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Gizzard myoglobin contents and feeding habits in avian species
Comparative Biochemistry and Physiology Part A 125 (2000) 33 – 43
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Gizzard myoglobin contents and feeding habits in avian species Yasunori Enoki *, Tomotoshi Morimoto Second Department of Physiology, Nara Medical Uni6ersity, Kashihara, Nara 634 -8521, Japan Received 24 May 1999; received in revised form 9 September 1999; accepted 22 September 1999
Abstract In an attempt to consider physiological function of myoglobin (Mb), we determined Mb contents of gizzard smooth muscles with special reference to feeding habits in 85 avian species of 19 orders. The Mb content in 44 species of herbivorous birds was 7.52 93.81 mg/g wet muscle, which was significantly higher than the value of 2.34 9 1.74 mg/g in 41 species of carnivorous ones (PB0.001). Buffering capacity, as determined by in vitro titration method, was 37.3 9 5.5 slykes/g in gizzard smooth muscles of 75 species and 60.7 9 10.5 slykes in breast skeletal muscles of 77 species (PB 0.001), which suggests a significantly higher dependence, almost comparable to cardiac muscles, of the gizzard muscular function on aerobic metabolism. Together with the fact that blood circulation in the gizzard is very low at resting, and might be further limited during activity, we conclude that the higher Mb content in gizzards of herbivorous birds is an adaptation, to allow storage and/or facilitated diffusion of oxygen, during process of high mechanical work required to grind down hard and fibrous vegetable food under the conditions of limited circulatory supply. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Birds; Buffering capacity; Capillarity; Circulation; Feeding habit; Gizzard; Myoglobin; Oxygen; Skeletal muscle; Smooth muscle
1. Introduction Locomotion by flight is undoubtedly the most characteristic functional property in most avian species. Absence of teeth in this class of animals has been interpreted as a morphological adaptation to this specific function, resulting in a lighter head without necessity of robust and heavy jaws for fixing the teeth and chewing muscles (Dilger, 1957; Welty, 1962; McLelland, 1979). Evolution of the gizzard or muscular stomach in the avian digestive system, allow for the mechanical diges-
* Corresponding author. Present address: Horai 4-28-5, Nara 631-0845, Japan. Tel./fax: + 81-742-461233.
tion of foods without teeth. Accumulated literature tells us that, in general, herbivorous birds have a gizzard with well-developed musculature, while carnivorous birds bear the organ with poorly-developed ones (Ziswiler and Farner, 1972; McLelland, 1979; Duke, 1986). The strong gizzard in the former category of birds has been considered to meet the increased mechanical work which is required to grind down the more fibrous, harder and much less digestible food of plant origin. In the latter group of birds, however, food of animal origin can be readily disposed of by chemical digestion with digestive juices secreted from the proventriculus (glandular stomach) and other digestive glands, and the rather weak musculature might be sufficient.
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Myoglobin, a single polypeptide heme protein with reversible oxygen binding ability, had long been believed to be present in skeletal and cardiac muscles but not in smooth muscle. Kennedy and Whipple were the first to suggest presence of the protein in gizzard smooth muscle of chicken by observing the absorption spectrum of an aqueous extract of the organ (Kennedy and Whipple, 1928). Later this possibility was further strengthened by Gro¨schel-Stewart et al. (1971) and also Blessing and Muller (1974), but their evidence was not conclusive. One of the present authors has presented conclusive evidence that Mb, which is structurally and functionally identical with those in the skeletal and cardiac muscles, is present in chicken gizzard smooth muscles (Enoki et al., 1984). We showed also that, although the presence of the protein was universal throughout other avian species, the concentration was highly variable fiom species to species. In view of the fact that the content appeared to be generally higher in herbivorous birds compared with carnivorous ones, we suggested that the variability might reflect feeding habit (Enoki et al., 1988). In this paper we present data on some eighty species of bird and provide solid support for the hypothesis. Data on the buffering capacity and capillarity of the muscles, which might be closely related to muscular metabolism, will be also presented.
2. Materials and methods
2.1. Materials Eighty five species of birds belonging to 19 different orders, reared and autopsied in Osaka Municipal Tennoji Zoological Garden (Osaka), were served for the present study. Common and scientific names of the birds are listed in Tables 2 and 3. Usually only one adult bird was used for any one species unless otherwise stated and the specimen from an animal with suspected gastrointestinal disorder was excluded. The gizzard was excised as quickly as possible after the animal’s death (usually within 6 h), its koilin lining, fascia and fat were trimmed off, and immediately frozen-stored at− 85°C until use. Chickens were obtained from a local poultry farm, decapitated after intraperitoneal pentobarbital sodium anesthesia (50 mg/kg body weight), and heart, proven-
triculus (glandular stomach), breast and leg muscles and gizzard were excised, and immediately used for the determination of myoglobin contents and other studies.
2.2. Determination of Mb contents in muscle A spectrophotometric method was used as described previously (Enoki et al., 1988), which was a modification of the procedure by Reynafarje (1963). Small pieces of muscle were excised fiom the specimens, chopped up and well mixed by the use of ophthalmological scissors and a razor blade on an ice-chilled glass plate, divided into tared and capped polypropylene micro-tubes and weighed on an electronic balance. Since a gradient of Mb content was suspected across the gizzard wall as suggested for canine and actually observed in human myocardial wall (Kirk and Honig, 1964; Lin et al., 1990), determinations were usually repeated at two locations of different depth, one submucosal and the other subserosal, and the results were averaged. The amount of muscle varied from 10 to 30 mg each depending on the Mb content as guessed by the sample colour. A 10 mM K2PO4 solution (0.8 ml) containing 1 mM Na-EDTA, pre-saturated with CO and ice-chilled, was added and the muscle sample was thoroughly homogenized with a metal microhomogenizer (NITION: Physcotron NS-310E) equipped with an NS-4 microshaft. The sample tube was held in ice water during homogenization. The homogenate was centrifuged at 10 000×g and 4°C in a refrigerated microcentrifuge (Tomy Seiko: MC15A), and the resultant supernate was transferred to a new microcentrifuge tube. The air space of the tube was flushed with CO after adding 0.3 ml of chloroform, and the capped tube with its content was vigorously hand-shaken for a few seconds. The final step was a modification of the original procedure to remove fatty ingredients which cause cloudiness in the sample and interfere with the following optical measurement. The supernatant aqueous layer, after centrifuging the mixture at 10 000×g and 4°C, was transferred into a microcuvette, the air space was gently flushed with CO stream, and a few crystals of sodium dithionite were added and the content was well mixed by several inversions. After the evolved bubbles had vanished on standing, the sample was scanned for absorbance in 530–580 nm wave length region. Mb content
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was derived as previously reported (Enoki et al., 1988); C=
5.696(A538 − A568)(0.800 +0.75W) W
where C: myoglobin content in mg/g wet muscle; A538 and A568: absorbances of the sample at 538 and 568 nm, respectively; and W: wet weight of muscle sample in g. In the gizzards with well-developed musculature as seen typically in herbivorous birds, the thick muscle portion was used for the determination unless otherwise stated, and usually five determinations were performed at two points each of different depth, one subserosal and the other submucosal, and the results were averaged. Carnivorous birds, on the contrary, have generally a poorlydeveloped and thin-walled gizzard, in which the determinations were usually done at one location.
2.3. Histochemical obser6ation of the capillarity in muscles Immediately after sacrifice of chicken, the gizzard was excised, cut into two pieces, and a tiny block (2–4 mm square) of the muscle was sampled from the thick muscle portion at the middle point of the muscle depth. The muscle block was frozen-embedded in AMES™ Tissue Tek OTC Compound (Lab Tec Products) in isopentane pre-cooled with liquid nitrogen. Serial 10 mm thick cross sections of the frozen muscle were prepared with a cryostat (Bright: model FS/FCS) at−20°C. The sections were stained histochemically for capillaries by Andersen’s amylase-PAS procedure (Andersen, 1975). Capillarity was expressed by counting the number of capillaries per mm2 of the tissue cross sectional area.
2.4. Buffering capacity (b) of muscles The method of Bate Smith (1938), which was later used by Castellini and Somero (1981) for measuring b of vertebrate muscles, was followed. Briefly, a muscle sample (0.5 g) was weighed into a specially designed Pyrex tube (12 ×110 mm) with a flat bottom, and was homogenized thoroughly with 10.0 ml of 0.9% saline by using a microhomogenizer (NITI-ON:
35
Physcotron NS-310E). The homogenates were adjusted to pH 6.0 with HCl when the initial pH was more alkaline, and were then titrated under magnetic stirring with 0.20 N NaOH in pH 6–7.5 range (20°C). The titrant was added drop by drop with a micrometer burette (Gilmont: S1200A), and the pH was measured by a TOA pH meter HM-5S equipped with a small-bore combined glass electrode(TOA: GS5018S). Strictly, the titration curve as a whole is not linear, but within a limited range such as pH 6–7, it can be safely considered as so. Therefore, from the titration curve, we obtained the b (slykes) as the amount of NaOH (m mols) required for a unit pH change of the homogenate of 1 g muscles.
2.5. Gel-filtration high performance liguid chromatography (HPLC) of gizzard muscle homogenates Gizzard muscles (20 mg) were homogenized in CO-saturated milliQ water (1 ml) in a capped microcentrifuge tube as described above, and centrifuged at 10 000× g (4°C) in a refrigerated microcentrifuge. The clear supernatant was used for gel-filtration HPLC analyses on a TOSO TSK-G3000 SWXL column (7.8× 300 mm). Isocratic elution was performed in 0.05 M phosphate (pH 7.0) containing 0.01% sodium azide and flow rate of 0.5 ml/min with a Waters 626 LC system, and the eluate’s absorbance was monitored at 215 nm. Retention times for the elution peaks were automatically determined and recorded. All chemicals were the analytical grade from Nacalai Tesque (Kyoto) and Wako (Osaka). Spectrophotometric measurements were carried out with a Hitachi spectrophotometer model 124 or a Union high sensitivity spectrophotometer model 401.
2.6. Statistical analysis After the test for equal variance (F-test), statistical significance of the difference between the means was assessed by either the two sample t test, or the two sample t test with Welch’s correction (Ichihara, 1990). The significance level was set at P B0.05.
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3. Results
3.1. Myoglobin contents of chicken skeletal, gizzard and cardiac muscles Table 1 shows the results obtained for four chickens. The contents in thick muscles of the gizzards were almost eight times those of the leg skeletal muscles, and more than four times those of the cardiac ventricles. It should be also noted that the gizzard thin muscles show a considerably lower content of Mb than the thick muscles.
3.2. Myoglobin contents in gizzard smooth muscles of 85 a6ian species Mb contents in gizzard thick muscles and breast skeletal muscles for comparison were determined in 85 species of birds. Table 2 represents the results for 41 mostly carnivorous species, including piscivorous, insectivorous and nectivorous birds. The gizzard Mb contents ranged from 0.58 mg/g wet muscle in grey-faced buzzard-eagle to 11.30 mg/g in northern white-rumpted swift. It is evident that most of the birds show a relatively low Mb content (B 5 mg/g) with an average9 S.D. of 2.3491.74 mg/g wet muscle (n =41). It should be noted that two species of swift, whitethroated spine-tailed swift and northern whiterumpled swift, show exceptionally high Mb, although they are undoubtedly insectivorous. The results for 44 species of herbivorous birds were summarized in Table 3, the values ranging from 1.03 mg/g in Gouldian finch to 17.33 in common coot. In contrast to the findings in Table 2, most of the birds show a relatively high level (5 mg/gB) of Mb, and the mean and S.D. were 7.529 3.81
mg/g wet muscle (n=44). Two species of the herbivores, blue-and-yellow macaw and roseringed parakeet, both belonging to the order of Psittaciformes, were granivorous, but they are reported to crush the feed into small readily digestible pieces with their strong bill to swallow, and have a poorly developed gizzard. Their low gizzard Mb might be a reflection of this feeding behavior. The difference between the means of the carnivores and the herbivores was highly significant (PB 0.001). A slight Mb concentration gradient, as suggested and found for myocardial Mb (Kirk and Honig, 1964; Lin et al., 1990), appears to be present in the gizzard muscular wall: the ratio of submucosal to subserosal Mb contents was 1.10 9 0.43 (n= 41) and 1.05 9 0.34 (n= 12) in herbivorous and carnivorous birds, respectively. The difference between the two categories of birds is not statistically significant.
3.3. Gel-filtration chromatographic obser6ations of the gizzard muscle extract The Mb peaks, identified by their characteristic Soret absorption and also by referring to the chromatographic behavior for the authentic chicken Mb, were found all through the avian species. Naturally, the peak heights (Mb contents) were much lower in the carnivorous species. The retention times for the Mb peaks were essentially identical with each other in the extracts from the gizzard smooth muscles (21.5890.08 min, n= 55), breast skeletal muscles (21.6190.14 min, n= 57) and cardiac muscles (21.61 min, n= 2), and the molecular weights estimated were around 1.8× 104.
Table 1 Myoglobin contents of chicken skeletal, cardiac and gastric smooth musclesa Sketetal muscles Breast
Cardiac muscles Leg
Atrium
Gastric smooth muscles Ventricle
Provontriculus
Gizzard
Thick muscle 0.449 0.09 (0.17)
Thin muscle 1.44b (0.55)
2.20 9 0.54 (0.85)
2.609 0.37 (1.00)
1.48 9 0.13 (0.57)
6.72 9 1.24 (2.58)
10.65 90.90 (4.10)
Means 9S.D. (mg/g wet muscle) for four chickens. Figures in parentheses are the ratios of Mb contents to that of the ventricular muscle. b Mean for two chickens. a
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Table 2 Myoglobin contents and buffering capacities of gizzard and breast muscles in carnivorous birdsa No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Birds
Grey-faced buzzard-eagle Red-necked phalarope King vulture Ancient murrelet White’s thrush Woodcock Wattled crane Ruddy kingfisher King penguin Greater flamingo Manchurian crane Scarlet ibis Black-headed gull Striated heron Grey heron White spoonbill White-crested laughing thrush Golden-breasted starling Brown hawk owl Roseate spoonbill Brown-eared bulbul Little egret Andean cock-of-the-rock Oriental scops owl Ural owl Rufous-tailed hummingbird Cattle egret White stork Sacred ibis Magellanic penguin Black-crowned night heron Great Indian hornbill Macaroni penguin Rockhopper penguin White-faced shearwater White-naped crane Swinhoe’s storm petrel Jungle nightjar Great crested grebe White-throated spine-tailed swift Northern white-rumped swift
Means9 S.D. (n)
Mb contents (mg/g wet muscle)
Buffering capaacities (slykes)
Gizzard muscle
Gizzard muscle
0.58 0.75 0.81 1.05 1.17 1.21 1.24 1.36 1.41 1.46 1.47 1.51 1.63 1.66 1.76 1.77 1.77 1.77 1.78 1.78 1.81 2.03 2.03 2.13 2.14 2.14 2.25 2.26 2.34 2.38 2.59 2.75 2.75 2.77 3.17 3.29 3.54 4.32 4.39 5.76 11.31
(2)b
(2) (2) (3) (4)
(2) (3)
(2)
Breast muscle 1.99 6.66 4.07 15.76 4.41 3.34 2.81 4.84 35.94 2.57 2.71 1.87 2.83 6.48 6.29 4.12 3.95 4.71 4.08 4.97 5.12 8.46 5.86 2.32 6.29 7.31 4.59 18.49
Breast muscle
47.1 52.3 20.7 32.6 36.7 39.2 30.6 37.8 31.9
43.7 53.1 69.4 51.3 53.3 60.6 45.8 54.7 61.6
31.2 33.3 40.1
63.2 58.2 55.9 64.7 57.2
33.3 31.7 34.2 39.1 38.4 33.4 39.5 44.9 44.9 39.1 41.5 47.3 31.2 27.2 39.2 31.6
33.1 51.8 60.1 64.7 63.7 70.4 60.8 71.5 72.7 54.6 53.3 67.1 51.9
(2) (2) (4) (2)
2.349 1.74 (41)
7.79 27.91 26.88 5.62 11.07 7.82 3.61 8.75 2.92 10.04 7.7797.51 (38)
28.9 27.9 37.6 34.1 35.7 37.6 31.8 33.9 44.5 36.3 9 6.3 (37)
59.7 48.4 66.5 54.7 67.2 43.4 64.4 43.9 34.3 44.5 56.6 99.8 (37)
a Latin names of birds: 1, Butastur indicus; 2, Phalaropus lobatus; 3, Sarcoramphus papa; 4, Synthliboramphus antiquus; 5, Zoothera dauma; 6, Scolopax rusticola; 7, Bugeranus carunculatus; 8, Halcyon coromanda; 9, Aptenodytes patagonicus; 10, Phoenicopterus ruber; 11, Grus japonensis; 12, Eudocimus ruber; 13, Larus ridibundus; 14, Ardeola striata; 15, Ardea cinerea; 16, Platalea leucorodia; 17, Carrulax leucolophus; 18, Cosmopsarus regius; 19, Ninox scutulata; 20, Platalea ajaja; 21, Hypsipetes amaurotis; 22, Egretta garzetta; 23, Rupicola peru6iana; 24, Otus sunia; 25, Strix uralensis; 26, Amazilia tzacatl; 27, Egretta ibis; 28, Ciconia ciconia; 29, Threskiornis aethiopicus; 30, Spheniscus magellanicus; 31, Nycticorax nycticorax; 32, Buceros bicornis; 33, Eudyptes chrysolophus; 34, Eudyptes chrysocome; 35, Calonectris leucomelas; 36, Grus 6ipio; 37, Oceanodroma monorhis; 38, Caprimulgus indicus; 39, Podiceps cristatus; 40, Hirundapus caudacuta; 41, Apus pacificus. b Figures in parentheses are numbers of birds.
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Table 3 Myoglobin contents and buffering capacities of gizzard and breast muscles in herbivorous birdsa No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Birds
Gouldian finch House sparrow White-faced whistling duck Red-billed whistling duck Blue-and-yellow macaw Ostrich Rose-ringed paraeet Rock pigeon Barnacle goose Spix’s guan Chestnut-bellied sandgrouse Chuker partridge Keel-billed toucan Bar-headed goose Red-crested pochard Black-necked swan European wigeon Emperor goose Green peafowl Ringed teal Baikal teal Mute swan Australian shoveller Mandarin duck Green-winged teal Brazilian teal Garganey Pintail Temminck’s tragopan Siamese fireback pheasant Philippine duck Bufflehead Silver pheasant Moorhen Gadwall Chicken White-eared pheasant Common peafowl Brown-eared pheasant California quail European pochard Grey junglefowl White-headed duck Common coot Means 9 S.D. (n)
Mb contents (mg/g wet muscle)
Buffering capacities (slykes)
Gizzard muscle
Breast muscle
Gizzard muscle
1.03b 1.24 (2) 1.26 1.28 1.33 1.43 1.71 5.23 5.24 5.82 5.99 6.09 6.09 6.11 (2) 6.31 6.33 6.37 6.46 6.64 (2) 6.74 7.01 7.53 (2) 7.57 7.78 (2) 7.81 (2) 7.84 (3) 7.86 8.08 (3) 8.59 (2) 8.66 8.97 9.25 9.67 (2) 9.74 (2) 9.99 10.54 (5) 10.66 10.67 11.32 11.54 (2) 11.94 15.37 16.66 17.33 7.529 3.81 (44)
2.53 2.63 3.06 6.81 4.02 6.51 4.44 0.87 4.46 0.58 4.65 0.76 4.65 5.91 2.39 9.39 4.38 3.87 0.99 1.32 3.24 3.34 3.99 5.56 3.12 3.51 2.01 3.88 0.92 0.21 1.91 7.94 0.45 1.54 4.54 0.44 0.71 0.61 0.29 0.68 6.02 0.63 9.65 8.74 3.36 92.55 (44)
Breast muscle
40.9 32.4 34.9 34.3 33.9 35.1 30.9
66.2 54.5 64.4 78.3 63.3 62.8 71.9 60.1 59.2 66.9 58.1 69.4 55.6 47.6
40.2 39.1
54.4 62.9
42.7 40.1 36.6 37.9 40.1 39.8 43.2 43.2 45.8 38.9 41.3 34.2 36.9 41.7 35.3 37.6 37.5 30.8
80.1 59.1 57.4 58.1 56.4 73.2 62.5 62.5 60.9 66.9 85.3 81.4 50.1 50.6 73.8 58.9 64.6 68.9
30.7
69.4
47.6 41.3 42.6 41.9 29.8
40.9 40.1 38.4 40.5 39.7 38.4 94.2 (38)
83.6 61.6 83.4 53.9 61.5 64.5 99.5 (40)
Latin names of birds: 1, Chloebia gouldiae; 2, Passer domesticus; 3, Dendrocygna 6iduata; 4, Dendrocygna autumnalis; 5, Ara ararauna; 6, Struthio camelus; 7, Psittacula krameri; 8, Columba li6ia; 9, Branta leucopsis; 10, Penelope jacquacu; 11, Pterocles exustus; 12, Alectoris chukar; 13, Ramphastos sulfuratus; 14, Anser indicus; 15, Netta rufina; 16, Cygnus melanocoryphus; 17, Anas penelope; 18, Anser canagicus; 19, Pa6o muticus; 20, Calonetta leucophrys; 21, Anas formosa; 22, Cygnus olor; 23, Anas rhynchotis; 24, Aix galericulata; 25, Anas crecca; 26, Amazonetta braziliensis; 27, Anas querquedula; 28, Anas acuta; 29, Tragopan temminckii; 30, Lophura diardi; 31, Anas luzonica; 32, Bucephala albeola; 33, Lophura nycthemera; 34, Gallinula chloropus; 35, Anas strepera; 36, Gallus gallus 6ar. domesticus; 37, Crossoptilon crossoptilon; 38, Pa6o cristatus; 39, Crossoptilon mantchuricum; 40, Lophortyx californica; 41, Aythya ferina; 42, Gallus sonneratii; 43, Oxyura leucocephala; 44, Fulica atra. b Figures in parentheses are numbers of birds. a
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Fig. 1. Titrations with NaOH of homogenates of gizzard (); cardiac (); and breast skeletal muscles ( ) from chicken.
3.4. Buffering capacities (b) of gizzard smooth muscles and breast skeletal muscles
was slightly but significantly higher in the herbivores than in the carnivores (PB 0.05).The value for the gizzard muscles in total of the 75 species, irrespective of their feeding habits, was 37.39 5.5 slykes, which was significantly lower than 60.79 10.5 slykes for the skeletal muscles in the total 77 species (PB 0.001).
An example of the titrations of homogenates of chicken gizzard, breast and cardiac muscles were shown in Fig. 1. The buffering capacities were estimated as 30.8, 68.9 and 37.5 slykes, respectively. The value for the proventriculus muscle was 30.6 slykes, and little different from that for the gizzard (data not shown). Compared with the gizzard muscle, a distinctly higher b value was found for the skeletal muscle (Tables 2 and 3). In carnivorous birds, the means and S.D. for gizzards and breast muscles were 36.3 9 6.3 (n =37) and 56.6 99.8 slykes (n =37) (P B 0.001), while the corresponding values in the herbivores were 38.4 94.2 (n=38) and 64.599.6 slykes (n =40) (PB 0.001). The buffering capacity of gizzards
3.5. Capillarity in chicken gizzard muscles As shown in Table 4, the mean capillary density in chicken gizzard muscles was 484 mm − 2, which was considerably lower than those in both ‘red’ and ‘white’ skeletal muscles in seven species of birds as reported previously by Snyder (1990), and almost comparable to those in ‘white’ or glycolytic skeletal muscles of mice.
Table 4 Capillarity of chicken gizzard thick muscles as compared with those of skeletal muscles in mice and birds Animals
Muscles
Capillarities (mm−2)
Remarks
Chicken Mice
Gizzard (thick) Skeletal (red)a (white)b Skeletal (red) (white)
484.3 939.5 (8) 1021.2 9196.8 (7) 401.3 923.0 (5) 804.3–842.6 723.6–773.3
Snyder (1990) Snyder (1990)
Birds (seven species)
a b
Gastrocnemius (deep), plantaris and diaphragm. Gastrocnemius (superficial), semimembranosus and biceps femoris.
40
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4. Discussion Mb is a monomeric heme protein with reversible oxygen binding ability of fairly high affinity for oxygen, but with neither homotropic nor heterotropic allosteric property (Antonini and Brunori, 1971). It is found in muscle cells of vertebrates which need continuous oxygen flow for maintenance of aerobic metabolism. Traditionally proposed main physiological functions of Mb are as follows (Suzuki and Imai, 1998); (1) short-term and long-term storage of oxygen; (2) Mb-facilitated oxygen diffusion; and (3) biochemical catalyst (Mb-mediated oxidative phosphorylation) (Wittenberg and Wittenberg, 1990). The proposals have not fully established yet, but have presented a very controversial area that remains vigorously studied. For example, a recent finding by Giardina et al. (1996) that lactate, an end product of glycolysis, lowered markedly oxygen affinity of Mb is far from the general knowledge so far.
4.1. Methodological comments on the present determinations We determined the Mb contents in the avian muscles by a modification of the Reynafarje’s procedure (Reynafarje, 1963; Enoki et al., 1988). Since the original method had been developed and used for the determinations in human and the other mammalian muscles, we revised the equation for the Mb contents in avian muscles based on the absorption data obtained for purified chicken Mb (Enoki et al., 1984, 1988). This method, albeit simple and widely used, could be in danger of interference by any coexisting substance(s), especially the other heme proteins, which have an absorption in the wave length region for the determination. The suspicion, however, could be safely discarded in view of the recent detailed discussions by Meng et al. (1993). Buffering capacity in muscles has been argued to be a possible measure to assess the dependence of muscle work on aerobic or anaerobic metabolism (Castellini and Somero, 1981; Hochachka and Mommsen, 1983). The present determinations by an in vitro homogenate titration technique can be performed conveniently, but may not be free from the possibility that the results may deviate somewhat from the true in vivo value since it can not measure the buffering
via bicarbonate system. The role of the bicarbonate system, however, has been generally considered to be negligible, in the intracellular buffering (Castellini and Somero, 1981). No detectable difference was found experimentally between the b values for human skeletal muscles by the in vitro and in vivo titration (Mannion et al., 1993). The HPLC results confirm and extend our previous finding in chicken (Enoki et al., 1984) that Mb is present all through gizzard, skeletal and cardiac muscles of avian species.
4.2. Feeding habits in birds To determine animal’s feeding habit in the field is difficult, requiring both behavioral observations and careful examination of the alimentary contents in captured animals (Ikeda, 1956; Haneda, 1962). In addition feeding habit varies both temporally (seasonal) and spatially (geographical). For example, the grey duck (Anas poecilorhyncha) feeds almost entirely (92%) on diets of plant origin, principally grass seeds, during autumn to spring seasons, but in the summer breeding period changes to 40% plants with the remainder of the diet being of animal origin such as insects (Haneda, 1962). To assess diet we relied completely on information from the literature (Ikeda, 1956; Haneda, 1962; Tollefson, 1978; Perrins and Middleton, 1984; Yoshii, 1988). Our assessment of diet was checked critically by a research group for avian ecological studies (Dr S. Yamagishi and his colleagues: personal communication). A comment should be added that the birds used in this studies were all artificially reared in a zoo during variable period. On the basis of a classical study by Brandes (1896), however, it will be reasonable to assume that no substantial change in gizzard morphology and probably the Mb contents should occur during the period.
4.3. Gizzard Mb contents in relation to feeding habit A number of structural adaptations have evolved coincident with flight in birds. Loss of teeth and the evolution of gizzard are most obvious, however the biolological implications may not be simple (Dilger, 1957; McLelland, 1979). First, weight saving, especially in the head region, which may be favourable for flight, results from loss of jaws and attendant chewing muscles. Ac-
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cording to Welty (1962), the head weight in pigeon is only 0.21% of the total body weight, while that in rat is 1.25%. Second, the changes may meet the need for rapid ingestion and digestion of food as a result of the higher metabolic rate demanded for the evolution of endothermy and flying in this class of animals. Finally, it should be mentioned that the development of the massive gastric mill brings the heavy musculatures closer to the center of gravity. Strong force generation has long been known in avian gizzard, a functional analogue of teeth. In addition a close correlation among food type and morphology and function of gizzard has been found (Ziswiler and Farner, 1972; McLelland, 1979; Duke, 1986). The well-developed musculatures, asymmetrically arranged in two pairs of thin and thick muscles, in gizzards of hard food eaters, i.e. herbivorous and some insectivorous birds, make it possible to perform a strong rotary crushing movement for grinding down ingested hard diet in the organ. Highly interlocking bundles and rich anastomoses in gizzard muscle fibers might also render the contraction further strong (Bennet and Cobb, 1969). Birds dependent on soft and readily digestible diets (carnivorous, piscivorous, frugivorous and nectivorous birds) may not need heavy milling work and consequently well-developed musculature. In fact, gizzard of a buzzard, a typical raptor, was shown to generate a less frequent and weak intraluminal pressure of 2 – 26 mmHg (Mangold, 1911). To the contrary, granivorous birds such as chicken, geese and duck showed a more frequent and much stronger pressure of 139–257 mmHg (Kato, 1914). Schorger (1960) reported that turkey gizzard could crush hickory nuts with a corresponding pressure of 75 kg. Although the energetics of smooth muscles, which are said a ‘high economy-low effficiency’ tissue, are not been fully understood (Paul, 1989), we can enumerate evidennce which suggests that the strong, repetitive and long-persisting contractions in gizzard smooth muscles are mostly supported by aerobic metabolism. In chicken and emu gizzard smooth muscles, Patak and Baldwin (1988) found higher activities of several oxidative enzymes, lower glycolytic and glycogenolytic enzymes activity and lower buffering capacities, as compared with the skeletal muscles. They argued that the metabolism might be highly aerobic glycolytic-lactate oxidative. The results and argu-
41
ments are consistent with the finding that glycogen stores in gizzards of chicken, pigeon and duck are much lower than the skeletal, cardiac and even uterine smooth muscles (Gro¨schel-Stewart and Zuber, 1990). These data suggest a lower contribution, if any, of anaerobic glycolysis to the energy source for the contraction (Gro¨schel-Stewart and Zuber, 1990). The morphological finding that the mitochondrial volume density is much higher in gizzard smooth muscles than in the other smooth muscles (Bennet and Cobb, 1969; Mackenson and Hikida, 1979) is also consistent with oxidative fuel use. The present results on the consistently lower buffering capacities in the gizzard muscles also support the general line of reasoning (Tables 2 and 3). Thus, it will be reasonable to think that the higher Mb content in the gizzards of the hard food eaters might be responsive to the higher oxygen demand for the more intensive grinding work. Another point should be discussed here concerning oxygen supply to the gizzard muscles during strong mechanical work. According to Kirk and Honig (1964) circulation through and, therefore, oxygen supply to cardiac muscles are greatly influenced by periodic changes in the intramyocardial tissue pressure consequent to the cardiac cycle. They suggest a depth-dependent elevation of Mb content in the myocardial wall to compensate for varied oxygen supply. Changes in Mb with depth have been found (Lin et al., 1990). The same situation may occur in the gizzards, also a hollow organ with massive muscular wall. The oxygen supply via circulation would be severely hampered during the powerful and long-lasting contraction of the organ. The fact that the organ has one of the poorest circulation of any organ even at rest (Sapirstein and Hartman, 1959) may further aggravate the situation. These circulatory factors may be reflected in the much higher Mb contents in the gizzard as compared with the cardiac muscles (Table 1). This appears also to be analogous to the situation in skeletal muscles of diving mammals and birds, in which the muscles are reflexly maintained almost ischemic during diving, and show enormously high Mb contents as shown in Table 2 (Dejours, 1981). Lower capillarisation in gizzard musculature (Table 4) may also have some concern with the circulatory characteristics. To conclude, the higher Mb contents in herbivorous avian gizzard muscles could be considered a functional adaptation, allowing stor-
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age and/or facilitated diffusion of oxygen, to secure the higher oxygen demand necessary to permit higher mechanical work under the conditions of limited circulatory supply. Comments are added on a few exceptional cases. Ostrich, a giant flightless and fast running bird, is mostly herbivorous, but the gizzard Mb content was as low as those in carnivorous birds (Table 3). The same explanation for the low Mb content in emu (Patak and Baldwin, 1988), another giant bird with the same locomotory behavior, will be possible. Huge proventriculus in ostrich (McLelland, 1979) might provide a space for temporal storage of ingested food prior to the later grinding and digestion. Exceptionally high gizzard Mb in two species of insectivorous swift (Table 2) may be concerned in any way with their restless long-lasting flight (Lack, 1974). Another exception in two herbivorous species of Psittacidae, blue-and-yellow macaw and rose-ringed parakeet (Table 3), appears to be explained reasonably as stated in Section 3.2 (Ziswiler and Farner, 1972; McLelland, 1979).
Acknowledgements We are deeply indebted to Dr S. Yamagishi and his colleagues (Osaka City University) for their information as to the feeding habits of birds. Thanks are also due to Osaka Municipal Tennoji Zoological Garden (Director: Y. Doi) for providing us the materials of the present research.
References Andersen, P., 1975. Capillary density in skeletal muscle of man. Acta Physiol. Scand. 95, 203–205. Antonini, E., Brunori, M., 1971. Hemoglobin and Myoglobin in their Reactions with Ligands. North Holland, Amsterdam, pp. 219–234. Bate Smith, E.C., 1938. The buffering of muscle in rigor: protein, phosphate and carnosine. J. Physiol. 92, 336–343. Bennet, T., Cobb, T.L.S., 1969. Studies on the avian gizzard: morphology and innervation of the smooth muscle. Z. Zellforsch. Mikroskop. Anat. 96, 173–185. Blessing, H.H., Muller, G., 1974. Myoglobin concentration in the chicken, especially in the gizzard. Comp. Biochem. Physiol. 47A, 535–540. .
Brandes, G., 1896. U8 ber den Einfluss vera¨nderter Erna¨hrung auf die Struktur des Vogelmagens. Biol. Zentrbl. 16, 825 – 838. Castellini, M.A., Somero, G.N., 1981. Buffering capacity of vertebrate muscle: correlations with potentials for anaerobic function. J. Comp. Physiol. B143, 191 – 198. Dejours, P., 1981. Principles of Comparative Respiratory Physiology, 2nd Edition. Elsevier/North Holland, Amsterdam, pp. 141 – 142. Dilger, W.C., 1957. The loss of teeth in birds. Auk 74, 103 – 104. Duke, G.E., 1986. Alimentary canal: anatomy, regulation of feeding and motility. In: Sturkie, P.D. (Ed.), Avian Physiology. Springer, New York, NY, pp. 269 – 288. Enoki, Y., Ohga, Y., Kawase, M., Nakatani, A., 1984. Identical myoglobin is present in both skeletal and smooth muscles of chicken. Biochim. Biophys. Acta 789, 334 – 341. Enoki, Y., Morimoto, T., Nakatani, A., Sakata, S., Ohga, Y., Kohzuki, H., 1988. Wide variation of myoglobin contents in gizzard smooth muscles of various avian species. Adv. Exp. Med. Biol. 222, 709 – 716. Giardina, B., Ascenzi, P., Clementi, M.E., De Sanctis, G., Rizzi, M., Coletta, M., 1996. Functional modulation by lactate of myoglobin. J. Biol. Chem. 271, 16999 – 17001. Gro¨schel-Stewart, U., Jaroschik, U., Schwalm, H., 1971. Chicken gizzard, a myoglobin containing smooth muscle. Experientia 27, 512 – 513. Gro¨schel-Stewart, U., Zuber, C., 1990. Is glycogen a major energy source in avian gizzard smooth muscle contraction? Experientia 46, 686 – 688. Haneda, K., 1962. Studies on the feeding type and the community of geese and ducks living in inland waters. XIII. Food of geese and ducks. Physiol. Ecol. 10, 98 – 129 (In Japanese). Hochachka, P.W., Mommsen, T.P., 1983. Protons and anaerobiosis. Science 219, 1391 – 1397. Ichihara, K., 1990. Statistics for bioscience — practical technique and theory. Nanko-Do, Tokyo. Ikeda, S., 1956. On the food habits of Japanese birds. Research Report of Japanese Wild Beasts and Birds: XV, pp. 1 – 95 (In Japanese). Kato, T., 1914. Druckmessungen im Muskelmagen der Vogel. Pflu¨gers. Arch. 159, 6 – 26. Kennedy, R.P., Whipple, G.H., 1928 – 1929. The hemoglobin of smooth and striated muscle of the fowl. Am. J. Physiol. 87, 192 – 195. Kirk, E.S., Honig, C.R., 1964. Nonuniform distribution of blood flow and gradients of oxygen tension within the heart. Am. J. Physiol. 207, 661 – 668. Lack, D., 1974. Swifts in a Tower. Chapman and Hall, London. Lin, L., Sylven, C., Sotonyi, P., Somogyi, E., Kaijser, L., Jansson, E., 1990. Myoglobin content and citrate
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synthase activity in different parts of the normal human heart. J. Appl. Physiol. 69, 899–901. Mackenson, C.A., Hikida, R.S., 1979. Avian smooth muscle: morphometric analysis and comparison of different types. Zoomorphology 92, 183–189. Mangold, E., 1911. Die funktionellen Schwankungen der motorischen Ta¨tigkeit des Raubvogelmagens. Pflu¨gers Arch. 139, 10–32. Mannion, A.F., Jakeman, P.M., Willan, P.L.T., 1993. Determination of human skeletal muscle buffer value by homogenate technique: methods of measurement. J. Appl. Physiol. 75, 1412–1418. McLelland, J., 1979. Digestive system. In: King, A.S., McLelland, J. (Eds.), Form and Function in Birds, vol. 1. Academic Press, London, pp. 69– 181. Meng, H., Bentley, T.B., Pittman, R.N., 1993. Myoglobin content of hamster skeletal muscles. J. Appl. Physiol. 74, 2194–2197. Patak, A., Baldwin, J., 1988. Why is there so much myoglobin in chicken gizzard and so little in emu gizzard? Comp. Biochem. Physiol. 89B, 27–30. Paul, R., 1989. Smooth muscle energetics. Ann. Rev. Physiol. 51, 331–349. Perrins, C.M., Middleton, A.L.A. (Eds.), 1984. The Encyclopaedia of Animals, vols. 7–9. Equinox, Oxford.
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Reynafarje, B., 1963. Simplified method for the determination of myoglobin. J. Lab. Clin. Med. 61, 138 – 145. Sapirstein, L.A., Hartman, F.A., 1959. Cardiac output and its distribution in the chicken. Am. J. Physiol. 196, 751 – 752. Schorger, A.W., 1960. The crushing of Carya nuts in the gizzard of the turkey. Auk. 77, 337 – 340. Snyder, G.K., 1990. Capillarity and diffusion distances in skeletal muscles in birds. J. Comp. Physiol. B160, 583 – 591. Suzuki, T., Imai, K., 1998. Evolution of myoglobin. Cell. Mol. Life Sci. 54, 979 – 1004. Tollefson, C.I., 1978. Diets for birds other than poultry. In: Rechcigl, M. (Ed.), Food habits of, and diets for, invertebrates and vertebrates-zoo diets. CRC Press, Cleveland, OH, pp. 349 – 361. Welty, J.C., 1962. The Life of Birds. Saunders, Philadelphia, PA, p. 3. Wittenberg, J.B., Wittenberg, B.A., 1990. Mechanisms of cytoplasmic hemoglobin and myoglobin function. Ann. Rev. Biophys. Biophys. Chem. 19, 217 – 241. Yoshii, T. (Ed.), 1988. Concise Dictionary of World Birds. Sansei-Do, Tokyo. Ziswiler, V., Farner, D.S., 1972. Digestion and the digestive system. In: Farner, D.S., King, J.R., Parkes, K.C. (Eds.), Avian Biology. II. Academic Press, New York, NY, pp. 360 – 430.
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Gizzard myoglobin contents and feeding habits in avian species Yasunori Enoki *, Tomotoshi Morimoto Second Department of Physiology, Nara Medical Uni6ersity, Kashihara, Nara 634 -8521, Japan Received 24 May 1999; received in revised form 9 September 1999; accepted 22 September 1999
Abstract In an attempt to consider physiological function of myoglobin (Mb), we determined Mb contents of gizzard smooth muscles with special reference to feeding habits in 85 avian species of 19 orders. The Mb content in 44 species of herbivorous birds was 7.52 93.81 mg/g wet muscle, which was significantly higher than the value of 2.34 9 1.74 mg/g in 41 species of carnivorous ones (PB0.001). Buffering capacity, as determined by in vitro titration method, was 37.3 9 5.5 slykes/g in gizzard smooth muscles of 75 species and 60.7 9 10.5 slykes in breast skeletal muscles of 77 species (PB 0.001), which suggests a significantly higher dependence, almost comparable to cardiac muscles, of the gizzard muscular function on aerobic metabolism. Together with the fact that blood circulation in the gizzard is very low at resting, and might be further limited during activity, we conclude that the higher Mb content in gizzards of herbivorous birds is an adaptation, to allow storage and/or facilitated diffusion of oxygen, during process of high mechanical work required to grind down hard and fibrous vegetable food under the conditions of limited circulatory supply. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Birds; Buffering capacity; Capillarity; Circulation; Feeding habit; Gizzard; Myoglobin; Oxygen; Skeletal muscle; Smooth muscle
1. Introduction Locomotion by flight is undoubtedly the most characteristic functional property in most avian species. Absence of teeth in this class of animals has been interpreted as a morphological adaptation to this specific function, resulting in a lighter head without necessity of robust and heavy jaws for fixing the teeth and chewing muscles (Dilger, 1957; Welty, 1962; McLelland, 1979). Evolution of the gizzard or muscular stomach in the avian digestive system, allow for the mechanical diges-
* Corresponding author. Present address: Horai 4-28-5, Nara 631-0845, Japan. Tel./fax: + 81-742-461233.
tion of foods without teeth. Accumulated literature tells us that, in general, herbivorous birds have a gizzard with well-developed musculature, while carnivorous birds bear the organ with poorly-developed ones (Ziswiler and Farner, 1972; McLelland, 1979; Duke, 1986). The strong gizzard in the former category of birds has been considered to meet the increased mechanical work which is required to grind down the more fibrous, harder and much less digestible food of plant origin. In the latter group of birds, however, food of animal origin can be readily disposed of by chemical digestion with digestive juices secreted from the proventriculus (glandular stomach) and other digestive glands, and the rather weak musculature might be sufficient.
1095-6433/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 ( 9 9 ) 0 0 1 6 1 - 0
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Myoglobin, a single polypeptide heme protein with reversible oxygen binding ability, had long been believed to be present in skeletal and cardiac muscles but not in smooth muscle. Kennedy and Whipple were the first to suggest presence of the protein in gizzard smooth muscle of chicken by observing the absorption spectrum of an aqueous extract of the organ (Kennedy and Whipple, 1928). Later this possibility was further strengthened by Gro¨schel-Stewart et al. (1971) and also Blessing and Muller (1974), but their evidence was not conclusive. One of the present authors has presented conclusive evidence that Mb, which is structurally and functionally identical with those in the skeletal and cardiac muscles, is present in chicken gizzard smooth muscles (Enoki et al., 1984). We showed also that, although the presence of the protein was universal throughout other avian species, the concentration was highly variable fiom species to species. In view of the fact that the content appeared to be generally higher in herbivorous birds compared with carnivorous ones, we suggested that the variability might reflect feeding habit (Enoki et al., 1988). In this paper we present data on some eighty species of bird and provide solid support for the hypothesis. Data on the buffering capacity and capillarity of the muscles, which might be closely related to muscular metabolism, will be also presented.
2. Materials and methods
2.1. Materials Eighty five species of birds belonging to 19 different orders, reared and autopsied in Osaka Municipal Tennoji Zoological Garden (Osaka), were served for the present study. Common and scientific names of the birds are listed in Tables 2 and 3. Usually only one adult bird was used for any one species unless otherwise stated and the specimen from an animal with suspected gastrointestinal disorder was excluded. The gizzard was excised as quickly as possible after the animal’s death (usually within 6 h), its koilin lining, fascia and fat were trimmed off, and immediately frozen-stored at− 85°C until use. Chickens were obtained from a local poultry farm, decapitated after intraperitoneal pentobarbital sodium anesthesia (50 mg/kg body weight), and heart, proven-
triculus (glandular stomach), breast and leg muscles and gizzard were excised, and immediately used for the determination of myoglobin contents and other studies.
2.2. Determination of Mb contents in muscle A spectrophotometric method was used as described previously (Enoki et al., 1988), which was a modification of the procedure by Reynafarje (1963). Small pieces of muscle were excised fiom the specimens, chopped up and well mixed by the use of ophthalmological scissors and a razor blade on an ice-chilled glass plate, divided into tared and capped polypropylene micro-tubes and weighed on an electronic balance. Since a gradient of Mb content was suspected across the gizzard wall as suggested for canine and actually observed in human myocardial wall (Kirk and Honig, 1964; Lin et al., 1990), determinations were usually repeated at two locations of different depth, one submucosal and the other subserosal, and the results were averaged. The amount of muscle varied from 10 to 30 mg each depending on the Mb content as guessed by the sample colour. A 10 mM K2PO4 solution (0.8 ml) containing 1 mM Na-EDTA, pre-saturated with CO and ice-chilled, was added and the muscle sample was thoroughly homogenized with a metal microhomogenizer (NITION: Physcotron NS-310E) equipped with an NS-4 microshaft. The sample tube was held in ice water during homogenization. The homogenate was centrifuged at 10 000×g and 4°C in a refrigerated microcentrifuge (Tomy Seiko: MC15A), and the resultant supernate was transferred to a new microcentrifuge tube. The air space of the tube was flushed with CO after adding 0.3 ml of chloroform, and the capped tube with its content was vigorously hand-shaken for a few seconds. The final step was a modification of the original procedure to remove fatty ingredients which cause cloudiness in the sample and interfere with the following optical measurement. The supernatant aqueous layer, after centrifuging the mixture at 10 000×g and 4°C, was transferred into a microcuvette, the air space was gently flushed with CO stream, and a few crystals of sodium dithionite were added and the content was well mixed by several inversions. After the evolved bubbles had vanished on standing, the sample was scanned for absorbance in 530–580 nm wave length region. Mb content
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was derived as previously reported (Enoki et al., 1988); C=
5.696(A538 − A568)(0.800 +0.75W) W
where C: myoglobin content in mg/g wet muscle; A538 and A568: absorbances of the sample at 538 and 568 nm, respectively; and W: wet weight of muscle sample in g. In the gizzards with well-developed musculature as seen typically in herbivorous birds, the thick muscle portion was used for the determination unless otherwise stated, and usually five determinations were performed at two points each of different depth, one subserosal and the other submucosal, and the results were averaged. Carnivorous birds, on the contrary, have generally a poorlydeveloped and thin-walled gizzard, in which the determinations were usually done at one location.
2.3. Histochemical obser6ation of the capillarity in muscles Immediately after sacrifice of chicken, the gizzard was excised, cut into two pieces, and a tiny block (2–4 mm square) of the muscle was sampled from the thick muscle portion at the middle point of the muscle depth. The muscle block was frozen-embedded in AMES™ Tissue Tek OTC Compound (Lab Tec Products) in isopentane pre-cooled with liquid nitrogen. Serial 10 mm thick cross sections of the frozen muscle were prepared with a cryostat (Bright: model FS/FCS) at−20°C. The sections were stained histochemically for capillaries by Andersen’s amylase-PAS procedure (Andersen, 1975). Capillarity was expressed by counting the number of capillaries per mm2 of the tissue cross sectional area.
2.4. Buffering capacity (b) of muscles The method of Bate Smith (1938), which was later used by Castellini and Somero (1981) for measuring b of vertebrate muscles, was followed. Briefly, a muscle sample (0.5 g) was weighed into a specially designed Pyrex tube (12 ×110 mm) with a flat bottom, and was homogenized thoroughly with 10.0 ml of 0.9% saline by using a microhomogenizer (NITI-ON:
35
Physcotron NS-310E). The homogenates were adjusted to pH 6.0 with HCl when the initial pH was more alkaline, and were then titrated under magnetic stirring with 0.20 N NaOH in pH 6–7.5 range (20°C). The titrant was added drop by drop with a micrometer burette (Gilmont: S1200A), and the pH was measured by a TOA pH meter HM-5S equipped with a small-bore combined glass electrode(TOA: GS5018S). Strictly, the titration curve as a whole is not linear, but within a limited range such as pH 6–7, it can be safely considered as so. Therefore, from the titration curve, we obtained the b (slykes) as the amount of NaOH (m mols) required for a unit pH change of the homogenate of 1 g muscles.
2.5. Gel-filtration high performance liguid chromatography (HPLC) of gizzard muscle homogenates Gizzard muscles (20 mg) were homogenized in CO-saturated milliQ water (1 ml) in a capped microcentrifuge tube as described above, and centrifuged at 10 000× g (4°C) in a refrigerated microcentrifuge. The clear supernatant was used for gel-filtration HPLC analyses on a TOSO TSK-G3000 SWXL column (7.8× 300 mm). Isocratic elution was performed in 0.05 M phosphate (pH 7.0) containing 0.01% sodium azide and flow rate of 0.5 ml/min with a Waters 626 LC system, and the eluate’s absorbance was monitored at 215 nm. Retention times for the elution peaks were automatically determined and recorded. All chemicals were the analytical grade from Nacalai Tesque (Kyoto) and Wako (Osaka). Spectrophotometric measurements were carried out with a Hitachi spectrophotometer model 124 or a Union high sensitivity spectrophotometer model 401.
2.6. Statistical analysis After the test for equal variance (F-test), statistical significance of the difference between the means was assessed by either the two sample t test, or the two sample t test with Welch’s correction (Ichihara, 1990). The significance level was set at P B0.05.
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3. Results
3.1. Myoglobin contents of chicken skeletal, gizzard and cardiac muscles Table 1 shows the results obtained for four chickens. The contents in thick muscles of the gizzards were almost eight times those of the leg skeletal muscles, and more than four times those of the cardiac ventricles. It should be also noted that the gizzard thin muscles show a considerably lower content of Mb than the thick muscles.
3.2. Myoglobin contents in gizzard smooth muscles of 85 a6ian species Mb contents in gizzard thick muscles and breast skeletal muscles for comparison were determined in 85 species of birds. Table 2 represents the results for 41 mostly carnivorous species, including piscivorous, insectivorous and nectivorous birds. The gizzard Mb contents ranged from 0.58 mg/g wet muscle in grey-faced buzzard-eagle to 11.30 mg/g in northern white-rumpted swift. It is evident that most of the birds show a relatively low Mb content (B 5 mg/g) with an average9 S.D. of 2.3491.74 mg/g wet muscle (n =41). It should be noted that two species of swift, whitethroated spine-tailed swift and northern whiterumpled swift, show exceptionally high Mb, although they are undoubtedly insectivorous. The results for 44 species of herbivorous birds were summarized in Table 3, the values ranging from 1.03 mg/g in Gouldian finch to 17.33 in common coot. In contrast to the findings in Table 2, most of the birds show a relatively high level (5 mg/gB) of Mb, and the mean and S.D. were 7.529 3.81
mg/g wet muscle (n=44). Two species of the herbivores, blue-and-yellow macaw and roseringed parakeet, both belonging to the order of Psittaciformes, were granivorous, but they are reported to crush the feed into small readily digestible pieces with their strong bill to swallow, and have a poorly developed gizzard. Their low gizzard Mb might be a reflection of this feeding behavior. The difference between the means of the carnivores and the herbivores was highly significant (PB 0.001). A slight Mb concentration gradient, as suggested and found for myocardial Mb (Kirk and Honig, 1964; Lin et al., 1990), appears to be present in the gizzard muscular wall: the ratio of submucosal to subserosal Mb contents was 1.10 9 0.43 (n= 41) and 1.05 9 0.34 (n= 12) in herbivorous and carnivorous birds, respectively. The difference between the two categories of birds is not statistically significant.
3.3. Gel-filtration chromatographic obser6ations of the gizzard muscle extract The Mb peaks, identified by their characteristic Soret absorption and also by referring to the chromatographic behavior for the authentic chicken Mb, were found all through the avian species. Naturally, the peak heights (Mb contents) were much lower in the carnivorous species. The retention times for the Mb peaks were essentially identical with each other in the extracts from the gizzard smooth muscles (21.5890.08 min, n= 55), breast skeletal muscles (21.6190.14 min, n= 57) and cardiac muscles (21.61 min, n= 2), and the molecular weights estimated were around 1.8× 104.
Table 1 Myoglobin contents of chicken skeletal, cardiac and gastric smooth musclesa Sketetal muscles Breast
Cardiac muscles Leg
Atrium
Gastric smooth muscles Ventricle
Provontriculus
Gizzard
Thick muscle 0.449 0.09 (0.17)
Thin muscle 1.44b (0.55)
2.20 9 0.54 (0.85)
2.609 0.37 (1.00)
1.48 9 0.13 (0.57)
6.72 9 1.24 (2.58)
10.65 90.90 (4.10)
Means 9S.D. (mg/g wet muscle) for four chickens. Figures in parentheses are the ratios of Mb contents to that of the ventricular muscle. b Mean for two chickens. a
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Table 2 Myoglobin contents and buffering capacities of gizzard and breast muscles in carnivorous birdsa No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Birds
Grey-faced buzzard-eagle Red-necked phalarope King vulture Ancient murrelet White’s thrush Woodcock Wattled crane Ruddy kingfisher King penguin Greater flamingo Manchurian crane Scarlet ibis Black-headed gull Striated heron Grey heron White spoonbill White-crested laughing thrush Golden-breasted starling Brown hawk owl Roseate spoonbill Brown-eared bulbul Little egret Andean cock-of-the-rock Oriental scops owl Ural owl Rufous-tailed hummingbird Cattle egret White stork Sacred ibis Magellanic penguin Black-crowned night heron Great Indian hornbill Macaroni penguin Rockhopper penguin White-faced shearwater White-naped crane Swinhoe’s storm petrel Jungle nightjar Great crested grebe White-throated spine-tailed swift Northern white-rumped swift
Means9 S.D. (n)
Mb contents (mg/g wet muscle)
Buffering capaacities (slykes)
Gizzard muscle
Gizzard muscle
0.58 0.75 0.81 1.05 1.17 1.21 1.24 1.36 1.41 1.46 1.47 1.51 1.63 1.66 1.76 1.77 1.77 1.77 1.78 1.78 1.81 2.03 2.03 2.13 2.14 2.14 2.25 2.26 2.34 2.38 2.59 2.75 2.75 2.77 3.17 3.29 3.54 4.32 4.39 5.76 11.31
(2)b
(2) (2) (3) (4)
(2) (3)
(2)
Breast muscle 1.99 6.66 4.07 15.76 4.41 3.34 2.81 4.84 35.94 2.57 2.71 1.87 2.83 6.48 6.29 4.12 3.95 4.71 4.08 4.97 5.12 8.46 5.86 2.32 6.29 7.31 4.59 18.49
Breast muscle
47.1 52.3 20.7 32.6 36.7 39.2 30.6 37.8 31.9
43.7 53.1 69.4 51.3 53.3 60.6 45.8 54.7 61.6
31.2 33.3 40.1
63.2 58.2 55.9 64.7 57.2
33.3 31.7 34.2 39.1 38.4 33.4 39.5 44.9 44.9 39.1 41.5 47.3 31.2 27.2 39.2 31.6
33.1 51.8 60.1 64.7 63.7 70.4 60.8 71.5 72.7 54.6 53.3 67.1 51.9
(2) (2) (4) (2)
2.349 1.74 (41)
7.79 27.91 26.88 5.62 11.07 7.82 3.61 8.75 2.92 10.04 7.7797.51 (38)
28.9 27.9 37.6 34.1 35.7 37.6 31.8 33.9 44.5 36.3 9 6.3 (37)
59.7 48.4 66.5 54.7 67.2 43.4 64.4 43.9 34.3 44.5 56.6 99.8 (37)
a Latin names of birds: 1, Butastur indicus; 2, Phalaropus lobatus; 3, Sarcoramphus papa; 4, Synthliboramphus antiquus; 5, Zoothera dauma; 6, Scolopax rusticola; 7, Bugeranus carunculatus; 8, Halcyon coromanda; 9, Aptenodytes patagonicus; 10, Phoenicopterus ruber; 11, Grus japonensis; 12, Eudocimus ruber; 13, Larus ridibundus; 14, Ardeola striata; 15, Ardea cinerea; 16, Platalea leucorodia; 17, Carrulax leucolophus; 18, Cosmopsarus regius; 19, Ninox scutulata; 20, Platalea ajaja; 21, Hypsipetes amaurotis; 22, Egretta garzetta; 23, Rupicola peru6iana; 24, Otus sunia; 25, Strix uralensis; 26, Amazilia tzacatl; 27, Egretta ibis; 28, Ciconia ciconia; 29, Threskiornis aethiopicus; 30, Spheniscus magellanicus; 31, Nycticorax nycticorax; 32, Buceros bicornis; 33, Eudyptes chrysolophus; 34, Eudyptes chrysocome; 35, Calonectris leucomelas; 36, Grus 6ipio; 37, Oceanodroma monorhis; 38, Caprimulgus indicus; 39, Podiceps cristatus; 40, Hirundapus caudacuta; 41, Apus pacificus. b Figures in parentheses are numbers of birds.
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Table 3 Myoglobin contents and buffering capacities of gizzard and breast muscles in herbivorous birdsa No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Birds
Gouldian finch House sparrow White-faced whistling duck Red-billed whistling duck Blue-and-yellow macaw Ostrich Rose-ringed paraeet Rock pigeon Barnacle goose Spix’s guan Chestnut-bellied sandgrouse Chuker partridge Keel-billed toucan Bar-headed goose Red-crested pochard Black-necked swan European wigeon Emperor goose Green peafowl Ringed teal Baikal teal Mute swan Australian shoveller Mandarin duck Green-winged teal Brazilian teal Garganey Pintail Temminck’s tragopan Siamese fireback pheasant Philippine duck Bufflehead Silver pheasant Moorhen Gadwall Chicken White-eared pheasant Common peafowl Brown-eared pheasant California quail European pochard Grey junglefowl White-headed duck Common coot Means 9 S.D. (n)
Mb contents (mg/g wet muscle)
Buffering capacities (slykes)
Gizzard muscle
Breast muscle
Gizzard muscle
1.03b 1.24 (2) 1.26 1.28 1.33 1.43 1.71 5.23 5.24 5.82 5.99 6.09 6.09 6.11 (2) 6.31 6.33 6.37 6.46 6.64 (2) 6.74 7.01 7.53 (2) 7.57 7.78 (2) 7.81 (2) 7.84 (3) 7.86 8.08 (3) 8.59 (2) 8.66 8.97 9.25 9.67 (2) 9.74 (2) 9.99 10.54 (5) 10.66 10.67 11.32 11.54 (2) 11.94 15.37 16.66 17.33 7.529 3.81 (44)
2.53 2.63 3.06 6.81 4.02 6.51 4.44 0.87 4.46 0.58 4.65 0.76 4.65 5.91 2.39 9.39 4.38 3.87 0.99 1.32 3.24 3.34 3.99 5.56 3.12 3.51 2.01 3.88 0.92 0.21 1.91 7.94 0.45 1.54 4.54 0.44 0.71 0.61 0.29 0.68 6.02 0.63 9.65 8.74 3.36 92.55 (44)
Breast muscle
40.9 32.4 34.9 34.3 33.9 35.1 30.9
66.2 54.5 64.4 78.3 63.3 62.8 71.9 60.1 59.2 66.9 58.1 69.4 55.6 47.6
40.2 39.1
54.4 62.9
42.7 40.1 36.6 37.9 40.1 39.8 43.2 43.2 45.8 38.9 41.3 34.2 36.9 41.7 35.3 37.6 37.5 30.8
80.1 59.1 57.4 58.1 56.4 73.2 62.5 62.5 60.9 66.9 85.3 81.4 50.1 50.6 73.8 58.9 64.6 68.9
30.7
69.4
47.6 41.3 42.6 41.9 29.8
40.9 40.1 38.4 40.5 39.7 38.4 94.2 (38)
83.6 61.6 83.4 53.9 61.5 64.5 99.5 (40)
Latin names of birds: 1, Chloebia gouldiae; 2, Passer domesticus; 3, Dendrocygna 6iduata; 4, Dendrocygna autumnalis; 5, Ara ararauna; 6, Struthio camelus; 7, Psittacula krameri; 8, Columba li6ia; 9, Branta leucopsis; 10, Penelope jacquacu; 11, Pterocles exustus; 12, Alectoris chukar; 13, Ramphastos sulfuratus; 14, Anser indicus; 15, Netta rufina; 16, Cygnus melanocoryphus; 17, Anas penelope; 18, Anser canagicus; 19, Pa6o muticus; 20, Calonetta leucophrys; 21, Anas formosa; 22, Cygnus olor; 23, Anas rhynchotis; 24, Aix galericulata; 25, Anas crecca; 26, Amazonetta braziliensis; 27, Anas querquedula; 28, Anas acuta; 29, Tragopan temminckii; 30, Lophura diardi; 31, Anas luzonica; 32, Bucephala albeola; 33, Lophura nycthemera; 34, Gallinula chloropus; 35, Anas strepera; 36, Gallus gallus 6ar. domesticus; 37, Crossoptilon crossoptilon; 38, Pa6o cristatus; 39, Crossoptilon mantchuricum; 40, Lophortyx californica; 41, Aythya ferina; 42, Gallus sonneratii; 43, Oxyura leucocephala; 44, Fulica atra. b Figures in parentheses are numbers of birds. a
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Fig. 1. Titrations with NaOH of homogenates of gizzard (); cardiac (); and breast skeletal muscles ( ) from chicken.
3.4. Buffering capacities (b) of gizzard smooth muscles and breast skeletal muscles
was slightly but significantly higher in the herbivores than in the carnivores (PB 0.05).The value for the gizzard muscles in total of the 75 species, irrespective of their feeding habits, was 37.39 5.5 slykes, which was significantly lower than 60.79 10.5 slykes for the skeletal muscles in the total 77 species (PB 0.001).
An example of the titrations of homogenates of chicken gizzard, breast and cardiac muscles were shown in Fig. 1. The buffering capacities were estimated as 30.8, 68.9 and 37.5 slykes, respectively. The value for the proventriculus muscle was 30.6 slykes, and little different from that for the gizzard (data not shown). Compared with the gizzard muscle, a distinctly higher b value was found for the skeletal muscle (Tables 2 and 3). In carnivorous birds, the means and S.D. for gizzards and breast muscles were 36.3 9 6.3 (n =37) and 56.6 99.8 slykes (n =37) (P B 0.001), while the corresponding values in the herbivores were 38.4 94.2 (n=38) and 64.599.6 slykes (n =40) (PB 0.001). The buffering capacity of gizzards
3.5. Capillarity in chicken gizzard muscles As shown in Table 4, the mean capillary density in chicken gizzard muscles was 484 mm − 2, which was considerably lower than those in both ‘red’ and ‘white’ skeletal muscles in seven species of birds as reported previously by Snyder (1990), and almost comparable to those in ‘white’ or glycolytic skeletal muscles of mice.
Table 4 Capillarity of chicken gizzard thick muscles as compared with those of skeletal muscles in mice and birds Animals
Muscles
Capillarities (mm−2)
Remarks
Chicken Mice
Gizzard (thick) Skeletal (red)a (white)b Skeletal (red) (white)
484.3 939.5 (8) 1021.2 9196.8 (7) 401.3 923.0 (5) 804.3–842.6 723.6–773.3
Snyder (1990) Snyder (1990)
Birds (seven species)
a b
Gastrocnemius (deep), plantaris and diaphragm. Gastrocnemius (superficial), semimembranosus and biceps femoris.
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4. Discussion Mb is a monomeric heme protein with reversible oxygen binding ability of fairly high affinity for oxygen, but with neither homotropic nor heterotropic allosteric property (Antonini and Brunori, 1971). It is found in muscle cells of vertebrates which need continuous oxygen flow for maintenance of aerobic metabolism. Traditionally proposed main physiological functions of Mb are as follows (Suzuki and Imai, 1998); (1) short-term and long-term storage of oxygen; (2) Mb-facilitated oxygen diffusion; and (3) biochemical catalyst (Mb-mediated oxidative phosphorylation) (Wittenberg and Wittenberg, 1990). The proposals have not fully established yet, but have presented a very controversial area that remains vigorously studied. For example, a recent finding by Giardina et al. (1996) that lactate, an end product of glycolysis, lowered markedly oxygen affinity of Mb is far from the general knowledge so far.
4.1. Methodological comments on the present determinations We determined the Mb contents in the avian muscles by a modification of the Reynafarje’s procedure (Reynafarje, 1963; Enoki et al., 1988). Since the original method had been developed and used for the determinations in human and the other mammalian muscles, we revised the equation for the Mb contents in avian muscles based on the absorption data obtained for purified chicken Mb (Enoki et al., 1984, 1988). This method, albeit simple and widely used, could be in danger of interference by any coexisting substance(s), especially the other heme proteins, which have an absorption in the wave length region for the determination. The suspicion, however, could be safely discarded in view of the recent detailed discussions by Meng et al. (1993). Buffering capacity in muscles has been argued to be a possible measure to assess the dependence of muscle work on aerobic or anaerobic metabolism (Castellini and Somero, 1981; Hochachka and Mommsen, 1983). The present determinations by an in vitro homogenate titration technique can be performed conveniently, but may not be free from the possibility that the results may deviate somewhat from the true in vivo value since it can not measure the buffering
via bicarbonate system. The role of the bicarbonate system, however, has been generally considered to be negligible, in the intracellular buffering (Castellini and Somero, 1981). No detectable difference was found experimentally between the b values for human skeletal muscles by the in vitro and in vivo titration (Mannion et al., 1993). The HPLC results confirm and extend our previous finding in chicken (Enoki et al., 1984) that Mb is present all through gizzard, skeletal and cardiac muscles of avian species.
4.2. Feeding habits in birds To determine animal’s feeding habit in the field is difficult, requiring both behavioral observations and careful examination of the alimentary contents in captured animals (Ikeda, 1956; Haneda, 1962). In addition feeding habit varies both temporally (seasonal) and spatially (geographical). For example, the grey duck (Anas poecilorhyncha) feeds almost entirely (92%) on diets of plant origin, principally grass seeds, during autumn to spring seasons, but in the summer breeding period changes to 40% plants with the remainder of the diet being of animal origin such as insects (Haneda, 1962). To assess diet we relied completely on information from the literature (Ikeda, 1956; Haneda, 1962; Tollefson, 1978; Perrins and Middleton, 1984; Yoshii, 1988). Our assessment of diet was checked critically by a research group for avian ecological studies (Dr S. Yamagishi and his colleagues: personal communication). A comment should be added that the birds used in this studies were all artificially reared in a zoo during variable period. On the basis of a classical study by Brandes (1896), however, it will be reasonable to assume that no substantial change in gizzard morphology and probably the Mb contents should occur during the period.
4.3. Gizzard Mb contents in relation to feeding habit A number of structural adaptations have evolved coincident with flight in birds. Loss of teeth and the evolution of gizzard are most obvious, however the biolological implications may not be simple (Dilger, 1957; McLelland, 1979). First, weight saving, especially in the head region, which may be favourable for flight, results from loss of jaws and attendant chewing muscles. Ac-
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cording to Welty (1962), the head weight in pigeon is only 0.21% of the total body weight, while that in rat is 1.25%. Second, the changes may meet the need for rapid ingestion and digestion of food as a result of the higher metabolic rate demanded for the evolution of endothermy and flying in this class of animals. Finally, it should be mentioned that the development of the massive gastric mill brings the heavy musculatures closer to the center of gravity. Strong force generation has long been known in avian gizzard, a functional analogue of teeth. In addition a close correlation among food type and morphology and function of gizzard has been found (Ziswiler and Farner, 1972; McLelland, 1979; Duke, 1986). The well-developed musculatures, asymmetrically arranged in two pairs of thin and thick muscles, in gizzards of hard food eaters, i.e. herbivorous and some insectivorous birds, make it possible to perform a strong rotary crushing movement for grinding down ingested hard diet in the organ. Highly interlocking bundles and rich anastomoses in gizzard muscle fibers might also render the contraction further strong (Bennet and Cobb, 1969). Birds dependent on soft and readily digestible diets (carnivorous, piscivorous, frugivorous and nectivorous birds) may not need heavy milling work and consequently well-developed musculature. In fact, gizzard of a buzzard, a typical raptor, was shown to generate a less frequent and weak intraluminal pressure of 2 – 26 mmHg (Mangold, 1911). To the contrary, granivorous birds such as chicken, geese and duck showed a more frequent and much stronger pressure of 139–257 mmHg (Kato, 1914). Schorger (1960) reported that turkey gizzard could crush hickory nuts with a corresponding pressure of 75 kg. Although the energetics of smooth muscles, which are said a ‘high economy-low effficiency’ tissue, are not been fully understood (Paul, 1989), we can enumerate evidennce which suggests that the strong, repetitive and long-persisting contractions in gizzard smooth muscles are mostly supported by aerobic metabolism. In chicken and emu gizzard smooth muscles, Patak and Baldwin (1988) found higher activities of several oxidative enzymes, lower glycolytic and glycogenolytic enzymes activity and lower buffering capacities, as compared with the skeletal muscles. They argued that the metabolism might be highly aerobic glycolytic-lactate oxidative. The results and argu-
41
ments are consistent with the finding that glycogen stores in gizzards of chicken, pigeon and duck are much lower than the skeletal, cardiac and even uterine smooth muscles (Gro¨schel-Stewart and Zuber, 1990). These data suggest a lower contribution, if any, of anaerobic glycolysis to the energy source for the contraction (Gro¨schel-Stewart and Zuber, 1990). The morphological finding that the mitochondrial volume density is much higher in gizzard smooth muscles than in the other smooth muscles (Bennet and Cobb, 1969; Mackenson and Hikida, 1979) is also consistent with oxidative fuel use. The present results on the consistently lower buffering capacities in the gizzard muscles also support the general line of reasoning (Tables 2 and 3). Thus, it will be reasonable to think that the higher Mb content in the gizzards of the hard food eaters might be responsive to the higher oxygen demand for the more intensive grinding work. Another point should be discussed here concerning oxygen supply to the gizzard muscles during strong mechanical work. According to Kirk and Honig (1964) circulation through and, therefore, oxygen supply to cardiac muscles are greatly influenced by periodic changes in the intramyocardial tissue pressure consequent to the cardiac cycle. They suggest a depth-dependent elevation of Mb content in the myocardial wall to compensate for varied oxygen supply. Changes in Mb with depth have been found (Lin et al., 1990). The same situation may occur in the gizzards, also a hollow organ with massive muscular wall. The oxygen supply via circulation would be severely hampered during the powerful and long-lasting contraction of the organ. The fact that the organ has one of the poorest circulation of any organ even at rest (Sapirstein and Hartman, 1959) may further aggravate the situation. These circulatory factors may be reflected in the much higher Mb contents in the gizzard as compared with the cardiac muscles (Table 1). This appears also to be analogous to the situation in skeletal muscles of diving mammals and birds, in which the muscles are reflexly maintained almost ischemic during diving, and show enormously high Mb contents as shown in Table 2 (Dejours, 1981). Lower capillarisation in gizzard musculature (Table 4) may also have some concern with the circulatory characteristics. To conclude, the higher Mb contents in herbivorous avian gizzard muscles could be considered a functional adaptation, allowing stor-
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age and/or facilitated diffusion of oxygen, to secure the higher oxygen demand necessary to permit higher mechanical work under the conditions of limited circulatory supply. Comments are added on a few exceptional cases. Ostrich, a giant flightless and fast running bird, is mostly herbivorous, but the gizzard Mb content was as low as those in carnivorous birds (Table 3). The same explanation for the low Mb content in emu (Patak and Baldwin, 1988), another giant bird with the same locomotory behavior, will be possible. Huge proventriculus in ostrich (McLelland, 1979) might provide a space for temporal storage of ingested food prior to the later grinding and digestion. Exceptionally high gizzard Mb in two species of insectivorous swift (Table 2) may be concerned in any way with their restless long-lasting flight (Lack, 1974). Another exception in two herbivorous species of Psittacidae, blue-and-yellow macaw and rose-ringed parakeet (Table 3), appears to be explained reasonably as stated in Section 3.2 (Ziswiler and Farner, 1972; McLelland, 1979).
Acknowledgements We are deeply indebted to Dr S. Yamagishi and his colleagues (Osaka City University) for their information as to the feeding habits of birds. Thanks are also due to Osaka Municipal Tennoji Zoological Garden (Director: Y. Doi) for providing us the materials of the present research.
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