Increase in oxidative capacity of pigeon pectoralis muscle before and after fledging

1778 Increase in oxidative capacity of pigeon pectoralis muscle before and after fledging Errin E. Rathgeber and Benjamin W.C. Rosser Abstract: Using the pectoralis muscle of the pigeon (Columba livia), we previously showed that at fledging, both fasttwitch oxidative glycolytic (FOG) and fast-twitch glycolytic (FG) fiber types retain a myosin isoform characteristic of the neonatal period, despite the birds being at adult body mass. Our aim here was to test the hypothesis that muscle fibers of the pigeon pectoralis increase their aerobic capacity both before and after fledging. Pigeons aged from 1 week after hatching through to adulthood were used. Adopting a microdensitometric technique from studies on mammalian muscle, the activity of succinate dehydrogenase (SDH) within individual fibers was quantified as optical density and used as an index of aerobic capacity. We demonstrate a strong linear correlation (r 2 = 0.936) between optical density of FOG fibers and time exposed to the SDH incubation medium. Optical density of FG fibers, however, could not be discerned from background staining. SDH activity in FOG fibers increased logarithmically (r 2 = 0.969) with the age of the birds, increasing 2.5-fold from 1 week through to adult. Thus, aerobic capacity did increase both before and after fledging. In addition, within FOG fibers, aerobic capacity and myosin content appear to have different timetables of development. Résumé : Nous avons déjà démontré, par examen du muscle pectoral du Pigeon biset (Columba livia), qu au moment de l envol, les deux types de fibres, fibres rapides oxydantes glycolytiques (FOG) et les fibres rapides glycolygiques (FG), contiennent un isoforme de la myosine présent durant la période néonatale, en dépit de la masse corporelle d adulte de ces oiseaux. Nous avons éprouvé l hypothèse selon laquelle les fibres musculaires du muscle pectoral du pigeon augmentent leur capacité aérobie avant et après l envol. Des pigeons gardés en élevage à partir de l âge de 1 semaine jusqu à l âge adulte ont été utilisés. Une méthode micro-densitométrique mise au point pour l étude des muscles de mammifères a été employée pour évaluer la densité optique, une mesure de l activité de la succinate déshydrogénase (SDH) dans des fibres individuelles utilisée comme indice de la capacité aérobie. Nous avons mis en lumière une forte corrélation linéaire (r 2 = 0,936) entre la densité optique des fibres FOG et la durée d exposition de la succinate déshydrogénase au milieu d incubation. La densité des fibres FG n a cependant pu être discernée par coloration du substrat. L activité de la succinate déshydrogénase dans les fibres FOG augmentait logarithmiquement (r 2 = 0,969) avec l âge des oiseaux, augmentation évaluée à 2,5 fois entre l âge de 1 semaine et l âge adulte. La capacité aérobie augmente donc avant et après l envol. En outre, dans les fibres FOG, le développement de la capacité aérobie et celui du contenu en myosine semblent suivre une chronologie différente. [Traduit par la Rédaction] Rathgeber and Rosser 1782 The pectoralis is the most massive muscle within those birds capable of flight (Hartman 1961). Extending from the sternum and ribs to the humerus (Raikow 1985), it provides all the power for the downstroke of the wing during flight and helps decelerate the wing during the upstroke (Biewener et al. 1992; Dial 1992). The avian pectoralis consists almost exclusively (99 100%) of fast-twitch fiber types in those species relying upon flapping or active flight (Rosser and George 1986; Rosser et al. 1996). Fast-twitch oxidative glycolytic (FOG) fibers are capable of sustained rapid contraction and fast-twitch glycolytic (FG) fibers of a more Received January 20, 1998. Accepted May 8, 1998. E.E. Rathgeber and B.W.C. Rosser. 1 Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada. 1 Author to whom all correspondence should be addressed (e-mail: Rosserb@duke.usask.ca). Can. J. Zool. 76: 1778 1782 (1998) powerful but fast-fatiguing contraction (Slater and Harris 1994). These fiber types differ in the activities of their energy-generating enzymes (Rosser and George 1986; Torrella et al. 1996) and in their constituent myosin heavychain (MyHC) isoforms (Rosser et al. 1996). Both energygenerating enzymes and MyHC isoforms have been directly correlated with the contractile properties of individual muscle fibers (see Nemeth 1990; Bottinelli et al. 1994; Reiser et al. 1996). The young of most avian species are essentially at their adult body mass before their first flight (Weathers 1992; Gill 1995). Body mass is strongly correlated with a number of parameters associated with avian flight (Rayner 1988; Pennycuick 1996), including the fiber type of the pectoralis muscle (Norberg 1990). We previously tested the hypothesis that FOG and FG fibers in the pectoralis reached a mature state in their MyHC content before the acquisition of flight (Rosser et al. 1998). The experimental model utilized was the pectoralis muscle of the domestic pigeon (Columba livia), which, in the adult, constitutes approximately 20% of the total body mass (Hartman 1961). Although pigeons attain their adult body mass prior to fledging (Abs 1983), we

Rathgeber and Rosser 1779 found that the MyHC composition of fibers in the pectoralis was not fully mature at fledging (Rosser et al. 1998). The activity of energy-generating enzymes, however, may be a more sensitive determinant of muscle maturation than the MyHC complement, as there can be a continuum of enzyme activities in fibers that contain the same MyHC isoforms (Nemeth 1990; Sieck et al. 1995). The activity of succinate dehydrogenase (SDH), an enzyme of the citric acid cycle, has been routinely measured within individual mammalian muscle fibers by a precise microdensitometry method (Blanco et al. 1988; Jasmin et al. 1995; Sieck et al. 1996). Although early microdensitometric methods were employed to estimate the radial distribution of enzyme activities within individual avian muscle fibers (Swatland 1984, 1985), the more recent methods (Sieck et al. 1995; Unguez et al. 1995) of quantifying activities across the entire crosssectional area of single fibers have not been applied to avian muscle. The purpose of this study was to test the hypothesis that muscle fibers in the avian pectoralis muscle increase their aerobic capacity both before and after fledging. The experimental model used was the pectoralis muscle of the domestic pigeon. Young pigeons are altricial, flightless, and confined to the nest until they are about 4 weeks of age (Vriends 1988). The muscle consists of readily identifiable populations of FG and FOG fibers from 1 week after hatching (George and Berger 1966; Rosser et al. 1998). We applied quantitative microdensitometry for SDH activity (after Blanco et al. 1988) to assess developmental changes in oxidative capacity. Pigeons and tissue preparation Domestic pigeons bred for racing were obtained from a local pigeon fancier. Pigeons aged 1, 2, 3, 4, 4½, 6, and 12 weeks after hatching and adults (at least 1 year old) were housed in an insulated, shielded loft located outdoors in Saskatoon (52 10 N, 106 40 W), Saskatchewan. While the 4-week-old birds had just begun to make weak sorties within the loft, all birds older than 4 weeks were independent and flying regularly outside the loft. All birds were collected by early fall, while the climate was still temperate, and each was killed by a large overdose of sodium pentobarbital injected into its abdominal cavity. Blocks of muscle were excised from the most superficial areas of the sternobrachialis portion of either the right or left pectoralis muscle of each bird. Blocks were removed from a location near the keel of the sternum, approximately midway between the cranial and caudal edges of the muscle. This area of the muscle consists entirely of fast-twitch fibers in mature birds (Kaplan and Goslow 1989; Rosser et al. 1996). Each block was approximately 0.5 0.5 2 3 cm and was cut so that its long axis ran parallel to the direction of the muscle fibers. Blocks of muscle were coated with Tissue Tek O.C.T. compound (Miles Inc., Elkhart, Indiana) and then quick-frozen in 2-methylbutane cooled to 160 C with liquid nitrogen (Dubowitz 1985). Blocks were stored at 80 C until sectioned. Sections 10 µm thick were cut from a block of each pectoralis in a cryostat maintained at 20 C. Two or three sections were picked up on each microscope slide. Between 60 and 100 serial sections were obtained from each muscle. We were able to follow the same fascicles and fibers with relative ease throughout our sections for up to 1 mm along the length of the muscle. This was not surprising, as the muscle fibers within the pectoralis muscle of the Japanese quail (Coturnix japonica), a species one-third the mass of the pigeon (Dunning 1993) but with a very similar fiber-type complement (Rosser et al. 1987), range in length from 8.8 to 33.2 mm (Trotter et al. 1992). Slides were kept at 20 C until staining for SDH activity, which was performed immediately after sectioning. Fiber typing Avian fast-twitch and slow fibers are commonly distinguished by their myosin ATPase activity (Rosser and George 1986; Torrella et al. 1993) and (or) their reactivity to antibodies against specific epitopes located on various MyHC isoforms (see Rosser et al. 1996; Meyers and Mathias 1997). Avian fast-twitch fibers are then usually classified as either FG or FOG on the basis of the biochemical pathways utilized to produce the energy for contraction (Rosser and George 1986; Rosser et al. 1996). Histochemical stains for mitochondrial oxidative enzymes are routinely used for this purpose. Intermediate fiber types, between FOG and FG fibers in their metabolic properties, have also been described in the pectoralis of many species (see Rosser and George 1986; Tobalske et al. 1997). In the pigeon pectoralis, however, from 1 week after hatching the muscle consists of clearly defined FG and FOG fiber types (George and Berger 1966; Rosser et al. 1998). Also, as all fibers are fast-twitch, use of a histochemical stain for a mitochondrial oxidative enzyme is sufficient to classify the fiber types (Rosser and George 1986). The smaller diameter fibers stain dark and the larger fibers light, as there is an inverse correlation between fiber diameter and mitochondrial density (George and Berger 1966). These fibers are then classified as FOG or FG type, respectively. Histochemistry SDH activity was measured using the technique described by Blanco et al. (1988). To determine the optimal conditions for avian muscle, serial sections were incubated at 30-s intervals from 30 to 540 s in an incubation medium at room temperature. The incubation medium consisted of 0.75 mm sodium azide, 1.0 mm 1-methoxyphenazine methosulphate, 48 mm succinic acid, 5.0 mm ethylenediaminetetraacetic acid, and 1.5 mm nitroblue tetrazolium in 100 mm phosphate buffer (ph 7.6). Reactions were stopped by immersing the slides in distilled water. Slides were then mounted in Aquamount mountant (BDH Ltd., Poole, England). SDH activity at zero time was determined using a slide that had not been in any incubation medium. Control slides were incubated for 150, 300, 450, and 600 s in an incubation medium in which 100 mm phosphate buffer replaced the succinic acid. SDH activity was expressed in units of optical density. The pectoralis from each pigeon in the developmental portion of this study was incubated for 8 min for SDH activity. This was done because we demonstrated that the reaction was linear over the first 9 min of incubation (see Results). This time period was also preferred in earlier studies (Blanco et al. 1988; Jasmin et al. 1995). Also in accordance with the previous studies, the optical densities of our control slides (without substrate) did not increase over time. Thus, their optical densities were averaged and then subtracted from the density obtained for each experimental slide. Image and data processing Using a 550 + 15 nm interference filter (Chroma Technology Inc., Brattleboro, Vermont) on a Leitz Ortholux microscope, images were acquired utilizing a Raster Ops Framegrabber card in a Quadra 800 Macintosh computer with IPLab Spectrum software (Scanalytics Inc., Fairfax, Virginia). Images were then converted to grayscale with 256 possible gray levels. The NIH image program (public domain program developed at the U.S. National Institutes of Health and available on the Internet by anonymous FTP from zippy.nimh.nih.gov) was used in a Macintosh Performa 5200 computer to measure the SDH mean optical density of fibers in the converted images. Data were plotted and regression or curve fits

1780 Can. J. Zool. Vol. 76, 1998 Fig. 1. Cross section of muscle fibers from the pectoralis of adult (A) and 4-week-old pigeons (B) incubated for 8 min for SDH activity. Arrowheads and arrows indicate FOG and FG fibers, respectively. Scale bars = 50 µm. Fig. 2. Relationship between SDH activity and incubation time. SDH activity, shown in units of optical density, was measured in serial sections of the same FOG fiber incubated for various lengths of time. Data for each time point are expressed as the mean optical density of the same fiber in two adjacent serial sections. SDH activity is linear over a 9-min period. Fig. 3. SDH activity of pigeon pectoralis FOG fibers during development. Three 4-week-old and three adult birds were studied. For other ages, one bird of each age was studied. Fifty fibers were measured in each bird. Data are expressed as the mean ± SE. SDH activity, indicated by optical density, increases logarithmically with age. calculated using the least squares algorithm of CA-Cricket Graph for Macintosh (Computer Associates International Inc., Islandia, New York). FG and FOG fiber types were clearly differentiated from each other by their SDH activity (Fig. 1). This concurs with the results of previous work (Rosser and George 1986; Torrella et al. 1993), as ambiguous intermediate types are not present in pigeon pectoralis (George and Berger 1966; Tobalske et al. 1997). SDH activity in FOG fibers of pigeon pectoralis increased linearly with incubation time over a 9-min period (Fig. 2). In fact, using a linear regression, a close correlation (r 2 = 0.936, p < 0.0001) was observed between optical density and time of incubation. Fibers in control slides, those incubated without succinic acid, demonstrated comparable optical densities regardless of duration of incubation. These results concurred with those of Blanco et al. (1988). Therefore, to account for background staining in the linear correlation of SDH activity and time, the mean optical density of the control slides was calculated and then subtracted from the optical densities for each test slide. Control slides incubated for 8 min were also available for the portion of the experiment examining SDH activity throughout development. The mean optical density of representative fibers on each control slide was subtracted from that of the respective experimental slide to correct for background staining. FG fibers in the pectoralis could not be assessed in this experiment. The optical density of the FG fibers could not be discerned from background staining. SDH enzyme activity in pigeon pectoralis FOG fibers increased logarithmically with the age of the bird (Fig. 3). There was a 2.5-fold increase from 1 week of age through to adulthood. Using a logarithmic curve, a high correlation (r 2 = 0.969, p < 0.0001) was obtained between mean optical

Rathgeber and Rosser 1781 density of the FOG fibers and age of the birds. Therefore, within FOG fibers, there is an increase in SDH activity from hatching through to adulthood. The technique used in this study corresponded to that utilized by Blanco et al. (1988) and Sieck et al. (1995). The composition of the incubation medium was identical with that used in these earlier studies. The only way in which the protocol differed was that the slides were not air-dried in the dark and mounted in glycerin jelly but, instead, wet-mounted in Aquamount in the light. Our results showing that SDH activity increased linearly with time concurred with those of both Blanco et al. (1988) and Jasmin et al. (1995). Small deviations from an exact correlation can be attributed to slight variation in SDH activity along the length of individual fibers (see Sieck et al. 1996). SDH activity has also been shown to increase linearly with section thickness (Blanco et al. 1988). Therefore, an additional source of error is inconsistent thickness of tissue sections due to minor aberrations during sectioning. Nevertheless, our results do clearly demonstrate that the method used by Blanco and colleagues (1988) to quantify SDH activity within individual mammalian muscle fibers can be applied to the study of pigeon FOG fibers. We were, however, unable to discern any correlation between SDH activity and FG fibers in the pigeon pectoralis. In fact, the optical densities of the FG fibers were not distinguishable from background staining. It has been demonstrated that pigeon FG fibers, in comparison with pigeon FOG fibers, have an extremely low capacity for oxidative metabolism (George and Berger 1966; Grinyer and George 1969; Kaplan and Goslow 1989; Mathieu-Costello et al. 1994; Mathieu-Costello and Agey 1997). Conversely, Sieck et al. (1995) were able to quantify SDH activity in mammalian type IIx and type IIb muscle fibers, which have moderate to low oxidative capacity. It would appear that the FG fibers in pigeon pectoralis might be less oxidative than mammalian IIx and IIb fibers. The FG fibers in pigeon pectoralis are used preferentially during takeoff and landing, but not in level flight (Goslow 1991). Endurance training has been shown to increase the activity of oxidative enzymes such as citrate synthase and SDH in skeletal muscle (Nemeth 1990; Proctor et al. 1995; Poole and Mathieu-Costello 1996). This increased oxidative enzyme activity reflects an overall increase in aerobic capacity of the muscle. The duration of exercise also appears to be a factor that determines the magnitude of the increase in aerobic capacity (Powers et al. 1994). While certain studies have shown a higher aerobic capacity in the pectoralis muscle of wild or exercised pigeons than in more sedentary controls (e.g., Mathieu-Costello et al. 1994), others have not (e.g., Viscor et al. 1992). Our results support the hypothesis that muscle fibers in the avian pectoralis increase their aerobic capacity both before and after fledging at 4 weeks of age. We observed an increase in oxidative capacity of the muscle from 1 week through to 4 weeks of age. After fledging, while SDH activity further increased in birds aged 6 and 12 weeks, maximum activity were not attained until the birds were older. During the development of the aerobic fibers in certain muscles of the chicken, an increase in oxidative enzyme activity was positively correlated with capillary density (Snyder 1995). Thus, in our study, with maturation of the muscle one might also expect an associated increase in muscle capillarity. Our results do show that at fledging, although pigeons are at adult body mass, the energy-generating capacity of the pectoralis muscle is not fully mature. Young pigeons are not strong fliers until approximately 6 12 weeks after hatching (K. King, personal communication), and pigeon fanciers do not advise training pigeons for racing until this age (Levi 1963). The observed increase in aerobic capacity following fledging concurs with a gradual attainment of more powerful flight performance. Determination of SDH activity reveals that FOG fibers are still immature 12 weeks after hatching. However, the MyHC composition of these fibers indicates that they have attained a mature profile by 6 weeks after hatching (Rosser et al. 1998). During normal development, expression of a neonatal MyHC wanes in all fibers shortly after fledging at 4 weeks. FOG fibers attain their mature MyHC profile and size by 6 weeks. Thus, it appears that during development the activity of energy-generating enzymes and the expression of MyHC isoforms in the pigeon pectoralis follow slightly different timetables. Examples of differential regulation of MyHC content and energy-generating enzymes in skeletal muscle fibers have previously been shown (Pette and Vrbova 1985; Sieck et al. 1995). Mr. Ken King of Saskatoon generously provided from his aviary all pigeons used in this study. In addition, we thank Mr. King for freely sharing his first-hand experience and in-depth knowledge of these birds. Dr. Sergey Federoff of our department kindly allowed us unlimited access to his Quadra 800 Macintosh computer and digitizing system, and Mr. Ross Cuddie assisted with its use. Ms. Cindy M. Farrar contributed her excellent technical skills to certain aspects of the study. We also thank Chroma Technology Inc. for their most considerate gift of an interference filter. 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