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Blood oxygen- and carbon dioxide-carrying properties in captive penguins: Effects of moulting and inter-specific comparison
Comparative Biochemistry and Physiology, Part A 168 (2014) 76–81
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Blood oxygen- and carbon dioxide-carrying properties in captive penguins: Effects of moulting and inter-specific comparison Valérie Maxime a,⁎, Sami Hassani b a b
Université de Bretagne Sud (UEB), Centre d'Enseignement et de Recherche Yves Coppens, LIMATB (EG2B), Campus de Tohannic, 56017 Vannes Cedex, France Océanopolis, Port de plaisance du Moulin Blanc, BP 411, 29275 Brest Cedex, France
a r t i c l e
i n f o
Article history: Received 23 July 2013 Received in revised form 19 October 2013 Accepted 6 November 2013 Available online 11 November 2013 Keywords: Acid–base balance Aptenodytes patagonicus Eudyptes chrysocome Moulting Penguins O2 storage Plasma ions Pygoscelis papua
a b s t r a c t Venous blood gas-carrying properties were compared in the three captive species of penguins (king, gentoo and rockhopper) at Océanopolis (France). Captivity permitted to control environmental influences. Given their different ecology and diving behaviour in the wild, it was wondered whether milder conditions and dive privation have repercussions on parameters determining oxygen storage and acid–base status of these birds. In addition, this work provided the opportunity to study the effects of moulting in king penguins. This annual event that imposes deep metabolic adjustments is liable to affect blood gas levels. Because of the regular food supply and probably also of the blood sampling conditions, the blood pH of captive penguins was low. This effect was increased in moulting penguins and supposedly due to both the decreased energetic metabolism and the production of uric acid resulting from new feather synthesis. The decrease in the anion gap also revealed the use of plasmatic albumin for this synthesis. The elevated venous PO2 in all birds is not likely due to stress caused by sampling conditions. The other data, in accordance with those in the literature, show neither major influence of captivity nor fundamental interspecific differences, despite potential diving aptitude. © 2013 Elsevier Inc. All rights reserved.
1. Introduction In sphaeniciformes, haematological values can be strongly influenced by site, season, species, age, sex and physiological and nutritional status (Villouta et al., 1997; Sergent et al., 2004). This underlines the great involvement of blood cells in environmental and physiological adaptations. Most of the previous studies devoted to blood respiratory properties were undertaken in wild birds and concern many geographically and biologically different species; for example: Aptenodytes forsteri (Ponganis et al., 2009), Aptenodytes patagonicus (Hawkey and Samour, 1988), Pygoscelis adeliae (Murrish, 1982), Pygoscelis papua and Pygoscelis antarctica (Milsom et al., 1973; Murrish, 1982), Eudyptula minor (Sergent et al., 2004), Eudyptes chrysocome (Karesh et al., 1999), and Spheniscus humboldti (Villouta et al., 1997). The overall analysis of these works does not indicate any fundamental differences between the species despite their different diving abilities. This could be explained not only by a variety of factors that can vary simultaneously but also by the dependence of oxygen storage on body mass and the variable distribution of oxygen between respiratory, blood and muscle compartments (Ponganis and Kooyman, 2000). The fact that captive penguins live for a long time in exactly the same conditions allows the researcher to eliminate the variations of surrounding effects. Taking
⁎ Corresponding author. Tel.: +33 297 017 169. E-mail address: [email protected] (V. Maxime). 1095-6433/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.11.002
advantage of this, this study compares the three penguin species kept at Océanopolis (the ocean discovery park in Brest, France). At the time of this experiment (April), about half of the king penguins at Océanopolis were moulting and the others were going later. Moulting is a physiological event liable to affect oxygen storage and consumption as well as the blood acid–base balance because of the deep metabolic adjustments that characterize this period of total fasting (Cherel et al., 1993; Cherel et al., 2005). Indeed, penguins replace their plumage entirely each year and wild birds spend a long time (14–40 days) fasting ashore because the consequent reduction in thermal insulation precludes staying in cold waters to feed (Groscolas and Cherel, 1992). Moulting involves the use of endogenous lipid reserves and body protein to fulfil the nutrient requirement for feather synthesis and the higher thermogenesis associated with the decrease in thermal insulation. Little work has been carried out on energy requirement during moulting and fasting: Le Maho and Despin (1976), Deswasmes et al. (1980), and Fahlman et al. (2004) show that resting energetic metabolism and heart rate decreased throughout the fast. Thus, in addition to an interspecific comparison, this study was also the opportunity to highlight the effects of moulting on the blood gas-carrying properties of king penguins. The three species of Océanopolis' penguins (king penguin — Aptenodytes patagonicus Miller, gentoo penguin — Pygoscelis papua Forster and rockhopper penguin — Eudyptes chrysocome Forster) were all born in captivity. In their territories of origin (Subantarctic islands and Antarctic peninsula for the first two species, Argentina and
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Chile for the third), these marine birds dive to feed and migrate and differ in the depths they frequently reach. Their ability to dive is positively related to body mass (Wilson, 1995). King penguins, being among the largest marine birds, have exceptional diving abilities, reaching a mean maximum depth of 304 m (Kooyman et al., 1992). Gentoo penguins dive to 156 m (Williams et al., 1992) and rockhopper penguins, the smallest of the three, to 66 m (Wilson et al., 1997). In captivity, penguins never dive and the consequent repercussions on blood gascarrying properties, if there are any, are unknown. Moreover, Océanopolis' penguins are exposed to mild conditions (no polar temperatures, no wind, regular and sufficient food) compared to wild ones. It is hypothesized that these opposing conditions, leading to a less drastic energetic adaptation for captive penguins, may concern the birds' blood properties, particularly when moulting making them temporarily more fragile. The penguins of Océanopolis are primarily destined to a daily public display and consequently they are preserved from all predictable causes of disturbance as much as possible. Thus, this study was carried out during a scheduled clinical examination of the overall population. Our goal was to describe parameters that are relevant in determining oxygen and carbon dioxide storage and their eventual consequences on acid–base status of blood in a comparative study between 1) moulting and non-moulting king penguins and 2) the three bird species kept at Océanopolis. 2. Materials and methods
77
and 6.086, respectively (Nicol, 1991). The total carbon dioxide content (CtCO2, mmol·L− 1) was calculated from: −
CtCO2 ¼ ðαCO2 PCO2 Þ þ ½HCO3 (Siggaard-Andersen et al., 1988). 2.3. Measured and derived oxygen storage capacity of blood The Radiometer analyser measured haematocrits (Hct, %) and calculated haemoglobin concentrations ([Hb], g·L−1) from Hct. These data were compared to those obtained by manual methods. Hct was measured after centrifugation of 70 μL microcapillaries for 5 min at 12,000 g. [Hb] was spectrophotometrically determined by the cyanmethaemoglobin method (kit Sigma 525A). Manually- and automatically-obtained Hct were identical. In contrast, [Hb] was systematically about 15% higher with the manual method. This difference came certainly from the equation used by the Radiometer analyser programme which includes constants for the human blood. Therefore, only manually-obtained [Hb] values were considered. The red blood cell count (RBC, L−1) was obtained by means of a Malassez haemocytometer following 1:200 dilution in Marcano solution (5 g sodium sulphate, 1 mL formaldehyde 40%, qs 100 mL distilled water). Mean red blood cell volume (MCV, μm3·cell−1) and mean cell haemoglobin concentration (MCHC, %) were respectively computed as follows: MCV ¼ Hct=RBC and MCHC ¼ ½Hb=Hct:
2.1. Animals and blood sampling 2.4. Electrolytic assessment The study was carried out on all penguins in captivity at Océanopolis: 9 king penguins; (7 ♂ and 2 ♀) weighing on average 12 kg and aged between 6 and 18 years old, 10 gentoo penguins (5 ♂ and 5 ♀) weighing on average 7 kg and aged between 1 and 21 years old and 10 rockhopper penguins (6 ♂ and 4 ♀) weighing on average 3 kg and aged between 1 and 21 years old. They were all born in captivity and were in good physical condition. They were fed daily ad libitum with thawed Atlantic herring Clupea harengus and Icelandic capelin Mallotus villotus except for the day before sampling and for moulting king penguins that refuse food. The slightly low mean body mass of king penguins reflects the moulting and consequent fasting status of some of them. Venous blood samples of 200 μL were quickly (b1 min) collected in heparinized (lithium heparin 100 IU/mL) syringes from the brachial vein of relatively calm manually-restrained birds. This sampling method was supposedly of minimal disturbance because the birds are used to regular human presence and manipulation. This also avoided the potential effects of sedation and anaesthesia on measured blood parameters. 100 μL of blood was immediately analysed by using a Radiometer ABL77 blood gas analyser (Brønshøj, Denmark) calibrated at 37 °C for a range of respiratory, haematological and ionic data. The time interval between sampling and analysis was always less than 5 min. The gas analyser calculated the measured gas partial pressure (pO2 and pCO2, mm Hg) using the temperature-correction factors given by Burnett and Noonan (1974) assuming a mean penguin temperature of 38.5 °C (Lenfant et al., 1969). The temperature of the birds was not taken to avoid additional disturbance. Moreover, solubility coefficients for O2 and CO2 for penguins in the literature are given at 38.5 °C. The remaining blood was stored on ice (24 h maximum) until required for a red blood cell count and manual checking of haematocrit and haemoglobin concentrations.
Plasma ion concentrations ([Na+], [K+], [Ca2+] and [Cl−], mmol·L−1) were measured by means of the Radiometer analyser's selective sensors. The anion gap (AG, mmol·L−1) was calculated from the following equation: þ
þ
−
−
AG ¼ ½Na þ ½K −½Cl −½HCO3 : 2.5. Statistical analysis Statistical analyses were conducted using Sigmastat 4.0 (Systat Software Inc.). All results were expressed as mean ± s.e.m. The effects of sex in gentoo and rockhopper penguins and of moulting in king penguins were tested by a t-test. The effects of sex in king penguins could not be performed in king penguins because of the low number of females. The inter-specific differences were tested by a one-way ANOVA analysis. Whenever significant effects were detected, multiple comparisons were conducted using the Tukey test. Differences were considered significant at p b 0.05. 3. Results Within gentoo and rockhopper species, no significant differences related to sex in any of the parameters measured. This was probably due to the low number of penguins studied. Therefore, the data are presented as means calculated from all subjects within each species including king penguins, taking the two sexes together. Values for moulting and non-moulting king penguins are shown separately when moulting status was a significant factor. 3.1. Acid–base status and ionic data
2.2. Derived acid base characteristics Using the temperature-corrected values of pH and partial pressure of carbon dioxide, the bicarbonate concentration ([HCO− 3 ], mmol·L− 1) was calculated from the Henderson–Hasselbalch equation assuming a CO2 solubility coefficient (αCO2) and an operational pK′ in penguin plasma at 38.5 °C of 0.0297 mmol·L− 1·mm Hg− 1
The mean key blood acid–base parameters are summarized in a Davenport diagram (Fig. 1) where pH, pCO2 and [HCO− 3 ] are simultaneously presented according to the Henderson–Hasselbalch equation. Rockhopper penguin blood was significantly more alkaline than one of the two other species. Gentoo and non-moulting king penguins' acid–base balances were very close as shown by the proximity of their points on
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the diagram. Although its pH was not significantly different to the others, the blood of moulting king penguins was very acidic. This low pH went with significantly high pCO2 and [HCO− 3 ]. Consequently, moulting was characterized by a 21% increase in CtCO2 (Fig. 2). Monovalent ion (Na+, K+, Cl−) data revealed neither fundamental inter-specific differences nor an effect of moulting (Table 1). On the other hand, [Ca2+] was about 12% higher in king penguins than in the two other species. The combination of plasma monovalent ion concentrations with [HCO− 3 ] to calculate AG, showed a 45% decrease in this parameter in the moulting king penguins (Fig. 3). 3.2. Haematological variables and blood oxygen carrying capacity
4. Discussion 4.1. Effects of moulting Moulting king penguins showed a notable but non-significant blood acidosis probably resulting from an adaptative reduction in energy expenditure to cope with the energetic limitations imposed by fasting (Le Maho and Despin, 1976; McCue, 2010). The significant high pCO2 suggesting a low ventilatory activity in moulting penguins supports this hypothesis. Indeed, ventilation is a significant part of total energetic metabolism in penguins (Stahel and Nicol, 1988). Additionally, feather synthesis during the moult may also contribute to blood acidosis given that this phenomenon is concomitant to a 1.5 to 2 times increase in
pCO2 (mm Hg)
[HCO3-] (mmol.l-1)
32
70
60 50
King p. (during moulting)
*
30
King p.
Rockhopper p.
28
+
40
26
* 34
32
30
28
26
Measured and calculated haematological data for the three species of penguins are summarized in Table 2 and compared with published ones. There was no inter-specific difference in [Hb]. However, Hct was about 19% lower in king penguins compared to the other two species. Consequently, king penguins differed by their significantly high MCHC. RBC and MCV data showed that birds with high red blood cell counts had small cells and vice versa. Thus, gentoo penguins had the least numerous and so far the largest red blood cells compared with those of the other two species. Microscopic observation of the cells confirmed these inter-specific differences. Venous pO2 was not significantly different between the three species (Fig. 4).
34
36
CtCO2 (mmol.l-1)
78
24 Gentoo p.
Rockhopper p.
King p.
Fig. 2. Total carbon dioxide content of blood in the three species of captive penguins studied. Values are mean ± standard error. n values are given in the legend of Fig. 1. The black bars correspond to non-moulting penguins whatever the species. The grey bar corresponds to moulting king penguins. The asterisk indicates a significant difference between moulting and non-moulting individuals of this species (p b 0.05).
plasma concentration of uric acid in king penguins (Cherel et al., 1988). This dual acidosis (respiratory and metabolic) was partially compensated by an alkalosis probably caused by an enhanced [HCO− 3 ] reabsorption by nephrons. The increase in plasma [HCO− 3 ] explained most of the significant increase in CtCO2, dissolved CO2 accounting for about 5% of the total. Moulting caused a significant decrease in the anion gap probably induced by a hypoalbuminaemia. Indeed, at physiological pH, the majority of the anion gap is due to the sum of the anionic charges on circulating proteins, mainly albumin (Kraute and Madias, 2007). The hypoalbuminaemia could result from an increased mobilization of circulating proteins for the synthesis of new feathers. This is corroborated by earlier studies demonstrating a significant decrease in plasma total protein, mainly the albumin fraction, during the moult (Cherel et al., 1988; Ghebremeskel et al., 1989, 1992). In addition, Groscolas and Cherel (1992) and Cherel et al. (1994) showed a drastic loss of total body proteins in moulting king penguins. According to these authors, protein reserves are used not only for new feather synthesis but also to face the increase in energy expenditure associated with the decrease in thermal insulation. However, proteins are considered to be a fuel of last resort, mobilized only after exhaustion of lipid reserves (McCue, 2010). Blood oxygen capacity seems not to be altered by moulting, contrary to data reported in previous studies. For example, Hawkey et al. (1989) demonstrated a clear decrease in nearly all indicators of blood capacity to catch, carry and release oxygen after moulting in rockhopper and magellanic penguins. Similarly, Sergent et al. (2004) reported low red blood-cell counts in moulting compared to non-moulting little penguins. The low plasma iron level indicative of anaemia in moulting
Gentoo p.
24 22 7,22
7,26
7,30
7,34
7,38
7,42
Table 1 Ion concentrations in blood plasma of the three species of captive penguins studied.
pH Fig. 1. Davenport diagram illustrating the blood acid–base status of captive king penguins (Aptenodytes patagonicus, n = 5 (non-moulting) + 4 (moulting)), gentoo penguins (Pygoscelis papua, n = 10) and rockhopper penguins (Eudyptes chrysocome, n = 10). Vertical and horizontal bars represent [HCO− 3 ] and pH standard errors respectively. The oblique line represents the theoretical mean buffer line drawn from the results of Lenfant et al., 1969 and Murrish, 1982. The cross indicates that the blood pH of rockhopper penguins is significantly higher than that of the other two species (p b 0.05). The asterisk indicates that the blood pCO2 and [HCO− 3 ] of moulting king penguins are significantly higher than those of non-moulting penguins (p b 0.05).
[Na+]
[K+]
[Cl−]
[Ca2+]
6.7 ± 0.8 6.5 ± 0.6 4.8 ± 0.4
119 ± 1 120 ± 1 117 ± 1
1.14 ± 0.03 1.15 ± 0.05 1.28 ± 0.02a,c
−1
(mmol·L Gentoo penguin Rockhopper penguin King penguin
152 ± 1 150 ± 0.4 151 ± 2
)
n values are given in the legend of Fig. 1. Moulting having no effect on these parameters in king penguins, the data of moulting and non-moulting subjects were merged into one mean (n = 9). a and c indicate a significant difference between king penguins and rockhopper and gentoo penguins respectively (p b 0.05).
V. Maxime, S. Hassani / Comparative Biochemistry and Physiology, Part A 168 (2014) 76–81
16
Anion Gap (mmol.l-1)
14
12
10
*
8
6
4 Gentoo p.
Rockhopper p.
King p.
Fig. 3. Blood anion gap calculated from AG = [Na+] + [K+] − [Cl−] − [HCO− 3 ] in the three species of captive penguins studied. Values are mean ± standard error. n values are given in the legend of Fig. 1. The black bars correspond to non-moulting penguins whatever the species. The grey bar corresponds to moulting king penguins. The asterisk indicates a significant difference between moulting and non-moulting individuals of this species (p b 0.05).
rockhopper, magellanic and gentoo penguins (Ghebremeskel et al., 1989, 1992) also reflected the weak aerobic energy expenditure associated with moult and fasting. It can be hypothesized that the blood oxygen capacity of non-moulting king penguins which were going to start moulting soon, had already decreased. Indeed, as discussed later, mean Hct of the overall king penguin colony was significantly low. Thus, protein synthesis in moulting penguins may be focused on the renewal of feathers rather than haemoglobin production. 4.2. Inter-specific differences Venous pO2 of the three studied species is elevated, on the whole, compared to the data in the literature obtained for catheterized birds. For gentoo penguins, pO2 was 49% higher in the present study than value reported by Murrish (1982). The difference is similar (56%) between the king penguins (present study) and the emperor penguins (A. forsteri — Ponganis et al., 2007). The conditions of blood sampling of the present experiment which potentially cause stress, could explain these differences. However, the avian spleen is relatively small and nonmuscular and changes in the number of circulating red cells in response
79
to stress are not likely to occur (Hawkey and Samour, 1988). This argument is confirmed by the similarity of our haematological results with those obtained in catheterized or anaesthetized penguins (Murrish, 1982; Nicol et al., 1988). The comparison of data obtained in captive birds with those of wild birds may be the reason for these differences. The Hct of king penguins was systematically lower than data reported in the literature for many penguin species and significantly lower than Hct measured in rockhopper and gentoo penguins in the same living conditions. This observation could be exclusively linked to the fact that moulting was in progress or imminent for all king penguins as previously discussed. Indeed, no hemolyse was observed during sampling. Moreover, king penguins were not anaemic because neither the number of red blood cells nor haemoglobin concentration was low. King penguins [Hb] fell within the interval value of those described for other species. A low Hct may indicate also a low red blood cell volume. MCV is indeed small in king penguins. This may be the consequence of a disruption of the body water balance leading to the shrinkage of blood cells. However, this hypothesis must be rejected at least for moulting birds, starvation inducing an increase in the water content of various tissues and organs (McCue, 2010). The low Hct of king penguins entails a high MCHC, among the highest reported in the literature. Plasma [Ca2+] was markedly higher in king than in gentoo and rockhopper penguins. King penguins being by far the largest and the best diver of the three studied species, it can be assumed that they have, on a mass specific basis, higher circulating [Ca2+] to ensure the calcification of a larger skeleton. Indeed, recently evolved penguins are very similar in their osteology characterized by heavy and solid bones for diving (Simpson, 1971). Since the three species were fed with the same diet, a nutritional origin to this difference is ruled out. Calcium metabolism in birds is associated with eggshell calcification during the period of reproduction. This explains higher circulating calcium levels in female Humboldt penguins compared to males (Smith et al., 2008). The majority of king penguins in the present study being males, the observed inter-specific difference cannot be attributed to egg production. Besides, samplings were performed before the egg-laying phase of king penguins. The venous blood pH of rockhopper penguins was the highest of those reported in the present study and the closest to the scarce data in the literature (Murrish, 1982). The overall acidity of blood in the Océanopolis penguins may be due to lactate production because the blood samples were obtained from restrained birds. It may be due also to their diet, probably more abundant and certainly more regular than in the natural environment. The metabolisation of rich food produces
Table 2 Haematological values in the three species of captive penguins studied (results of the present study and published data). Hb (g·L−1)
Hct (%)
RBC (1012 L−1)
MCV (μm3 cell−1)
MCHC (%)
Gentoo penguin Present study Milsom et al. (1973) Murrish (1982) Hawkey and Samour (1988) Hawkey et al. (1989)
Captive Wild Wild Wild Wild
170 ± 11 164 167 157 172
43 ± 2 43 46 45 45
1.62 ± 0.06b – – 1.61 2.24
251.3 ± 12.4b,c – – 258 206
36.3 ± 0.7 38.4 36.3 37.7 37.8
Rockhopper penguin Present study Hawkey and Samour (1988) Hawkey et al. (1989)
Captive Wild Wild
163 ± 8 179 164
43 ± 2 46 45
2.11 ± 0.11 1.94 2.36
203.2 ± 10.2 234 195
34.7 ± 0.6 39.1 36.6
King penguin Present study Hawkey and Samour (1988)
Captive Wild
160 ± 7 169
36 ± 1a,c 45
1.97 ± 0.13 1.58
185.7 ± 10.1 288
39.9 ± 1.1a,c 37.5
Hb, haemoglobin; Hct, haematocrit, RBC, red blood cell count; MCV, mean corpuscular volume; MCHC, mean corpuscular haemoglobin concentration. Values are mean ± standard error. n values are given in the legend of Fig. 1. Moulting having no effect on these parameters in king penguins, the data of moulting and non-moulting subjects were merged into one mean (n = 9). a and c indicate a significant difference between king penguins and rockhopper and gentoo penguins respectively (p b 0.05). b indicates a significant difference between rockhopper and gentoo penguins (p b 0.05).
80
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80
wild. The other results were within the range of data reported in the literature on wild birds and consequently an effect of diving privation on oxygen storage cannot be concluded. This baseline data may be used for further investigations focused on the health and respiratory physiology of these, or other captive and wild populations of penguins.
pO2 (mm Hg)
75
70
References
65
60
Gentoo p.
Rockhopper p.
King p.
Fig. 4. Oxygen partial pressure (pO2) of venous blood in the three species of captive penguins studied. Values are mean ± standard error. n values are given in the legend of Fig. 1. Moult having no effect on these parameters in king penguins, the data of moulting and non-moulting subjects were merged into one mean (n = 9). No significant differences were observed (p b 0.05).
more acid nutrients. The rockhopper penguins' blood could be the most buffered. The carbonic acid–bicarbonate system is the most important buffer for maintaining blood acid–base balance. Haemoglobin and extra-cellular proteins perform a minor role. However, neither [HCO− 3 ] nor [Hb] and the anion gap presented inter-specific differences in non-moulting birds. Therefore, the more alkaline blood of rockhopper penguins probably results from a relatively higher basal ventilatory activity than the other two species. Although not significantly different, pCO2 was indeed lower in rockhopper penguins. The most striking feature of the gentoo penguin haematology was the compensation of the low red cell count by a high cell volume. It follows that the blood oxygen carrying capacity of gentoo penguins, assessed by [Hb], Hct and pO2 values, was similar to that of rockhopper penguins. Large erythrocyte size may be an adaptation to a cold environment as it contributes to a high blood viscosity to reduce blood flow and heat loss at low temperatures (Block and Murrish, 1974). Since the three penguin species were acclimatized for a long time to the same temperature conditions, this possibility is not convincing. An explanation of this inter-specific difference may be sought in its impact on dynamics of gas exchanges. Indeed, transport processes through membranes are dependent on the surface area-to-volume ratio. This universal rule was verified for the oxygen conductance of avian erythrocytes (Nguyen Phu et al., 1986). Thus, the erythrocyte oxygen transport in gentoo penguins is expected to be lower than that of rockhopper and king penguins. This suggests the lower aerobic energy metabolism of gentoo penguins. This hypothesis is in agreement with the observations of Hawkey et al. (1989) who showed a decrease in red blood cell count concomitant to an increase in mean red blood cell volume in moulting (therefore fasting and energy limited) rockhopper penguins. In conclusion, this work takes advantage of the opportunity to have three different species of penguins simultaneously under the same conditions, something that is not possible in the wild. Captive birds are not exposed to environmental variations (low temperature, erratic food, etc.) that enable us to dismiss these factors in the interpretation. Given their different ecology and diving behaviour in the wild, it is wondered whether milder conditions and the impossibility to dive in captivity have repercussions on parameters which determine oxygen storage and acid–base status in the three studied species. This study was the occasion to analyse these parameters particularly in king penguins during their annual moult and to observe that they undergo adaptations already described in wild birds (decreased energetic metabolism, energy and proteins diverted for feather synthesis). Due to their living conditions and probably to the blood sampling method, captive birds had pO2 and pH respectively higher and lower compared to those in the
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Serum anion gap: uses and limitations in clinical medicine. Clin. J. Am. Soc. Nephrol. 2, 162–174. Le Maho, Y., Despin, B., 1976. Réduction de la dépense énergétique au cours du jeûne chez le manchot royal Aptenodytes patagonicus. C. R. Acad. Sci. D 283, 979–982. Lenfant, C., Kooymann, G.L., Elsner, R., Drabek, M., 1969. Respiratory function of blood of the Adélie penguin Pygoscelis adeliae. Am. J. Physiol. 216, 1598–1600. McCue, M.D., 2010. Starvation physiology: reviewing the different strategies animals use to survive a common challenge. Comp. Biochem. Physiol. A 156, 1–18. Milsom, W.K., Johansen, K., Millard, R.W., 1973. Blood respiratory properties in some Antarctic birds. Condor 75, 472–474. Murrish, D.E., 1982. Acid–base balance in three species of Antarctic penguins exposed to thermal stress. Physiol. Zool. 55, 137–143. Nguyen Phu, D., Yamaguchi, K., Scheid, P., Piiper, J., 1986. Kinetics of oxygen uptake and release by red blood cells of chicken and duck. J. Exp. Biol. 125, 15–27. Nicol, S., 1991. Respiratory properties of the blood of the little penguin Eudyptula minor. Comp. Biochem. Physiol. A 89, 383–386. Nicol, S.C., Melrose, W., Stahel, C.D., 1988. Haematology and metabolism of the blood of the little penguin, Eudyptula minor. Comp. Biochem. Physiol. A 89, 383–386. Ponganis, P.J., Kooyman, G.L., 2000. Diving physiology of birds: a history of studies on polar species. Comp. Biochem. Physiol. A 126, 143–151. Ponganis, P.J., Stockard, T.K., Meir, J.U., Williams, C.L., Ponganis, K.V., Van Dal, R.P., Howard, R., 2007. Returning on empty: extreme blood O2 depletion underlies dive capacity of emperor penguins. J. Exp. Biol. 210, 4279–4285. Ponganis, P.J., Stockard, T.K., Meir, J.U., Williams, C.L., Ponganis, K.V., Howard, R., 2009. O2 store management in diving emperor penguins. J. Exp. Biol. 212, 217–224. Sergent, N., Rogers, T., Cunningham, M., 2004. Influence of biological and ecological factors on hematological values in wild little penguins, Eudyptula minor. Comp. Biochem. Physiol. A 138, 333–339. Siggaard-Andersen, O., Wimberley, P.D., Fogh-Andersen, N., Gøthgen, I.H., 1988. Measured and derived quantities with modern pH and blood gas equipment: calculation algorithms with 54 equations. Scand. J. Clin. Lab. Investig. 189, 7–15. Simpson, G.G., 1971. Review of the fossil penguins from Seymour Island. Philos. R. Soc. Lond. B Biol. 178, 357–487. Smith, K.M., Karesh, W.B., Majluf, P., Paredes, R., Zavalaga, C., Hoogesteijn, R.A., Stetter, M., Breselton, W.E., Puche, H., Cook, R.A., 2008. Health evaluation of
V. Maxime, S. Hassani / Comparative Biochemistry and Physiology, Part A 168 (2014) 76–81 free-ranging Humboldt penguin (Spheniscus humboldti) in Peru. Avian Dis. 52, 130–135. Stahel, C.D., Nicol, S.C., 1988. Ventilation and oxygen extraction in the little penguin (Eudyptula minor), at different temperatures in air and water. Respir. Physiol. 71, 387–398. Villouta, G., Hargreaves, R., Riveros, V., 1997. Hematological and clinical biochemistry findings in captive Humboldt (Spheniscus humboldti). Avian Pathol. 26, 851–858.
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Williams, T.D., Briggs, D.R., Croxall, J.P., Naito, Y., Kato, A., 1992. Diving pattern and performance in relation to foraging ecology in the gentoo penguin, Pygoscelis papua. J. Zool. (Lond.) 227, 211–230. Wilson, R.P., 1995. The Penguins. In: Williams, T.D. (Ed.), Foraging Ecology. Oxford University Press, Oxford, pp. 81–106. Wilson, R.P., Bost, C.A., Pütz, K., Charassins, J.F., Culik, B.M., Adelung, D., 1997. Southern rockhopper penguin Eudyptes chrysocome chrysocome foraging at Possession Island. Polar Biol. 17, 323–329.
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Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa
Blood oxygen- and carbon dioxide-carrying properties in captive penguins: Effects of moulting and inter-specific comparison Valérie Maxime a,⁎, Sami Hassani b a b
Université de Bretagne Sud (UEB), Centre d'Enseignement et de Recherche Yves Coppens, LIMATB (EG2B), Campus de Tohannic, 56017 Vannes Cedex, France Océanopolis, Port de plaisance du Moulin Blanc, BP 411, 29275 Brest Cedex, France
a r t i c l e
i n f o
Article history: Received 23 July 2013 Received in revised form 19 October 2013 Accepted 6 November 2013 Available online 11 November 2013 Keywords: Acid–base balance Aptenodytes patagonicus Eudyptes chrysocome Moulting Penguins O2 storage Plasma ions Pygoscelis papua
a b s t r a c t Venous blood gas-carrying properties were compared in the three captive species of penguins (king, gentoo and rockhopper) at Océanopolis (France). Captivity permitted to control environmental influences. Given their different ecology and diving behaviour in the wild, it was wondered whether milder conditions and dive privation have repercussions on parameters determining oxygen storage and acid–base status of these birds. In addition, this work provided the opportunity to study the effects of moulting in king penguins. This annual event that imposes deep metabolic adjustments is liable to affect blood gas levels. Because of the regular food supply and probably also of the blood sampling conditions, the blood pH of captive penguins was low. This effect was increased in moulting penguins and supposedly due to both the decreased energetic metabolism and the production of uric acid resulting from new feather synthesis. The decrease in the anion gap also revealed the use of plasmatic albumin for this synthesis. The elevated venous PO2 in all birds is not likely due to stress caused by sampling conditions. The other data, in accordance with those in the literature, show neither major influence of captivity nor fundamental interspecific differences, despite potential diving aptitude. © 2013 Elsevier Inc. All rights reserved.
1. Introduction In sphaeniciformes, haematological values can be strongly influenced by site, season, species, age, sex and physiological and nutritional status (Villouta et al., 1997; Sergent et al., 2004). This underlines the great involvement of blood cells in environmental and physiological adaptations. Most of the previous studies devoted to blood respiratory properties were undertaken in wild birds and concern many geographically and biologically different species; for example: Aptenodytes forsteri (Ponganis et al., 2009), Aptenodytes patagonicus (Hawkey and Samour, 1988), Pygoscelis adeliae (Murrish, 1982), Pygoscelis papua and Pygoscelis antarctica (Milsom et al., 1973; Murrish, 1982), Eudyptula minor (Sergent et al., 2004), Eudyptes chrysocome (Karesh et al., 1999), and Spheniscus humboldti (Villouta et al., 1997). The overall analysis of these works does not indicate any fundamental differences between the species despite their different diving abilities. This could be explained not only by a variety of factors that can vary simultaneously but also by the dependence of oxygen storage on body mass and the variable distribution of oxygen between respiratory, blood and muscle compartments (Ponganis and Kooyman, 2000). The fact that captive penguins live for a long time in exactly the same conditions allows the researcher to eliminate the variations of surrounding effects. Taking
⁎ Corresponding author. Tel.: +33 297 017 169. E-mail address: [email protected] (V. Maxime). 1095-6433/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.11.002
advantage of this, this study compares the three penguin species kept at Océanopolis (the ocean discovery park in Brest, France). At the time of this experiment (April), about half of the king penguins at Océanopolis were moulting and the others were going later. Moulting is a physiological event liable to affect oxygen storage and consumption as well as the blood acid–base balance because of the deep metabolic adjustments that characterize this period of total fasting (Cherel et al., 1993; Cherel et al., 2005). Indeed, penguins replace their plumage entirely each year and wild birds spend a long time (14–40 days) fasting ashore because the consequent reduction in thermal insulation precludes staying in cold waters to feed (Groscolas and Cherel, 1992). Moulting involves the use of endogenous lipid reserves and body protein to fulfil the nutrient requirement for feather synthesis and the higher thermogenesis associated with the decrease in thermal insulation. Little work has been carried out on energy requirement during moulting and fasting: Le Maho and Despin (1976), Deswasmes et al. (1980), and Fahlman et al. (2004) show that resting energetic metabolism and heart rate decreased throughout the fast. Thus, in addition to an interspecific comparison, this study was also the opportunity to highlight the effects of moulting on the blood gas-carrying properties of king penguins. The three species of Océanopolis' penguins (king penguin — Aptenodytes patagonicus Miller, gentoo penguin — Pygoscelis papua Forster and rockhopper penguin — Eudyptes chrysocome Forster) were all born in captivity. In their territories of origin (Subantarctic islands and Antarctic peninsula for the first two species, Argentina and
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Chile for the third), these marine birds dive to feed and migrate and differ in the depths they frequently reach. Their ability to dive is positively related to body mass (Wilson, 1995). King penguins, being among the largest marine birds, have exceptional diving abilities, reaching a mean maximum depth of 304 m (Kooyman et al., 1992). Gentoo penguins dive to 156 m (Williams et al., 1992) and rockhopper penguins, the smallest of the three, to 66 m (Wilson et al., 1997). In captivity, penguins never dive and the consequent repercussions on blood gascarrying properties, if there are any, are unknown. Moreover, Océanopolis' penguins are exposed to mild conditions (no polar temperatures, no wind, regular and sufficient food) compared to wild ones. It is hypothesized that these opposing conditions, leading to a less drastic energetic adaptation for captive penguins, may concern the birds' blood properties, particularly when moulting making them temporarily more fragile. The penguins of Océanopolis are primarily destined to a daily public display and consequently they are preserved from all predictable causes of disturbance as much as possible. Thus, this study was carried out during a scheduled clinical examination of the overall population. Our goal was to describe parameters that are relevant in determining oxygen and carbon dioxide storage and their eventual consequences on acid–base status of blood in a comparative study between 1) moulting and non-moulting king penguins and 2) the three bird species kept at Océanopolis. 2. Materials and methods
77
and 6.086, respectively (Nicol, 1991). The total carbon dioxide content (CtCO2, mmol·L− 1) was calculated from: −
CtCO2 ¼ ðαCO2 PCO2 Þ þ ½HCO3 (Siggaard-Andersen et al., 1988). 2.3. Measured and derived oxygen storage capacity of blood The Radiometer analyser measured haematocrits (Hct, %) and calculated haemoglobin concentrations ([Hb], g·L−1) from Hct. These data were compared to those obtained by manual methods. Hct was measured after centrifugation of 70 μL microcapillaries for 5 min at 12,000 g. [Hb] was spectrophotometrically determined by the cyanmethaemoglobin method (kit Sigma 525A). Manually- and automatically-obtained Hct were identical. In contrast, [Hb] was systematically about 15% higher with the manual method. This difference came certainly from the equation used by the Radiometer analyser programme which includes constants for the human blood. Therefore, only manually-obtained [Hb] values were considered. The red blood cell count (RBC, L−1) was obtained by means of a Malassez haemocytometer following 1:200 dilution in Marcano solution (5 g sodium sulphate, 1 mL formaldehyde 40%, qs 100 mL distilled water). Mean red blood cell volume (MCV, μm3·cell−1) and mean cell haemoglobin concentration (MCHC, %) were respectively computed as follows: MCV ¼ Hct=RBC and MCHC ¼ ½Hb=Hct:
2.1. Animals and blood sampling 2.4. Electrolytic assessment The study was carried out on all penguins in captivity at Océanopolis: 9 king penguins; (7 ♂ and 2 ♀) weighing on average 12 kg and aged between 6 and 18 years old, 10 gentoo penguins (5 ♂ and 5 ♀) weighing on average 7 kg and aged between 1 and 21 years old and 10 rockhopper penguins (6 ♂ and 4 ♀) weighing on average 3 kg and aged between 1 and 21 years old. They were all born in captivity and were in good physical condition. They were fed daily ad libitum with thawed Atlantic herring Clupea harengus and Icelandic capelin Mallotus villotus except for the day before sampling and for moulting king penguins that refuse food. The slightly low mean body mass of king penguins reflects the moulting and consequent fasting status of some of them. Venous blood samples of 200 μL were quickly (b1 min) collected in heparinized (lithium heparin 100 IU/mL) syringes from the brachial vein of relatively calm manually-restrained birds. This sampling method was supposedly of minimal disturbance because the birds are used to regular human presence and manipulation. This also avoided the potential effects of sedation and anaesthesia on measured blood parameters. 100 μL of blood was immediately analysed by using a Radiometer ABL77 blood gas analyser (Brønshøj, Denmark) calibrated at 37 °C for a range of respiratory, haematological and ionic data. The time interval between sampling and analysis was always less than 5 min. The gas analyser calculated the measured gas partial pressure (pO2 and pCO2, mm Hg) using the temperature-correction factors given by Burnett and Noonan (1974) assuming a mean penguin temperature of 38.5 °C (Lenfant et al., 1969). The temperature of the birds was not taken to avoid additional disturbance. Moreover, solubility coefficients for O2 and CO2 for penguins in the literature are given at 38.5 °C. The remaining blood was stored on ice (24 h maximum) until required for a red blood cell count and manual checking of haematocrit and haemoglobin concentrations.
Plasma ion concentrations ([Na+], [K+], [Ca2+] and [Cl−], mmol·L−1) were measured by means of the Radiometer analyser's selective sensors. The anion gap (AG, mmol·L−1) was calculated from the following equation: þ
þ
−
−
AG ¼ ½Na þ ½K −½Cl −½HCO3 : 2.5. Statistical analysis Statistical analyses were conducted using Sigmastat 4.0 (Systat Software Inc.). All results were expressed as mean ± s.e.m. The effects of sex in gentoo and rockhopper penguins and of moulting in king penguins were tested by a t-test. The effects of sex in king penguins could not be performed in king penguins because of the low number of females. The inter-specific differences were tested by a one-way ANOVA analysis. Whenever significant effects were detected, multiple comparisons were conducted using the Tukey test. Differences were considered significant at p b 0.05. 3. Results Within gentoo and rockhopper species, no significant differences related to sex in any of the parameters measured. This was probably due to the low number of penguins studied. Therefore, the data are presented as means calculated from all subjects within each species including king penguins, taking the two sexes together. Values for moulting and non-moulting king penguins are shown separately when moulting status was a significant factor. 3.1. Acid–base status and ionic data
2.2. Derived acid base characteristics Using the temperature-corrected values of pH and partial pressure of carbon dioxide, the bicarbonate concentration ([HCO− 3 ], mmol·L− 1) was calculated from the Henderson–Hasselbalch equation assuming a CO2 solubility coefficient (αCO2) and an operational pK′ in penguin plasma at 38.5 °C of 0.0297 mmol·L− 1·mm Hg− 1
The mean key blood acid–base parameters are summarized in a Davenport diagram (Fig. 1) where pH, pCO2 and [HCO− 3 ] are simultaneously presented according to the Henderson–Hasselbalch equation. Rockhopper penguin blood was significantly more alkaline than one of the two other species. Gentoo and non-moulting king penguins' acid–base balances were very close as shown by the proximity of their points on
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the diagram. Although its pH was not significantly different to the others, the blood of moulting king penguins was very acidic. This low pH went with significantly high pCO2 and [HCO− 3 ]. Consequently, moulting was characterized by a 21% increase in CtCO2 (Fig. 2). Monovalent ion (Na+, K+, Cl−) data revealed neither fundamental inter-specific differences nor an effect of moulting (Table 1). On the other hand, [Ca2+] was about 12% higher in king penguins than in the two other species. The combination of plasma monovalent ion concentrations with [HCO− 3 ] to calculate AG, showed a 45% decrease in this parameter in the moulting king penguins (Fig. 3). 3.2. Haematological variables and blood oxygen carrying capacity
4. Discussion 4.1. Effects of moulting Moulting king penguins showed a notable but non-significant blood acidosis probably resulting from an adaptative reduction in energy expenditure to cope with the energetic limitations imposed by fasting (Le Maho and Despin, 1976; McCue, 2010). The significant high pCO2 suggesting a low ventilatory activity in moulting penguins supports this hypothesis. Indeed, ventilation is a significant part of total energetic metabolism in penguins (Stahel and Nicol, 1988). Additionally, feather synthesis during the moult may also contribute to blood acidosis given that this phenomenon is concomitant to a 1.5 to 2 times increase in
pCO2 (mm Hg)
[HCO3-] (mmol.l-1)
32
70
60 50
King p. (during moulting)
*
30
King p.
Rockhopper p.
28
+
40
26
* 34
32
30
28
26
Measured and calculated haematological data for the three species of penguins are summarized in Table 2 and compared with published ones. There was no inter-specific difference in [Hb]. However, Hct was about 19% lower in king penguins compared to the other two species. Consequently, king penguins differed by their significantly high MCHC. RBC and MCV data showed that birds with high red blood cell counts had small cells and vice versa. Thus, gentoo penguins had the least numerous and so far the largest red blood cells compared with those of the other two species. Microscopic observation of the cells confirmed these inter-specific differences. Venous pO2 was not significantly different between the three species (Fig. 4).
34
36
CtCO2 (mmol.l-1)
78
24 Gentoo p.
Rockhopper p.
King p.
Fig. 2. Total carbon dioxide content of blood in the three species of captive penguins studied. Values are mean ± standard error. n values are given in the legend of Fig. 1. The black bars correspond to non-moulting penguins whatever the species. The grey bar corresponds to moulting king penguins. The asterisk indicates a significant difference between moulting and non-moulting individuals of this species (p b 0.05).
plasma concentration of uric acid in king penguins (Cherel et al., 1988). This dual acidosis (respiratory and metabolic) was partially compensated by an alkalosis probably caused by an enhanced [HCO− 3 ] reabsorption by nephrons. The increase in plasma [HCO− 3 ] explained most of the significant increase in CtCO2, dissolved CO2 accounting for about 5% of the total. Moulting caused a significant decrease in the anion gap probably induced by a hypoalbuminaemia. Indeed, at physiological pH, the majority of the anion gap is due to the sum of the anionic charges on circulating proteins, mainly albumin (Kraute and Madias, 2007). The hypoalbuminaemia could result from an increased mobilization of circulating proteins for the synthesis of new feathers. This is corroborated by earlier studies demonstrating a significant decrease in plasma total protein, mainly the albumin fraction, during the moult (Cherel et al., 1988; Ghebremeskel et al., 1989, 1992). In addition, Groscolas and Cherel (1992) and Cherel et al. (1994) showed a drastic loss of total body proteins in moulting king penguins. According to these authors, protein reserves are used not only for new feather synthesis but also to face the increase in energy expenditure associated with the decrease in thermal insulation. However, proteins are considered to be a fuel of last resort, mobilized only after exhaustion of lipid reserves (McCue, 2010). Blood oxygen capacity seems not to be altered by moulting, contrary to data reported in previous studies. For example, Hawkey et al. (1989) demonstrated a clear decrease in nearly all indicators of blood capacity to catch, carry and release oxygen after moulting in rockhopper and magellanic penguins. Similarly, Sergent et al. (2004) reported low red blood-cell counts in moulting compared to non-moulting little penguins. The low plasma iron level indicative of anaemia in moulting
Gentoo p.
24 22 7,22
7,26
7,30
7,34
7,38
7,42
Table 1 Ion concentrations in blood plasma of the three species of captive penguins studied.
pH Fig. 1. Davenport diagram illustrating the blood acid–base status of captive king penguins (Aptenodytes patagonicus, n = 5 (non-moulting) + 4 (moulting)), gentoo penguins (Pygoscelis papua, n = 10) and rockhopper penguins (Eudyptes chrysocome, n = 10). Vertical and horizontal bars represent [HCO− 3 ] and pH standard errors respectively. The oblique line represents the theoretical mean buffer line drawn from the results of Lenfant et al., 1969 and Murrish, 1982. The cross indicates that the blood pH of rockhopper penguins is significantly higher than that of the other two species (p b 0.05). The asterisk indicates that the blood pCO2 and [HCO− 3 ] of moulting king penguins are significantly higher than those of non-moulting penguins (p b 0.05).
[Na+]
[K+]
[Cl−]
[Ca2+]
6.7 ± 0.8 6.5 ± 0.6 4.8 ± 0.4
119 ± 1 120 ± 1 117 ± 1
1.14 ± 0.03 1.15 ± 0.05 1.28 ± 0.02a,c
−1
(mmol·L Gentoo penguin Rockhopper penguin King penguin
152 ± 1 150 ± 0.4 151 ± 2
)
n values are given in the legend of Fig. 1. Moulting having no effect on these parameters in king penguins, the data of moulting and non-moulting subjects were merged into one mean (n = 9). a and c indicate a significant difference between king penguins and rockhopper and gentoo penguins respectively (p b 0.05).
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16
Anion Gap (mmol.l-1)
14
12
10
*
8
6
4 Gentoo p.
Rockhopper p.
King p.
Fig. 3. Blood anion gap calculated from AG = [Na+] + [K+] − [Cl−] − [HCO− 3 ] in the three species of captive penguins studied. Values are mean ± standard error. n values are given in the legend of Fig. 1. The black bars correspond to non-moulting penguins whatever the species. The grey bar corresponds to moulting king penguins. The asterisk indicates a significant difference between moulting and non-moulting individuals of this species (p b 0.05).
rockhopper, magellanic and gentoo penguins (Ghebremeskel et al., 1989, 1992) also reflected the weak aerobic energy expenditure associated with moult and fasting. It can be hypothesized that the blood oxygen capacity of non-moulting king penguins which were going to start moulting soon, had already decreased. Indeed, as discussed later, mean Hct of the overall king penguin colony was significantly low. Thus, protein synthesis in moulting penguins may be focused on the renewal of feathers rather than haemoglobin production. 4.2. Inter-specific differences Venous pO2 of the three studied species is elevated, on the whole, compared to the data in the literature obtained for catheterized birds. For gentoo penguins, pO2 was 49% higher in the present study than value reported by Murrish (1982). The difference is similar (56%) between the king penguins (present study) and the emperor penguins (A. forsteri — Ponganis et al., 2007). The conditions of blood sampling of the present experiment which potentially cause stress, could explain these differences. However, the avian spleen is relatively small and nonmuscular and changes in the number of circulating red cells in response
79
to stress are not likely to occur (Hawkey and Samour, 1988). This argument is confirmed by the similarity of our haematological results with those obtained in catheterized or anaesthetized penguins (Murrish, 1982; Nicol et al., 1988). The comparison of data obtained in captive birds with those of wild birds may be the reason for these differences. The Hct of king penguins was systematically lower than data reported in the literature for many penguin species and significantly lower than Hct measured in rockhopper and gentoo penguins in the same living conditions. This observation could be exclusively linked to the fact that moulting was in progress or imminent for all king penguins as previously discussed. Indeed, no hemolyse was observed during sampling. Moreover, king penguins were not anaemic because neither the number of red blood cells nor haemoglobin concentration was low. King penguins [Hb] fell within the interval value of those described for other species. A low Hct may indicate also a low red blood cell volume. MCV is indeed small in king penguins. This may be the consequence of a disruption of the body water balance leading to the shrinkage of blood cells. However, this hypothesis must be rejected at least for moulting birds, starvation inducing an increase in the water content of various tissues and organs (McCue, 2010). The low Hct of king penguins entails a high MCHC, among the highest reported in the literature. Plasma [Ca2+] was markedly higher in king than in gentoo and rockhopper penguins. King penguins being by far the largest and the best diver of the three studied species, it can be assumed that they have, on a mass specific basis, higher circulating [Ca2+] to ensure the calcification of a larger skeleton. Indeed, recently evolved penguins are very similar in their osteology characterized by heavy and solid bones for diving (Simpson, 1971). Since the three species were fed with the same diet, a nutritional origin to this difference is ruled out. Calcium metabolism in birds is associated with eggshell calcification during the period of reproduction. This explains higher circulating calcium levels in female Humboldt penguins compared to males (Smith et al., 2008). The majority of king penguins in the present study being males, the observed inter-specific difference cannot be attributed to egg production. Besides, samplings were performed before the egg-laying phase of king penguins. The venous blood pH of rockhopper penguins was the highest of those reported in the present study and the closest to the scarce data in the literature (Murrish, 1982). The overall acidity of blood in the Océanopolis penguins may be due to lactate production because the blood samples were obtained from restrained birds. It may be due also to their diet, probably more abundant and certainly more regular than in the natural environment. The metabolisation of rich food produces
Table 2 Haematological values in the three species of captive penguins studied (results of the present study and published data). Hb (g·L−1)
Hct (%)
RBC (1012 L−1)
MCV (μm3 cell−1)
MCHC (%)
Gentoo penguin Present study Milsom et al. (1973) Murrish (1982) Hawkey and Samour (1988) Hawkey et al. (1989)
Captive Wild Wild Wild Wild
170 ± 11 164 167 157 172
43 ± 2 43 46 45 45
1.62 ± 0.06b – – 1.61 2.24
251.3 ± 12.4b,c – – 258 206
36.3 ± 0.7 38.4 36.3 37.7 37.8
Rockhopper penguin Present study Hawkey and Samour (1988) Hawkey et al. (1989)
Captive Wild Wild
163 ± 8 179 164
43 ± 2 46 45
2.11 ± 0.11 1.94 2.36
203.2 ± 10.2 234 195
34.7 ± 0.6 39.1 36.6
King penguin Present study Hawkey and Samour (1988)
Captive Wild
160 ± 7 169
36 ± 1a,c 45
1.97 ± 0.13 1.58
185.7 ± 10.1 288
39.9 ± 1.1a,c 37.5
Hb, haemoglobin; Hct, haematocrit, RBC, red blood cell count; MCV, mean corpuscular volume; MCHC, mean corpuscular haemoglobin concentration. Values are mean ± standard error. n values are given in the legend of Fig. 1. Moulting having no effect on these parameters in king penguins, the data of moulting and non-moulting subjects were merged into one mean (n = 9). a and c indicate a significant difference between king penguins and rockhopper and gentoo penguins respectively (p b 0.05). b indicates a significant difference between rockhopper and gentoo penguins (p b 0.05).
80
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80
wild. The other results were within the range of data reported in the literature on wild birds and consequently an effect of diving privation on oxygen storage cannot be concluded. This baseline data may be used for further investigations focused on the health and respiratory physiology of these, or other captive and wild populations of penguins.
pO2 (mm Hg)
75
70
References
65
60
Gentoo p.
Rockhopper p.
King p.
Fig. 4. Oxygen partial pressure (pO2) of venous blood in the three species of captive penguins studied. Values are mean ± standard error. n values are given in the legend of Fig. 1. Moult having no effect on these parameters in king penguins, the data of moulting and non-moulting subjects were merged into one mean (n = 9). No significant differences were observed (p b 0.05).
more acid nutrients. The rockhopper penguins' blood could be the most buffered. The carbonic acid–bicarbonate system is the most important buffer for maintaining blood acid–base balance. Haemoglobin and extra-cellular proteins perform a minor role. However, neither [HCO− 3 ] nor [Hb] and the anion gap presented inter-specific differences in non-moulting birds. Therefore, the more alkaline blood of rockhopper penguins probably results from a relatively higher basal ventilatory activity than the other two species. Although not significantly different, pCO2 was indeed lower in rockhopper penguins. The most striking feature of the gentoo penguin haematology was the compensation of the low red cell count by a high cell volume. It follows that the blood oxygen carrying capacity of gentoo penguins, assessed by [Hb], Hct and pO2 values, was similar to that of rockhopper penguins. Large erythrocyte size may be an adaptation to a cold environment as it contributes to a high blood viscosity to reduce blood flow and heat loss at low temperatures (Block and Murrish, 1974). Since the three penguin species were acclimatized for a long time to the same temperature conditions, this possibility is not convincing. An explanation of this inter-specific difference may be sought in its impact on dynamics of gas exchanges. Indeed, transport processes through membranes are dependent on the surface area-to-volume ratio. This universal rule was verified for the oxygen conductance of avian erythrocytes (Nguyen Phu et al., 1986). Thus, the erythrocyte oxygen transport in gentoo penguins is expected to be lower than that of rockhopper and king penguins. This suggests the lower aerobic energy metabolism of gentoo penguins. This hypothesis is in agreement with the observations of Hawkey et al. (1989) who showed a decrease in red blood cell count concomitant to an increase in mean red blood cell volume in moulting (therefore fasting and energy limited) rockhopper penguins. In conclusion, this work takes advantage of the opportunity to have three different species of penguins simultaneously under the same conditions, something that is not possible in the wild. Captive birds are not exposed to environmental variations (low temperature, erratic food, etc.) that enable us to dismiss these factors in the interpretation. Given their different ecology and diving behaviour in the wild, it is wondered whether milder conditions and the impossibility to dive in captivity have repercussions on parameters which determine oxygen storage and acid–base status in the three studied species. This study was the occasion to analyse these parameters particularly in king penguins during their annual moult and to observe that they undergo adaptations already described in wild birds (decreased energetic metabolism, energy and proteins diverted for feather synthesis). Due to their living conditions and probably to the blood sampling method, captive birds had pO2 and pH respectively higher and lower compared to those in the
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