Prostaglandins & other Lipid Mediators 84 (2007) 66–78

n − 6 and n − 3 fatty acids ratio and vitamin E in porcinematernal diet influence the antioxidant status and immune

cell eicosanoid response in the progeny

Charlotte Lauridsen a,∗, Jan Stagsted b, Søren Krogh Jensen a

a Department of Animal Health, Welfare and Nutrition, Aarhus University, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmarkb Department of Product Quality, Aarhus University, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark

Received 25 January 2007; received in revised form 10 April 2007; accepted 18 April 2007Available online 25 April 2007

Abstract

Five groups of lactating sows were fed diets containing 8% of either added rapeseed oil, fish oil or sunflower oil and 60 mg vitaminE/kg feed, or the diets with sunflower oil and fish oil, respectively, supplemented with 500 mg vitamin E/kg. Supplementation ofvitamin E to the sows increased the concentration of �-tocopherol of the muscle, and addition of sunflower oil decreased theactivity of glutathione peroxidase in liver cytosol compared to fish oil and rapeseed oil. The composition of fatty acids of alveolarmacrophages (AM) of piglets was influenced by the dietary fat sources provided the sows, i.e., the ratio of n − 6:n − 3 fatty acidswas highest in AM of piglets suckling sows of the sunflower oil treatments, and lowest in AM of piglets suckling sows fed fish oilwith the rapeseed oil treatment in between. The ex vivo synthesis of prostaglandin E2 and thromboxane B2 in AM of piglets sucklingsows fed sunflower oil was elevated compared to piglets suckling sows fed fish oil. Vitamin E supplementation to sows enhancedthe synthesis of these eicosanoids, and also the concentration of �-tocopherol in the AM of the piglets.© 2007 Elsevier Inc. All rights reserved.

Keywords: Pigs; Fat source; Vitamin E; Macrophages; Eicosanoids

1. Introduction

The content of fatty acids and vitamin E in sow milk is influenced by dietary fat source [1], and vitamin E level[2] provided the sow. Milk is an efficient vehicle of transferring nutrients from the mother to the newborn, and fat hasa particularly important role in infant nutrition as it provides a major portion of the energy as well as the fatty acidsneeded for growth of organ structural lipids and storage of fats. In addition, the transfer of vitamin E with the milk tothe new-born is essential, as piglets are born almost vitamin E deficient [3], and are not capable of synthesizing vitaminE themselves. High levels of PUFA in the diet are thought to increase the vitamin E requirement as accumulation ofPUFA in tissues may make those tissues vulnerable to lipid peroxidation, especially if peroxidation overwhelms the

Abbreviations: AM, alveolar macrophages; GSH-PX, glutathione peroxidase; MUFA, monounsaturated fatty acids; PUFA, polyunsaturatedfatty acids; PGE2, prostaglandin E2; LTB4, leukotriene B4; SAFA, saturated fatty acids; SOD, superoxide dismutase; TXB2, thromboxane B2; UNS,unsaturated fatty acids

∗ Corresponding author. Tel.: +45 89 99 12 38; fax: +45 89 99 11 66.E-mail address: Charlotte.lauridsen@agrsci.dk (C. Lauridsen).

1098-8823/$ – see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.prostaglandins.2007.04.003

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normal antioxidant mechanisms [4]. Increased intake of PUFA without adequate antioxidant protection could resultin increased free radicals and lipid oxidative by-products. Little is known about the combined effect of vitamin E andPUFA on the antioxidant status of the suckling piglet.

After weaning from the sow, the vitamin E status of pigs often decreases to a critical low level [2], and thispostweaning decline has been associated with onset of vitamin E deficiency [5]. In addition, a decline in the apparentfat digestibility is recognized at weaning [6], which may lead to deficiency of some of the essential fatty acids. Theimportance of providing adequate amounts of essential n − 6 and n − 3 fatty acids is well recognized. Studies ofnon-human primates and newborn infants have shown that docosahexanoic acid (DHA) is essential for the normalfunctional development of the retina and the brain, especially in premature babies [7]. In addition, substantial literatureis available on the impact of these fatty acids on inflammation and immunity of other animals and humans. For piglets,the postweaning period is of special concern as piglets often suffer from a growth check characterized by amongstother factors increased susceptibility to enteric pathogens to cause diseases [8]. Vitamin E is important for developmentand maturation of the immune system, and vitamin E deficiency has been found to predispose pigs to Escherichia coliinfection [9], whereas dietary supplementation with vitamin E showed improved cellular and humoral immunity in pigs[10,11]. In a recent study [12] we tried to enrich alveolar macrophages obtained from weaned piglets with vitamin Ethrough supplementation of the feed with up to 300 mg all-rac-�-tocopheryl acetate, but little or no effect was obtainedwith regard to the synthesis of eicosanoids and cytokines ex vivo or the concentration of vitamin E in the immune cells,probably because of impaired uptake of the lipid soluble vitamin during the postweaning period. Dietary fatty acidmodulation of the sow milk influenced the composition of fatty acids in liver and adipose tissue after weaning [13],and it is therefore likely that immune cell fatty acid composition and content of lipid soluble vitamins, and hence thecellular responses, of the weaned piglets would also be affected by the dietary changes during the suckling period,thus offering a potential nutritional strategy for influencing the resistance towards infections post weaning. However,no difference (0.52 and 0.87 �g of �-tocopherol per 107 cells) was obtained in the �-tocopherol content of alveolarmacrophages at weaning of piglets suckling sows fed 70 or 250 IU/kg of all-rac-�-tocopheryl acetate, respectively.

The immune system is sensitive to modulation by both dietary (n − 3) polyunsaturated fatty acids (PUFA) andvitamin E through changes in the eicosanoid production [14]. Substitution of menhaden fish oil for lard in sows late-gestation and lactation diet greatly elevated the content of n − 3 fatty acids in the nursing pig immune cells and generallyreduced in vitro eicosanoid release by pig immune cells [15]. Findings have suggested significant interactions betweenvitamin E and n − 3 fatty acids that affect the immune system in rats and that further research in this area is warranted[16]. Studies in dogs have suggested that although a ratio of dietary n − 6 to n − 3 fatty acids of 1.4:1 depresses thecell-mediated immune response and PGE2 production, it increases lipid oxidation and lowers vitamin E concentration[17].

Based on the existing literature listed above, the developed hypothesis of the present study was that substantialsupplementation of sow diets with vitamin E in combination with different n − 6 and n − 3 fatty acid ratio wouldinfluence the tocopherol content and fatty acids profile of immune cells, and accordingly their ex vivo eicosanoidsynthesis, of the progeny after the suckling period. In addition, it was hypothesized that the provision of dietary fattyacids and vitamin E while suckling would influence the nutritional and antioxidative responses of the progeny beforeand after weaning. Two other papers related to the present study describe in detail the influence of sow milk fatty acidcomposition during lactation on the transfer of specific fatty acids to the progeny [13] and on their lipid absorptioncapacity [18]. In this study, piglets were suckling sows fed diets containing either sunflower oil (rich in n − 6 PUFA)or fish oil (rich in n − 3 PUFA) or rapeseed oil (rich in both n − 6 and n − 3 fatty acids), and at two different levels ofvitamin E.

2. Materials and methods

2.1. Animals and diets

The experiment complied with the guidelines of the Danish Ministry of Justice with respect to animal experimentationand care of animals under study. At day 108 of gestation, fifteen sows (alternate Danish Landrace × Danish Yorkshire),were randomly selected from the herd at the Research Centre Foulum, and were assigned to one of five dietarytreatments. The sows were arranged in a randomized complete block design with three blocks and five sows per block,corresponding to five treatments per block. Within each block, sows had father of the same breed, same number of parity,

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Table 1Analyzed chemical composition of diets (sow diets 1–3 and piglet diet, means)

Fat source included Sow diets Piglet diet

Rapeseed oil Fish oil Sunflower oil Animal fat

Dry matter, DM (%) 88.0 88.7 86.8 89.4Crude protein, % of DM 20.0 19.8 20.1 24.3Crude fat, % of DM 10.3 9.8 9.1 9.3Ash, % of DM 5.8 5.9 5.8 5.87Crude Fibre, % of DM 4.2 4.2 4.1 4.1EDOMa 89.6 89.5 89.7 89.5

�-Tocopherol (mg/kg) 65.3 62.3 61.3 103Retinol (mg/kg) –b 2.92

Fatty acids (g/kg DM)c

C8:0 – – –C10:0 0.09 0.08 0.08 <0.01C12:0 0.83 0.72 0.64 0.01C14:0 0.59 3.5 0.40 0.17C 16:0 9.4 16.4 10.9 1.83C16:1 0.35 3.0 0.15 0.20C18:0 2.1 2.3 3.8 0.96C 18:1 47.4 16.5 18.8 2.48C18:2 27.4 13.2 53.3 1.41C 18:3, n − 3 7.7 2.2 2.1 0.17C 18:3, n − 6 – 0.01 – <0.01C 18:4 1.5 0.03C 20:0 0.47 0.18 0.42 0.02C 20:1 1.2 3.6 0.26 0.16C20:2 0.09 0.26 – 0.02C20:3 – 0.11 – –C22:1 0.36 5.0 – 0.14C22:0 0.35 0.27 0.63 –C24:0 0.17 – –C20:4 – 0.26 – 0.02C 20:5n − 3 – 4.4 – 0.13C22:5n − 3 – 0.56 – –C 22:6n − 3 – 7.1 – 0.20

a EDOM: Enzyme digestibility of organic matter [21].b Sow diets were not analyzed for retinol.c Content of selected fatty acids (if number of samples where the given fatty acid is presented is more than 2).

and were mated with a boar of the Duroc breed. Sows with piglets were housed individually in pens (2.2 m × 2.4 m)with partly slatted floor, partly solid concrete combined with slats of iron grates. Sows and piglets were providedmoderate quantities of straw bedding. Sows farrowed in crates, 2.4 m length and flexible in width (0.6–0.8 m). Thecreep area for piglets (1.0 m × 1.3 m) was partly covered, and heating lamps were installed during the first week afterbirth.

The five dietary treatments of the sows consisted of a basal diet (without supplementary fat) and addition of 8%rapeseed oil (diet 1), a basal diet and addition of 8% fish oil (diet 2 and 4), a basal diet and addition of 8% sunfloweroil (diet 3 and 5). Diet 1–3 contained a control level of vitamin E (60 mg/kg), whereas diet 4 and 5 was added 500 mgvitamin E/kg feed. The oil sources were provided by KFK (Korn- og foderstofkompagniet, Viby, Denmark), and thevitamin E was provided as all-rac-�-tocopheryl acetate by Roche a/s (Basel, Switzerland). The experimental feed wasprovided the sows from day 108 of gestation and until weaning (4 weeks after farrowing), and the feeding strategy ofthe sows are described in detail elsewhere [1]. The experimental diets were mixed at the Research Centre Foulum andprovided as pellets. The diets were formulated in order to supply an equal amount of digestible essential amino acidsper MJ NE. The chemical composition of the isocaloric diets (diet 1–3) is given in Table 1. The content of dry matter,crude protein, crude fat, and ash in the diets was determined according to the AOCS [19] procedures, and the crude

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fibre content and the enzyme digestibility of organic matter (EDOM) were analyzed after the methods of Aaman andHesselman [20] and of Boisen and Fernandez [21], respectively. Dietary lipids were acidified in 3 N boiling HCl for 1 h,air-dried and Soxhlet extracted with petroleum ether overnight according to the method of Stoldt [22]. Long-chain fattyacids (>C:8) were determined by gas chromatography (GC, capillary) after saponification with NaOH with a knownamount of internal standard (C17) and methylation methanol/BF3. After methylation, the esters were extracted withheptane as described by Rothenberg and Andersen [23]. �-Tocopherol and retinol content of the diets was determinedby high pressure liquid chromatography (HPLC) after saponification with KOH and subsequent extraction into heptaneas previous described in details [24,25].

No creep feed was given to the piglets during the suckling period. After weaning (day 28 of age), all piglets hadfree access to the same diet, which was fed until the end of the experiment at 49 days of age. The weaner diet wasa standard pelleted diet, which was added 5% animal fat and 60 mg all-rac-�-tocopheryl acetate/kg feed, and thechemical composition is presented in Table 1.

2.2. Sampling and analytical procedures

Milk samples from each sow were obtained at day 4, 18 and 25 of lactation (by hand-milking from the teats, andsamples of milk from different teats were pooled). Milk samples were stored at −20 ◦C until analysis of the contentof fatty acids and �-tocopherol. Analysis of fatty acids in milk was performed by GC according to a previouslydescribed method [26]. Briefly, milk samples were thawed and gently mixed in a water bath held at 40 ◦C. Fat wasextracted from the milk according to the method of Bligh and Dyer [27]. To 500 mg milk was added 0.50 mL water,1.00 mL chloroform with 5.000 mg C17 as internal standard and 2.00 mL methanol. This monophasic mixture wasshaken for 1 min, then 1.00 mL water and 2.00 mL chloroform was added, and the mixture was shaken again for1 min, whereafter the mixture was centrifuged for 10 min at 1000 × g and exactly 1.00 mL of the lower (chloroform)phase was taken out and trans esterified into fatty acid methyl esters after saponification with NaOH and esterified withbortrifluorid methanol complex and separated on GC as described by Rothenberg and Andersen [23]. �-Tocopherol andretinol in milk samples were analyzed by HPLC after saponification and extraction into heptane as previous described[28,29].

At days 25, 35, and 49 after birth, blood samples of three piglets per litter was obtained from vena jugularis bypuncture into heparinized vacuum tubes. After centrifugation at 1000 × g for 10 min at 4 ◦C, the plasma sampleswere stored at −80 ◦C until analysis. On the same days, one piglet per litter was killed by intravenous injection ofpentobarbital. After bleeding of the animals, samples of the liver, the heart, and the hip muscle were obtained. Tissuesamples were stored at −20 ◦C until analysis.

Packed cell volume (PCV) was determined using heparin-stabilized blood in capillary tubes centrifuged at 1200 rpmfor 5 min (Sigma 201 m, B.Braun, Osterode/Harz, Germany). Concentration of hemoglobin was measured using spec-trophotometric determination of the hemoglobincyanide (J.T. Baker BV, Deventer, The Netherlands). Erythrocytehemolysis in vitro was performed photometrically (540 nm) after incubation of red blood cells with 0.9% NaCl, 0.6%NaCl, or distilled water, each for 30 min at 25 ◦C. An erytrocyte lipid peroxidation test (ELP) was carried out asdescribed by Fontaine and Vally [30] and modified by Jensen et al. [31]. The activity of gluthathione peroxidase(GSH-Px, E.C. 1.11.1.9) was determined spectrophotometrially in blood plasma by the method of Gunzler et al. [32]as modified by Agergaard and Jensen [33] at 366 nm and 37 ◦C. The results were expressed in millikatals per milliliterof plasma (mKat/mL).

2.3. Isolation and analysis of immune cells

Immediately after removing, lungs were filled with approximately 60 mL of ice-cold Hank’s through the trachea.The lungs were gently exercised, and the content was filtered through gauze into containers on ice. This process wasrepeated twice (total recovery was ≈100 mL). The tubes containing the alveolar macrophages (AM) were spun down(500 × g for 15 min), and washed twice with Minimal Essential Medium (MEM, Gibco) with 2 mM l-glutamine, 10 mMN-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid (HEPES, Gibco), penicillin (100,000 U/L) and streptomycin(0.17 mmol/L), pH 7.4. The cells were then spun at 500 × g for 10 min and resuspended in 1 mL of MEM. The cells werestained with tryphan blue and counted on a hemocytometer, and resuspended at a final concentration of 5 × 106 cell/mLof MEM. The AM preparations were verified by Weight-Giemsa staining [34], and adherence properties. Samples of

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the cell suspension were resuspended in 10 mM EDTA and stored at −80 ◦C until analysis of fatty acid compositionand �-tocopherol.

To each well of a 12-well cell culture plate, 0.5 mL (2.5 × 106) of the AM cell suspension was added in additionto 0.5 mL of MEM with 10% calf serum/FBS. Plates were placed in an incubator at 37 ◦C with 5% CO2 for 4 h toallow cells to adhere. After washing the adhered cells three times with MEM, the number of adhered cells per wellwas estimated by counting the cells not adhered (dead and alive) as described above. Then 0.5 mL of MEM with10% serum/FBS was added to each well, and thereafter 0.5 mL/well of MEM alone (controls) or containing calciumionophore A231857 (2 �g/mL) was added to wells in order to stimulate eicosanoid production. After 1 h, supernatantswere collected in 1.5-mL polyprolene micro-centrifuge tubes, spun (10.000 rpm, 3 min), and transferred into a newtube, and stored at −80 ◦C until analysis of eicosanoids: The concentration of prostaglandin E2 (PGE2), thromboxaneB2 (TXB2) and leukotriene B4 (LTB4) in the cell supernatants was determined using commercially available enzymeimmunoassay kits for each eicosanoid (Caymann Chemicals Co., Ann Arbor, MI).

2.4. Analysis of antioxidants and fatty acids

Analysis of fatty acids in AM suspensions was performed by GC as methyl esters [26] after extraction of the lipidsaccording to the method of Bligh and Dyer [27]. The concentration of �-tocopherol in plasma, organs, tissues samplesand alveolar macrophage suspensions was analyzed by HPLC according to Jensen et al. [24]. Vitamin A concentrationwas analyzed by HPLC in milk [29], and plasma and liver [28]. Samples of the liver were prepared into cytosol andmicrosomes as described by Lauridsen and Jakobsen [35] for determination of GSH-PX (cytosol) and SOD (cytosoland microsomes). GSH-PX and SOD was determined as previously described [36].

2.5. Statistical analysis

The statistical analysis of data was accomplished using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC).Analyses of data obtained on the piglets were based on the following model:

Yfdbi = μ + αf + βd + (αβ)fd + Ufbi + εfdpi

in which Yfdpi is the dependent variable, μ is the overall mean, αf is the systematic effect of dietary treatment of sows,βd is the systematic effect of day of sampling, (αβ)fd is the systematic effect of interaction between dietary treatmentand day. Ufbi refers to the effect of sow/block (i.e., dependency between sow and block) and εfdbi denotes the errorterm. The pen was considered as the experimental unit. The results of the statistical analysis are expressed as leastsquare means and S.E.

3. Results

3.1. Sow milk

The concentration of fatty acids in milk for sows reflected the dietary treatments of sows as for example considerablymore C18:2n − 6 was present in sow milk for sunflower oil fed sows, whereas milk from the fish oil supplementedsows contained more C20:5, C22:5n − 3 and C22:6, and rapeseed oil fed sows showed the highest concentration ofC18:1 (Table 2). The milk of sows fed 500 mg vitamin E/kg diet contained approximately twice as much �-tocopherolthan sows fed 60 mg vitamin E/kg diet (6.0 versus 2.8 mg/kg milk of high and low dietary vitamin E, respectively).

Addition of vitamin E had no significant influence on the proportion of fatty acids in milk of sows compared withthe use of fish oil or sunflower oil without extra vitamin E, except with regard to the ratio of n − 6 to n − 3 fatty acids,which was lower in milk of sows fed sunflower oil and vitamin E compared with sunflower oil alone (Table 2).

3.2. Vitamin E and antioxidative enzyme activities

Supplemental vitamin E to the sows increased the concentration of �-tocopherol in muscle of piglets, and tended toincrease the concentration of �-tocopherol in heart of piglets, but no effects were observed for �-tocopherol concen-tration in plasma and liver (Table 3). An influence of dietary treatments was observed with regard to the antioxidant

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Table 2Proportion of fatty acids (%), and concentration (mg/kg milk) of �-tocopherol and retinol in sow milk (pooled between samples of day 4, 18, 25)a

Sow diets S.E.

Rapeseed oil Fish oil Sunflower oil Fish oil + vitamin E Sunflower oil + vitamin E

C10:0 0.21 0.21 0.18 0.22 0.21 0.03C12:0 1.11 0.34 0.30 0.36 0.32 0.29C14:0 5.47 b,c 6.13 b,c 3.98 a,c 6.25 c 4.80 a,b 0.56C16:0 24.8 a 26.6 a,b 27.9 b 28.0 b 27.4 b 0.85C16:1 9.58 a 12.0 b 9.00 a 12.6 b 10.2 a 0.69C18:0 5.18 a 5.37 a,b 6.47 c 4.83 a 6.02 b,c 0.58C18:1 32.6 c 25.2 a,b 27.7 b 24.2 a 26.9 a,b 1.17C18:2n − 6 13.5 b 11.0 a,b 21.2 c 10.0 a 19.0 c 1.35C18:3n − 3 5.03 b 2.00 a 1.38 a 1.83 a 1.57 a 0.61C20:1 0.45 a 1.37 b 0.17 a 1.30 b 0.36 a 0.21C20:2 0.23 0.27 0.28 0.23 0.29 0.03C20:3n − 6 0.43 a 0.47 a,b 0.72 c 0.44 a,b 0.66 b,c 0.08C20:4n − 6 0.12 b 0.14 b 0.06 a 0.12 b 0.07 a 0.01C20:5n − 3 0.20 a 3.50 b 0.10 a 3.73 b 0.74 a 0.30C22:1 – – –C22:5n − 6 0.04 a 0.05 a 0.08 b 0.04 a 0.06 a,b 0.01C22:5n − 3 0.29 a 0.80 b 0.20 a 0.91 b 0.32 a 0.07C22:6n − 3 0.59 a 4.53 b 0.16 a 4.77 b 1.01 a 0.47SAFA 36.9 38.8 38.8 39.8 38.9 1.24MUFA 42.6 b 39.5 a 38.5 a 38.1 a 37.4 a 1.16PUFA 20.4 22.7 24.0 22.0 23.7 1.69Total 100 100 100n − 6 14.1 b 11.6 a,b 22.0 c 10.6 a 19.8 c 1.37n − 3 6.1 b 10.8 c 1.8 a 11.3 c 3.63 a 0.96Ratio n − 6/n − 3 2.97 1.17 a 12.4 c 1.03 a 8.21 b 1.04�-Tocopherol 3.31 a 3.33 a 2.22 a 5.46 b 6.62 b 0.50Retinol 0.40 a 0.58 b 0.35 a 0.50 b 0.53 b 0.05

a Means within a row of sow diets with different letters were statistically different (P < 0.05).

enzymes in liver, as sunflower oil decreased the activity of GSH-PX and tended to decrease the activity of SOD in thecytosol (Table 3). Age of the piglets had a major impact on the concentration of �-tocopherol in plasma and tissues,which was much lower after weaning compared with the suckling period. Age of piglets had no influence on the con-centration of retinol in plasma or liver, or on the activity of antioxidant enzymes. Dietary treatments of sows or age ofpiglets had no impact on the biochemical blood responses; erythrocyte hemolysis in vitro (0.9% NaCl: 0.69 ± 0.41%;0.6% NaCl: 38.8 ± 25.6%;), haemoglobin (6.22 ± 1.69 mmol/L), packed cell volume (36.3 ± 4.66%) or erythrocytelipid peroxidation (0.020 ± 0.009 at extinction at 562 nm).

When piglets from sows fed rapeseed oil were deleted from the dataset, and the other four groups were analyzedaccording to a three-parametric statistical model (vitamin E-level, fat type and age), �-tocopherol in liver (P = 0.039) andheart (P = 0.056) was elevated by vitamin E supplementation of sows, and the concentration decreased with increasingage of the piglets. With regard to muscle tissue, an interaction (P = 0.043) between dietary vitamin E supplementationof sows and age of piglets was observed, and piglets from sows fed fish oil had a higher �-tocopherol concentration inmuscle than piglets from sows fed sunflower oil (4.73 versus 3.27 mg �-tocopherol/kg muscle for fish oil and sunfloweroil, respectively, S.E. = 0.74, P = 0.009).

3.3. Fatty acid profile and vitamin E in immune cells

The concentration of n − 3 fatty acids was affected by dietary treatments, and this was mainly due to the contentof C20:5n − 3 and C22:5n − 3, which was higher in AM from piglets suckling sows fed fish oil compared with otherdietary treatments. A significant interaction of dietary treatments of sows and age of piglets was observed on the totalconcentration of n − 6 (and C18:2n − 6) as the concentration in AM from piglets suckling sows fed fish oil and rapeseedoil increased with age, whereas the opposite effect was observed for the sunflower oil treatments and the fish oil with

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Table 3Effect of dietary treatment of sows and age (days) of piglets on concentration of �-tocopherol and retinol and antioxidant enzymes in plasma and liver of pigletsa

Age Rapeseed oil Fish oil Sunflower oil Fish oil + vitamin E Sunflower oil + vitamin E S.E. Diet Age

25 35 49 25 35 49 25 35 49 25 35 49 25 35 49

�-Tocopherol (mg/L plasma or mg/kg)Plasma 2.16 1.13 0.91 3.58 0.83 0.56 1.32 0.87 0.88 3.71 1.21 0.73 3.77 0.91 1.00 0.70 0.220 <0.001Liver 6.30 3.26 4.96 8.96 5.37 2.59 12.2 3.22 4.13 14.1 4.43 4.25 17.2 5.32 4.85 2.73 0.332 <0.001Muscle 4.67 3.65 3.50 4.76 4.85 3.16 3.75 2.68 2.23 8.00 8.14 3.69 7.36 5.20 2.62 0.84 <0.001 <0.001Heart 7.76 6.70 5.72 9.17 7.79 4.62 6.30 5.78 4.50 8.39 9.09 5.02 12.0 8.07 5.81 1.43 0.112 0.001

Retinol (mg/L plasma or mg/kg)Plasma 0.13 0.22 0.18 0.19 0.19 0.21 0.11 0.16 0.20 0.24 0.23 0.22 0.21 0.18 0.19 0.04 0.177 0.586Liver 43.1 45.5 55.1 74.8 74.8 56.7 45.0 60.7 78.9 69.5 66.0 72.4 63.7 60.1 82.7 12.5 0.20 0.210

GSH-PX (mKat/mL plasma or m units/mg protein in of liver cytosol)Plasma 2.80 3.01 1.88 4.14 4.14 3.80 2.34 1.92 5.23 3.09 1.88 3.26 1.92 2.72 1.88 1.36 0.89 0.432Cytosol 54.7 72.2 113 47.3 47.3 63.0 33.9 42.5 45.4 69.3 80.5 95.4 57.3 70.0 55.2 13.6 0.006 0.158

SOD (m units/mg protein in liver mirosomes or cytosol)Microsomes 14.4 39.4 15.4 12.5 12.5 11.3 15.4 16.1 16.8 14.0 11.8 15.4 7.58 12.7 12.6 10.4 0.464 0.918Cytosol 10.7 14.4 17.6 15.1 15.1 11.1 9.18 16.4 8.97 15.5 9.13 9.01 6.55 7.75 8.93 4.58 0.086 0.661

a Interactions between dietary treatments of sows and age of piglets were not obtained on the shown responses.

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vitamin E supplementation (Table 4). When the rapeseed oil treatment was excluded from the data, the same trendwas seen on C20:4n − 6 (P = 0.001). The ratio of C18:2n − 6 to C20:4n − 6 tended (P = 0.067) to be lower in AM ofpiglets suckling sows fed sunflower oil (0.76, S.E. = 0.13) compared with piglets from sows fed rapeseed and fish oil(averaging 1.08, S.E. = 0.14). In addition, the ratio between n − 6 and n − 3 fatty acids in AM (Table 4) was influencedby a significant interaction between dietary treatments and age of piglets. The ratio of n − 6 to n − 3 fatty acids onday 25 of piglets suckling sows provided the dietary fat sources without supplemental vitamin E reflected the range inthe ratio obtained in milk, e.g., from 0.23 (fish oil) to 11.6 (sunflower oil). The dietary fat sources for the sows or theage of the piglets had no impact on the concentration of SAFA, MUFA and PUFA, but the ratio of MUFA:PUFA wasgenerally higher at day 49 of age than at other ages.

No significant effect of dietary treatments or age of piglets could be obtained with regard to the concentration of�-tocopherol in AM (Table 4). However, when piglets suckling sows fed rapeseed oil was deleted from the dataset, andthe other four groups were analyzed according to a three-parametric statistical model (vitamin E-level, fat type andage), dietary vitamin E supplementation of sows (P = 0.05) resulted in an increased concentration of �-tocopherol inAM, and concentrations of �-tocopherol decreased with age (P = 0.05), whereas the fat type (fish oil versus sunfloweroil) had no impact on the AM �-tocopherol concentration. In addition, an interaction between vitamin E and age of thepiglets was observed on the concentration of C18:1 (P = 0.025). As also described above (when using the statisticalmodel with rapeseed oil included), fat source and age of piglets influenced the concentration of C18:2n − 6, C20:4n − 6,C20:5n − 3, and C22:6n − 3, whereas vitamin E supplementation had no impact on the concentration of these fattyacids.

3.4. Immune cell eicosanoids

Piglets suckling fish oil fed sows had a lower production of PGE2 and TXB2 than piglets suckling sunflower oilfed sows, however, vitamin E supplementation to the sows seemed to increase the synthesis of these eicosanoids inboth fish oil and sunflower oil suckled piglets (Fig. 1). The synthesis of leukotriene was in general higher in stimulatedAM than in non-stimulated AM, but dietary treatments had no impact on the synthesis of this eicosanoid (Fig. 1). Fornon-stimulated cells obtained at day 35 of age, the LTB4 synthesis was lower than by AM from piglets aged 28 or 49of age.

4. Discussion

The present data demonstrate that the n − 6 and n − 3 fatty acid content of immune cells of the progeny is easilyinfluenced by the maternal dietary provision of these fatty acids. Fritsche et al. [15] showed that when comparedto dietary inclusion of lard from day 107 of gestation, dietary menhaden oil significantly reduced the concentrationof arachidonic acid in alveolar macrophages of suckling piglets aged 18–21 days. In addition, an increase in theproportion of C20:5n − 3, C22:5n − 3 and C22:6n − 3 was found by provision of fish oil leading to a reduction inthe basal release of eicosanoids by AM of the suckling pigs [15]. In accordance, the present data show that whencompared to provision of sunflower oil, the fish oil led to markedly reduction in the contribution of arachidonicacid of AM relative to sunflower oil. Interestingly, and also in accordance with other reports [15], the contributionof C20:4n − 6 in AM was higher or similar to the level of C18:2n − 6 in AM irrespective of dietary treatments,whereas C18:2n − 6 was the most dominating fatty acid in sow’s milk and in adipose tissues of the suckling piglets,but not in liver [18]. In addition, C18:3n − 3 was provided with the sow milk but was only present to a diminishingdegree in the immune cells relative to C18:3n − 6. This demonstrates that the dietary linoleic acid provided withthe milk was converted via �-linolenic (C18:3n − 6) and di-homo-�-linolenic (C20:3n − 6) acids to arachidonic acidunder desaturation and elongation processes by the piglet cells. In agreement with the literature [14], our resultsfurther indicate that the dietary long-chained n − 3 PUFA inhibited the conversion of C18:2n − 6 to C20:4n − 6, asthe ratio of C18:2n − 6 to C20:4n − 6 in AM was lower for the sunflower oil compared with fish oil and rapeseedoil.

In the assessment of the influence of the n − 3 fatty acids on the eicosanoid production of the AM, the n − 6 fattyacids must be considered because these fatty acids serve as precursors for the 2-series prostanoids. Previous studies haveshown that dietary supplementation with fish oil reduces the PGE2 and TXB2 production by macrophages of mouse[37], and piglets [15], as also found in the present investigation. Prostanoids of the n − 3 series are not normally present

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Table 4Effect of dietary treatment of sows and age (days) of piglets on fatty acid concentration (�g fatty acids/g protein) and �-tocopherol (�g/g protein) of AM of piglets

Age (days) Rapeseed oil Fish oil Sunflower oil Fish oil + vitamin E Sunflower oil + vitamin E S.E. P-value

25 35 49 25 35 49 25 35 49 25 35 49 25 35 49 Diet Age Int

C10:0 0.28 0.56 0.30 0.30 1.43 0.41 0.71 0.50 0.31 0.01 0.81 0.81 0.39 0.53 0.01 0.29 0.021 0.009 0.004C14:0 2.13 1.95 5.30 2.02 1.62 4.18 3.41 2.63 1.54 3.08 5.56 2.53 3.43 3.64 1.68 1.44 0.739 0.988 0.107C16:0 47.4 53.6 110 41.4 48.8 74.4 86.7 68.7 41.8 55.7 134 48.4 92.1 102 41.9 36.1 0.828 0.546 0.246C16:1 2.74 3.61 7.08 3.39 2.89 4.42 4.65 3.23 2.19 6.66 8.02 2.58 5.84 5.14 1.61 2.38 0.626 0.624 0.351C18:0 8.64 12.7 25.8 13.6 17.6 16.0 17.9 13.8 14.0 22.3 21.4 15.1 20.9 17.4 12.5 4.81 0.729 0.999 0.118C18:1 16.4 17.8 44.9 15.4 21.8 27.3 26.8 25.6 20.0 28.7 42.6 19.5 29.3 30.3 16.3 8.93 0.785 0.743 0.163C18:2 7.69 8.65 18.8 5.32 9.00 10.3 22.3 11.4 9.51 10.2 14.3 9.81 22.7 15.8 8.09 4.11 0.078 0.530 0.013C18:3n − 6 4.56 5.42 11.9 3.78 2.91 4.09 7.56 4.80 5.50 17.3 11.1 2.84 15.4 5.60 5.52 4.79 0.291 0.251 0.362C18:3n − 3 0.31 0.14 0 0.21 0.20 0.75 0.05 0.52 0.09 0.13 0.49 0.28 0 0.20 0 0.31 0.271 0.197 0.296C20:3n − 6 0.49 0.32 2.29 0.54 0.48 1.80 2.68 1.04 1.50 0.62 0.96 1.76 0.51 1.03 1.23 0.61 0.294 0.020 0.219C20:4n − 6 6.78 10.8 21.6 6.17 8.78 13.1 28.6 17.5 11.5 9.20 11.4 9.84 29.4 20.3 13.9 6.07 0.329 0.988 0.215C20:5n − 3 3.93 3.71 4.73 12.5 12.6 7.45 – 1.34 4.52 14.9 10.5 7.53 0.89 2.56 3.21 2.53 0.011 0.943 0.469C22:5n − 3 2.60 2.38 5.21 5.54 2.58 6.58 4.06 3.96 4.46 8.65 6.47 2.55 3.78 3.21 3.43 1.42 0.658 0.566 0.267C22:6n − 3 2.92 4.09 6.47 9.11 7.60 5.88 2.62 3.26 4.84 14.0 8.54 4.76 4.27 3.50 4.03 1.95 0.002 0.413 0.117SAFA 60.4 69.9 144 58.2 69.1 97.3 111 87.3 58.7 84.0 165 67.0 119 125 57.2 41.4 0.939 0.765 0.197MUFA 19.2 21.4 51.9 18.8 24.6 31.8 31.5 28.9 22.2 35.3 50.7 22.0 35.1 35.4 17.9 11.0 0.647 0.726 0.067PUFA 32.2 37.2 72.7 45.7 46.7 57.5 72.5 47.1 46.0 77.9 64.4 48.0 78.7 57.1 41.9 15.2 0.576 0.407 0.178SAFA:UNS 1.02 1.13 1.16 0.91 0.99 1.09 1.03 1.10 0.83 0.75 1.28 0.99 1.10 1.24 0.92 0.18 0.807 0.141 0.563MUFA:PUFA 0.51 0.56 0.69 0.41 0.54 0.56 0.42 0.57 0.48 0.45 0.75 0.47 0.49 0.61 0.44 0.10 0.631 0.033 0.473Total n − 6 22.5 26.9 56.7 18.4 23.1 36.8 65.7 38.0 32.1 40.6 38.4 33.2 69.7 47.6 31.2 12.6 0.073 0.438 0.048Total n − 3 32.6 21.1 21.6 86.7 74.8 52.7 5.92 13.0 26.0 73.3 35.1 54.7 8.56 19.9 17.3 17.3 0.001 0.619 0.474Ratio n − 6:n − 3 1.60 4.51 2.87 0.23 0.40 1.04 11.6 3.34 1.21 11.0 1.35 0.60 8.81 2.70 1.64 1.94 0.009 0.029 0.028�-tocopherol 0.16 0.35 0.68 0.35 0.24 0.29 0.60 0.25 0.27 0.57 0.34 0.10 0.87 0.47 0.27 0.18 0.315 0.112 0.086

C. Lauridsen et al. / Prostaglandins & other Lipid Mediators 84 (2007) 66–78 75

Fig. 1. Effect of dietary treatment of sows and age of piglets on ex vivo synthesis of eicosanoids (pg/106) by non-stimulated or calcium ionophorestimulated alveolar macrophages of piglets. Interactions between dietary treatments of sows and age of piglets were not obtained on the shownresponses.

in significant amount but can be synthesized from C20:5n − 3. Although the eicosanoids derived from C20:4n − 6 andC20:3n − 3 have very similar molecular structures, the biologic effects are markedly different [17], and the n − 3 derivedeicosanoids are considered to be less inflammatory [38]. The variation among piglets with regard to the production ofeicosanoids by alveolar macrophages was large, which agrees with the results of du Manoir et al. [39], who observed

76 C. Lauridsen et al. / Prostaglandins & other Lipid Mediators 84 (2007) 66–78

that alveolar macrophages functions of pigs were variable both on an individual basis and over time. Accordingly, wethink that the large variation may also be the reason why no clear stimulating effect of the calcium ionophore wasobserved on the eicosanoid production except for the leukotriene B4 synthesis. In agreement with a previous study [2],we observed an effect of age of piglets on the leukotriene B4 of AM at 35 days, but this response was not influencedby the dietary treatments.

Vitamin E and other lipid antioxidants modulate the biosynthetic pathways in the arachidonic acid metabolismat a number of different points [40]. Vitamin E inhibits both fatty acid release and lipoxygenase activity, but notcyclooxygenase. Indeed low concentrations of some antioxidants enhance endoperoxide biosynthesis by prolong-ing cyclooxygenase activity. In accordance with previous studies [40], vitamin E supplementation had no effecton the incorporation of arachidonic acid into AM. However, vitamin E supplementation at a level of 500 mg all-rac-�-tocopheryl acetate/kg for sows enhanced the synthesis of PGE2 and TXB2 by alveolar macrophages of theprogeny, which may be due to the protecting effect on cyclooxygenase by this antioxidant [40]. In contrast, whenusing up to 250 mg/kg of all-rac-�-tocopheryl acetate for lactating sows, we obtained no effect on the eicosanoidsynthesis by the immune cells of the progeny [2], and 900 mg/kg of all-rac-�-tocopheryl acetate for rats didnot influence the prostaglandin production by isolated immune cells [16]. Accordingly, supplementation of vita-min E at a level of 300 mg/kg diet for weaners had no influence on the ex vivo synthesis of eicosanoids andcytokines by AM [12]. In the present study and also in previous studies [2,12], the dietary vitamin E supple-mentation for sows or weaners increased the concentration of �-tocopherol in the alveolar macrophages with afactor 1.5–1.6 times the basic dietary level, and it is therefore interesting that an effect on the eicosanoid syn-thesis was observed in the present study but not in the previous pig studies [2,12]. It cannot be excluded thatthe biosynthesis of eicosanoids through an increased protection of cyclooxygenase would be influenced by thestereoisomer form of �-tocopherol present in the AM, indeed sows discriminate between natural and synthetic vita-min E [3], whereby the RRR-form of the �-tocopherol is preferentially incorporated into immune cells [2]. Indeed,Diplock et al. [41] demonstrated a stereospecific influence of �-tocopherol on the biosynthesis of prostaglandin E2in vitro.

Changes in the AM synthesis of eicosanoids may cause an influence on the differentiation and functionof T-cells, B-cells, NK cells and macrophages [14], but our data are insufficient to assess whether or notthe enhancing effect of substantially higher dietary vitamin E levels or on the other hand the limiting effectobserved by fish oil inclusion of maternal diets on the ex vivo eicosanoid synthesis by immune cells wouldbe beneficial or not in case of, e.g., infectious diseases of pigs. In future, combination of such in vitro stud-ies with in vivo tests is required to elucidate the actual impact on the immune and inflammatory responsesin pigs.

According to previous results [2], supplemental vitamin E in sows diets increased the concentration of �-tocopherolin sow milk, and in liver, muscle and immune cells of the progeny. The effect seemed to be most notable in pigletssuckling sows fed supplemental vitamin E and sunflower oil rather than fish oil (and supplemental vitamin E). However,fish oil increased �-tocopherol concentration in muscle relative to sunflower oil. It has been demonstrated that fish oilor n − 3 PUFA-rich diets reduce �-tocopherol of plasma and membranes and increase oxidative stress [16]. Althoughno dietary influence on the used measures of lipid oxidation (e.g., erythrocyte lipid peroxidation or hemolysis in vitro)was obtained, it should be noted that addition of sunflower oil decreased the activity of GSH-PX and tended to increasethe activity of SOD in the liver cytosol compared with rapeseed oil or fish oil. The changes in the antioxidative enzymescould be ascribed to the changes in the hepatic fatty acid profile [13], as the need for antioxidant protection may beincreased when sunflower oil was fed, as also indicated by the lower concentration of �-tocopherol observed in musclewhen compared to fish oil.

5. Conclusion

Provision of rapeseed oil, fish oil or sunflower oil to sows changed the n − 6 to n − 3 ratio in immune cells ofthe progeny, and influenced the eicosanoid synthesis ex vivo. Supplementation with vitamin E to maternal diet ofsows increased the vitamin E concentration of AM and enhanced the immune cell eicosanoid synthesis. Thus, bothmaternal n − 3 PUFA and vitamin E intake may impact the immunological and inflammatory responses of the newborn,but further studies are required to determine the appropriate vitamin E supplementation level during n − 3 PUFAintake.

C. Lauridsen et al. / Prostaglandins & other Lipid Mediators 84 (2007) 66–78 77

Acknowledgements

For technical expertise the authors wish to thank Elsebeth Lyng Pedersen and Anna Stouby. This study was financiallysupported by the Danish Research Council.

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