J Ornithol (2006) 147:531–542 DOI 10.1007/s10336-006-0076-2

O R I G I N A L A RT I C L E

Effect of endurance flight on haematocrit in migrating birds Lukas Jenni Æ Susanne Mu¨ller Æ Fernando Spina Æ ˚ ke Lindstro¨m Anders Kvist Æ A

Received: 30 August 2005 / Revised: 31 January 2006 / Accepted: 9 April 2006 / Published online: 5 August 2006
Dt. Ornithologen-Gesellschaft e.V. 2006

Abstract The effects of an endurance flight on the haematocrit, the percentage of packed red blood cells per blood volume, were examined within the framework of six possible factors explaining possible changes in the haematocrit. Two approaches were adopted: (1) the haematocrit was studied in four species of passerine birds which landed on an Italian island after having crossed the Mediterranean Sea on their spring migration in a non-stop flight; (2) the haematocrit was evaluated in six individual red knots after a flight of 1, 2, 4 and 10 h in a wind tunnel and the data thus obtained compared with data on resting birds with or without food. In the four passerine species, the haematocrit decreased from 51% in fat birds to 48% in lean birds. In lean birds, the haematocrit dropped from 48% in birds with well-developed breast muscles to 36% in birds with emaciated breast muscles. In the red

Communicated by R. Holberton L. Jenni (&) Æ S. Mu¨ller Swiss Ornithological Institute, 6204 Sempach, Switzerland e-mail: [email protected] F. Spina Istituto Nazionale per la Fauna Selvatica, 40064 Ozzano dell’Emilia, Bologna, Italy ˚ ke Lindstro¨m A. Kvist Æ A Department of Animal Ecology, Ecology Building, 223 62 Lund, Sweden Present Address: S. Mu¨ller Du¨rrmattweg 18, 4144 Arlesheim, Switzerland

knots, the haematocrit was dependent on body mass in flying and resting birds. The haematocrit decreased from about 51% pre-flight to about 49% within 1 h of flight and remained at this level for up to 10 h of flight. Taking the results from the passerines and the red knots together, it seems that the haematocrit drops by a few percentage points within 1 h after the onset of flight, decreases very slowly with decreasing body mass and decreases more steeply in very lean birds having entered stage III of fasting. This indicates that dehydration is not an underlying factor in decreased haematocrit because if this were the case we would expect an increase with endurance flight. We found no effect of the presence of blood parasites on haematocrit. With the onset of flight, haemodilution may be adaptive, because it reduces blood viscosity and, thereby, energy expenditure by the heart, or it may be a sign of water conservation as an insurance against the risk of dehydration during long non-stop flights. During endurance flight, a reduction in the haematocrit may be adaptive, in that oxygen delivery capacity is adjusted to the decreased oxygen needs as body mass decreases. A decreasing haematocrit would also allow birds to reduce heart beat frequency and/or heart size, because blood viscosity decreases disproportionally with decreasing haematocrit. However, when energy stores are about to come to an end and birds increase protein breakdown, the haematocrit decreases even further, and birds probably become anaemic due to a reduced erythropoiesis. Keywords Bird migration Æ Blood parasites Æ Energy stores Æ Fasting Æ Haematocrit Æ Haemoconcentration Æ Haemodilution Æ Wind tunnel

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Introduction Migratory endurance flights are exceptional among vertebrates for three reasons: they may last for many hours or even days; they are performed without food or water intake, thus relying exclusively on body stores outside the flight muscles; the birds have to fly at a very high metabolic rate (well above the maximum sustainable rate of exercising small mammals; Butler and Woakes 1990). Thus, the high energy requirements of the flight muscles need to be met through the aerobic catabolism of fuels, a process requiring an adequate oxygen and fuel delivery. The blood has to transport oxygen, bound to the red blood cells, and metabolites (fuel) in the plasma that are soluble or bound to carrier proteins. Blood viscosity sets an upper limit on the proportion of erythrocytes in total blood (Smith et al. 2000) and also on the concentration of metabolites and carrier proteins in the plasma, such as fatty acids bound to albumin (Jenni-Eiermann and Jenni 1992). Thus, the haematocrit, the percentage of packed red blood cells per blood volume, is probably a finely tuned blood parameter that may change at the onset of flight, but also during a migratory endurance flight as the requirements and conditions change. Several studies suggest that a migratory endurance flight results in a decreased haematocrit. Data collected from Bar-tailed Godwits Limosa lapponica indicate that there may have been a mild flight-induced anaemia after a 4000-km flight (Piersma et al. 1996; Landys-Ciannelli et al. 2002). During autumnal migration, haematocrits were low in Goldcrests Regulus regulus and Blue Tits Parus caeruleus at the beginning of a refuelling cycle (Merila¨ and Svensson 1995; Svensson and Merila¨ 1996). In Garden Warblers Sylvia borin and Willow Warblers Phylloscopus trochilus crossing the Sahara in the autumn, the haematocrits showed a tendency to be lower in southern Algeria than in central Algeria (Bairlein and Totzke 1992). However, evidence for a change in the haematocrit during an endurance flight is still very rare or circumstantial, and the factors underlying such changes – when they occur – remain largely unanswered. The actual changes that occur in the haematocrit are a result of changes in the number or size of the red blood cells and/or changes in plasma volume. Changes in the number of blood cells reflect the balance between the production of new blood cells and their breakdown, while changes in plasma volume reflect the redistribution of fluid between the blood, other compartments of the body and the surrounding environ-

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ment. A number of selective pressures affecting oxygen and nutrient requirements, water balance and susceptibility to parasite infections may influence the optimal haematocrit for an individual bird in a specific situation. The objective of this study was to determine whether the haematocrit changes during the course of a long flight during which energy stores slowly decrease and, if it does, what are the underlying factors explaining the change. As such, we measured the haematocrit in four species of passerines following the completion of their natural migratory flight over the Mediterranean Sea and in a wader species, the red knot Calidris canutus, flying for up to 10 h in a wind tunnel. Six possible underlying factors were evaluated for their likelihood in providing an explanation of changes in the haematocrit. One possible factor explaining an increase in the haematocrit is a loss of plasma volume caused by dehydration during flight. An increased haematocrit has been found in severely dehydrated birds (e.g. Vleck and Priedkalns 1985), but as yet it has not been observed as a consequence of flight-incurred dehydration as birds are good conservers of plasma volume (Carmi et al. 1993, 1994). If dehydration were to be a factor in affecting the haematocrit, we would expect high haematocrits to occur in birds with low energy stores, i.e. in birds that were possibly dehydrated after having completed a long non-stop flight. A second factor that would result in an increase in haematocrit is a loss of plasma volume as a response to exercise, the latter being a phenomenon commonly observed in mammals (e.g. humans and greyhound dogs) and which is partly explained by the withdrawal of fluid to the active muscle cells and the interstitial ˚ strand and Rodahl 1986; Gillen et al. 1991; fluid (e.g. A Ernst et al. 1991; Neuhaus et al. 1992; Collodel et al. 1997). However, no change or an increase in the haematocrit has also been observed in humans as a response to exercise (e.g. Harrison 1985; O’Toole et al. 1999). If the withdrawal of fluid to the active muscle cells would affect the haematocrit, we would expect high haematocrits to occur a short time after the onset of flight. A third proposal for explaining a decrease in the haematocrit is that an increase in plasma volume is an adaptive response to increased nutrient and metabolite transport requirements during an endurance flight. An increased plasma volume may facilitate blood flow by decreasing blood viscosity and could serve to maintain osmolality when many nutrients and metabolites are being transported in the blood. The latter is suspected to be a factor explaining the

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slightly reduced haematocrit in birds during egg production (Bell et al. 1965; Morton 1994). An analogue may be the decreased haematocrit observed in athletes under certain circumstances (e.g. Harrison 1985; O’Toole et al. 1999) and during endurance training (e.g. Harrison 1985; Gillen et al. 1991; Ernst et al. 1991). An increase in plasma volume may also be a consequence of water retention (metabolic water, water absorbed from intestinal contents and from protein breakdown) during the early stages of a long migratory flight as a buffer against possibly dehydrating conditions later during that same flight. A decrease in the haematocrit would then reflect a general increase in body water which, in turn, would also affect tissues and interstitial fluid. If blood flow is to be facilitated or water is to be stored as a risk insurance strategy, we would expect the haematocrit to decrease rapidly with the onset of flight. A fourth factor explaining changes in the haematocrit is that migrating birds may be adversely affected by blood parasites, which destroy red blood cells and have been shown to decrease haematocrit levels (Dein 1986; Dawson and Bortolotti 1997). The deleterious effects of blood parasites on the haematocrit may be expected to be most pronounced at the end of long non-stop flights, when erythropoiesis may be compromised by fasting (see below). In this case we would expect to find a low haematocrit in birds infected with appreciable numbers of circulating blood parasites, possibly more so in birds with a poor body condition. Fifthly, the haematocrit may decrease during endurance flight because birds cannot maintain erythropoiesis under the conditions of prolonged fasting combined with a high energy expenditure that occur during an endurance flight. Erythrocyte synthesis might be particularly difficult when fat stores are depleted and birds must increase body protein breakdown for use as a fuel substrate (Jenni et al. 2000). The increase in body protein breakdown as a direct consequence of the depletion of fat stores parallels the situation observed in inactive, long-term fasting birds and is termed phase III of fasting (Le Maho et al. 1981; Cherel et al. 1988b; Boismenu et al. 1992). Indeed, a decreased haematocrit occurs in nutritionally stressed, fasting and egg-laying birds and is thought to be caused by a reduced production of red blood cells, while the normal breakdown of erythrocytes continues (Jones 1983; Boismenu et al. 1992). If erythropoiesis cannot be uphold when protein breakdown increases, we would expect to find a low haematocrit in birds with very low fat and low protein stores. The final possible explanation of a decreased haematocrit to be examined in our study is that the

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number of red blood cells may decrease as an adaptation to decreasing oxygen needs as body mass and power requirements drop during endurance flight. This phenomenon is similar to that proposed for the adaptation of flight muscle mass (Pennycuick 1998; Schwilch et al. 2002). Migrating birds may enhance their oxygen delivery system through an increase in the haematocrit (Wingfield et al. 1990) as part of their physiological changes in preparation for an endurance flight; such adaptations would include the accumulation of energy stores or the increase in the activity of oxidative enzymes (see Lundgren and Kiessling 1985; Driedzic et al. 1993). For example, Piersma et al. (1996) and Landys-Ciannelli et al. (2002) present evidence that Bar-tailed Godwits increase their haematocrit and blood haemoglobin level in preparation for a 2-day non-stop migratory flight. If this explanation is valid, we would expect to find the haematocrit to drop continuously with decreasing energy stores (body mass). These factors may of course occur in combination and, therefore, their effects on the haematocrit may either cancel each other out or, alternatively, reinforce each other. However, their predicted effects on the haematocrit will also differ in their time course (at the beginning or end of flight or continuously during flight), which may also help to distinguish between them. We examined haematocrit in relation to fat and protein stores and to an acute blood parasite infection in four species of small passerine birds migrating from tropical Africa to a small island in the northern Mediterranean Sea in the spring. In particular, we examined whether the haematocrit is maintained when birds enter phase III of fasting with a greatly increased protein breakdown. Because the migrants were captured after an over-water flight during which no stopover was possible, we are confident that the migrants had completed a non-stop flight of at least 500 km. As the birds arrived with very different levels of energy stores, we assumed that a cross-sectional examination reflects processes in the individual bird during the course of the endurance flight, a process which is otherwise very difficult to observe directly. In red knots flying for up to 10 h in a wind tunnel, we examined whether haematocrit changed with flight duration and whether this change differed from birds resting or feeding. As the haematocrit was not determined before flight, we again depended on a crosssectional design, but in this experiment the same individual was examined several times under different flight, fasting or feeding regimes.

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Materials and methods Passerines The birds were caught in mist nets on the island of Ventotene, which is situated 50 km off the Tyrrhenian coast of Italy (4048¢N, 1325¢E). In the spring, tropical migrants arrive at this small island (1.3 km2) from the northeast, indicating that they have come from North Africa and have crossed at least 500 km of the Mediterranean Sea. Most of the migrants were caught within about 1 or a few hours of their arrival on the island. There was no open water available to the birds on the island. Four species of tropical migrants, all insectivores, were investigated during the peak spring migration 17 April–13 May 2000: the barn swallow Hirundo rustica, pied flycatcher Ficedula hypoleuca, whinchat Saxicola rubetra and redstart Phoenicurus phoenicurus. The arriving birds displayed considerable interspecific variation with respect to remaining fuel stores, which correlated with the distance flown since the last species-specific refuelling areas located south of the Sahara (Pilastro and Spina 1997). Considerable intraspecific variation in fuel stores was also noted. Two types of energy stores were estimated in live birds. Fat stores were estimated through visual inspection of the subcutaneous fat deposits between the furcula and on the abdomen (9-level score; Kaiser 1993). These fat scores have been found to correlate well with the amount of fat extracted from whole birds (Kaiser 1993 and own unpublished data). Protein stores were estimated by visually scoring the thickness of the breast muscle (four levels: 0 = breast muscle emaciated and its cross-section shaped concavely; 3 = breast muscle bulging and shaped convexly; Bairlein 1995). Because fat content of the breast muscle is negligible (1.9% in 84 small passerines from Ventotene; unpublished data), muscle scores represent an estimate of breast muscle protein mass. Fat and muscle scores were measured by four experienced and inter-calibrated ringers. Blood was collected from the brachial vein into heparinized microcapillary tubes. One drop of blood from each bird was used for a blood smear, prepared according to the standards of the Swiss Tropical Institute, Basel. The remaining blood was centrifuged. The haematocrit was determined in microcapillary tubes filled with more than 10 mm of blood (mean: 40.6 mm; range: 11–67). Blood smears were fixed with methanol, stained with Giemsa and carefully examined over their entire surface for blood parasites under a microscope (magnification: 100· and 500·) for approximately

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30 min until 100 leucocytes had been found. The percentage of birds with blood parasites found in their blood smears was 14% in the redstart (out of a total n=64), 17% in the barn swallow (n=84) and pied flycatcher (n=65) and 50% in the whinchat (n=151). Haemoproteus and Plasmodium were the most frequent blood parasites found, while Trypanosoma and microfilaria, which are typically difficult to find in blood smears, were detected in five and one bird, respectively. For this study, we used the presence or absence of any blood parasite found in blood smears as an indicator of an acute infection with blood parasites. Since we were not interested in birds with very low parasitaemia, we worked under the assumption that blood smears screened for a fixed time (30 min in the present study) are an appropriate tool by which to detect acute infections, although we must accept the fact that infections detectable at only certain times of day may have been missed (see Cooper and Anwar 2001). We examined the haematocrit as a function of energy stores remaining after a long non-stop flight. We assumed that differences in energy stores between individuals reflect the decrease in energy stores that occurs within that individual during prolonged flight. Although inferring the changes that occur within an individual from the variation of a sample group can cause methodological problems (e.g. Lindstro¨m and Piersma 1993), we propose that for this type of study this approach is justified because the variation in energy stores between individuals was large and thus overruled possible problems of intraspecific variation in bird size. We used fat score as the primary indicator of remaining energy stores. Although fat is the main fuel that powers an endurance flight, protein is also catabolized. We found that fat score and muscle score were correlated and that there was little variation in muscle score within each fat score. However, in birds with no or little fat stores remaining, protein catabolism increases dramatically to a high level (Jenni et al. 2000). Thus, we found that muscle score was more variable when the fat score was 0 (no visible subcutaneous fat stores left). Therefore, in birds with a fat score of 0 we used muscle score as an additional indicator of remaining energy stores. In order to make a statement on the passerines migrating over the Mediterranean Sea as a whole, we analysed the four species together in a mixed model with the species as the random factor. However, to show the differences between species, we also present the data for each species.

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Red knot Red knots of both the canutus and the islandica subspecies, all adults, were kept in an aviary (3·1.5·2 m) under a 12:12-h (long-day, LD) photoperiod (lights on: 09:00 h local time) in the wind tunnel building in Lund (for further information on bird keeping, see Lindstro¨m et al. 2000; Jenni-Eiermann et al. 2002). Blood was sampled from individuals in three different physiological states: (1) flight in the wind tunnel; (2) resting while fasting; (3) feeding. For each of these physiological states, one blood sample was taken at 0, 1, 2, 4 or 10 h following the lights being put on. We analysed samples from six birds during flight and compared these with the same six individuals during resting while fasting. Samples taken from feeding birds are from the same six birds as well as from seven additional ones. Each bird was used repeatedly for the alternating flight, resting and feeding experiments. The intervals between experiments for any one individual bird was usually 1–7 days, but occasionally the between-experiment interval was as long as 30 days. The aim was to sample each individual at least once in each of the different activity states (flight, resting or feeding) and time of day ( = duration of the experiment). Because all birds would not fly for long enough periods in the wind tunnel, it was not possible to follow this procedure strictly, and each individual was used for one to nine flight experiments, one to eight resting experiments and one to five feeding experiments. The evening before the day of flight, food was removed between 18:00 and 22:00 h. Each flight in the wind tunnel started at 09:00 h, irrespective of flight duration. Before the 1-, 2- and 4-h flights, the birds had access to water until the flight started. Before the 10-h flights, the birds had no access to water during the last hour before the flight because the 10-h flights were part of a study measuring energy expenditure using doubly labelled water (DLW) (Kvist et al. 2001). During the last hour before flight, the injected DLW diluted in the body, and the birds were kept in darkness where they were inactive. We have no reason to assume that the initial water balance was markedly different before the 10-h flights. In the wind tunnel, the birds flew continuously at 15 m s–1. The 1-h flights were non-stop. During the 2-, 4- and 10-h flights, the birds were briefly taken out of the wind tunnel for 40–80 s and weighed after 1, 2 and 4 h, where applicable. For each flight experiment, only one blood sample was taken at the end of flight. During the resting experiments, the birds were treated the same as the flying birds. Instead of flight,

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however, the bird was transferred to a cage (1·0.7·0.7 m) where it remained without food, but with water. For each resting experiment, only one blood sample was taken at the end of the pre-determined resting period of 0, 1, 2, 4 or 10 h. Because food was withdrawn the evening before the experiment, the birds were actually fasting for an additional 11–15 h. During fasting and resting, the birds typically remained motionless or walked around in the cage. Feeding birds had continuous access to food and water before and during the experiment and may have eaten during the night preceding the experiment, as shown in captive waders. Blood samples were taken from the brachial vein within 1–5 min of the end of the flight. Samples from resting and feeding birds were taken within 20 min of the intended end time. The haematocrit was determined in two to seven heparinized 50-ll capillary tubes filled with more than 20 mm of blood and the mean taken for analysis. The data were analysed in a mixed model analysis (residual maximum-likelihood analysis, REML; Patterson and Thomson 1971) in GENSTAT 5, release 6.1. The individuals were included as random factor, thus accounting for differences between individuals.

Results Passerines When all of the species were taken together, the haematocrit significantly decreased with decreasing fat score (Fig. 1) but showed no significant variation with the presence of blood parasites (Table 1). Sex did not significantly explain variations in the haematocrit when it was included as an additional factor (p=0.43). Haematocrit in birds with a fat score of 0 was more variable than in birds with higher fat scores (Fig. 1). This variation was partly explained by variations in the muscle score. In birds with a fat score of 0, the haematocrit decreased significantly with decreasing muscle score (Fig. 1; Table 1). The statistical significance of the decrease in the haematocrit with decreasing fat score may be a result of the birds having entered the stage of increased protein breakdown (phase III of fasting). When birds with a fat score of 0 and 1 (n=159), were excluded, the same pattern emerged – i.e. the haematocrit was not significantly related to the presence of blood parasites (p=0.3) but to fat score (p=0.04, n=205; mixed model analysis with species as random factor). When each species was evaluated separately (Table 1; Fig. 2), the same general pattern emerged.

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from 48% (SD: 4.5, n=54) in birds with a muscle score of 2, to 46% (SD: 7.1, n=59) in birds with a muscle score of 1 and, finally, to 36% (SD: 7.0, n=7) in birds with a muscle score of 0. Red knots

Fig. 1 Mean haematocrit levels (±SD) of all species taken together as a function of fat score and, for birds with a fat score of 0, as a function of muscle score. Sample sizes are given above the graph

Infection by parasites did not have a noticeable effect on the haematocrit in any of the four species. Haematocrit values decreased with decreasing fat score, except in the redstart, in which they remained stable. Sex did not explain variations in the haematocrit in any species when it was included as an additional factor (p>0.2). In individuals with a fat score of 0, haematocrit values decreased with muscle score, although significance was only attained in the whinchat, probably because of the larger sample size for this species. In summary, mean haematocrit in fat birds (fat score >2) was 51% (SD: 4.4%, n=134), with species means varying between 49 and 53%. In lean birds (fat score: 1 or 2), the mean haematocrit was 49% (SD: 4.2%, n=110; species means: 48.5–51%). In very lean birds (fat score: 0), the haematocrit showed additional variation with muscle score: the haematocrit dropped

Our analysis of all the flight and resting experiments revealed that the haematocrit varied significantly with status (resting, flying) and body mass, while the duration of the experiment and all interaction terms were not significant (Table 2). Birds flying in the wind tunnel had a significantly lower haematocrit than resting birds. For each individual, the haematocrit was positively related to its body mass, irrespective of whether the bird was flying or resting (Fig. 3). The common slope of the haematocrit on body mass in a model containing only the individuals as random factor and body mass as a fixed factor was 0.163 (±0.035 SE). When the data on the resting birds was analyzed without body mass taken into account, the haematocrit did not vary significantly with the duration of resting (resting duration as a categorical variable; mixed model analysis with individuals as the random factor and the duration of resting as the fixed factor; Wald statistic: 4.58, df=4, p=0.33, n=37; Fig. 4). In a similar analysis of the data from birds flying in the wind tunnel, including the starting point at time 0 (same data as for resting birds), the haematocrit varied significantly with flight duration (mixed model analysis; flight duration: Wald statistic: 15.34, df=4, p=0.004, n=45). The haematocrit decreased from 52.1% pre-flight to 48.0–49.7% after 1–4 h of flight and 49.6% after 10 h of flight (estimates from the mixed model analysis; Fig. 4). In an additional analysis with 13 individuals, feeding birds showed no significant change in their haematocrit with increased duration of feeding (mixed model

Table 1 Dependence of haematocrit values on the presence of blood parasites and on the fat or muscle score for all species taken together and for each species separately. A separate analysis is given for all individuals and for individuals with a fat score of 0 only All individuals

All speciesb Whinchat Pied flycatcher Barn swallow Redstart

Fat score = 0

Blood parasites (df=1) p (F)a

Fat score (df=5) p (F)

n

Blood parasites (df=1) p (F)

Muscle score (df=2) p (F)

n

0.79 0.96 0.45 0.87 0.32

<0.001 (8.4) 0.003 (3.7) 0.013 (3.4) 0.005 (3.7) 0.97 (0.1)

364 151 65 84 64

0.33 0.29 0.49 0.44 0.08

<0.001 (10.6) 0.002 (7.0) 0.10 (2.5) 0.21 (1.6) 0.34 (1.1)

120 53 29 28 10

(0.1) (0.003) (0.6) (0.03) (1.0)

a

(0.5) (1.1) (0.5) (0.6) (4.4)

The p- and F-values of the main effects from a type I two-way ANOVA (mixed model analysis when all species are taken together) are given. Sex and all interaction terms were not significant and excluded from the models

b

Mixed model analysis with species as random factor

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Fig. 2 Mean haematocrit levels (±SD) for each of the four species considered separately as a function of fat score and, for birds with a fat score of 0, as a function of muscle score. Sample sizes are given above the graph

Table 2 Effect of body mass, status (resting or flying in a windtunnel), time of resting or flight and interaction terms on the haematocrit in red knots tested in a mixed model analysis (deviance: 242.4, df = 59) with the individual as a random factor. Effects including the variable status (resting, flying) are given for resting birds (versus flying birds) Fixed effects

Effects ± SEa

Wald statistica

p

Mass Status (flying, resting) Duration of experiment (Duration of experiment)2 Status · duration Mass · duration Mass · (duration)2 Mass · status Constant

0.188±0.056 1.214±0.645 –0.307±0.407 0.039±0.033 –0.228±0.178 0.003±0.038 –0.001±0.003 –0.079±0.069 49.95±1.32

23.62 6.88 0.02 2.46 2.72 0.02 0.34 1.34

<0.001 0.009 0.89 0.12 0.10 0.90 0.56 0.25

a

The effect ± SE and Wald statistics are given with their significance levels. The degrees of freedom were 1 for all variables and interaction terms

analysis; feeding duration: Wald statistic: 2.40, df=4, p=0.79, n=54). The haematocrit after 10 h was marginally higher than at the start of the experiment (Fig. 4). The change in the haematocrit that occurred during 10 h of flight, resting or feeding matched the change in the haematocrit predicted from the change in body mass. Feeding birds gained mass (measurements are

missing) and showed a slight increase in their haematocrit (Fig. 4). Resting birds without food lost mass (measurements missing) and showed a slight decrease in their haematocrit. Birds flying for 10 h lost even more body mass (10.33±1.51 g) and, at the end of flight, had the lowest haematocrit of the three groups.

Discussion If we assume that the cross-sectional data obtained from the passerines reflect changes in the haematocrit in the individual bird during the course of an endurance flight, we can conclude that the haematocrit decreased from 51% in fat birds to 48% in lean birds. This is in accordance with observations on other migrants which suggest that endurance flight is associated with a slight decrease in haematocrit (Merila¨ and Svensson 1995; Piersma et al. 1996; Svensson and Merila¨ 1996; Landys-Ciannelli et al. 2002). Because the birds were caught within 1 or a few hours of their arrival on the island, we cannot assess any changes that might have occurred between landing and capture. From the red knot data (Table 2), we can conclude that the haematocrit changes with body mass and is lower in flying birds than in resting ones. Haematocrit seems to drop by about 3% during the first hour of

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Fig. 3 Haematocrit of six individual red knots (represented by different symbols) plotted against their body mass at the end of the fasting (small symbols) or flying experiments (large symbols). Note the general relationship between haematocrit and body mass as well as differences between individuals

Fig. 4 Mean haematocrit ± SE of knots flying in a wind tunnel (dots), fasting (open circles) or feeding (triangles) over a period of 1, 2, 4 or 10 h. Given are the estimates from a mixed model analysis with individuals as the random factor and the duration of the experiment as the fixed factor (see Results). Note that the data used to estimate the haematocrit at 0 h for the flying and resting birds were the same, but the data for the feeding birds were different

flight (Fig. 4); that it also decreased thereafter during flight was not apparent from Fig. 4. This may be due to the fact that the haematocrit was not measured at the beginning of the flight period, but only at the end. Therefore, Fig. 4 does not represent the change in haematocrit within single flights, but is confounded with the effects of variations in body mass between experiments within individuals. Apparently, in our data set individual variation in body mass between

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experiments overrides any effects of a decrease in individual body mass during flight within the experiments. Overall, however, the changes in the haematocrit of the feeding, resting and flying birds at least qualitatively match changes in body mass. Taking the results from the passerines and the red knots together, it would appear that the haematocrit drops by a few percentage points within 1 h after the onset of flight (Fig. 4), decreases very slowly with decreasing body mass (Figs. 1–3) and decreases more steeply in very lean birds having entered stage III of fasting (Figs. 1, 2; knots never reached stage III of fasting). This interpretation assumes that red knots and passerines do not differ to any considerable extent with respect to their changes in haematocrit as a response to the same causes, although they may differ in various other aspects, such as body water regulation, diet and the quantitative composition of their fuel stores. In passerines, sex did not appear to affect the haematocrit. Many previous studies have shown that females have lower haematocrits than males (see Prinzinger and Misovic 1994), probably due to the stimulating effect of androgens and the inhibiting effect of oestrogens on erythropoiesis (Wingfield et al. 1990; Morton 1994). However, in the present study birds were caught during the spring migration – before the breeding season – and about 60% were in their second calendar year, thus before their first breeding season. Of the six factors presented as possible explanations for a change in the haematocrit after a long flight (see Introduction), the first, an effect of dehydration, is unlikely, because we observed a low – and not the expected high – haematocrit in birds with low energy stores, i.e. in birds after a long non-stop flight that were possibly dehydrated. Birds are good plasma volume conservers (Carmi et al. 1993, 1994), and there is no indication to date of small- or medium-sized migrants suffering from dehydration after long flights in temperate areas (Landys et al. 2000; own unpublished data from Ventotene). Red knots flying for 10 h in the wind tunnel did not show any signs of dehydration (A. Kvist et al., unpublished data). The second explanation, haemoconcentration by the withdrawal of fluid to the active muscle cells, is also unlikely, because we observed lower haematocrits in flying knots, not higher ones as the haemoconcentration would imply. It is astonishing that birds apparently react to endurance exercise with a decrease in their haematocrit, and not with an increase as mammals ˚ strand and Rodahl 1986; Gillen et al. generally do (A 1991; Ernst et al. 1991; Neuhaus et al. 1992; Collodel et al. 1997).

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The third explanation – increasing plasma volume to facilitate blood flow by haemodilution or to buffer the risk of oncoming dehydration – is supported by the knot data. Within 1 h after the onset of flight, the haematocrit decreased. This may be adaptive in two ways. First, it reduces blood viscosity and, thereby, energy expenditure by the heart. Plasma volume may expand because of the water recruited from extravascular volumes, such as water from catabolized protein tissue. Second, migrants flying long distances non-stop may store, rather than dissipate, any surplus water (metabolic water, water absorbed from intestinal contents or protein breakdown) early during flight as a risk insurance against possible dehydration later on. Such water may be stored in tissues and interstitial fluid and also entail a higher plasma volume. Indeed, the knots flying for 10 h in the wind tunnel showed increased body water content (A. Kvist et al., unpublished DLW measurements). The fourth explanation, an effect of blood parasites, is unlikely. We found no effect of an acute infection with blood parasites on haematocrit levels in passerines. We did not account for blood-feeding ectoparasites, but there is no indication that lean birds at Ventotene with their lower haematocrits have more ectoparasites than fat birds. The fifth explanation, an inability to uphold erythropoiesis when energy stores are low and protein is catabolized to obtain energy for flight, is supported by our data from the passerines (the red knots did not attain low energy stores). Birds that decrease fat stores below a score of 2 increase protein catabolism (decrease muscle score) and enter a state which is comparable to phase III of fasting in inactive long-term fasting birds (Jenni et al. 2000). We found that concurrently with this increase in protein catabolism, haematocrit levels steeply decreased and attained low values (36%) in passerines whose breast muscles were emaciated (muscle score: 0). This parallels findings in birds subjected to severe nutritional stress or long-term fasting. Quelea Quelea quelea females laying eggs under severe nutritional stress had decreased haematocrit values of below 30% (Jones 1983). Nutritionally stressed captive red knots had lower haematocrit values than their well-fed conspecifics (Piersma et al. 2000). In long-term fasting geese Anser anser and Chen caerulescens, the haematocrit decreased from 41–43% to 31–32% in conjunction with decreasing energy stores (Le Maho et al. 1981; Boismenu et al. 1992). However, the haematocrit remained virtually unchanged in long-term fasting penguins (Cherel and Le Maho 1985; Cherel et al. 1988a). In nutritionally stressed or fasting birds, the reason for the decrease in

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the haematocrit is thought to be a reduced production of red blood cells, while normal breakdown of erythrocytes continues (Le Maho et al. 1981; Jones 1983; Boismenu et al. 1992). The sixth explanation, an adaptation of oxygen delivery to decreasing oxygen needs, is also supported by our data. In passerines, we found that the haematocrit decreased slightly and continuously with decreasing fat stores. In individual red knots, the haematocrit closely followed body mass irrespective of whether the bird was flying or resting (Fig. 3). During endurance flight, body mass decreases, thus resulting in decreased total body oxygen requirements. Birds have several possibilities by which to reduce oxygen delivery. They may decrease the number of erythrocytes (haematocrit), the size of the heart (affecting cardiac stroke volume), the frequency of the heart beat, the extraction rate of oxygen or the volume of the cardiac stroke. As shown by the passerine data, it is possible that these birds decreased their haematocrit during the endurance flight, possibly by reducing erythropoiesis, to adapt oxygen delivery capacity to the decreasing total oxygen needs as body mass decreases during flight. A decrease in the haematocrit has the additional advantage that blood viscosity is decreased, thereby enabling an increased blood flow (Smith et al. 2000); the reason for this being that avian erythrocytes are less deformable and tend to be larger than mammalian erythrocytes, and thus have more difficulties traversing the capillary bed (Smith et al. 2000). Therefore, a decrease in the haematocrit would allow a concomitant reduction in heart size or heart beat frequency. There is indeed evidence that birds during an endurance flight may also reduce heart size or heart beat frequency as a function of decreased total body oxygen requirements (Butler et al. 1998; Battley et al. 2000). If the oxygen delivery system is reduced during an endurance flight, it needs to be upgraded after a long flight and in preparation for the following endurance flight. Bar-tailed Godwits have been shown to increase the haematocrit and haemoglobin concentration during stopovers (Landys-Ciannelli et al. 2002); mean cell haemoglobin concentration also increases (LandysCiannelli et al. 2002), as well as heart size, the latter possibly as a consequence of high haematocrits (Piersma et al. 1996). The close relationship between haematocrit and body mass in the knots (Fig. 3) supports this hypothesis. As mentioned in the Introduction, several of the factors discussed above may have counteracting effects on the haematocrit and, subsequently, mask changes in the two constituents, erythrocytes and plasma. We cannot exclude that we may have missed such

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counteracting effects in our study because we did not measure changes in the number of erythrocytes and plasma volume. Further studies investigating changes in blood constituents (e.g. plasma volume, number of erythrocytes, proportion of old versus new erythrocytes; Landys-Ciannelli et al. 2002) are needed to more closely reveal the underlying causes for changes in the haematocrit. However, a change in the haematocrit has direct effects on blood viscosity which in itself is an important physiological parameter during endurance exercise. There are two striking parallels between the changes in the haematocrit observed in the present study and those occurring during long-term fasting and during endurance flight. First, changes in the haematocrit observed in this study during an endurance flight parallel those observed in inactive long-term fasting birds (Le Maho et al. 1981; Boismenu et al. 1992): the haematocrit decreased slowly with decreasing body mass until protein catabolism increased (phase III of fasting) and haematocrit dropped to low levels. Thus, we present yet another piece of evidence that adds to the emerging pattern that endurance flight in birds shows many similarities to inactive long-term fasting and can be regarded as a rapid, high energy-expenditure fasting (see Jenni et al. 2000). Second, there are striking parallels between changes in the haematocrit and changes in flight muscle mass, both being involved in generating power during flight. Both are increased prior to long flights to upgrade the flight machinery for the high body mass (for haematocrit, see Landys-Ciannelli et al. 2002; for flight muscles, see Lindstro¨m et al. 2000; Landys-Ciannelli et al. 2003). Both are subsequently decreased during flight, concomitantly with the decrease in body mass caused by losing energy stores (for haematocrit, see Piersma et al. 1996; Landys-Ciannelli et al. 2002; this study; for flight muscles, see Biebach 1998; Battley et al. 2000; Lindstro¨m et al. 2000; Schwilch et al. 2002). The observed reduction in both flight muscles and haematocrit (assuming red blood cell volume; see Le Maho et al. 1981) entails a decrease in capacity. It appears that the reduction in flight muscles during phase II of fasting is adaptive with respect to flight capability on both theoretical grounds (Pennycuick 1998) and empirically (Schwilch et al. 2002). This is also likely to be the case for the haematocrit. In long-term fasting geese the decrease in the haematocrit is due to a decrease in red blood cell volume, while total plasma volume remains constant, resulting in a constant amount of red blood cells per unit body mass (Le Maho et al. 1981).

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However, when energy stores come to an end and birds increase protein breakdown (phase III of fasting), flight muscle catabolism increases strongly and flight capability is likely to be reduced (Schwilch et al. 2002). Similarly, birds entering phase III of fasting begin to suffer from anaemia, as very low haematocrits are generally regarded as a sign of anaemia (Ots et al. 1998). These strong decreases in flight muscle mass and haematocrit are probably not adaptive with respect to flight capability, but are an adaptation to whole-body metabolism when fat stores are exhausted (reviewed in Cherel et al. 1988b).

Zusammenfassung Auswirkungen des Langstreckenfluges auf den Ha¨matokrit bei Zugvo¨geln Wir untersuchten die Auswirkungen des Langstreckenfluges auf den Ha¨matokrit (den Prozentsatz der Roten Blutko¨rperchen am Blutvolumen) im Lichte von 6 Hypothesen. Wir untersuchten 4 Arten von Singvo¨geln, die nach einem Non-stop-Flug u¨ber das Mittelmeer im Fru¨hling auf einer Italienischen Insel landeten. Bei 6 Knutts maßen wir den Ha¨matokrit nach Flu¨gen von 1, 2, 4 und 10 h im Windtunnel und verglichen die Werte mit solchen nach entsprechend langem Fasten oder Fressen. Bei den 4 Singvogelarten nahm der Ha¨matokrit von 51% bei fetten Individuen auf 48% bei mageren Individuen ab. Unter den mageren Individuen fiel der Ha¨matokrit von 48% bei Vo¨geln mit gut entwickelter Flugmuskulatur auf 36% bei Vo¨geln mit stark abgemagerten Flugmuskeln. Der Ha¨matokrit fliegender und nicht-fliegender Knutts war positiv mit dem Ko¨rpergewicht korreliert. Innerhalb der ersten Flugstunde nahm der Ha¨matokrit von 51% auf 49% ab und blieb auf diesem Wert bis zu 10 h Flug. Betrachtet man die Ergebnisse der Singvo¨gel und der Knutts zusammen, so fa¨llt der Ha¨matokrit innerhalb von 1 h nach Flugbeginn um wenige Prozent, nimmt mit abnehmendem Ko¨rpergewicht langsam ab und geht stark zuru¨ck bei sehr mageren Vo¨geln, die Stadium III des Fastens erreicht haben. Dies zeigt, dass Dehydration kein Grund fu¨r die Vera¨nderung des Ha¨matokrits ist, da wir mit zunehmender Flugdauer eine Erho¨hung des Ha¨matokrits erwarten wu¨rden. Wir fanden auch keinen Effekt von Blutparasiten auf den Ha¨matokrit. Mit Flugbeginn kann eine Blutverdu¨nnung adaptiv sein, da sie die Blutviskosita¨t und damit den Energieaufwand des Herzens senkt oder da sie als Wasserspeicher im Hinblick auf das Risiko einer Dehydration im Verlaufe des Fluges wirken kann.

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Wa¨hrend eines Langstreckenfluges mag eine langsame Reduktion des Ha¨matokrits adaptiv sein, da dadurch die Sauerstoffzufuhr an das abnehmende Ko¨rpergewicht angepasst wird. Ein abnehmender Ha¨matokrit wu¨rde es den Vo¨geln auch erlauben, die Herzschlagrate und/oder die Herzgro¨ße zu reduzieren, da die Blutviskosita¨t mit abnehmendem Ha¨matokrit u¨berproportional abnimmt. Wenn aber die Energiereserven ans Ende kommen und die Vo¨gel ihren Proteinabbau stark erho¨hen, nimmt der Ha¨matokrit stark ab und die Vo¨gel werden ana¨misch, da wohl die Bildung roter Blutko¨rperchen behindert ist. Acknowledgements We thank Y. Endriss, Swiss Tropical Institute, Basel, for introducing SM into the art of making blood smears, for providing a working place and for much helpful advice. The wind tunnel study was supported by a PIONIER grant to Theunis Piersma from the Netherlands Organization for Scientific Research (NWO), and by grants from the Crafoord Foundation and the Swedish Natural Science Research Council ˚ L), Knut and Alice Wallenberg Foundation (to Thomas (to A Alerstam), and the Swedish Council for Planning and Coordi˚ L). Bernard nation of Research (to Thomas Alerstam and A Spaans, Anita Koolhaas, Anne Dekinga, Maurine W. Dietz, Martin Green, Mikael Rose´n, Anders Hedenstro¨m and Anders Forslid kindly helped with practical matters. The wind tunnel work was carried out under license from the Lund/Malmo¨ Ethical Committee (no. M161-97). We are indebted to P.J. Butler, M. Landys-Ciannelli and R.L. Holberton for helpful comments on an earlier version.

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