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APPETITE, OBESITY AND METABOLISM
1Centre 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
Submitted 3 March 2003 ; accepted in final form 30 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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.
Prehatching (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 nonselective. At hatch, 53% of the
arachidonic acid (20:4n-6) of the initial yolk had been incorporated into
embryo lipid compared with 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 with 72% of the
total FA and 58-66% of the n-3 polyunsaturated FA. The high partitioning of
yolk FA toward 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. bird development; fatty acid transfer; fatty acid oxidation; n-3 polyunsaturated fatty acid; n-6 polyunsaturated fatty acid
Because 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 FA during the prehatching 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 FA from embryonic fat depots during hatching.
The selective use of particular FA for different purposes during development is also of considerable interest. For example, high proportions of docosahexaenoic acid (22:6n-3) 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 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), whereas 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 FA (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 semialtricial 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). Because 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 FA 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 FA and of the individual FA during development of the king penguin. The proportion of the FA transferred to the embryo, as opposed to retention in the residual yolk, and the partitioning of the transferred FA among the alternative fates of oxidation, incorporation into structural lipid, and formation of fat stores, were quantified. Moreover, these values were determined for the whole incubation, the prehatching period, and for hatching, and the mobilization of FA from the embryonic fat stores during hatching was estimated.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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 very limited impact on the local population (30,000 breeding pairs) given that natural mortality is several thousand eggs or chicks per year.
Eggs at laying (n = 8), embryos at day 49 of incubation (prehatching, n = 8), and fully hatched chicks (n = 8) were obtained from eggs laid in the wild under natural conditions of diet and habitat and 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 wk 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 was based on determination of mass balances and required results typical of the species, so 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 the weighing of the newly-laid egg in 100 pairs. 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. 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 after laying. Day 49 of incubation was selected as the prehatching 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 eggshell but that most of them were starting the hatching process (M.-A. Thil, F. Decrock, and R. Groscolas, unpublished data). The latter fact could be inferred 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 transferred from the parent to a laboratory incubator (37.5°C and 100% relative humidity) until the chicks had completely freed themselves from the eggshell 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 eggshell. On average, chicks were fully hatched 2 days later, after 53 ± 1 days of incubation as under natural conditions.
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 eight trios, the comparison of two 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 ± 3.8g(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 eggshell 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 eggshell. 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, and residual yolk was 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). Butylated hydroxytoluene was added to the solvent as an antioxidant at the final concentration of 0.05%. The extraction was repeated three 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 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 being dried under nitrogen and being sprayed 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 was 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 ATWAX 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 (Nu-check Prep, Ely-sian, 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.
Calculations and Statistics
Values are means ± SE. They are presented either at the three stages of incubation or for a given period of incubation (prehatching = 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. Extraembryonic tissues (allantois, amnion, etc.) were not taken into account in this study, their FA considered as lost and thus allocated as oxidized. From previous data on the lipid content of extraembryonic tissues (Decrock and Groscolas, unpublished data), this could have overestimated 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 as percentage. 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 did not have the same relative transfer, relative oxidation was not the reciprocal of relative incorporation. Transfer, oxidation, and incorporation as calculated above do 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 transferred 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.
Percentage values were transformed to arcsin before statistical analysis to make the variance independent of the mean. Comparison of several means was performed using a one-factor ANOVA followed by a Student-Newman-Keuls 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-Whitney U-test when the equality of variance failed. The criterion of significance was P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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 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.
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Metabolic Fate of Total Yolk FA
The metabolic fate of total FA is illustrated Figs. 1, 2, 3 for the whole incubation (days 0-53), for the prehatching 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, 2, 3). Throughout the whole incubation (Fig. 1), 87% of yolk FA were transferred, i.e., 65% prehatching (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% prehatching 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 prehatching (68.5 ± 1.5%) and hatching (31.5 ± 1.5%). In contrast, all the net incorporation of FA in the embryo occurred prehatching. As a consequence, the proportion of transferred FA that was incorporated in the embryo (24%) and that was oxidized (76%) prehatching (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 was stored in adipose tissues prehatching, this proportion significantly declined (P < 0.001) to 36% posthatching (Figs. 1 and 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 FA
FA in yolk and embryo. A total of 40 FA were identified in the yolk at day 0 (Table 2). Oleic (18:1n-9), palmitic (16:0), stearic (18:0), and docosahexaenoic (22: 6n-3) were the major FA. Total n-3 polyunsaturated FA (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 twofold lower weight percentage of palmitoleic acid (16:1n-7; P < 0.001) and by its severalfold higher weight percentage 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, 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).
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Transfer of yolk FA. The relative transfer of individual yolk FA differed significantly both prehatching (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 FA (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 FA, this selectivity being mostly observed during the prehatching period and concerning principally minor FA.
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Incorporation of FA in the embryo. The relative incorporation in the embryo of FA averaged 15.8 ± 0.7% and 14.6 ± 0.8% prehatching and during the whole incubation, respectively. The relative incorporation of individual FA differed markedly both prehatching (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 prehatching (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 prehatching (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 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 underincorporation of 16: 1n-7.
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Oxidation of FA. The relative oxidation of total FA averaged 49.2 ± 1.0% prehatching, 22.8 ± 1.4% during hatching, and 71.9 ± 1.6% during the whole incubation. The relative oxidation of individual FA differed markedly prehatching (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). Prehatching 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%) 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 compared with the prehatching period.
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Docosahexaenoic vs. arachidonic acid. To further focus on n-3 and n-6 PUFA, the metabolic fate of docosahexaenoic (22:6n-3) and arachidonic acid (20: 4n-6), i.e., the two major FA of each series in the embryo, is compared in Fig. 7 for the prehatching 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 their incorporation in the embryo is considered, 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).
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FA mobilization from fat stores during hatching. The FA composition of fat stores (embryo TAG) pre- and posthatching is shown in Table 3 together with the fractional mobilization of FA during hatching. The weight percentage of only two FA (18:0 and 22:6n-3) was slightly different between pre- and posthatching (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).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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 25% of the lipid that
was originally present in the egg
(17). By contrast, only 13% of
the original yolk FA remained within the residual yolk of the king penguin,
indicating that a much greater proportion of yolk lipid is delivered to the
embryo before hatching in this species.
Lin et al. (12)
demonstrated that 50% of the mass of FA originally present in the egg of
the domestic chicken was recovered in the lipids of the hatchling plus
residual yolk, indicating that about one-half of the yolk's initial content of
FA is oxidized for energy during embryonic development. Assuming that the
residual yolk of the domestic chicken accounts for 25% of the initial yolk FA
content (17), it can be
calculated from the data of Lin et al.
(12) that 65% of the FA
transferred from the yolk during embryonic development is oxidized for energy.
Similarly, 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%) of transferred FA
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
FA. Also, it is evident that hatching is an energetically costly process in
this species. Although hatching represents 6% of the incubation period,
it accounts for 32% of the oxidized FA. In part, this is due to the high
maintenance cost over the 3-day hatching period, because 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 that, 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 FA 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 16% of the FA oxidized
over this period, the remainder being supplied directly from the yolk.
Reductions in the fat stores of the chicken embryo, by as much as 25%, have
also been 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 1) the transfer of a high proportion of yolk FA before hatching, 2) the oxidation of an exceptionally high proportion of the transferred lipid, and 3) the substantial mobilization of the embryo's fat stores during hatching.
Transfer and Metabolic Fates of Individual FA
Although there was some selectivity in the transfer of individual FA from the yolk of the king penguin, this was largely confined to very minor FA. Most of the major FA 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 FA 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 FA 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).
To some extent, the FA 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 FA. There were, however, some notable differences, the most salient being the fourfold 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 FA. Conversely, the relative oxidation of 20:4n-6 during prehatching development was far less than that of the other FA. 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 FA (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 FA. It seems that the levels of 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 FA 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 FA into structural lipid (12).
There was a clear distinction between the prehatching 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 total FA, no such difference was observed during the hatching period. Possibly, the rate of oxidation during hatching is so high as to override 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 FA that are achieved in the phospholipids of the embryonic liver, heart, and skeletal muscle, despite the very high C20-22 n-3/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). 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).
For two FA, 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 FA were very minor components. The reason for synthesizing 22:0 and 22:4n-6 during development is not clear, although the latter FA is detected in the phospholipid of the heart and skeletal muscle of the king penguin embryo at concentrations ranging from 0.6 to 1.3% (wt/wt) (6). Some biosynthesis of 22:4n-6 also occurs during embryonic development of the chicken (3, 12). Because 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 FA 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, because the recovery of 18:2n-6 in the embryo is very similar to the average for total FA, whereas substantial conversion of this FA would result in a lower relative recovery.
Incorporation of FA Into Fat Stores and Mobilization During Hatching
The two main polyunsaturated FA 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 one-half of the 22:6n-3 incorporated into the embryo lipids before 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 FA among the plasma lipid classes of the embryo. We previously showed 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). Because the triacylglycerol fraction of plasma lipoproteins is the primary substrate for lipoprotein lipase expressed mainly in the embryonic adipose tissue (25), triacylglycerol FA such as 22:6n-3 will be diverted into the fat stores for reesterification. 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).
The FA 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 nonspecific with regard to the individual FA; there was little evidence for any preferential release of particular FA. This contrasts markedly with the profound selectivity of FA mobilization that has been observed in other systems. The release of FA 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 length. Thus highly unsaturated FA such as 18:4n-3, 20:4n-6, and 20:5n-3 are preferentially released, whereas long-chain saturated and monounsaturated FA are selectively retained. 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 FA from adipose tissue. However, the functional significance of this nonspecificity in FA release is not clear, and the mechanisms for regulating the selectivity of mobilization are not understood (23).
Perspectives
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 FA 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 FA that is oxidized for energy. Future comparative studies of FA partitioning using species at opposite ends of the ranges of egg size, yolk FA profile, incubation period, and hatchling maturity would help to explain how embryonic physiology adapts to developmental mode.
The mechanisms for regulating the partitioning of FA 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 FA into 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 FA
between 
-oxidation and membrane biogenesis
(32).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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2-adrenergic/arachidonic pathway in ventricular
cardiomyocytes. Regulation by the 1-adrenergic/cAMP pathway.
J Biol Chem 274:
628-637, 1999.
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