Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

Endogenous production of n-3 and n-6 fatty acids in mammalian cells

Polyunsaturated fatty acids (PUFA) are important components of mammalian diets, and the beneficial effects of n-3 PUFA on human development and cardiovascular health have been well documented. Caenorhabditis elegans is one of the few animals known to be able to produce linoleic (LA, 18:2n-6) and alpha-linolenic (ALA, 18:3n-3) essential fatty acids. These essential PUFA are generated by the action of desaturases that successively direct the conversion of monounsaturated fatty acids (MUFA) to PUFA. The cDNA coding sequences of the C. elegans Delta(12) and n-3 fatty acid desaturases were each placed under the control of separate constitutive eukaryotic promoters and simultaneously introduced into HC11 mouse mammary epithelial cells by adenoviral transduction. Phospholipids from transduced cells showed a significant decrease in the ratios of both MUFA:PUFA and n-6:n-3 fatty acids relative to control cultures. The fatty acid profile of transduced cellular phospholipids revealed significant decreases in MUFA and arachidonic acid (20:4n-6), and increases in LA, ALA, and eicosapentaenoic acid (20:5n-3). The fatty acid composition of triacylglycerols derived from transduced cells was similarly, but less dramatically, affected. These results demonstrate the functionality of C. elegans fatty acid desaturase enzymes in mammalian cells. Expression of these desaturases in livestock might act to counterbalance the saturating effect that rumen microbial biohydrogenation has on the fatty acid profile of ruminant products, and allow for the development of novel, land-based dietary sources of n-3 PUFA.     

Isolation and characterization of a Δ-9 fatty acid desaturase gene from the oleaginous yeast Cryptococcus curvatus CBS 570

The oleaginous yeast Cryptococcus curvatus is of industrial interest because it can accumulate triacylglycerols up to 60% of the cell dry weight. We are aiming at genetic modification of fatty acid biosynthesis for the production of tailor-made triacylglycerols in C. curvatus. As a first step in the development of a transformation and expression system a gene encoding the Δ-9 fatty acid desaturase of C. curvatus (CBS 570) was cloned. The 1470 bp gene encodes a protein of 493 amino acids with a calculated molecular mass of 55 kDa. The gene shows strong similarity to previous cloned Δ-9 desaturase genes from rat and Saccharomyces cerevisiae, 62 and 72%, respectively. Expression of the Δ-9 desaturase gene was studied. Supplementation of the growth medium with oleic acid (C18:1(c9)) showed a strong repression (90%) on the mRNA level, while supplementation with petroselinic acid (C18:1(c6)) had no effect on the amount of mRNA.     

Endogenous production and elevated levels of long-chain n-3 fatty acids in the milk of transgenic mice

n-3 Polyunsaturated fatty acids (n-3 PUFA) are important for the normal development and functioning of all organisms. Mammals lack the n-3 fatty acid desaturase required for the synthesis of α-linolenic acid (18:3n-3), and are therefore dependent on dietary sources to obtain this essential fatty acid. Currently, the richest source of dietary long-chain n-3 PUFA, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), are triacylglycerides extracted from rapidly declining marine resources. The nematode Caenorhabditis elegans synthesizes a wide range of PUFA and possesses the only known example of an n-3 fatty acid desaturase enzyme in the animal kingdom. Transgenic mice expressing the C. elegans n-3 desaturase under the control of the lactation-induced goat β-casein mammary gland promoter were generated via pronuclear microinjection. Significant increases in n-3 PUFA, decreases in n-6 PUFA, and an overall decrease in the n-6:n-3 PUFA ratio were observed in the milk produced by transgenic mice. Neonate mice consuming milk from transgenic females accumulated increased levels of docosahexaenoic acid in their brains. This transgenic model may provide useful information to address some basic questions of neonatal nutrition, and demonstrates one of the steps that would be required to increase the n-3 PUFA content of milk and dairy products endogenously. Increasing the proportion of n-3 PUFA in milk fat would help to improve the nutritional composition of an important component of the North American diet.     

Efficient expression, purification and characterization of mouse salivary alpha-amylase secreted from methylotrophic yeast, Pichia pastoris

We constructed a secretion vector of mouse salivary alpha-amylase, pPAM, using the AOX1 promoter-terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris. Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (approximately 240 microg/ml) of mouse alpha-amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90-fold higher than that of Saccharomyces cerevisiae. Growth temperature of cells was critical for efficient production of alpha-amylase. P. pastoris transformants secreted both core-glycosylated and non-glycosylated alpha-amylase molecules with a glycosylated:non-glycosylated ratio of about 20:80. Both glycosylated and non-glycosylated alpha-amylases were purified separately to apparent homogeneity. The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non-glycosylated alpha-amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse alpha-amylase secreted by P. pastoris.     

6-phosphofructokinase from Pichia pastoris: purification,kinetic and molecular characterization of the enzyme

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate. However, in comparison with the enzymes from Saccharomyces cerevisiae and Kluyveromyces lactis, the activation ratio of 6-phosphofructokinase from Pichia pastoris by AMP is several times higher, the ATP inhibition is stronger and the apparent affinity to fructose 6-phosphate is significantly lower. Aqueous two-phase affinity partitioning with Cibacron Blue F3G-A did not reflect remarkable structural differences of the nucleotide binding sites of the Pfks from Pichia pastoris and Saccharomyces cerevisiae. The structural organisation of the active enzyme seems to be different in comparison with hetero-octameric 6-phosphofructokinases from other yeast species. The enzyme was found to be a hetero-oligomer with an molecular mass of 975 kDa (sedimentation equilibrium measurements) consisting of two distinct types of subunits in an equimolar ratio with molecular masses of 113 kDa and 98 kDa (SDS-PAGE), respectively, and a third non-covalently complexed protein component (34 kDa, SDS-PAGE). The latter seems to be necessary for the catalytic activity of the enzyme. Sequencing of the N-terminus (VTKDSIXRDLEXENXGXXFF) and of peptide fragments by applying MALDI-TOF PSD, m/z 1517.3 (DAMNVVNH) and m/z 2177.2 [AQNCNVC(L/I)SVHEAHTM] gave no relevant information about the identity of this protein.     

Isolation of Delta12 and omega3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and alpha-linolenic acids in Saccharomyces cerevisiae

Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Delta12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited omega3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced alpha-linolenic acid. Co-expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and alpha-linolenic acids. The yield of alpha-linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; alpha-linolenic acid reached 8.7% when exogenous linoleic acid was available.     

Effect of season on fatty acid composition and n-3/n-6 ratios of zander and carp muscle lipids in Altinapa Dam Lake

In this study, seasonal variations on total fatty acid composition of carp, Cyprinus carpio, and zander, Sander lucioperca, muscle lipids in Altinapa Dam Lake were determined. Fish samples in 2 seasons (summer and winter) were obtained from Altinapa Dam Lake in Konya, Turkey. Polyunsaturated fatty acids were found to be higher during the cold season than in the hot both in zander and carp. Whereas the Docosahexaenoic acid was high in zander both in summer and winter, in carp it was high only during wintertime. Zander contained more n-3 fatty acids during the cold season compared to carp. Especially, the n-3:n-6 ratios in zander were 3.89 and 3.84 in summer and winter, respectively. In conclusion, seasonal variations affected fatty acid composition of zander and carp in Altinapa Dam Lake.     

Biomarker of long-chain n-3 fatty acid intake and breast cancer: Accumulative evidence from an updated meta-analysis of epidemiological studies

Abstract

Objective: We aimed to summarize the up-to-date epidemiology evidence on biomarkers of long-chain (LC) n-3 fatty acid (FA) intake in relation to breast cancer (BC).

Methods: Epidemiology studies determining FA levels in biospecimen (circulating blood or adipose tissue (AT)) were identified from PubMed, EMBASE, and Cochrane Library databases until March 2018. Multivariate-adjusted risk ratios (RRs) with 95% confidence intervals (CIs) were pooled using a random-effect model. Difference in biospecimen proportions of LC n-3 FA between BC cases and non-cases were analyzed as a standardized mean difference (SMD).

Results: Thirteen cohort and eleven case-control studies were eligible for the present meta-analysis. The estimated SMD was -0.14 (95% CI: -0.27, -0.11) for LC n-3 FA and -0.27 (95% CI: -0.42, -0.11) for LC n-3/n-6 FA ratio. When comparing the top tertiles with the bottom baseline levels, circulating LC n-3 FA was significantly associated with a lower risk of BC (RR: 0.84, 95% CI: 0.74, 0.96), but not AT (RR: 1.02, 95% CI: 0.70, 1.48). Significant inverse dose-response associations were observed for each 1% increment of circulating 20:5n-3 and 22:6n-3.

Conclusion: This meta-analysis highlights that circulating LC n-3 FA as a biomarker of intake may be an independent predictive factor for BC, especially 20:5n-3 and 22:6n-3.     

Effects of extruded linseed supplementation on n-3 fatty acids and conjugated linoleic acid in milk and cheese from ewes

The objective of this study was to assess the effects of dietary supplementation of extruded linseed on animal performance and fatty acid (FA) profile of ewe milk for the production of n-3 FA- and conjugated linoleic acid-enriched cheeses. A Manchega ewe flock (300 animals) receiving a 60:40 forage:concentrate diet was divided into 3 groups supplemented with 0, 6, and 12 g of extruded linseed/100 g of dry matter for the control, low, and high extruded linseed diets, respectively. Bulk and individual milk samples from 5 dairy ewes per group were monitored at 7, 14, 28, 45, and 60 d following supplementation. Manchego cheeses were made with bulk milk from the 3 treatment groups. Milk yield increased in dairy ewes receiving extruded linseed. Milk protein, fat, and total solids contents were not affected by linseed supplementation. Milk contents of α-linolenic acid increased from 0.36 with the control diet to 1.91% total FA with the high extruded linseed diet. Similarly, cis-9 trans-11 C18:2 rose from 0.73 to 2.33% and its precursor in the mammary gland, trans-11 C18:1, increased from 1.55 to 5.76% of total FA. This pattern occurred with no significant modification of the levels of trans-10 C18:1 and trans-10 cis-12 C18:2 FA. Furthermore, the high extruded linseed diet reduced C12:0 (−30%), C14:0 (−15%) and C16:0 (−28%), thus significantly diminishing the atherogenicity index of milk. The response to linseed supplementation was persistently maintained during the entire study. Acceptability attributes of n-3-enriched versus control cheeses ripened for 3 mo were not affected. Therefore, extruded linseed supplementation seems a plausible strategy to improve animal performance and nutritional quality of dairy lipids in milk and cheese from ewes.     

The effects of consuming docosahexaenoic acid (DHA)-enriched eggs on serum lipids and fatty acid compositions in statin-treated hypercholesterolemic male patients

The purpose of this study was to investigate the effects of consuming a novel docosahexaenoic acid (DHA)-enriched shell egg on the serum lipid levels and ω − 3 polyunsaturated fatty acids (n − 3 PUFA) of serum phospholipid in statin-treated hypercholesterolemic patients. Fifteen subjects were randomly divided into two treatment groups and consumed either two control or two novel DHA-enriched eggs during two organized breakfast periods of 21 consecutive days each using a double-blinded, cross-over design. The novel enriched eggs from feeding the specialty ration provided 217 mg of DHA and 629 mg of total n − 3 PUFA per day. Total serum cholesterol levels were unchanged with either egg consumption and no significant alterations in lipid levels were found due to a treatment effect. The novel egg group exhibited a significant rise in eicosapentaenoic acid (EPA) plus DHA levels in serum phospholipid (by 23%) which can be related to a reduced risk for fatal ischemic heart disease. Consumption of this novel egg offers an alternative food option for more than doubling current sub-optimal DHA intakes in North America.     

Effects of dietary fats differing in n-6:n-3 ratio fed to high-yielding dairy cows on fatty acid composition of ovarian compartments, follicular status, and oocyte quality

The objectives were to determine the incorporation of dietary encapsulated fats differing in n-6:n-3 ratio into milk fat, plasma, and various ovarian compartments and to examine the effects on ovarian follicular status, preovulatory follicle characteristics, and oocyte quality. Twenty-four multiparous Israeli Holstein cows, averaging 114 d in milk, were assigned to 1 of 3 treatment groups: 1) control (n = 7), in which cows were fed a lactating cow diet; 2) E-FLAX (n = 8), in which cows were fed a lactating cow diet that consisted of 1 kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% flaxseed oil, providing 242.2 g of C18:3n-3 (low n-6:n-3 ratio); or 3) E-SUN (n = 9), in which cows were fed a lactating cow diet that consisted of 1 kg/d of encapsulated fat (3.8% of dry matter) containing 40.8% sunflower oil, providing 260.0 g of C18:2n-6 (high n-6:n-3 ratio). Ovaries were monitored by ultrasonography for follicular status, and after synchronization, follicles >7 mm were aspirated and evaluated. Ovum pickup was performed (19 sessions for the control and E-FLAX groups and 11 for the E-SUN group), and in vitro maturation and oocyte fertilization were conducted. The E-FLAX treatment increased the proportions of C18:3n-3 (5.8 fold), C20:5n-3, and C22:5n-3 (approximately 4-fold) in milk fat as compared with the other 2 treatments. The proportion of C18:3n-3 fatty acid in plasma increased dramatically with the E-FLAX treatment, from 1.43 and 1.49% in the control and E-SUN groups, respectively, to 7.98% in the E-FLAX group. Consequently, the n-6:n-3 ratio in plasma was reduced from approximately 42 in the control and E-SUN groups to 6.74 in the E-FLAX group. Proportions of C18:3n-3 in follicular fluid and granulosa cells were approximately 5-fold higher in the E-FLAX group than in the other 2 groups. The percentage of C18:2n-6 in cumulus-oocyte complexes of cows in the E-SUN group was 54% higher than that in the E-FLAX group and was 2.4-fold higher than that in the control group; the proportion of C18:3n-3 in the E-FLAX group was 4.73% and was not detected in the other groups. The average numbers of 2- to 5-mm follicles on d 5 and 9 of the cycle were higher in the E-FLAX group than in the E-SUN group, whereas the average numbers of follicles ≥10 mm on d 5, 9, and 13 were higher in the E-SUN group than in the other 2 groups. The estrous cycles of the cows were synchronized and PGF_2α was injected on d 16 to 17 of the cycle. The interval from PGF_2α injection to behavioral estrus was longer in the E-FLAX group than in the E-SUN group, and the beginning of the luteal phase of the subsequent cycle was delayed. Concentrations of estradiol in follicular fluid of the preovulatory follicles were higher in the E-SUN group than in the E-FLAX group. The number of follicles aspirated by ovum pickup was higher in the E-FLAX group than in the control group, and the cleavage rate in the E-FLAX group was higher than in the control group, but not the E-SUN group. In conclusion, dietary n-3 fatty acids influenced the follicular status and increased the cleavage rate of oocytes as compared with those of control cows. These findings could be related to modifications of the fatty acid composition in plasma and ovarian compartments in response to dietary supplementation.     

Fatty acid composition of milk from multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation

Multiparous cows (n = 59) were blocked by expected calving date and previous milk yield and assigned randomly to treatments to determine effects of bovine somatotropin (bST; Posilac, Monsanto Animal Agricultural Group, St. Louis, MO) and source of dietary fat on milk fatty acid composition during the first 140 d in milk. Diets were provided from calving and included whole, high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; Alifet USA, Cincinnati, OH; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids (15.7% 18:3, 1.3% 20:5, and 1.3% 22:6). Treatments were derived from a 2 × 2 combination of supplemental fat source (SS, AF) and with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST administered every 10 d from 12 to 70 d in milk and at 14-d intervals thereafter. Milk fatty acid composition was determined in samples collected from 32 cows (8 complete blocks) during wk 2, 8, and 20 of lactation. Data were analyzed as repeated measures using mixed model procedures to determine the effects of diet, bST, week of lactation, and their interactions. Proportions of 18:3 (4.02 vs. 3.59 ± 0.16%), 20:5 (0.52 vs. 0.41 ± 0.02%), and 22:6 (0.11 vs. 0.02 ± 0.02%) were greater and the n-6/n-3 fatty acid ratio (7.40 vs. 8.80 ± 0.30) was reduced in milk from cows fed AF compared with SS. Proportions of de novo-synthesized fatty acids increased and preformed fatty acids decreased as lactation progressed, but bST administration delayed this shift in origin of milk fatty acids. Transfer efficiency of 18:3, 20:5, and 22:6 from AF to milk fat averaged 36.2, 4.9, and 5.2%, respectively. These efficiencies increased as lactation progressed, but were delayed by bST. Apparent mammary Δ⁹-desaturase activity and milk conjugated linoleic acid (cis-9, trans-11 conjugated linoleic acid) content increased through the first 8 wk of lactation. Based on the product-to-substrate ratio of 14:1/14:0 fatty acids in milk, there was an interaction of diet and bST because bST decreased apparent Δ⁹-desaturase activity in SSY cows but increased it in AFY cows (0.10, 0.09, 0.08, and 0.09 ± 0.01 for SSN, SSY, AFN, and AFY, respectively). Feeding Alifet-Repro increased n-3 fatty acids in milk and bST prolonged the partitioning of dietary fatty acids into milk fat.     

Effects of increased supplementation of n-3 fatty acids to transition dairy cows on performance and fatty acid profile in plasma, adipose tissue, and milk fat

The objective of this study was to determine the effects of feeding an increased amount of extruded flaxseed with high proportions of n-3 fatty acids (FA) to transition dairy cows on performance, energy balance, and FA composition in plasma, adipose tissue, and milk fat. Multiparous Israeli-Holstein dry cows (n = 44) at 256 d of pregnancy were assigned to 2 treatments: (1) control cows were fed prepartum a dry-cow diet and postpartum a lactating-cow diet that consisted of 5.8% ether extracts; and (2) extruded flaxseed (EF) cows were supplemented prepartum with 1 kg of extruded flaxseed (7.9% dry matter)/cow per d, and postpartum were fed a diet containing 9.2% of the same supplement. The EF supplement was fed until 100 d in milk. On average, each pre- and postpartum EF cow consumed 160.9 and 376.2 g of C18:3n-3/d, respectively. Postpartum dry matter intake was 3.8% higher in the EF cows. Milk production was 6.4% higher and fat content was 0.4% U lower in the EF group than in the controls, with no differences in fat and protein yields. Energy balance in the EF cows was more positive than in the controls; however, no differences were observed in concentrations of nonesterified fatty acids and glucose in plasma. Compared with controls, EF cows had greater proportions of C18:3n-3 in plasma and adipose tissue. The proportion of n-3 FA in milk fat was 3.7-fold higher in the EF cows, and the n-6:n-3 ratio was decreased from 8.3 in controls to 2.3 in the EF cows. Within-group tests revealed that the C18:3n-3 content in milk fat in the EF cows was negatively correlated with milk fat percentage (r = –0.91) and yield (r = –0.89). However, no decrease in de novo synthesis of less than 16-carbon FA was found in the EF group, whereas C16:0 yields were markedly decreased. It appears that the enrichment of C18:3n-3 in milk fat was limited to approximately 2%, and the potential for increasing this n-3 FA in milk is higher for cows with lower milk fat contents. In conclusion, feeding increased amounts of C18:3n-3 during the transition period enhanced dry matter intake postpartum, increased milk production, decreased milk fat content, and improved energy balance. Increased amounts of EF considerably influenced the FA profile of plasma, adipose tissue, and milk fat. However, the extent of C18:3n-3 enrichment in milk fat was limited and was negatively correlated with milk fat content and yield.     

Effects of dietary fish oil replacement with flaxseed oil on tissue fatty acid composition and expression of desaturase and elongase genes

BACKGROUND: This study was conducted to evaluate the effects of total or partial substitution of dietary fish oil (FO) by flaxseed oil (FlaxO) in Jade Tiger hybrid abalone on fatty acid composition of muscle, gonad and digestive gland, and the expression of desaturase and elongase genes. Abalone were fed five different experimental diets in which FO (control diet) was serially replaced by 25%, 50%, 75% and 100% FlaxO respectively. RESULTS: Muscle, gonad and digestive gland of abalone fed the control diet and the diets containing 25%, 50% and 75% FlaxO showed significantly higher (P < 0.05) levels of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) compared to those fed the 100% FlaxO. The results also showed that Δ‐6 desaturase and elongase gene expression in muscle was increased in a graded manner by increasing dietary FlaxO. The expression of both genes was higher in abalone fed the FlaxO‐substituted diets compared to the abalone fed FO. CONCLUSION: The replacement of FO with FlaxO in commercial abalone diets at levels of 25–75% can improve the composition of health‐benefiting n‐3 polyunsturated fatty acids in tissues of cultured hybrid abalone, and achieve similar outcomes to FO supplementation. Copyright © 2011 Society of Chemical Industry     

Change in α‐tocopherol contents,lipid oxidation and fatty acid profile in eggs enriched with linolenic acid or very long‐chain ω3 polyunsaturated fatty acids after different processing methods

The influences of dietary supplementation with α‐tocopheryl acetate (α‐TA) and of processing (by hard‐boiling and scrambling) of eggs enriched with ω3 fatty acids, either very long‐chain ω3 polyunsaturated fatty acids (VLC ω3 PUFAs) or linolenic acid (LNA), on fatty acid composition, α‐tocopherol content and lipid oxidation (thiobarbituric acid (TBA) values) were studied. Four dietary treatments were formulated from a basal diet containing 40 g kg^?1 linseed oil (LO) or fish oil (FO) combined with either 0 or 100 mg α‐TA kg^?1 of feed. Eggs from LO treatments were enriched with LNA and those from FO treatments were rich in VLC ω3 PUFAs. Neither processing nor dietary supplementation with α‐TA modified greatly the fatty acid profile of eggs. Dietary supplementation with α‐TA increased the α‐tocopherol content of eggs (187.2 versus 407.9 µg g^?1 dry matter). Eggs from FO treatments showed lower α‐tocopherol content than those from LO treatments (273.5 versus 321.6 µg g^?1 dry matter), and processing of eggs enriched with VLC ω3 PUFA reduced the α‐tocopherol content by a significant 16%. Moreover, processing of eggs increased lipid oxidation two‐ to nine‐fold. Oxidation levels of hard‐boiled eggs were 30.4% higher than those of scrambled eggs. TBA values in hard‐boiled and scrambled eggs were significantly reduced when 100 mg α‐TA kg^?1 of feed supplemented the diet only in those eggs enriched with VLC ω3 PUFA (from FO treatments). Copyright © 2003 Society of Chemical Industry