Articles in PresS. Am J Physiol Regul Integr Comp Physiol (June 5, 2003). 10.1152/ajpregu.00105.2003
THE METABOLIC FATE OF YOLK FATTY ACIDS IN THE
DEVELOPING KING PENGUIN EMBRYO
René Groscolas1, Françoise Fréchard1, Frédéric Decrock1 and Brian K. Speake2
1
Centre d'Ecologie et Physiologie Energétiques, Centre National de la Recherche Scientifique,
67087 Strasbourg, France; and 2Avian Science Research Center, Scottish Agricultural
College, Ayr KA6 5HW, United Kingdom
Running title: fatty acid metabolism in penguin embryo
Correspondence, proofs and reprint requests to:
R. Groscolas
1
Centre d'Ecologie et Physiologie Energétiques, CNRS, 23 rue Becquerel, 67087 Strasbourg,
France
Tel: (33) 388 10 69 23
Fax: (33) 388 10 69 06
E-mail: [email protected]
Copyright (c) 2003 by the American Physiological Society.
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Abstract: This study examines the metabolic fate of total and individual yolk fatty acids (FA)
during the embryonic development of the king penguin, a seabird characterized by prolonged
incubation (53 days) and hatching (3 days) periods, and a high n-3/n-6 polyunsaturated FA
ratio in the egg. Of the ~15 g of total FA initially present in the egg lipid, 87% was transferred
to the embryo by the time of hatching, the remaining 13% being present in the internalized
yolk sac of the chick. During the whole incubation, 83% of the transferred FA was oxidized
for energy, with only 17% incorporated into embryo lipids. Pre-hatching (days 0-49), the fat
stores (triacylglycerol) accounted for 58% of the total FA incorporated into embryo lipid.
During hatching (days 49-53), 40% of the FA of the fat stores was mobilized, the mobilization
of individual FA being unselective. At hatch, 53% of the arachidonic acid (20:4n-6) of the
initial yolk had been incorporated into embryo lipid compared to only 15% of the total FA
and 17-24% of the various n-3 polyunsaturated FA. Similarly, only 32% of the yolk’s initial
content of 20:4n-6 was oxidized for energy during development compared to 72% of the total
FA and 58-66% of the n-3 polyunsaturated FA. The high partitioning of yolk FA towards
oxidization and the intense mobilization of fat store FA during hatching most likely reflect the
high energy cost of the long incubation and hatching periods of the king penguin. The
preferential partitioning of 20:4n-6 into the structural lipid of the embryo in the face of its low
content in the yolk may reflect the important roles of this FA in tissue function.
Key words: bird development, fatty acid transfer, fatty acid oxidation, n-3 PUFA, n-6 PUFA
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INTRODUCTION
The primary importance of yolk lipids as nutrients for the avian embryo is well established
(26). Most notably, the oxidation of fatty acids provides almost all of the energy required for
embryonic development, while the intensive synthesis of cell membranes during tissue growth
also depends on the supply of lipids from the yolk (26). Additionally, in some avian species,
lipid derived from the yolk is used to form adipose tissue in the embryo (7, 26). The lipid
stored in adipose tissue may represent a source of energy that can be rapidly mobilized to
enable the chick to cope with the demands of hatching and of early post hatch life (7).
Since the avian embryo develops as a closed system, using only nutrients that are prepackaged in the egg before laying, it is ideally suited for quantifying the utilization of lipid
during development and the partitioning of lipid between its main functions of energy
provision, cell membrane synthesis and adipose tissue deposition. Although there is a
considerable amount of information relating to the distribution of yolk lipid among these
different fates in the embryo (2, 3, 12, 17), all such studies to date have been restricted to a
single precocial avian species, the domestic chicken. This limitation has precluded any
assessment of the effects of interspecies differences in yolk composition, incubation time or
developmental mode (altricial vs precocial) on the pattern of lipid utilization by the embryo.
Moreover, the comparison of the metabolic fate of yolk fatty acids during the pre-hatching
and hatching periods, i.e. when the rate of energy expenditure of the embryo differs markedly,
has not been made previously, and no study has examined the mobilization of fatty acids from
embryonic fat depots during hatching.
The selective use of particular fatty acids for different purposes during development is
also of considerable interest. For example, high proportions of docosahexaenoic acid (22:6n3) are incorporated into the membrane phospholipids of the developing brain and retina
whereas the phospholipids of the heart, liver and several other tissues become enriched with
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arachidonic acid (20:4n-6) (11, 16, 26). These long chain polyunsaturates perform important
functions during tissue development. Thus, phospholipids that contain 22:6n-3 impart unique
biophysical properties to neuronal membranes to facilitate neurotransmission (24), while
20:4n-6 has regulatory roles relating to eicosanoid synthesis and signal transduction (10, 19).
It is noteworthy that, during development of the chicken, yolk-derived 22:6n-3 and 20:4n-6
are recovered in the embryonic tissues to a far greater extent than the other fatty acids (3, 12,
14), indicating that these essential polyunsaturates are selectively used for cell membrane
synthesis and are diverted away from oxidative metabolism.
The embryonic development of the semi-altricial king penguin (Aptenodytes
patagonicus) displays a range of features that contrast markedly with the chicken, thus
forming the basis for a comparative assessment of lipid utilization and partitioning. Eggs of
the king penguin require a long incubation period of 53 ± 1 days compared with only 21 days
for development of the chicken embryo (28). Since long incubation periods incur greater
energy costs (30), this may affect the proportion of yolk lipid that is allocated for oxidation.
The extended hatching period (3 days) of the king penguin (28) is also likely to be
energetically expensive, possibly relying on the mobilization of fatty acids from adipose
tissue. A salient feature of the yolk lipids of the king penguin is the exceptionally high
proportion of long chain n-3 polyunsaturates such as 22:6n-3 and 20:5n-3, resulting in a very
high n-3/n-6 ratio (5). This extreme composition, which is due to the maternal diet of n-3 rich
myctophid fishes (5), could possibly influence the selective partitioning of the various
polyunsaturates among different fates during development.
The aim of this study was to determine the metabolic fates of total yolk fatty acid and
of the individual fatty acids during development of the king penguin. The proportion of the
fatty acids transferred to the embryo, as opposed to retention in the residual yolk, and the
partitioning of the transferred fatty acids among the alternative fates of oxidation,
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incorporation into structural lipid and formation of fat stores, were quantified. Moreover,
these values were determined for the whole incubation, the pre-hatching period, and for
hatching, and the mobilization of fatty acids from the embryonic fat stores during hatching
was estimated. (sentence deleted)
MATERIALS AND METHODS
Egg incubation and embryo sampling
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
1999-2000. The project was approved by the ethical committee of the Institut Français pour la
Recherche et la Technologie Polaires. The use of 24 eggs had a very limited impact on the
local population (30,000 breeding pairs) given that natural mortality is several thousands eggs
or chicks per year.
Eggs at laying (n = 8), embryos at day 49 of incubation (pre-hatching, n = 8), and
fully-hatched chicks (n =8) 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 marked
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 weeks during the 53 ± 1 days of
incubation (28, 31). The mass of the egg differs markedly among pairs, from 250 to 350 g
(average = 303 g) (28). Our study being based on the determination of mass balances, and
requiring results typical of the species, the mass of the selected eggs was as close as possible
to the average mass. Thus, only eggs with a mass ranging from 290 to 310 g at laying were
selected. This required (phrase deleted) the weighing of the newly-laid egg in 100 pairs.
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Within 12 h of laying, the egg was briefly removed from the brood pouch of the incubating
bird (there is no nest in this species and the egg is incubated on the feet), weighed to the
nearest 5 g using a spring scale and a linen bag, 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. (word deleted)
Females were banded at first relief. To compensate for possible egg loss or infertility, 35 eggs
and pairs were marked.
Eggs at day 0 were collected within 12 h following laying. Day 49 of incubation was
selected as the pre-hatching stage from previous observations in the field and during
incubation of king penguin eggs in a laboratory incubator. Notably, it was found that after 49
days of incubation no chick had begun to break the egg-shell, but that most of them were
starting the hatching process (Thil, M.-A., Decrock, F., and Groscolas, R., unpublished data).
The latter fact could be infered from the observation that during weighing of the eggs at ± 1
mg on a sensitive balance and under laboratory conditions a steady value was difficult to
obtain because of repeated movements of the embryo inside the egg. The embryo at day 49
was therefore likely to be at the stage of internal pipping, with an about ± 1 day individual
variability. Fully-hatched chicks were obtained from eggs transfered from the parent to a
laboratory incubator (37.5°C and 100% relative humidity) until the chicks had completely
freed themselves from the egg shell without assistance, as occurs under natural conditions.
This treatment was required because in the field chicks can be fed by their parents before
being fully-hatched, which would yield errors in the determination of the nutrient balance of
the egg during hatching. Eggs were removed from the parents after the beginning of hatching,
when a small hole (< 0.5 cm in diameter) was detected in the egg-shell. On average, chicks
were fully hatched 2 days later, after 53 ± 1 days of incubation as under natural conditions.
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In order to calculate mean ± SE losses or gains of total or individual FA for statistical
comparisons, eggs were randomly associated into groups of three (1 egg for day 0, 1 egg for
day 49 and 1 for the fully-hatched chick) at the time of sampling, before any measurement.
This procedure yielded 8 trios, the comparison of 2 eggs within a trio giving one individual
value of FA loss or gain during the corresponding period of incubation. The mass at laying of
eggs did not differ significantly according to the incubation stage for which they were
sampled (P = 0.53). It averaged 304.8 ± 3.8 g (day 0), 300.0 ± 3.4 g (day 49) and 303.6 ± 2.4
g (fully-hatched chicks).
Lipid analysis
The mass of the whole egg was measured at ± 0.1 g immediately after sampling, the
egg-shell remains being collected and added to the chick to obtain egg mass after hatching.
The eggs were then dissected into the following components: embryo, yolk, albumen, and
egg-shell. At day 49, allantoic fluid was not separated from albumen. Their weights were
determined at ± 1 mg. Embryo and newly-hatched chicks were killed by cervical dislocation,
residual yolk being removed from the abdomen of the hatched chicks after incision. Yolks
were homogenized by slow stirring using a glass rod, and whole embryos or chicks were
ground until completely homogenous using a blender at + 4°C to limit water loss.
Total lipids were extracted in triplicate on 1- and 2-g aliquots of yolk and embryo,
respectively, using chloroform-methanol 2:1 (vol/vol) (8). BHT was added to the solvent as
an antioxidant at the final concentration of 0.05%. The extraction was repeated 3 times to
ensure a complete recovery of lipids. The three extracts were pooled and washed with 0.88%
(wt/vol) KCl to remove non-lipid components. Then the organic phase was evaporated under
vacuum and the mass of the lipid extract was measured at ± 1 mg. The lipid contents of the
yolk and of the embryo were close to 17 and 2.5% of the fresh mass, respectively. The
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coefficient of variation (CV) of the determination of the lipid content was 3.2 and 4.3% for
yolk and embryo, respectively.
The total mass of FA in the fat depots of the embryo was considered as equal to the
mass of FA in the embryo's triacylglycerols (TAG), the form of storage of lipids in fat depots,
the TAG content of other tissues being considered as negligible (5). TAG from a known
amount of total lipids were separated from other lipids using thin-layer chromatography on
silica gel G. The developing solvent was hexane-diethyl ether-acetic acid 70:30:1
(vol/vol/vol). After drying under nitrogen and spraying with primulin the TAG band was
scraped into vials for preparation of FA methyl esters. Transmethylation of total lipids (yolk
and embryo) and of TAG (embryo) was performed using 14% boron fluoride in methanol. A
known amount of total lipids or the whole TAG band were added with a known amount of
nonadecanoic acid (19:0) as an internal FA standard. The resulting FA methyl esters were
separated by gas-liquid chromatography with use of a Chrompack CP 9001 chromatograph
equipped with an AT-WAX capillary column (0.25 mm ID x 30 m, 0.25 µm film thickness;
Alltech, Templeuve, France) and a flame ionization detector (Chrompack, Les Ulis, France).
The column was maintained at 195°C and helium was used as the carrier gas. FA peaks were
identified by comparison with retention times of standard FA methyl ester mixtures (Nucheck Prep, Elysian, MN) and from previous analyses (21). They were quantified with an
integrator (model SP 4290, Spectra-Physics, Les Ulis, France) and by comparison with the
19:0 standard. The whole procedure allowed determination of the FA composition (weight
percentage) of total lipids in yolk and embryo, and of embryo TAG, together with the total
amount (mg) of total and individual FA in yolk, embryo and embryo fat depots. The mass of
structural FA was calculated as (mass of FA in the embryo minus mass of FA in embryo
TAG).
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Calculations and statistics
Values are means ± SE. They are presented either at the 3 stages of incubation or for a given
period of incubation (pre-hatching = day 0 to day 49; hatching = day 49 to day 53; whole
incubation = day 0 to day 53). In the latter case, means ± SE were calculated using data
obtained after egg pairing (see "Egg incubation and embryo sampling"). During a given
period, the mass of FA transferred or oxidized was calculated as the mass of FA lost from the
yolk, or from the yolk + embryo, respectively. Extra-embryonic tissues (allantois, amnion, etc)
were not taken into account in this study, their FA being considered as lost and thus allocated
as oxidized. From previous data on the lipid content of extra-embryonic tissues (Decrock and
Groscolas, unpublished data), this could have over-estimated the oxidation of FA during
whole incubation by no more than 0.7%. Incorporation was defined as the amount of FA
recovered in the embryo. To compare the metabolic fate of individual FA during a given
period of incubation, four parameters were calculated and expressed in %. Relative transfer,
incorporation, and oxidation are the portions of the mass of a given FA present in the yolk at
day 0 that were transferred, incorporated in the embryo, and oxidized, respectively. Note that
since individual FA had not the same relative transfer, relative oxidation was not the
reciprocal of relative incorporation. Transfer, oxidation and incorporation as calculated above
does not give a straightforward measurement of the utilization of FA. For example, because
the avian embryo is able to desaturate and elongate FA (15, 18), a portion of incorporated FA
might be derived from a precursor FA in the egg yolk. Similarly, a portion of an FA
synthesized in the embryo could be transfered back to the yolk. Thus, relative transfer,
incorporation and oxidation represent a net balance between anabolic, catabolic, and transport
processes. To compare the mobilization of individual FA from fat stores during hatching,
fractional mobilization of a given FA was calculated as the part of its mass that was lost
(mobilized) from embryo TAG.
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Percent values were transformed to arcsin before statistical analysis to make the
variance independent of the mean. Comparison of several means was performed using a onefactor ANOVA followed by a S.N.K. or Kruskal-Wallis test according to whether
homoscedasticity and equality of variance were simultaneously observed or not. Comparison
between two means was done using paired or unpaired t-test, as appropriate, or Mann and
Whitney U-test when the equality of variance failed. The criterion of significance was P <
0.05.
RESULTS
Fresh mass of egg components
The mass of the egg decreased significantly throughout incubation (P < 0.001), by 19 % from
laying (day 0) to the end of hatching (day 53; Table 1). The mass of the yolk represented 22 ±
1% of the mass of the egg at day 0 and decreased by 35 and 72 % (P < 0.001) during the prehatching period (days 0-49) and the whole incubation (days 0-53), respectively. The mass of
the newly-hatched chicks (183 g on an average) represented 60 ± 2 % of the mass of the egg
at laying. Hatching (days 49-53) was associated with a slight (22 g) but significant (P < 0.01)
increase in the mass of the embryo.
Metabolic fate of total yolk fatty acids
The metabolic fate of total fatty acids (FA) is illustrated Figs. 1-3 for the whole
incubation (days 0-53), for the pre-hatching period (days 0-49), and for hatching (days 49-53),
respectively. At laying, the egg contained on average 15 g of total FA (Figs. 1-3). Throughout
the whole incubation (Fig. 1), 87% of yolk FA were transferred, i.e. 65% pre-hatching (Fig. 2)
and 22% during hatching (Fig. 3), the remaining 13% being in the residual yolk. The transfer
of FA partitioned between 74.8 ± 2.7% pre-hatching and 25.2 ± 2.7% during hatching.
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Of the yolk FA transferred throughout the whole incubation, the bulk (83%) was
oxidized to yield energy, 17% being incorporated in the embryo (Fig. 1). The oxidation of FA
partitioned between pre-hatching (68.5 ± 1.5%) and hatching (31.5 ± 1.5%). In contrast, all
the net incorporation of FA in the embryo occured pre-hatching. As a consequence, the
proportion of transferred FA that was incorporated in the embryo (24%) and that was oxidized
(76%) pre-hatching (Fig. 2) was significantly (P < 0.001) higher and lower, respectively, than
during hatching (- 6% and 106%, respectively; Fig. 3).
A net loss of FA from the embryo was observed during hatching (Fig. 3). It resulted
from a mobilization of FA from fat stores that was not fully compensated by the accretion of
FA in structural lipids. The mass of stored FA significantly decreased by 40% during hatching
(P < 0.001). Thus, whereas 57% of FA incorporated in the embryo were stored in adipose
tissues pre-hatching, this proportion significantly declined (P < 0.001) to 36% post-hatching
(Figs. 1-2). The accretion of FA in structural lipids partitioned between 72.1 ± 3.9% prehatching and 27.9 ± 3.9% during hatching.
Metabolic fate of individual fatty acids
FA in yolk and embryo. A total of forty FA were identified in the yolk at day 0 (Table 2).
Oleic (18:1n-9), palmitic (16:0), stearic (18:0) and DHA (22:6n-3) were the major FA. Total
n-3 PUFA represented 11.2 ± 0.4% of yolk FA, a contribution 3.5 times higher than that of
total n-6 PUFA (3.2%). Long-chain monounsaturated FA (20:1 to 24:1) contributed 3.5% of
yolk FA. The FA composition of the newly-hatched chick broadly resembled that of the yolk
(Table 2). However, it differed by its 2-fold lower weight % of palmitoleic acid (16:1n-7; P <
0.001) and by its several-fold higher weight % of two n-6 PUFA (22:4n-6 and 20:4n-6) and of
two minor long-chain saturated FA (20:0 and 22:0) (P < 0.001). Similar differences were
observed for the embryo at day 49 (not shown). The mass of all FA, except 22:4n-6 and 22:0,
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was lower in the embryo (days 49 and 53) than in the yolk (day 0). The mass of all embryonic
FA remained unchanged during hatching, except for a significant increase in 20:0, 22:0 and
22:4n-6 (P < 0.05).
Transfer of yolk FA. The relative transfer of individual yolk FA differed significantly both
pre-hatching (P < 0.001) and during the whole incubation (P < 0.01), the range between
extreme values being 2 and 1.3 fold, respectively. Very-long chain monounsaturates (20:1 to
24:1) and 22:4n-6 tended to have low relative transfer, the relative transfer of the other n-6
PUFA being similar to that of n-3 PUFA. When only major fatty acids (weight % in the yolk
at day 0 > 0.5%, n = 13) are considered, relative transfer ranged from 46.9 to 72.0 % prehatching (i.e., a 1.5-fold difference; P < 0.05; Fig. 4), and from 73.9 to 89.2% for the whole
incubation (P < 0.05; data not shown). The relative transfer of major n-6 and n-3 PUFA did
not differ from that of total FA (P > 0.34). Overall, there was therefore some selectivity in the
transfer of individual yolk fatty acids, this selectivity being mostly observed during the prehatching period and concerning principally minor FA.
Incorporation of FA in the embryo. The relative incorporation in the embryo of FA averaged
15.8 ± 0.7% and 14.6 ± 0.8% pre-hatching and during the whole incubation, respectively. The
relative incorporation of individual FA differed markedly both pre-hatching (range: 1.5 ± 1.1
to 450.3 ± 107.6%, P < 0.001) and during the whole incubation (range: 0.0 ± 0.0 to 562.8 ±
136.1%, P <0.001). The relative incorporation of the 13 major yolk FA differed by 5.7 fold
pre-hatching (range: 8.9 ± 0.6 to 51.0 ± 3.2 %, P < 0.001, Fig. 5), and by 6.7 fold during the
whole incubation (range: 8.0 ± 0.9 to 53.3 ± 2.6%, P < 0.001). 16:1n-7 and 20:4n-6 had the
lowest and highest relative incorporation, respectively. In contrast to 20:4n-6, 18:2n-6 was not
preferentially incorporated in the embryo, its relative incorporation being similar to that of
total FA both pre-hatching (P = 0.09) and during the whole incubation (P = 0.48). Prehatching, the relative incorporation of major n-3 PUFA was either similar (20:5n-3) or
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slightly higher (22:5n-3, 22:6n-3; P < 0.01) than that of total FA. The relative incorporation of
all FA, except 20:0, 22:0 and 22:4n-6, was slightly negative (net loss) during hatching.
Overall, the incorporation in the embryo of individual FA was highly selective, with notably a
preferential incorporation of some n-6 PUFA (20:4n-6 and 22:4n-6) and very-long chain
saturated FA (20:0 and 22:0), and an under-incorporation of 16:1n-7.
Oxidation of FA. The relative oxidation of total FA averaged 49.2 ± 1.0% pre-hatching, 22.8
± 1.4% during hatching, and 71.9 ± 1.6% during the whole incubation. The relative oxidation
of individual FA differed markedly pre-hatching (range: - 412.6 ± 111.4 to 67.2 ± 5.4%, P <
0.001) and during hatching (range: -78.1 ± 38.0 to 27.8 ± 2.4%, P < 0.001). Pre-hatching and
among the 13 major yolk FA, relative oxidation differed by 4.7 fold (range: 13.1 ± 4.0 to 61.5
± 2.5 %, P < 0.001, Fig. 6), the relative oxidation of 20:4n-6 being significantly lower than
that of total FA (P < 0.001). During hatching, the relative oxidation of these 13 FA differed by
2.3 fold (range: 12.2 ± 3.6 to 28.4 ± 3.7%, P < 0.01), that of 20:4n-6 (18.9 ± 3.1%) being no
longer lower than for total FA (P = 0.27). Overall, the oxidation of yolk FA was highly
selective, this selectivity being blunted during hatching in comparison to the pre-hatching
period.
DHA vs ARA. To further focus on n-3 and n-6 PUFA, the metabolic fate of DHA (22:6n-3)
and ARA (20:4n-6), i.e. the two major FA of each series in the embryo, is compared in Fig. 7
for the pre-hatching period. Whereas the proportion of each FA present in the yolk that was
transferred was similar (61-64 %), the proportion of transferred 22:6n-3 that was oxidized was
3.2 times higher (P < 0.001) than that of 20:4n-6. When considering their incorporation in the
embryo, the two FA also differed by their partitioning between structural lipids and fat stores,
the percentage of stored 20:4n-6 being 3.7 times lower than that of 22:6n-3 (P < 0.001).
FA mobilization from fat stores during hatching. The FA composition of fat stores (embryo
TAG) pre- and post-hatching is shown in Table 3 together with the fractional mobilization of
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FA during hatching. The weight % of only two FA (18:0 and 22:6n-3) was slightly different
between pre- and post-hatching (P < 0.05). The fractional mobilization of all individual FA
was similar to that of total FA, except for two minor saturated FA (20:0 and 22:0), the
fractional mobilization of which was negative and significantly lower that that of total FA (P
< 0.001).
DISCUSSION
Transfer and metabolic fates of total yolk fatty acid. Although lipid is transferred very
rapidly from the yolk to the embryo during the second half of the avian embryonic period, this
process is incomplete at hatch when the yolk sac containing some residual lipid is retracted
into the abdomen of the chick (26). This internalized yolk functions as a nutrient source for
the chick for several days after hatching and, in the case of the domestic chicken, contains
approximately 25% of the lipid that was originally present in the egg (17). By contrast, only
13% of the original yolk fatty acid remained within the residual yolk of the king penguin,
indicating that a much greater proportion of yolk lipid is delivered to the embryo prior to
hatching in this species.
Lin et al. (12) demonstrated that about 50% of the mass of fatty acid originally present
in the egg of the domestic chicken was recovered in the lipids of the hatchling plus residual
yolk, indicating that about half of the yolk’s initial content of fatty acid is oxidized for energy
during embryonic development. Assuming that the residual yolk of the domestic chicken
accounts for 25% of the initial yolk fatty acid content (17), it can be calculated from the data
of Lin et al. (12) that approximately 65% of the fatty acid transferred from the yolk during
embryonic development is oxidized for energy. Similarly, about 60% of the lipid transferred
from yolk to embryo during development of the turtle Emydura macquarii is allocated to
energy production (29). It is clear from the present work that a much greater proportion (83%)
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of transferred fatty acid is allocated to energy production during development of the king
penguin than is the case for the chicken. This finding is consistent with the reported low
energetic efficiency of embryonic growth in the king penguin compared with other birds (1).
The metabolic cost of avian embryo development is equal to the sum of the energy required to
synthesize new tissue (i.e. growth), to maintain existing tissue, and to support any physical
activity, particularly the muscular movements that occur during the hatching process (30).
The long incubation period of the king penguin egg, entailing a high maintenance cost (1, 30),
may partly explain why oxidation for energy accounts for such a major proportion of the
transferred fatty acid. Also, it is evident that hatching is an energetically costly process in this
species. Although hatching represents about 6% of the incubation period, it accounts for 32%
of the oxidized fatty acid. In part, this will be due to the high maintenance cost over the 3-day
hatching period, since the embryo has attained its maximum weight by this time. In addition,
it has been suggested that the physical effort required to break out of the shell is exceptionally
high in the case of penguin embryos (30). Penguins lay eggs with thick and heavy shells
which, for a given shell thickness, are the strongest of all bird eggs (30). It has been estimated
that physical activity accounts for 20-25% of the metabolic rate over the hatching period of
the Adelie penguin (Pygoscelis adeliae), representing 9% of the total energy expenditure of
development (30).
Although 22% of the fatty acid content of the initial yolk was transferred to the
embryo during the hatching period, representing 25% of the total amount transferred during
incubation, this was clearly insufficient to satisfy the energy needs of the hatching embryo.
Additional energy was provided by the mobilization of the embryo’s fat stores, which
declined dramatically by 40% during hatching. This mobilization accounted for about 16% of
the fatty acid oxidized over this period, the remainder being supplied direct from the yolk.
Reductions in the fat stores of the chicken embryo, by as much as 25%, have also been
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observed during hatching (7). Whether the rate of FA transfer from the yolk is too slow to
provide sufficient energy during hatching, and thus must be complemented by FA
mobilization from embryonic fat stores, should be considered in further studies.
In summary, it is suggested that embryonic development of the king penguin entails a
particularly high energy cost, due to the long incubation period and the special demands of
hatching. This cost is met by (a) the transfer of a high proportion of yolk fatty acids prior to
hatching, (b) the oxidation of an exceptionally high proportion of the transferred lipid, and (c)
the substantial mobilization of the embryo’s fat stores during hatching.
Transfer and metabolic fates of individual fatty acids. Although there was some
selectivity in the transfer of individual fatty acids from the yolk of the king penguin, this was
largely confined to very minor fatty acids. Most of the major fatty acids displayed similar
rates of transfer, consistent with the evidence that yolk lipid droplets are taken up nonspecifically into the yolk sac membrane by endocytosis (26). Nevertheless, during
development of the chicken embryo, there is a marked preferential transfer of 22:6n-3 from
the yolk, presumably to ensure the delivery of adequate amounts of this polyunsaturate to the
relevant tissues of the embryo (3, 12, 14, 17, 26). There was, however, no preferential transfer
of 22:6n-3 from the yolk of the king penguin, most likely because the very high provision of
this fatty acid in the yolk obviates the need for such a mechanism. Some studies have
suggested that 20:4n-6 may also be preferentially transferred from the yolk during
development of the chicken embryo (3, 12). However, no such preferential transfer of 20:4n-6
was evident during development of the king penguin. The explanation for the low relative
transfer of very-long monounsaturates is not clear. Possibly, this could reflect any selective
recycling of these fatty acids from the liver back to the yolk via the bile, similar to the
recycling of cholesteryl ester that occurs in the king penguin embryo (5).
18
To some extent, the fatty acid composition of the newly hatched king penguin was
similar to that of the initial yolk, particularly with regard to the percentage contributions of
many of the major fatty acids. There were, however, some notable differences, the most
salient being the 4-fold greater proportion of 20:4n-6 in the hatchling compared with the egg.
Over 53% of the mass of 20:4n-6 originally present in the egg was recovered in the lipids of
the hatchling, compared with an average recovery of 15% for total fatty acid. Conversely, the
relative oxidation of 20:4n-6 during pre-hatching development was far less than that of the
other fatty acids. Such biomagnification of 20:4n-6 during yolk-to-embryo transfer has also
been observed during development of the chicken, where 73% of the egg’s initial content of
this polyunsaturate was recovered in the chick compared with a 51% recovery of total fatty
acid (12). A large biomagnification of 22:6n-3 also occurred during development of the
chicken (3, 12). The hatchlings of a turtle (29) and of several lizards (27) also displayed
higher proportions of 20:4n-6, and particularly of 22:6n-3, than were present in the egg.
However, the recovery of 22:6n-3 in the penguin hatchling was only slightly greater than the
average for total fatty acid. It seems that the levels 20:4n-6 and 22:6n-3 typically present in
the chicken’s egg represent a precious resource to be preferentially used for cell membrane
synthesis as opposed to oxidation. Although the selective incorporation of 20:4n-6 into
structural lipid is clearly an important feature of king penguin development, there is obviously
much less of a need here to spare 22:6n-3 for this purpose due to the very high content of this
fatty acid in the yolk. It is noteworthy that chicken embryos developing within eggs enriched
with 22:6n-3, as a result of supplementing the hen’s diet with fish oil, do not preferentially
incorporate this fatty acid into structural lipid (12).
There was a clear distinction between the pre-hatching and hatching periods with
regard to the selective diversion of 20:4n-6 away from the fate of oxidation. Whereas the
relative oxidation of 20:4n-6 during the first 49 days of incubation was far less than that of
19
total fatty acid, no such difference was observed during the hatching period. Possibly, the rate
of oxidation during hatching is so high as to overide the previous selectivity.
The preferential incorporation of 20:4n-6 into structural lipid of the developing king
penguin is attested by the high proportions of this fatty acid that are achieved in the
phospholipids of the embryonic liver, heart and skeletal muscle, despite the very high C20-22 n3/n-6 ratio in the yolk (5, 6). Presumably, this reflects the important roles of 20:4n-6 in signal
transduction and eicosanoid synthesis that have been demonstrated in many cell types (10,
19). It is, in fact, well recognized from studies in mammals that 20:4n-6 is selectively
incorporated into cell phospholipids, thereby avoiding the fate of oxidation (33). (sentence
deleted) The mechanisms for this selective partitioning of 20:4n-6 have been explained in
terms of the substrate specificities of enzymes involved in the synthesis and remodeling of
phospholipids (13, 33). (3 sentences deleted).
For two fatty acids, 22:0 and 22:4n-6, the amounts recovered in the hatchling were
greater than those in the initial yolk, indicating biosynthesis during development. However,
even in the hatchling, these two fatty acids were very minor components. The reason for
synthesizing 22:0 and 22:4n-6 during development is not clear, although the latter fatty acid is
detected in the phospholipid of the heart and skeletal muscle of the king penguin embryo at
concentrations ranging from 0.6 – 1.3 %(w/w) (6). Some biosynthesis of 22:4n-6 also occurs
during embryonic development of the chicken (3, 12). Since the amount of 22:4n-6 recovered
in the hatchling of the king penguin represents at most only 6% of the mass of its precursor,
20:4n-6, that was present in the yolk, the synthesis of the former fatty acid from the latter will
only minimally affect the estimations of the partitioning of 20:4n-6 as described above. The
further possibility, that the high recovery of 20:4n-6 in tissue lipids may partly be due to its
biosynthesis from 18:2n-6, should also be considered. This seems unlikely, however, since the
20
recovery of 18:2n-6 in the embryo is very similar to the average for total fatty acid, whereas
substantial conversion of this fatty acid would result in a lower relative recovery.
Incorporation of fatty acids into fat stores and mobilization during hatching. The two
main polyunsaturated fatty acids of the embryo, 20:4n-6 and 22:6n-3, differed markedly in
their relative distribution between structural lipid and the fat stores. Whereas more than half
of the 22:6n-3 incorporated into the embryo lipids prior to hatching was recovered in the fat
stores, 20:4n-6 was overwhelmingly directed into structural lipid. The explanation for this
differential partitioning may lie in the distribution of these two fatty acids among the plasma
lipid classes of the embryo. We have previously shown that 22:6n-3 is a major component of
plasma triacylglycerol in the king penguin embryo, in contrast to 20:4n-6 which is mainly
transported in plasma phospholipid (5). Since the triacylglycerol fraction of plasma
lipoproteins is the primary substrate for lipoprotein lipase expressed mainly in the embryonic
adipose tissue (25), triacylglycerol fatty acids such as 22:6n-3 will be diverted into the fat
stores for re-esterification. As a consequence of lipoprotein lipase action, the 20:4n-6 present
in plasma phospholipid will be recovered in the lipoprotein remnants or else transferred to
high-density lipoprotein, both of these fates initially favoring uptake of 20:4n-6 by the liver
(33). (sentence deleted)
The fatty acid composition of the fat stores showed very little change between the
beginning and the end of the hatching process. Thus, the massive mobilization of the fat stores
during hatching is relatively unspecific with regard to the individual fatty acids: there was
little evidence for any preferential release of particular fatty acids. This contrasts markedly
with the profound selectivity of fatty acid mobilization that has been observed in other
systems. The release of fatty acids from mammalian adipose tissue, whether studied in vitro
(20, 22) or in vivo (4, 9, 21), conforms to a well defined pattern in which the relative
mobilization increases with the degree of unsaturation and decreases with increasing chain
21
length. Thus, highly unsaturated fatty acids such as 18:4n-3, 20:4n-6 and 20:5n-3 are
preferentially released, whereas long chain saturated and monounsaturated fatty acids are
selectively retained. (sentence deleted) Apart from the selective retention of the long-chain
saturates, 20:0 and 22:0, the mobilization of the fat stores of the king penguin embryo during
hatching appears to provide a unique exception to the general rules describing the differential
release of fatty acids from adipose tissue. However, the functional significance of this nonspecificity in fatty acid release is not clear, and the mechanisms for regulating the selectivity
of mobilization are not understood (23).
Perspectives
(sentence deleted) Topics such as parental investment of energy for egg formation, the
energy budgets of development, and the use of energy for different developmental functions
are of major interest with regard to the physiological adaptations of the embryo to different
developmental strategies (30). The avian embryo is ideal for such comparative studies
because of major interspecies differences in egg size, incubation periods, and the degree of
tissue maturation at hatch (30). Evaluating the alternative fates of yolk fatty acid during
development provides a useful insight into the physiological strategies of the embryo. The
present work has shown that the embryo of the king penguin is an extreme case with regard to
the exceptionally high proportion of transferred fatty acid that is oxidized for energy. Future
comparative studies of fatty acid partitioning using species at opposite ends of the ranges of
egg size, yolk fatty acid profile, incubation period and hatchling maturity would help to
explain how embryonic physiology adapts to developmental mode.
The mechanisms for regulating the partitioning of fatty acids among different
metabolic functions in the embryo should also be considered for investigation. The relative
activities and substrate affinities of enzymes that control the incorporation of fatty acids into
22
alternative pathways may provide a basis for such explanations. For example, comparison of
the properties of carnitine palmitoyl transferase I with those of various acyltransferases
involved in phospholipid synthesis in the embryonic tissues may help to explain the
distribution of fatty acids between β-oxidation and membrane biogenesis (32).
23
ACKNOWLEDGMENTS
We are grateful to Institut Polaire Français (IPEV) for financial support (program 119), and to
Terres Australes and Antarctiques Françaises for logistical support. We thank E. Mioskowski
for assistance in the sample analyses. F.F. was the recipient of a grant from the Bettencourt
Schueller Found. BKS was supported by a grant from the Scottish Executive Environment
and Rural Affairs Department.
24
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28
FIGURE LEGEND
Figure 1. Metabolic fate of total yolk fatty acids during the whole incubation in the king
penguin.
Values (means ± SE, n = 8) represent the net balance between days 0 and 53 of incubation.
Transferred = lost from the yolk; non-transferred = in residual yolk; oxidized = lost from the
egg (yolk + embryo); fat stores = embryo triacylglycerols; structure = lipids other than
triacylglycerols.
Figure 2. Metabolic fate of total yolk fatty acids during the pre-hatching period in the king
penguin.
Values (means ± SE, n = 8) represent the net balance between days 0 and 49 of incubation.
See Fig. 1 legend for further explanation.
Figure 3. Metabolic fate of total yolk fatty acids during hatching in the king penguin.
Values (means ± SE, n = 8) represent the net balance between days 49 and 53 of incubation.
See Fig. 1 legend for further explanation.
Figure 4. Relative transfer of individual yolk fatty acids during the pre-hatching period in the
king penguin.
Values are means (mass lost from the yolk between days 0-49 x 100 / mass in the yolk at day
0; n = 8) and T-bars show SE. Only major fatty acids (weight % in yolk at day 0 > 0.5%) were
considered. They are designed as in Table 2 footnote and arranged according to increasing
relative transfer. The horizontal dashed line shows relative transfer of total yolk fatty acids.
Values not sharing a common letter (a-c) are significantly different (P < 0.05).
29
Figure 5. Relative incorporation of individual yolk fatty acids during the pre-hatching period
in the king penguin.
Values are means (mass incorporated in the embryo between days 0-49 x 100 / mass in the
yolk at day 0; n = 8) and T-bars show SE. Fatty acids are arranged according to increasing
relative incorporation and the horizontal dashed line shows relative incorporation of total yolk
fatty acids. Other explanation as in Fig. 4 legend. Values not sharing a common letter (a-d)
are significantly different (P < 0.05).
Figure 6. Relative oxidation of individual yolk fatty acids during the pre-hatching period in
the king penguin.
Values are means (mass lost from the egg between days 0-49 x 100 / mass in the yolk at day
0; n = 8) and T-bars show SE. Fatty acids are arranged according to increasing relative
oxidation and the horizontal dashed line shows relative oxidation of total yolk fatty acids.
Other explanation as in Fig. 4 legend. Values not sharing a common letter (a-g) are
significantly different (P < 0.05).
Figure 7. Comparison of the metabolic fate of yolk arachidonic (ARA, 20:4n-6) and
docosahexaenoic (DHA, 22:6n-3) acids during the pre-hatching period in the king penguin.
Values are means ± SE (n = 8) and T-bars show SE. Oxidized and incorporated were
calculated as % of transferred fatty acids; structure and fat stores were calculated as % of
incorporated fatty acids. See Fig. 1 legend for further explanation. *Significantly different
from DHA, P < 0.001.
30
Table 1. Fresh mass of egg and egg components at 3 stages of incubation in the king penguin
__________________________________________________________________
Day 0
__________
Day 49
__________
Day 53
__________
Egg, g
304.8 ± 3.8a
268.2 ± 3.9b
246.6 ± 4.1c
Yolk, g
65.7 ± 2.0a
42.9 ± 2.3b
18.1 ± 1.7c
Embryo, g
-----
161.3 ± 3.3a
183.2 ± 5.2b
Albumen, g
194.6 ± 10.0a
10.0 ± 1.5b
-----
Egg shell, g
42.9 ± 1.1a
38.2 ± 1.1b
38.5 ± 1.5b
__________________________________________________________________
Values are means ± SEM (n = 8). Days 49 and 53 correspond to the beginning and end of
hatching, respectively. Within a row, values that do not share the same superscript letter (a, b,
c) are significantly different (P < 0.01).
31
Table 2. Composition and mass of fatty acids in yolk (day 0) and embryo (day 53) of the king
penguin.
_________________________________________________________________________________________
FA
YOLK AT DAY 0
EMBRYO AT DAY 53
_____________________________
_____________________________
Weight %
Mass, mg
Weight %
Mass, mg
________________________________________________________________________________________
14:0
0.66 ± 0,05
100.0 ± 7.7
0.65 ± 0.04
14.5 ± 1.7
15:0
0.15 ± 0.00
22.4 ± 0.6
0.13 ± 0.01
2.9 ± 0.3
16:0
21.35 ± 0.31
3205.0 ± 122.0
20.20 ± 0.35
447.2 ± 36.9
16:1n-9
0.54 ± 0.03
82.1 ± 5.3
0.36 ± 0.02*
8.1 ± 1.0
16:1n-7
3.01 ± 0.07
450.7 ± 15.4
1.62 ± 0.14*
36.6 ± 5.2
16:1n-5
0.10 ± 0.00
15.4 ± 0.8
0.18 ± 0.02*
3.9 ± 0.5
Iso 17:0
0.16 ± 0.00
23.6 ± 1.0
0.09 ± 0.01*
2.0 ± 0.2
a-iso 17:0
0.09 ± 0.00
13.0 ± 0.4
0.07 ± 0.01
1.5 ± 0.2
16:2n-4
0.22 ± 0.01
32.5 ± 2.0
0.23 ± 0.03
5.1 ± 0.8
17:0
0.48 ± 0.02
72.2 ± 3.2
0.39 ± 0.01
8.7 ± 0.8
17:1n-8
0.35 ± 0.01
52.9 ± 1.6
0.47 ± 0.04
10.3 ± 1.1
Iso 18:0
0.25 ± 0.00
36.8 ± 0.8
0.11 ± 0.00*
2.3 ± 0,2
18:0
13.01 ± 0.46
1949.3 ± 85.5
11.52 ± 0.29
250.5 ± 12.6
18:1n-9
35.88 ± 0.71
5384.7 ± 211.3
32.50 ± 0.76*
717.7 ± 58.7
18:1n-7
3.39 ± 0.06
507.2 ± 15.9
2.84 ± 0.16
63.6 ± 7.2
18:1n-5
0.58 ± 0.02
86.4 ± 2.4
0.23 ± 0.02*
5.1 ± 0.6
18:2n-6
1.67 ± 0.04
250.3 ± 10.7
1.77 ± 0.07
38.6 ± 2.5
18:2n-4
0.07 ± 0.01
9.8 ± 1.2
0.04 ± 0.01
1.0 ± 0.3
18:3n-3
0.28 ± 0.01
41.4 ± 1.9
0.14 ± 0.02*
3.2 ± 0.7
18:4n-3
0.05 ± 0.01
7.8 ± 1.0
0.02 ± 0.00
0.5 ± 0.1
18:4n-1
0.12 ± 0.01
18.0 ± 2.0
0.06 ± 0.01*
1.3 ± 0.3
20:0
0.08 ± 0.01
11.4 ± 1.3
0.40 ± 0.04*
8.3 ± 0.2
20:1n-11
1.07 ± 0.13
156.7 ± 13.0
1.02 ± 0.12
22.5 ± 3.1
20:1n-9
1.76 ± 0.15
262.3 ± 17.6
1.34 ± 0.10
29.6 ± 3.1
20:1n-7
0.12 ± 0.01
17.2 ± 1.0
0.08 ± 0.01
1.8 ± 0.2
20:1n-5
0.08 ± 0.01
12.5 ± 0.6
0.02 ± 0.00*
0.5 ± 0.1
20:2n-6
0.07 ± 0.00
9.6 ± 0.3
0.06 ± 0.00
1.3 ± 0.1
20:3n-6
0.10 ± 0.01
14.9 ± 0.9
0.15 ± 0.01
3.2 ± 0.3
20:4n-6
1.39 ± 0.02
207.4 ± 4.8
5.17 ± 0.41*
109.8 ± 3.8
20:4n-3
0.14 ± 0.01
21.8 ± 2.0
0.08 ± 0.01*
1.8 ± 0.3
20:4n-1
0.11 ± 0.02
17.0 ± 1.9
0.13 ± 0.01
2.7 ± 0.2
20:5n-3
2.86 ± 0.17
426.5 ± 22.0
3.34 ± 0.27
74.2 ± 9.0
32
Table 2
(continued)
22:0
0.00 ± 0.00
0.0 ± 0.0
0.17 ± 0.01*
3.6 ± 0.1
22:1n-11
0.27 ± 0.04
39.8 ± 4.3
0.12 ± 0.02*
2.7 ± 0.4
22:1n-9
0.10 ± 0.01
14.6 ± 1.4
0.14 ± 0.01
3.0 ± 0.2
21:5n-3
0.04 ± 0.00
5.9 ± 0.6
0.00 ± 0.00
0.0 ± 0.0
22:4n-6
0.02 ± 0.00
3.1 ± 0.4
0.64 ± 0.06*
13.6 ± 0.7
22:5n-3
1.43 ± 0.07
213.7 ± 8.9
2.33 ± 0.21*
52.5 ± 7.5
22:6n-3
6.38 ± 0.36
950.8 ± 40.9
7.96 ± 0.23*
176.0 ± 14.9
24:1n-9
0.06 ± 0.02
9.8 ± 3.0
0.04 ± 0.00
0.9 ± 0.1
other FA
1.53 ± 0.12
226.5 ± 15.7
3.21 ± 0.24*
68.2 ± 2.2
Total FA
100.00 ± 0.00
14983.1 ± 454.0
100.00 ± 0.00
2200.8 ± 158.4
_________________________________________________________________________________________
Values are means ± SE (n = 8). Fatty acids are identified by no. of carbon atoms:no. of double
bonds, position of 1st double bond from the methyl end of the molecule. Other FA consisted
of FA with retention times between those of 14:0 and 18:0. *Significantly different from
weight % in yolk at day 0, P < 0.01.
33
Table 3. Fatty acid composition of adipose tissue at the beginning and end of hatching
in the king penguin chick, and fractional mobilization of fatty acids during hatching.
______________________________________________________________
Beginning of
End of
Fractional
hatching
hatching
mobilization
(weight %)
(weight %)
(%)
_____________________________________________________________
FA
14:0
0.82 ± 0.05
0.90 ± 0.01
34.6 ± 8.9
15:0
0.16 ± 0.01
0.17 ± 0.01
39.2 ± 9.9
16:0
24.64 ± 0.44
25.09 ± 0.17
39.0 ± 9.6
16:1n-9
0.46 ± 0.03
0.43 ± 0.05
36.7 ± 16.6
16:1n-7
2.39 ± 0.11
2.32 ± 0.09
38.8 ± 13.2
16:1n-5
0.13 ± 0.01
0.09 ± 0.01
55.6 ± 9.0
iso 17:0
0.10 ± 0.01
0.08 ± 0.02
51.1 ± 8.0
a-iso 17:0
0.04 ± 0.00
0.03 ± 0.01
46.9 ± 12.8
16:2n-4
0.26 ± 0.04
0.31 ± 0.01
26.9 ± 14.3
17:0
0.35 ± 0.01
0.34 ± 0.00
43.3 ± 7.9
17:1n-8
0.27 ± 0.01
0.24 ± 0.02
44.8 ± 10.6
iso 18:0
0.14 ± 0.01
0.12 ± 0.01
47.0 ± 8.4
18:0
7.75 ± 0.21
9.24 ± 0.51*
32.3 ± 7.7
18:1n-9
36.47 ± 0.79
35.12 ± 1.35
41.1 ± 10.1
18:1n-7
3.36 ± 0.11
3.50 ± 0.05
37.1 ± 10.7
18:1n-5
0.37 ± 0.02
0.30 ± 0.03
52.0 ± 8.3
18:2n-6
2.22 ± 0.07
2.06 ± 0.04
43.1 ± 10.8
18:2n-4
0.05 ± 0.01
0.05 ± 0.01
29.9 ± 25.5
18:3n-3
0.29 ± 0.02
0.27 ± 0.01
39.7 ± 13.5
18:4n-3
0.02 ± 0.00
0.02 ± 0.01
ND
18:4n-1
0.07 ± 0.01
0.07 ± 0.01
16.2 ± 38.3
20:0
0.16 ± 0.02
0.49 ± 0.18
-54.1 ± 12.8#
20:1n-11
1.03 ± 0.13
1.25 ± 0.14
29.9 ± 10.1
20:1n-9
1.91 ± 0.13
2.19 ± 0.22
35.6 ± 7.4
20:1n-7
0.08 ± 0.01
0.08 ± 0.00
34.2 ± 11.4
20:1n-5
0.01 ± 0.00
0.02 ± 0.01
ND
34
Table 3 (continued)
20:2n-6
0.08 ± 0.01
0.08 ± 0.01
42.8 ± 4.9
20:3n-6
0.09 ± 0.01
0.08 ± 0.02
49.0 ± 10.5
20:4n-6
1.21 ± 0.05
1.12 ± 0.06
45.8 ± 7.6
20:3n-3
0.01 ± 0.00
0.01 ± 0.00
ND
20:4n-3
0.12 ± 0.02
0.10 ± 0.00
44.4 ± 18.0
20:4n-1
0.06 ± 0.01
0.04 ± 0.03
62.1 ± 16.6
20:5n-3
2.07 ± 0.21
1.83 ± 0.06
45.6 ± 11.6
22:0
0.03 ± 0.01
0.09 ± 0.03
-42.6 ± 17.2#
22:1n-11
0.15 ± 0.02
0.20 ± 0.02
21.6 ± 13.8
22:1n-9
0.12 ± 0.01
0.16 ± 0.02
28.3 ± 6.9
22:4n-6
0.11 ± 0.01
0.13 ± 0.02
34.0 ± 6.7
22:5n-3
2.19 ± 0.15
2.57 ± 0.20
27.2 ± 14.0
22:6n-3
9.31 ± 0.53
7.69 ± 0.18*
48.8 ± 8.6
24:1n-9
0.06 ± 0.01
0.07 ± 0.02
38.8 ± 12.2
Other FA
0.85 ± 0.07
1.04 ± 0.08
23.6 ± 10.6
Total FA
100.00 ± 0.00
100.00 ± 0.00
40.1 ± 9.4
__________________________________________________________________________
Values are means ± SE (n =8). ND: not determinable. *Significantly different from weight %
at the beginning of hatching, P < 0.05. #Significantly different from fractional mobilization of
total FA, P < 0.001.
35
64.2 ± 4.4%
Structure
1.375 ± 0.057 g
Incorporated
in the embryo
2.201 ± 0.158 g
16.9 ± 0.9%
35.8 ± 4.4%
Transferred
12.960 ± 0.459 g
86.6 ± 1.4%
83.1 ± 0.9%
Available in the
yolk at day 0
14.983 ± 0.454 g
Oxidized
10.759 ± 0.365 g
13.4 ± 1.4%
Non-transferred
2.026 ± 0.236 g
FIG 1
Fat stores
0.826 ± 0.146 g
36
FIG 2
42.5 ± 3.8%
Structure
0.991 ± 0.084 g
Incorporated
in the embryo
2.350 ± 0.083 g
24.3 ± 0.7%
57.5 ± 3.8%
Transferred
9.693 ± 0.216 g
64.9 ± 1.4%
75.7 ± 0.7%
Oxidized
7.343 ± 0.188g
Available in the
yolk at day 0
14.983 ± 0.454 g
35.1 ± 1.4%
Non-transferred
5.292 ± 0.378 g
Fat stores
1.358 ± 0.116 g
37
FIG 3
Structure
0.383 ± 0.245 g
Incorporated
in the embryo
- 0.149 ± 0.136 g
-5.8 ± 4.1%
Transferred
3.267 ± 0.272 g
21.6 ± 1.3%
Available in the
yolk at day 0
14.983 ± 0.454 g
105.8 ± 4.1%
Oxidized
3.416 ± 0.240 g
Fat stores
– 0.532 ± 0.124
38
39
40
41