 53 days of age, corresponding to standard and heavy body weights, respectively. Histopathology. A 1.0 cm 3 0.5 cm section of breast muscle was collected from the right pectoralis major muscle from each bird. Muscle samples were fixed by immersion in 10% neutral buffered formalin, routinely processed, and sectioned at 4–6 mm. Two serial sections of muscle were evaluated. The initial section was stained with hematoxylin and eosin, and the adjacent section was evaluated immunohistochemically with commercially available antisera for muscle-specific actin (Dako, Carpentaria, CA). Subjective assessment of the muscle sections was based on the following categories: increased refractility, loss of crossstriations, nuclear pyknosis, myodegeneration, and fragmentation. Subjective lesion categories are illustrated in Fig. 1. Subjective scores assigned for each category were as follows: 1 5 normal, 2 5 mild, 3 5 moderate, 4 5 marked, and 5 5 severe. Histomorphometric analysis. The following parameters were assessed histomorphometrically for quantitative and statistical evaluation between genetic strains, heat treatment, and age groups: number of capillaries, number of vessels, and number of myofibers; myofiber diameter; muscle cross-sectional area; and interfasicular edema. For each section, the number of capillaries, larger vessels, and myofibers were
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Fig. 1. Pectoralis major muscle; broiler chicken. Extensive regions of myodegeneration and necrosis in broiler chickens raised in hot (30 to 35 C) environmental conditions illustrate categories used for subjective assessment of myofibers. HE. Fig. 2. Pectoralis major muscle; broiler chicken. Standardized microscopic field used for assessment of capillary and vascular density, and direct count of myofibers. The numbers of capillaries (arrows), vessels (arrowhead) and muscle fibers were counted in five contiguous 4003 fields. Note increased interfasicular edema. HE. Fig. 3. Pectoralis major muscle; broiler chicken. Photomicrographs were converted to a 16-bit grayscale image, and the ImageJ red-filter threshold was applied. There is minimal enhancement of the myofiber margins. HE. Fig. 4. Pectoralis major muscle; broiler chicken. The ImageJ red-filter threshold was adjusted such that the myofibers were red-enhanced and the interfasicular space remained white. Myofiber cross-sectional area was determined by limiting the digital measurement to the red-enhanced myofibers within a defined area. Interfasicular space was determined by subtracting the myofiber cross-sectional area from the total area. HE.
directly counted in five 4003 microscopic fields by a single pathologist in a blinded fashion (Fig. 2). Assessment of each microscopic field was standardized by selecting solid fields of myofibers containing no adipose tissue or fascial planes. Myofibers along the margins that were partially observed in the field of view were counted as 0.5 fibers. Myofiber diameter, muscle cross-sectional area, and interfasicular edema were morphometrically quantitated for statistical comparison using Image J version 1.30 (National Institutes of Health, available at http://rsb.info. nih.gov/ij). Briefly, a high-resolution, 4080 3 3072, digital photomicrograph of a representative cross section of each muscle sample was taken at 4003 magnification using an Olympus DP71 digital camera system mounted to an Olympus BX41 microscope. To assess individual myofibers, the Image J linear measurement tool was used to measure the cross-sectional diameter of five contiguous myofibers. The shortest distance across each myofiber was measured to minimize variability associated with stage of muscle contraction in neighboring fibers. To
determine the amount of total myofiber cross-sectional area and interfasicular edema, each photomicrograph was converted to a 16-bit grayscale image. Using a polygon shape tool, a polygon was set to a fixed area that was limited to myofibers that were completely visible in the field, and the total area within the polygon was measured. The red-filter threshold was set, and by adjusting the threshold margin, the myofibers were red-enhanced, while the surrounding interfasicular edema remained in grayscale (Figs. 3–4). The measurement was limited to the red-enhanced threshold areas, and the area inside the polygon was measured, representing the myofiber cross-sectional area. The difference between the total area and the cross-sectional area represents the interfasicular edema. The proportion of myofiber cross-sectional area to interfasicular edema was calculated. In addition, the capillary to fiber ratio, capillary to vessel ratio, vessel to fiber ratio, sum of all capillaries and vessels, and sum of capillaries and vessels to fiber ratio were determined.
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Table 1. Histomorphometric assessment of myofiber diameter, myofiber and vascular density, and vascular to myofiber ratios.A Average number (five 4003 microscopic fields) Myofiber diameter
Capillaries
Blood vessels
Myofibers
Age (days) 39 53
525* 619*
70* 54*
8.2* 4.6*
238* 150*
Temperature (C) 20 to 25 30 to 35
597** 547**
61 64
6.3 6.4
176* 211*
Strain A B C D
577 556 570 585
62 62 62 63
7.0 6.6 5.6 6.2
197 193 187 197
Ratio Capillary/ vessel
Vessel/ fiber
Capillaries + vessels
Capillaries + vessels/fiber
8.5* 11.7*
0.036 0.035
78* 59*
0.34* 0.43*
0.36*** 0.33***
13.83 16.54
0.039 0.032
67 70
0.41 0.36
0.35 0.35 0.35 0.33
15.68 15.66 14.56 14.85
0.039 0.034 0.034 0.034
69 69 67 69
0.39 0.38 0.39 0.38
Capillary/ fiber
30* 39*
A
*P $ 0.0001; **P $ 0.001; ***P $ 0.05.
Statistical analysis. Least squares means analysis was used to detect statistically significant differences in subjective myofiber scores, histomorphometric parameters and ratios among age and heat treatment groups, and genetic broiler strains A–D. Interactions between age, temperature, and strain were also evaluated.
RESULTS
While subjective assessment of myofibers did not statistically differ among age or temperature treatment groups, the incidence of myodegeneration, fragmentation of individual myofibers with loss of cross-striations and increased refractility was slightly elevated in birds reared in hot environmental conditions. Histomorphometric evaluation of muscle sections revealed that as broilers age, muscle fibers significantly increase in diameter through hypertrophy, and vascular support is marginalized (Table 1). Capillary density decreased by 23% (P $ 0.0001), and vessel density decreased by 44% (P $ 0.0001) as the broilers aged from 39 to 53 days. Likewise, the number of myofibers visible in five 4003 fields decreased by 37% (P $ 0.0001), and the average myofiber diameter increased from 84.5 to 99.7 mm (P $ 0.0001) from 39 to 53 days. The vascular support relative to the number of myofibers was also evaluated for each age group. The number of capillaries per myofiber increased from 0.30 to 0.36 (P $ 0.001), and the number of capillaries and vessels per myofiber increased from 0.34 to 0.43 (P $ 0.0001). However, the number of vessels per myofiber did not significantly change with age, and the ratio of capillaries to vessels increased from 8.5:1 to 11.7:1 (P $ 0.001), indicating that the capillaries are the primary contributor to vascular support in growing myofibers. Myofiber diameter and number of myofibers in five 4003 microscopic fields significantly differed between birds reared in thermoneutral versus hot ambient conditions (Table 1). Broilers raised in thermoneutral environments had an average fiber diameter of 96.1 mm, while those in heat-stressed environments had an average fiber diameter of only 88.1 mm (P $ 0.001). Due to the overall reduction in myofiber diameter, more myofibers were visible per field of view in heat-stressed broilers as compared to the non-heatstressed broilers (P $ 0.0001). The number of capillaries visible in five 4003 fields was higher in heat-stressed birds (n 5 64) as compared to broilers reared in thermoneutral conditions (n 5 61). However, the number of capillaries per myofiber (n 5 0.33) and the number of capillaries and vessels per myofiber (n 5 0.36) were
significantly lower in heat-stressed birds (P $ 0.05) and (P $ 0.0001), respectively. There were no significant differences in the numbers of capillaries or blood vessels between temperature treatment groups. Similarly, differences in the number of vessels per fiber, total number of capillaries and vessels, total vascular support per fiber, and ratio of capillaries to vessels were not significant. Our data show that myofiber cross-sectional area differs slightly with age and among temperature treatment groups when expressed as a percentage of the total area (P 5 0.09) (Table 2). Individual myofibers are larger in older birds and in birds reared in thermoneutral conditions. The cross-sectional area of an individual myofiber is directly proportional to the radius of the myofiber. However, the absolute number of myofibers in a fixed area defined by using the ImageJ polygon tool is indirectly proportional to myofiber size and is reflected in the overall myofiber cross-sectional area. As broilers aged from 39 to 53 days, the total myofiber crosssectional area decreased by 14.7%. Total myofiber cross-sectional area was 10.9% lower in broilers raised in thermoneutral versus hot environmental conditions. There was slightly more interfasicular edema in the 39-day-old broiler chickens and in those birds raised in hot environments, although these differences were not significant. No significant differences in any of the histomorphometric parameters were observed between genetic broiler strains (Tables 1 and 2). DISCUSSION
It is well documented that muscle deposition is associated with higher levels of protein metabolism, which in turn elevates the demand for oxygen and other critical macronutrients to support enhanced growth (13,23). Others have demonstrated that radial expansion of myofibers increases the myofiber diameter and effectively marginalizes the capillary and vessel supply to the muscle (16). These findings are corroborated by the current results, which show that myofiber diameter increases with age, while the absolute number of myofibers and capillaries in a microscopic field decreases. However, the number of capillaries per myofiber and capillaries and vessels per myofiber is slightly higher in 53-day-old broilers. Therefore, the reduction in absolute numbers of capillaries is not directly proportional to myofiber growth. The increased ratio of capillaries to myofibers may be necessary to support adequate
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Evaluation of capillary and myofiber density
Table 2.
Quantitative histomorphometric assessment of myofiber cross-sectional area and interfasicular space.A Parameters (mm2) Total area (TA)
Myofiber cross-sectional area (CSA)
Interfasicular space
CSA % of TA
Age (days) 39 53
197 823.14* 170 174.94*
189 784.65* 162 337.50*
8038.49 7837.45
95.98*** 95.28***
Temperature (C) 20 to 25 30 to 35
179 434.99** 189 281.74**
171 746.44** 181 095.71**
7688.55 8186.03
95.59 95.67
Strain A B C D
183 185 182 185
176 178 174 176
7846.57 7015.88 7690.08 9196.64
95.75 96.24 95.61 94.92
905.99 426.86 354.96 757.56
059.42 410.98 664.89 560.92
A
*P $ 0.0001; **P $ 0.05; ***P 5 0.09.
macronutrient delivery, as diffusion distance across the myofiber increases with radial growth. While slight increases in vascular support may be sufficient for sedentary birds reared in optimal conditions, the capillary density may not be sufficient to support enhanced demand for oxygen during increased exertion thus predisposing the bird to microischemia and muscle damage. The incidence and economic significance of exertional ischemic myodegeneration and necrosis has been extensively studied in the pectoralis minor (deep pectoral) muscles of high-yield broiler chickens. With increased exertion (i.e., wing flapping), circulation increases and myofibers can expand up to 20% in volume (19). The pectoralis minor muscles are surrounded by an inelastic fascial membrane and located immediately adjacent to the sternum and pectoralis major; thus muscle expansion may lead to compartmentalization syndrome, restricting blood flow with subsequent ischemic necrosis. Recent field studies have indicated that deep pectoral myopathy, commonly referred to as ‘‘green muscle disease,’’ poses a significant negative economic impact on the high-yield broiler industry with projected U.S. industry losses of $16 million annually. Gross lesions can develop within a matter of hours following an acute bout of muscle exertion, irreversibly damaging the pectoralis minor muscles and decreasing breast muscle yield at slaughter (17). Our research group recently developed an experimental protocol for the induction of deep pectoral myopathy in broiler chickens via encouraged wing flapping (18). Forty-five day-old broiler chickens subjected to 10 or 20 cycles of encouraged wing flapping had deep pectoral myopathy indices of 7% and 33%, respectively (18). Gross lesions were subtle 24 hr after deep pectoral myopathy induction, but progressively increased to severe myonecrosis with significant hemorrhage over a course of 11 days (18). Histologically there is extensive interfasicular edema and early myonecrosis within as little as 3 hr postexertion (Joiner, pers. obs., unpubl. data), which can impact meat quality and consumer palatability. So therein lies the challenge of contemporary high-yield broiler chickens: meeting consumer demand without compromising meat quality and breast muscle yield. As genetic selection of contemporary broiler chickens evolved to support increased demand for higher growth rates and productivity, internal (metabolic) heat production has increased substantially. To offset internal metabolic heat production, high-yield broiler chickens are reared in lower ambient temperatures in order to fully express genetic potential for increased breast muscle yield and quality. It has been hypothesized that heat-stressed broilers cannot synthesize protein as efficiently as birds reared in thermoneutral conditions,
and therefore muscle growth by hypertrophy is limited. Broilers acclimate to hot conditions by reducing feed intake resulting in depressed growth rate, lower overall body weight, and poor breast muscle yield (2,3,5,6,7). Our data support these previous observations, by illustrating the corresponding histologic features of depressed muscle growth. Myofiber diameter was drastically reduced in birds reared in hot ambient conditions, while the absolute numbers of capillaries and blood vessels did not significantly differ between thermal treatment groups. The number of capillaries per myofiber and capillaries and vessels per myofiber was slightly less in birds exposed to hot ambient conditions due to the significant increase in absolute numbers of small diameter myofibers visible in each 4003 microscopic field. Collectively, these findings suggest that limited myofiber growth during heat stress is due to diminished radial myofiber growth (i.e., decreased protein deposition in the expanding myofiber) and not necessarily due to decreased vascular support and delivery of essential macronutrients. Histomorphometric assessment of myofiber and capillary density provided further evidence suggesting that diminished relative vascular support may contribute to an increased incidence of pectoral myopathy in high-yield, broiler chickens. While older birds and birds reared in thermoneutral ambient conditions had larger individual myofibers, the total myofiber cross-sectional area within a fixed dimension decreased significantly. However, the number of capillaries per unit area did not substantially increase with age or in the thermoneutral temperature treatment group. This effectively increases the diffusion distance of critical macronutrients from the capillary to the myofiber. Marginal capillary support may be sufficient in sedentary birds; however, as contemporary fast-growing, high-yield broilers are subjected to acute episodes of muscle exertion (i.e., preslaughter handling) or acute heat stress (i.e., transportation to processing facilities), the marginal capillary support cannot provide adequate nutrient support for the larger myofibers, thus predisposing the bird to microischemia and myonecrosis. Collectively our findings show that myofiber diameter significantly increases with age and individual myofibers are larger in birds reared in thermoneutral ambient conditions, as compared to younger counterparts, and birds reared in hot environmental conditions. Substantial increases in vascular support were not evident in birds with larger myofibers, suggesting that muscle growth may be approaching a critical mass in contemporary highyield broilers. Further enhancement of muscle development with subsequent generation of high levels of internal (metabolic) heat could lead to ischemic myopathies even in optimal environmental
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conditions. It is plausible that the effects of acute exertion and external heat stress in contemporary high-yield broiler chickens could be ameliorated with progressive acclimation to increased environmental stressors. Studies investigating the effects of acute versus sustained, progressive heat stress in high-yield broiler strains are ongoing. REFERENCES 1. Allouh, M. Z., Z. Yablonka-Reuveni, and B. W. Rosser. Pax7 reveals a greater frequency and concentration of satellite cells at the ends of growing skeletal muscle fibers. J Histochem. Cytochem. 56:77–87. 2008. 2. Cahaner, A., and F. Leenstra. Effects of high temperature on growth and efficiency of male and female broilers from lines selected for high weight gain, favorable feed conversion, and high or low fat content. Poult. Sci. 71:1237–1250. 1992. 3. Cahaner, A., Y. Pinchasov, I. Nir, and Z. Nitsan. Effects of dietary protein under high ambient temperature on body weight, breast meat yield, and abdominal fat deposition of broiler stocks differing in growth rate and fatness. Poult. Sci. 74:968–975. 1995. 4. Cheema, M. A., M. A. Qureshi, and G. B. Havenstein. A comparison of the immune response of a 2001 commercial broiler with a 1957 randombred broiler strain when fed representative 1957 and 2001 broiler diets. Poult. Sci. 82:1519–1529. 2003. 5. Cooper, M. A., and K. W. Washburn. The relationships of body temperature to weight gain, feed consumption, and feed utilization in broilers under heat stress. Poult. Sci. 77:237–242. 1988. 6. Deeb, N., and A. Cahaner. The effects of naked neck genotypes, ambient temperature, and feeding status and their interactions on body temperature and performance of broilers. Poult. Sci. 78:1341–1346. 1999. 7. Deeb, N., and A. Cahaner. Genotype-by-environment interaction with broiler genotypes differing in growth rate. 3. Growth rate and water consumption of broiler progeny from weight-selected versus nonselected parents under normal and high ambient temperatures. Poult. Sci. 81:293–301. 2002. 8. Griffin, H. D., and C. Goddard. Rapidly growing broiler (meat-type) chickens: their origin and use for comparative studies of the regulation of growth. Int. J. Biochem. 26:19–28. 1994. 9. Halevy, O., A. Geyra, M. Barak, Z. Uni, and D. Sklan. Early posthatch starvation decreases satellite cell proliferation and skeletal muscle growth in chicks. J. Nutr. 130:858–864. 2000. 10. Halevy, O., A. Krispin, Y. Leshem, J. P. McMurtry, and S. Yahav. Early-age heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281:R302–309. 2001. 11. Halevy, O., and O. Lerman. Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor-alpha. J. Cell Physiol. 154:566–572. 1993. 12. Halevy, O., Y. Piestun, I. Rozenboim, and Z. Yablonka-Reuveni. In ovo exposure to monochromatic green light promotes skeletal muscle cell
proliferation and affects myofiber growth in posthatch chicks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290:R1062–1070. 2006. 13. Halevy, O., S. Yahav, and I. Rozenboim. Enhancement of meat production by environmental manipulation in embryo and young broilers. World’s Poult. Sci. J. 62:485–497. 2006. 14. Hawke, T. J., and D. J. Garry. Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91:534–551. 2001. 15. Hocking, P. M. Effects of body weight at sexual maturity and the degree and age of restriction during rearing on the ovarian follicular hierarchy of broiler breeder females. Br. Poult. Sci. 34:793–801. 1993. 16. Hoving-Bolink, A. H., R. W. Kranen, R. E. Klont, C. L. Gerritsen, and K. H. de Greef. Fibre area and capillary supply in broiler breast muscle in relation to productivity and ascites. Meat Sci. 56:397–402. 2000. 17. Lien, R. J., S. F. Bilgili, J. B. Hess, and K. S. Joiner. Finding answers to ‘‘Green Muscle Disease.’’ WATT Poultry USA May:15–18. 2011. 18. Lien, R. J., S. F. Bilgili, J. B. Hess, and K. S. Joiner. Induction of deep pectoral myopathy in broiler chickens via encouraged wing flapping. J. Appl. Poult. Sci. 21:556–562. 2012. 19. Martindale, L., W. G. Siller, and P. A. Wight. Effects of subfascial pressure in experimental deep pectoral myopathy of the fowl: an angiographic study. Avian Pathol. 8:425–436. 1979. 20. Moss, F. P., and C. P. Leblond. Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170:421–435. 1971. 21. McDevitt, R. M., G. M. McEntee, and K. A. Rance. Bone breaking strength and apparent metabolisability of calcium and phosphorus in selected and unselected broiler chicken genotypes. Br. Poult. Sci. 47:613–621. 2006. 22. Morris, M. P. Ascites in broilers. Poult. Int. October:26–32. 1992. 23. Piestun, Y., M. Harel, M. Barak, S. Yahav, and O. Halevy. Thermal manipulations in late-term chick embryos have immediate and longer term effects on myoblast proliferation and skeletal muscle hypertrophy. J. Appl. Physiol. 106:233–240. 2009. 24. Olkowski, A. A., C. Wojnarowicz, S. Nain, B. Ling, J. M. Alcorn, and B. Laarveld. A study on pathogenesis of sudden death syndrome in broiler chickens. Res. Vet. Sci. 85:131–140. 2008. 25. Rath, N. C., G. R Huff, W. E Huff, and J. M. Balog. Factors regulating bone maturity and strength in poultry. Poult. Sci. 79:1024–1032. 2000. 26. Schmidt, C. J., M. E. Persia, E. Feierstein, B. Kingham, and W. W. Saylor. Comparison of a modern broiler line and a heritage line unselected since the 1950s. Poult. Sci. 88:2610–2619. 2009. 27. U.S. Department of Agriculture Foreign Agricultural Service. Livestock and Poultry: World Markets and Trade [Internet]. Livestock and Poultry: World Market and Trade Circular Archives. 2012 [cited 2012 July 31]. Available from: http://www.fas.usda.gov/livestock-and-poultryworld-markets-and-trade
ACKNOWLEDGMENTS We gratefully acknowledge the histotechnology expertise of Beth Landreth, Kristina Cammack, and Scott Kincaid and the technical assistance provided by William Clements and Charlotte Wilson.
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AVIAN DISEASES 58:377–382, 2014
Evaluation of Capillary and Myofiber Density in the Pectoralis Major Muscles of Rapidly Growing, High-Yield Broiler Chickens During Increased Heat Stress K. S. Joiner,AC G. A. Hamlin,A R. J. Lien,B and S. F. BilgiliB A B
Department of Pathobiology, College of Veterinary Medicine, 166 Greene Hall, Auburn University, AL Department of Poultry Science, College of Agriculture, 201 Poultry Science Building, Auburn University, AL Received 4 December 2013; Accepted 5 March 2014; Published ahead of print 6 March 2014
SUMMARY. Skeletal muscle development proceeds from early embryogenesis through marketing age in broiler chickens. While myofiber formation is essentially complete at hatching, myofiber hypertrophy can increase after hatch by assimilation of satellite cell nuclei into myofibers. As the diameter of the myofibers increases, capillary density peripheral to the myofiber is marginalized, limiting oxygen supply and subsequent diffusion into the myofiber, inducing microischemia. The superficial and deep pectoralis muscles constitute 25% of the total body weight in a market-age bird; thus compromise of those muscle groups can have profound economic impact on broiler production. We hypothesized that marginal capillary support relative to the hypertrophic myofibers increases the incidence of microischemia, especially in contemporary high-yield broilers under stressing conditions such as high environmental temperatures. We evaluated the following parameters in four different broiler strains at 39 and 53 days of age when reared under thermoneutral (20 to 25 C) versus hot (30 to 35 C) environmental conditions: capillary density, myofiber density and diameter, and degree of myodegeneration. Our data demonstrate that myofiber diameter significantly increased with age (P $ 0.0001), while the absolute numbers of capillaries, blood vessels, and myofibers visible in five 4003 microscopic fields decreased (P $ 0.0001). This is concomitant with marginalization of vascular support in rapidly growing myofibers. The myofiber diameter was significantly lower with hot environmental temperatures (P $ 0.001); therefore, the absolute number of myofibers visible in five 4003 microscopic fields was significantly higher. The incidence and subjective degree of myodegeneration characterized by loss of cross-striations, myocyte hyperrefractility, sarcoplasmic vacuolation, and nuclear pyknosis or loss also increased in hot conditions. Differences among strains were not observed. RESUMEN. Evaluacio´n de la densidad de capilares y miofibras en el mu´sculo pectoral superficial en pollos de engorde de ra´pido crecimiento y de alto rendimiento bajo condiciones aumentadas de estre´s por calor. El desarrollo del mu´sculo esquele´tico comprende desde de la embrioge´nesis temprana hasta la edad de la comercializacio´n en los pollos de engorde. Mientras que la formacio´n de las miofibras esta´ pra´cticamente concluida al momento de la eclosio´n, se puede incrementar la hipertrofia miofibrilar despue´s de la eclosio´n debido a la asimilacio´n de los nu´cleos de las ce´lulas sate´lite dentro de las miofibras. Como el dia´metro de las miofibras aumenta, la densidad capilar perife´rica a la miofibras se concentra en los ma´rgenes, lo que limita el suministro de oxı´geno y su difusio´n posterior a las miofibras, lo que induce microisquemia. Los mu´sculos pectorales superficiales y profundos constituyen el 25% del peso corporal total en un ave a la edad de mercado, por lo que el dan˜o de esos mu´sculos puede tener un profundo impacto econo´mico en la produccio´n de pollos de engorde. Se establecio´ la hipo´tesis de que el aporte capilar que esta marginalizado relacionado con las miofibras hipertro´ficas aumenta la incidencia de microisquemia, especialmente en pollos de engorde de alto rendimiento que se encuentran bajo condiciones de estre´s, tales como altas temperaturas ambientales. Se evaluaron diferentes para´metros en cuatro lı´neas diferentes de pollo de engorde a los 39 y 53 dı´as de edad que fueron criados en condiciones de termoneutralidad (20 a 25 C) y en condiciones de calor ambiental (30 a 35 C), los para´metros incluyeron: densidad capilar, dia´metro y densidad miofibrilar y el grado de miodegeneration. Los datos demuestran que el dia´metro de la miofibra aumento´ significativamente con la edad (P $ 0.0001), mientras que el nu´mero absoluto de capilares, vasos sanguı´neos y fibras musculares visibles en cinco campos microsco´picos de 400 3 disminuyeron (P $ 0.0001). Esto fue concomitante con la marginacio´n del soporte vascular en las miofibrillas de ra´pido crecimiento. El dia´metro de la miofibra fue significativamente menor en temperaturas ambientales calientes (P $ 0.001), por lo tanto, el nu´mero absoluto de fibras musculares visibles en cinco campos microsco´picos de 400 3fue significativamente mayor. La incidencia y el grado subjetivo de miodegeneration caracterizada por la pe´rdida de estrı´as cruzadas, hiperrefractibilidad de miocitos, vacuolizacio´n sarcopla´smica y picnosis o pe´rdida de nu´cleos tambie´n aumentaron en condiciones de calor. No se observaron diferencias entre las cepas. Key words: broiler chicken, capillary density, histomorphometrics, ischemia, myodegeneration, skeletal muscle
With the advent of industrial-scale agriculture during the early 20th century, intensive selection to improve broiler (chicken meat) production occurred and continues to occur at an astounding rate. Expansion of the broiler industry is driven by ever-increasing global consumer demand for sustainable, relatively inexpensive, and healthy protein sources. Vertical integration, advances in live production C
Corresponding author. E-mail: [email protected] The authors declared that they had no conflicts of interest with respect to their authorship or the publication of this article. This research was funded by the U.S.-Israel Binational Agricultural Research and Development Fund.
(breeding, nutrition, environmental control, and health programs), processing technologies (speed and automation), product diversification, and global trade have provided a market advantage for poultry as compared to other meat sources. According to the USDA Foreign Animal Service data on world markets and trade, approximately 81 million metric tons of broiler meat was produced worldwide in 2011, with a projected increase of 2.7% for FY2012 (27). Genetic selection for enhanced breast muscle development plays an important role in the high productivity and sustained economic efficiency of broiler chickens. Breast muscle (pectoralis major) development proceeds from early embryogenesis through marketing age. While myofiber formation is
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essentially complete at hatching, myofiber hypertrophy proceeds following hatch by the assimilation of satellite cell nuclei into the radially expanding myofiber (20). Satellite cells rapidly proliferate during the early growth phase; however, upon withdrawal from the cell cycle and differentiation, the number of satellite cells diminishes to less than 5% of total myofiber nuclei and toward the end of the growth phase become largely quiescent (1,14). Several growth factors including members of the fibroblast growth factor family, insulinlike growth factor I and II, and hepatocyte growth factor are able to stimulate or inhibit satellite cell proliferation and differentiation (13). Recently investigators have shown that nutritional or environmental treatments (heat conditioning or monochromatic green-light illumination) during late-term embryogenesis or first days posthatch increase breast muscle growth and weight at marketing age (9,10,11,12,13). Collectively, the increase in breast muscle growth is due to changes at the cellular and molecular levels, leading to increased satellite cell proliferation and differentiation with subsequent hyperplasia in the late term embryo (23). This is further enhanced by increased hypertrophy in the neonatal chick (13). A comparison between contemporary high-yield broiler chickens and a 1950s heritage line demonstrated that a market weight of approximately 2.3 kg is attained in 5 wk in the modern high-yield broiler, whereas it requires 16 wk in the heritage broiler line (26). Likewise, breast muscle mass increases at a rate of 6.1 g/day in the high-yield broiler as compared to 1.6 g/day in heritage line broilers. While the overall metabolism of the modern broiler has evolved, resulting in birds that are highly efficient at converting feed to body mass and increased muscle yield, so too have undesirable traits arisen, presumably due to increased stress from rapid growth rates (8). Rapid growth has been associated with sudden death due to cardiovascular failure, ascites, reduced adaptive immune function, poor reproductive performance, and reduction in bone strength due to alterations in calcium and phosphorous metabolism (4,15, 21,22,24,25). Enhanced breast muscle development is a major factor in genetic selection of contemporary broiler lines, and unfortunately the detrimental effects of rapid growth are often exacerbated in the pectoralis muscle groups. Increased radial growth of myofibers impairs the gas exchange capacity due to margination of the supporting capillaries and resultant increased diffusion distance of oxygen and various metabolites across the myofiber diameter (16). Functional ischemia is grossly evident at processing, as affected muscles are frequently pale and flaccid and may be discolored red or yellow-green due to associated hemorrhage or inflammatory infiltrates, respectively. This invariably diminishes carcass quality, resulting in economic loss. Vascular compromise with insufficient oxygen supply to muscles is exacerbated during periods of increased internal and/or external heat stress. Increased sarcoplasmic protein deposition during radial myofiber hypertrophy elevates oxygen demand and increases internal metabolic heat production. The heat load on rapidly growing birds is further intensified as expanding geographical spread of hot climatic conditions and energy costs for environmental cooling systems continue to increase. The effects of both internal metabolic heat production and high environmental temperatures synergistically contribute to the increasing prevalence of myofiber damage and less than optimal breast meat yield and quality. The present study was designed to evaluate the capillary and myofiber density in the pectoralis major muscles of standard yield and rapidly growing highyield broiler chickens reared in either thermoneutral or hot environmental conditions using histomorphometric assessment.
MATERIALS AND METHODS Chickens. One hundred forty-four, 1-day-old broiler chicks were obtained from two separate commercial poultry breeding companies, 1 and 2 (coded nomenclature to protect proprietary rights). Chicks were obtained from similarly aged breeder flocks so that chick weights and maternal immunity effects were comparable. Thirty-six standard yield broiler chickens were selected from each of the commercial poultry breeding companies 1 and 2 and designated strain A and strain B, respectively. Standard yield strains of broilers have been bred for several decades with the main emphasis placed on rapid growth and secondary emphasis placed on breast meat yield and other traits related to productive and reproductive performance. Thirty-six high-yield broiler chickens were also selected from each of the commercial breeding companies 1 and 2 and correspondingly designated strain C and strain D. High-yield strains have been developed during the past decade of intensive genetic selection pressure for high breast meat yield. Chickens were individually identified by wing banding and were reared in 12, 3.0 m 3 3.7 m, environmentally controlled rooms, each divided into two pens and bedded with pine shavings. The chickens were fed a standard commercial ration with ad libitum access to water. Experimental procedures and animal care were performed in compliance with all applicable federal and institutional animal use guidelines. Auburn University is an Association for Assessment and Accreditation of Laboratory Animal Care International–accredited institution. Study design. The experimental strategy was to evaluate capillary density and myofiber dynamics in each genetic strain A–D, subjected to two types of heat load: internal heat production due to metabolism and environmental temperature effects. Metabolic heat production is exacerbated in broilers reared to heavy body weight (about 3.5 kg) as compared to standard body weight (about 2.5 kg). Environmental heat load increases when broilers are reared in high ambient temperatures (30 to 35 C) versus thermoneutral ambient temperatures (20 to 25 C). Total heat stress in rapidly growing broilers is often a combination of both. Therefore, chickens from each strain A–D were reared to standard or heavy body weights in either high temperatures (30 to 35 C) or under thermoneutral (20 to 25 C) conditions with a 4 3 2 factorial arrangement of strains and temperature treatments. Seventy-two birds (18 from each strain A–D) were placed in 12 thermoneutral pens, and 72 birds (18 from each strain A–D) were placed in 12 high-temperature pens with a stocking density of six birds per pen. Temperatures in the thermoneutral treatment group were maintained at 29.4 to 32.2 C during week 1, 26.6 to 29.4 C during week 2, 23.8 to 26.6 C during week 3, and 20 to 25 C thereafter. Temperatures in the hot treatment group were maintained at 30 to 35 C throughout the trial. Nine birds from each strain A–D in the thermoneutral treatment group (n 5 36), and nine birds from each strain A–D in the hot treatment group (n 5 36) were humanely euthanatized and necropsied at 39 days of age and at 53 days of age, corresponding to standard and heavy body weights, respectively. Histopathology. A 1.0 cm 3 0.5 cm section of breast muscle was collected from the right pectoralis major muscle from each bird. Muscle samples were fixed by immersion in 10% neutral buffered formalin, routinely processed, and sectioned at 4–6 mm. Two serial sections of muscle were evaluated. The initial section was stained with hematoxylin and eosin, and the adjacent section was evaluated immunohistochemically with commercially available antisera for muscle-specific actin (Dako, Carpentaria, CA). Subjective assessment of the muscle sections was based on the following categories: increased refractility, loss of crossstriations, nuclear pyknosis, myodegeneration, and fragmentation. Subjective lesion categories are illustrated in Fig. 1. Subjective scores assigned for each category were as follows: 1 5 normal, 2 5 mild, 3 5 moderate, 4 5 marked, and 5 5 severe. Histomorphometric analysis. The following parameters were assessed histomorphometrically for quantitative and statistical evaluation between genetic strains, heat treatment, and age groups: number of capillaries, number of vessels, and number of myofibers; myofiber diameter; muscle cross-sectional area; and interfasicular edema. For each section, the number of capillaries, larger vessels, and myofibers were
Evaluation of capillary and myofiber density
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Fig. 1. Pectoralis major muscle; broiler chicken. Extensive regions of myodegeneration and necrosis in broiler chickens raised in hot (30 to 35 C) environmental conditions illustrate categories used for subjective assessment of myofibers. HE. Fig. 2. Pectoralis major muscle; broiler chicken. Standardized microscopic field used for assessment of capillary and vascular density, and direct count of myofibers. The numbers of capillaries (arrows), vessels (arrowhead) and muscle fibers were counted in five contiguous 4003 fields. Note increased interfasicular edema. HE. Fig. 3. Pectoralis major muscle; broiler chicken. Photomicrographs were converted to a 16-bit grayscale image, and the ImageJ red-filter threshold was applied. There is minimal enhancement of the myofiber margins. HE. Fig. 4. Pectoralis major muscle; broiler chicken. The ImageJ red-filter threshold was adjusted such that the myofibers were red-enhanced and the interfasicular space remained white. Myofiber cross-sectional area was determined by limiting the digital measurement to the red-enhanced myofibers within a defined area. Interfasicular space was determined by subtracting the myofiber cross-sectional area from the total area. HE.
directly counted in five 4003 microscopic fields by a single pathologist in a blinded fashion (Fig. 2). Assessment of each microscopic field was standardized by selecting solid fields of myofibers containing no adipose tissue or fascial planes. Myofibers along the margins that were partially observed in the field of view were counted as 0.5 fibers. Myofiber diameter, muscle cross-sectional area, and interfasicular edema were morphometrically quantitated for statistical comparison using Image J version 1.30 (National Institutes of Health, available at http://rsb.info. nih.gov/ij). Briefly, a high-resolution, 4080 3 3072, digital photomicrograph of a representative cross section of each muscle sample was taken at 4003 magnification using an Olympus DP71 digital camera system mounted to an Olympus BX41 microscope. To assess individual myofibers, the Image J linear measurement tool was used to measure the cross-sectional diameter of five contiguous myofibers. The shortest distance across each myofiber was measured to minimize variability associated with stage of muscle contraction in neighboring fibers. To
determine the amount of total myofiber cross-sectional area and interfasicular edema, each photomicrograph was converted to a 16-bit grayscale image. Using a polygon shape tool, a polygon was set to a fixed area that was limited to myofibers that were completely visible in the field, and the total area within the polygon was measured. The red-filter threshold was set, and by adjusting the threshold margin, the myofibers were red-enhanced, while the surrounding interfasicular edema remained in grayscale (Figs. 3–4). The measurement was limited to the red-enhanced threshold areas, and the area inside the polygon was measured, representing the myofiber cross-sectional area. The difference between the total area and the cross-sectional area represents the interfasicular edema. The proportion of myofiber cross-sectional area to interfasicular edema was calculated. In addition, the capillary to fiber ratio, capillary to vessel ratio, vessel to fiber ratio, sum of all capillaries and vessels, and sum of capillaries and vessels to fiber ratio were determined.
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Table 1. Histomorphometric assessment of myofiber diameter, myofiber and vascular density, and vascular to myofiber ratios.A Average number (five 4003 microscopic fields) Myofiber diameter
Capillaries
Blood vessels
Myofibers
Age (days) 39 53
525* 619*
70* 54*
8.2* 4.6*
238* 150*
Temperature (C) 20 to 25 30 to 35
597** 547**
61 64
6.3 6.4
176* 211*
Strain A B C D
577 556 570 585
62 62 62 63
7.0 6.6 5.6 6.2
197 193 187 197
Ratio Capillary/ vessel
Vessel/ fiber
Capillaries + vessels
Capillaries + vessels/fiber
8.5* 11.7*
0.036 0.035
78* 59*
0.34* 0.43*
0.36*** 0.33***
13.83 16.54
0.039 0.032
67 70
0.41 0.36
0.35 0.35 0.35 0.33
15.68 15.66 14.56 14.85
0.039 0.034 0.034 0.034
69 69 67 69
0.39 0.38 0.39 0.38
Capillary/ fiber
30* 39*
A
*P $ 0.0001; **P $ 0.001; ***P $ 0.05.
Statistical analysis. Least squares means analysis was used to detect statistically significant differences in subjective myofiber scores, histomorphometric parameters and ratios among age and heat treatment groups, and genetic broiler strains A–D. Interactions between age, temperature, and strain were also evaluated.
RESULTS
While subjective assessment of myofibers did not statistically differ among age or temperature treatment groups, the incidence of myodegeneration, fragmentation of individual myofibers with loss of cross-striations and increased refractility was slightly elevated in birds reared in hot environmental conditions. Histomorphometric evaluation of muscle sections revealed that as broilers age, muscle fibers significantly increase in diameter through hypertrophy, and vascular support is marginalized (Table 1). Capillary density decreased by 23% (P $ 0.0001), and vessel density decreased by 44% (P $ 0.0001) as the broilers aged from 39 to 53 days. Likewise, the number of myofibers visible in five 4003 fields decreased by 37% (P $ 0.0001), and the average myofiber diameter increased from 84.5 to 99.7 mm (P $ 0.0001) from 39 to 53 days. The vascular support relative to the number of myofibers was also evaluated for each age group. The number of capillaries per myofiber increased from 0.30 to 0.36 (P $ 0.001), and the number of capillaries and vessels per myofiber increased from 0.34 to 0.43 (P $ 0.0001). However, the number of vessels per myofiber did not significantly change with age, and the ratio of capillaries to vessels increased from 8.5:1 to 11.7:1 (P $ 0.001), indicating that the capillaries are the primary contributor to vascular support in growing myofibers. Myofiber diameter and number of myofibers in five 4003 microscopic fields significantly differed between birds reared in thermoneutral versus hot ambient conditions (Table 1). Broilers raised in thermoneutral environments had an average fiber diameter of 96.1 mm, while those in heat-stressed environments had an average fiber diameter of only 88.1 mm (P $ 0.001). Due to the overall reduction in myofiber diameter, more myofibers were visible per field of view in heat-stressed broilers as compared to the non-heatstressed broilers (P $ 0.0001). The number of capillaries visible in five 4003 fields was higher in heat-stressed birds (n 5 64) as compared to broilers reared in thermoneutral conditions (n 5 61). However, the number of capillaries per myofiber (n 5 0.33) and the number of capillaries and vessels per myofiber (n 5 0.36) were
significantly lower in heat-stressed birds (P $ 0.05) and (P $ 0.0001), respectively. There were no significant differences in the numbers of capillaries or blood vessels between temperature treatment groups. Similarly, differences in the number of vessels per fiber, total number of capillaries and vessels, total vascular support per fiber, and ratio of capillaries to vessels were not significant. Our data show that myofiber cross-sectional area differs slightly with age and among temperature treatment groups when expressed as a percentage of the total area (P 5 0.09) (Table 2). Individual myofibers are larger in older birds and in birds reared in thermoneutral conditions. The cross-sectional area of an individual myofiber is directly proportional to the radius of the myofiber. However, the absolute number of myofibers in a fixed area defined by using the ImageJ polygon tool is indirectly proportional to myofiber size and is reflected in the overall myofiber cross-sectional area. As broilers aged from 39 to 53 days, the total myofiber crosssectional area decreased by 14.7%. Total myofiber cross-sectional area was 10.9% lower in broilers raised in thermoneutral versus hot environmental conditions. There was slightly more interfasicular edema in the 39-day-old broiler chickens and in those birds raised in hot environments, although these differences were not significant. No significant differences in any of the histomorphometric parameters were observed between genetic broiler strains (Tables 1 and 2). DISCUSSION
It is well documented that muscle deposition is associated with higher levels of protein metabolism, which in turn elevates the demand for oxygen and other critical macronutrients to support enhanced growth (13,23). Others have demonstrated that radial expansion of myofibers increases the myofiber diameter and effectively marginalizes the capillary and vessel supply to the muscle (16). These findings are corroborated by the current results, which show that myofiber diameter increases with age, while the absolute number of myofibers and capillaries in a microscopic field decreases. However, the number of capillaries per myofiber and capillaries and vessels per myofiber is slightly higher in 53-day-old broilers. Therefore, the reduction in absolute numbers of capillaries is not directly proportional to myofiber growth. The increased ratio of capillaries to myofibers may be necessary to support adequate
381
Evaluation of capillary and myofiber density
Table 2.
Quantitative histomorphometric assessment of myofiber cross-sectional area and interfasicular space.A Parameters (mm2) Total area (TA)
Myofiber cross-sectional area (CSA)
Interfasicular space
CSA % of TA
Age (days) 39 53
197 823.14* 170 174.94*
189 784.65* 162 337.50*
8038.49 7837.45
95.98*** 95.28***
Temperature (C) 20 to 25 30 to 35
179 434.99** 189 281.74**
171 746.44** 181 095.71**
7688.55 8186.03
95.59 95.67
Strain A B C D
183 185 182 185
176 178 174 176
7846.57 7015.88 7690.08 9196.64
95.75 96.24 95.61 94.92
905.99 426.86 354.96 757.56
059.42 410.98 664.89 560.92
A
*P $ 0.0001; **P $ 0.05; ***P 5 0.09.
macronutrient delivery, as diffusion distance across the myofiber increases with radial growth. While slight increases in vascular support may be sufficient for sedentary birds reared in optimal conditions, the capillary density may not be sufficient to support enhanced demand for oxygen during increased exertion thus predisposing the bird to microischemia and muscle damage. The incidence and economic significance of exertional ischemic myodegeneration and necrosis has been extensively studied in the pectoralis minor (deep pectoral) muscles of high-yield broiler chickens. With increased exertion (i.e., wing flapping), circulation increases and myofibers can expand up to 20% in volume (19). The pectoralis minor muscles are surrounded by an inelastic fascial membrane and located immediately adjacent to the sternum and pectoralis major; thus muscle expansion may lead to compartmentalization syndrome, restricting blood flow with subsequent ischemic necrosis. Recent field studies have indicated that deep pectoral myopathy, commonly referred to as ‘‘green muscle disease,’’ poses a significant negative economic impact on the high-yield broiler industry with projected U.S. industry losses of $16 million annually. Gross lesions can develop within a matter of hours following an acute bout of muscle exertion, irreversibly damaging the pectoralis minor muscles and decreasing breast muscle yield at slaughter (17). Our research group recently developed an experimental protocol for the induction of deep pectoral myopathy in broiler chickens via encouraged wing flapping (18). Forty-five day-old broiler chickens subjected to 10 or 20 cycles of encouraged wing flapping had deep pectoral myopathy indices of 7% and 33%, respectively (18). Gross lesions were subtle 24 hr after deep pectoral myopathy induction, but progressively increased to severe myonecrosis with significant hemorrhage over a course of 11 days (18). Histologically there is extensive interfasicular edema and early myonecrosis within as little as 3 hr postexertion (Joiner, pers. obs., unpubl. data), which can impact meat quality and consumer palatability. So therein lies the challenge of contemporary high-yield broiler chickens: meeting consumer demand without compromising meat quality and breast muscle yield. As genetic selection of contemporary broiler chickens evolved to support increased demand for higher growth rates and productivity, internal (metabolic) heat production has increased substantially. To offset internal metabolic heat production, high-yield broiler chickens are reared in lower ambient temperatures in order to fully express genetic potential for increased breast muscle yield and quality. It has been hypothesized that heat-stressed broilers cannot synthesize protein as efficiently as birds reared in thermoneutral conditions,
and therefore muscle growth by hypertrophy is limited. Broilers acclimate to hot conditions by reducing feed intake resulting in depressed growth rate, lower overall body weight, and poor breast muscle yield (2,3,5,6,7). Our data support these previous observations, by illustrating the corresponding histologic features of depressed muscle growth. Myofiber diameter was drastically reduced in birds reared in hot ambient conditions, while the absolute numbers of capillaries and blood vessels did not significantly differ between thermal treatment groups. The number of capillaries per myofiber and capillaries and vessels per myofiber was slightly less in birds exposed to hot ambient conditions due to the significant increase in absolute numbers of small diameter myofibers visible in each 4003 microscopic field. Collectively, these findings suggest that limited myofiber growth during heat stress is due to diminished radial myofiber growth (i.e., decreased protein deposition in the expanding myofiber) and not necessarily due to decreased vascular support and delivery of essential macronutrients. Histomorphometric assessment of myofiber and capillary density provided further evidence suggesting that diminished relative vascular support may contribute to an increased incidence of pectoral myopathy in high-yield, broiler chickens. While older birds and birds reared in thermoneutral ambient conditions had larger individual myofibers, the total myofiber cross-sectional area within a fixed dimension decreased significantly. However, the number of capillaries per unit area did not substantially increase with age or in the thermoneutral temperature treatment group. This effectively increases the diffusion distance of critical macronutrients from the capillary to the myofiber. Marginal capillary support may be sufficient in sedentary birds; however, as contemporary fast-growing, high-yield broilers are subjected to acute episodes of muscle exertion (i.e., preslaughter handling) or acute heat stress (i.e., transportation to processing facilities), the marginal capillary support cannot provide adequate nutrient support for the larger myofibers, thus predisposing the bird to microischemia and myonecrosis. Collectively our findings show that myofiber diameter significantly increases with age and individual myofibers are larger in birds reared in thermoneutral ambient conditions, as compared to younger counterparts, and birds reared in hot environmental conditions. Substantial increases in vascular support were not evident in birds with larger myofibers, suggesting that muscle growth may be approaching a critical mass in contemporary highyield broilers. Further enhancement of muscle development with subsequent generation of high levels of internal (metabolic) heat could lead to ischemic myopathies even in optimal environmental
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conditions. It is plausible that the effects of acute exertion and external heat stress in contemporary high-yield broiler chickens could be ameliorated with progressive acclimation to increased environmental stressors. Studies investigating the effects of acute versus sustained, progressive heat stress in high-yield broiler strains are ongoing. REFERENCES 1. Allouh, M. Z., Z. Yablonka-Reuveni, and B. W. Rosser. Pax7 reveals a greater frequency and concentration of satellite cells at the ends of growing skeletal muscle fibers. J Histochem. Cytochem. 56:77–87. 2008. 2. Cahaner, A., and F. Leenstra. Effects of high temperature on growth and efficiency of male and female broilers from lines selected for high weight gain, favorable feed conversion, and high or low fat content. Poult. Sci. 71:1237–1250. 1992. 3. Cahaner, A., Y. Pinchasov, I. Nir, and Z. Nitsan. Effects of dietary protein under high ambient temperature on body weight, breast meat yield, and abdominal fat deposition of broiler stocks differing in growth rate and fatness. Poult. Sci. 74:968–975. 1995. 4. Cheema, M. A., M. A. Qureshi, and G. B. Havenstein. A comparison of the immune response of a 2001 commercial broiler with a 1957 randombred broiler strain when fed representative 1957 and 2001 broiler diets. Poult. Sci. 82:1519–1529. 2003. 5. Cooper, M. A., and K. W. Washburn. The relationships of body temperature to weight gain, feed consumption, and feed utilization in broilers under heat stress. Poult. Sci. 77:237–242. 1988. 6. Deeb, N., and A. Cahaner. The effects of naked neck genotypes, ambient temperature, and feeding status and their interactions on body temperature and performance of broilers. Poult. Sci. 78:1341–1346. 1999. 7. Deeb, N., and A. Cahaner. Genotype-by-environment interaction with broiler genotypes differing in growth rate. 3. Growth rate and water consumption of broiler progeny from weight-selected versus nonselected parents under normal and high ambient temperatures. Poult. Sci. 81:293–301. 2002. 8. Griffin, H. D., and C. Goddard. Rapidly growing broiler (meat-type) chickens: their origin and use for comparative studies of the regulation of growth. Int. J. Biochem. 26:19–28. 1994. 9. Halevy, O., A. Geyra, M. Barak, Z. Uni, and D. Sklan. Early posthatch starvation decreases satellite cell proliferation and skeletal muscle growth in chicks. J. Nutr. 130:858–864. 2000. 10. Halevy, O., A. Krispin, Y. Leshem, J. P. McMurtry, and S. Yahav. Early-age heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281:R302–309. 2001. 11. Halevy, O., and O. Lerman. Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor-alpha. J. Cell Physiol. 154:566–572. 1993. 12. Halevy, O., Y. Piestun, I. Rozenboim, and Z. Yablonka-Reuveni. In ovo exposure to monochromatic green light promotes skeletal muscle cell
proliferation and affects myofiber growth in posthatch chicks. Am. J. Physiol. Regul. Integr. Comp. Physiol. 290:R1062–1070. 2006. 13. Halevy, O., S. Yahav, and I. Rozenboim. Enhancement of meat production by environmental manipulation in embryo and young broilers. World’s Poult. Sci. J. 62:485–497. 2006. 14. Hawke, T. J., and D. J. Garry. Myogenic satellite cells: physiology to molecular biology. J. Appl. Physiol. 91:534–551. 2001. 15. Hocking, P. M. Effects of body weight at sexual maturity and the degree and age of restriction during rearing on the ovarian follicular hierarchy of broiler breeder females. Br. Poult. Sci. 34:793–801. 1993. 16. Hoving-Bolink, A. H., R. W. Kranen, R. E. Klont, C. L. Gerritsen, and K. H. de Greef. Fibre area and capillary supply in broiler breast muscle in relation to productivity and ascites. Meat Sci. 56:397–402. 2000. 17. Lien, R. J., S. F. Bilgili, J. B. Hess, and K. S. Joiner. Finding answers to ‘‘Green Muscle Disease.’’ WATT Poultry USA May:15–18. 2011. 18. Lien, R. J., S. F. Bilgili, J. B. Hess, and K. S. Joiner. Induction of deep pectoral myopathy in broiler chickens via encouraged wing flapping. J. Appl. Poult. Sci. 21:556–562. 2012. 19. Martindale, L., W. G. Siller, and P. A. Wight. Effects of subfascial pressure in experimental deep pectoral myopathy of the fowl: an angiographic study. Avian Pathol. 8:425–436. 1979. 20. Moss, F. P., and C. P. Leblond. Satellite cells as the source of nuclei in muscles of growing rats. Anat. Rec. 170:421–435. 1971. 21. McDevitt, R. M., G. M. McEntee, and K. A. Rance. Bone breaking strength and apparent metabolisability of calcium and phosphorus in selected and unselected broiler chicken genotypes. Br. Poult. Sci. 47:613–621. 2006. 22. Morris, M. P. Ascites in broilers. Poult. Int. October:26–32. 1992. 23. Piestun, Y., M. Harel, M. Barak, S. Yahav, and O. Halevy. Thermal manipulations in late-term chick embryos have immediate and longer term effects on myoblast proliferation and skeletal muscle hypertrophy. J. Appl. Physiol. 106:233–240. 2009. 24. Olkowski, A. A., C. Wojnarowicz, S. Nain, B. Ling, J. M. Alcorn, and B. Laarveld. A study on pathogenesis of sudden death syndrome in broiler chickens. Res. Vet. Sci. 85:131–140. 2008. 25. Rath, N. C., G. R Huff, W. E Huff, and J. M. Balog. Factors regulating bone maturity and strength in poultry. Poult. Sci. 79:1024–1032. 2000. 26. Schmidt, C. J., M. E. Persia, E. Feierstein, B. Kingham, and W. W. Saylor. Comparison of a modern broiler line and a heritage line unselected since the 1950s. Poult. Sci. 88:2610–2619. 2009. 27. U.S. Department of Agriculture Foreign Agricultural Service. Livestock and Poultry: World Markets and Trade [Internet]. Livestock and Poultry: World Market and Trade Circular Archives. 2012 [cited 2012 July 31]. Available from: http://www.fas.usda.gov/livestock-and-poultryworld-markets-and-trade
ACKNOWLEDGMENTS We gratefully acknowledge the histotechnology expertise of Beth Landreth, Kristina Cammack, and Scott Kincaid and the technical assistance provided by William Clements and Charlotte Wilson.
