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Changes in the lipid and fatty acid composition of the yolk during embryonic development of the alligator (Alligator mississipiensis)
Comp. Biochem. Physiol. Vol. 96B, No. 1, pp. 183-187, 1990 Printed in Great Britain
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc
C H A N G E S IN THE LIPID A N D F A T T Y ACID COMPOSITION OF THE YOLK D U R I N G E M B R Y O N I C D E V E L O P M E N T OF THE A L L I G A T O R
(ALLIGATOR MISSISSIPIENSIS) R. C. NOBLE,*D. C. DEEMING,tM. W. J. FERGUSONtand R. MCCARTNEY* *Department of Poultry Science, The West of Scotland College, Auchincruive, Ayr KA6 5HW, Scotland (Tel: 0292 520331); and ~Department of Cell and Structural Biology, The University of Manchester. Coupland lIl Building, Coupland Street, Manchester M13 9PL, England (Tel: 061 275 6775) (Received 11 October 1989) Changes in absolute and relative amounts of the yolk lipid fractions and their fatty acids were determined for alligator embryos at various stages during incubation. 2. Lipid absorption was particularly active after day 56 of incubation. 3. The major lipid components at the start of incubation were triacylglyceride and phosphoglyceride. The levels of both these fractions decreased considerably during incubation whilst cholesterol ester increased to become a major lipid component of the yolk at hatching. 4. All lipid fractions displayed high levels of palmitoleic acid and in the phosphoglyceride there were high levels of C20 and C22 polyunsaturated fatty acids. 5. The fatty acid composition of the triacylglyceride and phosphoglyceride remained relatively unchanged during incubation but there was extensive esterification of cholesterol with oleic acid Abstract--l.
INTRODUCTION
The egg of the alligator, in c o m m o n with its avian, amphibian and other reptilian counterparts, contains an extensive yolk mass which supplies the developing embryo with a large proportion of its nutrients (Romanoff, 1960; Noble and Moore, 1966; Thompson, 1981; Manolis et al., 1987). The yolk size, the proportional distribution of the major nutrients within the yolk and the rate of utilization of the components during embryonic development differ widely between species. However, the major feature of the yolks of all species is a high initial lipid content and rapid lipid utilization during the later stages of embryo development when growth is maximal. In avian eggs the yolk is the major energy source for the developing embryo (Freeman and Vince, 1974); importantly, assimilation of lipid over the later part of development is characterized by extensive and, in many cases, distinctive compositional and metabolic changes that are intimately related to the uptake of essential tissue components (Noble et al., 1984; Noble and Shand, 1985; Noble, 1987). Transfer of the yolk lipid, therefore, constitutes a major part of the interrelated chain of events required for the successful emergence of the embryo (Noble, 1987). Although the yolk lipids of the crocodilian egg have also been shown to perform a vital quantitative role as both a nutrient and energy source for the developing embryo (Manolis et al., 1987), precise lipid analyses at different stages of development, as exist for avians are unavailable for crocodilians. However, factors which bring about the embryological changes within the alligator egg are becoming ever more important as a result of their captive breeding for conservation and commercial purposes (Ferguson, 1985). The specific
objective of the present study therefore was to provide detailed information for the alligator embryo on yolk lipid assimilation and its metabolism at different stages of embryonic development.
MATERIALS AND METHODS
Eggs of Alligator mississippiensis were collected from several nests of wild alligators at the Rockefeller Wildlife Refuge, LA, USA, on the first or second day after laying and were immediately transported by air to Manchester, England. On arrival at the laboratory (day 3/4 depending on the clutch) the eggs were weighed and placed in an incubator set at 30°C which was accurate to +0.01°C (Vinden Scientific Ltd., Bolton, England). The relative humidity in the incubator was maintained as close as possible to 100% RH. The investigation used yolks from eggs described in another study (Deeming and Ferguson, 1989). Yolk sacs were carefully excised from four fertile eggs at days 8, 32, 40, 48, 56, 64 and 75 (hatching) of incubation. Following weighing, the yolk sacs were individually homogenized and a known weight of the homogenate was removed. The total lipid from the sample was extracted by homogenization and refluxing in a suitable excess of chloroform:methanol (2:1, v/v) according to well established procedures (Christie, 1984). All lipid extracts were then stored in chloroform at -20°C to await analysis. The lipids were fractionated into their major classes on thin layer chromatoplates of silica gel G, thickness 0.25mm, using a solvent system of hexane:diethyl ether: formic acid (80: 20:1, v/v/v). Following visualization and identification of the separated bands under u.v. light after spraying with a 0.1% (w/v) solution of 2,7-dichlorofluorescein in methanol, the phosphoglycerides were eluted from the silica gel by washing with 3 x 5 ml of chloroform: methanol: water (5 : 5:1, v/v/v) and the remaining lipid fractions with 3 × 5 ml of diethyl ether. The identified lipid fractions were then transmethylated by refluxing with 183
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Table 1. Total weights of the eggs, yolks and yolk lipid and the lipid composition (major fractions, incubation Embryo age (days) 8 32 40 48 56 Egg weight (g) 69.6 ± 0.99 70.2± 1.45 71.1±4.65 69.8+_2.99 72.7+_3.23 Yolk weight (g) 31.4 +_0.32 29.6_+0.64 26.9+_2.58 24.3+_1.79 23.1±2.03 Weight lipid/yolk (g) 6.44 +_0.05 6.37_+0.46 5.82 ± 0.59 5.17+_0.44 6.24+_0.22 Lipid composition (%) Cholesterol ester 1.48 +_0.06 1.97_%0.16 2.39±0.25 4.55+_0.19 3.76+_0.18 Triacylglyceride 69.5 +_0.70 69.1-+0.07 68.1+_0.21 67.9_+1.12 72.5±0.55 Free fatty acid 1.60 +_0.08 1.53-+0.07 1.38+-0.03 2.15±0.23 1.26±0.04 Free cholesterol 7.68 ± 0.21 6.04_+0.46 5.28+0.48 5.89_+0.28 5.66±0.47 Phosphoglyceride 19.8 _+0.75 21.4+ 0.50 22.0+0.56 19.5±1.00 16.9-+0.44 Values are means +_SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; ***P < 0.001.
methanohtoluene:sulphuric acid (20:10:1, v/v/v) in the presence of a pentadecanoic acid standard (Christie et al., 1970). Gas liquid chromatography of the methyl fatty acid derivatives on a packed column of 15% CP Sil 84 on Chromosorb WHP (Chrompak UK Ltd., Middleburg, The Netherlands) then enabled quantification of both the relative proportions of the major long chain fatty acids present and the absolute amount of the lipid associated with each fraction. Quantification of the fatty acids was by electronic integration. The amount of the free cholesterol was determined by charring and subsequent densitometry using a liquid scintillation counter (Shand and Noble, 1980). Identification of lipid and fatty acid fractions was by comparison with known standards. All solvents were distilled before use and where necessary, operations were performed under an atmosphere of nitrogen.
RESULTS The weights o f the whole eggs, their yolks, the lipid contents of the yolks a n d their compositions (major classes, weight per cent o f the total lipid present) over the 75 days of i n c u b a t i o n period are given in Table 1. The weight range o f the eggs c o n f o r m e d to t h a t expected for m a t u r e alligators. W h e r e a s over the first 56 days of i n c u b a t i o n the weight of the yolk showed only a slight decrease, thereafter there was a rapid decline in weight. This p a t t e r n o f yolk weight change was parallelled by a similar reduction in the a m o u n t of yolk lipid a n d by substantial changes in lipid composition. The principal c o m p o n e n t o f the yolk lipid initially a n d t h r o u g h o u t the i n c u b a t i o n period was triacylglyceride a c c o m p a n i e d by substantial levels also of phosphoglyceride. W h e r e a s the level of cholesterol ester present initially within the yolk lipid was very small, during the second half of the incubation period its level increased markedly, so t h a t by day 75 o f i n c u b a t i o n cholesterol ester accounted for over 2 0 % of the total yolk lipid. This large increase
% of total lipid) of the yolk during 64 75 75.1+_3.40 64.7+_0.58 15.6+_1.41 3.08"**±0.21 4.25+_0.46 0.938***+ 0.076 8.47+_0.32 20.8***±2.08 67.6+-0.56 59.4**±2.49 2.81+-0.17 6.72***_+0.39 5.32_+0.46 5.17"*_+0.60 15.8_+0.30 7.89***_+0.49
in the p r o p o r t i o n o f the cholesterol ester occurred mainly at the expense of a decrease in the level of phosphoglyceride a n d latterly a decrease also in the level o f triacylglyceride. The level of unesterified fatty acid in the yolk lipid h a d also increased markedly by the 75th day o f incubation. The fatty acid compositions of the cholesterol ester, triacylglyceride, phosphoglyceride a n d unesterified fatty acid fractions of the yolks at the various stages of the i n c u b a t i o n period are given in Tables 2, 3 a n d 4, respectively. The fatty acids listed in the Tables accounted for 9 7 - 9 8 % of the total fatty acids present, the remaining 2 - 3 % consisting of trace a m o u n t s only of a selection of saturated and unsaturated fatty acids of chain lengths varying between 14 a n d 20 c a r b o n atoms. By far the m a j o r fatty acid present initially within the cholesterol ester was palm±tic which accounted for more t h a n half of the total fatty acids present (Table 2). However, as i n c u b a t i o n proceeded the p r o p o r t i o n of palm±tic acid u n d e r w e n t a m a r k e d decrease particularly over the last part of the incubation period. A t the same time there was a large increase in the level of oleic acid such t h a t by the end of the i n c u b a t i o n period it was the m a j o r fatty acid present (Table 2). Levels of total p o l y u n s a t u r a t e d fatty acids also decreased during the i n c u b a t i o n period, this arising in the m a i n from a disproportionately large decrease t h a t occurred early in the incubation (Table 2). Over the complete i n c u b a t i o n period there was a tendency for the levels of linoleic and linolenic acids to increase. The c o n c e n t r a t i o n of stearic acid remained low t h r o u g h o u t incubation. The m a j o r fatty acids of the triacylglyceride fraction of the yolk (Table 3) were palm±tic, palmitoleic a n d oleic. T o g e t h e r they accounted for some 7 5 - 8 0 % o f the total fatty acids present. A notable feature of the triacylglyceride was the presence t h r o u g h o u t in-
Table 2. Fatty acid compositions (major acids, weight % of total) of the cholesterol ester fraction of the yolk during incubation 32 40 48 56 64 75 Embryo age (days) 8 Acid: Palm±tic 54.4 +_0.97 43.8 + 1.79 33.2+ 4.29 31.9+- 1.50 19.1_+1.33 28.0_+2.26 14.0"**± 0,75 9.79+_0,53 Palmitoleic 8.39 ± 1.30 9.25+0.65 6.22± 0.58 6.42±0.34 6.74_+0.51 8.72± 0 . 5 1 3.88+0.15 Stearic 5.87 +_ 1.12 6.21+_0.90 2.68+_1.02 6.15+_0.76 3.50+_0.37 5.75+_0.24 Oleic 13.3 ± 2.80 27.4±1.14 41.1+_3.66 37.3+_2.43 53.9+_0.33 39.8+_3.26 57.9"**_+1.14 5.72* +_0.48 Linoleic 3.11 +_0.49 5.16 ± 0.29 5.61+_0.48 7.04± 0.80 7.60+_0.44 3.72+_0.90 3,59*±0.74 Linolenic 0.87 ± 0.45 1.20+_0.10 4.11+_0.65 3.55+_0.79 4.64±0.88 2.69±0.53 1.45"*±0.15 Arachidonic 8.85 ± 1.69 4.68+_0.78 3.83±0.39 3.16+_0.76 1.81+_0.21 4.85± 0.70 tr tr tr 4.35 ± 1.79 tr*** Docosapentaenoic 3.62 +_0.86 2.05 -!-_0.56 1.55+_0.13 Docosahexaenoic 1.24 ± 0.23 2.22±0.54 4.08+-0.27 2.09+_0.17 2.63+_0.39 1.88±0.69 Values are means + SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; ***P < 0.001.
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Table 3. Fatty acid compositions (major acids, weight % of total) of the triacylglyceride fraction of the yolk during incubation Embryo aged (days) 8 32 40 48 56 64 75 Acid: Palmitic 28.9_ 0.22 28.3+ 0.29 27.2+ 0.43 25.5+ 0.85 24.9_ 0.49 26.0+ 1.72 25.3***+ 0.41 Palmitoleic 18.6 _+0.16 17.2+ 0.27 17.6+ 1.48 15.7_.+0.27 16.1+ 0.09 17.9+ 0.70 15.8 + 1.07 Stearic 6.29 + 0.38 9.56__+0.55 6.41+ 0.22 8.22+0.55 9.10+0.32 8.94___0.68 9.27**+ 0.67 Oleic 32.8 + 0.56 34.3+ 0.37 33.8__.0.90 34.5_+0.98 34.6+0.96 34.5+ 0.71 35.0 + 0.85 Linoleic 6.37 + 0.17 5.83_+0.07 8.82+ 0.58 8.51_+0.06 9.09+ 0.24 7.45+ 0.89 7.82* __.0.57 Linolenic 4.41 + 0.19 2.64+ 0.09 4.62+ 0.62 5.83+ 0.02 3.97+ 0.55 3.25+ 0.49 3.24 + 0.55 Arachidonic 1.04 + 0.03 0.83+ 0.02 0.66+ 0.12 0.96+ 0.07 0.74+ 0.03 0.62+ 0.12 1.09 _+0.05 Docosapentaenoic 0.68 + 0.02 0.52+0.02 0.59+ 0.07 0.56+ 0.06 0.44+0.01 0.45_+0.04 0.79 + 0.06 Docosahexaenoic 0.99 + 0.09 0.85+ 0.01 0.82+ 0.19 0.98_+0.04 0.97+ 0.09 0.94_+_+0.10 1.73"* + 0.12 Values are means + SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; ***P < 0.001. c u b a t i o n o f high levels of palmitoleic (Table 3). The fatty acid c o m p o s i t i o n of the triacylglyceride fraction r e m a i n e d fairly c o n s t a n t t h r o u g h o u t the i n c u b a t i o n period. However, a small but significant reduction in the level of palmitic acid occurred with a compensatory increase in the level o f stearic acid (Table 3). The m a j o r fatty acids present in the phosphoglyceride fraction (Table 4) were palmitic a n d oleic. The fatty acid c o m p o s i t i o n was characterized by high levels o f polyunsaturates, in particular C20 a n d C22 p o l y u n s a t u r a t e d fatty acids. Thus a l t h o u g h linoleic acid accounted for only 4 % of the total fatty acids, arachidonic a n d docosahexaenoic acids accounted for over 10 a n d 11% respectively (Table 4). As in the case o f the triacylglyceride a n d to a lesser extent the cholesterol ester, the phosphoglyceride displayed high c o n c e n t r a t i o n s o f palmitoleic (Table 4). The fatty acid c o m p o s i t i o n of the phosphoglyceride did n o t change to any appreciable extent during i n c u b a t i o n (Table 4). The unesterified fatty acid fraction present in the yolk at day 8 o f i n c u b a t i o n c o n t a i n e d high levels o f stearic, palmitoleic a n d oleic. The level of palmitic acid r e m a i n e d u n c h a n g e d during the i n c u b a t i o n period b u t those of palmitoleic a n d oleic decreased m a r k e d l y with a p r o p o r t i o n a l increase occurring in the level of p o l y u n s a t u r a t e d fatty acids, in particular the C20 a n d C22 polyunsaturates. DISCUSSION A t day 8 of i n c u b a t i o n total yolk accounted for some 4 5 % o f the whole weight of the alligator egg; o f the total yolk weight, some 2 1 % was lipid. These figures for the yolk a n d its lipid c o n t e n t are in general agreement with previous observations for unincub a t e d eggs o f the crocodilians a n d a range o f reptil-
ians (Steward a n d Castillo, 1984; Wilhoft, 1986; Manolis et al., 1987). They are, however, very different from respective values t h a t have been o b t a i n e d for avians (Romanoff, 1960; N o b l e a n d Moore, 1966; Noble, 1986a) where the p r o p o r t i o n by weight o f the yolk in the egg is very m u c h lower ( 3 0 - 3 3 % ) but the level o f the lipid in the yolk very m u c h higher (60-65%). The slow decline in the weight o f the yolk o f the alligator egg over the first 50-60 days of i n c u b a t i o n a n d the sudden weight loss thereafter, such t h a t by the 75th day only 10% of the initial yolk weight remained, also accorded with previous observations in crocodiles (Manolis et al., 1987). A l t h o u g h weight loss o f the yolk is associated with the absorption of a range o f dietary c o m p o n e n t s (Romanoff, 1960; F r e e m a n a n d Vince, 1974), it is clear from b o t h the present a n d previous results t h a t the majority o f the dry m a t t e r loss is due to the removal o f lipid. However, the p r o p o r t i o n of lipid within the yolk steadily increased as i n c u b a t i o n proceeded. Thus by the end of the i n c u b a t i o n period lipid accounted for some 31% of the total weight o f the remaining yolk material c o m p a r e d to a level o f only 21% at the beginning o f incubation. In the chick e m b r y o where investigations o f yolk lipid removal have been extensive (Noble, 1986b, 1987), the increase in overall lipid c o n c e n t r a t i o n per unit weight o f yolk as i n c u b a t i o n proceeds is due to the increasing d e v e l o p m e n t o f the lipid rich yolk sac m e m b r a n e (Noble a n d Moore, 1967a). In the chick the extensive redistribution of the yolk lipid between the contents a n d the yolk sac memb r a n e a n d its subsequent a b s o r p t i o n by the e m b r y o is not a c c o m p a n i e d by any change in the relative p r o p o r t i o n s of the two m a j o r lipid fractions, i.e. the triacylglyceride a n d phosphoglyceride within the yolk (Noble a n d Moore, 1964). This was clearly n o t the
Table 4. Fatty acid compositions (major acids, weight % of total) of the phosphoglyceride and free fatty acid fractions of the yolk at the 8th and 75th days of incubation Phospholipid Free fatty acids Embryo aged (days) 8 75 8 75 Acid: Palmitic 32.7 _+ 1.27 34.4 _+1.21 27.6 _+1.30 25.3 _+ 1.58 Palmitoleic 8.02 _+1.30 7.88 4- 0.37 17.0 _+0.51 9.84***_+0.80 Stearic 7.67 _+0.52 9.04 + 0.57 5.38 _+0.49 8.53***+_0.16 Oleic 21.1 + 0.39 17.3"*+ 0.92 35.4 _+ 1.52 25.8**+ 0.79 Linoleic 4.32 _+0.75 4.29 _+1.09 3.39 + 0.39 5.61" _+0.77 Linolenic 2.93 + 0.22 1.89 + 0.45 6.06 + 0.75 2.51"*+ 0.35 Arachidonic 11.0 _+2.04 11.5 _+0.34 2.75 _+0.46 12.9"**_+0.91 Docopentaenoic 2.22 _-+0.30 1.70 + 0.11 0.56 + 0.13 2.54***__+0.22 Docosahaenoic 10.1 + 0.90 11.5 _ 0.57 1.87 _+0.14 6.42***___0.17 Values are means + SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; P < 0.001. CBPB 96/1--M
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case during yolk lipid absorption in the alligator where there was a substantial difference in the relative proportion of triacylglyceride and phosphoglyceride between days 8 and 75 of incubation. However, whereas there were substantial reductions in both the relative and absolute levels of triacylglyceride and phosphoglyceride in the yolk during incubation, the amount of cholesterol ester increased substantially from 95 to 195 mg/yolk. As a result, the cholesterol ester rose from being a minor lipid component at day 8 of incubation to one that accounted for more than 20% of the total yolk lipid at day 75. Although only a small increase in cholesterol ester content was observed in comparable investigations of whole yolk changes in the chick embryo during incubation (Noble and Moore, 1964), when the yolk of the chick was divided into contents and yolk sac membrane a significant increase in the level of cholesterol ester within the lipid of the yolk sack membrane was observed over the latter part of incubation (Noble and Moore, 1967a). This increase, however, was still far short of that presently observed in the whole yolk of the alligator. With the exceptions of some small changes, the fatty acid composition of the alligator yolk triacylglyceride remained fairly constant during incubation. Thus, although it has been previously suggested that yolk lipid assimilation may be associated with some preferential absorption of triacylglyceride species (Isaaks et al., 1964), the present results for the alligator conform to the vast majority of results for chick and other avian embryos in which no evidence of any preferential removal of triacylglyceride species has been obtained (Budowski et al., 1961; Noble and Moore, 1967a; Noble, 1987). Compared with the chick embryo, the triacylglyceride within the yolk of the alligator egg showed very high levels of palmitoleic and very low levels of linoleic acid. The general distribution of the other acids was quite similar. Compared with the chick embryo (Noble and Moore, 1964, 1966), the phosphoglyceride of the yolk of the alligator egg showed several distinct features. A low level of stearic and, in particular, linoleic acid was accompanied by very much higher levels of C20 and C22 polyunsaturated fatty acids throughout incubation. In the chick embryo, and in the alligator, the fatty acid composition of the yolk phosphoglyceride remained largely unchanged over the incubation period (Noble and Moore, 1964, 1966). However, separation of the chick embryo yolks into contents and membrane showed differences in the distributions of arachidonic and docosahexaenoic acid as incubation proceeded (Noble and. Moore, 1967a,b). With regard to both arachidonic and docosahexaenoic acids, there was a preferential uptake into the yolk sac membrane of yolk phosphatidyl ethanolamine species that were rich in docosahexaenoic acid (Noble and Moore, 1967b). With regard to arachidonic acid uptake, subsequent investigations (Noble and Shand, 1985) also revealed the presence within the yolk sac membrane of appreciable A-6 desaturation activity, which is responsible for the first step in the conversion of linoteic to arachidonic acid. Both these features were associated with enhancement of the C20 and C22 polyunsaturated fatty acid level within the absorbed yolk lipid in order to satisfy
an increased requirement during embryo development (Noble and Cocchi, 1989). The high levels of C20 and C22 polyunsaturated fatty acids present within the phosphoglycerides of the aligator egg yolk throughout incubation poses several questions. For instance, the assurance of a continuous and very rich source of C20 and C22 polyunsaturated fatty acids for the start of incubation would reduce the necessity for mechanisms similar to those present in the chick embryo. It is also possible that the unusual polyunsaturated fatty acid pattern with the egg yolk of the alligator is accommodated by a wholly different range of phosphoglyceride species from that of the chick embryo (Noble and Moore, 1967b). The large accumulation of cholesterol ester within the yolk of the alligator during incubation was accompanied by a sharp increase in its content of oleic acid. In the chick embryo the level of oleic acid within the cholesterol ester fraction of the whole yolk also progressively increased during incubation (Tsuji et al., 1955; Noble and Moore, 1964). This change in whole yolk lipid composition was also almost wholly associated with events taking place in the yolk sac membrane rather than the contents (Noble and Moore, 1967a; Noble et al., 1984). Extensive synthesis and accumulation of cholesterol esters by the yolk sac membrane occurred as a vital part of the transfer of the lipid from the yolk contents into the chick embryo (Noble, 1986b, 1987). It is clear that the overall features of yolk lipid assimilation by the alligator and avian display extensive similarities. However, with regard to two of the major features, namely the cholesterol ester and polyunsaturated fatty acid metabolism, the extent of change in the alligator is far more extreme. The alligator yolk lipids do possess features that are unique. Thus the presence of very high levels of palmitoleic acids in the major lipid fractions throughout incubation is quite distinctive. Compared to the avian where yolk lipid uptake occurs by non-specific engulfment by the membrane (Lambson, 1970), the sudden rise in the level of unesterified fatty acids within the yolk of the alligator just prior to hatching may implicate an increased role for lipolytic breakdown in lipid assimilation. In the avian the lipid metabolic events that take place in the yolk are an essential and integral part of yolk assimilation. In the alligator these events may assume an even greater importance. Acknowledgements--We thank Ted Joanen, Larry McNease and David Richard of the Rockefeller Wildlife Refuge, Louisiana Department of Wildlife and Fisheries, USA for their invaluable assistance over several years with the collection and transportation of eggs of Alligator mississipensis. D.C.D. is funded by the University of Manchester Research Support Fund. REFERENCES
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Lipids in alligator yolk Deeming D. C. and Ferguson M. W. J. (1989) Effects of incubation temperature on the growth and development of Alligator mississipiensis. J. comp. Physiol. (in press). Ferguson M. W. J. (1985) Reproductive biology and embryology of the crocodilians. In Biology of the Reptilia, Vol. 14 Development A (Edited by Gans C., Billet F. and Maderson P. F. A.), pp. 329-491. John Wiley & Sons, New York. Freeman B. M. and Vince M. A. (1974) Development of the Avian Embryo. Chapman & Hall, London. Isaaks R. E., Davies R. E., Ferguson T. M., Reiser R. and Couch J. R. (1964) Studies on avian fat composition. 2. The selective utilisation of fatty acids by the chick embryo. Poult. Sci. 43, 113-120. Lambson R. O. (1970) An electron microscopic study of the entodermal cells of the yolk sac of the chick during incubation and after hatching. Am. J. Anat. 129, 1-20. Manolis S. C., Webb G. J. W. and Dempsey K. E. (1987) Crocodile egg chemistry. In Wildlife Management: Crocodiles and Alligators (Edited by Webb G. J. W., Manolis S. C. and Whitehead P. J.), pp. 445-472. Surrey Beatty, Sydney. Noble R. C. (1986a) Egg lipids. In Egg Quality: Current Patterns and Recent Advances (Edited by Wells R. G. and Belyavin C. G.), pp. 159-177. Butterworths, London. Noble R. C. (1986b) Lipid metabolism in the chick embryo. Proc. Nutr. Soc. 45, 17-25. Noble R. C. (1987) Lipid metabolism in the chick embryo: some recent ideas. J. exp. Zool. (Suppl. I), 65-73. Noble R. C. and Cocchi M. (1989) The relationship between the supply and demand for essential polyunsaturated fatty acids during mammalian and avian embryonic development. Res. Devl Agric. 6, 65~59. Noble R. C., Connor K. and Smith W. K. (1984) The synthesis and accumulation of cholesterol esters by the developing embryo of the domestic fowl. Poult. Sci. 63, 558-564.
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Noble R. C. and Moore J. H. (1964) Studies on the lipid metabolism of the chick embryo. Can. J. Biochem. 42, 1729-1741. Noble R. C. and Moore J. H. (1966) Some aspects of the lipid metabolism of the chick embryo. In Physiology of the Domestic Fowl (Edited by Horton-Smith C. and Amoroso E. D.), pp. 86-101. Oliver & Boyd, Edinburgh. Noble R. C. and Moore J. H. (1967a) The partition oflipids between the yolk and yolk sac membrane during the development of the chick embryo. Can. J. Biochem. 45, 949-958. Noble R. C. and Moore J. H. (1967b) The transport of phospholipids from the yolk to the yolk-sac membrane during the development of the chick embryo. Can. J. Biochem. 45, 1125-1133. Noble R. C. and Shand J. H. (1985) Unsaturated fatty acid compositional changes and desaturation during the embryonic development of the chicken (Gallus domesticus). Lipids 20, 278-282. Ramanoff A. L. (1960) The Avian Embryo. MacMillan, New York. Shand J. H. and Noble R. C. (1980) Quantification of lipid mass by a liquid scintillation counting procedure following charring on thin-layer plates. Analyt. Biochem. 101, 427-434. Stewart J. R. and Castillo R. E. (1984) Nutritional provision of the yolk of two species of viviparous reptiles. Physiol. Zool. 57, 377 383. Thompson J. (1981) A study of the sources of nutrients for embryonic development in a viviparous lizard. Sphenomorphus quoyii. Comp. Biochem. Physiol. 70A, 509-518. Tsuji F. I., Brin M. and Williams H. H. (1955) Lipid phosphorus and cholesterol changes in the hen's egg during incubation. Arch. Biochem. Biophys. 56, 290--296. Wilhoft D. C. (1986) Eggs and hatching components of the snapping turtle (Chelydra serpentina). Comp. Biochem. Physiol. 84A, 483-486.
0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc
C H A N G E S IN THE LIPID A N D F A T T Y ACID COMPOSITION OF THE YOLK D U R I N G E M B R Y O N I C D E V E L O P M E N T OF THE A L L I G A T O R
(ALLIGATOR MISSISSIPIENSIS) R. C. NOBLE,*D. C. DEEMING,tM. W. J. FERGUSONtand R. MCCARTNEY* *Department of Poultry Science, The West of Scotland College, Auchincruive, Ayr KA6 5HW, Scotland (Tel: 0292 520331); and ~Department of Cell and Structural Biology, The University of Manchester. Coupland lIl Building, Coupland Street, Manchester M13 9PL, England (Tel: 061 275 6775) (Received 11 October 1989) Changes in absolute and relative amounts of the yolk lipid fractions and their fatty acids were determined for alligator embryos at various stages during incubation. 2. Lipid absorption was particularly active after day 56 of incubation. 3. The major lipid components at the start of incubation were triacylglyceride and phosphoglyceride. The levels of both these fractions decreased considerably during incubation whilst cholesterol ester increased to become a major lipid component of the yolk at hatching. 4. All lipid fractions displayed high levels of palmitoleic acid and in the phosphoglyceride there were high levels of C20 and C22 polyunsaturated fatty acids. 5. The fatty acid composition of the triacylglyceride and phosphoglyceride remained relatively unchanged during incubation but there was extensive esterification of cholesterol with oleic acid Abstract--l.
INTRODUCTION
The egg of the alligator, in c o m m o n with its avian, amphibian and other reptilian counterparts, contains an extensive yolk mass which supplies the developing embryo with a large proportion of its nutrients (Romanoff, 1960; Noble and Moore, 1966; Thompson, 1981; Manolis et al., 1987). The yolk size, the proportional distribution of the major nutrients within the yolk and the rate of utilization of the components during embryonic development differ widely between species. However, the major feature of the yolks of all species is a high initial lipid content and rapid lipid utilization during the later stages of embryo development when growth is maximal. In avian eggs the yolk is the major energy source for the developing embryo (Freeman and Vince, 1974); importantly, assimilation of lipid over the later part of development is characterized by extensive and, in many cases, distinctive compositional and metabolic changes that are intimately related to the uptake of essential tissue components (Noble et al., 1984; Noble and Shand, 1985; Noble, 1987). Transfer of the yolk lipid, therefore, constitutes a major part of the interrelated chain of events required for the successful emergence of the embryo (Noble, 1987). Although the yolk lipids of the crocodilian egg have also been shown to perform a vital quantitative role as both a nutrient and energy source for the developing embryo (Manolis et al., 1987), precise lipid analyses at different stages of development, as exist for avians are unavailable for crocodilians. However, factors which bring about the embryological changes within the alligator egg are becoming ever more important as a result of their captive breeding for conservation and commercial purposes (Ferguson, 1985). The specific
objective of the present study therefore was to provide detailed information for the alligator embryo on yolk lipid assimilation and its metabolism at different stages of embryonic development.
MATERIALS AND METHODS
Eggs of Alligator mississippiensis were collected from several nests of wild alligators at the Rockefeller Wildlife Refuge, LA, USA, on the first or second day after laying and were immediately transported by air to Manchester, England. On arrival at the laboratory (day 3/4 depending on the clutch) the eggs were weighed and placed in an incubator set at 30°C which was accurate to +0.01°C (Vinden Scientific Ltd., Bolton, England). The relative humidity in the incubator was maintained as close as possible to 100% RH. The investigation used yolks from eggs described in another study (Deeming and Ferguson, 1989). Yolk sacs were carefully excised from four fertile eggs at days 8, 32, 40, 48, 56, 64 and 75 (hatching) of incubation. Following weighing, the yolk sacs were individually homogenized and a known weight of the homogenate was removed. The total lipid from the sample was extracted by homogenization and refluxing in a suitable excess of chloroform:methanol (2:1, v/v) according to well established procedures (Christie, 1984). All lipid extracts were then stored in chloroform at -20°C to await analysis. The lipids were fractionated into their major classes on thin layer chromatoplates of silica gel G, thickness 0.25mm, using a solvent system of hexane:diethyl ether: formic acid (80: 20:1, v/v/v). Following visualization and identification of the separated bands under u.v. light after spraying with a 0.1% (w/v) solution of 2,7-dichlorofluorescein in methanol, the phosphoglycerides were eluted from the silica gel by washing with 3 x 5 ml of chloroform: methanol: water (5 : 5:1, v/v/v) and the remaining lipid fractions with 3 × 5 ml of diethyl ether. The identified lipid fractions were then transmethylated by refluxing with 183
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Table 1. Total weights of the eggs, yolks and yolk lipid and the lipid composition (major fractions, incubation Embryo age (days) 8 32 40 48 56 Egg weight (g) 69.6 ± 0.99 70.2± 1.45 71.1±4.65 69.8+_2.99 72.7+_3.23 Yolk weight (g) 31.4 +_0.32 29.6_+0.64 26.9+_2.58 24.3+_1.79 23.1±2.03 Weight lipid/yolk (g) 6.44 +_0.05 6.37_+0.46 5.82 ± 0.59 5.17+_0.44 6.24+_0.22 Lipid composition (%) Cholesterol ester 1.48 +_0.06 1.97_%0.16 2.39±0.25 4.55+_0.19 3.76+_0.18 Triacylglyceride 69.5 +_0.70 69.1-+0.07 68.1+_0.21 67.9_+1.12 72.5±0.55 Free fatty acid 1.60 +_0.08 1.53-+0.07 1.38+-0.03 2.15±0.23 1.26±0.04 Free cholesterol 7.68 ± 0.21 6.04_+0.46 5.28+0.48 5.89_+0.28 5.66±0.47 Phosphoglyceride 19.8 _+0.75 21.4+ 0.50 22.0+0.56 19.5±1.00 16.9-+0.44 Values are means +_SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; ***P < 0.001.
methanohtoluene:sulphuric acid (20:10:1, v/v/v) in the presence of a pentadecanoic acid standard (Christie et al., 1970). Gas liquid chromatography of the methyl fatty acid derivatives on a packed column of 15% CP Sil 84 on Chromosorb WHP (Chrompak UK Ltd., Middleburg, The Netherlands) then enabled quantification of both the relative proportions of the major long chain fatty acids present and the absolute amount of the lipid associated with each fraction. Quantification of the fatty acids was by electronic integration. The amount of the free cholesterol was determined by charring and subsequent densitometry using a liquid scintillation counter (Shand and Noble, 1980). Identification of lipid and fatty acid fractions was by comparison with known standards. All solvents were distilled before use and where necessary, operations were performed under an atmosphere of nitrogen.
RESULTS The weights o f the whole eggs, their yolks, the lipid contents of the yolks a n d their compositions (major classes, weight per cent o f the total lipid present) over the 75 days of i n c u b a t i o n period are given in Table 1. The weight range o f the eggs c o n f o r m e d to t h a t expected for m a t u r e alligators. W h e r e a s over the first 56 days of i n c u b a t i o n the weight of the yolk showed only a slight decrease, thereafter there was a rapid decline in weight. This p a t t e r n o f yolk weight change was parallelled by a similar reduction in the a m o u n t of yolk lipid a n d by substantial changes in lipid composition. The principal c o m p o n e n t o f the yolk lipid initially a n d t h r o u g h o u t the i n c u b a t i o n period was triacylglyceride a c c o m p a n i e d by substantial levels also of phosphoglyceride. W h e r e a s the level of cholesterol ester present initially within the yolk lipid was very small, during the second half of the incubation period its level increased markedly, so t h a t by day 75 o f i n c u b a t i o n cholesterol ester accounted for over 2 0 % of the total yolk lipid. This large increase
% of total lipid) of the yolk during 64 75 75.1+_3.40 64.7+_0.58 15.6+_1.41 3.08"**±0.21 4.25+_0.46 0.938***+ 0.076 8.47+_0.32 20.8***±2.08 67.6+-0.56 59.4**±2.49 2.81+-0.17 6.72***_+0.39 5.32_+0.46 5.17"*_+0.60 15.8_+0.30 7.89***_+0.49
in the p r o p o r t i o n o f the cholesterol ester occurred mainly at the expense of a decrease in the level of phosphoglyceride a n d latterly a decrease also in the level o f triacylglyceride. The level of unesterified fatty acid in the yolk lipid h a d also increased markedly by the 75th day o f incubation. The fatty acid compositions of the cholesterol ester, triacylglyceride, phosphoglyceride a n d unesterified fatty acid fractions of the yolks at the various stages of the i n c u b a t i o n period are given in Tables 2, 3 a n d 4, respectively. The fatty acids listed in the Tables accounted for 9 7 - 9 8 % of the total fatty acids present, the remaining 2 - 3 % consisting of trace a m o u n t s only of a selection of saturated and unsaturated fatty acids of chain lengths varying between 14 a n d 20 c a r b o n atoms. By far the m a j o r fatty acid present initially within the cholesterol ester was palm±tic which accounted for more t h a n half of the total fatty acids present (Table 2). However, as i n c u b a t i o n proceeded the p r o p o r t i o n of palm±tic acid u n d e r w e n t a m a r k e d decrease particularly over the last part of the incubation period. A t the same time there was a large increase in the level of oleic acid such t h a t by the end of the i n c u b a t i o n period it was the m a j o r fatty acid present (Table 2). Levels of total p o l y u n s a t u r a t e d fatty acids also decreased during the i n c u b a t i o n period, this arising in the m a i n from a disproportionately large decrease t h a t occurred early in the incubation (Table 2). Over the complete i n c u b a t i o n period there was a tendency for the levels of linoleic and linolenic acids to increase. The c o n c e n t r a t i o n of stearic acid remained low t h r o u g h o u t incubation. The m a j o r fatty acids of the triacylglyceride fraction of the yolk (Table 3) were palm±tic, palmitoleic a n d oleic. T o g e t h e r they accounted for some 7 5 - 8 0 % o f the total fatty acids present. A notable feature of the triacylglyceride was the presence t h r o u g h o u t in-
Table 2. Fatty acid compositions (major acids, weight % of total) of the cholesterol ester fraction of the yolk during incubation 32 40 48 56 64 75 Embryo age (days) 8 Acid: Palm±tic 54.4 +_0.97 43.8 + 1.79 33.2+ 4.29 31.9+- 1.50 19.1_+1.33 28.0_+2.26 14.0"**± 0,75 9.79+_0,53 Palmitoleic 8.39 ± 1.30 9.25+0.65 6.22± 0.58 6.42±0.34 6.74_+0.51 8.72± 0 . 5 1 3.88+0.15 Stearic 5.87 +_ 1.12 6.21+_0.90 2.68+_1.02 6.15+_0.76 3.50+_0.37 5.75+_0.24 Oleic 13.3 ± 2.80 27.4±1.14 41.1+_3.66 37.3+_2.43 53.9+_0.33 39.8+_3.26 57.9"**_+1.14 5.72* +_0.48 Linoleic 3.11 +_0.49 5.16 ± 0.29 5.61+_0.48 7.04± 0.80 7.60+_0.44 3.72+_0.90 3,59*±0.74 Linolenic 0.87 ± 0.45 1.20+_0.10 4.11+_0.65 3.55+_0.79 4.64±0.88 2.69±0.53 1.45"*±0.15 Arachidonic 8.85 ± 1.69 4.68+_0.78 3.83±0.39 3.16+_0.76 1.81+_0.21 4.85± 0.70 tr tr tr 4.35 ± 1.79 tr*** Docosapentaenoic 3.62 +_0.86 2.05 -!-_0.56 1.55+_0.13 Docosahexaenoic 1.24 ± 0.23 2.22±0.54 4.08+-0.27 2.09+_0.17 2.63+_0.39 1.88±0.69 Values are means + SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; ***P < 0.001.
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Table 3. Fatty acid compositions (major acids, weight % of total) of the triacylglyceride fraction of the yolk during incubation Embryo aged (days) 8 32 40 48 56 64 75 Acid: Palmitic 28.9_ 0.22 28.3+ 0.29 27.2+ 0.43 25.5+ 0.85 24.9_ 0.49 26.0+ 1.72 25.3***+ 0.41 Palmitoleic 18.6 _+0.16 17.2+ 0.27 17.6+ 1.48 15.7_.+0.27 16.1+ 0.09 17.9+ 0.70 15.8 + 1.07 Stearic 6.29 + 0.38 9.56__+0.55 6.41+ 0.22 8.22+0.55 9.10+0.32 8.94___0.68 9.27**+ 0.67 Oleic 32.8 + 0.56 34.3+ 0.37 33.8__.0.90 34.5_+0.98 34.6+0.96 34.5+ 0.71 35.0 + 0.85 Linoleic 6.37 + 0.17 5.83_+0.07 8.82+ 0.58 8.51_+0.06 9.09+ 0.24 7.45+ 0.89 7.82* __.0.57 Linolenic 4.41 + 0.19 2.64+ 0.09 4.62+ 0.62 5.83+ 0.02 3.97+ 0.55 3.25+ 0.49 3.24 + 0.55 Arachidonic 1.04 + 0.03 0.83+ 0.02 0.66+ 0.12 0.96+ 0.07 0.74+ 0.03 0.62+ 0.12 1.09 _+0.05 Docosapentaenoic 0.68 + 0.02 0.52+0.02 0.59+ 0.07 0.56+ 0.06 0.44+0.01 0.45_+0.04 0.79 + 0.06 Docosahexaenoic 0.99 + 0.09 0.85+ 0.01 0.82+ 0.19 0.98_+0.04 0.97+ 0.09 0.94_+_+0.10 1.73"* + 0.12 Values are means + SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; ***P < 0.001. c u b a t i o n o f high levels of palmitoleic (Table 3). The fatty acid c o m p o s i t i o n of the triacylglyceride fraction r e m a i n e d fairly c o n s t a n t t h r o u g h o u t the i n c u b a t i o n period. However, a small but significant reduction in the level of palmitic acid occurred with a compensatory increase in the level o f stearic acid (Table 3). The m a j o r fatty acids present in the phosphoglyceride fraction (Table 4) were palmitic a n d oleic. The fatty acid c o m p o s i t i o n was characterized by high levels o f polyunsaturates, in particular C20 a n d C22 p o l y u n s a t u r a t e d fatty acids. Thus a l t h o u g h linoleic acid accounted for only 4 % of the total fatty acids, arachidonic a n d docosahexaenoic acids accounted for over 10 a n d 11% respectively (Table 4). As in the case o f the triacylglyceride a n d to a lesser extent the cholesterol ester, the phosphoglyceride displayed high c o n c e n t r a t i o n s o f palmitoleic (Table 4). The fatty acid c o m p o s i t i o n of the phosphoglyceride did n o t change to any appreciable extent during i n c u b a t i o n (Table 4). The unesterified fatty acid fraction present in the yolk at day 8 o f i n c u b a t i o n c o n t a i n e d high levels o f stearic, palmitoleic a n d oleic. The level of palmitic acid r e m a i n e d u n c h a n g e d during the i n c u b a t i o n period b u t those of palmitoleic a n d oleic decreased m a r k e d l y with a p r o p o r t i o n a l increase occurring in the level of p o l y u n s a t u r a t e d fatty acids, in particular the C20 a n d C22 polyunsaturates. DISCUSSION A t day 8 of i n c u b a t i o n total yolk accounted for some 4 5 % o f the whole weight of the alligator egg; o f the total yolk weight, some 2 1 % was lipid. These figures for the yolk a n d its lipid c o n t e n t are in general agreement with previous observations for unincub a t e d eggs o f the crocodilians a n d a range o f reptil-
ians (Steward a n d Castillo, 1984; Wilhoft, 1986; Manolis et al., 1987). They are, however, very different from respective values t h a t have been o b t a i n e d for avians (Romanoff, 1960; N o b l e a n d Moore, 1966; Noble, 1986a) where the p r o p o r t i o n by weight o f the yolk in the egg is very m u c h lower ( 3 0 - 3 3 % ) but the level o f the lipid in the yolk very m u c h higher (60-65%). The slow decline in the weight o f the yolk o f the alligator egg over the first 50-60 days of i n c u b a t i o n a n d the sudden weight loss thereafter, such t h a t by the 75th day only 10% of the initial yolk weight remained, also accorded with previous observations in crocodiles (Manolis et al., 1987). A l t h o u g h weight loss o f the yolk is associated with the absorption of a range o f dietary c o m p o n e n t s (Romanoff, 1960; F r e e m a n a n d Vince, 1974), it is clear from b o t h the present a n d previous results t h a t the majority o f the dry m a t t e r loss is due to the removal o f lipid. However, the p r o p o r t i o n of lipid within the yolk steadily increased as i n c u b a t i o n proceeded. Thus by the end of the i n c u b a t i o n period lipid accounted for some 31% of the total weight o f the remaining yolk material c o m p a r e d to a level o f only 21% at the beginning o f incubation. In the chick e m b r y o where investigations o f yolk lipid removal have been extensive (Noble, 1986b, 1987), the increase in overall lipid c o n c e n t r a t i o n per unit weight o f yolk as i n c u b a t i o n proceeds is due to the increasing d e v e l o p m e n t o f the lipid rich yolk sac m e m b r a n e (Noble a n d Moore, 1967a). In the chick the extensive redistribution of the yolk lipid between the contents a n d the yolk sac memb r a n e a n d its subsequent a b s o r p t i o n by the e m b r y o is not a c c o m p a n i e d by any change in the relative p r o p o r t i o n s of the two m a j o r lipid fractions, i.e. the triacylglyceride a n d phosphoglyceride within the yolk (Noble a n d Moore, 1964). This was clearly n o t the
Table 4. Fatty acid compositions (major acids, weight % of total) of the phosphoglyceride and free fatty acid fractions of the yolk at the 8th and 75th days of incubation Phospholipid Free fatty acids Embryo aged (days) 8 75 8 75 Acid: Palmitic 32.7 _+ 1.27 34.4 _+1.21 27.6 _+1.30 25.3 _+ 1.58 Palmitoleic 8.02 _+1.30 7.88 4- 0.37 17.0 _+0.51 9.84***_+0.80 Stearic 7.67 _+0.52 9.04 + 0.57 5.38 _+0.49 8.53***+_0.16 Oleic 21.1 + 0.39 17.3"*+ 0.92 35.4 _+ 1.52 25.8**+ 0.79 Linoleic 4.32 _+0.75 4.29 _+1.09 3.39 + 0.39 5.61" _+0.77 Linolenic 2.93 + 0.22 1.89 + 0.45 6.06 + 0.75 2.51"*+ 0.35 Arachidonic 11.0 _+2.04 11.5 _+0.34 2.75 _+0.46 12.9"**_+0.91 Docopentaenoic 2.22 _-+0.30 1.70 + 0.11 0.56 + 0.13 2.54***__+0.22 Docosahaenoic 10.1 + 0.90 11.5 _ 0.57 1.87 _+0.14 6.42***___0.17 Values are means + SE. Significance of difference from value at day 8: *P < 0.05; **P < 0.01; P < 0.001. CBPB 96/1--M
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case during yolk lipid absorption in the alligator where there was a substantial difference in the relative proportion of triacylglyceride and phosphoglyceride between days 8 and 75 of incubation. However, whereas there were substantial reductions in both the relative and absolute levels of triacylglyceride and phosphoglyceride in the yolk during incubation, the amount of cholesterol ester increased substantially from 95 to 195 mg/yolk. As a result, the cholesterol ester rose from being a minor lipid component at day 8 of incubation to one that accounted for more than 20% of the total yolk lipid at day 75. Although only a small increase in cholesterol ester content was observed in comparable investigations of whole yolk changes in the chick embryo during incubation (Noble and Moore, 1964), when the yolk of the chick was divided into contents and yolk sac membrane a significant increase in the level of cholesterol ester within the lipid of the yolk sack membrane was observed over the latter part of incubation (Noble and Moore, 1967a). This increase, however, was still far short of that presently observed in the whole yolk of the alligator. With the exceptions of some small changes, the fatty acid composition of the alligator yolk triacylglyceride remained fairly constant during incubation. Thus, although it has been previously suggested that yolk lipid assimilation may be associated with some preferential absorption of triacylglyceride species (Isaaks et al., 1964), the present results for the alligator conform to the vast majority of results for chick and other avian embryos in which no evidence of any preferential removal of triacylglyceride species has been obtained (Budowski et al., 1961; Noble and Moore, 1967a; Noble, 1987). Compared with the chick embryo, the triacylglyceride within the yolk of the alligator egg showed very high levels of palmitoleic and very low levels of linoleic acid. The general distribution of the other acids was quite similar. Compared with the chick embryo (Noble and Moore, 1964, 1966), the phosphoglyceride of the yolk of the alligator egg showed several distinct features. A low level of stearic and, in particular, linoleic acid was accompanied by very much higher levels of C20 and C22 polyunsaturated fatty acids throughout incubation. In the chick embryo, and in the alligator, the fatty acid composition of the yolk phosphoglyceride remained largely unchanged over the incubation period (Noble and Moore, 1964, 1966). However, separation of the chick embryo yolks into contents and membrane showed differences in the distributions of arachidonic and docosahexaenoic acid as incubation proceeded (Noble and. Moore, 1967a,b). With regard to both arachidonic and docosahexaenoic acids, there was a preferential uptake into the yolk sac membrane of yolk phosphatidyl ethanolamine species that were rich in docosahexaenoic acid (Noble and Moore, 1967b). With regard to arachidonic acid uptake, subsequent investigations (Noble and Shand, 1985) also revealed the presence within the yolk sac membrane of appreciable A-6 desaturation activity, which is responsible for the first step in the conversion of linoteic to arachidonic acid. Both these features were associated with enhancement of the C20 and C22 polyunsaturated fatty acid level within the absorbed yolk lipid in order to satisfy
an increased requirement during embryo development (Noble and Cocchi, 1989). The high levels of C20 and C22 polyunsaturated fatty acids present within the phosphoglycerides of the aligator egg yolk throughout incubation poses several questions. For instance, the assurance of a continuous and very rich source of C20 and C22 polyunsaturated fatty acids for the start of incubation would reduce the necessity for mechanisms similar to those present in the chick embryo. It is also possible that the unusual polyunsaturated fatty acid pattern with the egg yolk of the alligator is accommodated by a wholly different range of phosphoglyceride species from that of the chick embryo (Noble and Moore, 1967b). The large accumulation of cholesterol ester within the yolk of the alligator during incubation was accompanied by a sharp increase in its content of oleic acid. In the chick embryo the level of oleic acid within the cholesterol ester fraction of the whole yolk also progressively increased during incubation (Tsuji et al., 1955; Noble and Moore, 1964). This change in whole yolk lipid composition was also almost wholly associated with events taking place in the yolk sac membrane rather than the contents (Noble and Moore, 1967a; Noble et al., 1984). Extensive synthesis and accumulation of cholesterol esters by the yolk sac membrane occurred as a vital part of the transfer of the lipid from the yolk contents into the chick embryo (Noble, 1986b, 1987). It is clear that the overall features of yolk lipid assimilation by the alligator and avian display extensive similarities. However, with regard to two of the major features, namely the cholesterol ester and polyunsaturated fatty acid metabolism, the extent of change in the alligator is far more extreme. The alligator yolk lipids do possess features that are unique. Thus the presence of very high levels of palmitoleic acids in the major lipid fractions throughout incubation is quite distinctive. Compared to the avian where yolk lipid uptake occurs by non-specific engulfment by the membrane (Lambson, 1970), the sudden rise in the level of unesterified fatty acids within the yolk of the alligator just prior to hatching may implicate an increased role for lipolytic breakdown in lipid assimilation. In the avian the lipid metabolic events that take place in the yolk are an essential and integral part of yolk assimilation. In the alligator these events may assume an even greater importance. Acknowledgements--We thank Ted Joanen, Larry McNease and David Richard of the Rockefeller Wildlife Refuge, Louisiana Department of Wildlife and Fisheries, USA for their invaluable assistance over several years with the collection and transportation of eggs of Alligator mississipensis. D.C.D. is funded by the University of Manchester Research Support Fund. REFERENCES
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