Concentrations of retinol, 3,4didehydroretinol ... - Wiley Online Library

DOI: 10.1111/j.1439-0396.2011.01219.x

ORIGINAL ARTICLE

Concentrations of retinol, 3,4-didehydroretinol, and retinyl esters in plasma of free-ranging birds of prey K. Mu¨ller1, J. Raila2, R. Altenkamp3, D. Schmidt4, R. Dietrich5, A. Hurtienne2, M. Wink6, O. Krone7, L. Brunnberg1 and F. J. Schweigert2 1 2 3 4 5 6 7

College of Veterinary Medicine, Small Animal Clinic, Freie Universita¨t Berlin, Germany Institute of Nutritional Science, University of Potsdam, Potsdam-Rehbru¨cke, Germany Institute of Biology and Zoology (Section Evolutionary Biology), Freie Universita¨t Berlin, Germany Bird Conservation Centre of the Nature and Biodiversity Conservation Union (NABU), Mo¨ssingen, Germany Berlin, Germany Institute of Pharmacy & Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany Leibniz-Institute for Zoo and Wildlife Research, Berlin, Germany

Keywords birds of prey, plasma, retinol, 3,4-didehydroretinol, retinyl esters Correspondence Kerstin Mu¨ller, Freie Universita¨t Berlin, College of Veterinary Medicine, Small Animal Clinic, Oertzenweg 19b, D-14163 Berlin, Germany. Tel: + 49-30-83-86-23-88; Fax: + 49-30-83-8625-21; E-mail: [email protected] Received: 13 April 2011; accepted: 10 July 2011

Summary This study investigated vitamin A compounds in the plasma of healthy free-ranging Central European raptors with different feeding strategies. Plasma samples of nestlings of white-tailed sea eagle [white-tailed sea eagle (WTSE), Haliaeetus albicilla) (n = 32), osprey (Pandion haliaetus) (n = 39), northern goshawk (Accipiter gentilis) (n = 25), common buzzard (Buteo buteo) (n = 31), and honey buzzard (Pernis apivorus) (n = 18) and adults of WTSE (n = 10), osprey (n = 31), and northern goshawk (n = 45) were investigated with reversed-phase-high-performance liquid chromatography (RP-HPLC). In WTSE, northern goshawks and common buzzards retinol were the main plasma component of vitamin A, whilst in ospreys and honey buzzards, 3,4-didehydroretinol predominated. The median of the retinol plasma concentration in the nestlings group ranged from 0.12 to 3.80 lM and in the adult group from 0.15 to 6.13 lM. Median plasma concentrations of 3,4-didehydroretinol in nestlings ranged from 0.06 to 3.55 lM. In adults, northern goshawks had the lowest plasma concentration of 3,4-didehydroretinol followed by WTSE and ospreys. The plasma of all investigated species contained retinyl esters (palmitate, oleate, and stearate). The results show considerable speciesspecific differences in the vitamin A plasma concentrations that might be caused by different nutrition strategies.

Introduction Vitamin A is a group of fat-soluble substances possessing the biological activity of all-trans-retinol, which occurs naturally only in animals including humans. To this group belong retinol (vitamin A1), 3,4-didehydroretinol (vitamin A2), as well as their retinyl esters and aldehyde (retinal, 3,4-didehydroretinal). The biological functions of vitamin A are manifold and involve sensory performances like 1044

vision, the embryonic development, cell differentiation, haematopoesis, growth, reproduction, immune functions, and development of tumour resistances (Moore, 1957; Olson, 1984; Wolf, 1984). Herbivores can meet their vitamin A requirements from provitamin A carotenoids such as a- and b-carotene or b-cryptoxanthin found in the vegetable diet, which are converted via retinal to retinol and retinyl esters in the enterocytes (Wolf, 1984). Carnivores ingest preformed vitamin A as retinol

Journal of Animal Physiology and Animal Nutrition 96 (2012) 1044–1053 ª 2011 Blackwell Verlag GmbH

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and retinyl esters with their prey (Underwood, 1984). The main storage tissues of retinol and retinyl esters are the liver and kidneys (Moore, 1957; Wolf, 1984; Raila et al., 2000). The mobilisation of vitamin A in the liver and the transport of retinol to peripheral organs are homeostatically regulated. The concentration of retinol in blood plasma remains constant independently of the hepatic vitamin A stores or the amount of vitamin A ingested with the diet. Therefore, retinol plasma concentrations give only vague information about the vitamin A status (Underwood et al., 1979). The transport of retinol from the liver to target tissues is mediated by retinol binding protein (RBP) (Blomhoff et al., 1990). On the other hand, only low concentrations of retinyl esters are found in the plasma of fasted humans and many mammal species although they are adequately supplemented with vitamin A. An increase in plasma retinyl esters can only be detected post-prandial after food intake (Krasinski et al., 1990) or during chronic hypervitaminosis A (Mallia et al., 1975; Smith and Goodman, 1976; Penniston et al., 2003). Carnivores seem to be an exception in this regard, because they physiologically transport lipoprotein bound retinyl esters (predominantly retinyl stearate, retinyl palmitate, and retinyl oleate) in the blood (Wilson et al., 1987; Schweigert, 1988). Although vitamin A2 (3,4-didehydroretinol) was mainly found in freshwater fish (Balasundaram et al., 1956), only few studies have dealt so far with the biological properties of 3,4-didehydroretinol, whose importance in mammals and birds seems to be negligible (Shantz and Brinkman, 1950; John et al., 1966). 3,4-Didehydroretinol has a lower biological activity than retinol (Shantz and Brinkman, 1950) but both compounds have the same binding affinity to cellular RBP (MacDonald and Ong, 1987). Gillam (1938) detected 3,4-didehydroretinol in livers of fish-eating mammals like the European otter (Lutra lutra) and harbour seal (Phoca vitulina), but not in the only investigated fish-eating bird species, the common kingfisher (Alcedo atthis). 3,4-Didehydroretinol was also found in different tissues of some amphibian species (Collins et al., 1953), in plasma, liver, and kidneys of American minks (Neovison vison) fed with fresh and saltwater fish (Ka¨kela¨ et al., 1999), and in liver and blubber of two freshwater seal species (Ka¨kela¨ et al., 1997). 3,4-Didehydroretinol and accordingly its oxygenated form (3,4-dehydroretinal) serve also as chromophores of visual pigments in some fish, amphibian, reptile (Bridges, 1972; Lythgoe, 1972) and some freshwater crustacean species (Suzuki and Eguchi, 1987). In chicken Journal of Animal Physiology and Animal Nutrition. ª 2011 Blackwell Verlag GmbH

Concentrations of retinol, 3,4-didehydroretinol, and retinyl esters

embryos, 3,4-dehydroretinol and all-trans-3,4-dehydroretinol acid were found in high concentrations (Scott et al., 1994). So far, only little information is available about the vitamin A metabolism in birds of prey. The aim of our study was therefore to determine the plasma concentrations of retinol, 3,4-didehydroretinol, and retinyl esters in healthy free-living nestlings and adults of piscivorous (osprey), carnivorous (northern goshawk) as well as piscivorous and carnivorous [white-tailed sea eagle (WTSE)] birds of prey. As an objective ‘health status’ is very difficult to evaluate in individual wild birds, we chose for our study for the most part territorial birds prior to or during the breeding season. In long-lived birds of prey, these birds should represent the ‘fittest’ part of any population (Newton, 1998). Additionally, we studied nestlings of the same species and of the predominantly carnivorous common buzzard and the almost exclusively insectivorous honey buzzard. Materials and methods Animals

Consent for blood sampling and bird captures were issued by the responsible authorities. Nestlings of WTSE (n = 32), ospreys (n = 39), northern goshawks (n = 25), common buzzards (n = 31), and honey buzzards (n = 18) included in the study were banded between 2004 and 2007 in the frame of monitoring projects undertaken in Germany over several years. In association with other scientific projects, adult birds of WTSE (n = 10), osprey (n = 31), and northern goshawk (n = 45) were also captured between 2004 and 2007 using different methods. WTSE was captured during their hunts on lakes in Mecklenburg-Western Pomerania (Germany) from August to October. The method used includes a floating fish snare (Cain and Hodges, 1989). As the birds were captured during hunting activities, it remained unclear if those birds were breeding. Adult ospreys were captured in June and July using a mist net (dho-gaza, 12.0 · 4.5 m; 210/2 denier, mesh aperture 60 mm, two pockets) (Schmidt, 1999). Breeding birds with 4 to 6 weeks old nestlings in the nest were provoked to attack a mounted specimen of an adult WTSE placed on the ground adjacent to the nesting site. When the ospreys attacked, they got caught in the net. Adult northern goshawks were captured in their breeding territories near the nesting site between December and March using a bal-chatri and a decoy pigeon (Bloom, 1987). 1045

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All birds underwent a thorough clinical examination. Complete haematology and blood chemistry were also performed. Only birds without any signs of disease or abnormalities in haematology and blood chemistry were included. After blood sampling, data of body mass, crop filling, and some biometrical measurements (length of the wing, beak, hypotarsus, tarsometatarsus, and foot span) were taken. The sex of the nestlings of WTSE, ospreys, common buzzards, and honey buzzards was determined by the amplification of sex-specific alleles by PCR after Kahn et al. (1998) and Becker and Wink (2002). The determination of the sex in northern goshawk nestlings as well as in osprey and in northern goshawk adults was performed based on biometrical measurements previously described (Glutz von Blotzheim et al., 1989; Bijlsma, 1997). The age determination of adult birds was possible if they had been banded as nestlings. Unbanded ospreys, northern goshawks, and WTSE older than one year were considered to be adults. The age was determined based on feather characteristics (northern goshawk and osprey) according to Glutz von Blotzheim et al. (1989) in WTSE according to Forsman (1999). An age estimation of WTSE nestlings was made according to the figures of Heinroth and Heinroth (1926), for the ospreys according to Glutz von Blotzheim et al. (1989). For both species, no guidelines for precise age determination are available. The age of northern goshawk, common buzzard, and honey buzzard nestlings was determined by wing length and weight (±1 day) according to Bijlsma (1997). In four osprey and two honey buzzard nestlings, precise age determination was not possible. Blood sampling

Approximately 1.5 ml blood was collected from the Vena ulnaris using a 24 G needle (Sterican, Fa. B. Braun, Melsungen, Germany), stored in a lithium heparin tube (Fa. Sarstedt, Nu¨mbrecht, Germany), and centrifuged with a manually operated centrifuge (Fa. Hettich, Tuttlingen, Germany) for 10 to 15 min at 1076 g immediately after blood sampling. The plasma was stored in the dark at 7 C and frozen at 75 C below zero no later than 10 h after sampling. High-performance liquid chromatography-analysis

All specimens were examined within three months after blood sampling. Concentrations of plasma 1046

retinol, 3,4-didehydroretinol, and retinyl esters were measured using a modified gradient reversed-phase HPLC-system (Waters, Eschborn, Germany) after the organic extraction. For the separation of the compounds, a reversed-phase C30 column (5 lm, 250 · 4.6 mm; YMC, Wilmington, DE, USA) in line with a C18 pre-column (Luna, Phenomenex, Germany) was applied as previously described (Schweigert et al., 2003). Retinol, 3,4-didehydroretinol, and the retinyl esters were identified by comparison of their retention time with external standards (retinol, retinyl palmitate: Sigma, Deisenhofen, Germany; retinyl oleate and retinyl stearate: Hoffmann-La Roche, Basel, Switzerland; 3,4-didehydroretinol: DSM, Heerlen, Netherlands), by the use of a photodiode array detector (PDA Model 996, Waters) and quantified by measuring the absorption at 325 nm. The following detection limits were determined: retinol 0.023 lM, 3,4-didehydroretinol 0.054 lM, retinyl oleate 0.029 lM, retinyl palmitate 0.034 lM, and retinyl stearate 0.026 lM. Accuracy and precision were verified using a standard reference material (SMR 968a fat-soluble vitamins in human serum; National Institute of Standards and Technology (NIST), Gaithersburg, USA). The recovery rate was 95%, and the coefficient of variability was less than 5%. Statistical analysis

Statistical analysis was performed with the software program SPSS 15.0 (SPSS, Chicago, IL, USA). The data distribution was verified with the Kolmogorov–Smirnov test (corrected after Lilliefors (1967). As the data were mostly not normally distributed, all data were indicated as median and ranges. Despite that mean and standard deviation (SD) were given for comparison of our data with those in the literature. The Mann–Whitney U test was used to evaluate species-, age-, and sex-specific differences. Differences were considered significant at p < 0.05. Results Plasma retinol in nestlings

Number, age, and weight of the nestlings are presented in Table 1 and corresponding values of adults in Table 2. The median retinol plasma concentration of the nestlings varied between 0.12 lM (osprey) and 3.80 lM (northern goshawk) (Table 3). Osprey and honey buzzard nestlings had the lowest retinol plasma concentrations of the investigated species. Apart from common buzzard and goshawk nestlings, significant differences were observed between the Journal of Animal Physiology and Animal Nutrition. ª 2011 Blackwell Verlag GmbH

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Table 1 Number, age, sex, and body mass of the nestlings Age in weeks/days

Body mass (g)

n Species

Sex

Known

Unknown

Median

Min

Max

n

Median

Min

Max

White-tailed sea eagle

Female Male Female Male Female Male Female Male Female Male

14 18 19 16 11 14 15 16 10 6

0 0 0 4 0 0 0 0 2 0

8 8 5 5 24 26 31 32 30 25

6 5 4 3 19 21 23 22 28 18

10 10 8 7 29 32 36 36 34 34

14 18 19 20 11 14 15 16 8 5

4905 3800 1654 1412 845 663 780 755 755 700

4100 2950 1274 890 690 560 655 405 620 480

5590 4840 1818 1612 1065 800 950 865 1005 845

Osprey Northern goshawk Common buzzard Honey buzzard

Age of white-tailed sea eagles and ospreys in weeks; age of northern goshawks, common buzzards, and honey buzzards in days, n, number of individuals; min, minimum; max, maximum.

Table 2 Number, age, sex, and body mass of adults Age

Body mass (g)

n Species

Sex

Known

Unknown

Median

Min

Max

n

Median

Min

Max


Female Male Female Male Female Male

1 2 9 8 9 9

4 3 10 4 16 11

2 3 9 11 4 5

2 2 3 9 1 2

2 4 17 14 16 15

5 5 19 12 25 20

5370 4170 1760 1423 1300 818

5255 3130 1375 1300 1065 720

6150 4670 1960 1605 1725 990

Osprey Northern goshawk

Age as calendar years, n, number of individuals; min, minimum; max, maximum.

Table 3 Retinol plasma concentrations (lmol/l) in nestlings of white-tailed sea eagle, osprey, northern goshawk, common buzzard, and honey buzzard as well as adults of white-tailed sea eagle, osprey, and northern goshawk Species

Age

Sex

n

Median

Min

Max

Mean

SD


Nestling Nestling Adult Adult Nestling Nestling Adult Adult Nestling Nestling Adult Adult Nestling Nestling Nestling Nestling

Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male

14 18 5 5 19 20 19 12 11 14 25 20 15 16 12 6

3.22 2.38 3.38 4.92 0.09 0.16 0.14 0.16 3.67 3.85 5.49 6.94 3.82 3.70 0.48 0.32

2.11 0.8 3.18 4.29 bdl 0.06 0.05 0.01 2.55 2.80 2.38 4.41 2.47 2.94 0.30 0.24

4.62 4.15 5.66 5.99 0.47 1.69 0.42 0.41 4.20 5.00 7.98 9.08 4.52 4.62 0.75 0.38

3.12 2.42 4.21 4.91 0.12 0.25 0.17 0.17 3.55 3.81 5.33 7.05 3.58 3.81 0.46 0.32

0.76 0.97 1.25 0.70 0.10 0.35 0.09 0.12 0.51 0.54 1.50 1.20 0.71 0.53 0.12 0.05

Osprey

Northern goshawk

Common buzzard Honey buzzard

n, number of individuals; min, minimum; max, maximum; SD, standard deviation; bdl, below detection limit.

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Table 4 3,4-Didehydroretinol plasma concentrations (lmol/l) in nestlings of white-tailed sea eagle, osprey, northern goshawk, common buzzard, and honey buzzard as well as adults of white-tailed sea eagle, osprey, and northern goshawk Species

Age

Sex

n

Median

Min

Max

Mean

SD




14 18 5 5 19 20 19 12 11 14 25 20 15 16 12 6

0.30 0.39 0.12 0.13 2.73 2.63 5.38 6.41 0.08 0.06 0.04 0.04 0.06 0.07 3.74 3.29

0.07 0.07 0.04 0.07 2.51 2.21 4.18 5.59 bdl bdl 0.01 0.01 bdl bdl 2.56 2.95

1.30 1.32 0.17 0.16 3.41 3.70 6.88 7.93 0.25 0.18 0.21 0.12 0.16 0.22 6.36 4.36

0.39 0.42 0.12 0.12 2.87 2.76 5.51 6.50 0.07 0.07 0.06 0.04 0.06 0.07 4.11 3.52

0.31 0.34 0.06 0.04 0.30 0.43 0.88 0.79 0.08 0.06 0.05 0.03 0.05 0.07 1.15 0.64

Osprey

Northern goshawk



Table 5 Total retinyl ester plasma concentrations (lmol/l) in nestlings of white-tailed sea eagle, osprey, northern goshawk, common buzzard and honey buzzard as well as adults of white-tailed sea eagle, osprey, and northern goshawk Species

Age

Sex

n

Median

Min

Max

Mean

SD




14 18 5 5 19 20 19 12 11 14 25 20 15 16 12 6

0.44 0.38 0.38 0.48 0.12 0.13 0.28 0.38 0.58 0.97 0.58 0.54 0.54 0.47 0.25 0.17

0.07 bdl 0.35 0.33 0.03 0.06 0.15 0.14 0.40 0.36 0.13 0.11 0.23 0.11 0.14 0.06

2.61 1.62 0.43 0.70 0.26 0.44 0.95 1.29 3.14 6.58 2.69 2.33 5.22 1.77 0.73 0.49

0.56 0.47 0.38 0.50 0.13 0.15 0.33 0.46 1.21 1.59 0.66 0.69 0.87 0.72 0.31 0.21

0.62 0.39 0.03 0.14 0.06 0.08 0.19 0.30 0.97 1.68 0.52 0.55 1.24 0.51 0.17 0.15

Osprey

Northern goshawk



other species (p < 0.01). Sex differences were only found in two species. Male osprey nestlings had higher retinol plasma concentrations than females (Mann–Whitney U test, p = 0.005; Table 3), whereas male honey buzzards had lower retinol plasma concentrations than females (p = 0.010).

higher retinol plasma concentrations than the females (p < 0.001; Table 3). No sex differences were found between adults of ospreys and WTSE (p > 0.05). Adult WTSE and goshawks had significantly higher retinol concentrations than the respective nestlings (p < 0.001; Table 3).

Plasma retinol in adults

Plasma 3,4-didehydroretinol in nestlings

For adult birds, northern goshawks had the highest plasma concentration, followed by WTSE and ospreys (Table 3). Male northern goshawks had

In the plasma of all investigated species and age groups, concentrations of 3,4-didehydroretinol were detected in different amounts (Table 4). The highest

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plasma concentrations of 3,4-didehydroretinol in the nestlings were found in honey buzzards, the lowest in common buzzards and goshawks. Apart from common buzzards and goshawks, significant species-specific differences were found (p < 0.001). Sex-specific differences were not observed in the nestlings group. Plasma 3,4-didehydroretinol in adults

Adult ospreys had the highest plasma concentration of 3,4-didehydroretinol followed by WTSE and goshawks (p < 0.001) (Table 4). Adult goshawks had lower 3,4-didehydroretinol plasma concentrations than adult WTSE (p < 0.001). Male ospreys showed significant higher 3,4-didehydroretinol plasma concentrations than females (p = 0.01, Table 4), whereas in goshawks and WTSE no sex-specific differences were found (p = 0.235 and 0.841). Goshawk and WTSE nestlings had higher 3,4-didehydroretinol plasma concentrations than adults (p < 0.001 und p = 0.003; Table 4), whereas adult osprey nestlings had lower concentrations than adults (p < 0.001; Table 4). Plasma retinyl esters in nestlings

In the plasma of all investigated species retinyl palmitate, retinyl oleate, and retinyl stearate were detected. In the nestling group, the highest total ester concentrations were determined in northern goshawks (Table 5). Osprey and honey buzzard nestlings revealed only small ester plasma concentrations. No sex-specific differences of the total ester plasma concentration in the nestlings group were obvious (p > 0.05). Plasma retinyl esters in adults

Adult ospreys had significantly lower total ester plasma concentrations than adult WTSE and northern goshawks (p = 0.033, 0.002, Table 5). No differences were found between northern goshawks and WTSE (p = 0.295). Osprey nestlings had a significant lower total ester concentration than the adults (p < 0.004; Table 5), whereas northern goshawk nestlings had a higher total ester plasma concentration than the adults (p = 0.005, Table 5). In WTSE, no significant age differences of the total ester plasma concentrations were observed. Palmitate was the main ester of the three detected esters in all investigated birds of prey. Journal of Animal Physiology and Animal Nutrition. ª 2011 Blackwell Verlag GmbH


Apart from northern goshawk nestlings, the oleate was the second most abundant ester, followed by stearate. Discussion This is the first study presenting data about vitamin A components in free-living birds of prey from Central Europe that perform different feeding strategies. Ospreys feed exclusively on fish year-round and WTSEs do so mainly during summer (Oehme, 1975; Glutz von Blotzheim et al., 1989). In contrast, the diet of northern goshawks is based mainly on birds, whereas common buzzards feed mainly on small mammals (Glutz von Blotzheim et al., 1989). Honey buzzards, which are phylogenetically not related to buzzards, are outstanding specialists that feed on different growth stages of several wasp species, bumblebees and to a lesser amount on other insects, amphibians, reptiles, and small bird nestlings (Glutz von Blotzheim et al., 1989). Our results represent for the first time concentrations of plasma vitamin A components in wild birds of prey. Vitamin A plasma concentration can strongly be influenced by dietary vitamin A intake (Crissey et al., 1998; Su¨nder and Flachowsky, 2001; Raila et al., 2002); therefore, the use of captive birds of prey will not be adequate to get information about the physiologic vitamin A plasma composition in free-ranging birds of prey. All birds in our study underwent a throughout clinical as well as complete blood examination, and based on these results, the investigated individuals were considered to be healthy. Additionally, the adult northern goshawks and ospreys were successful breeding birds in the year of capture. Breeding birds are the fittest segment of an adult bird population because these birds are able to conquer and defend a breeding territory as well as they are able to foster chicks (Newton, 1979, 1998)Only few studies investigated the vitamin A plasma concentration in birds of prey. Schweigert et al. (1991) investigated plasma of one to three individuals of different species of birds of prey kept in captivity. Because of the low number of investigated birds, species-, age-, and sex-specific differences could not be addressed. The retinol plasma concentration of our investigated nestlings differed, apart from the common buzzard and northern goshawks species specifically. Significant speciesspecific differences were also observed in the adult birds. Retinol plasma concentrations comparable to our investigated common buzzard and northern goshawk nestlings were found in the plasma of black 1049


footed penguins (Spheniscus demersus) held in captivity and in free-living individuals of other penguin species (Ghebremeskel et al., 1989, 1991, 1992; Wallace et al., 1996; Crissey et al., 1998). In contrast, the plasma of osprey and honey buzzard nestlings contained only small amounts of retinol. This difference is remarkable, especially owing to the fact that ospreys like penguins are almost exclusively piscivorous. A possible explanation might be that the ospreys investigated here nourish on freshwater fish, whereas the food basis of penguins is saltwater fish. Fresh-water fish store mainly 3,4-didehydroretinol, whereas many saltwater fish species accumulate mainly retinol (Lall and Parazo, 1995). Only in WTSEs age-specific differences of the retinol plasma concentrations were observed. Other studies about age-specific differences in retinol plasma concentrations of birds were not available for the authors. Sex-specific differences were found in osprey and honey buzzard nestlings as well as in northern goshawk adults. Like demonstrated for macaroni penguins (Eudyptes chrysolophus) (Ghebremeskel et al., 1991), the retinol plasma concentration of male osprey nestlings and male northern goshawk adults was higher than the retinol plasma concentration of the females respectively. In contrast, female honey buzzard nestlings revealed a higher retinol plasma concentration than the males. The reasons for these sex differences remained unclear. Schweigert et al. (1991) found retinyl esters in the plasma of birds of prey and Ciconiiformes. In fasted humans and rats, elevated plasma retinyl esters are associated with hypervitaminosis A because in these species, the unspecific transport of vitamin A bound to lipoproteins occurs only if the RBP transport capacity is exceeded during chronic high vitamin A intake (Mallia et al., 1975; Smith and Goodman, 1976). In carnivorous animals like dogs, the transport of retinyl esters by lipoproteins is physiological (Schweigert et al., 1990). The retinyl ester concentrations found in the plasma of our investigated birds of prey show that this transport mechanism might also be considered for carnivorous and piscivorous bird species. Only few bird species were investigated so far, therefore, a comparison is not possible. The presence of retinyl esters in bird plasma (mainly retinyl palmitate) was mentioned by Ganguly et al. (1952) in chicken fed with fish oil. Kerti et al. (2002) and Schweigert et al. (1991) found only small amounts of retinyl esters in quail plasma. Schweigert et al. (1991) detected higher retinyl ester plasma concentrations in most of the carnivorous 1050

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and piscivorous bird species kept in captivity. In contrast, the plasma of the likewise carnivorous owls contained only traces of retinyl esters. Retinyl esters were also detected in the plasma of Humboldt penguins (Wallace et al., 1996; Crissey et al., 1998). Wallace et al. (1996) investigated free-living penguins in two different seasons and could only detect small amounts of retinyl esters during the breeding season, whereas no retinyl esters were found in September. In this species, the retinyl ester plasma concentration might depend on the nutrition. In addition to retinol also 3,4-didehydroretinol was detected in the plasma of all investigated birds of prey species. So far, only few studies report the occurrence of 3,4-didehydroretinol in bird plasma. Given that 3,4-didehydroretinol was found in high amounts in liver and other organs of freshwater fish (Moore, 1957; Lall and Parazo, 1995), the detection in osprey plasma is not unexpected. Already Lederer and Rathmann (1938) assumed that the absence of vitamin A2 in the liver of mammals and other terrestrial animals can be traced back to the lack of vitamin A2 in nutrition. Interestingly, the presence of 3,4-didehydroretinol is not mentioned in several studies about penguins (Gulland et al., 1988; Ghebremeskel et al., 1989, 1991, 1992; Monroe, 1993; Wallace et al., 1996; Crissey et al., 1998). This might be attributed to the fact that saltwater fish mostly store retinol (Lall and Parazo, 1995). Nevertheless, it would be expected to find 3,4-didehydroretinol in these penguins, because also plasma of other saltwater fish-eating animals, like harbour seals (P. vitulina) (Gillam, 1938), ringed seals (Pusa hispida spp.) (Ka¨kela¨ et al., 1997), and minks (N. vison) (Ka¨kela¨ et al., 1999), contained 3,4-didehydroretinol. Possibly the studies about penguins did not distinguish between the two components or could not separate them. On the other hand, also Lovern et al. (1939) did not find 3,4-didehydroretinol in the liver of a herring gull (Larus argentatus), eight great skuas (Stercorarius skua), and a northern gannet (Morus bassanus) that fed on saltwater fish, whereas they could find it in several fish species. The high 3,4-didehydroretinol content of osprey plasma might be caused by the fish diet, but the reason of the high content of this component in honey buzzard plasma remains unknown. Honey buzzards feed on several developmental stages of wasps and other insects and to a considerable lesser part on amphibians, reptiles, small birds in the nestling stage and rarely on small mammals (Glutz von Blotzheim et al., 1989). The wasp brood and the liver of one toad we investigated did not contain 3,4-didehydroretinol Journal of Animal Physiology and Animal Nutrition. ª 2011 Blackwell Verlag GmbH

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(data not presented). We did also not detect any of the described precursors of 3,4-didehydroretinol, like b-carotene (Morton and Creed, 1939; Gross and Budowski, 1966), astaxanthin (Gross and Budowski, 1966), lutein (Barua et al., 1973; Goswami and Barua, 1981b), or anhydrolutein (Gross and Budowski, 1966; Barua and Das, 1975) in any or elevated concentrations in comparison with the other birds of prey. If a conversion of retinol to 3,4-didehydroretinol is possible is discussed controversial. Although it was several times postulated that this is impossible (Morcos and Salah, 1951; Moore, 1957; Goswami and Barua, 1981a), in rainbow trout, a conversion of retinol to 3,4-didehydroretinol was demonstrated (Lambertsen and Braekkan, 1969). Also in the eyes of rats (Yoshikami et al., 1969), tadpoles of bullfrogs (Rana catesbeiana) (Tsin et al., 1985) and hybrids of Lepomis species (Naito and Wilt, 1962) as well as human keratocytes (To¨rma¨ and Vahlquist, 1985; Rollman et al., 1993) a conversion was shown. Two biosynthetic pathways of 3,4-didehydroretinol have been described for birds. The one uses anhydrolein, which is an artificial carotenoid and not available in nature (Budowski et al., 1963), the other is the dehydrogenation of retinol in chicken embryos (Thaller and Eichele, 1990). Further studies are needed to show which synthetic pathway is used by honey buzzards. Owing to the fact that 3,4-didehydroretinol was not detected in other bird species so far, no further comparison with literature data is possible. Our study shows that free-ranging birds of prey species with a high retinol plasma concentrations (northern goshawk, common buzzard) had a low 3,4-didehydroretinol plasma, whereas free-ranging birds of prey species with a low retinol plasma concentration had a high 3,4-didehydroretinol plasma concentration (osprey, honey buzzard). Acknowledgements We would like to thank G. Dietrich, St. Fischer, A. Hallau, U. Hain, S. Herold, B. Heuer, H. D. Martens, W. Nachtigall, M.-C. Rothmund, R. Scho¨nbrodt, Z. Shamaev, P. So¨mmer, D. Stoewe, H. Tauchnitz and F. Ziesemer for their generous help in capturing the birds and sampling the specimens. H. Sauer-Gu¨rth (IPMB) assisted sexing the birds by PCR. D. Schmidt was supported by a grant from the Deutsche Ornithologen-Gesellschaft. K. Mu¨ller was supported by a grant from the Freie Universita¨t of Berlin, Germany. We thank I. Ruhnke for the critical proofreading of the manuscript. Journal of Animal Physiology and Animal Nutrition. ª 2011 Blackwell Verlag GmbH


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