Production of new unsaturated lipids during wood decay by ligninolytic basidiomycetes

Lipids were analyzed by gas chromatography-mass spectrometry for a 7-week in vitro decay of eucalypt wood by four ligninolytic basidiomycetes. The sound wood contained up to 75 mg of lipophilic compounds per 100 g of wood. Hydrolysis of sterol esters, which represented 38% of total wood lipids, occurred during the fungal decay. The initial increase of linoleic and other free unsaturated fatty acids paralleled the decrease of sterol esters. Moreover, new lipid compounds were found at advanced stages of wood decay that were identified from their mass spectra as unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid. These dicarboxylic acids were especially abundant in the wood treated with Ceriporiopsis subvermispora (up to 24 mg per 100 g of wood) but also were produced by Phlebia radiata, Pleurotus pulmonarius, and Bjerkandera adusta. We hypothesize that three main alkylitaconic acids (tetradecylitaconic, cis-7-hexadecenylitaconic, and hexadecylitaconic acids) are synthesized by fungi in condensation reactions involving palmitic, oleic, and stearic acids. We suggest that both wood unsaturated fatty acids (present in free form or released from esters during natural decay) and unsaturated metabolites synthesized by fungi could serve as a source for peroxidizable lipids in lignin degradation by white rot basidiomycetes.     

Oxidative degradation of model lipids representative for main paper pulp lipophilic extractives by the laccase–mediator system

Different model lipids-alkanes, fatty alcohols, fatty acids, resin acids, free sterols, sterol esters, and triglycerides-were treated with Pycnoporus cinnabarinus laccase in the presence of 1-hydroxybenzotriazole as mediator, and the products were analyzed by gas chromatography. The laccase alone decreased the concentration of some unsaturated lipids. However, the most extensive lipid modification was obtained with the laccase-mediator system. Unsaturated lipids were largely oxidized and the dominant products detected were epoxy and hydroxy fatty acids from fatty acids and free and esterified 7-ketosterols and steroid ketones from sterols and sterol esters. The former compounds suggested unsaturated lipid attack via the corresponding hydroperoxides. The enzymatic reaction on sterol esters largely depended on the nature of the fatty acyl moiety, i.e., oxidation of saturated fatty acid esters started at the sterol moiety, whereas the initial attack of unsaturated fatty acid esters was produced on the fatty acid double bonds. In contrast, saturated lipids were not modified, although some of them decreased when the laccase-mediator reactions were carried out in the presence of unsaturated lipids suggesting participation of lipid peroxidation radicals. These results are discussed in the context of enzymatic control of pitch to explain the removal of lipid mixtures during laccase-mediator treatment of different pulp types.     

Fatty acid and sterol specificity in the biosynthesis of steryl esters by enzyme preparations from spinach leaves

Acetone powders prepared from the 20,000g participate fraction of spinach (Spinacia oleracea L.) leaves catalyzed the formation of steryl esters from free sterol and 1,2-diacylglycerol as the acyl donor. There was no sterol specificity when cholesterol, sitosterol, and campesterol were compared. When rates of sterol ester biosynthesis were compared using different 1,2-diacylglycerols it was found that the shorter chain fatty acids and the more unsaturated fatty acids were preferred. When the substrate concentration of diacylglycerol was varied, the maximal velocities obtained with the different substrates were dipalmitoleoyl- >dilinolenoyl- >dioleoyl- >dilinoleoyl-glycerol. It was demonstrated by silver nitrate thin-layer chromatography that the fatty acids of the supplied diacylglycerols were transferred to the sterol. When diacylglycerol mixtures were supplied, it was found that unsaturated diacylglycerols greatly stimulated conversion of saturated diacylglycerols to saturated steryl esters. For an equimolar mixture of dipalmitoyl-, dioleoyl-, dilinoleoyl-, and dilinolenoyl-glycerol, about equal amounts of the four steryl ester species were synthesized.     

Integrated enzymatic production of specific structured lipid and phytosterol ester compositions

Mixtures of specific structured lipids and phytosterol esters, valuable food components, were synthesized by an enzymatic one-pot process in organic-solvent-free medium starting from a mixture of phytosterol, caprylic acid and sunflower oil. Nine biocatalysts, seven commercially available lipases and two air-dried solid state (SSF) fermentation preparations of Aspergillus oryzae NRRL 6270 (AoSSF) and Aspergillus sojae NRRL 6271 (AsSSF), were screened for lipase activity in the transesterification reactions of sunflower oil with caprylic acid and for sterol esterase activity in the direct esterification of phytosterols with free fatty acids. The best process variant using a sequence of sterol esterase (AoSSF)-catalyzed esterification reaction of the free fatty acids and phytosterols, followed by water removal in vacuum and lipase-catalyzed transesterification with immobilized lipase from Rhizomucor miehei (Lipozyme) resulted in 92.1% conversion to phytosterol esters and 44.1% conversion to triacylglycerols containing two caprylic esters.     

Skin surface lipids of the mole Scalopus aquaticus

Skin surface lipids of the mole Scalopus aquaticus were found to consist principally of squalene (70%), wax esters (15%), and sterol esters (5%), together with small amounts of triglycerides, free fatty acids, free fatty alcohols, and free sterols. Analysis of the fatty acids occurring free and as wax esters and sterol esters showed these to consist of approximately equal amounts of saturated and monounsaturated compounds. The saturated fatty acids consisted predominantly of odd-carbon anteiso and even-carbon straight-chain compounds, with minor amounts of even-carbon iso-branched chains. The unsaturated fatty acids had double bond positions that would have been produced by delta 9-desaturation of C14, C16 and C18 straight chain saturated precursors. Both the free and the esterified fatty alcohols had chain structures corresponding with those of the fatty acids but of somewhat greater average chain length. Discovery of a major proportion of squalene in the sebum of this animal extends the number of non-human species that have this characteristic to four, all of which inhabit a damp environment, suggesting that squalene conveys some biological advantage under these conditions.     

Lipid composition of the fat body and haemolymph and its relation to lipid release in Oncopeltus fasciatus

Lipid composition of the fat body and haemolymph of male milkweed bug, Oncopeltus fasciatus, was determined. Triglycerides were the predominant lipids of the fat body while diglycerides accounted for the major lipid in the haemolymph. Sterols, sterol esters, and non-esterified fatty acids were present in both fat body and haemolymph besides triglycerides and diglycerides. Only traces of monoglycerides were detected.Gas chromatographic analysis of the fatty acids revealed a difference in the fatty acid composition between fat body and haemolymph glycerides and sterol esters. Oleate and linoleate were the predominant unsaturated fatty acids in both fat body and haemolymph lipids and in the milkweed seeds as well.When fat body was labelled in vivo and in vitro with ¹⁴C-palmitate, the fatty acid was incorporated largely into the triglycerides. When the prelabelled fat body was incubated with a medium containing haemolymph the fat body released lipids mainly as diglycerides. Some radioactivity was observed in the triglycerides and non-esterified fatty acids also.Electrophoretic analysis of the incubation medium containing the haemolymph revealed that the released lipids were bound to three haemolymph lipoprotein bands. Lipid mobilization, release, and transport in Oncopeltus are discussed in relation to studies on other insects.     

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial distribution of membrane and storage lipids was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and fluctuations in the amount and composition of phospholipids, free sterols, steryl esters, diand triacylglycerols, and free fatty acids were investigated across the sapwood-heartwood boundary. The individual compounds were identified and quantified by thin layer chromatography, enzymatic and colorimetric assays, and by capillary gas chromatography. Phospholipids show a significant decrease towards the boundary area, and in the heartwood only trace amounts can be detected. The same pattern is observed for free sterols in the sapwood; in the heartwood, however, they reach maximum values with increasing depth of the trunk. Steryl esters exhibit a complementary behaviour by accumulating at the periphery of the heartwood. No concentration changes are found in the total amounts of diacylglycerols and free fatty acids. In contrast, the triacylglycerol concentration declines steadily across the trunk. With regard to qualitative composition, free fatty acids and the fatty acid moieties of the esterified constituents vary in their chain length from 14 to 24 carbon atoms and have up to three double bonds. A radial gradient in the ratio saturated/unsaturated fatty acids can be observed: except for the phospholipid fraction the relative amounts of unsaturated fatty acids increase in centripetal direction. Seven phospholipids were identified: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and phosphatidic acid, which constitutes the major proportion. In the sterol group, sitosterol is the most abundant component. The composition of the esterified sterols remains constant across the trunk cross-section, whereas the relative frequencies of individual free sterols change markedly.     

Fungal Degradation of Lipophilic Extractives in Eucalyptus globulus Wood

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl₂-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way, Phlebia radiata, Funalia trogii, Bjerkandera adusta, and Poria subvermispora strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives. Ophiostoma piliferum, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.     

A system for manipulating the membrane Fatty Acid composition of soybean cell cultures by adding tween-Fatty Acid esters to their growth medium : basic parameters and effects on cell growth

The development of a system for modifying the membrane fatty acid composition of cultured soybean cells (Glycine max [L.] Merr.) is described. Tween-fatty acid esters carrying specific fatty acids were synthesized and added to the medium of suspension cultures. Cells transferred large quantities of exogenous fatty acids from Tweens to all acylated membrane lipids; up to 50% of membrane fatty acids were exogenously derived. C15 to C20 saturated fatty acids and C16, C18, and C20 unsaturated fatty acids with either cis or trans double bonds were incorporated into lipids. Cells elongated saturated fatty acids of C16 or less, and unsaturated fatty acids with cis double bonds were further desaturated. No other types of modifications were observed. Growth ceased in cells treated with excessive concentrations of Tween-fatty acid esters, but frequently not for several days. Cessation of cell growth was correlated with changes in membrane fatty acid composition resulting from incorporation of large amounts of exogenous fatty acids into membrane lipids, although cells tolerated large variations in fatty acid composition. Maximum tolerable Tween concentrations varied widely according to the fatty acid supplied. Potential uses of this system and implications of the observed modifications on the pathway of incorporation are discussed.     

Durability improvement of wood by treatment with Methyl Alkenoate Succinic Anhydrides (M-ASA) of vegetable origin

Methyl Alkenoate Succinic Anhydride (M-ASA) is the product of the reaction between methyl esters of fatty acids and maleic anhydride. Crude M-ASA was synthesized from rapeseed oil methyl esters. The main compounds in this adduct are methyl oleate succinic anhydride (30%), methyl linoleate succinic anhydride (24%), unreacted methyl esters (41%) and unreacted maleic anhydride (4%). The treatment of wood at high temperature with crude M-ASA conferred protection against fungal decay and insects. Biological tests were carried out on Scots pine (Pinus sylvestris) sapwood and beech (Fagus sylvatica) according to European standards. M-ASA treatment was efficient against mould fungi (BS 3900), blue staining (EN 152), white and brown rot fungi (EN 113), longhorn beetle larvae (EN 46 and 47) and termites (EN 117). This treatment delayed the degradation of wood by soft rot (ENV 807) but it did not prevent it. Therefore, M-ASA combines all the necessary conditions to fulfil the requirements of the biological use classes 2 and 3, but not for class 4.     

Agonistic interactions, cuticular and hemolymphatic lipid variations during the foraging period in spider females Brachypelma albopilosa (Theraphosidae)

Agonistic behaviour and lipid state were examined in tarantula Brachypelma albopilosa females during the foraging period. Modulation of the agonistic behaviour of females was not connected to their body size. Results show that the agonistic pattern of females differed significantly from the predation pattern at the behavioural and lipid levels. Aggressive-foraging females had low predation behaviour. Quantitative lipid changes were observed in relation to agonistic behaviour and predation. The total lipid index was studied by colorimetric methods, and lipid compounds were analyzed by gas chromatography-mass spectrometry in cuticle and hemolymph of females. The lipid components were free fatty acids, methyl esters, cholesterol, and long-chain aliphatic hydrocarbons. Methyl esters were much more abundant in cuticular lipids; unsaturated free fatty acids (linoleic and oleic acids) and methyl esters (methyl linoleate and methyl stearate) predominated in the hemolymph. Spider aggression was positively correlated with lipid concentration (cholesterol, fatty acids, methyl esters and hydrocarbons) in the hemolymph and the levels of cuticular fatty acids. Lipid levels are hypothesized to have evolved as a regulatory factor of predation and agonistic behaviours in tarantula females.     

Lipid Composition of the Arthrospores, Yeastlike Cells, and Mycelium of the Fungus Mucor hiemalis

The fungus Mucor hiemalisF-1156, which is believed to be monomorphic, was found to be able to grow dimorphically in a liquid medium that is free of chemical agents influencing morphogenesis. The growing mycelium produced arthrospores in large amounts. The lipids of the mycelium, yeastlike budding cells, and arthrospores differed in the contents of saturated and unsaturated fatty acids and in the proportion of polar and neutral lipids. The arthrospores contained more monoenoic fatty acids in the total lipids, more triacylglycerides and sterol esters in the neutral lipids, and more phosphatidylcholine and phosphatidylethanolamine in the polar lipids than the yeastlike cells. These differences in the lipid composition of different types of fungal cells should be taken into account in the studies of the lipogenesis of M. hiemalis.     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell I. Separation and partial characterization of altered lipids

Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl₄-treated rats, the presence of a component (the “D” spot) with an R_f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes.Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of ¹⁴C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of ¹⁴C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot.The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column.On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl₄ (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl₄ free radicals to double bonds has occurred.     

Aliphatic Chains of Esterified Lipids in Isolated Eyespots of Euglena gracilis var. bacillaris

Isolated eyespot granules of Euglena gracilis Klebs var. bacillaris Pringsheim contained approximately 6% lipids (based on protein). Separation of the lipid extracts by thin layer chromatography revealed four major fractions: wax esters, triacylglycerols, free fatty acids, and phospholipids. Methanolysis of each fraction yielded between 27 and 29 different fatty acids ranging from 12:0 to 22:6. Acetates of the fatty alcohols of the wax fraction consisted of 11:0 to 18:0 carbon chains, with 14:0 being the major component; unsaturated alcohols were not detected.     

Lipids of Rhizopus arrhizus Fischer

The lipids of Rhizopus arrhizus Fischer mycelia and sporangiospores were extracted, isolated, and separated by thin-layer, liquid, and gas chromatography. Structural confirmations of the compounds were made by a gas chromatographmass spectrometer combination. The n-heptane fraction contained squalene (1%) as a major hydrocarbon constituent. Other major lipid classes detected were free fatty acids, naturally occurring methyl esters of fatty acids, triglycerides, sterols, and polar lipids. The polar lipids (44.4%) were found in the highest concentrations, and the triglycerides (22.1%), sterols (16.7%), and free fatty acids (11.7%) were present in lesser concentrations. This is the first report of naturally occuring methyl esters of long-chain fatty acids being present in fungal mycelium. There appears to be a preference for incorporation of unsaturated acids into the complex lipids, with the exception of the triglycerides. The major saturated fatty acids in the mycelium were palmitic (C(16)) and arachidic (C(20)), whereas the major unsaturated acids were oleic (C(18:1)) and linoleic (C(18:2)), respectively.     

Wood-inhabiting ligninolytic basidiomycetes in soils: Ecology and constraints for applicability in bioremediation

Ligninolytic basidiomycetes associated with wood decay are a physiologically distinct group of saprotrophic fungi capable of decomposition of all major components of wood. Although wood is their natural substrate, several species can survive in soil if suitable substrates are available, the soil-colonizing ability of species in the genera Phanerochaete, Pleurotus and Trametes being comparable to that of the soil-inhabiting basidiomycetes utilizing plant litter. Wood-inhabiting ligninolytic basidiomycetes (WLB) in soil interact with soil microflora, and some species are very efficient competitors affecting both the soil microbial biomass and the composition of the indigenous microbial community. The extracellular enzymes utilized by saprotrophic basidiomycetes for nutrient acquisition participate in the interspecific interactions with other soil biota but are also involved in the transformation of soil organic matter – lignocellulose and humic compounds. Bioremediation research has significantly enriched our knowledge of the ecology of basidiomycetes, but it has also identified several limitations for practical applicability of WLB at a field scale, and has led to the conclusion that natural attenuation or bioaugmentation are todays methods of choice for on site treatment.     

Sterols and fatty acids from three species of mangrove

The hydrolysis of steryl esters on thin-layer chromatographic plates by porcine pancreatic lipase is described. The sterols and fatty acids produced were separated on the same plate, recovered, and analysed by gas-liquid chromatography for their compositions. Synthetic cholesteryl esters containing various saturated and unsaturated fatty acids and synthetic steryl oleates with various sterols were lipolysed along with steryl esters of Acanthus ilicifolius, Bruguiera gymnorhiza and Rhizophora mucronata mangrove leaves. The major sterol was sitosterol which was accompanied by cholesterol, campesterol, stigmasterol and 28-isofucosterol. In addition, stigmast-7-en-3β-ol was present in R. mucronata leaves. The component fatty acids found in all three species were 16:0, 18:0, 18:1, 18:2 and 18:3. The relative proportions of the sterols and fatty acids were significantly different from the chemotaxonomic standpoint. The results obtained by carrying out plate lipolysis for 45 min at 40° compared well with those produced by conventional chemical hydrolysis.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Changes of lipid composition in non-cultured and cultured porcine embryos

The objective of the present study was to evaluate the lipid composition of cultured and non-cultured pig embryos during cleavage using histochemical methods. The authors studied pig zygotes as well as 2-to 4-cell embryos, morulae, and blastocysts that were either non-cultured or cultured in NCSU-23 medium. To detect different types of lipids, the authors used the Churukian method with Oil red O, the Sudan black B method, the Cain method with Nile blue sulfate, and the modified osmium tetroxide-ethanol treatment. In the zygotic lipid droplets, diverse classes of unsaturated and saturated lipids were found, with particularly high levels of unsaturated hydrophobic lipids, mainly triglycerides and other esters, free fatty acids, and phospholipids. In the zygotic cytoplasm, the authors observed high levels of fatty acids and phospholipids. The total lipid content remained constant up to the morula stage, decreasing later at the blastocyst stage, but the overall amount of unsaturated lipids declined earlier, at the 2-to 4-cell stage. The amount of free fatty acids and phospholipids decreased during cleavage in both non-cultured and cultured porcine embryos. The main differences between the non-cultured and cultured embryos were the more pronounced reduction in the amount of unsaturated hydrophobic lipids in droplets and the cytoplasmic free fatty acids observed in the cultured morula and the lower content of phospholipids in the cytoplasm of the cultured 2-to 4-cell embryos relative to the non-cultured embryos. The decrease in the unsaturated lipid, free fatty acid, and phospholipid content during in vivo development and the differences in the amount of these types of lipids between developmentally matched cultured and non-cultured pig embryos correlate well with modifications of lipid and carbohydrate metabolism.