Effects of divergent selection for 8-week body weight on postnatal enzyme activity pattern of 3 fiber types in fast muscles of male broilers (Gallus gallus domesticus)

PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Effects of divergent selection for 8-week body weight on postnatal enzyme activity pattern of 3 fiber types in fast muscles of male broilers (Gallus gallus domesticus) R. Dahmane Gošnak,* I. Eržen,† A. Holcman,‡ and D. škorjanc§1 *Faculty of Health Sciences, †Institute of Anatomy, Faculty of Medicine, and ‡Biotechnical Faculty, University of Ljubljana, Ljubljana 1000, Slovenia; and §Faculty of Agriculture and Life Sciences, University of Maribor, Hoče 2311, Slovenia of the BF muscles of the SGL had significantly (P ≤ 0.001) lower SDH:GPDH activity ratios than those of the FGL. A step decrease in the SDH:GPDH activity of O, OG, and G fibers in the PP of both lines occurred, and this differed significantly between SGL and FGL (P ≤ 0.001). Age and line effects influenced the diameter of the 3 fiber types in the BF muscle only. In contrast to this response, all 3 fiber types of the PP muscles reached similar diameters in both lines during the growth process from wk 3 to 12. From the results of this study, we concluded that the activities of metabolic enzymes in skeletal muscle fibers are under the influence of muscle type, age, and selection pressure. Microphotometry is a suitable method for the evaluation of enzyme activity measured in a single muscle fiber. The method enables precise estimation of enzyme activities, especially in muscles composed of populations of different metabolic fiber types.

Key words: divergent selection, cockerel, microphotometry, muscle fiber 2010 Poultry Science 89:2651–2659 doi:10.3382/ps.2010-00641

INTRODUCTION Long-term divergent selection for BW at 8 wk and older in chickens is a well-established model for study of specific postnatal changes in BW and growth rates. It is also used to study correlations between the increase in muscle mass and muscle fiber characteristics, and the relationships between muscle growth and meat quality (Dunnington and Siegel, 1996; Dransfield and Sosnicki, 1999; Rehfeldt et al., 2000). In chicken the number of muscle fibers does not increase after hatching (Smith, 1963), and postnatal growth of muscles is accompanied by longitudinal and transverse growth of muscle fibers (Burke and Henry, ©2010 Poultry Science Association Inc. Received January 13, 2010. Accepted September 6, 2010. 1 Corresponding author: [email protected]

1997). In previous studies related to muscle growth and fiber type characteristics, it was reported that fiber growth differs among different muscles and fiber types and within different lines of chickens (Smith, 1963; Aberle et al., 1979; Iwamoto et al., 1993; Ono et al., 1993). Two histochemical approaches allow the separation of different muscle fiber types. One method is based upon the activity of myofibrillar actomyosin adenosine triphosphatase and the other upon reference enzymes of anaerobic and aerobic energy metabolism. Primarily, 2 types of muscle fibers have been distinguished by use of a histochemical assay for myofibrillar actomyosin adenosine triphosphatase activity: type I fibers and type II fibers (Padykula and Herman, 1955). Detailed study of the acid stability of myofibrillar adenosine triphosphate activity has shown a delineation of subtypes of fast fibers, termed types IIA, IIB, and IID/X, in mammalian skeletal muscles (Schiaffino et al., 1986; Termin

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ABSTRACT A divergent selection experiment was conducted for 8-wk BW in chickens. At 3, 6, 9, and 12 wk of age, samples of pectoralis profundus (PP) and biceps femoris (BF) muscles from fast-growing and slow-growing lines were used to estimate the enzyme activities and muscle fiber diameter. Microphotometric measurements made in situ of succinate dehydrogenase (SDH, EC 1.3.99.1) and glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.99.5) were completed on serial sections of PP and BF muscles from male chickens, in order to examine the ratio of SDH:GPDH activity in single fibers. On the basis of the SDH:GPDH activity ratios, muscle fibers were divided using cluster analysis into 3 populations of different fiber types (O = oxidative, OG = oxidative-glycolytic, and G = glycolytic). Cockerels of the SGL attained an 8.1-fold increase and those of the FGL a 6.8-fold increase in BW at 12 wk compared with that at 3 wk of age. The O, OG, and G type fibers

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colytic system in the cytosol (Bücher and Klingenberg, 1958). Both enzymes incorporate cytochrome-reducing dehydrogenases and transfer electrons to tetrazolium salts. Therefore, they can be assessed quantitatively in situ by use of the microphotometric method. The present study focuses on the analysis of single fibers using microphotometric determination of enzyme activity by endpoint measurements in 2 muscles. Based on the enzyme activity ratio of SDH:GPDH, muscle fibers were divided by using cluster analysis into 3 types: oxidative (O), intermediary oxidative-glycolytic (OG), and glycolytic (G). The ratio of SDH:GPDH activity within the fiber type was estimated during growth from 3 to 12 wk of age. In addition, the specific increase in the diameter of each type of fiber was estimated in breast and leg muscles of fast and slow growing lines of chickens.

MATERIALS AND METHODS All animal procedures and care were performed in accordance with Zakon o zaščiti živali, Uradni list Republike Slovenije, 510-05/91-1/20, 19 November 1999 (the Slovenian Law Regulating the Protection of Animals) of the Republic of Slovenia.

Animals The origin, selection procedure, and response to selection of the fast- and slow-growing chicken lines used in this study have been described previously by Terčič and Holcman (2008). In short, the experiment was conducted at the test station of the Zootechnical Department (Biotechnical faculty, University of Ljubljana, Slovenia) using fast- and slow-growing lines of chickens derived from long-term divergent selection for BW at 8 wk of age. The base population for divergent selection comprised animals from a Slovenian commercial Prelux-bro line (Zootechnical Department, Biotechnical Faculty, University of Ljubljana, Groblje, Slovenia). Two lines were formed at 8 wk of age by selection of animals of the greatest and the least BW. Within each line, 10 males and 50 females were randomly mated to establish a fast-growing line (FGL) and a slow-growing line (SGL). After formation of the selected first generation, the lines were closed and progeny for each generation were gained from 2 or 3 hatches. Growing chickens were housed on a deep litter system. Selected parents of each line were later moved to separate pens within the same house. The pens were equipped with pan feeders and bell drinkers. The chickens were given access to water and feed ad libitum. The lights were on for 23 h/d. From 3 to 12 wk of age, the light was gradually reduced from 20 lx to a minimum of 10 lx. The chickens were reared at 33°C, and the temperature was gradually decreased to 22°C at 3 wk of age and thereafter decreased to 20°C at 12 wk of age. After 8 wk of age, animals of the SGL were given continued access to food and water ad libitum, whereas

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et al., 1989). Chicken skeletal muscle fibers have been classified into 3 (A = high activity of NADH-diaphorase and alkali-stable ATPase; B = low activity of NADHdiaphorase and high alkali-stable ATPase; C = high activity of NADH-diaphorase and low alkali-stable ATPase; Suzuki, 1978) and 4 [W = white (low SDHase and fast twitch); I = intermediate (moderate SDHase and fast twitch); R = red (high SDHase and fast twitch); S = slow tonic (alkali labile/acid stable mATPase activity); Rosser and George, 1986] types. Both classification systems for fiber type in chickens are based on the speed of contraction and the metabolism of specific fibers. Moreover, 5 fiber types have been described in the muscles of chicken by Remignon and coworkers (1994). Three are slow-twitch (types I, IIIA, and IIIB), whereas 2 are fast-twitch fibers (types IIA and IIB). Type III fibers are specific to birds and have the characteristic of being multiply innervated; the other 3 types (I, IIA, and IIB) are common to mammals and birds. Fiber types differ not only in the isoform pattern of their myofibrillar protein but also in their metabolic profiles (for review, see Pette and Staron, 1990). Different fiber types have also been distinguished based on histochemical reactions for enzymes of aerobic oxidative metabolism; for example, succinate dehydrogenase, nicotinamide adenine dinucleotide tetrazolium reductase, and cytochrome oxidase. Major fiber types derived from differences in the activities of these enzymes (Ogata and Mori, 1964) reflect differences in mitochondrial content and therefore primarily relate to differences in aerobic oxidative potential (Hoppeler et al., 1987). The fiber types can also be delineated histochemically on the basis of glycogen phosphorylase or mitochondrial glycerolphosphate dehydrogenase. Glycerolphosphate oxidase (α-glycerolphosphate dehydrogenase) is present in amounts that are directly proportional to the activities of glycolytic enzymes in skeletal muscle (Pette and Hoffer, 1980). Therefore, the fiber type can be defined by the activity ratio of enzymes that represent different systems, mainly aerobic and anaerobic. A previous study has shown no significant difference in the activities of the mitochondrial enzymes of oxidative and glycolytic metabolism between the muscles of fast- and slow-growing chickens (Remignon et al., 1995). However, that study was performed on muscle homogenates. The aim of the present study was to investigate the effect of divergent selection on the metabolic activity of muscle fibers in the muscles of chickens. For this purpose, 2 representative enzymes of different metabolic pathways were chosen. Succinate dehydrogenase (SDH; EC 1.3.99.1), which is bound in the inner mitochondrial membrane, is a mitochondrial flavoprotein and a reference enzyme for the citric acid cycle (Reichmann and Pette, 1984). Glycerol-3-phosphate dehydrogenase (α-GPDH; EC 1.1.99.5) is located in the outer part of the mitochondrial inner membrane and is a mitochondrial flavoprotein that is functionally related, by its key role in the glycerolphosphate shuttle, to the gly-

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those of the FGL were given restricted access to food (to approximately 60% of ad libitum consumption). A starter diet was fed from hatching to the end of wk 2 and a finisher diet from wk 3 to 12. The CP and ME contents were 234 g and 13.0 MJ/kg for the starter diet and 205 g and 13.4 MJ/kg for the finisher diet.

the sections were embedded in glycerol gelatin. Control sections were incubated in the same medium without substrate.

Tissue Samples

We applied a microscope photometer system, which consisted of a microscope photometer UMSP 30 (Opton, Oberkochen, Germany) equipped with a system processor MSP 65, a personal computer, and a special software for absorbency measurement within a polygonal area corresponding to the delineated muscle fibers (Stirn-Kranjc et al., 2000). An objective lens with 10fold magnification was used for the measurements, and the final reaction product was assessed at a wavelength of 585 nm (Butcher and Altman, 1973). The measuring diaphragm was 16 μm in diameter, and it was moved stepwise by 0.25 μm on the x and y coordinates within the delineated polygons. The software enabled loading of traces of polygons (i.e., muscle fibers) to estimate the muscle fiber diameter and to repeat the measurement along the same coordinates several times. Before we applied the end-point measurement of SDH and GPDH activity, we tested its reliability in the following way: we selected 4 successive tissue sections from each experimental group. One section was stained for SDH and one for GPDH to find fibers with different levels of the enzyme activities: very low, intermediate, and high. The remaining 2 sections were used for the kinetic measurement of either SDH or GPDH in the 3 fibers, defined and delineated in the parallel (already stained) section. We placed a drop of the incubation medium on the unstained section under the microscope and measured absorbency within the delineated polygons in constant time intervals of 3 min during incubation from 0 to 40 min and at intervals of 5 min from 40 to 60 min of incubation. For both enzymes, absorbency increased with time up to 30 min of incubation. The difference in absorbency between the 3 fiber types remained constant up to 60 min of incubation. Because the staining intensity differed essentially among different experimental groups, we chose sections stained for 60 min for endpoint measurements, for SDH, GPDH and no-dehydrogenase activities. In all samples, the no-dehydrogenase blank was beyond the sensitivity of the method, and sections stained for SDH and GPDH showed no metachromasia. In approximately 50 fibers of every muscle, we measured the average absorbency within the delineated polygons, estimated the fiber diameter, and calculated the ratio in absorbency between SDH and GPDH activity, given that the measurements of both enzymes were made in the same fibers on successive sections. Within the boundary of a muscle fiber, the density of mitochondria declines from the subsarcolemmal compartment to the central core of the fiber. Therefore, to

SDH and GPDH Histochemistry The preparation and composition of the assay mixture for SDH activity were as described previously (Reichmanm and Pette, 1984). The mixture contained (final concentrations) 60 mM K2HPO4/KH2PO4 buffer with 5 mM EDTA (pH 7.6), 50 mM sodium succinate, 1.5 mM nitro blue tetrazolium chloride, 1 mM NaCN, and 0.2 mM phenazine methosulfate. The incubation medium was kept at 37°C. After being washed in H2O, the sections were embedded in glycerol gelatin. The assay mixture for GPDH was prepared according to the methods of Kugler (1991). The final incubation medium consisted of 15 mM l-glycerol 3-phosphate (dicyciohexylammonium salt), 0.4 mM menadione (2-methyl-1,4-naphtoquinone), 5 mM nitro blue tetrazolium chloride dissolved in dimethyl formamide, 0.1 mM MgCl2, and 7.5% polyvinyl alcohol. The final pH of the incubation medium was 7.5 and it was kept at 37°C. Sections of both investigated muscles were incubated in medium that contained substrate (for measurement of the enzyme activity) or lacked it (for measurement of “no-dehydrogenase activity”). One area outside the muscle cross-section was selected for measurement of the blind reaction. After being washed in distilled H2O,

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Animals were randomly taken from the FGL or SGL that had been selected through 16 generations for high and low BW at 8 wk of age. Seven cockerels representing each age and line were weighed and killed by decapitation at 3, 6, 9, and 12 wk posthatch. Muscle samples were excised from the pectoralis profundus (PP) and biceps femoris (BF) muscles within 45 to 60 min postmortem, and thin longitudinal fiber bundles were dissected and frozen quickly in a slightly stretched position in liquid nitrogen until assay. Serial muscle cross-sections were cut at 10 μm using a cryostat microtome (Reichert-Young 2800 Frigocut, Leica, Germany) at −25°C. Variation in section thickness was reduced by using a motor-driven microtome with a constant speed of muscle sectioning. The muscle samples were cut in composite blocks of the corresponding muscle from chickens of 3, 6, 9, and 12 wk of age to avoid any uncontrollable variation in thickness, processing conditions, or temperature (Nemeth and Pette, 1981). Cross-sections of the composite blocks were mounted on glass coverslips, air-dried, and used for microphotometry.

Microphotometric Determination of Enzyme Activity

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eliminate errors caused by differences in the distribution of mitochondrial density, and consequently differences in SDH and GPDH activities within fibers, the integrated absorbency of the reaction product values was divided by the number of fiber areas scanned (n) within the measured fiber. Subtraction of the blind reaction activity was automatically performed. Correction for the no-dehydrogenase samples was not necessary because the absorbency in all fibers was beyond the sensitivity of the method.

Morphometric Characteristics of Muscle Fibers



DIAmin =

4 ´ Area . p ´ DIAmax

All fibers with diameters 3 to 5 times larger than normal were classified as “giant” fibers, according to Dransfield and Sosnicki (1999) and were excluded from further statistical analyses.

Statistical Analyses Based on the SDH:GPDH ratio, muscle fibers were categorized using cluster analysis into O, OG, or G types. Cluster analysis was performed by the k-means cluster analysis procedure using the clustering approach of SPSS 15.0 for Windows (SPSS Inc., Chicago, IL). This procedure tries to identify relatively homogeneous groups of different muscle fiber types according to the SDH:GPDH ratio as continuous data. However, the kmeans algorithm requires that the number of clusters be specified in advance; we decided on 3 clusters in the current study. The SDH:GPDH ratio of each muscle fiber is assigned to the cluster on the basis of its shorter distance to the cluster mean. The algorithm iteratively estimated the cluster means using the initial set of means and classified cases based on their distances to the centers. By repeating this step until cluster means did not change between successive steps, the procedure developed means of 3 final permanent cluster centers with the specific number of muscle fibers. This procedure for muscle fiber type differentiation was applied within specific age, muscle, and line. An ANOVA was performed using the GLM procedure of SPSS 15.0 for Windows software. The SDH:GPDH ratio and DIAmin of the muscle fibers were analyzed as fixed effects at different ages (3, 6, 9 and 12 wk), and for the 2 lines (FGL, SGL), the muscle of origin (PP,

Yijklm = µ + Ai + DLij + Mijk + FTijkl + eijklm, where Yijklm is the estimated SDH:GPDH ratio or DIAmin, µ is the overall mean, Ai is the fixed effect of the specific age (i = 1, 2, 3, 4), DLij is the fixed effect of the jth divergent line (j = 1, 2), Mijk is the fixed effect of the kth muscle (k = 1, 2), FTijkl is the fixed effect of the lth specific fiber type (l = 1, 2, 3), and eijklm is the residual error. The values in tables are presented as mean ± standard error. Multiple comparisons of the observed means were based on the Duncan multiple range post hoc test, which was used to determine differences between time points. An unpaired Student’s t-test was applied to evaluate the difference between the 2 lines. Significant differences were declared at P < 0.05, P ≤ 0.01, and P ≤ 0.001.

RESULTS BW Changes in BW of cockerels are summarized in Table 1. Significant differences in live BW between the lines were already present at 3 wk of age and persisted from 3 to 12 wk of age. Cockerels from the SGL showed significantly lower BW than those from the FGL (P ≤ 0.001) in each subsequent week. The cockerels of the SGL attained an 8.1-fold increase in BW from 3 to 12 wk of age. Surprisingly, the FGL cockerels showed only a 6.8-fold increase in BW in the same growth period. Moreover, the difference in BW between the 2 lines was greater at 3 wk than at 12 wk of age (3.2-fold vs. 2.7-fold).

Aerobic Versus Anaerobic Metabolism Enzyme activity ratios of SDH:GPDH in the muscle fibers of the PP and BF muscles are presented in Table 2. Three types of fiber could be distinguished in the PP and BF muscles based on their enzyme activity ratios in both lines of chicken. The BF muscle of the SGL cockerels exhibited statistically (P ≤ 0.001) lower SDH:GPDH ratios than did that of FGL cockerels for each specific type of fiber, but especially for O and OG fibers, from wk 3 to wk 12. The same was found for G fibers but only at wk 3. Thereafter, from wk 6 no significant difference between the lines for this fiber type was detected in either muscle. During the investigated growth period, the O fibers in the BF muscle of the FGL had a significantly (P ≤ 0.01) higher oxidative potential compared with those of the SGL, and they remained high throughout the experiment. In contrast, in both muscles of the SGL, oxidative activity decreased gradually to a significant level (P < 0.05) and reached its lowest value at wk 12.

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The polygonal area of each fiber cross-section was used to calculate an equivalent ellipsis. The maximal distance between 2 points on the boundary of the muscle fiber was designated the maximal diameter (DIAmax). With the use of DIAmax and the area of the ellipse, the minimal diameter (DIAmin) was calculated using the equation of Sullivan and Pittman (1984):

BF), and the effect of muscle fiber type (O, OG, G) using the following statistical model:

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DIVERGENT SELECTION AND MUSCLE FIBER CHARACTERISTICS Table 1. Comparison of the BW (g) of the fast-growing (FGL) and slow-growing (SGL) lines FGL

SGL

Week of age

n

Mean ± SE

n

Mean ± SE

3 6 9 12

7 7 7 7

592 1,639 2,863 4,066

24a 33b 33c 53d

7 7 7 7

187 581 941 1,522

± ± ± ±

± ± ± ±

Line effect

6a 15b 43c 45d

*** *** *** ***

a–dValues

within the column with different superscript letters differ statistically (P < 0.05). ***P ≤ 0.001.

Muscle Fiber Diameter Muscle fiber diameters are presented in Table 3. Divergent selection induced pronounced increases of fiber diameter in PP muscles of both lines. Nevertheless, in the FGL, fibers of types O and G reached their maxi-

mum size at 6 wk of age (P < 0.05) and later did not significantly increase in diameter. In contrast to this growth pattern, the diameter of all 3 fiber types in the PP muscles of the SGL gradually increased significantly (P < 0.05) during the period from 3 to 12 wk of age. The FGL showed significantly (P < 0.05) smaller diameters of fibers of types O and OG at 12 wk of age. Moreover, the relative increase in fiber diameter calculated during the period from 3 to 12 wk showed marked differences in the rate of fiber growth among fiber types and lines. In the PP muscles of the FGL, fibers of types O, OG, and G increased in diameter from 3 to 12 wk by 1.4-, 1.6-, and 1.5-fold, respectively. A much greater rate of increase in the diameter of all 3 fiber types was noted in the PP muscles of the SGL: the diameter of O, OG, and G fibers increased in the investigated period by 1.8-, 1.9-, and 2.2-fold, respectively. The hind limb BF muscle of the FGL and SGL was found to be composed of all 3 fiber types. However, throughout the investigated period, a significant difference was observed between lines in the diameter of each type of fiber. The FGL had fibers of a significantly larger diameter than those of SGL cockerels (P ≤ 0.001). In contrast, the BF muscle of the SGL had markedly smaller diameters of all 3 fiber types, and they attained their growth plateau with respect to fiber diameter at

Table 2. Values (mean ± SE) of the SDH/GPDH activity ratios in 3 muscle fibers types of pectoralis profundus and biceps femoris muscles of male fast-growing line (FGL) and slow-growing line (SGL) broilers1 Pectoralis profundus Muscle fiber type Oxidative (O)   Week 3   Week 6   Week 9   Week 12 Intermediary (OG)   Week 3   Week 6   Week 9   Week 12 Glycolytic (G)   Week 3   Week 6   Week 9   Week 12 a–dValues

FGL 2.01 1.83 1.89 1.64 1.26 1.17 0.99 0.88 0.71 0.73 0.60 0.45

  ± ± ± ±   ± ± ± ±   ± ± ± ±

SGL

Biceps femoris Line effect

FGL

SGL  

0.07a 0.04b 0.11b 0.09c

1.63 1.87 1.48 1.24

± ± ± ±

0.05a 0.04b 0.04c 0.04d

*** NS *** ***

       

2.40 2.27 2.43 2.44

± ± ± ±

0.08a 0.07b 0.05ac 0.09ac

0.02a 0.01b 0.02c 0.02d

1.07 1.24 0.87 0.67

± ± ± ±

0.01a 0.01b 0.01c 0.01d

*** *** *** ***

       

1.34 1.23 1.46 1.21

± ± ± ±

0.03a 0.03b 0.03c 0.02b

0.91 0.90 0.72 0.75

0.02a 0.01a 0.01b 0.01c

0.60 0.75 0.56 0.37

± ± ± ±

0.02a 0.01b 0.01a 0.01c

*** NS ** ***

       

0.67 0.61 0.71 0.65

± ± ± ±

0.02a 0.01a 0.02a 0.01a

0.33 0.58 0.52 0.57

1.30 1.44 1.21 1.13

± ± ± ±   ± ± ± ±   ± ± ± ±

within a muscle fiber type with different superscript letters differ significantly within the column (P < 0.05). = succinate dehydrogenase; GPDH = glycerol-3-phosphate dehydrogenase. NS = P ≥ 0.05; **P ≤ 0.01; ***P ≤ 0.001. 1SDH

Line effect

0.02a 0.03b 0.02c 0.03d

*** *** *** ***

0.01a 0.01a 0.01b 0.01c

*** *** *** ***

0.01a 0.01b 0.01a 0.01b

*** NS *** ***

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Changes in enzyme activity ratio in different types of muscle fiber were observed with aging in the PP muscle of FGL and SGL. The activity of the O, OG, and G fibers of the SGL decreased significantly (P < 0.05) and progressively from wk 3 to 12. Furthermore, the SDH:GPDH activity ratio was significantly (P ≤ 0.001) lower in the PP of the SGL than in that of the FGL. A sustained significant difference between the lines in enzyme activity was observed, except for type O fibers at 6 wk. Interestingly, in the PP of the FGL, some delay in reduction of the enzyme activity ratio of the G fibers was demonstrated. At wk 9, a significant decrease in the enzyme activity of type G fibers was observed, whereas type O fibers showed a bimodal decrease. The first significant decrease (P < 0.05) in O fibers was recorded after 3 wk and the second after 9 wk of age. A step decrease in the SDH:GPDH ratio of intermediary OG fibers in the PP occurred in both lines and this differed significantly (P ≤ 0.001).

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9 wk of age. The relative growth of each type of fiber from 3 to 12 wk of age indicated that the O, OG, and G fibers in the BF muscle of the FGL increased in diameter by 2.5-, 2.2-, and 2.0-fold, respectively. The BF fiber types of the SGL showed much lower growth rate potential compared with those of the FGL. In addition, only the OG and G fibers enlarged during the aforementioned study period; they increased in diameter by 1.6- and 1.3-fold, respectively.

DISCUSSION

Table 3. Diameters (µm; mean ± SE) of different muscle fiber types of pectoralis profundus and biceps femoris muscles of male fastgrowing line (FGL) and slow-growing line (SGL) broilers Pectoralis profundus Muscle fiber type Oxidative (O)   Week 3   Week 6   Week 9   Week 12  Intermediary (OG)   Week 3   Week 6   Week 9   Week 12  Glycolytic (G)   Week 3   Week 6   Week 9   Week 12 a–dValues

FGL 22.5 31.8 33.6 32.0 23.8 29.2 32.6 37.1 24.0 26.8 31.9 37.0

  ± ± ± ±   ± ± ± ±   ± ± ± ±

SGL

Biceps femoris Line effect

FGL

SGL

0.9a 1.7b 2.4b 2.7b

22.3 30.1 34.7 40.8

± ± ± ±

1.1a 1.4b 1.6c 1.6d

NS NS NS *

       

17.2 24.0 29.7 42.2

± ± ± ±

1.8a 1.6b 1.7c 1.6d

11.3 16.3 9.6 9.2

0.3a 0.8b 1.2c 1.2d

21.6 29.4 31.7 41.0

± ± ± ±

0.8a 0.8b 1.3c 1.0d

* NS NS *

       

16.1 21.9 28.7 35.3

± ± ± ±

0.6a 0.7b 1.2c 1.0d

11.8 16.7 19.0 18.7

0.2a 0.7a 0.9b 0.9b

17.3 27.5 31.8 38.1

± ± ± ±

0.6a 0.6b 1.0c 1.2d

*** NS NS NS

       

16.4 22.2 33.2 33.5

± ± ± ±

0.4a 0.5b 0.8c 0.9c

15.0 17.3 20.1 19.3

  ± ± ± ±   ± ± ± ±   ± ± ± ±

within a muscle fiber type with different superscript letters differ significantly within the column (P < 0.05). *P ≤ 0.01; ***P ≤ 0.001; NS = P ≥ 0.05.

Line effect

0.4a 0.9b 0.3a 0.3a

*** *** *** ***

0.3a 0.6b 0.7c 0.5c

*** *** *** ***

0.5a 0.6b 0.6c 0.7c

* *** *** ***

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The BW of the chickens studied here confirm the results from previous studies on the effects of long-term divergent selection of chickens on the basis of BW at 8 wk. From these studies it is known that birds in the FGL show a more pronounced increase in BW than birds of the SGL during postnatal growth (Siegel, 1963; Dunnington and Siegel, 1985; Remignon et al., 1995; Park et al., 2006). In the present study, comparison of FGL and SGL has shown that the BW of the FGL were, on average, 2.7- to 3.2-fold higher than those of the SGL (Table 1). Although significant changes in BW first occur at 3 wk of age and progress during posthatch development, the FGL exhibited greater BW than the SGL at all ages. Animals with a higher growth potential deposit more skeletal muscle tissue because of 2 processes: hyperplasia and hypertrophy. The increase in muscle weight is therefore determined by the number of muscle fibers and the intensity of their longitudinal and transverse growth. It has been suggested that selection based on higher growth rate increases, in the muscle fibers of fast-growing lines, the proliferative activity of satellite cells (Merly et al., 1998) that are under the control of chicken growth hormone (Hodik et al., 1997). In addition, myoblasts respond to insulin-like growth factor-I, which stimulates DNA synthesis (Duclos et al., 1996).

Selection of chickens over 16 generations results in larger fiber cross-sectional area of the pectoralis major muscle, as seen on postmortem examination, together with higher levels of insulin-like growth factor-I and myostatin mRNA compared with those of chickens with low growth rate (Guernec et al., 2003). In other words, muscle fiber size is the result of posthatch enlargement of fibers, which is dependent upon activity of myogenic precursor satellite cells, differences in the levels of stimulatory hormones or growth factors, or altered responsiveness of the cells to these factors (Duclos et al., 1996). Iwamoto et al. (1993) divided muscle growth process in chickens into at least 2 phases: the early phase that lasts from hatching to 2 wk of age and the subsequent phase of marked growth that occurs up to 15 wk. The latter stage also contains 2 phases: sexual prematurity (from 15 to 20 wk of age) and sexual maturation (to 35 wk). Iwamoto et al. (1993) reported that muscle fibers have the potential to grow up to 80 µm in diameter. From the results of the present study, it is clear that different types of fiber reach their growth plateaus at different ages, depending on the type of muscle and the growth potential of the cockerel in the early stage. Differences in diameter between fiber types were demonstrated in the deepest region of the pectoralis thoracius muscle at 6 wk of age in White Leghorn (Suzuki, 1978). It appears that divergent selection has different influences on fiber size and enzyme activity in white breast PP muscle compared with white leg BF muscle. Interestingly, the effects of age and line had a significant influence on the diameter of all 3 types of fiber only in the BF muscle. We found selective hypertrophy of O, OG, and G fibers in the BF muscle of chickens that were selected for rapid growth. In contrast to this response, during growth from wk 3 to 12, all 3 fiber types in PP muscles reached similar diameters in both lines. These findings are in agreement with a previous

DIVERGENT SELECTION AND MUSCLE FIBER CHARACTERISTICS

cross-sections, especially in the muscles of FGL cockerels, and they had a diameter more than 3 times greater than that of average fibers. They were excluded from the population of muscle fibers involved in further statistical analysis. The present study provides a means, by using ratios of SDH:GPDH activity measured in situ, of distinguishing 3 types of fibers; these distinctions have been made on the basis of the metabolic profile of the fibers. Our data demonstrate that both muscles of the FGL and SGL contain 3 types of fibers. The importance of this finding is that the present method enables us to measure alterations in enzyme activity within the fibers. This could be a convenient method for studying the process of transformation between muscle fiber types during the growth or aging of chickens. It has been reported that in the newly hatched chick all fibers exhibit predominantly aerobic metabolism (Bass et al., 1970; Ashmore and Doerr, 1971). The β (red) fibers are constantly “red” at any age, but the α (white) fibers have the capacity to transform from a “white” to a “red” fiber. During growth of a chicken, the extent of transformation of fast fiber types depends on the type of muscle (Bass et al., 1970; Ashmore and Doerr, 1971). During postnatal development, a rapid loss of oxidative capacity was detected in chicken fast-white muscle (pars posterior of latissimus dorsi). However, the slow-red muscle (pars anterior of latissimus dorsi) did not change its metabolic type markedly during development (Bass et al., 1970). Although many studies have been published on the growth of chickens, little is known about the effect of divergent selection for BW at 8 wk on changes in enzyme activity patterns in skeletal muscle tissue. To our knowledge, only one study has directly analyzed enzyme activity in the pectoralis major and anterior latissimus dorsi muscles of male chickens of 2 lines from hatching to adulthood (Remignon et al., 1995). The authors found that the rate of development of the muscles, as indicated by myosin isoform patterns and reference enzymes of the citric acid cycle and fatty acid oxidation, were not altered substantially by selection. Furthermore, during normal maturation and hypertrophy, muscles with anaerobic fiber types were not affected by changes in lactate dehydrogenase and citrate synthase activities. Nevertheless, citrate synthase activity per unit muscle mass was positively correlated with muscle capillary density for the muscles with aerobic fiber types (Snyder, 1995). These studies were performed on muscle homogenate and not by using in situ measurements of dehydrogenases. They were not able to distinguish changes in aerobic and anaerobic enzyme activities in a single muscle fiber during muscle growth. Moreover, detection of enzyme activities in homogenates is suitable only for homogeneous muscle tissue, in which the prevalence of a specific fiber type defines the enzymatic activity of the whole muscle (Punkt et al., 1989). In BF muscles, the O, OG, and G fibers of FGL cockerels were significantly more oxidative with a higher

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study by Smith and Fletcher (1988), but are in contrast to those of Roy et al. (2006). It has been reported that the largest diameter of type IIB fibers (50.5 µm) was noted in the pectoralis muscle at 95 d in a group of broilers given feed of a low nutritional value. However, the results of the present study indicate that the PP muscle of the FGL showed specific fiber types of much larger diameter in the early stages of growth. The present results for the diameter of type G fibers at wk 12 in the PP muscle agree with the previous data on the diameter of IIB fibers in pectoralis muscle, but are in contrast to those found in chickens of the New Hampshire breed at wk 10 to 15, in which an average diameter of 30.2 to 38.3 µm was recorded (Ono et al., 1993). Indeed, the present results confirm and extend previous observations on the size of different types of fiber in chicken muscles. Moreover, the significance of the observed difference in diameter between types of fiber is not entirely clear. It may be associated with the differences caused by the functional differentiation of muscle. A probable explanation is that, in the BF muscle, differences in fiber diameter between lines are influenced by the selection for rapid growth and increased body mass at 8 wk of age. However, the cockerels of the FGL were significantly heavier than those of the SGL, but no consistent pattern in the fiber diameter of the PP muscle was noted. Muscle weights that differ between lines result in variation either in muscle fiber number or in fiber size. It is generally accepted that the total number of muscle fibers in a chicken (Smith, 1963) or a quail (Fowler et al., 1980) is fixed at hatching. No report has been found on the total number of fibers in the PM and BF muscles of chickens. The reason for this may be that the size of the whole muscle (e.g., the PM) makes it impossible to section using conventional cryo-microtomes. However, it has been reported that male broilers have a higher density of fibers per unit area in the pectoralis muscle compared with Leghorn-type chickens (Scheuermann et al., 2004), and it has been suggested that selection for growth favors factors that promote selective radial hypertrophy in the sartorius muscle (Aberle and Stewart, 1983). The basic mechanism of hypertrophy and possible postnatal hyperplasia in the skeletal muscles of chickens remains unclear. It is therefore tempting to speculate, based on our results, that divergent selection for muscle weight at 8 wk of age increases the size of the PM in fast-growing compared with slow-growing cockerels because of muscle fiber numbers and not solely because of an increase in fiber diameter. However, selection for higher growth rates induces increases in fiber diameter over enlargement of the population of small fibers in breast muscles (Dransfield and Sosnicki, 1999). Stephan and Dzapo (1996) reported that selection for meatiness increases the number of giant fibers, and these findings could be interpreted as an indicator of disarranged metabolism of the skeletal muscle, and consequently lower meat quality. In the present study, such giant fibers were observed on tissue

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ACKNOWLEDGMENTS The authors gratefully acknowledge the technical staff of the Zootechnical Department of the Biotechnical faculty of the University of Ljubljana (Slovenia) for the careful breeding of the experimental lines of chicken. Special software for measurement of enzyme activity within polygons corresponding to individual muscle fibers was developed by M. Ambrož (Governmental Agency for Informatics, Ljubljana, Slovenia).

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SDH:GPDH ratio than those of the SGL during wk 3 to 12. In contrast to the high ratio of SDH:GPDH in fibers of BF muscle, the O, OG, and G fibers in the PP muscle showed lower ratios of SDH:GPDH activity and a progressive decline in oxidative capacity during aging. This oxidative decline was observed in the PP muscle of the FGL as well as in the SGL, but to a lesser extent in the BF muscle. It is obvious from the present study that the process of transformation of the fiber type from red to white in muscle during animal growth is encompassed not only by changes in the proportion of fiber types but also in differentiation within the fiber based on its metabolic profile. Every 3 wk, a significant decline in oxidative capacity (measured in the intermediary OG fiber type) was detected in the PP muscle. This classification is supported by the significant differences in the ratios of SDH:GPDH activity in the 3 populations of fibers. Although this ratio showed pronounced variability among the fibers of each population, very little overlap was observed between the 3 fiber populations. A specific distribution of these 2 mitochondrial dehydrogenases in the tibialis anterior muscles of the mouse and rabbit have been shown previously using microphotometry. Reichmann and Pette (1984) reported that this activity ratio might be regarded as a sensitive measure of metabolic differentiation in muscle. In conclusion, divergent selection for BW at 8 wk induced pronounced changes at specific ages in BW, fiber size, and the activities of SDH and GPDH in muscle fibers between FGL and SGL cockerels. At all ages, birds of FGL exhibited greater BW than those of the SGL. The effects of age and line significantly influenced the diameter of all 3 fiber types in the BF muscle only. In contrast to this response, all 3 fiber types in the PP muscles reached similar diameters in both lines during growth from wk 3 to 12. In BF muscles, the O, OG, and G fibers of FGL cockerels were significantly more oxidative with a higher SDH:GPDH ratio than those of the SGL during wk 3 to 12. In contrast with the BF muscle, the O, OG, and G fibers of the PP muscle showed lower ratios of SDH:GPDH activity and a progressive decline in oxidative capacity during aging.

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