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Plasma metabolites in macaroni penguins (Eudyptes chrysolophus) arriving on land for breeding and moulting
Comp.Biochem.Physiol.Vol.99A,No. 1/2,pp. 245--250,1991
0300-9629/91 $3.00+ 0.00 © 1991 PergamonPressplc
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PLASMA METABOLITES IN MACARONI PENGUINS ON LAND FOR BREEDING A N D MOULTING
(EUDYPTES CHRYSOLOPHUS) ARRIVING
K. GHEBREIVlESKEL,*t T. D. WILLIAMS,~ G. WILLIAMS,* D. A. GARDNER§ and M. A. CRAWVORD*~" *Department of Nutritional Biochemistry, Instituteof Zoology, Zoological Society of London, Regents Park, London N W I 4RY, U.K. Telephone: 071-722-3333, Fax: 071-483-4436; SBritish Antarctic Survey, Natural Environment Research Council, High Cross Madingley Road, Cambridge CB3 0ET, U.K.; and §Department of Chemical Pathology, University College Hospital, London, U.K.
(Received 7 September 1990) Abstract--l. Plasma metabolites and nutrients of macaroni penguins (Eudypteschrysolophus)arriving on land in good condition, following intense feeding, for breeding and moulting were investigated. 2. The pre-breeding female birds had significantlyhigher concentrations of totallipid,iron, magnesium, calcium, inorganic phosphate and alkaline phosphatase, and lower concentrations of total cholesterol, all-tram retinol (vitamin A) than the pre-breeding males. 3. With the exception of iron, which was lower in the male, the various parameters measured in male and female pre-moult penguins did not differ. 4. Some of the plasma metabolite concentrations of the pre-moult birds differedsubstantiallyfrom those of their male and female pre-breeding counterparts. 5. The observed differenceswere thought to be the resultof mobilization in response to physiological demands for egg formation and moulting.
6. However, it is possible that discrepancies were partly due to selective ingestion of nutrients in preparation for higher physiological demands.
INTRODUCTION Reproduction and moulting are accompanied by an increased demand and utilization of nutrients in birds. Consequently birds have developed various behavioural and metabolic strategies to ensure the supply of nutrients, and to reduce the strain associated with the higher requirements. One effective strategy shown by some birds, is a separation of the two events by a time interval. Regardless of environment, moulting and reproduction do not overlap in altrices (King et al., 1965; Morton et al., 1969; Payne, 1969; Wolf, 1969). Because most incubating and all moulting penguins (Sphenisciformes) abstain from eating (Croxall, 1982), without becoming torpid (Cherel et al., 1988), the annual physiological cycles of these species often result in a severe deficit of nutrients. Moreover, penguins need to expend additional energy in order to maintain thermal homeostasis in the temperate, sub-Antarctic and Antarctic milieu. The extent of nutritional and metabolic stress that penguins endure during moulting and reproduction is manifested in a substantial loss of body weight. Mean reduction in body weight during incubation was 35--40% in macaroni penguins (Eudyptes chrysolophus) (Croxall, 1982); 40.5% in male and 21.8% in female emperor penguins (Aptenodytes tPresent address: Institute of Brain Chemistry and Human Nutrition, The Hackney Hospital, Homerton High Street, London E9 6BE, U.K.
forsteri) (Groscolas, 1982), and 47% in king penguins (Aptenodytes patagonicus) (Cherel, 1988a); weight loss in emperor, adelie (Pygoscelis adeliae), gentoo (Pygoscelis papua) and chinstrap (Pygoscelis antarctica) penguins during incubation has also been documented (Croxall, 1982). Weight loss during the "moult-fast" is as striking as that in the "incubation-fast". Losses of 50% in macaroni penguins, and 52-60.1% in rockhopper penguins (Eudyptes chrysocome) (Williams et al., 1977; Strange, 1982), 45.1 and 46.5% in adult and juvenile emperor penguins (Groscolas, 1982), 45% in yellow eyed penguins (Megdyptes antipodes), 50% in white flippered penguins (Eudyptula minor) (Sparks and Soper, 1987), have been documented. A drastic decrease in body weight during moulting in penguins has also been reported by Stonehouse (1967), Warham (1974), Le Maho et al. (1976) and Croxall (1982). Before breeding and moulting, penguins resort to excessive food consumption and fat deposition in order to meet the high nutrient expenditure that ensues. However, it is not understood whether penguins depend on selective feeding, or random consumption followed by mobilization of specific nutrients in order to meet the specialized nutrient demands of feather formation and egg production. The study was undertaken to investigate concentrations of plasma metabolites in "pre-breeding" and "pre-moulting" macaroni (Eudyptes chrysolophus) penguins arriving on land, in good condition, following intensive feeding. In particular, we consider
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K. GHEBREMESKELet al.
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differences between male and female birds with respect to the physiological demands of egg production and feather replacement (moulting). MATERIALSAND METHODS Penguins
Healthy, free-living adult macaroni penguins arriving on land for (1) breeding, and (2) moulting were caught on Bird Island, South Georgia (54°0'S 38°02'W). The pre-breeding birds (eight female and eight male) were captured between October and 4 November 1987; and the pre-moulting birds (seven female and seven male) were collected between 6 and 15 March 1988. Blood was taken from the brachial vein into heparinized plastic containers. The plasma was promptly separated from the red blood cells by centrifugation and stored at -20°C until required for analyses. Analytical methods
Alpha-tocopherol (vitamin E) and all-trans retinol (vitamin A) concentrations were determined by the use of a Varian high performance liquid chromatograph (HPLC) equipped with a variable wave-length UV-9050 and fluorometer (Fluorichrom II) detectors (Varian Ltd, Palo Alto, CA, U.S.A.) in series. Absorption area was integrated by a Varian 4290 integrator. The eluting solvents and column conditions employed have been described by Ghebremeskel and Williams (1988). Total lipid and cholesterol were assayed colorimetrically by means of kits supplied by Boehringer Mannheim (Boehringer Mannheim GmbH, Mannheim, W. Germany). Some of the plasma components were analysed by means of clinical Technicon SMAC analyser. An ion selective electrode was used for assaying sodium, potassium and magnesium. Calcium, inorganic phosphate, iron, urea, urate and creatinine were determined by the o-cresolphthalein complexone, phosphomolybdate, ferrozine, diacetyl monoxine, phosphotungstate and alkaline picrate methods, respectively. Total protein was measured by the Biuret method, albumin by means of bromocresol green (BCG) and globulin was estimated by difference. The activities of alkaline phosphatase and aspartate transaminase were determined by means of p-nitrophenylphosphate and coupled aspartate/MDH/NADH, respectively. Data analysis
The unpaired Student's t-test was employed to investigate statistical differences between pre-mating and pre-moulting plasma biochemical values between and within groups. RESULTS
Macaroni penguins that had arrived for breeding but not yet mated were classified as "pre-breeding"; and those that had arrived at the moulting area but not yet started to shed off their old plumage were regarded as "pre-moulting". Table 1 shows the plasma biochemical values of male and female pre-breeding penguins. The female penguins had significantly higher concentrations of total lipid (P < 0.005), iron (P < 0.005), magnesium (P <0.001), calcium (P<0.001), inorganic phosphate (P < 0.001) and alpha-tocopherol/total cholesterol (P < 0.05). Conversely, the levels of total cholesterol (P < 0.005), all-trans retinol (vitamin A) ( P < 0 . 0 0 1 ) and alpha-tocopherol/total lipid (P < 0.05) were higher in the males. There was no statistical difference in total protein, albumin, globulin, urea, urate, creatinine and alpha-tocopherol
values between the two sexes. Moreover, the activities of aspartate transaminase and alkaline phosphatase were similar in the male and female (P > 0.05). The results of the plasma chemical analyses of the pre-moult birds are shown in Table 2. With the exception of iron which was higher in the female (P < 0.05), the analyses were similar (P > 0.05) in all the pre-moult penguins. Since 19 of 20 analyses were similar in male and female pre-moult birds, the two sets of data were combined before comparison was made with the values of their pre-breeding counterparts. The concentrations of total lipid (P <0.001), urea (P < 0.001), magnesium (P < 0.001), calcium (P < 0.001), inorganic phosphate (P < 0.001), alkaline phosphatase (P <0.01) and iron (P <0.001) were higher in the pre-breeding female than in the pre-moult penguins. However, the female prebreeding birds gave lower values for total protein ( P < 0 . 0 2 ) , albumin ( P < 0 . 0 1 ) , all-trans retinol (P < 0.001), creatinine (P < 0.02), sodium (P < 0.02) and alpha-tocopherol/total lipid (P < 0.001). Values for urate, globulin, potassium, aspartate transaminase, alpha-tocopherol, total cholesterol and alphatocopherol/total cholesterol were similar (P > 0.05) in the pre-breeding female and pre-moult penguins. The pre-moult birds differed from pre-breeding males in showing higher values for total protein (P < 0.02), albumin (P < 0.05), globulin (P < 0.05), creatinine (P < 0.02), all-trans retinol (P < 0.005), iron (P <0.001), magnesium (P <0.005), calcium (P < 0.001), alpha-tocopherol/total lipid (P < 0.001) and alpha-tocopherol/total cholesterol (P <0.001), and lower values for total lipid (P <0.001) and cholesterol (P < 0.001). The concentrations of urea, urate, sodium, potassium, inorganic phosphate, alpha-tocopherol and the activities of alkaline phosphatase and aspartate transaminase were, however, comparable (P > 0.05) in the pre-breeding male and pre-moult penguins. DISCUSSION The investigation indicates that the biochemical preparation for reproduction starts before macaroni penguins arrive on land for mating. This preparation is manifested in a significant increase or decrease in the concentrations of some plasma nutrients that are essential for egg production. The concentrations of lipid in the plasma of the females arriving for breeding was higher than that of their male counterparts by 64%. However, the concentrations of total cholesterol and all-trans retinol (vitamin A) were lower in the female by 25 and 44% respectively. The results support the observation of Groscolas (1982) that the level of plasma total lipid in emperor penguins was 80% higher in females than in males during their arrival at the breeding rookery. Although there was no difference in plasma total cholesterol between male and female arriving birds Groscolas reported a 40% reduction in the concentration of cholesterol in the female from the onset of breeding to copulation. Griminger (1976) and Hazelwood (1972) reported higher levels of lipid in mature female chicken during the periods of egg
Plasma metabolites in macaroni penguins
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Table 1. Plasma biochemistry of pre-breeding male and female free-living macaroni (Eudyptes chrysolophus) penguins Male (n = 8) Female (n = 7) Range Mean ± SD Range Mean ± SD Total lipid (g/I) 11.4-21.1 13.7 ± 3.1 13.7-31.1 22.4 ± 5.4 Total cholesterol (g/l) 2.9-4.2 3.6 ± 0.4 1.3-3.4 2.7 ± 0.7 All-trans retinol (mg/I) 1.1-2.2 1.6 ± 0.3 0.7-1.1 0.9 ± 0.1 Alpha-tocopherol (rag/l) 22.0-37.8 32.6 ± 5.4 15.8--43.6 32.0 ± 9.6 Tocopherol/lipid (mg/g) 1.8-2.9 2.4 ± 0.4 0.5--2.2 1.5 ± 0.6 Toeopherol/cholesterol (mg/g) 7.5-10.0 9.0 ± 0.9 4.9-18.2 12.6 ± 4.2 Total protein (g/I) 45.0-55.0 49.8 ± 3.5 41.0-55.0 48.9 ± 5.2 Albumin (g/I) 17.0-23.0 19.1 ± 2.2 15.0-20.0 17.7 ± t.8 Globulin (g/l) 28.0-36.0 30.6 + 2.2 25.0-36.0 31.1 + 4.1 Urea (mmol/l) 0.5-2.6 1.3 + 0.7 1.3-2.2 1.9 ± 0.3 Urate (#mol/l) 227-603 410.3 ± 130.6 177-1174 482.4 ± 325 Creatinine (pmol/I) 39.0-79.0 53.0 ± 11.9 19.0-81.0 47.0 ± 20 Aspartate transaminase (IU/I) 239-410 304.3 ± 56.5 194-365 255.5 ± 59.8 Iron (/~mol/l) 13.0-45.0 23.3 ± 9.4 10.0-189 105.0 ± 57.5 Sodium (mmol/l) 151-160 154.5 ± 2.9 134-159 152.1 ± 7.7 Potassium (mmol/l) 4.6-7.7 5.9 ± 1.0 4.1--6.6 5.3 _ 0.8 Magnesium (mmol/1) 0.9-1.2 1.0 + 0.1 1.3-2.0 1.6 ± 0.3 Calcium (mmol/1) 2.2-2.7 2.4 + 0.2 4.7-6.2 5.4 ± 0.6 Inorganic phosphate (mmol/I) 0.8-1.3 1.1 ± 0.2 1.4-2.8 2.2 ± 0.5 Alkaline phosphatase (IU/I) 58-218 131.6±46.5 164-1424 464.5±438
formation. T h e increase in p l a s m a lipid was partly the result o f p r o t e i n - l i p i d complex t h a t t r a n s p o r t s lipids f r o m the liver to the ovary for deposition (Christie a n d M o o r e , 1972). Striking increases in p l a s m a lipid, p r o t e i n a n d cholesterol have also been observed in o t h e r a n i m a l s such as reptiles d u r i n g vitellogenesis (Clark, 1967; Lawerence, 1987). The r e m a r k a b l y low values of all-trans retinol a n d cholesterol in females relative to males m a y indicate a substantial or complete transfer o f these n u t r i e n t s to the developing yolk. There was n o rise in alphat o c o p h e r o l c o n c o m i t a n t with the increase o f p l a s m a total lipid (Fig. 1), implying t h a t the elevation in lipid c o n c e n t r a t i o n was the result of de novo synthesis in the liver r a t h e r t h a n o f mobilization. A l t h o u g h statistically n o t significant, the total p l a s m a protein a n d a l b u m i n were lower, a n d u r e a a n d u r a t e were higher in the female t h a n in the male breeding p e n g u i n s (Fig. 2). These differences between the sexes could n o t have been r a n d o m variations since the total protein a n d its catabolic p r o d u c t s showed consistent trends. It is possible t h a t the a m i n o acids required for yolk synthesis were being transferred to the o v a r y a n d those n o t needed for egg
f o r m a t i o n utilized for energy purposes, giving rise to increased c o n c e n t r a t i o n s o f urate a n d urea. M a c d o n ald a n d Riddle (1945) reported a rise in p l a s m a protein in the egg laying pigeon; whereas Sturkie a n d N e w m a n (1951a) f o u n d n o difference in the p l a s m a p r o t e i n o f laying a n d n o n - l a y i n g hens. G r o s c o l a s et al. (1975) observed a n increase in p l a s m a a m i n o a d d s , mainly serine d u r i n g egg f o r m a t i o n , in e m p e r o r penguins. The discrepancy between the various c o m m u n i c a t i o n s m a y be due to species differences or m o r e likely to the discrepancies o n the time o n which samples were taken. It is possible t h a t the mobilization a n d transfer o f a m i n o acids need to be p r o t r a c t e d a n d gradual in o r d e r to m a i n t a i n p l a s m a haemostasis. This m a y m a k e it r a t h e r difficult to gauge the extent a n d m o d e o f p r o t e i n utilization in birds t h a t do n o t fast d u r i n g breeding. Calcium, inorganic p h o s p h a t e , iron a n d magnesium c o n c e n t r a t i o n s o f the p l a s m a o f female maca r o n i penguins were substantially greater t h a n those in males (Fig. 3). In the females there was also high activity o f alkaline p h o s p h a t a s e , p e r h a p s due to the active t u r n o v e r o f calcium a n d p h o s p h a t e . Burley a n d V a d e h r a 0 9 8 9 ) reported m o r e p h o s p h o r u s in the
Table 2. Plasma biochemistry of pre-moult male and female free-living macaroni (Eudyptes chrysolophus) penguins M a l e ( N = 7)
Total lipid (g/l) Total cholesterol (g/l) All-trans retinol (mg/1) Alpha-tocopherol (rag/l) Tocopherol/lipid (mg/g) Tocopherol/cholesterol (rag/g) Total protein (g/l) Albumin (g/l) Globulin (g/l) Urea (mmol/l) Urate (#mol/l) Creatinine (/~mol/l) Aspartate transaminase (IU/I) Iron (/zmol/I) Sodium (mmol/l) Potassium (mmol/l) Magnesium (mmol/l) Calcium (retool/l) Inorganic phosphate (mmol/l) Alkaline phosphatase (IU/I)
Range 8.5-12.1 2.6-3.9 1.8-3.5 27.5-43.8 3.0-4.7 10.2-14.7 49.0-63.0 17.0-24.0 28.0-39.0 0.3-1.5 28.0-539 48.0-82.0 246-1230 24.0-41.0 148-168 4.6-6.3 0.9-1.3 2.6-3.0 0.7-1.7 64-208
Mean ± SD 10.1 _ 1.2 3.0 ± 0.4 2.3 _+0.6 37.1 _+5.5 3.7 _ 0.5 12.4 + 1.4 55.9 ± 5.4 21.9 ± 2.7 34.0 ± 3.6 0.9 ± 0.4 320 ± 146 65.0 ± 9.4 441.4 + 324 35.6 ± 5.8 159.4 ± 6.1 5.3 ± 0.5 1.2 ± 0.1 2.9 ± 0.1 1.4 _+0.4 132.6 ± 47.0
Female ( N = 7)
Range 7.9-10.4 2.2-2.9 1.7-3.2 29.2-51.4 3.2-4.9 l 1.7-17.6 46.0-66.0 17.0-29.0 29.0-39.0 0.4-1.7 31.0-543 51.0-83 214-530 34.0-59.0 154-161 4.3-7.2 1..0-1.3 2.9-3.0 0.8-1.5 90.0-215
Mean ± SD 9.3 _+0.8 2.6 _+0.3 2.5 _+0.5 36.1 _+7.2 3.9 ± 0.6 13.9 ± 1.9 57.0 ± 7.1 22.6 ± 4.1 34.4 ± 3.5 1.1 ± 0.5 319.9 ± 142 66.1 ± 10.6 326.8 ± 103 44.6 ± 8.2 157.4 ± 2.4 5.4 ± 1.0 1.1 ± 0.1 2.8 ± 0.1 1.2 ± 0.2 143.4 ± 35.0
K. GHEBREMESKELet al.
248 4-
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Fig. 1. Plasma lipid, cholesterol, vitamin A and E in male and female macaroni penguins arriving for breeding. Male N = 8, female N = 7. plasma of laying than non-laying birds. Serum calcium in pullets rose from 10 mg % to 16-30 mg % during the 10 days before the commencement of laying, the increase was mainly due to the appearance of complex phospholipoproteins which bind large quantities of calcium (Simkiss and Taylor, 1976). According to Hazelwood (1972) the egg shell calcium is laid down during the last 15-16hr of egg shell formation. Moreover, because the shell gland does not store significant quantities of calcium the ion must be extracted continuously from the blood (Sturkie and Mueller, 1976). The activity of ovarian oestrogenic hormone was responsible for the elevation in the concentrations of calcium and phosphorus during ovulation in the pigeon (Fleischmann and Fried, 1945). Assenmacher (1973) reported that the concentration of calcium in the plasma of laying hens is regulated by female hormones. Before and during egg laying, oestrogen produces changes in protein-bound calcium (Urist, 1959), iron and calcium transporting serum proteins (Beuving and Gruber, 1971), and total protein (Sturkie and Newman, 1951b). The concentrations of the plasma metabolites investigated in the pre-moult macaroni penguins was broadly comparable to those of the pre-moult rockhopper and magellanic (Spheniscus magellanicus) penguins (Ghebremeskel et al., 1989a; Williams et al., 1989). However, the concentrations of all-trans retinol (vitamin A) (Ghebremeskel et al., 1989b), urea, potassium, inorganic phosphate and alphatocopherol of the magellanics, and of creatinine, 60-
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Fig. 3. Plasma iron, magnesium, calcium, inorganic phosphate, potassium and alkaline phosphatase in male and female macaroni penguins on arrival for breeding. Male N = 8, female N = 7. potassium, alkaline phosphatase and all-trans retinol of the rockhopper were different from those of the pre-moult macaronis. The discrepancies may be due to species variability and/or dietary habits. The plasma of the pre-moult macaroni penguins had more total protein, albumin and globulin than that of the pre-breeding male. This was likely to have been the result of mobilization of these nutrients in preparation for the formation of feathers and associated tissues. Although mobilization of the cation binding proteins would in part explain the relatively higher calcium, iron and magnesium levels in the pre-moult birds, a structural and or functional role of calcium, iron magnesium during feather synthesis could not be excluded. Concentrations of total lipid and total cholesterol were higher in the pre-breeding male than in premoulting birds, whereas the plasma of the latter group had more all-trans retinol. This observation suggests that all-trans retinol may have some vital function during feather growth in macaroni penguins. Lower concentrations of total lipids also characterize the period of feather synthesis in emperor penguins (Groscolas, 1982). Cher¢l et al. (1988a, b) reported that nutrient mobilization during breeding and moulting in penguins was under hormonal control. However, it is possible that birds such as penguins that fast during breeding and moulting may selectively ingest nutrients to reduce the stress that ensues from the higher requirement of certain nutrients for feather growth and egg formation. Indeed, Moss (1972) found that birds were more selective for nitrogen and phosphorus if less of the nutrients were available; moreover hens were particularly selective in spring before the commencement of laying. Regulation of calcium intake in relation to demands for egg formation was observed by Hughes and Wood-Gush (1971). West and Meng (1968) contended that birds were capable of selective fatty acid deposition depending on the conditions of captivity and also on the normal seasonal shifts of their physiological state in the wild. The pre-moult nutrient status of penguins is known to be a reflection of intensive feeding during the period preceding the moult. Nevertheless, the condition of birds arriving on land for breeding, and the substantial but variable nutrient requirement for reproduction (pairing to chick feeding) would be
Plasma metabolites in macaroni penguins expected to influence pre-moult nutrient status and survival. Van Heezik and Davis (1990) reported that the low body weights of adult yellow-eyed penguins (Megadyptes antipodes) at Nugget Point during the 1985-86 breeding season was followed by a high chick mortality due to starvation, delayed onset of moult of adults and juveniles, and higher death of the moulting birds. The data presented demonstrate that there was a remarkable distinction in the concentrations of plasma metabolites between the male and the female macaroni penguins arriving on land for breeding. Although the differences are clearly related to eggformation, the mode of control is not evident. "Premoulting" male and female birds however, showed no such differences, indicating that both sexes have similar nutrient requirements for feather formation. The variation in the levels of some plasma parameters between the "pre-breeding" and the "pre-moulting" macaroni penguins might be a reflection of the different nutrients required for breeding and feather growth. The results of the investigation have significance to the nutritional management and clinical monitoring of captive and wild penguins. REFERENCES
Assenmacher I, (1973) The peripheral endocrine glands. In: Avian Biology (Edited by Farner D. S., King J. R. and Parkes K. C.), Vol. III, pp. 183-286. Academic Press, New York. Beuving G. and Gruber M. (1971) Induction of phosvitin synthesis in roosters by estradiol injection. Biochim. Biophys. Acta. 232, 529-536. Burley R. W. and Vadehra D. V. (1989) The Avian Egg: Chemistry and Biology. A Wiley-Interscience publication, New York. Clark N. B. (1967) Influence of estrogen upon serum calcium, phosphate and protein concentrations of fresh water turtles. Comp. Biochem. Physiol. 20, 823-834. Cherel Y., Robin J.-P. and Le Maho Y. (1988) Physiology and biochemistry of long term fasting in birds. Can. J. Zool. 66, 159-166. Cherel Y., Leloup J. and Le Maho Y. (1988a) Fasting in king penguin--II. Hormonal and metabolic changes during molt. Am. J. Physiol. 254, R178-R184. Cherel Y., Robin J.-P., Walch O., Karmann H., Netchitailo P. and Le Maho Y. (1988b) Fasting in king penguin--I. Hormonal and metabolic changes during breeding. Am. J. Physiol. 254, R170-R177. Christie W. W. and Moore J. H. (1972) The lipid components of plasma, liver, ovarian follicles in the domestic chicken. Comp. Biochem. Physiol. 41B, 287-295. Croxall J. P. (1982) Energy cost of incubation and moult in petrels and penguins. J. Anim. Ecol. 51, 177-194. Fleischmann W. and Fried I. A. (1945) Studies on the mechanism of hypercholesterolemia and hypercalcemia induced by estrogen in immature chicks. Endocrinology 36, 406-415. Ghebremeskel K. and Williams G. (1988) Plasma retinol and alpha-tocopherol levels in captive wild animals. Comp. Biochem. Physiol. 89B, 279-283. Ghebremeskel K., Williams G., Keymer I. F., Horsley D. T. and Gardner D. A. (1989a) Plasma chemistry of rockhopper (Eudyptes crestatus), magellanic (Spheniscus magellanicus) and gentoo (Pygoscelis papua) wild penguins in relation to moult. Comp. Biochem. Physiol. 92A, 43-47. Ghebremeskel K., Williams G., Keymer I. F. and Horsley D. T. (1989b) Liver and plasma retinol (vitamin A) in CBPA ~lll2-...Q
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wild, and liver retinol in captive penguins (Spheniscidae). d. ZooL, Lond. 219, 245-250. Griminger P. (1976) Lipid metabolism. In: Avian Physiology (Edited by Sturkie, P. D.), pp. 253-261, Springer, New York. Groscolas R., Carpentier C. et Lemonnier F. (1975) Variation de la concentration des acides amines libres du plasma au cours du cycle reproducteur chez manchot empereur, Aptenodytes forsteri. Comp. Biochem. Physiol. 51B, 57-67. Groscolas R. (1982) Changes in plasma lipids during breeding molting, and starvation in male and female emperor penguins (Aptenodytes forsteri). Physiol. ZooL 55, 45-55. Hazelwood R. L. (1972) The Intermediary Metabolism of Birds In: Avian biology (Edited by Famer D. S., King J. R. and Parkes K. C.), Vol. II, pp. 471-526. Academic Press, New York. Hughes B. O. and Wood-Gush D. G. M. (1971) A specific appetite for calcium in domestic chickens. Anim. Behav. 19, 490-499. King J. R., Farrier D. S. and Morton M. L. (1965) The lipid reserves of white-crowned sparrows on the breeding ground in central Alaska. Auk 82, 236-252. Lawerenc¢ K. (1987) Seasonal variations in blood biochemistry of long term captive Mediterranean tortoises (Testudo graeca and Testudo hermanni). Res. Vet. Sci. 43, 379-383. Le Maho Y., Delclitte P. and Chatonnet J. (1976) Thermoregulation in fasting emperor penguins under natural conditions. Am. J. Physiol. 231, 913-922. Macdonald M. R. and Riddle O. (1945) The effect of reproduction and estrogen administration on the partition of calcium, phosphorus, and nitrogen in pigeon plasma. J. Biol. Chem. 159, 445-464. Morton M. L., King J. R. and Farner D. S. (1969) Postnuptial and postjuvenal molt in white-crowned sparrow in central Alaska. Condor 71, 376-385. Moss R. (1972) Food selection by Red Grouse (Lagopus lagopus scoticus) in relation to chemical composition. J. Anita. Ecol. 9, 837-847. Payne R. B. (1969) Overlap of breeding and molting schedules in a collection of African birds. Condor 71, 140-145. Simkiss K. and Taylor T. G. (1976) Shell formation. In: Physiology and Biochemistry of the Domestic Fowl (Edited by Bell D. J. and Freeman B. M.), ch. 55. Academic Press, New York. Sparks J. and Soper T. (1987) Penguins, p. 109. David and Charles Publishers Ltd., London. Stonehouse B. (1967) The general biology and thermal balance of penguins. Adv. Ecol. Res. 4, 131-196. Strange I. J. (1982) Breeding ecology of the rockhopper penguin (Eudyptes crestatus) in the Falkland Islands. Le Gerfaut 72, 137-188. Sturkie P. D. and Newman H. J. (1951a) Effects of estrogen and thyroxine upon plasma proteins and blood volume in the fowl. Endocrinology 49, 565-570. Sturkie P. D. and Newman H. J. (1951b) Proteins of chickens as influenced by time of laying, ovulation, number of blood samples taken and plasma volume. Poultry sci. 30, 240-248. Sturkie P. D. and Mueller W. J. (1976) Reproduction in the female and egg production. In Avian Physiology (Edited by Sturkie P. D.), pp. 311-330. Springer, New York. Urist M. R. (1959) The effect of calcium deprivation upon the blood, adrenal cortex, ovary and skeleton in domestic fowl, Rec. Prog. Horm. Res. 15, 455-477. Van Hcczik Y. and Davis L. (1990) Effects of food variability on growth rates, fledging sizes and reproductive success in the yellow-eyed penguin (Magadyptes antipides). Ibis 132, 354-365. Warham J. (1974) The Fiordland crested penguin Eudyptes crestatus. Ibis. 116, 1-27. West G. C. and Meng M. S. (1968) Seasonal changes in body weight and fat and the relation of fatty acid
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composition to diet in the willow ptarmigan. Wilson Bull. 80, 426-441. Williams G., Ghebremeskel K., Keymer I. F. and Horsley D. T. (1989) Plasma alpha-tocopherol, total lipids and total cholesterol in the wild rockhopper, magellanic and gentoo penguins before and after moulting. Vet. Rec. 124, 585-586.
Williams A. J., Siegfried W. R., Burger A. E. and Berruti A. (1977) Body composition and energy metabolism of moulting eudyptid penguins. Comp. Biochem. Physiol. 56A, 27-30. Wolf L. L. (1969) Breeding and molting periods in a Costa Rican population of the Andean sparrow. Condor 71, 212-219.
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PLASMA METABOLITES IN MACARONI PENGUINS ON LAND FOR BREEDING A N D MOULTING
(EUDYPTES CHRYSOLOPHUS) ARRIVING
K. GHEBREIVlESKEL,*t T. D. WILLIAMS,~ G. WILLIAMS,* D. A. GARDNER§ and M. A. CRAWVORD*~" *Department of Nutritional Biochemistry, Instituteof Zoology, Zoological Society of London, Regents Park, London N W I 4RY, U.K. Telephone: 071-722-3333, Fax: 071-483-4436; SBritish Antarctic Survey, Natural Environment Research Council, High Cross Madingley Road, Cambridge CB3 0ET, U.K.; and §Department of Chemical Pathology, University College Hospital, London, U.K.
(Received 7 September 1990) Abstract--l. Plasma metabolites and nutrients of macaroni penguins (Eudypteschrysolophus)arriving on land in good condition, following intense feeding, for breeding and moulting were investigated. 2. The pre-breeding female birds had significantlyhigher concentrations of totallipid,iron, magnesium, calcium, inorganic phosphate and alkaline phosphatase, and lower concentrations of total cholesterol, all-tram retinol (vitamin A) than the pre-breeding males. 3. With the exception of iron, which was lower in the male, the various parameters measured in male and female pre-moult penguins did not differ. 4. Some of the plasma metabolite concentrations of the pre-moult birds differedsubstantiallyfrom those of their male and female pre-breeding counterparts. 5. The observed differenceswere thought to be the resultof mobilization in response to physiological demands for egg formation and moulting.
6. However, it is possible that discrepancies were partly due to selective ingestion of nutrients in preparation for higher physiological demands.
INTRODUCTION Reproduction and moulting are accompanied by an increased demand and utilization of nutrients in birds. Consequently birds have developed various behavioural and metabolic strategies to ensure the supply of nutrients, and to reduce the strain associated with the higher requirements. One effective strategy shown by some birds, is a separation of the two events by a time interval. Regardless of environment, moulting and reproduction do not overlap in altrices (King et al., 1965; Morton et al., 1969; Payne, 1969; Wolf, 1969). Because most incubating and all moulting penguins (Sphenisciformes) abstain from eating (Croxall, 1982), without becoming torpid (Cherel et al., 1988), the annual physiological cycles of these species often result in a severe deficit of nutrients. Moreover, penguins need to expend additional energy in order to maintain thermal homeostasis in the temperate, sub-Antarctic and Antarctic milieu. The extent of nutritional and metabolic stress that penguins endure during moulting and reproduction is manifested in a substantial loss of body weight. Mean reduction in body weight during incubation was 35--40% in macaroni penguins (Eudyptes chrysolophus) (Croxall, 1982); 40.5% in male and 21.8% in female emperor penguins (Aptenodytes tPresent address: Institute of Brain Chemistry and Human Nutrition, The Hackney Hospital, Homerton High Street, London E9 6BE, U.K.
forsteri) (Groscolas, 1982), and 47% in king penguins (Aptenodytes patagonicus) (Cherel, 1988a); weight loss in emperor, adelie (Pygoscelis adeliae), gentoo (Pygoscelis papua) and chinstrap (Pygoscelis antarctica) penguins during incubation has also been documented (Croxall, 1982). Weight loss during the "moult-fast" is as striking as that in the "incubation-fast". Losses of 50% in macaroni penguins, and 52-60.1% in rockhopper penguins (Eudyptes chrysocome) (Williams et al., 1977; Strange, 1982), 45.1 and 46.5% in adult and juvenile emperor penguins (Groscolas, 1982), 45% in yellow eyed penguins (Megdyptes antipodes), 50% in white flippered penguins (Eudyptula minor) (Sparks and Soper, 1987), have been documented. A drastic decrease in body weight during moulting in penguins has also been reported by Stonehouse (1967), Warham (1974), Le Maho et al. (1976) and Croxall (1982). Before breeding and moulting, penguins resort to excessive food consumption and fat deposition in order to meet the high nutrient expenditure that ensues. However, it is not understood whether penguins depend on selective feeding, or random consumption followed by mobilization of specific nutrients in order to meet the specialized nutrient demands of feather formation and egg production. The study was undertaken to investigate concentrations of plasma metabolites in "pre-breeding" and "pre-moulting" macaroni (Eudyptes chrysolophus) penguins arriving on land, in good condition, following intensive feeding. In particular, we consider
245
K. GHEBREMESKELet al.
246
differences between male and female birds with respect to the physiological demands of egg production and feather replacement (moulting). MATERIALSAND METHODS Penguins
Healthy, free-living adult macaroni penguins arriving on land for (1) breeding, and (2) moulting were caught on Bird Island, South Georgia (54°0'S 38°02'W). The pre-breeding birds (eight female and eight male) were captured between October and 4 November 1987; and the pre-moulting birds (seven female and seven male) were collected between 6 and 15 March 1988. Blood was taken from the brachial vein into heparinized plastic containers. The plasma was promptly separated from the red blood cells by centrifugation and stored at -20°C until required for analyses. Analytical methods
Alpha-tocopherol (vitamin E) and all-trans retinol (vitamin A) concentrations were determined by the use of a Varian high performance liquid chromatograph (HPLC) equipped with a variable wave-length UV-9050 and fluorometer (Fluorichrom II) detectors (Varian Ltd, Palo Alto, CA, U.S.A.) in series. Absorption area was integrated by a Varian 4290 integrator. The eluting solvents and column conditions employed have been described by Ghebremeskel and Williams (1988). Total lipid and cholesterol were assayed colorimetrically by means of kits supplied by Boehringer Mannheim (Boehringer Mannheim GmbH, Mannheim, W. Germany). Some of the plasma components were analysed by means of clinical Technicon SMAC analyser. An ion selective electrode was used for assaying sodium, potassium and magnesium. Calcium, inorganic phosphate, iron, urea, urate and creatinine were determined by the o-cresolphthalein complexone, phosphomolybdate, ferrozine, diacetyl monoxine, phosphotungstate and alkaline picrate methods, respectively. Total protein was measured by the Biuret method, albumin by means of bromocresol green (BCG) and globulin was estimated by difference. The activities of alkaline phosphatase and aspartate transaminase were determined by means of p-nitrophenylphosphate and coupled aspartate/MDH/NADH, respectively. Data analysis
The unpaired Student's t-test was employed to investigate statistical differences between pre-mating and pre-moulting plasma biochemical values between and within groups. RESULTS
Macaroni penguins that had arrived for breeding but not yet mated were classified as "pre-breeding"; and those that had arrived at the moulting area but not yet started to shed off their old plumage were regarded as "pre-moulting". Table 1 shows the plasma biochemical values of male and female pre-breeding penguins. The female penguins had significantly higher concentrations of total lipid (P < 0.005), iron (P < 0.005), magnesium (P <0.001), calcium (P<0.001), inorganic phosphate (P < 0.001) and alpha-tocopherol/total cholesterol (P < 0.05). Conversely, the levels of total cholesterol (P < 0.005), all-trans retinol (vitamin A) ( P < 0 . 0 0 1 ) and alpha-tocopherol/total lipid (P < 0.05) were higher in the males. There was no statistical difference in total protein, albumin, globulin, urea, urate, creatinine and alpha-tocopherol
values between the two sexes. Moreover, the activities of aspartate transaminase and alkaline phosphatase were similar in the male and female (P > 0.05). The results of the plasma chemical analyses of the pre-moult birds are shown in Table 2. With the exception of iron which was higher in the female (P < 0.05), the analyses were similar (P > 0.05) in all the pre-moult penguins. Since 19 of 20 analyses were similar in male and female pre-moult birds, the two sets of data were combined before comparison was made with the values of their pre-breeding counterparts. The concentrations of total lipid (P <0.001), urea (P < 0.001), magnesium (P < 0.001), calcium (P < 0.001), inorganic phosphate (P < 0.001), alkaline phosphatase (P <0.01) and iron (P <0.001) were higher in the pre-breeding female than in the pre-moult penguins. However, the female prebreeding birds gave lower values for total protein ( P < 0 . 0 2 ) , albumin ( P < 0 . 0 1 ) , all-trans retinol (P < 0.001), creatinine (P < 0.02), sodium (P < 0.02) and alpha-tocopherol/total lipid (P < 0.001). Values for urate, globulin, potassium, aspartate transaminase, alpha-tocopherol, total cholesterol and alphatocopherol/total cholesterol were similar (P > 0.05) in the pre-breeding female and pre-moult penguins. The pre-moult birds differed from pre-breeding males in showing higher values for total protein (P < 0.02), albumin (P < 0.05), globulin (P < 0.05), creatinine (P < 0.02), all-trans retinol (P < 0.005), iron (P <0.001), magnesium (P <0.005), calcium (P < 0.001), alpha-tocopherol/total lipid (P < 0.001) and alpha-tocopherol/total cholesterol (P <0.001), and lower values for total lipid (P <0.001) and cholesterol (P < 0.001). The concentrations of urea, urate, sodium, potassium, inorganic phosphate, alpha-tocopherol and the activities of alkaline phosphatase and aspartate transaminase were, however, comparable (P > 0.05) in the pre-breeding male and pre-moult penguins. DISCUSSION The investigation indicates that the biochemical preparation for reproduction starts before macaroni penguins arrive on land for mating. This preparation is manifested in a significant increase or decrease in the concentrations of some plasma nutrients that are essential for egg production. The concentrations of lipid in the plasma of the females arriving for breeding was higher than that of their male counterparts by 64%. However, the concentrations of total cholesterol and all-trans retinol (vitamin A) were lower in the female by 25 and 44% respectively. The results support the observation of Groscolas (1982) that the level of plasma total lipid in emperor penguins was 80% higher in females than in males during their arrival at the breeding rookery. Although there was no difference in plasma total cholesterol between male and female arriving birds Groscolas reported a 40% reduction in the concentration of cholesterol in the female from the onset of breeding to copulation. Griminger (1976) and Hazelwood (1972) reported higher levels of lipid in mature female chicken during the periods of egg
Plasma metabolites in macaroni penguins
247
Table 1. Plasma biochemistry of pre-breeding male and female free-living macaroni (Eudyptes chrysolophus) penguins Male (n = 8) Female (n = 7) Range Mean ± SD Range Mean ± SD Total lipid (g/I) 11.4-21.1 13.7 ± 3.1 13.7-31.1 22.4 ± 5.4 Total cholesterol (g/l) 2.9-4.2 3.6 ± 0.4 1.3-3.4 2.7 ± 0.7 All-trans retinol (mg/I) 1.1-2.2 1.6 ± 0.3 0.7-1.1 0.9 ± 0.1 Alpha-tocopherol (rag/l) 22.0-37.8 32.6 ± 5.4 15.8--43.6 32.0 ± 9.6 Tocopherol/lipid (mg/g) 1.8-2.9 2.4 ± 0.4 0.5--2.2 1.5 ± 0.6 Toeopherol/cholesterol (mg/g) 7.5-10.0 9.0 ± 0.9 4.9-18.2 12.6 ± 4.2 Total protein (g/I) 45.0-55.0 49.8 ± 3.5 41.0-55.0 48.9 ± 5.2 Albumin (g/I) 17.0-23.0 19.1 ± 2.2 15.0-20.0 17.7 ± t.8 Globulin (g/l) 28.0-36.0 30.6 + 2.2 25.0-36.0 31.1 + 4.1 Urea (mmol/l) 0.5-2.6 1.3 + 0.7 1.3-2.2 1.9 ± 0.3 Urate (#mol/l) 227-603 410.3 ± 130.6 177-1174 482.4 ± 325 Creatinine (pmol/I) 39.0-79.0 53.0 ± 11.9 19.0-81.0 47.0 ± 20 Aspartate transaminase (IU/I) 239-410 304.3 ± 56.5 194-365 255.5 ± 59.8 Iron (/~mol/l) 13.0-45.0 23.3 ± 9.4 10.0-189 105.0 ± 57.5 Sodium (mmol/l) 151-160 154.5 ± 2.9 134-159 152.1 ± 7.7 Potassium (mmol/l) 4.6-7.7 5.9 ± 1.0 4.1--6.6 5.3 _ 0.8 Magnesium (mmol/1) 0.9-1.2 1.0 + 0.1 1.3-2.0 1.6 ± 0.3 Calcium (mmol/1) 2.2-2.7 2.4 + 0.2 4.7-6.2 5.4 ± 0.6 Inorganic phosphate (mmol/I) 0.8-1.3 1.1 ± 0.2 1.4-2.8 2.2 ± 0.5 Alkaline phosphatase (IU/I) 58-218 131.6±46.5 164-1424 464.5±438
formation. T h e increase in p l a s m a lipid was partly the result o f p r o t e i n - l i p i d complex t h a t t r a n s p o r t s lipids f r o m the liver to the ovary for deposition (Christie a n d M o o r e , 1972). Striking increases in p l a s m a lipid, p r o t e i n a n d cholesterol have also been observed in o t h e r a n i m a l s such as reptiles d u r i n g vitellogenesis (Clark, 1967; Lawerence, 1987). The r e m a r k a b l y low values of all-trans retinol a n d cholesterol in females relative to males m a y indicate a substantial or complete transfer o f these n u t r i e n t s to the developing yolk. There was n o rise in alphat o c o p h e r o l c o n c o m i t a n t with the increase o f p l a s m a total lipid (Fig. 1), implying t h a t the elevation in lipid c o n c e n t r a t i o n was the result of de novo synthesis in the liver r a t h e r t h a n o f mobilization. A l t h o u g h statistically n o t significant, the total p l a s m a protein a n d a l b u m i n were lower, a n d u r e a a n d u r a t e were higher in the female t h a n in the male breeding p e n g u i n s (Fig. 2). These differences between the sexes could n o t have been r a n d o m variations since the total protein a n d its catabolic p r o d u c t s showed consistent trends. It is possible t h a t the a m i n o acids required for yolk synthesis were being transferred to the o v a r y a n d those n o t needed for egg
f o r m a t i o n utilized for energy purposes, giving rise to increased c o n c e n t r a t i o n s o f urate a n d urea. M a c d o n ald a n d Riddle (1945) reported a rise in p l a s m a protein in the egg laying pigeon; whereas Sturkie a n d N e w m a n (1951a) f o u n d n o difference in the p l a s m a p r o t e i n o f laying a n d n o n - l a y i n g hens. G r o s c o l a s et al. (1975) observed a n increase in p l a s m a a m i n o a d d s , mainly serine d u r i n g egg f o r m a t i o n , in e m p e r o r penguins. The discrepancy between the various c o m m u n i c a t i o n s m a y be due to species differences or m o r e likely to the discrepancies o n the time o n which samples were taken. It is possible t h a t the mobilization a n d transfer o f a m i n o acids need to be p r o t r a c t e d a n d gradual in o r d e r to m a i n t a i n p l a s m a haemostasis. This m a y m a k e it r a t h e r difficult to gauge the extent a n d m o d e o f p r o t e i n utilization in birds t h a t do n o t fast d u r i n g breeding. Calcium, inorganic p h o s p h a t e , iron a n d magnesium c o n c e n t r a t i o n s o f the p l a s m a o f female maca r o n i penguins were substantially greater t h a n those in males (Fig. 3). In the females there was also high activity o f alkaline p h o s p h a t a s e , p e r h a p s due to the active t u r n o v e r o f calcium a n d p h o s p h a t e . Burley a n d V a d e h r a 0 9 8 9 ) reported m o r e p h o s p h o r u s in the
Table 2. Plasma biochemistry of pre-moult male and female free-living macaroni (Eudyptes chrysolophus) penguins M a l e ( N = 7)
Total lipid (g/l) Total cholesterol (g/l) All-trans retinol (mg/1) Alpha-tocopherol (rag/l) Tocopherol/lipid (mg/g) Tocopherol/cholesterol (rag/g) Total protein (g/l) Albumin (g/l) Globulin (g/l) Urea (mmol/l) Urate (#mol/l) Creatinine (/~mol/l) Aspartate transaminase (IU/I) Iron (/zmol/I) Sodium (mmol/l) Potassium (mmol/l) Magnesium (mmol/l) Calcium (retool/l) Inorganic phosphate (mmol/l) Alkaline phosphatase (IU/I)
Range 8.5-12.1 2.6-3.9 1.8-3.5 27.5-43.8 3.0-4.7 10.2-14.7 49.0-63.0 17.0-24.0 28.0-39.0 0.3-1.5 28.0-539 48.0-82.0 246-1230 24.0-41.0 148-168 4.6-6.3 0.9-1.3 2.6-3.0 0.7-1.7 64-208
Mean ± SD 10.1 _ 1.2 3.0 ± 0.4 2.3 _+0.6 37.1 _+5.5 3.7 _ 0.5 12.4 + 1.4 55.9 ± 5.4 21.9 ± 2.7 34.0 ± 3.6 0.9 ± 0.4 320 ± 146 65.0 ± 9.4 441.4 + 324 35.6 ± 5.8 159.4 ± 6.1 5.3 ± 0.5 1.2 ± 0.1 2.9 ± 0.1 1.4 _+0.4 132.6 ± 47.0
Female ( N = 7)
Range 7.9-10.4 2.2-2.9 1.7-3.2 29.2-51.4 3.2-4.9 l 1.7-17.6 46.0-66.0 17.0-29.0 29.0-39.0 0.4-1.7 31.0-543 51.0-83 214-530 34.0-59.0 154-161 4.3-7.2 1..0-1.3 2.9-3.0 0.8-1.5 90.0-215
Mean ± SD 9.3 _+0.8 2.6 _+0.3 2.5 _+0.5 36.1 _+7.2 3.9 ± 0.6 13.9 ± 1.9 57.0 ± 7.1 22.6 ± 4.1 34.4 ± 3.5 1.1 ± 0.5 319.9 ± 142 66.1 ± 10.6 326.8 ± 103 44.6 ± 8.2 157.4 ± 2.4 5.4 ± 1.0 1.1 ± 0.1 2.8 ± 0.1 1.2 ± 0.2 143.4 ± 35.0
K. GHEBREMESKELet al.
248 4-
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Fig. 1. Plasma lipid, cholesterol, vitamin A and E in male and female macaroni penguins arriving for breeding. Male N = 8, female N = 7. plasma of laying than non-laying birds. Serum calcium in pullets rose from 10 mg % to 16-30 mg % during the 10 days before the commencement of laying, the increase was mainly due to the appearance of complex phospholipoproteins which bind large quantities of calcium (Simkiss and Taylor, 1976). According to Hazelwood (1972) the egg shell calcium is laid down during the last 15-16hr of egg shell formation. Moreover, because the shell gland does not store significant quantities of calcium the ion must be extracted continuously from the blood (Sturkie and Mueller, 1976). The activity of ovarian oestrogenic hormone was responsible for the elevation in the concentrations of calcium and phosphorus during ovulation in the pigeon (Fleischmann and Fried, 1945). Assenmacher (1973) reported that the concentration of calcium in the plasma of laying hens is regulated by female hormones. Before and during egg laying, oestrogen produces changes in protein-bound calcium (Urist, 1959), iron and calcium transporting serum proteins (Beuving and Gruber, 1971), and total protein (Sturkie and Newman, 1951b). The concentrations of the plasma metabolites investigated in the pre-moult macaroni penguins was broadly comparable to those of the pre-moult rockhopper and magellanic (Spheniscus magellanicus) penguins (Ghebremeskel et al., 1989a; Williams et al., 1989). However, the concentrations of all-trans retinol (vitamin A) (Ghebremeskel et al., 1989b), urea, potassium, inorganic phosphate and alphatocopherol of the magellanics, and of creatinine, 60-
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umol/lOmlumol/dl wmol/1 IU/dl
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I ~I IU/dl
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Fig. 3. Plasma iron, magnesium, calcium, inorganic phosphate, potassium and alkaline phosphatase in male and female macaroni penguins on arrival for breeding. Male N = 8, female N = 7. potassium, alkaline phosphatase and all-trans retinol of the rockhopper were different from those of the pre-moult macaronis. The discrepancies may be due to species variability and/or dietary habits. The plasma of the pre-moult macaroni penguins had more total protein, albumin and globulin than that of the pre-breeding male. This was likely to have been the result of mobilization of these nutrients in preparation for the formation of feathers and associated tissues. Although mobilization of the cation binding proteins would in part explain the relatively higher calcium, iron and magnesium levels in the pre-moult birds, a structural and or functional role of calcium, iron magnesium during feather synthesis could not be excluded. Concentrations of total lipid and total cholesterol were higher in the pre-breeding male than in premoulting birds, whereas the plasma of the latter group had more all-trans retinol. This observation suggests that all-trans retinol may have some vital function during feather growth in macaroni penguins. Lower concentrations of total lipids also characterize the period of feather synthesis in emperor penguins (Groscolas, 1982). Cher¢l et al. (1988a, b) reported that nutrient mobilization during breeding and moulting in penguins was under hormonal control. However, it is possible that birds such as penguins that fast during breeding and moulting may selectively ingest nutrients to reduce the stress that ensues from the higher requirement of certain nutrients for feather growth and egg formation. Indeed, Moss (1972) found that birds were more selective for nitrogen and phosphorus if less of the nutrients were available; moreover hens were particularly selective in spring before the commencement of laying. Regulation of calcium intake in relation to demands for egg formation was observed by Hughes and Wood-Gush (1971). West and Meng (1968) contended that birds were capable of selective fatty acid deposition depending on the conditions of captivity and also on the normal seasonal shifts of their physiological state in the wild. The pre-moult nutrient status of penguins is known to be a reflection of intensive feeding during the period preceding the moult. Nevertheless, the condition of birds arriving on land for breeding, and the substantial but variable nutrient requirement for reproduction (pairing to chick feeding) would be
Plasma metabolites in macaroni penguins expected to influence pre-moult nutrient status and survival. Van Heezik and Davis (1990) reported that the low body weights of adult yellow-eyed penguins (Megadyptes antipodes) at Nugget Point during the 1985-86 breeding season was followed by a high chick mortality due to starvation, delayed onset of moult of adults and juveniles, and higher death of the moulting birds. The data presented demonstrate that there was a remarkable distinction in the concentrations of plasma metabolites between the male and the female macaroni penguins arriving on land for breeding. Although the differences are clearly related to eggformation, the mode of control is not evident. "Premoulting" male and female birds however, showed no such differences, indicating that both sexes have similar nutrient requirements for feather formation. The variation in the levels of some plasma parameters between the "pre-breeding" and the "pre-moulting" macaroni penguins might be a reflection of the different nutrients required for breeding and feather growth. The results of the investigation have significance to the nutritional management and clinical monitoring of captive and wild penguins. REFERENCES
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