Contribution of shivering in leg muscles to heat production in ...

Contribution of shivering in leg muscles to heat production in Japanese quail E. DONSTEVENS, J. FERGUSON, AND V.G. THOMAS Department of Zoology, University of Guelph, Guelph, Ont., Canada NlG 2 W1 AND

E. HOHTOLA Department of Zoology, University of Oulu, Oulu, Finland Received September 18, 1985 STEVENS, E. D., J. FERGUSON, V. G. THOMAS, and E. HOHTOLA.1986. Contribution of shivering in leg muscles to heat production in Japanese quail. Can. J. Zool. 64: 889-892. We estimated heat production in Japanese quail (Coturnix coturnix japonica) by measuring oxygen uptake using open-circuit respirometry as ambient temperature was decreased gradually from 26 to 3.5"C. At the same time, the intensity of shivering was estimated in both the leg muscles and the flight muscles by measuring electromyograms. Metabolic heat production increased in a linear fashion as ambient temperature decreased. Shivering intensity increased at the same linear rate in the leg muscles as in the flight muscles as ambient temperature decreased. The leg muscles produce a substantial fraction (about 114) of the total shivering heat production at low ambient temperatures. Shivering occurred in bursts; the onset of a burst in the leg muscles was precisely synchronized with the onset of a burst in the flight muscles. V. G. THOMAS et E. HOHTOLA.1986. Contribution of shivering in leg muscles to heat STEVENS, E. D., J. FERGUSON, production in Japanese quail. Can. J. Zool. 64: 889-892. Nous avons estimk la production de chaleur chez des cailles japonaises (Coturnix coturnix japonica) en mesurant la consommation d'oxygkne au moyen d'un respiromktre a circuit ouvert, tout en abaissant graduellement la tempkrature ambiante de 26 a 3,5"C. L'enregistrement des klectromyogrammes dans les muscles des pattes et les muscles du vol a permis d'kvaluer en mCme temps I'intensitk du frissonnement. La production mktabolique de chaleur est fonction linkaire de la baisse de tempkrature. L'intensitk du frissonnement augmente selon le mCme taux linkaire dans les muscles des pattes et les muscles du vol a mesure que baisse la tempkrature. Les muscles des pattes sont responsables d'une fraction importante (environ 114) de la production de chaleur totale due au frissonnement lorsque la tempkrature ambiante est faible. Le frissonnement se produit par secousses; le dkclenchement du frissonnement dans les muscles des pattes est parfaitement synchronisk avec le dkclenchement du frissonnement dans les muscles du vol. [Traduit par la Revue]

Introduction Most authorities agree that birds in cold environments increase heat production by shivering rather than by nonshivering themogenesis (see George 1984; Hohtola 1982). B~ thermogenesis, we refer specifically to faculative thermogenesis involved in thermost&ic heat - production. For example, Calder and King (1974) stated that "the metabolic response to cold stress is produced almost entirely by shivering of the skeletal muscle in birds." A good correlation between the integrated electromyogram (EMG) and metabolic heat production (oxygen has been in birds et al. 1970;Hudson et al. 1974; Chaplin 1976; Dawson et al. 1976; Arieli et al. 1979; Hohtola 1982). Moreover, brown adipose tissue, the tissue responsible for much of the nonshivering thermogenesis in mammals, is absent in birds (Johnston 1971; however, see Oliphant (1983) for an opposing view). Furthermore, injection of noradrenaline (usually used to estimate the capacity to increase nonshivering heat production in mammals) either causes a decrease or has no effect on heat production in birds at low ambient temperature (Hart 1962; Hissa et al. 1975.) Thus, any discussion of thermogenesis in birds must focus on shivering by the skeletal muscles. In most species of birds, the muscles that move the wings form the largest muscle mass (Hartman 1961). Accordingly, most studies of shivering in birds have involved electromyographic measurements of activity in the pectoral muscles. However, most species of birds have a relatively large muscle mass in the legs when newly hatched. Some workers have argued that heat production by leg muscles may contribute substantially to total heat production in very young birds

(Ederstrom and Brumleve 1969; Hudson et al. 1974; Aulie 1 9 7 6 ~19766). , In the present study, We quantified shivering in the 1% and flight muscles in adult birds (Japanese quail) and show that heat production of leg muscles contributes substantially to total shivering themogenesis in this species-

Materials and methods AdultmaleJapanesequail (Coturnixcoturnixjaponica)werehoused indoors at 26°C in individual cages with a 16 h light : 8 h dark photoperiod for at least 4 weeks prior to experimentation. Food and water were supplied ad libitum. Food was withdrawn 12 h prior to experimentation to avoid effect of specific dynamic action. During each trial a single unrestrained bird was placed in an 8-L cyclindrical aluminum metabolic chamber (21 cm diameter. 23 cm deep). Chamber temperature was controlled with a water jacket and was monitored continuously with thermocouples. The chamber was insulated (10 cm polystyrene) and the temperature of the air entering the chamber was adjusted prior to going into the chamber. Oxygen uptake was measured using open-circuit respirometry. Dry C02-free air was drawn into the chamber, C 0 2 and water produced by the bird were removed, and the oxygen concentration in the outflow air was measured with an Applied Electrochemistry analyzer (model S-3A). Oxygen uptake was calculated from equations given by Hill (1972) modified for the case in which dry C02-free air flow rate is measured after passing through the respirometer. vo2 =

flow X (0.2094 - 0 2 out) 1 - 0.2094

where flow was the flow rate of dry C02-free air after passing through the respirometer and the sensor (600 mllmin). The intensity of shivering was estimated by measuring EMG's of the flight and leg muscles with pin electrodes. Differential signals were

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measured using three pin electrodes (made from 00 insect pins; diameter, 0.25 mm) spaced 3 mm apart and with 3 mm of uninsulated tips inserted into the muscle. One set of EMG electrodes was placed in the right flight (pectoralis) muscle 1 cm lateral to the keel and 1 cm posterior to the furcula and another in the right leg muscle just posterior to and midway along the femur. A band pass filter (Tektronix model 122)was used to reduce the interference below 80 Hz and above 1 kHz. The signals were displayed, stored, and analysed with a Nicolet digital oscilloscope. At each ambient temperature we took 16 samples, each 20 s long. For each sample we calculated the root mean square (RMS) in microvolts and used the average of the RMS for the 16 samples as the estimator of the intensity of shivering. Leads were directed posteriorly and did not interfere with the normal posture of the bird. For each trial, a bird was weighed, cloacal temperature was measured with a thermocouple, electrodes were attached, and the bird was placed into the dark chamber at a temperature of about 26°C. The chamber was closed and the bird was left 1 h to adjust. At the end of the hour, the 16 EMG samples were taken and 16 values of oxygen uptake were calculated for the same time. Temperature was then decreased about 5°C over a period of 10 min and the bird was left 50 min to adjust. The procedure was continued until ambient temperature was about 3.5"C. Only one bird was tested per day. After all six birds had been tested, they were killed for analysis of muscle weights. True body weight was measured as the body weight minus the weight of the intestinal contents. Flight muscles (pectoralis and supracoracoideus) and leg muscles (the entire mass of muscles originating from the pelvic bones, femur, and tibiotarsus) were removed bilaterally and were weighed. The muscles were freeze-dried, reweighed, and homogenized in a Wiley mill. The crude protein content of the muscles was estimated using a Kjeldahl nitrogen analysis (Horwitz 1980) and was used to indicate the content of contractile protein. For comparison, one trial was performed on a pigeon over the same range ambient temperatures with EMG recordings from both pectoral and leg muscles.

0

0

5

10 15 20 25 Ambient temperature (OC)

30

FIG. 1. The effect of ambient temperature on oxygen uptake in adult male Japanese quail. The line is a least-squares regression forced to intersect the abscissa at 40.8"C, the measured cloacal temperature. Data are from six quail.

Results Metabolic heat production There was a significant ( P < 0.001) linear increase in metabolic heat production as ambient temperature decreased from 26 to 3OC in all birds tested (Fig. 1). There are insufficient data at the higher temperatures to delineate exactly the lower critical temperature. The slope using the combined data from all . h- ."C- and has been calculated to birds is 0.14 mL 0 2gintersect the abscissa at the measured cloacal temperature (mean + SD, 40.8 ? 0.49"C, N = 17). Using a least-squares fit not forced to intersect the abscissa at 40.8OC yields an intercept of 43.1°C when all the data are used and an intercept of 38.7"C if the six points at the highest ambient temperatures are omitted. It is likely that the lower critical temperature was in the range of 24-28°C for these quail under these conditions.

'. '

'

Shivering There was a significant increase in the intensity of shivering in both the leg (EMG = 10.3 - 0.36Ta; SEE = 0.0646) and flight (EMG = 14.2 - 0.47Ta; SEE = 0.0795) muscles as ambient temperature (T,) decreased (Fig. 2). The slopes of these two lines were significantly different from zero (P = 0.001), but were not significantly different from one another ( P = 0.37). The intensity of shivering increased 6- to 10-fold as ambient temperature decreased from 26 to 3.5OC in both flight muscles (increase from 1.12 to 12.72 pV) and leg muscles (increase from 1.51 to 9.15 pV) . Peak to peak amplitude in the cold was 400-700 pV. The EMGs differed from many others reported in the literature for birds in that shivering occurred in bursts rather than continuously (Fig. 3) The duration of the burst was approxi-

0

5

10 15 20 25 Ambient temperature (OC)

30

FIG.2. The effect of ambient temperature on shivering EMG in adult male Japanese quail. Each point is the mean RMS value of 320 s of muscles EMG signal. The regression lines for leg (a)and flight (0) were not significantly different from one another and the line shown is the regression line for all data.

FIG. 3. Typical pattern of bursts in flight and leg muscles during shivering. Note the synchronous onset of bursts. The regular signal on the top trace is interference from the heart. The ambient temperature was 6°C.

STEVENS ET AL.

TABLE1 . Characteristics of the flight and leg muscles of the Japanese quail used in the present study

Muscle

Wet weight (g/ 100 g)

Dry weight (g/ 100 g)

Protein (mg/ 100 g)

13.8220.33 4.6220.13 18.4420.44

4.0920.09 1.2920.04 5.3820.12

53.0923.31 17.082 1.15 70.17

70.3820.46 72.0420.19

62.2522.38 65.7522.47

6.9320.13

2.0020.04

22.742 1.81

71.1620.28

57.7823.27

Water

Protein

(% wet weight)

(% dry weight)

-

Pectoralis Supracoracoideus Total flight Leg

NOTE: Values are means f SEM for six quail; mean (-CSEM) body weight was 161.5 2 17.0 g.

Tremor

muscles. Presumably, the fat was due to the ad libitum feeding 0 regime and to the inopportunity for much exercise in the cages.

Flight / EMG

FIG. 4. Typical pattern of bursts to show the synchrony of burst intensity between flight and leg muscles. The raw EMG signal was rectified and integrated (Grass polygraph integrator, model 7P10B). Tremor was measured with an accelerometer attachedto the EMG leads on the flight muscle. Note the strong positive correlation between the intensity of the integrated signal from the flight and the leg muscles. The ambient temperature was 6°C.

mately equal to the duration of the interval between bursts. As ambient temperature decreased, both the frequency and the intensity of bursts increased. The onset of a burst of shivering in the flight muscles was precisely synchronized with the onset of a burst in the leg muscles. However, within any particular burst, the peaks of the high-frequency electrical activity in the flight muscles were not correlated with those in the leg muscles (Fig. 4). The results for shivering in the pigeon were similar to those reported by Hohtola (1982) in that shivering was continuous rather than occurring in bursts and gradually increased in intensity as ambient temperature decreased. There was no measurable shivering in the leg muscles at any ambient temperature. Characteristics of muscles studied While the pectoral muscles were the largest combined muscle mass (18%), the mass of muscle in the thigh constituted a substantial portion (7%) of total body mass (Table I). There were small (but statistically significant) differences in water content and in protein content between the muscles studied. In each of the six birds examined, water content was greatest in the supracoracoideus and least in the pectoralis. Crude protein was significantly less in the leg muscle than in either flight muscle. The leg muscles appeared darker red than the flight muscles and there was more subcutaneous fat in the leg than in the flight

Discussion The concentratio~of protein in the different muscles studied were similar. The small differences in protein concentration reflect the fact that the leg muscles contained more fat than the flight muscles. It is also likely that the muscle fibers of the leg of these quail contained a relatively greater proportion of aerobic fibers than the flight muscles, especially since the birds were caged for some time. The important point for our present analysis is that the protein values are similar. Furthermore, the bulk of protein in muscle is contractile protein and, thus, its measurement can be used as an index of a particular muscle to produce heat by shivering. Thus, if we calculate the relative contribution of leg muscles (leg/(leg + total flight)) based on wet weight, the leg contributed 27% of total heat production. Based on protein content, the contribution was 24% of the total. Although the leg muscles of most birds are small, there are several species in addition to the quail that have relatively large muscles. In five species of grouse (Galliformes: Tetraoninae), the leg muscles account for 7- 11% of body weight (Thomas and Popko 1981;Thomas 1982; V .G. Thomas, unpublished). Leg muscles make up 9- 13% of the body weight of Canada geese (Branta canadensis) (Mainguy and Thomas 1985) and snow geese (Chen caerulescens) (Thomas 1983). Certain raptorial species exposed to northern winters have large leg muscle masses, such as red-tailed hawks (Buteo jamaicensis; 14- 16% body weight). Common loons (Gavia immer) are active underwater piscivores whose leg muscles make up 14% of body weight (V .G. Thomas, unpublished). In these and related species, the leg muscles may contribute to shivering heat production. In this regard, Ederstrom and Brumleve (1969) showed that the pheasant (Phasianus colchicus) has a vascular countercurrent heat exchanger that reduces heat loss below the knee where there is no insulation, while the muscles above the knee are maintained at a high temperature. They concluded that "since the pheasant is a cursorial bird with well-developed leg muscles, these may well be an important source of heat production." Many publications on shivering in birds report that shivering is continuous, but several report that shivering occurs in bursts: e.g., house wren (Troglodytes aedon) (Odum 1942), blackcapped chickadee (Parus atricapillus) (Chaplin 1976), and . et al. willow ptarmigan (Lagopus lagopus) (Aulie 1 9 7 6 ~ )West (1968) report that in evening grosbeaks (Hesperiphona vespertina) and common grackles (Quiscalus quiscula), shivering is continuous but fluctuates in intensity. Aulie (1976 b) generalized from two species to argue that bursts are characteristic of arctic birds. This is unlikely, however, because Hissa et al.

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(1983) reported that shivering is continuous in the common capercaillie, Tetrao urogallus. The bursts occurred at the same time in the leg muscles as in the flight muscles. However, the pattern of impulses within each burst was not identical between muscles, presumably because the specific motor units fired randomly depending on other synaptic inputs. The fact that the bursts in leg and flight muscle are synchronous raises some interesting questions regarding the control system. Although birds possess a temperature-regulation center in the hypothalamus, the current view is that the control of shivering may involve a spinal cord mechanism. For example, Gorke and Pierau (1979) observed no difference in the general shivering EMG pattern between normal and spinal pigeons. Moreover, shivering does not increase when hypothalamic temperature is decreased. Shivering heat, production depends on the number of motoneurons firing and their firing rate. It has been suggested that the motoneurons are progressively recruited in a manner similar to that for voluntary movements (i.e., progressive recruitment of larger motoneurons) (Klussman 1969). The excitability of motoneurons increases with a decrease in temperature and this in itself could provide partial regulation of shivering heat production. The intensity and pattern of shivering may be controlled by neural connections within the spinal cord, but can be influenced by supraspinal events; e.g., shivering ceases with the perception of any alarm stimulus. Thus, it seems reasonable to suggest that the pattern of shivering depends on neural networks within the spinal cord in birds, but that these can be inhibited by supraspinal inputs.

Acknowledgments We thank Dr. R. Hissa and Ben Rosser for comments at various stages of the project. The quail were donated by M . Wernaat of the Halton Region Conversation Authority. ARIELI, A., A. BERMAN, and A. MELTZER. 1979. Cold thermogenesis in the summer acclimatized and cold-acclimated domestic fowl. Comp. Biochem. Physiol. C, 63: 7-12. AULIE,A. 1976a. The pectoral muscles and the development of thermoregulation in chicks of willow ptarmigan (Lagopus lagopus). Comp. Biochem. Physiol. A, 53: 343-346. 19766. The shivering pattern in an arctic (willow ptarmigan) and a tropical bird (bantam hen). Comp. Biochem. Physiol. A, 53: 347-350. CALDER, W. A., and J. R. KING.1974. Thermal and caloric relations of birds. In Avian biology. Vol . 4. Edited by D. S . Farner and J . R . King. Academic Press, New York. pp. 260-4 13. CHAPLIN, S. B. 1976. The physiology of hypothermia in the blackcapped chickadee. J. Comp. Physiol. 112: 335-344. DAWSON, W. R., A. F. BENNETT, and J. W. HUDSON. 1976. Metabolism and thermoregulation in hatchling ring billed gulls. Condor, 78: 49-60.

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