metabolic responses to flight and fasting in night-migrating passerines

J Comp Physiol B (1991) 161:465-474 Journal of Comparat ive ~'~"~,'~176176

and Environ-

Physiology B " " Physiology

�9 Springer-Verlag 1991

Metabolic responses to flight and fasting in night-migrating passerines Susanne Jenni-Eiermann* and Lukas Jenni

Schweizerische Vogelwarte, CH 6204 Sempach, Switzerland

Accepted April 2, 1991

Summary. 1. Small passerine migrants achieve endurance flight while fasting, together with one of the highest mass-specific energy rates. Metabolic responses to flight and fasting were examined in three species of free-living migrants (Sylvia borin, Ficedula hypoleuca, Erithacus rubecula) by measuring plasma concentrations of glucose, uric acid, triglycerides, glycerol, free fatty acids (FFA), and fl-hydroxybutyrate 0G-OHB) in three main physiological situations (feeding, overnight fasting, nocturnal flight) and while changing between these situations.

2. Overnight-fasted birds showed low triglyceride and uric acid levels. Contrary to mammals, FFA and glycerol levels were not increased in agreement with published data on birds. The transition from feeding to fasting (post-feeding) was distinguished by a temporary rise in FFA and a drop in glucose levels.

3. Birds utilize fat during migratory flight, indicated by high levels of FFA, glycerol, and fl-OHB. For the first time, high triglyceride levels were found in an exercising vertebrate. The use of protein during flight was demon- strated by high uric acid levels.

4. Birds kept inactive after flight showed a more pro- nounced reduction of the fat and protein utilization and post-exercise ketosis than naturally landed birds.

5. Differences among the three species in the metabolic pattern suggest that the garden warbler shows the greatest metabolic adaption to endurance flight, having the highest levels of fat metabolites and the highest body fat reserves.

Key words: Bird flight - Fasting - Fat metabolism - Protein utilization - Bird migration

Introduction

Each year migrant birds fly to their winter quarters and back. In long-distance migrants this involves single j our-

Abbreviations: FAS overnight fasted ; FED feeding; FED30, FEDgO without food for 30 and 90 min, respectively; FFA free fatty acids; F L Y flying; FL Y30, FL }160 inactive for 30 and 60 min, respectively, after flight; L A N naturally landed after flight; f l-OHB fl-hydroxybutyrate

* To whom offprint requests should be sent

neys of 4000-10000 kin. Many migrants fly at night. Recoveries of ringed birds show that non-stop flight occurs during a whole night, or even longer when oceans have to be crossed. For small passerine migrant birds this is an astonishing physical performance, because they achieve endurance locomotion while fasting, together with one of the highest mass-specific energy requirements. The minimum power input during flight is 8-12 times resting metabolic rate [reviewed in Masman and Klaassen (1987)] and 2.3 times the maximum rate of exercising small mammals (Butler and Woakes 1990).

Some metabolic adaptations to migration are known. The most obvious is the deposition of large fat reserves which may account for 50 % of live body mass. The flight muscles double their ability to oxidize fatty acids (Marsh 1981) by switching from a high glycolytic and anaerobic capacity to a high oxidative capacity (Lundgren and Kiessling 1985, 1986). The respiratory quotient of birds flying in wind tunnels is nearly 0.7, indicating that fat is the main fuel [reviewed in Rothe et al. (1987)]. The high mass-specific energy content and the production of water during oxidation affirm the advantage of fat as an energy reserve compared with protein and carbohydrate. How- ever, it is not clear how a sufficient energy supply for the flight muscles is achieved during long-lasting flights. From marathon runners it is known that the fat supply from adipose tissues to the muscles is a limiting factor (Paul 1975; Guppy 1988; Newsholme 1988).

Certainly, fat provides most of the energy during flight (Ramenofsky 1990). Only recently, studies on body composition have indicated the importance of protein during migration. An increase in lean dry body mass is observed before long migrations (Piersma 1990; Piersma and Jukema 1990) and related in part to hypertrophy of the flight muscles [reviewed in Ramenofsky (1990)]. This could be a response to the higher body mass (fat reserves) to be transported (Fry et al. 1972; Marsh 1984), but it might also serve as protein reserve (Evans 1972; Ward and Jones 1977; Piersma 1990). However, until now protein utilization during flight has not been measured directly.

Changes in carbohydrate metabolism and stores during the migratory season are equivocal [reviewed in Berthold (1975) and Marsh (1983)], yet it is agreed that glycogen is quantitatively unimportant as energy storage for long-distance flight (Marsh 1983, 1984).

466 S. Jenni-Eiermann and L. Jenni: Metabolic responses to flight and fasting in birds

The aim of this study was to examine direct responses of the fat, protein, and carbohydrate metabolism in free- living small night migrants to the three main physiological situations which dominate the migratory period: (1) endurance flight, (2) feeding, and (3) overnight fasting during stop-overs. Each situation presents different combinations of activity and nutritional status. Therefore, it was possible to define the metabolic responses specific to flight and to investigate the problems of fuel supply and protein utilization during endurance flight. Transitions between all three situations were used to study how the metabolism switches from one situation to the other. Finally, three species of migrants were compared in order to investigate whether there might be different degrees of adaptations to migration.

Materials and methods

Animals. The studies were carried out on bird ringing stations on the Alpine pass Col de Bretolet and at Lake NeucMtel, Switzerland. Free-living garden warblers (Sylvia borin), robins (Erithaeus rubeeula), and pied flycatchers (Fieedula hypoleuca) were caught in mist nets during their autumn migration periods in 1986, 1987, and 1988 at both sites. Only birds which had finished their juvenile or post-breeding moult and were thus potentially ready tbr migration (Jenni 1984) were selected,

On Col de Bretolet birds were caught (1) during the night on their migratory flight mainly in high nets (up to 8 m above ground) and (2) around dawn while resting and beginning to feed mainly in low nets (up to 2 m above ground) placed in bushes (Jenni 1984). These latter birds were all migrating the night before since recaptures of ringed birds indicating a stay of more than 1 day hardly occur. At Lake Neuchfitel, birds were caught only during the day while resting and feeding; frequent recaptures indicate that most birds stayed for more than one day.

Physiological situations. The birds flying at night (FLY) combine two physiological states: activity and fasting. Since we were interest- ed in the responses specific to flight. We compared the FLY sample with free-living birds actively feeding during the day (FED) and with birds kept inactive and without food for 10-12 h overnight and blood-sampled before dawn (FAS) (Fig. 1). The latter two samples were taken during the migratory season but from birds resting for several days. In order to corroborate observations the metabolic changes (a) from FED to FAS, (b) from FLY to FAS, and (c) from FLY to FED were also studied, (a) by keeping FED birds inactive and without food for 30 rain (FED30) or 90 120 min (FED90) and (b) by keeping FLY birds inactive for 30 min (FLY30) or 60 min (FLY60); study of (c) was possible because robins caught within 20 min around dawn at the Alpine pass were all migrating the previous night and interrupted migration naturally at dawn to rest and feed (LAN). Their metabolic pattern, therefore, represents the changes from FLY or FLY30 towards the FED situation. (b) and (c) allowed the study of metabolic changes after flight under two different conditions: no activity and fasting (b), activity and onset of feeding (c).

Since the number of caPtures differed between the species and the study sites, not all situations could be controlled for the three species. Situations FLY, FLY60, FAS, FED, and FED90 were examined for all three species, FLY30 for the pied flycatcher, FED30 for the garden warbler, and LAN for the robin only.

Blood sampling. Blood was collected as quickly as possible after the bird flew into the mist net for the situations FLY, FED, and LAN. The time lapse from the moment the bird flew into the net to the termination of blood sampling ranged between 3 and 15 min for the night captures (FLY) and 3-30 rain for daylight captures. Birds from situations FLY30, FLY60, FED30, FED90, and FAS were

\ 6r

\ ffr

Fig. 1. Schematic outline of the physiological situations. FLY = birds caught during active night migration on the Alpine pass Col d~ Bretolet; FLY30 and FLY60 = FLY birds kept inactive and blood- sampled after 30 and 60 rain, respectively; FED = birds caught at Lake Neuchfitel during the day while resting and feeding; FED30 and FED90 = FED birds kept inactive and blood-sampled after 30 and 90-120 rain, respectively; FAS=bi rds from Lake Neuchfitel caught in the afternoon, kept overnight, and blood-sampled before dawn; LAN = birds caught on Col de Bretolet within 20 rain around dawn after landing from a migratory flight the previous night. For further informations see text

kept singly in cotton bags and blood was sampled at a predeter- mined time after capture according to the experiment. Of some FED garden warblers and robins, a second blood sample was collected 90 rain later (FED90) or the next morning before dawn (FAS).

Blood was sampled by puncturing the vena ulnaris and the blood drops were collected with a capillary system (Microvette R CB300 Fluore, Sarstedt). All birds were later released. On average, 71-73 gl (garden warbler, robin) or 40 gl (pied flycatcher) of blood was collected. The blood was centrifuged and the plasma stored at - 2 0 ~ until analysis.

Experimentalprocedures. As the amount of blood collected varied, not all metabolites could be determined in all individuals.

Glucose was determined in hemolyzed blood by the clinical laboratory of the Kantonsspital Basel using a standard enzymatic UV-test. The values were expressed in plasma concentrations by taking account of different hematocrits according to species and physiological situation (unpublished data). All other metabolites were determined in the plasma in our institute using standard test combinations modified for small amounts of plasma (3-10 gl per determination): enzymatic UV-tests for glycerol (Boehringer Mannheim) and fl-hydroxybutyrate (Sigma Diagnostics), enzymatic colorimetric tests for uric acid, free fatty acids (Boehringer Mann- helm), and triglycerides including free glycerol (Roche Diagnostica, Basel).

Effects of diurnal variation. Glucose, uric acid, and triglyceride levels of samples from FED birds of all species showed a significant steep increase in the morning, which leveled off during the day depending on the metabolite and the species (unpublished data). In order to get a sample of feeding birds as homogeneous as possible, data were selected from the stabilizing period.

Effects of time span between capture and blood sampling. In FED, FLY, and LAN birds, no significant (P > 0.1) linear or higher order dependence of any metabolite level on time lapse after capture could be detected. The only exception were the triglyeeride levels of the FLY garden warblers which decreased significantly with time lapse after capture. This effect is discussed in the Results section.

Statistics. For each species and each metabolite, the means within four groups of physiological situations were compared (planned

S. Jenni-Eiermann and L. Jenni: Metabolic responses to flight and fasting in birds 467

comparisons): (1) the means of the three main situations FLY, FED, FAS, (2) the means of the transitional situations FED, FED30, and FED90, and (3) FLY, FLY30, and FLY60. (4) The 15- transitional situation LAN was compared with the FLY and FLY60 samples. 12-

In all samples, no significant deviation from a normal distribu- tion could be detected (Kolmogorov-Smirnov goodness-of-fit test). 9- Samples of a group, of which the variances were considered horn- 6- ogeneous [Fm,~-test , P>0.05; Sokal and Rohlf (1981)], were tested by ANOVA and, if significant, further analyzed by comparing each 3- mean value with each other [planned non-orthogonal comparisons; Sokal and Rohlf (1981)]. Samples of a group of which the variances 0 were heterogeneous were tested for differences by the non-parame- tric Kruskal-Wallis test; if significant, each mean was compared with the others by the approximate test of equality of means (Sokal 15- and Rohlf 1981). In FLY pied flycatchers uric acid and glycerol levels changed significantly in the course of the night, and in FED -~ 12- robins FFA increased significantly with the season. This was taken E into account by procedures explained in the Results section. Data E 9- obtained from the same individual were compared by the t-test for paired comparisons, o 6-

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Results

Plasma metabolite levels in different physiological situations

Glucose. Compared with the F E D situation, the glucose level was significantly reduced in the FLY samples of pied flycatcher and garden warbler (Fig. 2). In the robin, almost no differences existed between the three main situations FLY, FAS, and FED. The high glucose level of the F E D birds decreased when they were fasted for 90-120 min.

Compared with F L Y birds, FLY60 pied flycatchers showed a significantly reduced glucose level. The glucose level of LAN robins was significantly lower than that o f the FLY sample, but not compared with the FLY60 sample.

Uric acid. Among the three main situations, FAS had lower uric acid concentrations than both F L Y and F E D birds (Fig. 3). The levels in FLY and F E D samples did not differ significantly, but F L Y garden warblers had a higher uric acid level ( P = 0.10) than the F E D birds.

The change f rom the F E D to the FED90 situation was not uniform among the species. In the robin a decrease occurred, whereas in the other species no significant changes were observed.

Uric acid levels of flying birds dropped with resting in all species, but significantly only in the pied flycatcher. In this species, uric acid levels of F L Y birds decreased significantly during the night. However, FLY30 and FLY60 birds were predominant ly sampled during the second part of the night; accounting for this decrease by ANCOVA, FLY30 and FLY60 birds still had uric acid levels 0.53 and 0.31 m m o l . 1-1, respectively, lower than the F L Y birds (P<0.001) .

The values of the L A N robins did not differ from the FLY sample nor f rom the FLY60 sample.

Triylyceride. The triglyceride levels (Fig. 4) showed a pattern similar to the uric acid levels. The mean values of the FAS samples were significantly lower than those

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of F E D and F L Y birds. In the garden warbler, the mean value of the F L Y sample was significantly higher than that of the FED. In the pied flycatcher and the robin, however, the values for F E D and F L Y were similar. In all species, FED90 birds showed decreased triglyceride levels like the FAS birds.

Triglyceride levels o f F L Y garden warblers were about 60% higher than in the other two species. This might be the reason why triglyceride levels decreased significantly with time lapse after capture (Fig. 5). This decrease continued to the FLY60 sample. Therefore, triglyceride levels of F L Y garden warblers were corrected according to the relationship shown in Fig. 5, to the value at 5 min after capture. The mean of these corrected values is shown in Fig. 4. A similar decrease within 15 rain after capture was not found in FLY robins and pied flycatchers, but FLY60 samples were also significantly lower.


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Fig. 3. Mean uric acid concentrations. See legend to Fig. 2 for details

LAN robins had a significantly lower triglyceride level than FLY and FLY60 samples.

The triglyceride values reported here include free glycerol. It was not possible to deduct glycerol for every individual since triglyceride and glycerol were determined concomitantly in only about half of the individuals. Thus, the mean glycerol levels were subtracted from the mean triglyceride values. The pattern remained approximately the same (Fig. 4).

Glycerol. Glycerol levels changed little (Fig. 6), with only FL Y birds showing elevated mean values.

The increased values of the FLY samples disappeared with resting: FLY30 and FLY60 birds showed low glycerol levels. The FLY30 and FLY60 pied flycatchers were sampled predominantly during the second half of the night, but a significant increase of glycerol occurred in F LY birds during the night. When correcting for this diurnal trend by ANCOVA, the difference between F LY and FLY30 samples was still the same, but the difference between FLY and FLY60 samples increased from 0.21

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s. Jenni-Eiermann and L. Jenni: Metabolic responses to flight and fasting in birds 469

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to 0.30 mmol �9 1 - t (ANCOVA, P<0.001) . The values of LAN robins were as high as in the FLY sample.

Free fatty acids (FFA). As with glycerol levels, FFA concentrations were higher in the FLY samples than in FED and FAS situations (Fig. 7). The F F A level increased post-feeding (FEDg0). In FED robins, F F A levels increased significantly over the season, but FED90 birds were sampled during the first part of the migratory season only; when comparing the common part of the season only, the difference between FED and FED90 birds became significant ( ~ + S D , FED: 0.94+_ 0.27 mmol �9 1-1 ; n = 17; FED90: 1.44 + 0.47 mmol- 1 - 1, n = 8 ; ANOVA, P=0.002) .

The elevated level of the FLY samples decreased with resting in both species examined. The LAN robins had a similar F F A level as FLY birds, but a significantly higher level than the FLY60 sampte.

fl-Hydroxybutyrate (fl-OHB). This was the metabolite which changed most dramatically (Fig. 8). It was low for

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FED, high for FLY, and very high for FAS samples of all species. A level similar to that of FAS birds (or even higher in the robin) was reached after 90 min of fasting.

The /? -OHB levels of the FLY30 and FLY60 birds increased sharply: they were even higher than those of the FAS and FED90 samples. This increase was also observed in LAN robins: they had a significantly higher fl-OHB level than during flight, but FLY60 birds had an even higher level.

Changes from FED to FED90 and FAS situations in the same individual

Changes in metabolite levels in the same individual showed a close correspondence with the data from the preceeding section (Table 1): All differences between FED and FEDg0 or FAS blood samples had the same sign as the values from the preceding section, except in two cases where the differences were very small and non-significant. The dimensions of the differences, however, did not always correspond with those of the preced-


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ing section. This migh t p a r t l y be due to the smal l s ample sizes. Howeve r , m o s t species-specific charac ter i s t ics were r epea ted in the ind iv idua l s s amp led twice, i.e., the h igher increase o f F F A a n d f l -OHB in the F E D g 0 rob ins than in the o the r species a n d the la rger uric ac id decrease o f F E D 9 0 and F A S robins .

Differences in metabolite levels among species

In the F L Y s i tua t ion , the levels o f all me tabo l i t e s differed a m o n g species (Table 2). The ga rden warb le r h a d the h ighes t values o f the three fat ca tabo l i t e s and t r igly- cerides, and the lowest g lucose a n d ur ic ac id levels o f all species du r ing flight. The r o b i n showed exact ly the op- pos i te p a t t e r n a n d the p i ed f lyca tcher r anged in be tween.

In the F A S s i tua t ion , no differences be tween species were a p p a r e n t , bu t some differences were de tec ted in the F E D s i tua t ion .

Table 1. Mean differences (d_ SD) in metabolite levels between FED and FED90 as well as between FED and FAS values sampled on the same individuals. *P<0.05, **P<0.01 for significant differences (paired t-test). For comparison, the differences (D) between the mean values of the corresponding situations from Figs. 2, 3, 4, 6, 7, and 8 are given

FED-FED90 FED-FAS

D d SD (n) D d SD (n)

Glucose G. warbler 1.12 1.25" ,++1.22 (8) Robin 1.34" 2.00* ,++1.52 (6) -0.31 -0.80 -++2.83 (4) Uric acid G. warbler 0.00 0.11 • (8) 0.29** 0.59** -/-0.22 (5) Robin 0.53** 0.21 _++0.25 (7) 0.75** 0.92 _+0.41 (3) Triglycerides G. warbler 1.37" 0.35* _+0.28 (6) 1.45" 1.56" __1.00 (6) Robin 2.27** 0.35* • (6) 1.85" 1.11; 1.66 (2) Glycerol G. warbler 0.03 0.02 ,++0.14 (9) 0.05 0.08 ___0.37 (5) Robin 0.03 -0.07 _+0.15 (5) 0.00 0.05 _+0.42 (5)

FFA G. warbler -0.22 -0.12 +_.0.44 (7) -0.05 -0.02 +0.18 (4) Robin -0.50**-0.63* +_,0.28 (4) 0.17 -0.07 _+1.04 (3) ~-OHB G. warbler -1.94"*-1.50"* ___0.26 (5) -2.01"* -I.83" +1.06 (5) Robin -4.03**-3.87** ,++1.70 (7) --2.18"* --1.69" _+1.35 (5)

Table 2. Species arranged from the smallest to the largest mean metabolite concentration. The values and the number of individuals are the same as those in Figs. 2, 3, 4, 6, 7, and 8. The means were tested for differences between species by ANOVA and further by planned non-orthogonal comparisons. Species separated by a com- ma are not significantly different from each other. Species to the left of the < sign are all significantly different from those to the right of the < sign (P < 0.05). Species in parentheses are not significantly different from any other species. G=garden warbler, P=pied flycatcher, R = robin

Situations

FLY FAS FED

Glucose G < P < R G, R, P R < G , P Uric acid G < R , P G, R, P G < P < R Triglycerides R, P < G P, R, G P, G, R Glycerol R<P , G G, R, P G, P, R FFA R < G, P G, R, P G(P) < R fl-OgB P(R)<G R, G, P R, G, P

Discussion

As far as is known, this is the first s tudy to inves t iga te p l a s m a me tabo l i t e s in free- l iving flying migran t s . F e w studies a re ava i lab le on cap t ive fas t ing passer ines [see Swain (1987)] and on ly scarce d a t a on glucose levels i m m e d i a t e l y ( G r o e b b e l s 1930) or some t ime (Fre l in 1974) af ter flight.

P l a s m a me tabo l i t e concen t r a t i ons c a n n o t genera l ly be equa ted wi th m e t a b o l i t e tu rnover . However , s tudies in h u m a n s and m a m m a l s ind ica te this for p l a s m a g lycerol and F F A concen t r a t i ons (Scow and Che rn i ck 1970; Hur - ley et al. 1986) and s tudies in b i rds for uric ac id (Mor i and G e o r g e 1978). F o r these metabo l i t e s , p l a s m a concen t r a t ions were a s sumed to be re la ted to tu rnover .


The following discussion deals with the metabolic responses to fasting, flight, and rest after flight. The feeding birds are merely used for comparison and will be discussed elsewhere.

Metabolic responses to fastin 9

In small insectivorous and omnivorous passerines, in- sects and fruits pass through the alimentary canal within about 0.5-2h (Stevenson 1933; Jordano 1987). The metabolic responses of the 0.5-2 h-fasted birds in our study, therefore, represent the transition from a fed to a fasted state.

The changes in metabolite levels of individual feeding birds sampled a second time 90 min or 10-12 h later in a fasted situation were in agreement with the differences in the respective mean values of birds sampled only once (Table 1). This indicates that the changes in the mean values are mainly due to the different physiological situation and not to other factors associated with individual differences or the experimental procedure.

Fat metabolism. Most triglycerides found postprandially in the blood originate directly or via synthesis in the liver from the diet and are transported to the peripheral tissues (Robinson 1970; Havel 1987). The reduction of plasma triglyceride levels within 30-90 rain (Fig. 4) indicates that the balance between entry into and removal from the blood changes, i.e., by a reduction of triglyceride release or a comparatively larger uptake by peripheral tissues. Overnight, the triglyceride levels were low, as found in other studies (Swain 1987; Marsh 1983). In gull chickens and male quails fasted for 2-4 days, however, only slight decreases in triglyceride levels have been found (Didier et al. 1983; Jeffrey et al. 1985).

During fasts triglycerides of the fat deposits are hy- drolysed to glycerol and FFA and released into the blood. Glycerol levels in the plasma are regarded as being proportional to glycerol turnover and FFA plasma levels proportional to FFA release and oxidation (Scow and Chernick 1970; Paul 1975; Dohm et al. 1986; Hurley et al. 1986; Elia et al. 1987; Billow 1988). In this study the plasma glycerol levels remained stable and the FFA concentrations were elevated only during 90-120 min of fasting and returned in overnight fasted birds to the FED level (Fig. 7). Assuming that FFA and glycerol plasma concentrations reflect turnover, it seems that our fasted birds did not mobilize their fat deposits to a greater extent during an overnight fast. Similar effects were found by other studies: In overnight fasted Vesper Spar- rows, Pooecetes gramineus, no significant changes in FFA levels were found (Swain 1987). In long-term fasting geese, glycerol levels remained at prefasting levels and FFA levels returned to prefasting levels within a few days of fasting (Le Maho et al. 1981). In other studies examin- ing fasting periods of more than 1 day, no changes (Mig- liorini et al. 1973; Didier et al. 1983) and higher FFA levels have been reported (Didier et al. 1983; Jeffrey et al. 1985; Cherel and Le Maho 1988; Cherel et al. 1988a, b).

Protein metabolism. Uric acid is the principal endproduct of nitrogen metabolism and is considered to be an indica- tor of protein catabolism (Mori and George 1978; Cherel et al. 1988a; Robin et al. 1988). The high levels of uric acid during feeding (Fig. 3) indicate the use of diet protein for energetic needs or transformation into other compounds, e.g., fat. Uric acid levels were reduced after 90 min of fasting in the robin and after an overnight fast in all species (Fig. 3). This indicates a low protein utilization during this normal fasting period. A similar drop in plasma uric acid was found in long-term fasting penguins (Cherel et al. 1988b). Only towards the end of a long starvation period did uric acid levels increase again, indicating the beginning of the critical final stage of starvation (e.g., Jeffrey et alo 1985; Cherel et al. 1988a, b).

Glucose and fi~OHB. Plasma glucose levels in birds are higher than in most mammals and are reported to be constant even under prolonged fasting despite low glycogen stores (Migliorini et al. 1973; Langslow 1978; Veiga et al. 1978; Le Maho et al. 1981 ; Didier et al. 1983 ; Jeffrey et al. 1985; Cherel and Le Maho 1988; Cherel et al. 1988a). However, other studies reported significantly decreased glucose levels during fasting (e.g., Veiga et al. 1978; Swain 1987; Cherel et al. 1988b). In this study, glucose levels decreased significantly after 90 min of fasting in the pied flycatcher and the robin but regained the initial values of the FED birds overnight (Fig. 2). The conflicting results on glucose homeostasis in fasting birds reported in the literature could be due to a relatively large individual variation (Swain 1987) or diet (e.g., Migliorini et al. 1973).

The fl-OHB level is a mirror image of the glucose level (Fig. 8). fl-OHB is synthesized predominantly during fasting from FFA and replaces part of the glucose, es- pecially in the brain (Robinson and Williamson 1980). The increase of/?-OHB during fasting was therefore expected and coincides with other studies (Scow and Chernick 1970; Cherel and Le Maho 1988; Elia et al. 1987; Cherel et al. 1988a).

Conclusions. Overnight fasted birds are distinct from both feeding and flying birds by reduced levels of fat and protein metabolites (except fl-OHB), in agreement with other bird studies. Hc,wever, in mammals fasting causes a rise in plasma FFA and glycerol concentrations (Scow and Chernick 1970; Elia et al. 1987). Further investiga- tions in birds are needed to show to what extent reduced energy needs [resting metabolism is reduced by 25% during the night; Aschoff and Pohl (1970)] and other energy sources (e.g., glycogen stores or triglycerides stored in the tissues of utilization) are responsible for the low fat metabolites in the plasma during overnight fasting, or whether the assumed relationship between plasma concentration and turnover does not hold in birds. In long-term fasting birds, lipids contribute up to 94% of the total energy expenditure (Cherel et al. 1988a); in overnight-fasting passerines, however, no data on the relative substrate utilization are available. In migrating birds, it was suggested that fat stores are saved during overnight fasting at the expense of glycogen (Pilo and George 1983).


The transition from feeding to fasting is distinguished by a temporary rise of FFA (and fl-OHB in robins) and a drop in glucose levels. Thus, the metabolic reaction to fasting in small passerines shows two similar phases within 10 h as in long-term fasting geese and penguins over several days (Cherel et al. 1988a).

Metabolic responses to flight

Fat metabolism. Mammals during sustained fasting and submaximal exercise show increased plasma levels of glycerol and FFA (Paul 1975; Hurley et al. 1986; F6ry and Balasse 1986; Dohm et al. 1986; Wolfe et al. 1990). The FFA are usually oxidized by the tissues (except the brain) and glycerol may be utilized in gluconeogenesis and contribute to replace glucose (Langslow 1978). From this it was to be expected that FFA and glycerol levels in our migrants would be highest during flight (Figs. 6 and 7) since fat is the main energy supply (Rothe et al. 1987). Levels of FFA also increased during treadmill exercise in the fowl (Brackenbury 1984).

The extremely high triglyceride levels of flying birds were a surprising result (Fig. 4). In mammals, prolonged heavy exercise provokes a fall in plasma triglyceride levels while moderate work does not change them (Ro- binson 1970; Keul 1975; Liesen et al. 1975; Paul 1975; Billow 1988; Lamon-Flava et al. 1989). In mammals, it is known that the triglycerides present during fasting originate from plasma FFA which enter the liver and are reesterified to triglycerides and subsequently delivered into the blood as very low density lipoproteins (Robin- son 1970; Wolfe 1982; Elia et al. 1987; Havel 1987; Wolfe et al. 1990).

Protein utilization. The high uric acid concentration during flight (Fig. 3) indicates an increased breakdown of nitrogenous substances (e.g., amino acids, purine nu- cleotides) as shown for exercising humans (Cerny 1975; Poortmans 1988; Sahlin and Katz 1988). This may be due to the higher activity of the muscles and concomitantly a higher protein and purine nucleotide turnover leading to a negative nitrogen balance (Poortmans 1988). The breakdown of protein delivers amino acids which can be oxidized or used as glucogenic precursors (Cerny 1975; Veiga et al. 1978; Dolny and Lemon 1988; Poort- mans 1988). The latter may contribute through gluconeogenesis to glucose homeostasis or they may be necessary for the breakdown of FFA by supporting the citrate cycle with its intermediates (Cerny 1975). The catabolism of proteins could therefore be essential for the oxidation of FFA (Gorman and Milne 1971).

fl~OHB and 9lucose. In fasted humans, the level of /~-OHB is the same pre-exercise and during exercise after an initial decrease at the beginning of exercise (F6ry and Balasse 1986; Hurley et al. 1986). In flying birds, however, an even lower /~-OHB level was observed than during overnight fasting (Fig. 8). Ketone bodies are known to reduce FFA release from adipose tissue in humans and dogs (Robinson and Williamson 1980). This could be a reason for the comparatively low ]?-OHB levels in flying birds and it would emphasize the role of glycerol and protein for gluconeogenesis and triglyceride resynthesis.

Glucose levels are decreased in flying garden warblers and pied flycatchers, but kept constant in flying robins (Fig. 2). Brackenbury (1984) also found decreasing glucose levels during treadmill exercise in the fowl. In fasting exercising humans, glucose levels are higher, stable, or decreasing during exercise (Dohm et al. 1986; Hurley et al. 1986; Wolfe et al. 1990).

Conclusions. During endurance flight, the metabolic pattern is characterized by high levels of all fat metabolites. This is in accordance with the finding that fat is quantitatively the main energy source during flight (Rothe et al. 1987). For the first time, high triglyceride levels have been found in an exercising vertebrate (Fig. 4). This might indicate an additional mechanism of fat supply during endurance flight which will be discussed elsewhere.

This study documents an increased protein utilization during flight (Fig. 3). Variation in lean dry mass found in birds during winter (Reinecke et al. 1982; Jenni and Jenni-Eiermann 1987) and migration (Fry et al. 1972; Marsh 1981, 1984; Piersma 1990; Piersma and Jukema 1990) suggested that proteins are utilized during fasting. In long-term fasting birds, protein is the factor limiting fasting duration (Cherel et al. 1988a).

However, it remains open to what extent protein utilization is metabolically essential during endurance flights or whether the flight muscles are reduced concomitantly with the mass (fat) loss in order to adapt continuously to the decreasing energy needs and keep the flight muscles close to maximum efficiency (Pennycuik 1975). Since not only the flight muscles but also other non-fat parts of the body vary in mass, protein breakdown might be necessary during endurance flights (Pier- sma 1990). If so, flight muscle hypertrophy during migration (Fry et al. 1972; Marsh 1981, 1983) would not only" satisfy increased power requirements because of the increased body mass (Fry et al. 1972; Marsh 1984) but also serve as a protein store (Evans 1972; Ward and Jones 1977).

The reserves of a small passerine before crossing the Sahara contain 70% fat and 30% lean mass (Biebach 1990); their contribution in energetic terms, however, is 94 % and 6 %, respectively (calculations after Zwarts et al. 1990). Thus, a small but necessary contribution of protein needs a comparatively large non-fat mass to be carried. Further studies investigating the metabolic role of proteins are needed in order to show whether protein could limit flight duration.

Metabolic responses after flight

Changes from the flying to the fastin 9 situation. The observed drop of FFA, glycerol, triglyceride, and uric acid levels (Figs. 3, 4, 6, and 7) in birds kept inactive for 60 rain after flight shows that fat and protein utilization decrease sharply. The fl-OHB level, however, increases in parallel to a decrease in glucose level (Figs. 2 and 8). This postexercise ketosis also occurs in humans (Robinson and Williamson 1980; F6ry and Balasse 1983; Dohm et al. 1986). Possible explanations for this/q-OHB rise are that the remaining reduced fat catabolism is diverted mainly to ketogenesis in order to maintain the combined


level of glucose plus/~-OHB and to decrease fat mobilization f rom adipose tissues (Robinson and Williamson 1980); or that fl-OHB inhibits glycolysis, thereby induc- ing replenishment of muscle glycogen stores (F6ry and Balasse 1983).

Changes from the flyin9 to the feeding situation. Robins end their nocturnal flight naturally at dawn and land at the Alpine pass. This presented the possibility of examin- ing the natural metabolic changes f rom endurance flight into the feeding phase.

Robins just landed showed a similar F F A and glycerol level as the flying robins (Figs. 6 and 7), but a decreased triglyceride level (Fig. 4). This could indicate that fat utilization is reduced mainly through a reduction of F F A resynthesis into triglycerides. The uric acid level is similar in the two samples (Fig. 3), indicating that protein utilization after flight continues to be high or that it merges into the feeding situation.

Conclusions. The metabol ism of birds kept inactive after flight adapts quickly to the new situation by reducing fat and protein utilization. The metabolic pat tern is inter- mediate between the flying and the overnight fasted birds with the exception of a high p -OHB and a low glucose level. Birds landing naturally at dawn do not reduce protein utilization, decrease fat catabolism only partially, and increase fl-OHB level less than birds kept inactive (Figs. 3, 4, 6, 7, and 8). This could be explained by the fact that birds after landing show some activity [e.g., dispersion into feeding areas; Bruderer and Jenni (1988)] which needs energy not yet provided by feeding. A cogent reason for landing and resting is not apparent in our data, the more so since landed birds continue locomotion.

Differences among species

Overnight-fasted birds showed similar metabolite levels among species (Table 2). The reason for this might be that these birds were all kept under the same condition for about 10-12 h. Therefore, differences due to diurnal feeding activity, diet composition, or food utilization are reduced. Such reasons could be responsible for the differences among species in F E D birds, e.g., diet protein content on uric acid level.

In flying birds, species differed markedly in metabolic pattern. The garden warbler, characterized by a high fat catabolism, a low protein utilization, and a low glucose level, seems to present the greatest metabolic adaptat ions to endurance flight, relying most strongly on fat reserves. Concomitantly, garden warblers, which winter south of the Sahara, migrate through Switzerland with the largest fat reserves (unpublished data). Robins showed an op- posite metabolic pattern, seeming to rely less on fat and more on glucose and protein during flight. Wintering in the Mediterranean area, they cross Switzerland with the lowest fat reserves of the three species. Pied flycatchers ranged in between regarding both the metabolic pat tern and fat reserves.

Further studies are needed to show whether this suggested relationship between fat reserves and fat supply during flight among species can be corroborated and

whether it is indicative of different flight strategies, e.g., flight durat ion during one night.

Acknowledgements. We thank U. Andres, Kantonsspital Basel, for his continuous interest, for generously providing materials, and for carrying out the glucose determinations; H.P. yon Hahn, Stiftung fiir experimentelle Alternsforschung, and Ciba-Geigy AG for giving us laboratory equipment; all persons helping at the ringing stations and enduring our special wishes; R. Schwilch for her help during the field work; P. Goeldlin for making the hut of the Mus6e Zoo- logique de Lausanne on Col de Bretolet available to us; Y. Cherel, R. Groscolas, Y. Le Maho, S. M6rikofer-Zwez, and J.-P. Robin for valuable comments on an earlier draft of the manuscript.

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metabolic responses to flight and fasting in night-migrating passerines

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