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Tissue-specific distribution of carotenoids and vitamin E in tissues of newly hatched chicks from various avian species
Comparative Biochemistry and Physiology, Part A 140 (2005) 506 – 511 www.elsevier.com/locate/cbpa
Tissue-specific distribution of carotenoids and vitamin E in tissues of newly hatched chicks from various avian species Filiz Karadasa,*, Nicholas A.R. Woodb, Peter F. Suraic, Nicholas H.C. Sparksd a
Department of Animal Science, University of Yu¨zu¨ncu¨ Yil, 65080, Van, Turkey b Waterfowl Consulting and Research, Sherborne, Dorset DT9 5BX, UK c Alltech (UK) Ltd., Alltech House, Ryhall Road, Stamford, Lincs PE9 1TZ, UK d Avian Science Research Centre, Animal Health Group, SAC, West Mains Road, Edinburgh, Scotland EH9 3JG, UK Received 4 December 2004; received in revised form 27 February 2005; accepted 1 March 2005
Abstract The aim of this study was to evaluate carotenoid and vitamin E distribution in egg and tissues of newly hatched chicks from wild mallard (Anas platyrhynchos), game pheasant (Phasianus colchicus), free-range guinea fowl (Numida meleagris), hen (Gallus domesticus) and domestic duck (Anas platyrhynchos) and intensively housed hens. Carotenoid concentrations in the egg yolk of free-range guinea fowl, pheasant and wild mallard were similar (61.3 – 79.2 Ag/g). Egg yolks from ducks and intensively housed hens were characterised by the lowest carotenoid concentration comprising 11.2 – 14.8 Ag/g. However, carotenoid concentration in eggs from free-range ducks and hens was less than half of that in free-range guinea fowl or pheasant. Depending on carotenoid concentration in the livers of species studied could be placed in the following descending order: free living pheasant > free-range guinea fowl > free-range hen > intensively housed hen > wild mallard > housed duck > freerange duck. The carotenoid concentrations in other tissues of free-range guinea fowl and pheasant were substantially higher than in the other species studied. Egg yolk of housed hens was characterised by the highest a- and g-tocopherol concentrations. In accordance with the atocopherol concentration in the egg yolk, the birds can be placed in the following descending order: intensively housed hen > wild mallard > freeliving pheasant > free-range duck > free-range hen = free-range guinea fowl > housed duck. The main finding of this work is species- and tissuespecific differences in carotenoid and vitamin E distribution in the various avian species studied. D 2005 Elsevier Inc. All rights reserved. Keywords: Carotenoids; Egg yolk; Tissues; Vitamin E, Free-range, Wild
1. Introduction Embryo development of birds is associated with unique changes in fatty acid profile of various tissues. In particular, exceptionally high proportions of polyunsaturated fatty acids are incorporated into the lipids of several embryonic tissues (Speake et al., 1998). This makes embryonic tissues highly susceptible to free radicals. Various antioxidants are expressed in embryonic tissues where they form an integrated antioxidant system, responsible ultimately for preventing the damaging effects of free radicals and toxic * Corresponding author. Tel.: +90 432 225 10 24/2701; fax: +90 425 225 11 04. E-mail address: [email protected] (F. Karadas). 1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2005.03.002
products of their metabolism (Surai, 2002). The antioxidant system of the chicken embryo is based on antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase and catalase (Surai, 1999a,b) and chain-breaking antioxidants such as vitamin E (Surai et al., 1999a,b), carotenoids (Surai and Speake, 1998; Surai et al., 2001a,b), ascorbic acid (Surai et al., 1996) and reduced glutathione (Surai, 1999a). Some of these can be synthesised in the tissues, but vitamin E and carotenoids as well as cofactors such as selenium have to be obtained from the yolk and derive ultimately from the maternal diet. In fact, increased vitamin E (Surai et al., 1999a,b), carotenoid (Surai and Speake, 1998) or selenium (Surai, 2000) in the diet of the hen can substantially increase their concentrations in the yolk and subsequently in the embryonic tissues, thereby
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decreasing their susceptibility to lipid peroxidation. However, the antioxidant roles of carotenoids during development are still not clear (Surai et al., 2001a,b; Surai, 2002). Conventional intensive poultry production utilises compounded feed balanced in major nutrients but in many cases is poor in carotenoids (Surai and Sparks, 2001). In contrast wild birds as well as free-range birds have access to various sources of natural carotenoids and, as a result, their egg yolks are richer in carotenoids in comparison to commercially produced eggs. It has been suggested (Surai et al., 2001a,b) that because carotenoids are more easily available in natural conditions in comparison to vitamin E they could play an important role in the antioxidant defence of the avian embryo, partly by promoting vitamin E recycling in the tissues. In fact, this possibility is supported by the demonstration of electron transfer from h-carotene to atocopheroxyl radical (Bohm et al., 1997). It has also been shown that one-electron-oxidized lycopene can be reduced by a-tocopherol whereas one-electron-oxidized y-tocopherol can be reduced by lycopene (Mortensen and Skibsted, 1997). This same study also indicated that, hand g-tocopherols are in equilibrium with lycopene, i.e. they can reduce the lycopene radical or can be reduced by lycopene depending on the conditions. In polar environments the vitamin E radical cation is deprotonated and TocO* does not react with carotenoids, whereas in nonpolar environments, the tocopherol radical is reduced to active tocopherol form by hydrocarbon carotenoids (Mortensen et al., 2001). Li et al. (1995) showed that the combination of a-tocopherol and h-carotene exhibited a significant synergistic effect during oxidation of linoleic acid in tert-butyl alcohol and the decay of a-tocopherol was decreased by about the half in the presence of h-carotene. These results could be a reflection of a-tocopherol regeneration by hcarotene (Yanishlieva et al., 1998). The results of these recent experiments form a connecting link between carotenoids and other (vitamin E and C) antioxidants, explaining some old results and providing an insight into new directions of research in this field. It is interesting that when h-carotene, a-tocopherol or ascorbic acid were used singly or in mixtures, the best protection against lipid peroxidation in model systems was achieved by the mixture of these antioxidants (Zhang and Omaye, 2001a,b). However, the potential role of carotenoid in avian embryonic development and its distribution in tissues of newly hatched chicks from various avian species, has received only limited attention. Such data would provide a better understanding of the roles of natural antioxidants, not only in domestic poultry, but also in wild birds (Blount et al., 2002a,b) in relation to their evolution and fitness parameters (Møller et al., 2000). For example, it has been suggested (Surai et al., 2001a,b; Surai, 2002) that while in some species male birds are investing carotenoids in plumage for better reproductive success, females invest carotenoids into egg yolk to provide benefit for the developing embryo.
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The aim of this study was to evaluate carotenoid and vitamin E distribution in tissues of newly hatched chicks from various avian species.
2. Materials and methods Eggs of wild mallard (Anas platyrhynchos) and game pheasant (Phasianus colchicus) were collected under licence from the NCC from a wetland habitat of lakelands and streams adjoining agricultural grassland in North Dorset, England (51 -ON, 2-30I W), where the parent birds subsisted on a naturally available diet of invertebrates, seeds, leaves and various parts of plants. Eggs of domesticated Peking-type duck (A. plathyrhynchos) and Guinea fowl (Numida meleagris) were laid by birds free ranging around a farmstead and all surrounding meadows and streams, while eggs from free ranging hens (Gallus domesticus) of a small or bantam sized type came from a similar neighbouring farmstead. Hen and duck eggs from housed birds were provided by commercial poultry companies. These last mentioned were fed a commercial feed balanced for the major nutrients and supplemented with vitamin E at the level of 60 mg/kg for chickens and 30 mg/ kg for ducks. The levels of vitamin E in the diets were confirmed analytically and they comprised 76.2 mg/kg and 41.2 mg/kg, respectively. The hatchlings were produced from these eggs by artificial incubation in a Western Turkey bator 7 force draught machine electrically controlled to run at 37.2 -C dry bulb, with automatic turning at 1 h intervals, until a few days prior to hatching when eggs were transferred to a nonturning machine (Funki 4200 metal-Klaekker Type 7). Egg yolk was analysed for vitamin E and carotenoid concentration in freshly laid or frozen eggs. Other eggs were artificially incubated under standard conditions. Hatched chicks were killed by cervical dislocation on the day of hatch and the liver, kidney, lung, leg and breast muscles, heart, skin, adipose tissue as well as the yolk sac membrane (YSM) were collected and stored frozen until analysis. Vitamin E (a- and g-tocopherol) was determined by HPLC as previously described (Surai et al., 2001c). In brief, the egg yolk (100 – 200 mg) or tissues (200 – 500 mg) were mixed with a 5% (w/v in H2O) solution of NaCl (0.7 mL) and ethanol (1 mL) and homogenised for 2 min. Hexane (5 mL) was then added and the mixture was further homogenised for 2 min. The hexane phase, containing the vitamin E and carotenoids, was separated by centrifugation and collected. The extraction was repeated twice more with 5 mL hexane. Hexane extracts were combined, evaporated and re-dissolved in a mixture of methanol/dichloromethane (1:1, v/v). Samples were injected into HPLC system (Shimadzu Liquid Chromatograph, LC-10AD, Japan Spectroscopic Co. LTD with JASCO Intelligent Spectrofluorometer 821-FP) fitted with a Spherisorb, type S30DS2, 3 AC18 reverse phase HPLC column, 15 cm 4.6 mm (Phase
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Separations Limited, UK). Chromatography was performed using a mobile phase of methanol/water (97:3, v/v) at a flow rate of 1.05 mL/min. Fluorescence detection of vitamin E used excitation at 295 nm and emission at 330 nm. Standard solutions of a-tocopherol and g-tocopherol in methanol were used for instrument calibration and tocol was used as an internal standard. Carotenoids were determined from the same extracts using the same HPLC system, but fitted with a Spherisorb, type S5NH2 5 Am reverse phase HPLC column, 25 cm4.6 mm (Phase Separations Limited, UK). Chromatography was performed using a mobile phase of methanol/water (97:3, v/ v) at a flow rate of 1.5 mL/min. Total carotenoids were detected at 445 nm as a single peak using lutein as a standard. Results are presented as meanTS.E. of measurements on samples from 10 replicate determinations. Statistical comparisons were performed using t-test and single factor ANOVA. Differences were considered significant at P < 0.05.
duck. On the one hand, there was species-specific differences in carotenoid distribution from egg yolk to the YSM and liver. For example, in free-range pheasant and guinea fowl, the carotenoid concentration in the YSM was 2-fold higher than that in the egg yolk and 1.6– 2 times higher in comparison to the liver. In contrast, in the chicken, the carotenoid concentrations in the liver and YSM were similar. On the other hand, ducks were characterised by low carotenoid transfer from YSM to the liver resulting in liver carotenoid concentration being 4- to 5-fold lower than in the YSM and significantly lower than in the egg yolk. The carotenoid concentrations in other tissues of freerange guinea fowl and pheasant were substantially higher than in the other species studied. However, carotenoid concentration in pheasant skin and fat was 5.5 and 2.5 times lower, respectively, in comparison to guinea fowl. The carotenoid concentrations in the tissues of free-range chickens were higher than in intensively housed hens with the exception of adipose tissue, where there was no significant difference. Similarly, in tissues from the wild mallard, carotenoid concentrations were higher than in freerange ducks.
3. Results 3.2. Vitamin E 3.1. Carotenoids Carotenoid concentrations in egg yolk and tissues of the newly hatched chicks are shown in Table 1. Carotenoid concentration in the egg yolk of free-range guinea fowl, pheasant and wild mallard were similar. However, carotenoid concentration in eggs from free-range ducks and chickens was less than half of that in free-range guinea fowl or pheasant. Egg yolks from intensively housed hens and ducks were characterised by the lowest carotenoid concentration. The liver of newly hatched free-living pheasants was characterised by the highest carotenoid concentration. Depending on carotenoid concentration in the livers, species could be placed in the following descending order: free living pheasant > free-range guinea fowl H free-range hen H intensively housed hen > wild mallard H free-range
Vitamin E in egg yolk was represented by tocopherols (Tables 2 and 3). Egg yolk of housed chicken was characterised by the highest a- and g-tocopherol concentrations. In fact, g-tocopherol proportion in the egg yolk was about 10% of that a-tocopherol in all the species except ducks where the g-tocopherol proportion in the egg yolk was only 3 – 5%. In accordance with the a-tocopherol concentration in the egg yolk, the birds can be placed in the following descending order: intensively housed hens > wild mallard > free-living pheasant > free-range duck > freerange hens = free-range guinea fowl > housed duck. In most species, except the free-range duck, the a-tocopherol concentration in the YSM was higher than that in the egg yolk. Similarly, the a-tocopherol concentration in the liver of newly hatched chicks was also higher than that in the initial yolk. On the one hand, the most effective transfer of
Table 1 Carotenoid concentration (Ag/g fresh tissue) in egg yolk and in tissues of newly hatched chicks (n = 10) Tissue
Housed Duck
Free-range Duck
Wild Mallard
Housed Chicken
Free-range Chicken
Free-range Guinea Fowl
Free-living Pheasant
Yolk Yolk sac membrane Liver Kidney Lung Heart Leg muscle Breast muscle Skin Fat
11.22 T 1.13a 18.14 T 1.36a 20.33 T 2.01a 1.22 T 0.11a 1.13 T 0.15a 2.01 T 0.22a 1.02 T 0.11a 2.11 T 0.26a 2.12 T 0.12a 0.28 T 0.05a
34.05 T 3.66b 56.94 T 5.12b 11.80 T 1.12b 4.75 T 0.44b 2.85 T 0.33b 3.46 T 0.26b 1.77 T 0.15b 3.12 T 0.32b 4.79 T 0.41b 0.88 T 0.10b
61.27 T 5.16c 95.67 T 8.22c 23.67 T 2.11c 6.24 T 0.55b 5.27 T 0.51c 3.59 T 0.28b 1.95 T 0.17b 4.96 T 0.55c 6.12 T 0.66c 1.11 T 0.10b
14.77 T 1.11a 29.66 T 2.33d 31.45 T 3.11d 2.88 T 0.23c 3.98 T 0.28b 2.78 T 0.26b 3.69 T 0.33c 4.01 T 0.35b,c 5.11 T 0.55c 3.41 T 0.28c
33.93 T 4.66b 56.28 T 5.12b 50.85 T 5.66e 5.75 T 0.66b 6.87 T 0.66c 4.75 T 0.58c 4.11 T 0.51c 6.82 T 0.55d 11.33 T 1.55d 2.62 T 0.33c
79.18 T 6.88c 164.62 T 17.55e 90.57 T 10.11f 23.07 T 2.16d 20.69 T 3.11d 14.12 T 1.66d 8.22 T 0.93d 18.40 T 1.88e 3.30 T 0.41a 6.86 T 0.77d
72.61 T 8.22c 179.07 T 19.33e 115.25 T 13.55f 19.97 T 2.44d 18.57 T 2.14d 18.44 T 1.77d 14.22 T 1.66e 22.30 T 2.33e 18.22 T 2.33e 16.80 T 2.13e
Values are means T S.E.M. (n = 10). Numbers with different superscript are significantly ( P < 0.05) different with respect to row.
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Table 2 a-Tocopherol concentration (Ag/g fresh tissue) in egg yolk and in tissues of newly hatched chicks (n = 10)
Yolk Yolk sac membrane Liver Kidney Lung Heart Leg Breast
Housed Duck
Free-range Duck
Wild Mallard
Housed Chicken
Free-range Chicken
Free-range Guinea Fowl
Free-living Pheasant
16.33 T 1.66a 38.22 T 2.88a 62.11 T 5.22a 2.11 T 0.33a 2.16 T 0.23a 2.01 T 0.28a 1.02 T 0.12a 2.16 T 0.22a
68.13 T 5.66b 52.72 T 5.22b 101.12 T 9.66b 4.22 T 0.33b 3.60 T 0.32b 3.00 T 0.22b 1.90 T 0.13b 3.60 T 0.34b
94.32 T 6.99c 157.81 T16.33c 231.97 T 21.33c 11.58 T 1.66c 11.23 T 1.12c 10.12 T 1.02c 7.57 T 0.66c 16.63 T 1.66c
154.21 T12.22d 211.33 T 14.55d 601.33 T 29.33d 16.41 T1.33c 20.13 T 2.02d 22.21 T1.66d 13.11 T1.22d 16.34 T 1.55c
36.35 T 3.22e 86.12 T 7.11e 92.52 T 8.55b 4.12 T 0.36b 3.31 T 0.32b 6.49 T 0.52e 3.58 T 0.33e 7.60 T 0.55d
31.07 T 2.77e 56.99 T 6.33b 59.14 T 4.25a 5.19 T 0.62b,d 3.28 T 0.22b 5.34 T 0.44e 2.55 T 0.32b,e 5.05 T 0.32e
77.02 T 6.33b 157.36 T 14.22c 173.39 T 16.22e 5.83 T 0.32d,e 3.74 T 0.33b 6.18 T 0.55e 6.55 T 0.74c 13.90 T 1.52c
Values are means T S.E.M. (n = 10). Numbers with different superscript are significantly ( P < 0.05) different with respect to row.
a-tocopherol from egg yolk to the liver was observed in housed hens, where the a-tocopherol concentration in the liver was 3.9 times higher than in the yolk. On the other hand, the lowest efficiency of vitamin E accumulation in the liver was seen in the free-range duck where the concentration of a-tocopherol in the liver was only 1.5 times higher than that in the egg yolk. The concentrations of atocopherol in peripheral tissues were highest in the commercial chicken, reflecting its highest level in the egg yolk and liver. However, the a-tocopherol concentration in peripheral tissues of birds has some tissue- and speciesspecific features. For example, wild mallard breast muscle and kidney were characterised by comparatively high atocopherol concentration comparable to that in the commercial chicken. In contrast, the a-tocopherol concentration in the egg yolk and liver of the housed chicken was 1.6- and 2.6-fold higher than that in mallards. Furthermore, the atocopherol concentration in the egg yolk of free-range chicken’s hens was two times lower than that in the freeliving pheasants; however, the a-tocopherol concentrations in their lung tissue and heart were the same. In housed hens, the g-tocopherol concentration in the egg yolk and tissues were highest, with free-living pheasants being in the second position and free-range guinea fowl having the lowest g-tocopherol concentration. Similar to gtocopherol, the liver of housed hens was much more effective in accumulating g-tocopherol in comparison to other species, with free-range hens and guinea fowls to follow. In the other species in this study, the g-tocopherol concentration in the liver was not dissimilar to that in the
egg yolk. The yolk sac membrane of the housed chicken also contained the highest g-tocopherol concentration with free-living pheasant and free-range hens also showing active accumulation of g-tocopherol in the YSM. In the other species studied, there was no difference in g-tocopherol concentration between yolk and YSM. Other peripheral tissues were characterised by comparatively low levels of gtocopherol with housed chicken tissues found to contain much more g-tocopherol than other species of birds.
4. Discussion The main finding of this work is species- and tissuespecific differences in carotenoid and vitamin E distribution in the various avian species studied. Of particular interest are the comparatively high carotenoid concentrations in the egg yolks of the free-living pheasant, free-range guinea fowl and wild mallard. They are similar to those in free-living gulls (Surai et al., 2001a,b; Royle et al., 2001; Blount et al., 2002a,b), higher than those in wild moorhen (Gallinula chloropus) (Surai et al., 2001d) or housed, free-range domestic and feral Canada geese (Speake et al., 1999), gannet and skua (Surai et al., 2001e), great tits (Horak et al., 2002) or captive falcons (Barton et al., 2002). However, much higher carotenoid concentrations were observed in other wild birds, including the American coot (Fulica americana), Surai et al., 2001d), pelican (Pelecanus erythrorhynchos) and cormorant (Phalacrocorax auritus) (Surai et al., 2001e), or gulls (Larus fuscus) after carotenoid
Table 3 Gamma-tocopherol concentration (Ag/g fresh tissue) in egg yolk and in tissues of newly hatched chicks (n = 10)
Yolk Yolk sac membrane Liver Kidney Lung Heart Leg Breast
Housed Duck
Free-range Duck
Wild Mallard
Housed Chicken
Free-range Chicken
Free-range Guinea Fowl
Free-living Pheasant
2.11 T 0.22a 3.12 T 0.52a 4.66 T 0.44a 0.32 T 0.05a 0.32 T 0.03a 0.58 T 0.06a 0.33 T 0.02a 0.41 T 0.05a
3.23 T 0.34b 3.84 T 0.35a 3.24 T 0.33b 0.64 T 0.05b 0.44 T 0.03a,b 0.73 T 0.08ab 0.39 T 0.05a 0.50 T 0.04a
3.15 T 0.33b 2.95 T 0.25a 3.43 T 0.36b 0.42 T 0.05a 0.54 T 0.06b,c 0.62 T 0.08a 0.38 T 0.03a 0.65 T 0.06b
15.33 T 1.66c 41.15 T 5.11b 57.84 T 4.05c 3.45 T 0.33c 4.88 T 0.44d 6.54 T 0.58c 2.01 T 0.33b 2.88 T 0.41c
3.34 T 0.33b 6.02 T 0.55c 7.68 T 0.63d 0.84 T 0.08d 0.63 T 0.06c 0.83 T 0.07b 0.54 T 0.05a,c 0.81 T 0.10b,d
2.58 T 0.21a,b 2.68 T 0.21a 4.42 T 0.36a,b 0.63 T 0.06b 0.38 T 0.05a,b 0.57 T 0.06a 0.27 T 0.02a 0.42 T 0.03a
7.75 T 0.66d 13.37 T 1.22d 8.86 T 0.56d 1.44 T 0.12e 0.99 T 0.10e 1.23 T 0.11b 0.82 T 0.10d 1.03 T 0.11d
Values are means T S.E.M. (n = 10). Numbers with different superscript are significantly ( P < 0.05) different with respect to row.
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dietary supplementation (Blount et al., 2002a,b). The carotenoid concentration in the egg yolk is a reflection of dietary provision and efficiency of transfer from feed to egg yolk (Surai, 2002; Surai et al., 2001a,b). Hence it is to expect that eggs from free-range birds having an access to green vegetation contain a higher concentration of carotenoid than eggs from housed birds (Speake et al., 1999). Our previous data on carotenoid concentration in the egg yolk of the housed hen, turkey, goose and duck fed similar diets (Surai et al., 1998) indicate significantly higher levels of carotenoids in the eggs from housed hens in comparison to other avian species studied. However, when chickens and ducks have access to range the levels of carotenoids in their egg yolks were similar. The striking feature is that in egg yolk from wild mallards, the carotenoid concentration was 1.8-fold higher than that in free-range ducks. A different picture was observed in free-range geese where carotenoid concentration was significantly higher than that in feral Canada geese (Speake et al., 1999). Clearly there are aspects of carotenoid metabolism in captive and wild birds that need further investigation. The efficiency of carotenoid transfer from egg yolk to the liver differed significantly between species studied. In this respect, free-range and wild ducks showed an unusual pattern, with the carotenoid concentration in the liver of newly hatched ducklings being lower than the initial yolk. In contrast, in the chicks from housed hens, for example, the carotenoid concentration in the liver was more than twice that in egg yolk, indicating an active carotenoid accumulation in this tissue during embryonic development. It seems likely that a redistribution of carotenoids from the egg yolk to the liver is mediated via the yolk sac membrane, and that this process is somehow delayed in these ducks in comparison to other species studied since carotenoid concentrations in the YSM of free-range and wild ducks were higher that those in the egg yolk and liver. From all the species studied, free-range guinea fowls and pheasant were characterised by the highest carotenoid concentrations in peripheral tissues. For example in the kidney, lung, heart and breast muscle of these two species carotenoid concentration is identical and several times higher than that in the other species studied. However, in leg muscle, fat and especially in skin of the free-living pheasant, the carotenoid concentration is much higher than that in free-range guinea fowl. Therefore, although in these two species carotenoid concentration in the initial yolk was the same, during embryonic development species-specific features in carotenoid distribution between various tissues were observed. Molecular mechanisms of such differences are not known at present and need further research. In this respect, duck fat was characterised by the lowest carotenoid concentration. Again, in wild duck egg yolk carotenoid concentration was almost twice that in free-range duck, but in the skin and fat this difference practically disappeared. Since carotenoids are involved in immunomodulation in birds (Blount et al., 2003) increased carotenoid concentration in tissues of wild
birds could have health-related consequences. Indeed, egglaying capacity is limited by carotenoid pigment availability in wild gulls (L. fuscus) (Blount et al., 2004). Housed hen eggs were characterised by the highest vitamin E concentration which is probably a reflection of vitamin E supplementation (60 mg/kg) in the feed. On the other hand, wild mallard was characterised by comparatively high vitamin E concentration even though the diet was natural and was not supplemented with vitamin E. It is interesting to note that in egg yolk of commercial ducks supplemented with vitamin E at the level of 30 mg/kg, the concentration of a-tocopherol was 4 times lower than that in feral ducks without vitamin E supplementation. Similar extremely high vitamin E concentrations (294 – 299 Ag/g) were found in wild pelican and cormorant egg yolk (Surai et al., 2001e). It is not known at present if this is a reflection of a high vitamin E diet or efficient vitamin E transfer from the diet to the egg yolk. In contrast to carotenoids, in all the species studied, vitamin E concentration in the liver was several-fold higher than in the egg yolk, indicating an active accumulation of vitamin E during embryonic development. In fact, vitamin E accumulation in the embryonic liver, which reaches a maximum at hatching time, was suggested to be an adaptive mechanism in birds to deal with overproduction of free radicals at the time of hatchling (Surai, 2002). It is interesting that the efficiency of vitamin E transfer from the yolk to the developing liver in the housed hen and duck was higher than in free-range or wild birds. It seems likely that selection to achieve the highest growth rate and egg production is associated with increased free radical production (Surai, 2002), and the increased accumulation of vitamin E in the liver could be considered as an adaptive mechanism. Wild ducks were characterised by increased a-tocopherol concentration in the peripheral tissues (kidney, lung, heart and muscles) in comparison to other species except housed chicken. For example, in peripheral tissues of mallards atocopherol concentrations were several times higher than those in pheasants. Eggs from housed hens were characterised by the highest g-tocopherol concentrations in the egg yolk which is probably a reflection of grain-based diet providing more g-tocopherol than diets of free-range or wild birds (Surai, 2002). In general, it seems likely that the role of gtocopherol in the feed is underestimated. For example, in granivorous birds, g-tocopherol concentrations in the diet could exceed those of a-tocopherol (e.g. zebra finch, Royle et al., 2003; or other birds in captivity fed on unsupplemented diets, Surai, 2002). On the other hand, g-tocopherol could be an important element of the antioxidant-prooxidant balance in the digestive tract (Surai et al., 2003, 2004), which is ultimately responsible for health maintenance in birds or in mammalian species, including humans. This study provides evidence of species-specific differences in carotenoid and vitamin E accumulation in the egg
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yolk and their distribution from the liver and peripheral tissues during embryonic development. In particular, freerange and wild birds are usually characterised by increased carotenoid concentrations in the egg yolk and embryonic tissues in comparison to captive birds. If as has been proposed (Surai, 2002) that all antioxidants in the body work together to provide an effective antioxidant defence during critical periods of ontogenesis including hatching process the maternal investment of carotenoids and vitamin E into the egg is an important element in maintaining progeny fitness and survival. More work is needed to address specific questions in relation to this important issue in birds.
Acknowledgments The authors gratefully acknowledge The Scientific and Technical Research Council of Turkey (TUBI˙TAK) for a NATO Science Fellowship for Filiz Karadas and SEERAD which provides support to SAC.
References Barton, N.W.H, Fox, N.C., Surai, P.F., Speake, B.K., 2002. Vitamins E and A, carotenoids and fatty acids of the egg yolk of raptors. J. Raptor Res. 36, 33 – 38. Blount, J.D., Surai, P.F., Houston, D.C., Møller, A.P., 2002a. Patterns of yolk enrichment with dietary carotenoids in gulls: the roles of pigment acquisition and utilization. Funct. Ecol. 16, 445 – 453. Blount, J.D., Surai, P.F., Nager, R.G., Houston, D.C., Møller, A.P., Trewby, M.L., Kennedy, M.W., 2002b. Carotenoids and egg quality in the lesser black-backed gull Larus fucus: a supplemental feeding study of maternal effect. Proc. R. Soc. Lond., B 269, 29 – 36. Blount, J.D., Metcalfe, N.B., Birkhead, T.R., Surai, P.F., 2003. Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Science 300, 125 – 127. Blount, J.D., Houston, D.C., Surai, P.F., Møller, A.P., 2004. Egg-laying capacity is limited by carotenoid pigment availability in wild gulls Larus fuscus. Proc. R. Soc. Biol. Lett. 271 (Suppl. 1), S79 – S81. Bohm, F., Edge, R., Land, E.J., McGarvey, D.J., Truscott, T.G., 1997. Carotenoids enhance vitamin E antioxidant efficiency. J. Am. Chem. Soc. 119, 621 – 622. Horak, P., Surai, P.F., Møller, A.P., 2002. Fat-soluble antioxidants in the eggs of great tits Parus major in relation to breeding habitat and laying sequence. Avian Sci. 2, 123 – 130. Li, L.L., Wu, L.M., Ma, L.P., Liu, J.C., Liu, Z.L., 1995. Antioxidant synergism and mutual protection of a-tocopherol and h-carotene in the inhibition of radical-initiated peroxidation of linoleic acid in solution. J. Phys. Org. Chem. 8, 774 – 780. Møller, A.P., Biard, C., Blount, J., Houston, D.C., Ninni, P., Saino, N., Surai, P., 2000. Carotenoid-dependent signals: indicators of foraging efficiency, immunocompetence or detoxification ability? Poult. Avian Biol. Rev. 11, 137 – 159. Mortensen, A., Skibsted, L.H., 1997. Importance of carotenoid structure in radical-scavenging reactions. J. Agric. Food Chem. 45, 2970 – 2977. Mortensen, A., Skibsted, L.H., Truscott, T.G., 2001. The interaction of dietary carotenoids with radical species. Arch. Biochem. Biophys. 385, 13 – 19. Royle, N.J., Surai, P.F., Hartley, I.R., 2001. Maternally derived androgens and antioxidants in bird eggs: complementary but opposing effects? Behav. Ecol. 12, 381 – 385.
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Royle, N.J., Surai, P.F., Hartley, I.R., 2003. Effect of variation in dietary intake on maternal deposition of antioxidants in zebra finch eggs. Funct. Ecol. 17, 472 – 481. Speake, B.K., Murray, A.M.B., Noble, R.C., 1998. Transport and transformation of yolk lipids during development of the avian embryo. Prog. Lipid. Res. 37, 1 – 32. Speake, B.K., Surai, P.F., Noble, R.C., Beer, J.V., Wood, N., 1999. Differences in egg lipid and antioxidant composition between wild and captive pheasants and geese. Comp. Biochem. Physiol., B 124 (1), 101 – 107. Surai, P.F., 1999a. Tissue-specific changes in the activities of antioxidant enzymes during the development of the chicken embryo. Br. Poult. Sci. 40, 397 – 405. Surai, P.F., 1999b. Vitamin E in avian reproduction. Poult. Avian Biol. Rev. 10, 1 – 60. Surai, P.F., 2000. Effect of the selenium and vitamin E content of the maternal diet on the antioxidant system of the yolk and the developing chick. Br. Poult. Sci. 41, 235 – 243. Surai, P.F., 2002. Natural Antioxidants in Avian Nutrition and Reproduction. Nottingham University Press, Nottingham, UK. Surai, P.F., Sparks, N.H.C., 2001. Comparative evaluation of the effect of two maternal diets on fatty acids, vitamin E and carotenoids in the chicken embryo. Br. Poult. Sci. 42, 252 – 259. Surai, P.F., Speake, B.K., 1998. Distribution of carotenoids from the yolk to the tissues of the chick embryo. J. Nutr. Biochem. 9, 645 – 651. Surai, P.F., Noble, R.C., Speake, B.K., 1996. Tissue-specific differences in antioxidant distribution and susceptibility to lipid peroxidation during development of the chick embryo. Biochim. Biophys. Acta 1304, 1 – 10. Surai, P., Ionov, I., Kuchmistova, E., Noble, R.C., Speake, B.K., 1998. The relationship between the levels of a-tocopherol and carotenoids in the maternal feed, yolk and neonatal tissues: comparison between the chicken, turkey, duck and goose. J. Sci. Food Agric. 76, 593 – 598. Surai, P.F., Noble, R.C., Speake, B.K., 1999a. Relationship between vitamin E content and susceptibility to lipid peroxidation in tissues of the newly hatched chick. Br. Poult. Sci. 40, 406 – 410. Surai, P.F., Speake, B.K., Noble, R.C., Sparks, N.H.C., 1999b. Tissuespecific antioxidant profiles and susceptibility to lipid peroxidation of the newly hatched chick. Biol. Trace Elem. Res. 68, 63 – 78. Surai, P.F., Speake, B.K., Sparks, N.H.C., 2001a. Carotenoids in avian nutrition and embryonic development: 1. Absorption, availability and levels in plasma and egg yolk. J. Poult. Sci. 38, 1 – 27. Surai, P.F., Speake, B.K., Sparks, N.H.C., 2001b. Carotenoids in avian nutrition and embryonic development: 2. Antioxidant properties and discrimination in embryonic tissues. J. Poult. Sci. 38, 117 – 145. Surai, P.F, Speake, B.K., Decrock, F., Groscolas, R., 2001c. Transfer of Vitamins E and A from yolk to embryo during development of the King Penguin. Physiol. Biochem. Zool. 74, 928 – 936. Surai, P.F., Speake, B.K., Wood, N.A.R., Blount, J.D., Bortolotti, G.R., Sparks, N.H.C., 2001d. Carotenoid discrimination by the avian embryo: a lesson from wild birds. Comp. Biochem. Physiol., B 128, 743 – 750. Surai, P.F., Bortolotti, G.R., Fidgett, A., Blount, J., Speake, B.K., 2001e. Effects of piscivory on the fatty acid profiles and antioxidants of avian yolk: studies on eggs of the gannet, skua, pelican and cormorant. J. Zool. Lond. 255, 305 – 312. Surai, K.P., Surai, P.F., Speake, B.K., Sparks, N.H.C., 2003. Antioxidant – prooxidant balance in the intestine: food for thought: 1. Prooxidants. Nutr. Genomics Funct. Foods 1, 51 – 70. Surai, K.P., Surai, P.F., Speake, B.K., Sparks, N.H.C., 2004. Antioxidant – prooxidant balance in the intestine: food for thought: 2. Antioxidants. Curr. Top. Nutraceutical Res. 2, 27 – 46. Yanishlieva, N.V., Aitzetmuller, K., Raneva, V.G., 1998. h-Carotene and lipid oxidation. Fett/Lipid 10, 444 – 462. Zhang, P., Omaye, S.T., 2001a. DNA strand breakage and oxygen tension: effects of beta-carotene, alpha-tocopherol and ascorbic acid. Food Chem. Toxicol. 39, 239 – 246. Zhang, P., Omaye, S.T., 2001b. Antioxidant and prooxidant roles for betacarotene, alpha-tocopherol and ascorbic acid in human lung cells. Toxicol. in Vitro 15, 13 – 24.
Tissue-specific distribution of carotenoids and vitamin E in tissues of newly hatched chicks from various avian species Filiz Karadasa,*, Nicholas A.R. Woodb, Peter F. Suraic, Nicholas H.C. Sparksd a
Department of Animal Science, University of Yu¨zu¨ncu¨ Yil, 65080, Van, Turkey b Waterfowl Consulting and Research, Sherborne, Dorset DT9 5BX, UK c Alltech (UK) Ltd., Alltech House, Ryhall Road, Stamford, Lincs PE9 1TZ, UK d Avian Science Research Centre, Animal Health Group, SAC, West Mains Road, Edinburgh, Scotland EH9 3JG, UK Received 4 December 2004; received in revised form 27 February 2005; accepted 1 March 2005
Abstract The aim of this study was to evaluate carotenoid and vitamin E distribution in egg and tissues of newly hatched chicks from wild mallard (Anas platyrhynchos), game pheasant (Phasianus colchicus), free-range guinea fowl (Numida meleagris), hen (Gallus domesticus) and domestic duck (Anas platyrhynchos) and intensively housed hens. Carotenoid concentrations in the egg yolk of free-range guinea fowl, pheasant and wild mallard were similar (61.3 – 79.2 Ag/g). Egg yolks from ducks and intensively housed hens were characterised by the lowest carotenoid concentration comprising 11.2 – 14.8 Ag/g. However, carotenoid concentration in eggs from free-range ducks and hens was less than half of that in free-range guinea fowl or pheasant. Depending on carotenoid concentration in the livers of species studied could be placed in the following descending order: free living pheasant > free-range guinea fowl > free-range hen > intensively housed hen > wild mallard > housed duck > freerange duck. The carotenoid concentrations in other tissues of free-range guinea fowl and pheasant were substantially higher than in the other species studied. Egg yolk of housed hens was characterised by the highest a- and g-tocopherol concentrations. In accordance with the atocopherol concentration in the egg yolk, the birds can be placed in the following descending order: intensively housed hen > wild mallard > freeliving pheasant > free-range duck > free-range hen = free-range guinea fowl > housed duck. The main finding of this work is species- and tissuespecific differences in carotenoid and vitamin E distribution in the various avian species studied. D 2005 Elsevier Inc. All rights reserved. Keywords: Carotenoids; Egg yolk; Tissues; Vitamin E, Free-range, Wild
1. Introduction Embryo development of birds is associated with unique changes in fatty acid profile of various tissues. In particular, exceptionally high proportions of polyunsaturated fatty acids are incorporated into the lipids of several embryonic tissues (Speake et al., 1998). This makes embryonic tissues highly susceptible to free radicals. Various antioxidants are expressed in embryonic tissues where they form an integrated antioxidant system, responsible ultimately for preventing the damaging effects of free radicals and toxic * Corresponding author. Tel.: +90 432 225 10 24/2701; fax: +90 425 225 11 04. E-mail address: [email protected] (F. Karadas). 1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2005.03.002
products of their metabolism (Surai, 2002). The antioxidant system of the chicken embryo is based on antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase and catalase (Surai, 1999a,b) and chain-breaking antioxidants such as vitamin E (Surai et al., 1999a,b), carotenoids (Surai and Speake, 1998; Surai et al., 2001a,b), ascorbic acid (Surai et al., 1996) and reduced glutathione (Surai, 1999a). Some of these can be synthesised in the tissues, but vitamin E and carotenoids as well as cofactors such as selenium have to be obtained from the yolk and derive ultimately from the maternal diet. In fact, increased vitamin E (Surai et al., 1999a,b), carotenoid (Surai and Speake, 1998) or selenium (Surai, 2000) in the diet of the hen can substantially increase their concentrations in the yolk and subsequently in the embryonic tissues, thereby
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decreasing their susceptibility to lipid peroxidation. However, the antioxidant roles of carotenoids during development are still not clear (Surai et al., 2001a,b; Surai, 2002). Conventional intensive poultry production utilises compounded feed balanced in major nutrients but in many cases is poor in carotenoids (Surai and Sparks, 2001). In contrast wild birds as well as free-range birds have access to various sources of natural carotenoids and, as a result, their egg yolks are richer in carotenoids in comparison to commercially produced eggs. It has been suggested (Surai et al., 2001a,b) that because carotenoids are more easily available in natural conditions in comparison to vitamin E they could play an important role in the antioxidant defence of the avian embryo, partly by promoting vitamin E recycling in the tissues. In fact, this possibility is supported by the demonstration of electron transfer from h-carotene to atocopheroxyl radical (Bohm et al., 1997). It has also been shown that one-electron-oxidized lycopene can be reduced by a-tocopherol whereas one-electron-oxidized y-tocopherol can be reduced by lycopene (Mortensen and Skibsted, 1997). This same study also indicated that, hand g-tocopherols are in equilibrium with lycopene, i.e. they can reduce the lycopene radical or can be reduced by lycopene depending on the conditions. In polar environments the vitamin E radical cation is deprotonated and TocO* does not react with carotenoids, whereas in nonpolar environments, the tocopherol radical is reduced to active tocopherol form by hydrocarbon carotenoids (Mortensen et al., 2001). Li et al. (1995) showed that the combination of a-tocopherol and h-carotene exhibited a significant synergistic effect during oxidation of linoleic acid in tert-butyl alcohol and the decay of a-tocopherol was decreased by about the half in the presence of h-carotene. These results could be a reflection of a-tocopherol regeneration by hcarotene (Yanishlieva et al., 1998). The results of these recent experiments form a connecting link between carotenoids and other (vitamin E and C) antioxidants, explaining some old results and providing an insight into new directions of research in this field. It is interesting that when h-carotene, a-tocopherol or ascorbic acid were used singly or in mixtures, the best protection against lipid peroxidation in model systems was achieved by the mixture of these antioxidants (Zhang and Omaye, 2001a,b). However, the potential role of carotenoid in avian embryonic development and its distribution in tissues of newly hatched chicks from various avian species, has received only limited attention. Such data would provide a better understanding of the roles of natural antioxidants, not only in domestic poultry, but also in wild birds (Blount et al., 2002a,b) in relation to their evolution and fitness parameters (Møller et al., 2000). For example, it has been suggested (Surai et al., 2001a,b; Surai, 2002) that while in some species male birds are investing carotenoids in plumage for better reproductive success, females invest carotenoids into egg yolk to provide benefit for the developing embryo.
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The aim of this study was to evaluate carotenoid and vitamin E distribution in tissues of newly hatched chicks from various avian species.
2. Materials and methods Eggs of wild mallard (Anas platyrhynchos) and game pheasant (Phasianus colchicus) were collected under licence from the NCC from a wetland habitat of lakelands and streams adjoining agricultural grassland in North Dorset, England (51 -ON, 2-30I W), where the parent birds subsisted on a naturally available diet of invertebrates, seeds, leaves and various parts of plants. Eggs of domesticated Peking-type duck (A. plathyrhynchos) and Guinea fowl (Numida meleagris) were laid by birds free ranging around a farmstead and all surrounding meadows and streams, while eggs from free ranging hens (Gallus domesticus) of a small or bantam sized type came from a similar neighbouring farmstead. Hen and duck eggs from housed birds were provided by commercial poultry companies. These last mentioned were fed a commercial feed balanced for the major nutrients and supplemented with vitamin E at the level of 60 mg/kg for chickens and 30 mg/ kg for ducks. The levels of vitamin E in the diets were confirmed analytically and they comprised 76.2 mg/kg and 41.2 mg/kg, respectively. The hatchlings were produced from these eggs by artificial incubation in a Western Turkey bator 7 force draught machine electrically controlled to run at 37.2 -C dry bulb, with automatic turning at 1 h intervals, until a few days prior to hatching when eggs were transferred to a nonturning machine (Funki 4200 metal-Klaekker Type 7). Egg yolk was analysed for vitamin E and carotenoid concentration in freshly laid or frozen eggs. Other eggs were artificially incubated under standard conditions. Hatched chicks were killed by cervical dislocation on the day of hatch and the liver, kidney, lung, leg and breast muscles, heart, skin, adipose tissue as well as the yolk sac membrane (YSM) were collected and stored frozen until analysis. Vitamin E (a- and g-tocopherol) was determined by HPLC as previously described (Surai et al., 2001c). In brief, the egg yolk (100 – 200 mg) or tissues (200 – 500 mg) were mixed with a 5% (w/v in H2O) solution of NaCl (0.7 mL) and ethanol (1 mL) and homogenised for 2 min. Hexane (5 mL) was then added and the mixture was further homogenised for 2 min. The hexane phase, containing the vitamin E and carotenoids, was separated by centrifugation and collected. The extraction was repeated twice more with 5 mL hexane. Hexane extracts were combined, evaporated and re-dissolved in a mixture of methanol/dichloromethane (1:1, v/v). Samples were injected into HPLC system (Shimadzu Liquid Chromatograph, LC-10AD, Japan Spectroscopic Co. LTD with JASCO Intelligent Spectrofluorometer 821-FP) fitted with a Spherisorb, type S30DS2, 3 AC18 reverse phase HPLC column, 15 cm 4.6 mm (Phase
508
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Separations Limited, UK). Chromatography was performed using a mobile phase of methanol/water (97:3, v/v) at a flow rate of 1.05 mL/min. Fluorescence detection of vitamin E used excitation at 295 nm and emission at 330 nm. Standard solutions of a-tocopherol and g-tocopherol in methanol were used for instrument calibration and tocol was used as an internal standard. Carotenoids were determined from the same extracts using the same HPLC system, but fitted with a Spherisorb, type S5NH2 5 Am reverse phase HPLC column, 25 cm4.6 mm (Phase Separations Limited, UK). Chromatography was performed using a mobile phase of methanol/water (97:3, v/ v) at a flow rate of 1.5 mL/min. Total carotenoids were detected at 445 nm as a single peak using lutein as a standard. Results are presented as meanTS.E. of measurements on samples from 10 replicate determinations. Statistical comparisons were performed using t-test and single factor ANOVA. Differences were considered significant at P < 0.05.
duck. On the one hand, there was species-specific differences in carotenoid distribution from egg yolk to the YSM and liver. For example, in free-range pheasant and guinea fowl, the carotenoid concentration in the YSM was 2-fold higher than that in the egg yolk and 1.6– 2 times higher in comparison to the liver. In contrast, in the chicken, the carotenoid concentrations in the liver and YSM were similar. On the other hand, ducks were characterised by low carotenoid transfer from YSM to the liver resulting in liver carotenoid concentration being 4- to 5-fold lower than in the YSM and significantly lower than in the egg yolk. The carotenoid concentrations in other tissues of freerange guinea fowl and pheasant were substantially higher than in the other species studied. However, carotenoid concentration in pheasant skin and fat was 5.5 and 2.5 times lower, respectively, in comparison to guinea fowl. The carotenoid concentrations in the tissues of free-range chickens were higher than in intensively housed hens with the exception of adipose tissue, where there was no significant difference. Similarly, in tissues from the wild mallard, carotenoid concentrations were higher than in freerange ducks.
3. Results 3.2. Vitamin E 3.1. Carotenoids Carotenoid concentrations in egg yolk and tissues of the newly hatched chicks are shown in Table 1. Carotenoid concentration in the egg yolk of free-range guinea fowl, pheasant and wild mallard were similar. However, carotenoid concentration in eggs from free-range ducks and chickens was less than half of that in free-range guinea fowl or pheasant. Egg yolks from intensively housed hens and ducks were characterised by the lowest carotenoid concentration. The liver of newly hatched free-living pheasants was characterised by the highest carotenoid concentration. Depending on carotenoid concentration in the livers, species could be placed in the following descending order: free living pheasant > free-range guinea fowl H free-range hen H intensively housed hen > wild mallard H free-range
Vitamin E in egg yolk was represented by tocopherols (Tables 2 and 3). Egg yolk of housed chicken was characterised by the highest a- and g-tocopherol concentrations. In fact, g-tocopherol proportion in the egg yolk was about 10% of that a-tocopherol in all the species except ducks where the g-tocopherol proportion in the egg yolk was only 3 – 5%. In accordance with the a-tocopherol concentration in the egg yolk, the birds can be placed in the following descending order: intensively housed hens > wild mallard > free-living pheasant > free-range duck > freerange hens = free-range guinea fowl > housed duck. In most species, except the free-range duck, the a-tocopherol concentration in the YSM was higher than that in the egg yolk. Similarly, the a-tocopherol concentration in the liver of newly hatched chicks was also higher than that in the initial yolk. On the one hand, the most effective transfer of
Table 1 Carotenoid concentration (Ag/g fresh tissue) in egg yolk and in tissues of newly hatched chicks (n = 10) Tissue
Housed Duck
Free-range Duck
Wild Mallard
Housed Chicken
Free-range Chicken
Free-range Guinea Fowl
Free-living Pheasant
Yolk Yolk sac membrane Liver Kidney Lung Heart Leg muscle Breast muscle Skin Fat
11.22 T 1.13a 18.14 T 1.36a 20.33 T 2.01a 1.22 T 0.11a 1.13 T 0.15a 2.01 T 0.22a 1.02 T 0.11a 2.11 T 0.26a 2.12 T 0.12a 0.28 T 0.05a
34.05 T 3.66b 56.94 T 5.12b 11.80 T 1.12b 4.75 T 0.44b 2.85 T 0.33b 3.46 T 0.26b 1.77 T 0.15b 3.12 T 0.32b 4.79 T 0.41b 0.88 T 0.10b
61.27 T 5.16c 95.67 T 8.22c 23.67 T 2.11c 6.24 T 0.55b 5.27 T 0.51c 3.59 T 0.28b 1.95 T 0.17b 4.96 T 0.55c 6.12 T 0.66c 1.11 T 0.10b
14.77 T 1.11a 29.66 T 2.33d 31.45 T 3.11d 2.88 T 0.23c 3.98 T 0.28b 2.78 T 0.26b 3.69 T 0.33c 4.01 T 0.35b,c 5.11 T 0.55c 3.41 T 0.28c
33.93 T 4.66b 56.28 T 5.12b 50.85 T 5.66e 5.75 T 0.66b 6.87 T 0.66c 4.75 T 0.58c 4.11 T 0.51c 6.82 T 0.55d 11.33 T 1.55d 2.62 T 0.33c
79.18 T 6.88c 164.62 T 17.55e 90.57 T 10.11f 23.07 T 2.16d 20.69 T 3.11d 14.12 T 1.66d 8.22 T 0.93d 18.40 T 1.88e 3.30 T 0.41a 6.86 T 0.77d
72.61 T 8.22c 179.07 T 19.33e 115.25 T 13.55f 19.97 T 2.44d 18.57 T 2.14d 18.44 T 1.77d 14.22 T 1.66e 22.30 T 2.33e 18.22 T 2.33e 16.80 T 2.13e
Values are means T S.E.M. (n = 10). Numbers with different superscript are significantly ( P < 0.05) different with respect to row.
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Table 2 a-Tocopherol concentration (Ag/g fresh tissue) in egg yolk and in tissues of newly hatched chicks (n = 10)
Yolk Yolk sac membrane Liver Kidney Lung Heart Leg Breast
Housed Duck
Free-range Duck
Wild Mallard
Housed Chicken
Free-range Chicken
Free-range Guinea Fowl
Free-living Pheasant
16.33 T 1.66a 38.22 T 2.88a 62.11 T 5.22a 2.11 T 0.33a 2.16 T 0.23a 2.01 T 0.28a 1.02 T 0.12a 2.16 T 0.22a
68.13 T 5.66b 52.72 T 5.22b 101.12 T 9.66b 4.22 T 0.33b 3.60 T 0.32b 3.00 T 0.22b 1.90 T 0.13b 3.60 T 0.34b
94.32 T 6.99c 157.81 T16.33c 231.97 T 21.33c 11.58 T 1.66c 11.23 T 1.12c 10.12 T 1.02c 7.57 T 0.66c 16.63 T 1.66c
154.21 T12.22d 211.33 T 14.55d 601.33 T 29.33d 16.41 T1.33c 20.13 T 2.02d 22.21 T1.66d 13.11 T1.22d 16.34 T 1.55c
36.35 T 3.22e 86.12 T 7.11e 92.52 T 8.55b 4.12 T 0.36b 3.31 T 0.32b 6.49 T 0.52e 3.58 T 0.33e 7.60 T 0.55d
31.07 T 2.77e 56.99 T 6.33b 59.14 T 4.25a 5.19 T 0.62b,d 3.28 T 0.22b 5.34 T 0.44e 2.55 T 0.32b,e 5.05 T 0.32e
77.02 T 6.33b 157.36 T 14.22c 173.39 T 16.22e 5.83 T 0.32d,e 3.74 T 0.33b 6.18 T 0.55e 6.55 T 0.74c 13.90 T 1.52c
Values are means T S.E.M. (n = 10). Numbers with different superscript are significantly ( P < 0.05) different with respect to row.
a-tocopherol from egg yolk to the liver was observed in housed hens, where the a-tocopherol concentration in the liver was 3.9 times higher than in the yolk. On the other hand, the lowest efficiency of vitamin E accumulation in the liver was seen in the free-range duck where the concentration of a-tocopherol in the liver was only 1.5 times higher than that in the egg yolk. The concentrations of atocopherol in peripheral tissues were highest in the commercial chicken, reflecting its highest level in the egg yolk and liver. However, the a-tocopherol concentration in peripheral tissues of birds has some tissue- and speciesspecific features. For example, wild mallard breast muscle and kidney were characterised by comparatively high atocopherol concentration comparable to that in the commercial chicken. In contrast, the a-tocopherol concentration in the egg yolk and liver of the housed chicken was 1.6- and 2.6-fold higher than that in mallards. Furthermore, the atocopherol concentration in the egg yolk of free-range chicken’s hens was two times lower than that in the freeliving pheasants; however, the a-tocopherol concentrations in their lung tissue and heart were the same. In housed hens, the g-tocopherol concentration in the egg yolk and tissues were highest, with free-living pheasants being in the second position and free-range guinea fowl having the lowest g-tocopherol concentration. Similar to gtocopherol, the liver of housed hens was much more effective in accumulating g-tocopherol in comparison to other species, with free-range hens and guinea fowls to follow. In the other species in this study, the g-tocopherol concentration in the liver was not dissimilar to that in the
egg yolk. The yolk sac membrane of the housed chicken also contained the highest g-tocopherol concentration with free-living pheasant and free-range hens also showing active accumulation of g-tocopherol in the YSM. In the other species studied, there was no difference in g-tocopherol concentration between yolk and YSM. Other peripheral tissues were characterised by comparatively low levels of gtocopherol with housed chicken tissues found to contain much more g-tocopherol than other species of birds.
4. Discussion The main finding of this work is species- and tissuespecific differences in carotenoid and vitamin E distribution in the various avian species studied. Of particular interest are the comparatively high carotenoid concentrations in the egg yolks of the free-living pheasant, free-range guinea fowl and wild mallard. They are similar to those in free-living gulls (Surai et al., 2001a,b; Royle et al., 2001; Blount et al., 2002a,b), higher than those in wild moorhen (Gallinula chloropus) (Surai et al., 2001d) or housed, free-range domestic and feral Canada geese (Speake et al., 1999), gannet and skua (Surai et al., 2001e), great tits (Horak et al., 2002) or captive falcons (Barton et al., 2002). However, much higher carotenoid concentrations were observed in other wild birds, including the American coot (Fulica americana), Surai et al., 2001d), pelican (Pelecanus erythrorhynchos) and cormorant (Phalacrocorax auritus) (Surai et al., 2001e), or gulls (Larus fuscus) after carotenoid
Table 3 Gamma-tocopherol concentration (Ag/g fresh tissue) in egg yolk and in tissues of newly hatched chicks (n = 10)
Yolk Yolk sac membrane Liver Kidney Lung Heart Leg Breast
Housed Duck
Free-range Duck
Wild Mallard
Housed Chicken
Free-range Chicken
Free-range Guinea Fowl
Free-living Pheasant
2.11 T 0.22a 3.12 T 0.52a 4.66 T 0.44a 0.32 T 0.05a 0.32 T 0.03a 0.58 T 0.06a 0.33 T 0.02a 0.41 T 0.05a
3.23 T 0.34b 3.84 T 0.35a 3.24 T 0.33b 0.64 T 0.05b 0.44 T 0.03a,b 0.73 T 0.08ab 0.39 T 0.05a 0.50 T 0.04a
3.15 T 0.33b 2.95 T 0.25a 3.43 T 0.36b 0.42 T 0.05a 0.54 T 0.06b,c 0.62 T 0.08a 0.38 T 0.03a 0.65 T 0.06b
15.33 T 1.66c 41.15 T 5.11b 57.84 T 4.05c 3.45 T 0.33c 4.88 T 0.44d 6.54 T 0.58c 2.01 T 0.33b 2.88 T 0.41c
3.34 T 0.33b 6.02 T 0.55c 7.68 T 0.63d 0.84 T 0.08d 0.63 T 0.06c 0.83 T 0.07b 0.54 T 0.05a,c 0.81 T 0.10b,d
2.58 T 0.21a,b 2.68 T 0.21a 4.42 T 0.36a,b 0.63 T 0.06b 0.38 T 0.05a,b 0.57 T 0.06a 0.27 T 0.02a 0.42 T 0.03a
7.75 T 0.66d 13.37 T 1.22d 8.86 T 0.56d 1.44 T 0.12e 0.99 T 0.10e 1.23 T 0.11b 0.82 T 0.10d 1.03 T 0.11d
Values are means T S.E.M. (n = 10). Numbers with different superscript are significantly ( P < 0.05) different with respect to row.
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dietary supplementation (Blount et al., 2002a,b). The carotenoid concentration in the egg yolk is a reflection of dietary provision and efficiency of transfer from feed to egg yolk (Surai, 2002; Surai et al., 2001a,b). Hence it is to expect that eggs from free-range birds having an access to green vegetation contain a higher concentration of carotenoid than eggs from housed birds (Speake et al., 1999). Our previous data on carotenoid concentration in the egg yolk of the housed hen, turkey, goose and duck fed similar diets (Surai et al., 1998) indicate significantly higher levels of carotenoids in the eggs from housed hens in comparison to other avian species studied. However, when chickens and ducks have access to range the levels of carotenoids in their egg yolks were similar. The striking feature is that in egg yolk from wild mallards, the carotenoid concentration was 1.8-fold higher than that in free-range ducks. A different picture was observed in free-range geese where carotenoid concentration was significantly higher than that in feral Canada geese (Speake et al., 1999). Clearly there are aspects of carotenoid metabolism in captive and wild birds that need further investigation. The efficiency of carotenoid transfer from egg yolk to the liver differed significantly between species studied. In this respect, free-range and wild ducks showed an unusual pattern, with the carotenoid concentration in the liver of newly hatched ducklings being lower than the initial yolk. In contrast, in the chicks from housed hens, for example, the carotenoid concentration in the liver was more than twice that in egg yolk, indicating an active carotenoid accumulation in this tissue during embryonic development. It seems likely that a redistribution of carotenoids from the egg yolk to the liver is mediated via the yolk sac membrane, and that this process is somehow delayed in these ducks in comparison to other species studied since carotenoid concentrations in the YSM of free-range and wild ducks were higher that those in the egg yolk and liver. From all the species studied, free-range guinea fowls and pheasant were characterised by the highest carotenoid concentrations in peripheral tissues. For example in the kidney, lung, heart and breast muscle of these two species carotenoid concentration is identical and several times higher than that in the other species studied. However, in leg muscle, fat and especially in skin of the free-living pheasant, the carotenoid concentration is much higher than that in free-range guinea fowl. Therefore, although in these two species carotenoid concentration in the initial yolk was the same, during embryonic development species-specific features in carotenoid distribution between various tissues were observed. Molecular mechanisms of such differences are not known at present and need further research. In this respect, duck fat was characterised by the lowest carotenoid concentration. Again, in wild duck egg yolk carotenoid concentration was almost twice that in free-range duck, but in the skin and fat this difference practically disappeared. Since carotenoids are involved in immunomodulation in birds (Blount et al., 2003) increased carotenoid concentration in tissues of wild
birds could have health-related consequences. Indeed, egglaying capacity is limited by carotenoid pigment availability in wild gulls (L. fuscus) (Blount et al., 2004). Housed hen eggs were characterised by the highest vitamin E concentration which is probably a reflection of vitamin E supplementation (60 mg/kg) in the feed. On the other hand, wild mallard was characterised by comparatively high vitamin E concentration even though the diet was natural and was not supplemented with vitamin E. It is interesting to note that in egg yolk of commercial ducks supplemented with vitamin E at the level of 30 mg/kg, the concentration of a-tocopherol was 4 times lower than that in feral ducks without vitamin E supplementation. Similar extremely high vitamin E concentrations (294 – 299 Ag/g) were found in wild pelican and cormorant egg yolk (Surai et al., 2001e). It is not known at present if this is a reflection of a high vitamin E diet or efficient vitamin E transfer from the diet to the egg yolk. In contrast to carotenoids, in all the species studied, vitamin E concentration in the liver was several-fold higher than in the egg yolk, indicating an active accumulation of vitamin E during embryonic development. In fact, vitamin E accumulation in the embryonic liver, which reaches a maximum at hatching time, was suggested to be an adaptive mechanism in birds to deal with overproduction of free radicals at the time of hatchling (Surai, 2002). It is interesting that the efficiency of vitamin E transfer from the yolk to the developing liver in the housed hen and duck was higher than in free-range or wild birds. It seems likely that selection to achieve the highest growth rate and egg production is associated with increased free radical production (Surai, 2002), and the increased accumulation of vitamin E in the liver could be considered as an adaptive mechanism. Wild ducks were characterised by increased a-tocopherol concentration in the peripheral tissues (kidney, lung, heart and muscles) in comparison to other species except housed chicken. For example, in peripheral tissues of mallards atocopherol concentrations were several times higher than those in pheasants. Eggs from housed hens were characterised by the highest g-tocopherol concentrations in the egg yolk which is probably a reflection of grain-based diet providing more g-tocopherol than diets of free-range or wild birds (Surai, 2002). In general, it seems likely that the role of gtocopherol in the feed is underestimated. For example, in granivorous birds, g-tocopherol concentrations in the diet could exceed those of a-tocopherol (e.g. zebra finch, Royle et al., 2003; or other birds in captivity fed on unsupplemented diets, Surai, 2002). On the other hand, g-tocopherol could be an important element of the antioxidant-prooxidant balance in the digestive tract (Surai et al., 2003, 2004), which is ultimately responsible for health maintenance in birds or in mammalian species, including humans. This study provides evidence of species-specific differences in carotenoid and vitamin E accumulation in the egg
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yolk and their distribution from the liver and peripheral tissues during embryonic development. In particular, freerange and wild birds are usually characterised by increased carotenoid concentrations in the egg yolk and embryonic tissues in comparison to captive birds. If as has been proposed (Surai, 2002) that all antioxidants in the body work together to provide an effective antioxidant defence during critical periods of ontogenesis including hatching process the maternal investment of carotenoids and vitamin E into the egg is an important element in maintaining progeny fitness and survival. More work is needed to address specific questions in relation to this important issue in birds.
Acknowledgments The authors gratefully acknowledge The Scientific and Technical Research Council of Turkey (TUBI˙TAK) for a NATO Science Fellowship for Filiz Karadas and SEERAD which provides support to SAC.
References Barton, N.W.H, Fox, N.C., Surai, P.F., Speake, B.K., 2002. Vitamins E and A, carotenoids and fatty acids of the egg yolk of raptors. J. Raptor Res. 36, 33 – 38. Blount, J.D., Surai, P.F., Houston, D.C., Møller, A.P., 2002a. Patterns of yolk enrichment with dietary carotenoids in gulls: the roles of pigment acquisition and utilization. Funct. Ecol. 16, 445 – 453. Blount, J.D., Surai, P.F., Nager, R.G., Houston, D.C., Møller, A.P., Trewby, M.L., Kennedy, M.W., 2002b. Carotenoids and egg quality in the lesser black-backed gull Larus fucus: a supplemental feeding study of maternal effect. Proc. R. Soc. Lond., B 269, 29 – 36. Blount, J.D., Metcalfe, N.B., Birkhead, T.R., Surai, P.F., 2003. Carotenoid modulation of immune function and sexual attractiveness in zebra finches. Science 300, 125 – 127. Blount, J.D., Houston, D.C., Surai, P.F., Møller, A.P., 2004. Egg-laying capacity is limited by carotenoid pigment availability in wild gulls Larus fuscus. Proc. R. Soc. Biol. Lett. 271 (Suppl. 1), S79 – S81. Bohm, F., Edge, R., Land, E.J., McGarvey, D.J., Truscott, T.G., 1997. Carotenoids enhance vitamin E antioxidant efficiency. J. Am. Chem. Soc. 119, 621 – 622. Horak, P., Surai, P.F., Møller, A.P., 2002. Fat-soluble antioxidants in the eggs of great tits Parus major in relation to breeding habitat and laying sequence. Avian Sci. 2, 123 – 130. Li, L.L., Wu, L.M., Ma, L.P., Liu, J.C., Liu, Z.L., 1995. Antioxidant synergism and mutual protection of a-tocopherol and h-carotene in the inhibition of radical-initiated peroxidation of linoleic acid in solution. J. Phys. Org. Chem. 8, 774 – 780. Møller, A.P., Biard, C., Blount, J., Houston, D.C., Ninni, P., Saino, N., Surai, P., 2000. Carotenoid-dependent signals: indicators of foraging efficiency, immunocompetence or detoxification ability? Poult. Avian Biol. Rev. 11, 137 – 159. Mortensen, A., Skibsted, L.H., 1997. Importance of carotenoid structure in radical-scavenging reactions. J. Agric. Food Chem. 45, 2970 – 2977. Mortensen, A., Skibsted, L.H., Truscott, T.G., 2001. The interaction of dietary carotenoids with radical species. Arch. Biochem. Biophys. 385, 13 – 19. Royle, N.J., Surai, P.F., Hartley, I.R., 2001. Maternally derived androgens and antioxidants in bird eggs: complementary but opposing effects? Behav. Ecol. 12, 381 – 385.
511
Royle, N.J., Surai, P.F., Hartley, I.R., 2003. Effect of variation in dietary intake on maternal deposition of antioxidants in zebra finch eggs. Funct. Ecol. 17, 472 – 481. Speake, B.K., Murray, A.M.B., Noble, R.C., 1998. Transport and transformation of yolk lipids during development of the avian embryo. Prog. Lipid. Res. 37, 1 – 32. Speake, B.K., Surai, P.F., Noble, R.C., Beer, J.V., Wood, N., 1999. Differences in egg lipid and antioxidant composition between wild and captive pheasants and geese. Comp. Biochem. Physiol., B 124 (1), 101 – 107. Surai, P.F., 1999a. Tissue-specific changes in the activities of antioxidant enzymes during the development of the chicken embryo. Br. Poult. Sci. 40, 397 – 405. Surai, P.F., 1999b. Vitamin E in avian reproduction. Poult. Avian Biol. Rev. 10, 1 – 60. Surai, P.F., 2000. Effect of the selenium and vitamin E content of the maternal diet on the antioxidant system of the yolk and the developing chick. Br. Poult. Sci. 41, 235 – 243. Surai, P.F., 2002. Natural Antioxidants in Avian Nutrition and Reproduction. Nottingham University Press, Nottingham, UK. Surai, P.F., Sparks, N.H.C., 2001. Comparative evaluation of the effect of two maternal diets on fatty acids, vitamin E and carotenoids in the chicken embryo. Br. Poult. Sci. 42, 252 – 259. Surai, P.F., Speake, B.K., 1998. Distribution of carotenoids from the yolk to the tissues of the chick embryo. J. Nutr. Biochem. 9, 645 – 651. Surai, P.F., Noble, R.C., Speake, B.K., 1996. Tissue-specific differences in antioxidant distribution and susceptibility to lipid peroxidation during development of the chick embryo. Biochim. Biophys. Acta 1304, 1 – 10. Surai, P., Ionov, I., Kuchmistova, E., Noble, R.C., Speake, B.K., 1998. The relationship between the levels of a-tocopherol and carotenoids in the maternal feed, yolk and neonatal tissues: comparison between the chicken, turkey, duck and goose. J. Sci. Food Agric. 76, 593 – 598. Surai, P.F., Noble, R.C., Speake, B.K., 1999a. Relationship between vitamin E content and susceptibility to lipid peroxidation in tissues of the newly hatched chick. Br. Poult. Sci. 40, 406 – 410. Surai, P.F., Speake, B.K., Noble, R.C., Sparks, N.H.C., 1999b. Tissuespecific antioxidant profiles and susceptibility to lipid peroxidation of the newly hatched chick. Biol. Trace Elem. Res. 68, 63 – 78. Surai, P.F., Speake, B.K., Sparks, N.H.C., 2001a. Carotenoids in avian nutrition and embryonic development: 1. Absorption, availability and levels in plasma and egg yolk. J. Poult. Sci. 38, 1 – 27. Surai, P.F., Speake, B.K., Sparks, N.H.C., 2001b. Carotenoids in avian nutrition and embryonic development: 2. Antioxidant properties and discrimination in embryonic tissues. J. Poult. Sci. 38, 117 – 145. Surai, P.F, Speake, B.K., Decrock, F., Groscolas, R., 2001c. Transfer of Vitamins E and A from yolk to embryo during development of the King Penguin. Physiol. Biochem. Zool. 74, 928 – 936. Surai, P.F., Speake, B.K., Wood, N.A.R., Blount, J.D., Bortolotti, G.R., Sparks, N.H.C., 2001d. Carotenoid discrimination by the avian embryo: a lesson from wild birds. Comp. Biochem. Physiol., B 128, 743 – 750. Surai, P.F., Bortolotti, G.R., Fidgett, A., Blount, J., Speake, B.K., 2001e. Effects of piscivory on the fatty acid profiles and antioxidants of avian yolk: studies on eggs of the gannet, skua, pelican and cormorant. J. Zool. Lond. 255, 305 – 312. Surai, K.P., Surai, P.F., Speake, B.K., Sparks, N.H.C., 2003. Antioxidant – prooxidant balance in the intestine: food for thought: 1. Prooxidants. Nutr. Genomics Funct. Foods 1, 51 – 70. Surai, K.P., Surai, P.F., Speake, B.K., Sparks, N.H.C., 2004. Antioxidant – prooxidant balance in the intestine: food for thought: 2. Antioxidants. Curr. Top. Nutraceutical Res. 2, 27 – 46. Yanishlieva, N.V., Aitzetmuller, K., Raneva, V.G., 1998. h-Carotene and lipid oxidation. Fett/Lipid 10, 444 – 462. Zhang, P., Omaye, S.T., 2001a. DNA strand breakage and oxygen tension: effects of beta-carotene, alpha-tocopherol and ascorbic acid. Food Chem. Toxicol. 39, 239 – 246. Zhang, P., Omaye, S.T., 2001b. Antioxidant and prooxidant roles for betacarotene, alpha-tocopherol and ascorbic acid in human lung cells. Toxicol. in Vitro 15, 13 – 24.