Modification of lipid composition and oxidation in porcine muscle and muscle microsomes as affected by dietary supplementation of n-3 with either n-9 or n-6 fatty acids and α-tocopheryl acetate

Animal Feed Science and Technology 113 (2004) 223–238

Modification of lipid composition and oxidation in porcine muscle and muscle microsomes as affected by dietary supplementation of n-3 with either n-9 or n-6 fatty acids and ␣-tocopheryl acetate A.I. Rey a,∗ , C.J. Lopez-Bote a , J.P. Kerry b , P.B. Lynch c , D.J. Buckley b , P.A. Morrissey b a

b

Departamento de Producción Animal, Facultad de Veterinaria, Universidad Complutense, Madrid 28040, Spain Department of Food and Nutritional Sciences, University College Cork, National University of Ireland, Cork, Ireland c Teagasc, Moorepark Research Centre, Fermoy, Co., Cork, Ireland

Received 23 December 2002; received in revised form 17 July 2003; accepted 9 August 2003

Abstract The effect of different oil combinations: olive oil (20 g/kg), sunflower oil (20 g/kg), linseed oil (5 g/kg) and either olive (15 g/kg) or sunflower oil (15 g/kg) and ␣-tocopheryl acetate supplementation (200 mg/kg feed) on fatty acid composition and lipid oxidation of pig muscle and muscle microsomes was investigated. The proportions of ␣-linolenic acid (P < 0.001), eicosapentaenoic acid (P < 0.001) and docosahexaenoic acid (P < 0.05) in neutral lipids were significantly greater when linseed oil was added to diets. However, the proportion of docosahexaenoic acid in polar lipids was not significantly affected by the addition of linseed oil to the diets. Adding n-3 fatty acids in the diet produced a higher (P < 0.05) linoleic acid proportion in muscle neutral lipids, particularly in those groups fed n-9 MUFA. Pigs fed n-9 and n-3 enriched diets had the closest n-6:n-3 ratio to human dietary recommendations. The addition of linseed oil produced a significantly (P < 0.05) lower proportion of oleic acid (C18:1n-9) in polar lipids of muscles from pigs fed olive and linseed oils (OL + LIN), but not in those fed sunflower and linseed when compared with groups fed diets not enriched with linseed oil. Supplementation with 200 mg ␣-tocopheryl acetate/kg feed also led to higher (P < 0.05) oleic acid concentrations in polar lipids. Muscle from the pigs fed sunflower and linseed oils had significantly (P < 0.05) higher Thiobarbituric acid Reactive Substances (TBARS) values throughout the study compared to those fed olive and linseed oils. ␣-Tocopheryl acetate supplementation consistently improved lipid stability. No interactions between ␣-tocopherol and oils were observed on lipid oxidation. Iron-induced peroxidation showed a similar trend to that ∗ Corresponding author. Tel.: +34-91-394-3785; fax: +34-91-394-3889. E-mail address: [email protected] (A.I. Rey).

0377-8401/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2003.08.007

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observed for TBARS development. Iron-induced peroxidation of muscle microsomes showed a similar trend to oxidation in muscle. No interactions were detected between different oils in muscle microsomes. The partial replacement of n-9 MUFA (as olive oil) by n-3 PUFA in diet, results in a small increase in muscle and membrane lipid oxidation and could be an interesting alternative in pig diet formulation. © 2003 Elsevier B.V. All rights reserved. Keywords: Lipid oxidation; ␣-Tocopherol; n-3 Fatty acid; n-6 Fatty acid; n-9 Fatty acid; Microsomes; Pig muscle

1. Introduction Meat quality largely depends on the feeding background of the meat producing nonruminant animals (Buckley et al., 1995). One of the main mechanisms of quality deterioration is lipid oxidation (Buckley et al., 1995), which is manifested by adverse changes in flavor, color, texture and nutritive value and by the possible production of toxic compounds (mainly malondialdehyde). Lipid oxidation depends on a number of factors, including the polyunsaturated fatty acid (PUFA) content, and the concentration of prooxidants and antioxidants (Lauridsen et al., 1999). These factors can be manipulated by dietary means. Recently, there has been an increased interest in the substitution of animal fat sources by vegetable oils in animal nutrition. Vegetable oils have been attributed with reducing the level of saturation in pig tissues due to their high unsaturated fatty acid concentration when compared with animal fat (Ellis and Isbell, 1926). In addition, some vegetable oils (linseed oil) are rich in n-3 PUFA (mostly C18:3n-3). ␣-Linolenic acid (C18:3n-3) might elongate in pig tissues to produce long chain n-3 PUFA, which have been found to improve the status of the cardiovascular system (by reducing platelet aggregation and serum triglycerides and cholesterol) and the immune response control (Wood and Enser, 1997). Conversely, a higher proportion of long chain n-6 PUFA derived from linoleic acid (C18:2n-6) results in a pro-inflammatory status, so it is recommended to maintain a n-6:n-3 ratio below 4 (Wood and Enser, 1997). This has led to studies to reduce the high level of n-6 fatty acids present in pig meat, by increasing the level of n-3 in the feed (Romans et al., 1995; Leskanick et al., 1997; Enser et al., 2000). However, some studies indicate that increasing dietary PUFA accelerates oxidative deterioration in pig meat (Monahan et al., 1992; Nurnberg et al., 1999). Therefore, not only oil source and inclusion level but also adequate combination with antioxidants must be considered when formulating pig diets. An alternative approach to produce meat with low levels of lipid oxidation, would be the inclusion of a high source of monounsaturated fatty acids (MUFA), such as olive oil (Lopez-Bote et al., 1997a; Rey et al., 1997) in pig feed. Various studies have increased the concentration of MUFA by replacing n-6 PUFA and saturated fatty acids (Miller et al., 1990; Myer et al., 1992; Lopez-Bote et al., 1997a,b; Lauridsen et al., 1999). However, some of these studies resulted in adverse effects for meat quality parameters (Miller et al., 1990; Myer et al., 1992). From an extensive review of the scientific literature, little information of the use of combinations of oils in pig rations was detected. There is a lack of data on the effects of dietary n-9 MUFA-enrichment (olive oil) combined with a source of n-3

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fatty acids on muscle composition and oxidation. Therefore, further research on fatty acid relationships and their possible effects on fresh meat quality characteristic is required. It is generally believed that lipid oxidation in muscle foods is initiated in the highly unsaturated phospholipid fraction of cellular membranes (microsomes and mitochondrias) (Gray and Pearson, 1987). Antioxidants, such as ␣-tocopherol are mainly incorporated in muscle membranes (Buckley et al., 1995). Therefore, changes that could happen in phospholipid composition and oxidation by feeding combinations of either n-9 and n-3 or n-6 and n-3 dietary fatty acids and supranutritional levels of Vitamin E (␣-tocopheryl acetate), have to be explored. The objectives of this study were (1) to assess the effect of the combination of different levels of dietary n-3 PUFA using linseed oil (no addition versus 5 g/kg), with either n-6 PUFA (sunflower oil) or n-9 MUFA (olive oil) on the fatty acid profile of pig muscle and (2) to study the effects of the different fatty acid combinations on the oxidative stability of fresh muscle and muscle membranes. 2. Material and methods 2.1. Chemicals All chemicals used were ‘AnalaR’ grade and were supplied by British Drug House Poole, Dorset, UK, Sigma Chemical, Poole, Dorset, UK or Rathburn Chemicals, Walkerburn, Peableshire, Scotland. The ␣-tocopheryl acetate used in the diets was obtained from Roche Products, Welwyn Garden City, England. 2.2. Animals and experimental diets Male Landrace x Large White pigs (n = 80) were selected at 50 kg live weight and randomly divided into eight groups (n = 10). Pigs were housed in an environmentally controlled slatted floor facility (Teagasc, Moorepark Pig Husbandry Unit, Fermoy, Co., Cork, Ireland). The experimental diets and water were provided ad libitum. Throughout the experiment, the animals were treated according to the guidelines for the care of experimental animals (NRC, 1985) and comparable to those laid down by the Scientist Centre for Animal Welfare (Maryland, USA). Ingredients, chemical composition and fatty acid concentrations of the experimental diets are shown in Table 1. Fat was added at 20 g sunflower oil/kg (SUN), 20 g olive oil/kg (OL), or 5 g linseed oil/kg (LIN) and either 15 g sunflower/kg (SUN + LIN) or 15 g olive oil/kg (OL + LIN), in order to obtain different n-9:n-3 and n-6:n-3 fatty acid ratios. Within each dietary treatment, one group was fed a low level of ␣-tocopheryl acetate (10 mg ␣-tocopheryl acetate/kg feed) and the other group received a supplemental level (200 mg ␣-tocopheryl acetate/kg feed). 2.3. Slaughter and samples collection After 42 days on experimental diets, pigs were slaughtered at 90 ± 2.90 kg live weight (Cappoquin Pork & Bacon, Cappoquin, Co., Waterford, Ireland). Carcasses were chilled

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Table 1 Ingredients, calculated metabolizable energy, ␣-tocopherol and fatty acid composition of the experimental diets ␣-Tocopheryl acetate (mg/kg)

Dietsa SUN

OL

SUN + LIN

OL + LIN

10

200

10

200

10

200

10

200

Ingredientsb (g/kg) Sunflower oil Olive oil Linseed oil Calculated ME (MJ/kg food) ␣-Tocopherol (mg/kg DM)

20 0 0 13.36 21.7

20 0 0 13.35 193.9

0 20 0 13.35 22.0

0 20 0 13.34 191.8

15 0 5 13.35 22.0

15 0 5 13.34 212.2

0 15 5 13.35 18.5

0 15 5 13.35 197.4

Fatty acids (g/100 g fatty acids) C16:0 C18:0 C18:1n-9 C18:2n-6 C18:3n-3

12.1 3.8 18.7 62.7 2.7

14.5 3.7 17.1 61.4 3.4

15.7 2.2 45.8 33.0 3.3

14.9 2.1 46.9 32.8 3.3

13.3 3.5 17.8 54.3 11.1

14.4 3.6 16.1 55.3 10.6

14.7 2.3 38.8 33.0 11.3

13.8 2.2 41.0 32.1 10.9

a Diets: SUN = 20 g sunflower oil/kg with 10 or 200 mg ␣-tococopheryl acetate/kg feed; OL = 20 g olive oil/kg with 10 or 200 mg ␣-tocopheryl acetate/kg feed; SUN + LIN = 15 g sunflower oil/kg + 5 g linseed oil/kg with 10 or 200 mg ␣-tocopheryl acetate/kg feed; OL + LIN = 15 g olive oil/kg + 5 g linseed oil/kg with 10 or 200 mg ␣-tocopheryl acetate/kg feed. b Ingredients (g/kg): barley, 251; wheat, 487; soya hy-pro, 220; limestone flour, 10; dicalcium phosphate, 5; NaCl, 3; lysine synthetic, 2.5; methionine synthetic, 0.5; threonine, 0.8; vitamin-mix, 1.5 (per ton finished diet: copper sulphate, 400 g; ferrous sulphate, 200 g; manganese oxide, 40 g; zinc oxide, 100 g; potassium iodate, 0.5 g; sodium selenite, 0.4 g; Vitamin A, 2 IU; Vitamin D3 , 0.5 IU; Vitamin K, 4 g; Vitamin B12 , 15 mg; riboflavin, 2 g; nicotinic acid, 10 g; Vitamin B1 , 2 g; Vitamin B6 , 3 g; copper, 100 g; iron, 40 g; manganese, 31 g; zinc, 80 g; iodine, 0.3 g; and selenium, 2 g.

(4 ◦ C) overnight and approximately 30 cm size samples from the thoracic location of longissimus muscle were taken, vacuum packed in low-oxygen permeable (45 ml O2 /m2 /24 h at STP) film and stored at −20 ◦ C until required. Thiobarbituric acid reactive substances (TBARS) were measured in fresh muscle. Oxidation in muscle homogenates and microsomal fractions (frozen samples) and ␣-tocopherol analyses were measured within 3 weeks of slaughter. Fatty acid composition was determined 3–5 weeks after slaughter. 2.4. Chemical analysis of feed and meat samples Determination of the compositional analysis of feeds was carried out according to the Association of Official Analytical Chemists’ procedures (AOAC, 1996). Fat was extracted from feed samples by a combination of chloroform and methanol (Bligh and Dyer, 1959). ␣-Tocopherol was extracted from feed and muscle as previously described by Sheehy et al. (1994). One milliliter volume of 20% homogenate in 1.15% KCl solution was mixed with 1% ethanolic pyrogallol and 50% aqueous KOH. Samples were saponified in a shaking water bath at 70 ◦ C for 30 min. ␣-Tocopherol was extracted by hexane and redissolved in absolute alcohol. The analyses were carried out by HPLC (Waters, Machery–Nagel Nucleosil C18 column, Waters 486 UV detector, Germany). The eluting solvent was methanol:water (97:3)

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at a flow rate of 2 ml/min. The ␣-tocopherol content of the muscle was determined by comparison of peak areas with those obtained for a standard solution (50 mg/ml absolute alcohol) of dl-␣-tocopherol. Neutral and polar lipids from the muscle samples were extracted using the method of Marmer and Maxwell (1981). All lipid samples were methylated in the presence of sodium methylate (0.1 N) and 14% boron trifloride (Slover and Lanza, 1979) to obtain fatty acid methyl esters which were analyzed using a Hewlett Packard HP-5890 (Avondale, PA, USA), gas chromatograph equipped with a flame ionization detector and a capillary column HP-Innowax (30 m × 0.32 mm, i.d. and 0.25 ␮m polyethylene glycol-film thickness). The carrier gas was nitrogen at a constant flow of 2.5 ml/min. The temperature program was as follows: injector and detector temperature 250 ◦ C; the initial column temperature was 170 ◦ C, which was maintained for 2 min; 170–210 ◦ C at 3.5 ◦ C/min; 210–250 ◦ C at 7 ◦ C/min, hold for 18 min. The split ratio was 50:1. Identification was made by comparison with retention times of the corresponding pure standards. Results were expressed as g/100 g total fatty acids. Thiobarbituric acid reactive substances (TBARS) were evaluated to determine the effects of experimental diets on oxidative stability in meat samples on days 0, 3, 6 and 9 of refrigerated (4 ◦ C) storage under retail display conditions. Pork chops of longissimus muscle weighing approximately 40 g were placed on polystyrene trays, over-wrapped with an oxygen permeable (6000–8000 ml O2 /m2 /24 h at STP) PVC wrap and stored at 4 ◦ C under fluorescent light (616 lx). Lipid oxidation was assessed on days 0, 3, 6 and 9 by the 2-thiobarbituric acid method of Salih et al. (1987). Thiobarbituric acid reactive substances (TBARS) were expressed as nmols malondialdehyde (MDA)/kg muscle. The susceptibility of muscle tissue homogenates to iron-induced lipid oxidation was determined by a modification of the method of Kronbrust and Mavis (1980), in which 1 mM FeSO4 was used as the catalyst of lipid oxidation. Homogenates (approximately 1 mg/ ml buffer) were incubated at 37 ◦ C in 40 mM Tris–malate buffer (pH 7.4) with 1 mM FeSO4 in a total volume of 10 ml. At fixed time intervals, 1 ml aliquots were removed for measurement of thiobarbituric acid-reactive substances (TBARS). TBARS were expressed as nM malondialdehyde (MDA)/mg protein. Protein content was measured by the procedure of Bradford (1976). Membrane-bound lipids (microsomes) were extracted by differential centrifugation to separate microsomal fractions (Asghar et al., 1990). In order to enhance microsomal aggregation, CaCl2 was added to the supernatant to give a final concentration of 8 mM (w/v). The peroxidative stability was determined by a modification of the method of Kronbrust and Mavis (1980) as previously described for muscle homogenates. 2.5. Statistical analysis The data were analyzed as a (2 × 2 × 2) factorial arrangement using the General Linear Models procedures of SAS (1999), in which treatment comparisons were made using the following non-orthogonal contrasts: (1) 10 mg ␣-tocopheryl acetate/kg feed groups versus 200 mg ␣-tocopheryl acetate/kg feed supplemented groups, (2) sunflower oil-enriched groups versus olive oil-enriched, (3) Non-linseed oil-enriched groups versus linseed oilenriched, (4) Linseed oil × olive or sunflower oil interaction, (5) ␣-tocopheryl acetate ×

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sunflower or olive oil interaction, (6) ␣-tocopheryl acetate × linseed oil interaction. Data were presented as the means of each group and the pooled standard deviation (Pooled S.D.) together with the significance levels of the main effects and interactions. 3. Results and discussion 3.1. Muscle composition No significant effect of dietary treatment was observed for crude protein (23 ± 0.08), total intramuscular fat (1.8 ± 0.19), neutral lipids (1.1 ± 0.17), and polar lipids (0.8 ± 0.04). The ␣-tocopherol content of muscle (Table 4) was affected by the ␣-tocopheryl acetate concentration of diets. Significantly (P < 0.02) higher ␣-tocopherol values were detected in the muscle from pigs fed sunflower oil-enriched diets than those fed the olive oil-enriched which may be due to the higher Vitamin E concentration of sunflower oil or to a better absorption of Vitamin E in presence of some fatty acids. 3.2. Muscle neutral lipids and phospholipids The influence of the experimental diets on the fatty acid composition of the neutral and polar lipids of M. longissimus is shown in Tables 2 and 3, respectively. Pigs fed olive oil-enriched diets (OL, OL + LIN) had a significantly greater proportion of C16:1n-9 (P < 0.01), C16:1n-7 (P < 0.01) and C18:1n-9 (P < 0.001), and a lower proportion of C18:2n-6 (P < 0.01) and C20:3n-9 (P < 0.05), in muscle neutral lipids (Table 2) than pigs fed sunflower oil-enriched diets (SUN, SUN + LIN). Changes were of a higher magnitude in the polar lipid fraction (Table 3). These results agree with Rhee et al. (1988) who reported that the n-9 MUFA content of pig muscle is related to the n-9 MUFA of the diet. The addition of an oil source rich in n-9 monounsaturated fatty acids (olive oil) instead of n-6 polyunsaturated fatty acids (sunflower oil) to the pig diets produced significantly (P < 0.0001) higher ␣-linolenic acid (C18:3n-3) and lower linoleic acid (C18:2n-6) contents in the polar lipid fraction of muscle, thus leading to a lower n-6:n-3 fatty acid ratio. This effect has also been observed by Pan and Storlien (1993) and Lopez-Bote et al. (1997b) and may be explained by the metabolic competition between n-6 and n-3 PUFA for the phospholipid sites. The addition of 0.5 g linseed oil as a source of n-3 fatty acid/kg diet (OL + LIN, SUN + LIN) led to an even lower n-6:n-3 fatty acid ratio, produced by significantly higher proportions of ␣-linolenic acid (C18:3n-3, P < 0.0001), eicosapentaenoic acid (C20:5n-3, P = 0.0001) and docosahexaenoic acid (C22:6n-3, P < 0.05) in neutral lipids (Table 2). In polar lipids, pigs fed linseed oil-enriched diets showed significantly greater values of C18:3n-3 (P < 0.0001), C20:5n-3 (P < 0.0001) but not C22:6n-3 (Table 3). Several authors (Cherian and Sim, 1995; Romans et al., 1995; Fontanillas et al., 1997; Matthews et al., 2000), using higher levels of ␣-linolenic acid, reported the efficient deposition of ␣-linolenic (C18:3n-3) and the elongation and desaturation to n-3 long chain fatty acids in pig tissues. However, other trials with linseed have failed to increase tissue C22:6n-3 (DHA) levels consistently (Enser et al., 2000). In the present study, when linseed oil was not added to the diets (OL, SUN), docosahexaenoic acid (C22:6n-3) was detected at approximately

Table 2 Fatty acid composition (g/100 g neutral lipids) of M. longissimus dorsi from pigs fed experimental diets ␣-Tococopheryl acetate

Dietsa

10 C14:0 C16:0 C16:1n-9 C16:1n-7 C17:0 C18:0 C18:1n-9 C18:1n-7 C18:2n-6 C18:3n-3 C20:0 C20:1n-9 C20:3n-9 C20:4n-6 C20:5n-3 C22:5n-3 C22:6n-3 UIc

1.36 23.50 0.37 3.44 0.24 12.62 41.37 4.02 9.16 0.51 0.18 0.82 0.41 0.91 0.14 0.52 0.42 0.81

OL 200 1.30 23.92 0.39 2.89 0.24 13.99 41.17 3.84 8.45 0.53 0.21 1.03 0.44 0.57 0.14 0.56 0.35 0.77

10 1.33 23.26 0.36 3.45 0.24 13.41 45.66 3.32 5.65 0.63 0.20 0.86 0.27 0.36 0.10 0.48 0.41 0.74

200 1.29 22.11 0.45 3.12 0.27 14.11 42.79 4.19 7.74 0.71 0.23 0.84 0.46 0.74 0.11 0.51 0.33 0.78

SUN + LIN

OL + LIN

10

10

1.27 22.34 0.42 2.75 0.27 13.80 39.32 3.60 10.88 1.34 0.22 0.90 0.44 1.03 0.26 0.58 0.58 0.86

200 1.32 24.34 0.33 2.85 0.27 12.85 40.07 3.54 9.91 1.21 0.21 0.85 0.44 0.75 0.21 0.47 0.38 0.81

1.32 23.83 0.45 3.32 0.24 13.21 42.04 3.93 7.42 1.06 0.18 0.87 0.34 0.60 0.19 0.56 0.46 0.78

Pooled S.D.

Probability of contrastsb

200

1

2

3

4

5

6

1.20 22.62 0.50 3.08 0.31 11.72 44.09 3.77 7.88 1.06 0.17 0.93 0.32 0.90 0.29 0.61 0.54 0.84

NSd

NS NS 0.005 0.007 NS NS <0.001 NS <0.001 NS NS NS 0.005 NS NS NS NS NS

NS NS NS 0.037 0.047 NS NS NS 0.015 <0.001 NS NS NS NS <0.001 NS 0.042 0.022

NS NS NS NS NS NS NS NS NS 0.0143 0.008 NS NS NS NS NS NS NS

NS NS 0.017 NS NS NS NS NS 0.038 NS NS NS NS <0.001 NS NS NS 0.043

NS NS NS 0.049 NS NS 0.012 NS NS NS NS NS NS NS NS NS NS NS

0.098 2.028 0.080 0.363 0.055 2.093 2.331 0.738 1.880 0.258 0.038 0.130 0.108 0.329 0.080 0.208 0.197 0.063

NS NS 0.019 NS NS NS NS NS NS NS NS NS NS NS NS NS NS

a Diets: SUN = 20 g sunflower/kg oil with 10 or 200 mg ␣-tococopheryl acetate/kg feed; OL = 20 g olive/kg oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed; SUN + LIN = 15 g sunflower/kg oil + 5 g linseed/kg oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed; OL + LIN = 15 g olive/kg oil + 5 g/kg linseed oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed. b Contrast: (1) 10 vs. 200 ␣-tocopheryl acetate; (2) OL vs. SUN; (3) LIN vs. no LIN; (4) LIN × OLSUN; (5) OLSUN × 200 ␣-tocopheryl acetate; (6) LIN × 200 ␣-tocopheryl acetate. c UI: unsaturation index (average number of double bonds per fatty acid residue). d NS: not statistical significant.

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SUN

229

230

Table 3 Fatty acid composition (g/100 g polar lipids) of M. longissimus dorsi from pigs fed experimental diets ␣-Tococopheryl acetate

Dietsa SUN

C14:0 C15:0 C16:0 C16:1 n-9 C17:0 C17:1 C18:0 C18:1 n-9 C18:1 n-7 C18:2 n-6 C18:3 n-3 C20:0 C20:1 n-9 C20:3 n-9 C20:4 n-6 C20:5 n-3 C22:4 n-6 C22:5 n-3 C22:6 n-3 UIc

2.00 0.18 22.08 0.50 0.51 0.85 10.38 12.45 2.69 34.44 0.64 0.10 0.25 0.55 7.39 1.02 0.95 1.65 1.38 1.44

200 1.75 0.17 22.19 0.39 0.40 1.18 11.08 13.53 3.08 31.40 0.54 0.16 0.53 0.77 6.78 1.67 0.75 2.07 1.56 1.44

10 1.93 0.17 21.13 0.69 0.45 0.81 9.20 19.10 3.16 28.53 0.94 0.07 0.26 0.45 6.94 1.67 0.63 2.11 1.77 1.45

200 1.52 0.19 19.94 0.66 0.45 0.86 9.69 21.52 3.42 27.20 0.91 0.12 0.36 0.53 7.44 1.11 0.98 1.89 1.21 1.42

SUN + LIN

OL + LIN

10

10

1.69 0.16 20.56 0.42 0.42 0.81 9.35 12.46 2.72 35.27 1.67 0.11 0.23 0.56 7.32 1.78 0.79 2.40 1.27 1.55

200 2.02 0.17 20.07 0.43 0.36 0.86 9.60 13.22 2.83 34.71 1.35 0.09 0.22 0.55 7.19 1.82 0.70 2.34 1.49 1.55

1.55 0.18 21.91 0.54 0.45 0.57 9.95 17.56 3.27 28.29 1.75 0.33 0.31 0.52 6.38 2.02 0.67 2.34 1.42 1.45

Pooled S.D.

Probability of contrastsb

200

1

2

3

4

5

6

1.62 0.20 22.14 0.71 0.54 0.76 9.26 18.40 3.10 27.67 1.72 0.08 0.28 0.41 6.63 2.00 0.65 2.41 1.42 1.45

NSd

NS NS NS <0.001 NS NS NS <0.001 <0.001 <0.001 0.003 NS NS NS NS NS NS NS NS NS

NS NS NS NS NS NS NS 0.021 NS NS <0.001 NS NS NS NS <0.001 0.004 <0.001 NS NS

NS NS 0.039 NS NS NS NS 0.045 NS NS NS NS NS NS NS NS NS NS NS NS

NS NS NS NS 0.048 NS NS NS NS NS NS NS NS NS NS 0.001 <0.001 NS 0.006 NS

NS NS NS NS NS NS NS NS 0.014 NS NS 0.032 NS NS NS NS NS NS NS NS

0.598 0.046 3.035 0.190 0.119 0.409 2.271 1.976 0.261 3.980 0.343 0.092 0.284 0.213 1.133 0.348 0.155 0.333 0.318 0.132

NS NS NS NS NS NS 0.017 0.030 NS NS NS NS NS NS NS NS NS NS NS

a Diets: SUN = 20 g sunflower/kg oil with 10 or 200 mg ␣-tococopheryl acetate/kg feed; OL = 20 g olive/kg oil with 10 or 200 mg ␣-tocopzheryl acetate/kg feed; SUN + LIN = 15 g sunflower/kg oil + 5 g/kg linseed oil with 10 or 200 mg ␣-Tocopheryl acetate/kg feed; OL + LIN = 15 g olive/kg oil + 5 g/kg linseed oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed. b Contrast: (1) 10 vs. 200 ␣-tocopheryl acetate; (2) OL vs. SUN; (3) LIN vs. no LIN; (4) LIN × OLSUN; (5) OLSUN × 200 ␣-tocopheryl acetate; (6) LIN × 200 ␣-tocopheryl acetate. c UI: unsaturation index (average number of double bonds per fatty acid residue). d NS: not statistical significant.

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OL

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80% in phospholipids and 20% in neutral lipids, while, the addition of linseed oil produced a higher level of C22:6n-3 in muscle neutral lipids (approximately 31% for SUN + LIN and 24.5% for OL + LIN), which could indicate a competitive exclusion of C22:6n-3 from the phospholipids. Furthermore, including linseed oil (5 g/kg) to the diets containing either olive (OL + LIN) or sunflower (SUN + LIN) oils also produced a significantly (P < 0.05) higher concentration of linoleic acid (C18:2n-6) in the neutral lipid fraction of intramuscular fat (Table 2). The C18:2n-6 was increased by 30% in muscle neutral lipids from pigs fed OL + LIN diet and by 19% in those fed SUN + LIN diet. In a previous experiment carried out in Iberian pigs fed in free-range conditions (with high dietary levels of n-9 and n-3 fatty acids) a high proportion of linoleic acid was found in muscle neutral lipids (Rey and Lopez-Bote, 2001). These authors could not explain these findings and suggested a possible metabolic competition among the fatty acid classes. The results obtained in the current study corroborate the theory that an enrichment of dietary n-3 and either n-9 or n-6 fatty acids produces a higher proportion of linoleic acid in muscle neutral lipids, more so in those groups fed n-9 MUFA than in those fed n-6 PUFA, contrary to what would be expected. The contents of linoleic acid for muscle neutral and polar lipids from the OL group were 16 and 84 g/100 g, respectively. These levels changed slightly (21 g and 79 g, respectively) when linseed oil was included in the diet (OL + LIN). The differences in the levels of linoleic acid for the SUN and SUN + LIN diets were even smaller. These changes in the fatty acid distribution between the neutral and polar lipids could suggest that linoleic acid was displaced from the phospholipids to the neutral lipids by the inclusion of n-3 PUFA. The OL + LIN group had a higher C18:3n-3 proportion (although differences were not statistically significant) in the polar lipids when compared with SUN + LIN group, which might explain the greater (although not statistically different), displacement of the n-6 PUFA from the phospholipids in the OL + LIN group than in SUN + LIN. The n-6:n-3 ratio from the pigs fed n-9 and n-3-enriched diets (OL + LIN) was the closest to human dietary recommendations. However, in polar lipids the addition of linseed oil produced a significantly (P < 0.05) lower proportion of oleic acid (C18:1n-9) in muscle samples from pigs fed olive and linseed oils (OL + LIN), but not in those fed sunflower and linseed when compared with those groups not enriched with linseed oil. These results may be due to a dilution effect by the different fatty acid proportions, since the distribution of C18:1n-9 between the polar and neutral lipids did not change to a great extent when adding linseed oil to the diets. Fuhrmann and Sallman (1996) described a slight competition between n-9 and n-3 fatty acids. The fact that the addition of linseed and olive oil to the diets produced a higher (P < 0.05) proportion of C16:0 in muscle polar lipids, while a contrary effect was observed for those groups fed linseed and sunflower oil (Table 3) is also of interest. The higher concentration of n-3 PUFA in phospholipids from those groups enriched with olive and linseed oil (OL + LIN), leads to changes in the relative proportion of saturated fatty acids. These changes allow the phospholipid to keep overall unsaturation (estimated by the insaturated index) in a narrow range. Conversely, lower C20:0 proportions were found in muscle neutral lipids from the groups fed OL + LIN diets (Table 2). Groups supplemented with 200 mg ␣-tocopheryl acetate/kg of feed had significantly lower (P < 0.02) level of C16:1n-7 in muscle neutral lipids and higher C18:1n-9 (P < 0.02)

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and C18:1n-7 (P < 0.05) proportions in muscle polar lipids (Tables 2 and 3) compared to groups supplemented with 10 mg ␣-tocopheryl acetate/kg of feed. Monahan et al. (1992) and Lopez-Bote et al. (1997a,b) did not find any effect on the fatty acid composition of pig muscle, using similar levels of ␣-tocopheryl acetate in diets. However, other authors found that the deficiency of ␣-tocopherol in chicken diets increased the levels of unsaturation, while the proportion of n-9 fatty acid decreased (Fuhrmann and Sallman, 1996). These results were attributed to a possible effect of the ␣-tocopherol on the -9 desaturase activity (Okayasu et al., 1977). These authors also found more marked effects in the phospholipid fraction, where ␣-tocopherol is principally incorporated, which is in agreement with findings of the current study. 3.3. Lipid oxidation of M. longissimus dorsi To evaluate the oxidative stability of M. longissimus during refrigerated storage, TBARS values were measured on day 0, 3, 6 and 9 (Table 4). Muscle from the pigs fed olive oil-enriched diets (OL) had significantly lower TBARS values after 3 (P < 0.005), 6 (P < 0.001) and 9 days (P < 0.001) of refrigerated storage than those fed sunflower oil-enriched (SUN). The addition of 5 g of linseed oil/kg diet significantly increased TBARS numbers on days 3, 6 and 9 (P < 0.0001) of refrigerated storage when compared with groups fed non-enriched linseed oil diets. The inclusion of linseed oil (OL + LIN and SUN + LIN) increased TBARS numbers by 30% on day 9. The highest TBARS numbers were found in the groups fed linseed oil and sunflower oil (SUN + LIN). This group had TBARS numbers which were 40% higher than those for the group fed sunflower oil only (SUN) at day 9. Nurnberg et al. (1999) reported that having a higher proportion of n-3 PUFA in pig muscle resulted in increased lipid oxidation. However, it is interesting to note that in the present study, the utilization of a n-9 and n-3-enriched diet (OL + LIN) only increased TBARS values by 10% by day 9 compared to a non linseed oil-enriched diet (OL). From an extensive review of the scientific literature, no reports were found on the effects of dietary linseed and olive oil combinations on pig muscle oxidation, and its comparison with other oil sources. Miller et al. (1990) and Myer et al. (1992) found inconsistent results in terms of off-flavours when the oleic acid content of muscle was increased by dietary canola. These inconsistencies were attributed to the variable content of PUFA in oils, mainly ␣-linolenic acid. However, studies using rapeseed oil, which has a very favorable ratio of linoleic acid to ␣-linolenic acid and a high content of MUFA, resulted in stability problems due to elevated muscle C18:3 levels (Rhee et al., 1988). Experiments in which olive oil was used as fat source, showed high lipid stability results (Lopez-Bote et al., 1997b; Rey et al., 1997). All these previous studies used different dietary fat sources with its particular n-3 fatty acid composition, but the fat source was not tested with different n-3 fatty acid levels. In the present research, raw meat from pigs fed a diet that included olive and linseed oil (OL + LIN) was less oxidized than that from pigs fed a diet enriched with sunflower and linseed oil. Linolenic acid (C18:3n-3) increased by 86% in muscle polar lipids and by 68% in neutral lipids when comparing OL and OL + LIN diets, while the increase was 160% in both neutral and polar muscle lipids when SUN and SUN + LIN groups were compared. These results are noteworthy and might partially explain the low rate of increase in muscle lipid oxidation for those groups fed linseed and olive

Table 4 ␣-Tocopherol concentration, iron-induced lipid peroxidation at 37 ◦ C and TBARS (refrigerated stored at 4 ◦ C) in raw muscle from the pigs fed experimental diets Dietsa SUN 10 ␣-Tocopherol (mg/kg muscle)

OL 200

10

200

SUN + LIN

OL + LIN

10

10

200

Pooled S.D.

200

Probability of contrastsb 1

2

3

4

5

6

1.03

2.26

1.23

2.71

0.99

2.62

0.32

<0.001

0.020

NSc

NS

NS

NS

Iron-induced lipid peroxidation Minutes MDA (nmol/mg protein) 0 0.71 0.41 0.58 30 1.29 1.00 0.96 60 1.42 1.18 1.18 90 1.72 1.40 1.67 120 2.19 1.88 2.20

0.39 0.78 1.02 1.16 1.68

0.76 1.26 2.06 2.35 2.66

0.62 1.13 1.44 1.74 2.24

0.70 1.16 1.26 1.77 2.12

0.43 0.93 1.33 1.66 1.92

0.363 0.402 0.459 0.634 0.742

0.014 0.032 0.035 0.013 NS

NS 0.034 0.004 NS NS

NS NS 0.005 0.012 NS

NS NS NS NS NS

NS NS NS NS NS

NS NS NS NS NS

TBARS refrigerated storage Day MDA (nmol/kg meat) 0 6.67 6.09 6.43 3 9.24 8.19 9.60 6 18.99 15.65 17.12 9 24.21 20.04 21.73

4.52 9.34 11.30 15.94

6.90 13.34 28.52 34.88

7.25 12.65 23.18 27.49

6.82 10.61 17.32 23.83

6.41 8.71 17.80 23.20

0.119 0.165 0.308 0.391

0.053 0.032 <0.001 <0.001

0.038 0.005 <0.001 <0.001

0.006 <0.001 <0.001 <0.001

NS <0.001 0.003 0.043

NS NS NS NS

NS NS NS NS

1.14

2.63

a Diets: SUN = 20 g sunflower/kg oil with 10 or 200 mg ␣-tococopheryl acetate/kg feed; OL = 20 g olive/kg oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed; SUN + LIN = 15 g sunflower/kg oil + 5 g linseed/kg oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed; OL + LIN = 15 g olive/kg oil + 5 g/kg linseed oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed. b Contrast: (1) 10 vs. 200 ␣-tocopheryl acetate; (2) OL vs. SUN; (3) LIN vs. no LIN; (4) LIN × OLSUN; (5) OLSUN × 200 ␣-tocopheryl acetate; (6) LIN × 200 ␣-tocopheryl acetate. c NS: not statistical significant.

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␣-Tococopheryl acetate

233

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oil (OL + LIN). The antioxidant effect of polyphenols found in olive oil might also be involved. The influence of ␣-tocopherol on lipid oxidation was also studied (Table 4). Muscles from the pigs fed 200 mg/kg ␣-tocopheryl acetate-supplemented diets had significantly lower TBARS values on day 3 (P < 0.032) day 6 (P < 0.001) and day 9 (P < 0.001) than the other dietary groups. No interaction effects were detected among Vitamin E and the different oil types on lipid oxidation of raw meat. Supranutritional Vitamin E increased lipid stability by an average of 15% in those groups fed sunflower oil (SUN + E, SUN + LIN + E) when compared with groups not supplemented with ␣-tocopheryl acetate (SUN, SUN + LIN). A similar increase was observed for pigs fed an olive oil-enriched diet supplemented with ␣-tocopheryl acetate (OL + E). However, in groups fed linseed and olive oil (OL + LIN) the lipid stability was only improved by 7% on dietary ␣-tocopheryl acetate supplementation (OL + LIN + E). The small differences found in lipid oxidation between OL + LIN and OL + LIN + E dietary groups may be explained by the higher C18:2n-6 PUFA (P < 0.05) present in muscle neutral lipids from pigs fed the OL + LIN + E diet (Table 2) and therefore, by the higher degree of unsaturation (P < 0.05) (Table 2) found in muscle neutral lipids from pigs fed the OL + LIN + E diet. In agreement with these findings, Lauridsen et al. (1999) reported that the proportion of PUFA in backfat increased when pigs fed rapeseed oil-enriched diets were supplemented with ␣-tocopheryl acetate. ␣-Tocopherol has been reported to be an effective antioxidant in the presence of different oils (Monahan et al., 1992; Dirinck et al., 1996). However, TBARS numbers were not lower when ␣-tocopheryl acetate was added to diets with a high content of MUFA (Cherian et al., 1996) or when there were not marked differences in PUFA concentration (Leskanick et al., 1997). Ferrous sulphate-induced lipid peroxidation was also measured in raw muscle (Table 4). A similar trend to that reported for TBARS values was detected. However, for induced peroxidation statistical differences were of lower magnitude. Lopez-Bote et al. (1997b) also described a similar tendency for the data either measured by induced peroxidation or TBARS development. 3.4. Lipid oxidation of muscle membranes Microsomal ferrous sulphate-induced lipid peroxidation was measured (Table 5). MDA values in microsomal iron-induced oxidation were higher than those for muscles tissue throughout the study. Membrane-bound polar lipids are the sites where lipid oxidation is believed to be initiated (Gray and Pearson, 1987), so oxidation values were expected to be higher than those reported for muscle tissues. Feeding pigs with olive oil produced a lower level of oxidation at time 0 (P = 0.009) and after incubating for 60 min (P = 0.002) compared to groups fed sunflower oil. No interaction effect between different oils was detected in muscle microsomes. These data are supported by the findings of Lauridsen et al. (1999), Asghar et al. (1990) and Lin et al. (1989) in chicken, and Lopez-Bote et al. (1997b) in rabbits. All these authors reported dietary oil administration affected fatty acid composition of muscle microsomes and hence lipid oxidation. In the present study, adding linseed oil to diets significantly (P < 0.02) increased the microsomal lipid oxidation. The addition of linseed oil had a greater effect on iron-induced oxidation of microsomes for

␣-Tocopheryl acetate

Dietsa SUN 10

SUN + LIN

OL + LIN

200

10

200

10

200

2.76 3.73 3.88 4.01 4.32

5.18 7.63 8.15 8.91 8.77

4.21 5.94 6.25 6.61 7.36

3.68 5.31 6.02 6.34 6.33

3.08 4.36 4.76 4.90 4.71

OL 200

10

Iron-induced lipid peroxidation Minutes MDA (nmols/mg protein) 0 3.78 2.74 3.17 15 5.09 4.55 4.74 30 5.50 4.92 5.04 60 6.27 5.81 5.28 120 5.03 4.57 5.00

Pooled S.D.

1.276 1.754 2.012 2.310 2.304

Probability of contrastsb 1

2

3

4

5

6

0.014 0.013 0.012 0.014 0.059

0.009 0.003 0.009 0.002 0.016

0.003 0.003 0.003 0.016 <0.001

NSc NS NS NS 0.030

NS NS NS NS NS

NS NS NS NS NS

a Diets: SUN = 20 g sunflower/kg oil with 10 or 200 mg ␣-tococopheryl acetate/kg feed; OL = 20 g olive/kg oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed; SUN + LIN = 15 g sunflower/kg oil + 5 g linseed/kg oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed; OL + LIN = 15 g olive/kg oil + 5 g/kg linseed oil with 10 or 200 mg ␣-tocopheryl acetate/kg feed. b Contrast: (1) 10 vs. 200 ␣-tocopheryl acetate; (2) OL vs. SUN; (3) LIN vs. no LIN; (4) LIN × OLSUN; (5) OLSUN × 200 ␣-tocopheryl acetate; (6) LIN × 200 ␣-tocopheryl acetate. c NS: not statistical significant.

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Table 5 Iron-induced lipid peroxidation measured during 2 h of muscle microsomes from pigs fed experimental diets

235

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groups fed sunflower oil than for groups fed olive oil similar to results for muscle oxidation. Lin et al. (1989) reported similar results for muscle microsomes from chicken fed diets enriched with olive oil or linseed oil. However, from an extensive review of the scientific literature, no reports on the effects on microsome oxidation by combination of a dietary source of n-3 fatty acids with either n-9 or n-6 fatty acids were detected. Microsomes from pigs fed a diet supplemented with 200 mg ␣-tocopheryl acetate/kg feed had significantly (P < 0.20) lower levels of lipid oxidation at all time points. In all dietary groups, Vitamin E decreased the extent of membrane-bound lipid oxidation by 20% after 90 min of incubation. Interestingly, the group fed olive oil enriched with linseed oil and supplemented with Vitamin E had a greater increase in microsomal lipid stability than in muscles when compared with the group fed olive oil and linseed oil with a basal level of Vitamin E. These results would again indicate that muscle neutral lipid composition could partially be responsible for the small differences found in muscle lipid stability between these groups. ␣-Tocopherol is principally incorporated in the membrane structure where it protects polyunsaturated fatty acids from oxidation (Buckley et al., 1995). No interaction effects were detected between ␣-tocopherol and oils. It can be concluded that dietary linseed oil (5 g/kg diet) as a source of n-3 PUFA in combination with either n-9 MUFA (olive oil), or n-6 PUFA (sunflower oil), has a favorable effect on fatty acid composition of neutral and polar lipids in decreasing the n-6:n-3 fatty acid ratio of pig muscle, and consequently in encouraging the formation of n-3 eicosanoics instead of n-6. The partial replacement of n-9 MUFA by n-3 PUFA in the diet produced a low increase in muscle and membrane lipid oxidation when compared with those groups fed either sunflower or sunflower and linseed oil and could be an interesting alternative in pig feeding formulation. ␣-Tocopheryl acetate supplementation improved the oxidative status of muscle and membrane-bound lipids and its addition to the diets in combination with sunflower or sunflower and linseed oil is recommended.

Acknowledgements This research was funded by the Commission of the European Community (Contract no. FAIR CT96 5067). The authors thank E. Beatty, D. Morrissey and A. Lynch for their technical assistance. The authors also thank personnel at Moorepark Research Centre.

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