Vol. 280, Issue 3, R843-R853, March 2001
Transfer of n-3 and n-6 polyunsaturated fatty acids
from yolk to embryo during development of the king penguin
Frederic
Decrock1,
René
Groscolas1,
Ruth J.
McCartney2, and
Brian K.
Speake2
1 Centre d'Ecologie et Physiologie Energétiques,
Centre National de la Recherche Scientifique, Associé à
l'Université Louis Pasteur, 67087 Strasbourg,
France; and 2 Avian Science Research Centre,
Scottish Agricultural College, Ayr KA6 5HW, United Kingdom
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ABSTRACT |
This study
examines the transfer of lipids from the yolk to the embryo of the king
penguin, a seabird with a high dietary intake of n-3 fatty acids.
The concentrations of total lipid, triacylglycerol (TAG), and
phospholipid (PL) in the yolk decreased by ~80% between days
33 and 55 of development, indicating intensive lipid
transfer, whereas the concentration of cholesteryl ester (CE) increased
threefold, possibly due to recycling. Total lipid concentration in
plasma and liver of the embryo increased by twofold from day
40 to hatching due to the accumulation of CE. Yolk lipids contained high amounts of C20-22 n-3 fatty acids
with 22:6(n-3) forming 4 and 10% of the fatty acid mass in TAG
and PL, respectively. Both TAG and PL of plasma and liver contained
high proportions of 22:6(n-3) (~15% in plasma and >20% in
liver at day 33); liver PL also contained a high proportion
of 20:4(n-6) (14%). Thus both 22:6(n-3) and 20:4(n-6),
which are, respectively, abundant and deficient in the yolk, undergo
biomagnification during transfer to the embryo.
docosahexaenoic acid; arachidonic acid; yolk sac membrane
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INTRODUCTION |
THE AMNIOTE EGG,
EVOLVED from ancestral reptiles, was the crucial adaptation that
enabled reproduction on land. The provisioning of the egg with
large amounts of lipid was central to this adaptation, supplying the
embryo with a concentrated source of energy to sustain development to
an advanced stage, thus avoiding the need for an aquatic larval phase.
The evolution of birds was accompanied by an increased reliance on yolk
lipids as the primary nutrients for the embryo. Consequently, the
embryonic development of modern avian species is characterized by an
array of highly specialized features that promote the efficient
transfer and use of yolk lipid (31).
Our current understanding of the transport and metabolism of yolk
lipids during avian development has been almost exclusively derived
from studies on the embryo of the domestic chicken. These have shown
that the lipids are mainly taken up intact from the yolk into the yolk
sac membrane (YSM) by phagocytosis. Within the YSM, the lipids are
remodeled by hydrolysis and reesterification and are then assembled
into lipoproteins of the very low density type (VLDL). The secretion of
these VLDL particles from the YSM into the embryonic circulation is
followed by their hydrolysis by lipoprotein lipase located in the
capillaries of the embryo's adipose tissue, heart, and skeletal
muscle. The fatty acids released by this action are used by the growing
tissues for energy, membrane synthesis, or lipid storage. The resultant
VLDL remnants are taken up by the embryonic liver; as a consequence,
the cytoplasm of the hepatocytes becomes engorged with lipid droplets.
Superimposed onto this background of bulk lipid transport is a series
of specialized features that relate to the preferential delivery of the
long-chain polyunsaturates, arachidonic [20:4(n-6)] and
docosahexaenoic [22:6(n-3)] acids, to certain tissues at defined
stages of development. These aspects of lipid use by the avian embryo
have been the subject of several recent review articles (14, 16,
28, 30, 31).
Some of the findings of these studies are particularly striking. The
sheer intensity of lipid uptake from the yolk during the second half of
the developmental period is complemented by the expression of extremely
high activities of a range of enzymes involved in lipid metabolism
(12, 23, 24, 29) and of high levels of the mRNA
transcripts that code for various apoproteins (8, 34, 35).
Also, many tissues of the embryo display highly characteristic lipid
and fatty acid compositions, partly as a result of the general process
of bulk lipid transfer and partly due to the specialized features
relating to the transport of 20:4(n-6) and 22:6(n-3)
(14, 28). It is important, however, to reiterate that
these findings have emanated almost entirely from studies on only one
of the 9,000 extant species of birds, namely the domestic chicken. As a
consequence, there is very little information relating to the potential
adaptations of the lipid transfer process to the great diversity in
diet, yolk fatty acid composition, reproductive strategies, and
developmental modes displayed by avian species.
The king penguin (Aptenodytes patagonicus) is a seabird with
an adult weight of ~12 kg. This species inhabits the sub-Antarctic region and breeds during the summer in very large colonies on remote
islands. It feeds exclusively at sea, and its diet during the breeding
season consists mainly of myctophid fishes (4). This diet
is characterized by very high levels of n-3 polyunsaturated fatty
acids, particularly 22:6(n-3) and eicosapentaenoic acid [20:5(n-3)], but provides relatively low amounts of n-6
polyunsaturates (19). The diet also provides large amounts
of very long chain monounsaturates, particularly gondoic
[20:1(n-9)], erucic [22:1(n-9)], and cetoleic
[22:1(n-11)] acids (19). The fatty acid profile of
this marine-based diet is markedly different from that of the granivorous chicken, where linoleic acid [18:2(n-6)] is the main polyunsaturate (28). Recent studies on eggs of the emperor
penguin (Aptenodytes forsteri), a species with a similar
diet to that of the king penguin, indicated that the proportion of
long-chain n-3 fatty acids in the yolk lipids was eight times
greater than in the yolk of the chicken (26). With yolk
n-3/n-6 ratios of 1.8 and 0.1 for the emperor penguin and
chicken, respectively (26), it is feasible that the
embryos of these two species may have adopted different strategies for
optimizing the delivery of 22:6(n-3) and 20:4(n-6) to the
relevant tissue sites.
Several other aspects of the king penguin's unique reproductive
strategy may also have a bearing on the composition and use of yolk
lipid and could provide contrasts with the situation described for the
chicken. The female king penguin produces only one egg per breeding
season, and the yolk lipid is mainly formed during a period of ~2 wk
while the female is ashore and fasting, using fatty acids mobilized
from adipose tissue that was accumulated during the previous feeding
period at sea (6, 32). By contrast, the domestic chicken
produces over 300 eggs per year, synthesizing the yolk lipid from the
concurrent dietary intake (28). Also, the chicken produces
precocial hatchlings, whereas penguin chicks are classified as
semialtricial, being dependent on their parents for food and warmth
(31). Thus embryonic development in the penguin produces
chicks that are in some ways less advanced than those of precocial
species. Another factor is the phylogenetic distance between the
different avian species. The period of major diversification of birds
occurred during the early Tertiary, and most of the present avian
orders had evolved by ~35 million years ago (31). The
lineages that gave rise to the Sphenisciformes (penguins) and
Galliformes (e.g., the chicken) have, therefore, been separate for a
considerable duration, presumably sufficient for any specialized
adaptations of the yolk lipid transfer process to become established.
To provide an adaptationary perspective for the process of yolk lipid
use, we attempted to delineate the main features of this transfer
during the embryonic development of the king penguin. This was
performed by determining the lipid content and fatty acid composition
of yolk, YSM, plasma, and liver at various stages of egg incubation
under natural conditions. Also, the provisioning of the yolk with fatty
acids during its formation was examined by measuring the composition of
the diet and adipose tissue of the breeding female.
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MATERIALS AND METHODS |
The penguin colony.
The samples for this study were obtained from a breeding colony of king
penguins at Baie du Marin, Crozet Archipelago, Indian Ocean
(46°26'S-51°52'E) in the austral summer of 1998/1999. The project was approved by the ethical committee of the Institut Français pour la Recherche et la Technologie Polaires and
conformed to the Agreed Measures for the Conservation of Antarctic and
Subantarctic Fauna. The use of 50 eggs had a very limited impact on the
local population (30,000 breeding pairs) given that natural mortality is several thousand chicks per year. Nevertheless, to reduce this impact, eggs laid in an unfavorable area where posthatching mortality is >90% due to recurrent flooding were mostly used.
Sampling of adults' food and adipose tissue.
Food was obtained by aspiration with a tube from the stomachs of six
adults that were feeding their newly hatched chicks. It was shown
previously that the composition of stomach contents is similar to that
of fresh food (19) and that the diet of king penguins
remains the same throughout the breeding season (4). Subcutaneous adipose tissue biopsies (0.2-0.5 g) were taken under general anesthesia (halothane inhalation) from seven fasting-courting females ~10 days before laying and when yolk was being deposited in
the oocyte. Food and adipose tissue samples were kept under 1:1
(vol/vol) chloroform-methanol at 
20°C until analysis.
Egg incubation and embryo sampling.
Embryos at different stages of development were obtained from eggs laid
in the wild under natural conditions of diet and habitat and which were
naturally incubated by their parents in the breeding colony. A few days
before laying, territorial king penguin pairs were located, and birds
were premarked from a distance with Nil blue. The pairs were observed
daily to determine the date of laying. The female departs to feed at
sea within hours of laying, and both parents alternate in incubating
the eggs, changing shifts roughly every 2 wk during the 53 ± 1 days of incubation (6, 32). The mass of the egg differs
markedly among pairs, from 250 to 350 g. To reduce possible bias
due to these differences, only eggs weighing 280-320 g were
selected. Thus within 12 h of laying, the egg was briefly removed
from the brood pouch of the incubating bird, weighed, and marked with
ink if its mass was within the selected range. It was immediately
returned to the incubating bird (male at this stage), the latter being
banded at flipper for further identification throughout incubation,
whereas females were banded at first relief. To compensate for possible egg loss or infertility, 80 eggs and pairs were marked, and 50 of these
eggs were subsequently used for the study.
Eggs were collected after 0, 27, 33, 40, and 47 days of natural
incubation; eggs between 49 and 51 days that were in the process of
hatching were also collected (hatching is completed after 53 ± 1 days but begins ~3 days earlier). These time points correspond to
~0, 50, 60, 75, 90, and 100% of the duration of embryonic
development. For the first five time points, embryos were killed by
decapitation immediately after removing the egg from the incubating
parent. Eggs containing embryos that had commenced hatching (detected by a hole in the shell ~1 cm diameter) were transferred from the parent to a laboratory incubator maintained at 37.5°C and 100% relative humidity until the chicks were fully hatched. Also, eight chicks were maintained for 2 days after hatching and provided with
drinking water but no food. At death, a blood sample was obtained from
the umbilical vein of the embryo using a needle and heparinized
syringe. Blood samples from hatched chicks were obtained from the neck
after decapitation. Plasma was separated by centrifugation for 15 min
at 4,500 g. Yolk and liver samples were dissected and
immediately stored frozen at 20°C in polypropylene tubes. The
excised YSM samples were washed thoroughly in saline at 4°C to remove
any adherent yolk and blotted dry before freezing. Samples were
maintained at 20°C during transport by air to Scotland, where they
were then stored at 80°C until analyzed. All analyses were
completed within 6 mo of sampling.
Because portions of the tissue samples were needed for a separate study
that required immediate freeze-clamping, it was not possible to
determine the fresh weights of embryos or tissues at the time of
sampling. However, in a previous study (Groscolas and Decrock,
unpublished data) using eggs in the same mass range, the fresh weights
(g) of the embryos at days 27, 33, 40,
47, and 53 were 15.5 (n = 1),
36.5 (n = 2), 88.0 (n = 1), 140.1 ± 3.5 (n = 5), and 177.5 ± 3.7 (n = 14), respectively. The fresh weights of the yolk
complex (yolk plus YSM) at days 0, 33,
47, and 53 were, respectively, 64.6 ± 2.1 (n = 9), 56.5 ± 4.2 (n = 8),
42.9 ± 2.9 (n = 8), and 23.8 ± 2.4 (n = 22).
Lipid analysis.
Samples were homogenized in a suitable excess of 2:1 (vol/vol)
chloroform-methanol with subsequent washing of the organic phase with
0.88% (wt/vol) KCl. The amount of total lipid in the samples was
determined gravimetrically on half the chloroform extract after
evaporation of solvent. Portions of the extracts were subjected to
thin-layer chromatography on silica gel G using a solvent system of
80:20:1 (vol/vol/vol) hexane-diethyl ether-formic acid to isolate the
major lipid classes [triacylglycerol (TAG), phospholipid (PL), free
fatty acid (FFA), cholesteryl ester (CE), free cholesterol (FC)].
Visualization of the bands, elution of the lipid classes from the
silica, and transmethylation to form fatty acid methyl esters were
performed as previously described (15). To determine the
fatty acid composition of the total lipid, the thin-layer
chromatography step was omitted and transmethylation was performed on a
portion of the initial extract. The fatty acid methyl esters were
analyzed by gas-liquid chromatography using a capillary column
(Carbowax, 30 m × 0.25 mm, film thickness 0.25 µm; Alltech,
Carnforth, UK) in a CP9001 Instrument (Chrompack, Middleburg, The
Netherlands) connected to an EZ Chrom Data Analysis System (Scientific
Software, San Ramon, CA). The column was maintained at 185°C for 2 min after injection of the sample and then increased at 5°C/min for 9 min and maintained at 230°C for a further 24 min. The peaks were
quantified by comparison with a 19:0 standard and were identified by
comparison with the retention times of standard fatty acid methyl ester
mixtures (Sigma, Poole, UK). The EZ Chrom software enabled the
expression of the fatty acid compositions in terms of weight
percentage. The amounts of the lipid classes were calculated from the
amount and composition of the fatty acyl groups derived from each
class, together with the acyl group contribution to the molecular
weight of these compounds. FC was determined using an
enzymatic-colorimetric assay kit based on the cholesterol oxidase
reaction (Boehringer, Lewes, UK).
Data analysis.
Values are means ± SE. Percent values were transformed to arcsin
before statistical analysis to make the variance independent of the
mean. Comparison between two developmental stages was performed using
the unpaired, two-tailed Student's t-test. Multiple
comparisons used the Peritz' F test.
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RESULTS |
Fatty acid composition of total lipid in adult's diet, maternal
adipose tissue, and the yolk.
The diet of the adult king penguin is rich in C20-22
n-3 polyunsaturates (21% of dietary fatty acids) and in
C20-22 monounsaturates (12%), but it is a very poor
source of all n-6 polyunsaturates (2%) (Table
1). Although the proportions of certain fatty acids [e.g., 16:0 and 22:6(n-3)] in the maternal adipose tissue were significantly different from those in the diet, these differences were relatively low in magnitude. Thus the fatty acid profile of the adipose tissue, where C20-22 n-3
polyunsaturates, C20-22 monounsaturates, and total
n-6 polyunsaturates, respectively, form 24, 13, and 2% of the
fatty acid mass, largely reflects that of the diet. In contrast, the
proportions of several fatty acids in the yolk lipids were markedly
different from those in the maternal adipose tissue. Thus, by
comparison with adipose tissue, the yolk lipid was relatively enriched
with 16:0, 18:0, 18:1(n-9), and 20:4(n-6); contained
relatively reduced levels of 20:1(n-9), 20:5(n-3), and
22:6(n-3); and was devoid of 22:1(n-11)/(n-9) and
24:1(n-9).
Transfer of lipid from yolk to embryo.
Changes in the concentrations of total lipid and of the acyl-lipid
classes (TAG, PL, CE) in the yolk, YSM, plasma, and liver are shown
throughout the embryonic period. The concentration of total lipid in
the wet yolk (Fig. 1) decreased
significantly (P < 0.001) between the beginning of
incubation and day 27, then increased transiently at
day 33 (P < 0.001) followed by a continuous decrease to the time of hatching (P < 0.001). Two days
after hatching, the concentration of lipid in the internalized yolk was
only 11% of that in the yolk of the newly laid egg. The concentrations of TAG and PL in the yolk changed parallel with the alterations in
total lipid content. Throughout development, the concentrations of TAG
and PL in the yolk were highly correlated (r = 0.98, P < 0.001). By contrast, the concentration of CE in
the yolk did not change significantly between the beginning and end of
incubation but then increased 3.1-fold (P < 0.002) by
2 days after hatching.
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Fig. 1.
Changes in the concentrations of total lipid,
triacylglycerol (TAG), phospholipid (PL), and cholesteryl ester (CE) in
the yolk during embryonic development of the king penguin. Values are
means ± SE of n = 8 yolks at each stage, except
days 0 and 55, when n = 10 and 4, respectively.
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The concentration of total lipid within the wet mass of the YSM (Fig.
2) increased 1.8-fold (P < 0.001) between days 27 and 33 of development,
did not change significantly between days 33 and
40, and then decreased by 50% (P < 0.001)
by the time of hatching. At its maximal concentration (at day
33), lipid formed 32% of the wet mass of this tissue. TAG and PL
in the YSM changed parallel with the total lipid content and were
significantly correlated (r = 0.98, P < 0.001) throughout development. Changes in CE concentration followed
a different course, increasing continuously throughout development. Two
days after hatching, the concentration of CE in the internalized YSM
was 3.2 times greater (P < 0.001) than at day
27 of embryonic life.
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Fig. 2.
Changes in the concentrations of total lipid, TAG, PL,
and CE in the yolk sac membrane (YSM) during embryonic development of
the king penguin. Values are means ± SE from n = 8 embryos, except days 27 and 53, when
n = 7 and 6, respectively.
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The concentration of total lipid in the plasma (Fig.
3) was constant from day 27 to
day 40 but then increased continuously to the hatching
period. Thus the plasma lipid concentration was 2.1 times
(P < 0.002) higher at hatching than at day
40. This was accompanied by a parallel increase in the
concentration of CE in the plasma, which had increased 3.2-fold
(P < 0.002) between day 40 and hatching.
The concentration of PL in plasma increased 2.0-fold (P < 0.01) between day 40 and hatching, but unlike CE, this
increase did not continue into the posthatch period. The plasma
concentration of TAG did not change significantly between days
27 and 47 but thereafter decreased sharply so that its
value 2 days after hatching was only 24% of that at day 47 (P < 0.02).
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Fig. 3.
Changes in concentrations of total lipid, TAG, PL, and CE
in the plasma during embryonic development of the king penguin. Values
are means ± SE from n = 3, 6, 5, 6, 8, and 8 embryos at days 27, 33, 40,
47, 53, and 55, respectively.
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Lipids formed an increasing proportion of the wet mass of the liver
(Fig. 4) throughout the period of
embryonic development from day 27 to hatching, with the most
pronounced elevation occurring after day 40. As a result,
the hepatic lipid concentration was 2.7 times higher (P < 0.001) at hatching than at day 27. This increase was
entirely due to the accumulation of CE in this tissue; this lipid class
changed parallel with the level of total lipid throughout development,
whereas the hepatic concentrations of TAG and PL showed no significant
change. At the time of hatching, CE formed 13% of the wet mass of the
liver.
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Fig. 4.
Changes in concentrations of total lipid, TAG, PL, and CE
in the liver during embryonic development of the king penguin. Values
are means ± SE from n = 8 embryos, except
days 27 and 53, when n = 4 and 7, respectively.
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Fatty acid compositions of lipid classes of yolk,
YSM, plasma, and liver.
Comparisons of the fatty acid compositions at days 33 and 55 (i.e., at 60% of the embryonic period and 2 days
after hatching) are presented. Day 33 was chosen instead of
day 27 to illustrate the data, because only three replicates
of plasma were available for day 27. In the yolk of the
newly laid egg, TAG, PL, CE, FC, and FFA, respectively, formed
61.4 ± 0.4, 26.8 ± 0.3, 3.8 ± 0.4, 6.7 ± 0.2,
and 1.3 ± 0.2% of the total lipid mass (n = 9).
There were no significant differences in the fatty acid profiles of the
lipid classes between day 0 (data not shown) and day
33, with the exception that 22:6(n-3) formed 7.9% of CE
fatty acids at day 0 compared with 9.5% at day
33 (P < 0.02). Thus the fatty acid profile of the
yolk lipid classes at day 33 presented in Table
2 is essentially representative of the
initial composition of the newly laid egg.
Whereas TAG followed by PL were the major lipid classes of the yolk at
day 33, their proportions at day 55 were greatly
reduced; these changes were commensurate with a dramatic increase in
the proportion of CE that formed almost half the total yolk lipid at
the latter stage (Table 2). Palmitic (16:0) and oleic
[18:1(n-9)] acids were the major fatty acids of yolk TAG, PL,
and CE at day 33. Particularly notable were the high
proportions of C20-22 n-3 polyunsaturates, the
substantial presence of 20:1(n-9), and the low levels of
C18 polyunsaturates in the three lipid classes, especially
when compared with data for chicken eggs (28). There were
no significant changes in the fatty acid compositions of TAG and PL
between days 33 and 55, apart from a minor
decrease in the proportion of palmitoleic acid [16:1(n-7)] in
TAG. The increased proportion of CE by day 55 was, however,
accompanied by changes in its fatty acid profile; thus the proportions
of 18:0 and 18:1(n-9) were increased and those of 14:0, 16:0,
16:1(n-7), 20:4(n-6), 20:5(n-3), and 22:6(n-3) were
decreased at this stage.
The YSM also displayed decreased proportions of TAG and PL and a
pronounced increase in the level of CE between days 33 and 55 (Table 3). The fatty acid
profiles of the TAG and PL of the YSM at day 33 were very
similar to those of the yolk at the same developmental stage; the only
significant differences (P < 0.01) shown by the YSM
were reduced proportions of 20:1(n-9) and 16:0 in TAG and PL,
respectively, and a slight increase in the level of 20:4(n-6) in
PL and of 18:3(n-3) in TAG. The compositional differences for CE
between the YSM and the yolk at day 33 were, however, more
marked; the CE of the YSM displayed significantly (P < 0.001) higher proportions of 18:0, 18:1(n-9), and 18:3(n-3) and lower proportions of 16:0, 20:4(n-6), 20:5(n-3), and
22:6(n-3). The changes in the compositions of TAG and PL of the
YSM between days 33 and 55 were relatively minor;
in both lipid classes there were increases in 20:1(n-9) and
22:6(n-3) and a decrease in 16:0. Differences in the composition
of CE between the two stages were limited to an increased proportion of
20:1(n-9) and decreases in the levels of 18:0 and 20:5(n-3)
at day 55.
Whereas TAG, PL, and CE each contributed ~30% of the total lipid of
the plasma at day 33, the plasma at day 55 was
characterized by the near depletion of TAG, a reduction in the
proportion of PL, and a doubling of the proportion of CE (Table
4). Most noteworthy were the differences
in fatty acid composition between the lipids of the plasma and those of
the YSM and yolk, particularly regarding the C20-22
polyunsaturates. For example, at day 33, the proportions of
22:6(n-3) in TAG and PL were, respectively, almost 3.0 (P < 0.001) and 1.5 (P < 0.01) times
greater in the plasma compared with the YSM. Increased
proportions of 20:5(n-3) (P < 0.001) and 22:5(n-3) (P < 0.01) were also
observed in the TAG of the plasma, whereas the proportion of
20:4(n-6) in TAG and PL of the plasma was, respectively, 2.3 and
1.8 times (P < 0.001) that of the YSM. Thus the plasma
TAG is enriched in all long-chain n-3 polyunsaturates and in
20:4(n-6), whereas plasma PL is rich in 20:4(n-6) as well as
in 22:6(n-3) when compared with the yolk and YSM. The fatty acid
profiles of the plasma lipids changed little between days 33 and 55, the main difference being a decreased proportion of
16:0 with an increase in 18:1(n-9) in TAG.
Almost half the total lipid of the liver at day 33 consisted
of CE, a proportion that had increased to over three quarters by
day 55 (Table 5). Although TAG
made a relatively minor contribution to the total lipid content of the
liver, its composition was dominated by the exceptionally high
proportion of 22:6(n-3), which was 6.1-fold greater than in yolk
TAG at day 33. The PL of the liver was also enriched in
22:6(n-3) as well as being the main repository for 20:4(n-6)
in this tissue. Between days 33 and 55, there was
a marked decrease in the proportion of 16:0 as well as a reduction in
the level of 22:6(n-3) in TAG, with increased contributions from
18:0 and 20:4(n-6). Changes in the composition of liver PL over
this time were relatively minor; for example, there was a slight
reduction in the proportion of 20:4(n-6). The major fatty acid of
liver CE at day 33 was 18:1(n-9), and the proportion of this monounsaturate was even higher at day 55. Reduced
levels of 20:5(n-3) and 22:6(n-3) were also observed in CE
over this period.
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DISCUSSION |
Yolk lipids.
The proportions of TAG, PL, and CE at 61.4, 26.8, and 3.6%,
respectively, of total lipid in the initial yolk of the king penguin differ somewhat from the profile in the yolk of the chicken where these
lipid classes, respectively, form ~70, 21, and 1% of the total
(31). As discussed previously (31), it is
possible that the "high TAG" pattern of the chicken's yolk may be
typical of species that produce precocial hatchlings, whereas altricial
species tend to lay eggs with relatively less TAG and more PL and CE. Penguins are usually classified as semialtricial, producing hatchlings that rely on their parents for food and warmth (31). Thus
the differences in yolk lipid class profile exhibited by the king penguin compared with the chicken may reflect their relative positions in the precocial-altricial continuum. It should, however, be noted that
yolk lipid classes of the emperor penguin show less displacement from
the pattern described for the chicken (26) and that
patterns of embryonic energy metabolism in penguins are in some ways
more typical of the precocial rather than the altricial mode
(1).
The fatty acid profiles of the yolk lipids of the king penguin are
markedly different from those of the chicken. This distinction is most
pronounced with regard to the C20-22 n-3
polyunsaturates that, in the case of the penguin, form 17 and 7%,
respectively, of the fatty acid mass of the yolk's PL and TAG compared
with values of ~6 and 0.2% in eggs of the chicken (26,
28). Moreover, the C20-22 n-3 fatty acids
of the chicken's yolk consist almost entirely of 22:6(n-3),
whereas those of the penguin are more varied due to the additional
presence of 20:5(n-3) and 22:5(n-3). A further characteristic
of the penguin eggs is that, in terms of absolute mass, the n-3
polyunsaturates are almost evenly distributed between PL and TAG. For
example, we calculate that 1 g of yolk lipid from the king penguin
egg contains ~28 mg of 22:6(n-3) in TAG and 27 mg in PL. This
contrasts with a typical chicken's egg, where 22:6(n-3) is almost
entirely confined to the PL fraction (26, 28). The
myctophid fishes that predominate in the diet of the king penguin are a
rich source of C20-22 n-3 polyunsaturates and of
long-chain monounsaturates (Table 1), thus also explaining the
characteristic presence of 20:1(n-9) in the yolk lipids of this
species. The fatty acid compositions of the yolk lipid classes of the
king penguin are very similar to those reported for the emperor penguin
(26). The main differences are that the proportions of
20:1(n-9) and 20:5(n-3) are about twice as high in the yolk of the king penguin. This may partly be due to differences in prey
species caught in distinct feeding areas by the sub-Antarctic king
penguin (4) and the Antarctic emperor penguin
(3).
Inasmuch as the yolk lipids of the king penguin are mainly deposited
during a 2-wk period of fasting during courtship ashore (6,
32), we compared the fatty acid composition of total lipid of
the newly laid egg with that for the adipose tissue of courting females
(Table 1). Subcutaneous adipose tissue accounts for ~80% of the fat
stores in penguins (6, 20), and its fatty acid profile is
therefore representative of that of the lipid that is mobilized during
fasting. Although the adipose tissue is also characterized by high
proportions of C20-22 polyunsaturates and
long-chain monounsaturates, reflecting the dietary intake during the
previous sojourn at sea, there were marked differences in detail
between the fatty acid profile of the yolk and that of the precursor
lipid in the adipose depot. A comparable spectrum of compositional
differences between the fatty acids of the yolk and of the parents'
adipose tissue has been reported for the emperor penguin
(26), and the same explanations may be offered. The greatly reduced proportions of the long-chain monounsaturates, 20:1(n-9), 22:1(n-11)/(n-9), and 24:1(n-9) in the
yolk lipids compared with the adipose stores may be due to the relative
resistance of these fatty acids to mobilization as discerned from in
vitro and in vivo studies on the adipose tissue of the emperor penguin (6). This is consistent with the concept that the relative mobilization of the various fatty acids from adipose TAG is directly proportional to the number of double bonds and inversely proportional to the chain length (18). On this basis, the relative
enhancement of 20:4(n-6) in the yolk may be favored by
the preferential release of this highly polyunsaturated fatty acid from
adipose TAG. However, the even greater degree of preferential
mobilization displayed by 20:5(n-3) (6, 18) fails to
augment the proportion of this fatty acid in the yolk. This may imply a
role for other factors, such as the fatty acyl substrate specificity of
the acyltransferases that synthesize yolk lipids in the maternal liver,
in enforcing the resultant fatty acid composition of the yolk. It may
be proposed that such selective mechanisms have evolved to benefit the
embryo, filtering out most of the long-chain monounsaturates, favoring the delivery of 20:4(n-6), although this fatty acid is deficient in the maternal diet and attenuating the provision of
C20-22 n-3 polyunsaturates that the diet contains
in excess.
Transfer of lipids from the yolk to the embryo.
Studies performed mainly on the chicken embryo have indicated that
lipid is transferred sequentially from the yolk to the YSM and
subsequently to the plasma and then to the embryonic tissues, with the
liver in particular accumulating large amounts of lipid (14, 16,
28, 30). These movements are accompanied by changes in the
proportions of the yolk lipid classes at each stage of the sequence.
Pivotal to these transformations is the action of lipoprotein lipase on
the VLDL released from the YSM (29). This enzyme depletes
the TAG from the core of the VLDL, thus converting the lipoprotein to a
smaller remnant particle consisting mainly of CE. Uptake of remnants by
the embryo's liver results in the hepatocytes becoming packed with CE
droplets (24). Thus CE accounts for most of the liver
lipid in total contrast to the original yolk where TAG and to a lesser
extent PL predominate. Also, the lipid transfer process is not
exclusively unidirectional; some CE from the liver is excreted as a
component of bile into the embryo's small intestine from where it
recycles into the yolk sac (14, 28). As a result, the
proportion of CE in the yolk and YSM of the chicken increases markedly
at the end of the developmental period (14, 28). The
cholesterol (free plus esterified) content of the embryonic tissues is
almost entirely derived from the yolk (14), with the
exception of the brain, where de novo synthesis accounts for a large
proportion of the cholesterol content (37).
The changes in the concentration of total lipid and in the proportions
of the lipid classes in the different yolk/embryo compartments during
the development of the king penguin are largely consistent with this
scheme. The decrease in yolk lipid concentration between days
0 and 27 and the transient increase at day
33 most likely reflect movements of water into and out of the
yolk. Thus, during the first trimester of chicken embryo development,
the yolk contents become diluted by an influx of water from the albumen
and are subsequently reconcentrated by the dissipation of fluid to
other compartments (2, 17, 22). Both the mass of yolk and
the concentration of lipid within the yolk of the penguin then decrease markedly during the second half of development, indicating intensive lipid transfer to the embryo. The transitory increase in the lipid content of the YSM, followed by the sustained elevation of the levels
of lipid in the plasma and liver, can be interpreted in terms of a
precursor-product relationship between the successive compartments of
yolk, YSM, and plasma and/or liver. The high degree of
correlation between TAG and PL in terms of their concentrations in the
yolk and also in the YSM throughout development suggests that these two
lipid classes are taken up from the yolk together. This fits with
evidence from the chicken embryo that indicates that the YSM takes up
lipid nonspecifically from the yolk by phagocytosis (14,
28). The constant concentration of CE in the yolk during the
embryonic period and the increase that occurs after hatching when the
yolk sac is internalized into the abdomen of the chick presumably
reflect the recycling of CE from the liver to the yolk via the bile. A
similar explanation may pertain to the accumulation of CE in the YSM.
The increasing concentration of lipid in the plasma of the king penguin
embryo from day 40 onward is comparable with data for the
chicken embryo (10, 38). In the case of the chicken, however, this concentration rises from ~6 to 20 mg lipid/ml plasma between days 15 and 18 but then decreases to half
this maximal value by the time of hatching at day 21. In the
penguin by contrast, this increase was sustained over the hatching
period, reaching a concentration of ~26 mg/ml by 2 days posthatch.
The basis for this difference is not clear but could possibly somehow
relate to the precocial versus semialtricial developmental modes of the two species. The dramatic changes in the proportions of the plasma lipid classes during development are also features common to penguin and chicken embryos. For example, the plasma lipids of the chicken embryo halfway through development contained TAG and CE at,
respectively, 43 and 19% of the total; however, by 1 day after
hatching, TAG formed only 2% of the total plasma lipid, whereas CE had
increased to 46% (10). The increasing prevalence of CE in
plasma as development advances could result from an imbalance between
the rates of remnant formation and hepatic uptake. However, it is
likely that transfer of surplus surface lipids to high-density
lipoprotein (HDL) during the conversion of VLDL to remnants and the
esterification of the transferred FC to form CE in the HDL could also
partly explain the progressive accumulation of CE in the plasma of
avian embryos (28).
Changes in the fatty acid profiles of the lipid classes during
transfer.
Regarding the rearrangement of the fatty acid compositions that
accompany the lipid transfer process, two salient themes may be
discerned, both of which correspond to events reported for the chicken
embryo. The first of these relates to the reduced proportion of FC and
the commensurate increase in CE after uptake of lipid from the yolk by
the YSM (data for day 33 in Tables 2 and 3). In the chicken
embryo, this rapid esterification of yolk-derived FC to form CE in the
YSM is catalyzed by the very high activity of acyl-CoA:cholesterol
acyltransferase expressed in this tissue and which synthesizes
cholesteryl oleate as the main product (23). Similarly,
the proportion of 18:1(n-9) in CE is greater in the YSM of the
penguin than in the initial yolk. The preferential use of
18:1(n-9) for cholesterol esterification in the YSM is subsequently reflected in the composition of the large amounts of CE
that accumulate in the liver of the penguin embryo, where cholesteryl
oleate forms >76% of the total mass of sterol ester.
The second theme relates to the C20-22
polyunsaturates. Very high proportions of both 20:4(n-6) and
22:6(n-3) are attained in the tissue PL of the chicken embryo, the
former mainly in brain, liver, and heart and the latter in brain and
retina, and it has been proposed that these polyunsaturates are
essential for the development and function of these tissues (7,
13, 28, 31). This achievement is impressive considering the
scarcity or absence of C20-22 polyunsaturates in the
chicken's diet and is dependent on successive maternal and embryonic
phases of synthesis of 20:4(n-6) and 22:6(n-3) from their
C18 precursors as well as the expression of specific
mechanisms to preferentially deliver these fatty acids to the
appropriate tissues of the embryo (28). Although such biomagnification is also evident in the penguin embryo, with the proportions of 20:4(n-6) and 22:6(n-3) being far higher in
the lipids of plasma and liver than in those of the yolk and YSM, both
the focus and the mechanism may differ from the situation in the
chicken. Because the diet of the chicken is characterized by a very low
n-3/n-6 ratio, the priority in this species has been to
evolve embryonic mechanisms that selectively transport 22:6(n-3)
to the developing brain and retina; additional 20:4(n-6) for the
embryo is also provided by the desaturation/elongation of
18:2(n-6) in the YSM (28). The king penguin faces the
opposite challenge, because its diet is abundant with 22:6(n-3)
but deficient in 20:4(n-6). In fact, the limited amount of
20:4(n-6) obtained from the diet must be treated as an
exceptionally valuable resource, because the penguin's food is also
deficient in 18:2(n-6) (Table 1), effectively precluding the
possibility of synthesizing 20:4(n-6) by desaturation either
maternally or in the YSM. Possibly, the augmented proportion of
20:4(n-6) in plasma PL of the penguin embryo may be achieved by
the selective partitioning of this fatty acid away from 
-oxidation
and into the PL of the prospective VLDL particles during their assembly
in the YSM (9, 10). Whatever the mechanisms that are
invoked, it is evident that the biomagnification of
20:4(n-6) is the net result of a sequence of events in
the mother and the embryo. From the present results, the progressive
increase in the proportion of 20:4(n-6) in total lipid on transfer
between the successive maternal and embryonic compartments can be
illustrated (Fig. 5). Whereas
20:4(n-6) forms only 0.7 and 0.4% of the total fatty acid mass in
the parent's diet and adipose tissue, respectively, maternal events
increase this proportion to 1.6% in the total lipid of the yolk, with
mechanisms in the embryo then augmenting the proportion of this
polyunsaturate to form 8.6% of the fatty acids in the total lipid of
the liver at day 33. However, it should be noted that at
day 55, 20:4(n-6) formed only 3.7 ± 0.2% of the
total hepatic fatty acid mass. In contrast to the situation for
20:4(n-6), the proportion of 22:6(n-3) is diminished
maternally during transfer of lipid from adipose tissue into yolk but
is then biomagnified in the penguin embryo, as shown by the data for
the embryonic plasma and liver at day 33 (Fig. 5). Again,
this biomagnification was less evident at day 55, with
22:6(n-3) forming only 8.6 ± 0.3% of the total fatty acids
of the posthatch liver compared with 19.7 ± 0.5% at day 33. As mentioned above, the attenuation of the n-3 content of the egg compared with the precursor lipid in the maternal adipose tissue may represent an attempt to prevent delivery of excessive amounts of this polyunsaturate to the embryo. However, the
biomagnification of 22:6(n-3) during embryonic transport in the
penguin embryo may seem superfluous in light of its ample supply, and
the significance of this finding awaits explanation.
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Fig. 5.
Biomagnification of docosahexaenoic [22:6(n-3)] and
arachidonic [20:4(n-6)] acids during lipid transfer between
successive maternal and embryonic compartments. Values are weight
percentages of 22:6(n-3) and 20:4(n-6) in the total lipid of
each compartment and are means ± SE of n = 6 diet
samples (adult stomach contents), 7 breeding females' adipose tissue,
10 yolks at day 0 and of 6 YSM, 8 plasma samples, and 8 livers at day 33. *Significantly different from previous
compartment (P < 0.05).
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Possibly in the penguin, the biomagnification of
22:6(n-3), in contrast to that of 20:4(n-6), may
represent a consequence rather than a purpose of the embryonic lipid
transfer mechanism. The plasma TAG of the penguin embryo contains a
high proportion of 22:6(n-3), whereas 20:4(n-60) is
largely conveyed by plasma PL. This differential partitioning of these
polyunsaturates among the plasma lipid classes is also observed
in the chicken embryo (10). The action of lipoprotein
lipase on plasma VLDL in the penguin embryo may tend to increase
the proportion of 22:6(n-3) in the TAG of the lipoprotein and its
remnants because TAG molecules that contain this fatty acid are
relatively resistant to hydrolysis by this enzyme (11,
29). As the plasma will not only contain pristine VLDL newly
released from the YSM, but also lipoprotein particles in various stages
of hydrolytic contraction culminating in remnant formation, the
proportion of 22:6(n-3) in total plasma TAG may therefore exceed
that of the nascent VLDL. The uptake of remnants by the liver will
result in delivery of TAG to this tissue; this TAG will be small in
amount because remnants consist mainly of CE, but it will be highly
enriched in 22:6(n-3). Thus the biomagnification of 22:6(n-3)
in the lipids of the plasma and liver of the embryo may simply be an
inevitable consequence of the substrate specificity of lipoprotein
lipase rather than a purposeful means of enriching the tissues with
this fatty acid. By contrast, in the chicken embryo, where
22:6(n-3) is a relatively scarce but essential nutrient, the
substrate specificity of lipoprotein lipase may have facilitated the
evolution of mechanisms for the preferential delivery of this fatty
acid to the brain and retina as previously proposed (28).
Perspectives
Many of the salient features of the process of lipid transfer that
have been highlighted by studies on the embryonic development of the
chicken (14, 28), turkey (5), pheasant
(27), gull (27), and pigeon (36)
are also shared by the king penguin. These include the rapid uptake of
lipid from the yolk during the second half of the developmental period,
the esterification of yolk-derived free cholesterol to form cholesteryl
oleate in the YSM, the accumulation of large amounts of CE in the
embryonic liver, and the enrichment of the hepatic TAG and PL with
22:6(n-3) and 20:4(n-6). The changing profile of the lipid
classes in the plasma during development and the distribution of
22:6(n-3) and 20:4(n-6) between these lipid classes are also
reminiscent of the pattern reported for the chicken embryo
(10). These similarities suggest the existence of a
mechanism of embryonic lipid utilization, which, in its central
features, is common to avian species. The distinguishing feature of the
king penguin is the high proportion of C20-22 n-3
polyunsaturates in the yolk lipids as a consequence of
piscivory. Adaptations to provide some synchrony between
dietary supply and embryonic demand for the various polyunsaturated
fatty acids are expressed both in the female parent and in the embryo, particularly with regard to the biomagnification of 20:4(n-6). A
major challenge for the penguin embryo will be to attune the influx of
n-3 polyunsaturates from the yolk to the requirements of the
developing brain and retina. Whereas the chicken embryo expresses
mechanisms that preferentially direct 22:6(n-3) for uptake by the
neural tissues (27, 28, 30), such mechanisms may be
redundant in the penguin. The tissue-specific partitioning of
yolk-derived 22:6(n-3) and, in particular, the means by which the
delivery of this fatty acid to the brain are regulated are topics
worthy of further investigation in this species. Questions for such
future work include the potential role of the embryo's adipose tissue
in mediating the transfer of 22:6(n-3) to the brain (25), the relative contribution of the various fatty acids
to energy production in the embryo (9), and the
antioxidant requirements of the polyunsaturate-enriched tissues
(33). The information obtained from such work on the king
penguin may provide general insights in the important area of the
requirements, transport, and metabolism of n-3 polyunsaturates
during development. Comparative studies using chicken eggs enriched
with n-3 fatty acids (9) could help to elucidate
species-specific embryonic adaptations to their characteristic yolk
fatty acid profiles. It is also worth noting that the biomagnification
of essential polyunsaturates performed by the YSM during avian
development is somewhat analogous to the preferential delivery of
n-3 and n-6 polyunsaturates to the fetus that is mediated by
the placenta during mammalian development (21).
| |
ACKNOWLEDGEMENTS |
We are grateful to the Scottish Executive Rural Affairs Department
and to Institut Français pour la Recherche et la Technologie Polaires (Program 119) for financial support. Logistical support was
provided by Terres Australes et Antarctiques Françaises.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
B. K. Speake, Avian Science Research Centre, Scottish Agricultural College, Auchincruive, Ayr KA6 5HW, UK (E-mail:
b.speake{at}au.sac.ac.uk).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 May 2000; accepted in final form 31 October 2000.
| |
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ABSTRACT
INTRODUCTION
