The esterification of cholesterol in the liver of the chick embryo.

The development of the chick embryo was characterised by the accumulation of large droplets of lipid in the cytoplasm of the embryonic liver, as revealed by electron microscopy. Analysis of the lipid composition of the livers indicated that this accumulation resulted from a dramatic increase in the cholesteryl ester content of the tissue during the the latter part of the embryonic period. This lipid is apparently derived from yolk cholesterol and may be taken up by the liver in the form of lipoprotein remnants. Significant levels of acyl-CoA: cholesterol acyltransferase (ACAT) activity were expressed in the liver throughout the second half of the developmental period, and this activity was maximal at the time when lipid transfer from the yolk was most intensive. The activity of microsomal cholesterol ester hydrolase (CEH) was very low throughout development, and no CEH activity was detected in the cytosolic fraction. In addition, substantial amounts of a cytosolic protein which inhibits CEH activity were present. Thus the relative activities of these enzymic systems are consistent with the net accumulation of cholesteryl ester which occurs in the liver during development.

Effects of adrenaline on the turnover of lipoprotein lipase in rat adipose tissue.

The mechanisms by which adrenaline brings about a reduction in the lipoprotein lipase activity of adipose tissue in vitro were investigated. The incorporation of [3H]leucine into lipoprotein lipase was measured during 1-h pulse incubations of rat epididymal fat bodies that had been preincubated for 4 h in the presence of glucose, insulin and dexamethasone. When adrenaline was added to the incubation medium at the start of the pulse, the incorporation of [3H]leucine was markedly reduced, suggesting that the rate of the enzyme's synthesis had decreased. On the other hand, the degradation of lipoprotein lipase, as measured by the loss of 3H-labelled enzyme protein during pulse-chase incubations of the epididymal fat bodies, was found to be significantly increased by the addition of adrenaline to the incubation medium at the start of the chase period. It is concluded that adrenaline is able both to inhibit the synthesis of lipoprotein lipase and to stimulate its degradation.

Regulation of the synthesis of lipoprotein lipase in adipose tissue by dexamethasone.

The incorporation of [3H]leucine into lipoprotein lipase during incubations of rat epididymal fat-bodies in vitro was significantly stimulated by dexamethasone, whereas total protein synthesis was unaffected. The stimulation by dexamethasone required the presence of insulin. The results suggest that dexamethasone, in the presence of insulin, may specifically induce lipoprotein lipase synthesis in adipose tissue.

Tissue-specific changes in lipid composition and lipoprotein lipase activity during the development of the chick embryo.

Lipoprotein lipase was present at a high specific activity in adipose tissue and heart of the chick embryo at the 14th day of development. The enzyme was also present in skeletal muscle but was absent from brain and liver. Major increases in the activity of lipoprotein lipase in adipose tissue and heart occurred from day 12 of development, concomitant with the beginning of the period of lipid uptake from the yolk. These results suggest that lipoprotein lipase may be involved in the utilisation of yolk-derived lipid by the tissues of the embryo. Relatively high levels of docosahexaenoic acid (22:6(n--3)) were present in the triacylglycerol isolated from plasma, adipose tissue, heart and liver. The relative proportions of this fatty acid in the triacylglycerol of the different tissues may be explicable in terms of the substrate specificity of lipoprotein lipase.

The transfer of docosahexaenoic acid from the yolk to the tissues of the chick embryo.

Changes in the amounts of the major fatty acids present in the lipids of the yolk complex and the embryo were delineated during embryogenesis of the chicken. The rates of transfer of palmitic, oleic, linoleic, linolenic and arachidonic acids from the lipids of the yolk complex were essentially identical. In contrast, docosahexaenoic acid (DHA) was preferentially transferred from the yolk complex at a rate which was significantly higher than that exhibited by the other major fatty acids. The rates of accumulation of both arachidonic acid and DHA in the lipids of the whole embryo were significantly greater than the rates observed for the C16 and C18 fatty acids, particularly between days 12 and 16 of the 21-day embryonic period. Analysis of the fatty acid composition of plasma lipid throughout development indicated that the triacylglycerol fraction contained relatively high proportions (up to approx. 14% w/w of total fatty acids) of DHA, but much lower proportions (approx. 3%) of arachidonic acid. In contrast, plasma phospholipid was enriched in arachidonic acid (up to approx. 18%), but contained much lower proportions (generally less than 3%) of DHA. A considerable amount of DHA was incorporated into adipose tissue triacylglycerol, so that by the time of hatching, the tissue represented a major store of this fatty acid. Over the hatching period, the amount of DHA in adipose triacylglycerol decreased dramatically, by up to 85%, whereas there was little or no change in the amounts of the other major fatty acyl components in this tissue. The amount of DHA as a component of brain phospholipid increased continuously throughout the developmental period studied. However, by the time of hatching, the amount of DHA in brain phospholipid represented less than 10% of the amount of this fatty acid originally present in the lipids of the yolk.

Tissue-specific differences in antioxidant distribution and susceptibility to lipid peroxidation during development of the chick embryo.

The purpose of this study was to determine the tissue-specific acquisition of antioxidant capacity during chick embryo development and to assess the effectiveness of this process in the prevention of lipid peroxidation. The transfer of alpha-tocopherol, carotenoids and ascorbic acid from the yolk/yolk sac membrane (YSM) to the developing chick embryo and the distribution of these antioxidant compounds between the embryonic tissues were investigated. The concentrations of alpha-tocopherol and carotenoids in the yolk decreased between day 15 of development and hatching at day 21, concomitant with an increase in the levels of these antioxidants in the YSM. The concentration of both these lipid-soluble antioxidants in the liver increased dramatically between day 18 of embryonic development and day 1 after hatching. The adipose tissue content of alpha-tocopherol also increased markedly during the late embryonic/early neonatal period. However, the levels of alpha-tocopherol in the liver were far higher than in any other tissue with particularly low levels observed for the brain. Also, carotenoids were undetectable in the developing brain. Ascorbic acid was not present in the initial yolk but high levels of this water-soluble antioxidant were detected in the YSM, particularly at the early stages of development. The concentration of ascorbic acid in the embryonic brain was far higher than in any other tissue. Homogenates of brain tissue were extremely susceptible to lipid peroxidation during incubation in vitro whereas extracts of liver, yolk and YSM were relatively resistant to lipid peroxidation, particularly in the absence of exogenous Fe2+.

The preferential mobilisation of C20 and C22 polyunsaturated fatty acids from the adipose tissue of the chick embryo: potential implications regarding the provision of essential fatty acids for neural development.

The aim of this study was to determine the relative mobilisation of the different fatty acyl components of the triacylglycerol (TAG) of the chick embryo's adipose tissue in the light of the specific requirements of the developing neural tissues of the embryo for C20-22 polyunsaturated fatty acids. Pieces of adipose tissue, obtained from embryos at various developmental stages, were incubated in vitro in Dulbecco's Medium containing serum albumen. The fatty acid compositions of the initial tissue TAG and of the free fatty acid (FFA) mobilised from the tissue during 1 h of incubation were determined and compared. The composition of the FFA released into the medium under conditions of basal (i.e., unstimulated) lipolysis was markedly different in several respects from that of the TAG from which it originated. The polyunsaturated fatty acids, 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3, were consistently found to be preferentially released into the medium, whereas the major fatty acyl constituents of the tissue, 16:0 and 18:1n-9, were selectively retained in the TAG. For example, at day 18 of development, the proportions (% w/w of fatty acids) of 20:5n-3 and 22:6n-3 released into the incubation medium were respectively 6.5 and 7.5 times higher than in the original tissue TAG. Glucagon stimulated the overall rate of mobilisation by approx. 2-fold and also partially suppressed the preferential mobilisation of C20-22 polyunsaturates. These results may be relevant to the elucidation of the means by which essential polyunsaturates are delivered from the yolk to the neural tissues of the embryo, with the implication of a mediatory role for the embryonic adipose tissue in this transfer.

Degradation of lipoprotein lipase in rat adipose tissue.

Pulse-chase studies have shown that the lipoprotein lipase protein of rat epididymal fat bodies is apparently rapidly degraded (43% in 3 h) during incubation at 37 degrees C under conditions where little degradation of the total adipose tissue protein is taking place.

Designer egg evaluation in a controlled trial.

OBJECTIVE: To evaluate the ability of designer eggs enriched in vitamin E, lutein, selenium (Se) and docosahexaenoic acid (DHA) to deliver micronutrients to the human in a palatable and visually acceptable form. DESIGN: Double-blind, placebo-controlled trial, two treatment groups balanced for sex and age. SETTING: Department of Biochemistry and Nutrition, SAC, Scotland. SUBJECTS: Forty healthy adult volunteers completed the study. Volunteers were recruited among staff of the Scottish Agricultural College Interventions: Volunteers consumed, for 8 weeks, either a designer egg or a normal table egg per day. Fasting blood samples were taken before and at the end of the study. RESULTS: Consumption of designer eggs enriched in vitamin E, lutein, Se and DHA significantly increased the levels of alpha-tocopherol, lutein and DHA in plasma as compared to the changes found after consumption of normal table eggs, with the largest increases found in plasma lutein (1.88-fold increase). The proportion of DHA was increased in all the main lipid classes of the plasma including triacylglycerol (2.3-fold), free fatty acids (1. 6-fold), cholesteryl ester (1.4-fold) and phospholipid (1.3-fold). Egg consumption did not change Se concentration in plasma, blood pressure, total plasma lipid concentrations or the concentrations of total cholesterol and HDL-cholesterol in plasma. CONCLUSION: Consumption of designer eggs enriched in vitamin E, lutein, DHA and Se as part of normal diet for 8 weeks effectively increased the blood levels of alpha-tocopherol, lutein and DHA. SPONSORSHIP: Scottish Office Agriculture, Environment, and Fisheries Department.

Selective excretion of yolk-derived tocotrienols into the bile of the chick embryo.

The aim of this study was to investigate the possibility of biodiscrimination between different forms of vitamin E during the development of the chick embryo. The vitamin E present in the initial yolk consisted of alpha-tocopherol (90%), (beta + gamma)-tocopherol (8%), alpha-tocotrienol (0.3%) and (beta + gamma)-tocotrienol (1.3%). In marked contrast, the vitamin E recovered from the bile of the day-16 embryo contained much higher proportions of alpha-tocotrienol (10%) and especially of (beta + gamma)-tocotrienol (42%). By the time of hatching, 56% of the vitamin E present in the bile was in the form of (beta + gamma)-tocotrienol. The residual yolk of the newly-hatched chick contained far greater proportions of alpha-tocotrienol (2.6%) and (beta + gamma)-tocotrienol (10%) than were present in the initial yolk. The results suggest that the liver of the embryo may selectively excrete tocotrienols as components of bile, whilst retaining the tocopherols within the hepatocytes. The increased proportions of tocotrienols in the residual yolk may result from the recycling of bile from the gall bladder to the yolk. The liver of the day-old chick contained alpha-tocopherol as the main form of vitamin E (90%) with only a small proportion (0.2%) of (beta + gamma)-tocotrienol. The alpha-tocopherol form was also the main vitamin E component in the brain (85%), heart (79%), lung (82%) and adipose tissue (91%) of the day-old chick. The present study suggests the occurrence of a high degree of biodiscrimination between tocopherols and tocotrienols during the development of the chick embryo.

Developmental changes in the levels of molecular species of triacylglycerol that contain docosahexaenoic acid in adipose tissue of the chick embryo.

Triacylglycerols from the initial yolk and from the adipose tissue of the chick embryo were subjected high-performance liquid chromatography in the silver ion mode to resolve molecular species of triacylglycerol on the basis of the degree of unsaturation. A total of 12 such species was resolved from the yolk samples, with only one of these species containing docosahexaenoic acid. In contrast, 17 species were resolved from adipose tissue at day 12 of embryo development, of which 8 contained docosahexaenoic acid. The major species containing this fatty acid consisted of docosahexaenoic acid in combination with palmitic and oleic acids. The amounts of the species consisting of C16 and C18 fatty acids in the adipose tissue increased continuously from day 12 until just before hatching at day 19 and then decreased slightly over the hatching period. In contrast, the amounts within the adipose depot of the species containing docosahexaenoic acid reached a maximal level by day 16 and then decreased dramatically to almost undetectable levels during the hatching period. The possibility that adipose tissue triacylglycerol in the chick embryo may function as a temporary store of docosahexaenoic acid for subsequent delivery to the developing neural tissues is suggested.

The fatty acid composition of brain phospholipids from chicken and duck embryos.

The effects of differences in the fatty acid composition of the lipids of egg yolk on the subsequent levels of arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) in the total phospholipids and in the isolated phospholipid classes of the embryonic brain were investigated by a comparison of two domesticated avain species, the chicken and the duck. The yolk phospholipids of chicken eggs contained similar proportions of 20:4n-6 and 22:6n-3 (approx. 6% wt/wt of total fatty acids). In marked contrast, the yolk phospholipids of commercially produced duck eggs contained an overwhelming preponderance of 20:4n-6 over 22:6n-3 (approx. 10% cf.1%). These differences between the yolks of the two species were only partly reflected in the fatty acid compositions of the total phospholipids of the embryonic brains at equivalent developmental stages. Typically, the chicken brain phospholipids contained approximate proportions of 20:4n-6 and 22:6n-3 of 8% and 17%, respectively, whereas both these polyunsaturates were present at approx. 11% in the duck samples. The brain phospholipids were resolved into their component phospholipid classes by high performance liquid chromatography. In both species, phosphatidylcholine contained only low levels of 20:4n-6 and 22:6n-3, whereas phosphatidylethanolamine displayed a high content of 22:6n-3. Phosphatidylserine was also rich in 22:6n-3 whereas phosphatidylinositol exhibited a high proportion of 20:4n-6. The results suggest that the relatively low level of 22:6n-3 in the yolk of duck eggs is partly compensated for by an enhanced efficiency in the incorporation of this fatty acid into the brain phospholipids, in comparison with the chicken.

The effects of dietary supplementation with docosahexaenoic acid on the phospholipid fatty acid composition of avian spermatozoa.

This study represents an attempt at the dietary manipulation of the fatty acid composition of chicken spermatozoa in order to enhance the levels of n-3 polyunsaturates at the expense of the n-6 fatty acids, which normally predominate in the lipids of avian semen. Male chickens were provided with either a control diet supplemented with maize oil or the test diet supplemented with fish oil (Tuna Orbital Oil) from 10 weeks of age. Semen samples were collected from the birds after 30 and 48 weeks of supplementation. The fish oil diet induced a significant but limited increase in the proportion of 22:6n-3 in the spermatozoan phospholipid in parallel with an equivalent decrease in the proportions of 20:4n-6 and 22:4n-6. However, since the maximal level of 22:6n-3 in the phospholipid that was achieved by fish oil feeding was less than 10% (wt/wt of fatty acids), these changes fell far short of representing a switch from the typical avian pattern to that more characteristic of the n-3 enriched mammalian semen. Analysis of the fatty acid compositions of the constituent classes of phospholipid in the spermatozoa indicated that, in both dietary states, the phosphatidylethanolamine fraction contained much greater proportions of n-6 and n-3 C20-22 polyunsaturates than the phosphatidylcholine fraction. The results indicate that the typical fatty acid profile of the spermatozoa of domesticated poultry, characterised by the predominance of C20-22 n-6 polyunsaturates, displays a considerable degree of resistance to manipulation by dietary means and does not adopt the "mammalian" type of profile following supplementation with n-3 fatty acids.

Carotenoid discrimination by the avian embryo: a lesson from wild birds.

The concentrations (microg/g wet yolk) of total carotenoids in eggs of the common moorhen (Gallinula chloropus), American coot (Fulica americana) and lesser black-backed gull (Larus fuscus), collected in the wild, were 47.5, 131.0 and 71.6, respectively. In contrast to data for eggs of the domestic chicken, beta-carotene was a significant component in the yolks of these three wild species, forming 25-29% by wt. of the total carotenoids present. The concentration of total carotenoids in the livers of the newly-hatched chicks was 5-10 times higher than in the other tissues and beta-carotene was again a major component, forming 37-58% of the hepatic carotenoids. In the newly-hatched gull, the proportions of both lutein and zeaxanthin were very low in the liver but high in the heart and muscle when compared with the yolk. By contrast canthaxanthin, echinenone and beta-carotene were very minor constituents of heart and muscle when compared with their proportions in the yolk of the gull. The proportions of lutein and zeaxanthin in the liver of the newly-hatched coot and moorhen were also far lower than in the yolk whereas the liver was relatively enriched with beta-cryptoxanthin, beta-carotene and (in the moorhen) echinenone. The results indicate that avian embryos discriminate between different carotenoids during their distribution from the yolk to the various tissues.

Acyltransferase activities in the yolk sac membrane of the chick embryo.

The activities of some enzymes of glycerolipid synthesis and fatty acid oxidation were measured in subcellular fractions of the yolk sac membrane (YSM), an extra-embryonic tissue that mediates the transfer of lipid from the yolk to the circulation of the chick embryo. The activities of monoacylglycerol acyltransferase and carnitine palmitoyl transferase-1 in the YSM (respectively, 284.8+/-13.2 nmol/min/mg microsomal protein and 145.6+/-9.1 nmol/min/mg mitochondrial protein; mean +/- SE; n = 4) at day 12 of development appear to be the highest yet reported for any animal tissue. Also, the carnitine palmitoyl transferase-1 of the YSM was very insensitive to inhibition by malonyl CoA. The maximal activities of glycerol-3-phosphate acyltransferase and diacylglycerol acyltransferase in the YSM (respectively, 26.7+/-2.2 and 36.1+/-2.1 nmol/min/mg microsomal protein) were also high compared with the reported values for various animal tissues. The very high enzymic capacity for glycerolipid synthesis supports the hypothesis that the yolk-derived lipids are subjected to hydrolysis followed by reesterification during transit across the YSM. The monoacylglycerol pathway appears to be the main route for glycerolipid resynthesis in the YSM. The results also suggest that the YSM has the capacity to perform simultaneously beta-oxidation at a high rate in order to provide energy for the lipid transfer process.

Changes in the size and docosahexaenoic acid content of adipocytes during chick embryo development.

The development of adipose tissue in the chick embryo was investigated using two groups of fertile eggs which differed by 1.7-fold in their initial yolk lipid levels. The triacylglycerol content of the subcutaneous adipose depot in both groups increased dramatically from day 12 of the 21-day embryonic period, attaining a maximal value just prior to hatching. During this period, the amount of triacylglycerol deposited in the adipose tissue was very highly correlated with the amount of lipid transferred from the yolk. The triacylglycerol content of the depot was also dependent on the initial yolk lipid content. During the hatching period, the amount of adipose triacylglycerol remained approximately constant in the group with the higher initial yolk lipid content but, in the case of the group with the lower initial yolk lipid levels, decreased by approximately 25%. The size distribution of adipocytes isolated from the tissue was determined by computerized image analysis microscopy. The mean adipocyte diameter increased from approximately 6 to 35 microns between days 12 and 19, irrespective of the initial yolk content, although development within the eggs with the lower initial yolk content resulted in a decrease in cell size over the hatching period. Both the triacylglycerol and phospholipid fractions of the isolated adipocytes contained substantial proportions (approximately 6%, w/w) of docosahexaenoic acid (DHA) at days 12 and 14, and lower levels of this fatty acid at the later stages. The amount (mg/depot) of DHA in adipose triacylglycerol decreased dramatically over the hatching period. The amount (mg/brain) of DHA in brain phospholipid increased by more than 5-fold between day 12 of development and hatching. A possible explanation for the data may be that DHA is preferentially mobilized from adipose tissue in order to deliver the fatty acid to the developing neural tissues in a form suitable for uptake.

Fatty acid composition of the adipose tissue and yolk lipids of a bird with a marine-based diet, the emperor penguin (Aptenodytes forsteri).

The emperor penguin (Aptenodytes forsteri) is an Antarctic seabird feeding mainly on fish and therefore has a high dietary intake of n-3 polyunsaturated fatty acids. The yolk is accumulated in the developing oocyte while the females are fasting, and a large proportion of the fatty acid components of the yolk lipids are derived by mobilization from the female's adipose tissue. The fatty acid composition of the total lipid of the yolk was characterized by high levels of n-3 polyunsaturated fatty acids. However, it differed in several respects from that of the maternal adipose tissue. For example, the proportions of 14:0, 16:1n-7, 20:1n-9, 22:1n-9, 20:5n-3, and 22:6n-3 were significantly greater in adipose tissue than in yolk. Thus adipose tissue lipids contained 7.6+/-0.3% and 8.0+/-0.3% (wt% of total fatty acids; mean +/- SE; n = 5) of 20:5n-3 and 22:6n-3, respectively, whereas the yolk total lipid contained 1.6+/-0.1 and 5.5+/-0.3% of these respective fatty acids. The proportions of 16:0, 18:0, 18:1n-9, 18:2n-6, and 20:4n-6 were significantly lower in the adipose tissue than in the yolk lipids. The proportions of triacylglycerol, phospholipid, free cholesterol, and cholesteryl ester in the yolk lipid were, respectively, 67.0+/-0.2, 25.4+/-0.3, 5.3+/-0.2, and 1.8+/-0.2% (wt% of total yolk lipid). The proportions of 20:4n-6, 20:5n-3, 22:5n-3, and 22:6n-3 were, respectively, 5.7+/-0.3, 2.8+/-0.2, 1.4+/-0.1, and 11.7+/-0.5% in phospholipid and 0.4+/-0.0, 1.2+/-0.1, 0.8+/-0.1 and 3.6+/-0.3% in triacylglycerol. About 95% of the total vitamin E in the yolks was in the form of alpha-tocopherol with gamma-tocopherol forming the remainder. Two species of carotenoids, one identified as lutein, were present.

Lipid composition of eggs of an oviparous lizard (Bassiana duperreyi).

Lipid analysis was performed on freshly ovulated eggs (n = 5) of the oviparous lizard Bassiana duperreyi. The fresh weight of the whole egg contents was 132.0 +/- 4.3 mg (mean +/- SE) of which lipid constituted 21.9 +/- 1.1% (w/w). Triacylglycerol formed an exceptionally high proportion (85.4 +/-0.5%, w/w) of the total lipid, whereas phospholipid, free cholesterol, cholesteryl ester, and free fatty acid, respectively, contributed 11.2 +/- 0.3, 1.4 +/- 0.1, 1.3 +/- 0.1, and 0.6 +/- 0.1% of the total lipid mass. Linoleic and alpha-linolenic acids were the major polyunsaturates of the triacylglycerol fraction, respectively, forming 16.3 +/- 0.1 and 8.3 +/- 0.1% (w/w) of the fatty acids. Linoleic acid was the major fatty acid (29.0 +/- 0.1%) of the total phospholipid, which also contained substantial amounts of arachidonic (6.4 +/- 0.1%) and eicosapentaenoic (3.0 +/- 0.1%) acids, but a relatively low proportion (1.6 +/- 0.1%) of docosahexaenoic acid. Phosphatidylcholine formed the major phospholipid class (73.8 +/- 2.3%) w/w of total phospholipid) and was enriched in linoleic acid, whereas phosphatidylethanolamine, which formed 20.4 +/- 1.9%(w/w) of total phospholipid, contained higher proportions of arachidonic and docosahexaenoic acids.

The esterification of cholesterol in the yolk sac membrane of the chick embryo.

The uptake of lipid from the yolk by the yolk sac membrane of the chick embryo is accompanied by the rapid esterification of a large proportion of the yolk cholesterol. This could arise from enhanced acyl-CoA:cholesterol acyltransferase (ACAT) activity and/or inhibition of cholesteryl ester hydrolase (CEH) activity. The activity of ACAT was therefore measured in microsomes obtained from yolk sac membranes at various stages of development. A high level of activity (up to 929 pmol of cholesteryl oleate formed per min per mg protein) was found during the second half of this period. Supplementation with exogenous cholesterol stimulated ACAT activity in microsomes obtained from the tissue at the earlier, but not at the later, stages of development suggesting that the enzyme became saturated with microsomal cholesterol as development proceeded. Correlating with this, the concentration of cholesterol in the microsomes increased 4-fold between 9 and 20 d of development. The activity of CEH was very low in the microsomes and could not be detected in the cytosolic fraction. The activity of a protein, which has been shown to function as an inhibitor of CEH, was found to be present at all stages of development. The high activity of ACAT, together with the low activity of CEH and an active CEH inhibitor protein is a combination well suited to promote an essentially unidirectional conversion of cholesterol to cholesteryl ester. This process may be a major determinant of the rate of lipid transfer from the yolk to the embryo.

Transfer of Vitamins E and A from yolk to embryo during development of the king penguin (Aptenodytes patagonicus).

Since the yolk lipids of the king penguin (Aptenodytes patagonicus) are rich in n-3 fatty acids, which are potentially susceptible to peroxidative damage, the yolk contents and yolk-to-embryo transfer of antioxidants and lipid-soluble vitamins were investigated under conditions of natural incubation in the wild. The concentration of vitamin E in the unincubated egg was 155 microg/g wet yolk, of which 88% was alpha-tocopherol and the rest was gamma-tocopherol. Vitamin A (2.9 microg/g) was present in the yolk entirely as retinol; no retinyl esters were detected. Throughout the latter half of the incubation period, vitamins E and A were taken up from the yolk into the yolk sac membrane (YSM) and later accumulated in the liver, with vitamin A being transferred in advance of vitamin E. In the YSM, vitamin A was present almost entirely as retinyl ester, indicating that the free retinol of the yolk is rapidly esterified following uptake. Retinyl esters were also the predominant form in the liver. The retinyl esters of the liver and YSM displayed different fatty acid profiles. At hatching, the brain contained relatively little vitamin E (4.7 microg/g) compared to the much higher concentration in the liver (482.9 microg/g) at this stage. Ascorbic acid was not detected in the yolk but was present at a high concentration in the brain at day 27 (404.6 microg/g), decreasing to less than half this value by the time of hatching. This report is the first to delineate the yolk-to-embryo transfer of lipid-soluble vitamins for a free-living avian species. The yolk fatty acids of the king penguin provide an extreme example of potential oxidative susceptibility, forming a basis for comparative studies on embryonic antioxidant requirements among species of birds whose yolk lipids differ in their degree of unsaturation.