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Muscle lipids, vitamins E and A, and lipid oxidation as affected by diet and RN genotype in female and castrated male Hampshire crossbreed pigs
Meat Science 60 (2002) 411–420 www.elsevier.com/locate/meatsci
Muscle lipids, vitamins E and A, and lipid oxidation as affected by diet and RN genotype in female and castrated male Hampshire crossbreed pigs A. Ho¨gberga,*, J. Pickovaa, J. Babola, K. Anderssonb, P.C. Duttaa a Department of Food Science, Swedish University of Agricultural Sciences, PO Box 7051, S-75007 Uppsala, Sweden Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, PO Box 7051, S-75007 Uppsala, Sweden
b
Received 4 March 2001; received in revised form 26 May 2001; accepted 25 June 2001
Abstract The objectives of this study were to investigate the polar and neutral lipid fatty acid composition, content of retinol, a-tocopherol, g-tocopherol and level of oxidation of the pig muscle (M. longissimus dorsi). Female and castrated male Hampshire crossbreeds were produced in two systems. One group was raised indoors with a more polyunsaturated diet and the other raised outdoors with a more saturated diet. The level of polyunsaturated fatty acids in muscle was higher in the indoor females compared with the outdoor females, indoor castrated males and outdoor castrated males. The increased level of polyunsaturated fatty acids in the muscle, which was accompanied by a relatively low content of a-tocopherol, increased the susceptibility to lipid oxidation in the form of MDA (malondialdehyde) in the indoor female pigs. Finally, the level of polyunsaturated fatty acids in the polar lipids was affected by the RN genotype, and this difference was dependent on sex. In conclusion, diet has a major effect on the fatty acid composition and oxidation stability in pork muscle, but additional factors such as sex and RN genotype might also contribute. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Malondialdehyde; Pork; Retinol; RN-genotype; Sex; Tocopherol
1. Introduction Fatty acid composition of feed is known to affect the quality of pig meat. Fatty acid composition as well as the oxidation stability of pig muscle, especially the effect of different feeding regimes, have been intensively studied (Irie & Sakimoto, 1992; Lauridsen, Andersen, Andersson, Danielsen, Engberg, & Jakobsen, 1999; Leskanich, Matthews, Warkup, Noble, & Hazzledine, 1997; Øverland, Taugbøl, Haug, & Sundstøl, 1996). However, in most of these studies, total lipids of the muscles have been investigated, not considering a possible difference among lipid fractions. Polyunsaturated fatty acids (PUFA) of polar lipids, especially C20 fatty acids, are important constituents of membranes and they function as precursors in the eicosanoid metabolism, whereas the neutral lipids serve mainly as a depot * Corresponding author. Tel.: +46-18-672011; fax: +46-18673080. E-mail address: [email protected] (A. Ho¨gberg).
of lipids used as energy source (Henderson & Tocher, 1987). Due to different functions, it is likely that different fatty acids are incorporated differently into lipid classes. Thus, it has been shown that the level of C18:3 n-3 was more susceptible to change due to diet in the neutral lipid fraction than in the polar lipid fraction (Ho¨gberg, Pickova, Dutta, Babol, & Bylund, 2001; Leskanich, 1999). Fatty acid composition of the muscle has been shown to be affected by other factors such as sex and RN genotype. In a previous study (Ho¨gberg et al., 2001), we observed that the fatty acid profile of the polar lipids was more polyunsaturated in female pigs compared with castrated male pigs. This indicated a difference in fatty acid metabolism between castrated male pigs and female pigs. However, an interaction between sex and rearing system was also found in the same study (Ho¨gberg et al., 2001). The RN genotype gives an increased content of glycogen in pig muscle (Enfa¨lt, Lundstro¨m, Karlsson, & Hansson, 1997). In addition, Nilze´n, Babol, Lundeheim,
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00153-X
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Enfa¨lt, and Lundstro¨m (2001) have shown that the RN genotype influenced the fatty acid composition in pig muscle. Pigs with the RN allele had a higher level of n-3 polyunsaturated fatty acids in the muscle. Therefore, it is of interest to further study the effects of the RN genotype effects on the fatty acid composition of different lipid classes. Finally, when measuring changes in fatty acid composition, it is important to measure the content of antioxidants, vitamins and the level of oxidation in the meat. Among the antioxidants a-tocopherol has been recognised as one of the most important for preventing oxidation changes in meat (Jensen, Lauridsen, & Bertelsen, 1998). g-Tocopherol is also a potent antioxidant (Lynch, 1991; Rey, Isabel, Cava, & Lopez-Bote, 1998), whereas retinol is known to have an impact on a-tocopherol status resulting in its decrease (Hoppe, Schoner, & Frigg, 1992). The objectives of this study were to investigate how different lipid classes in pig muscle are affected by dietary fatty acids, RN genotype and sex using commonly produced genders, castrated males and female pigs. In addition, the content of retinol, a-tocopherol, g-tocopherol and the level of lipid oxidation were studied.
2. Materials and methods 2.1. Animals and treatments The pigs were raised at the Swedish University of Agricultural Sciences, Experimental Station in Bjertorp, Sweden, from 28 to 107 kg live weight. The growing/ finishing period span from July to November. The animals studied were a subsample of 40 animals selected from a total of 80 crossbreeds [(Swedish LandraceSwedish Yorkshire)(Hampshire)]. The animals were divided in two groups of 40 animals each. In production system 1 (further on called indoors) pigs were raised indoors in five pens, eight animals in each, and fed feed 1 (Tables 1 and 2). In production system 2 (further on called outdoor) pigs were raised outdoors together in one group in an area of about 6000 m2 clay soil overgrown with grass. The outdoor pigs were given feed 2 (Tables 1 and 2). Feed 2 had an Table 1 Analysed nutrient content of diets to the indoor (feed 1) and outdoor pigs (feed 2)
MJ/kg Crude protein (%) Lysine (%) Metionine (%) Treonine (%)
Feed 1
Feed 2
12.0 15.0 0.80 0.22 0.53
12.6 16.8 0.86 0.28 0.60
intentionally higher energy content than feed 1 to compensate for the increased energy expenditure for the outdoor environment. The outdoor pigs had access to huts with straw. All pigs were fed restrictedly in accordance with the standard feeding regimen for growing pigs in Sweden (Andersson, 1985). The daily feed allowances for the outdoor pigs in MJ of ME (metabolisable energy) were 16.5, 19.0, 24.1, 29.0 at 25, 30, 40, 50, 60 kg live weight, respectively, and 34.1 from 60 kg until slaughter. The feed allowances for the indoor pigs were 5% less than Table 2 Fat content, fatty acid composition, vitamin E content and retinol content of diets fed to indoor (feed 1) and outdoor pigs (feed 2)a Feed 1
Feed 2
Fat content (%) 12:0 (mg/100 g) 14:0 Unknown 16:0 16:1 tr 16:1 17:0 Unknown 18:0 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2 tr 18:2 n6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 22:0 22:1 20:5 n-3 24:0 24:1 22:6 n-3
5.3 56.4 43.6 1.4 791.2 3.3 16.8 5.9 0.6 247.0 26.1 1410.0 83.5 7.4 1.7 1541.3 1.4 177.7 17.9 30.8 3.1 10.8 8.5 2.5 7.3 3.6 3.0
3.4 12.1 13.9 0.5 475.1 1.7 9.5 2.9 0.0 80.6 5.6 918.1 44.9 1.8 0.6 1183.1 0.8 88.2 8.0 22.1 1.7 6.7 3.5 nd 4.6 2.1 nd
SAFA (mg/100 g) MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans n-6/n-3
1176.3 1560.7 10.1 4.5 5.5 1729.1 1545.8 183.2 31.1 8.4
601.3 1002.0 2.6 2.5 nd 1273.9 1185.7 88.2 7.9 13.4
19.9 9.0 7.3
25.3 4.9 8.1
a-Tocopherol g-Tocopherol Retinol (mg/g) a
SAFA, saturated fatty acids; MUFA, mono saturated fatty acids; PUFA, polyunsaturated fatty acids; HUFA, highly polyunsaturated fatty acids (18 carbon atoms or more not including 18:2 n-6 and 18:3 n-3); nd, not detectable.
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duction system and taken from the front part of M. Longissimus dorsi (LD) 48 h post-mortem and were frozen and stored at 80 C until analysis. Samples for analysis of MDA were collected from the same muscle and stored at 20 C for 12 months.
for the outdoor pigs. The lysine level was approximately 0.85% of air-dried feed and the total amounts of methionine and threonine were calculated to be 31 and 65% of the dietary lysine level respectively (Table 1). The amount of feed was adjusted every 2nd week depending on the mean weight of each group. The pigs were slaughtered at a commercial abattoir (Scan Farmek, Skara, Sweden). The carcasses were submitted to standard slaughter procedures and divided into joints 2 days after slaughter. Samples were chosen randomly within each sex and RN genotype and pro-
2.2. Analytical procedures The assumed RN genotype of each individual pig was determined with the method of Dalrymple and Hamm (1973), by enzymatic determination of the concentration
Table 3 Intramuscular fat content and fatty acid composition (%, least squares mean and standard error) in neutral and polar lipids of intramuscular fat from indoor (feed 1) and outdoor (feed 2) pigs Fatty acid
(a) Neutral lipid
(b) Polar lipid
Indoor (n=20)
Outdoor (n=20)
S.E.
P-value
IMF 12:0 14:0 Unknown 16:0 16:1 9 tr 16:1 n-7 17:0 18:0 Unknown 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2 9.12tr 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 20:3 n-6 20:4 n-6 20:3 n-3 20:5 n-3 24:0 22:4 n-6 22:5 n-3 22:6 n-3
1.88 Trace 1.34 nd 23.08 0.19 2.76 0.21 12.08 nd 0.24 40.73 3.49 nd 0.14 8.92 Trace 0.82 0.21 0.77 0.38 Trace 0.39 Trace Trace nd Trace Trace Trace
1.55 Trace 1.36 nd 23.86 0.21 3.23 0.19 12.03 nd 0.18 42.93 4.02 nd 0.15 6.16 Trace 0.43 0.22 0.81 0.28 Trace 0.38 Trace Trace nd Trace Trace Trace
0.12
0.05
0.02 nd 0.34 0.03 0.09 0.01 0.31 nd 0.02 0.41 0.07 nd 0.01 0.46
0.65 nd 0.12 0.71 0.001 0.26 0.62 nd 0.06 0.001b,c 0.001 nd 0.13 0.001b
0.04 0.01 0.02 0.02
0.001b 0.76 0.13 0.001
0.02 Trace Trace nd
0.78c Trace Trace Nd
SAFA MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans
37.10 47.84 1.46 1.09 0.35 11.15 9.97 1.15 0.57
38.03 51.31 1.15 1.00 0.23 7.79 7.13 0.65 0.54
0.63 0.50 0.06 0.5 0.01 0.55 0.50 0.05 0.04
0.31 0.001b,c 0.001b,c 0.23 0.001b 0.001b 0.001 0.001 0.59
8.62
10.95
0.17
0.001
n-6/n-3
Indoor (n=20)
Outdoor (n=20)
S.E.
P-value
nd Trace 1.15 19.04 0.15 0.27 0.25 8.69 4.31 nd 9.12 2.59 2.67 0.26 30.34 0.27 0.82 nd Tracea 0.48 0.95 9.30 0.20 0.80 Trace 1.23 1.78 0.79
nd Trace 1.43 20.18 0.20 0.51 0.29 7.50 3.41 nd 9.40 2.64 2.47 0.30 30.67 0.33 0.72 nd Trace 0.44 0.98 9.40 0.20 0.63 Trace 1.52 1.73 0.52
nd
nd
0.34 0.43 0.02 0.02 0.01 0.19 0.28 nd 0.25 0.07 0.19 0.02 0.39 0.01 0.02 nd
0.56 0.07 0.10 0.001 0.01 0.03 0.03 nd 0.43 0.58 0.48 0.21 0.57 0.001 0.003 nd
0.02 0.02 0.25 0.01 0.24
0.07 0.41 0.79 0.46 0.001
0.04 0.06 0.03
0.001 0.56 0.000
28.24 14.90 15.83 13.85 3.56 46.94 42.56 4.38 0.50
28.24 15.26 15.70 13.89 3.10 47.16 43.34 3.82 0.55
0.05 0.35 0.35 0.31 0.09 0.52 0.47 0.09 0.03
1.00 0.47 0.79 0.93 0.002 0.77 0.27 0.00 0.18
9.87
11.41
0.23
0.001
Trace: Identified fatty acids below 0.15% are shown as trace but included in sum of SAFA, PUFA n-6, PUFA n-3, HUFA n-6 and HUFA n-3, respectively. a Abbreviations: see Table 2. b Shown in Table 9 for each sex in the outdoor and indoor rearing system. c Significant effect of the intramuscular fat content when included as a covariate (P <0.05).
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of residual glycogen (including glucose and glucose-6phosphate) in homogenised muscle tissue. Animals with a concentration less than 35 mmol/mg of M. Longissimus dorsi were considered as not carrying the RN allele, while animals with a concentration above 35 mmol/mg were considered as carriers of the RN allele. Since there is some overlap in the distribution of glycogen content between RN and rn+ animals, a diagnostic DNA test for the causative mutation (Milan et al., 2000) was carried out on the four individuals with glycogen concentrations between 30 and 40 mmol to avoid misclassifications.
The intra muscular fat content (IMF) of M. Longissimus dorsi (LD) was analysed with Soxhlet analysis (Soxtec System H+ equipment, Tecator AB, Ho¨gana¨s, Sweden). In the Soxhlet analysis and for all analyses below, meat without connective tissue and subcutanous fat was used. For fatty acid analyses the method was used as described by Ho¨gberg et al. (2001). In brief, 215 g of minced muscle were homogenised and total lipids were separated into neutral lipid and polar lipid fractions. Fatty acid methyl esters (FAME) from both neutral and polar lipids were prepared before gas chromatograph
Table 4 Fatty acid composition (%, least squares mean and standard error) of neutral and polar lipids in intramuscular fat from castrated male pigs and female pigsa Fatty acid
IMF 12:0 14:0 Unknown 16:0 16:1 9 tr 16:1 n-7 17:0 18:0 Unknown 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2tr 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 20:3 n-6 20:4 n-6 20:3 n-3 20:5 n-3 24:0 22:4 n-6 22:5 n-3 22:6 n-3 SAFA MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans n-6/n-3 a b c d
(a) Neutral lipid
(b) Polar lipid
Castrates (n=18)
Female pigs (n=22)
S.E.
1.76 Trace 1.34 nd 23.59 0.17 3.04 0.20 12.48 nd 0.21 42.28 3.80 nd 0.15 6.65 Trace 0.55 0.21 0.80 0.31 Trace 0.35 Trace Trace nd Trace Trace Trace
1.67 Trace 1.36 nd 23.34 0.23 2.95 0.20 11.91 nd 0.21 41.38 3.72 nd 0.15 8.43 Trace 0.70 0.21 0.77 0.36 Trace 0.41 Trace Trace nd Trace Trace Trace
38.00 50.13 1.21 0.96 0.26 8.40 7.58 0.80 0.53
37.13 49.03 1.40 1.14 0.33 10.54 9.52 1.01 0.58
0.61 0.48 0.06 0.05 0.01 0.54 0.49 0.04 0.04
9.77
9.77
0.16
Castrates (n=18)
Female pigs (n=22)
S.E.
P-value
nd Trace 1.29 19.75 0.18 0.43 0.30 7.95 3.82 nd 9.74 2.61 2.64 0.26 30.55 0.31 0.82 nd 0.18 0.45 0.97 8.79 0.19 0.71 Trace 1.37 1.76 0.58
nd Trace 1.30 19.45 0.17 0.35 0.25 8.25 3.90 nd 8.77 2.62 2.50 0.30 30.46 0.30 0.72 nd 0.16 0.46 0.95 9.90 0.21 0.72 Trace 1.38 1.75 0.74
nd
nd
0.33 0.42 0.02 0.02 0.01 0.18 0.28 nd 0.25 0.07 0.19 0.02 0.39 0.01 0.02 nd 0.01 0.02 0.02 0.02 0.01 0.23
0.98 0.63 0.66 0.01 0.001 0.26 0.83 nd 0.01 0.90 0.63 0.25 0.86d 0.45 0.004 nd 0.03 0.78 0.48 0.003 0.13 0.83d
0.32 0.11b,c 0.02b,c 0.03 0.004b 0.01b 0.01 0.002 0.39
28.26 15.75 15.14 13.31 3.24 46.51 42.44 4.07 0.51
0.90
10.57
P-value 0.12
0.02 nd 0.33 0.03 0.09 0.01 0.31 nd 0.20 0.39 0.07 nd 0.01 0.05
0.61 0.52 nd 0.61 0.22 0.50 0.76 0.20 nd 1.00 0.11b,c 0.41 nd 0.86 0.01b
0.04 0.01 0.02 0.02 Trace 0.02 Trace
0.01b 0.80 0.14 0.04 Trace 0.07c Trace
nd
nd
Abbreviations, see Tables 2 and 3. Shown in Table 9 for each sex in the free-range and indoor rearing system. Significant effect of the intramuscular fat content when included as a covariate (P <0.05). Significant interaction between sex and RN genotype. See Table 6.
0.04 0.06 0.03
0.78 0.86 0.002d
28.21 14.40 16.39 14.42 3.41 47.59 43.45 4.14 0.53
0.48 0.35 0.34 0.30 0.09 0.49 0.45 0.09 0.25
0.94 0.01 0.02 0.01 0.21d 0.14d 0.12d 0.60d 0.61
10.72
0.22
0.65d
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For the analysis of retinol and a- and g-tocopherol a modified method by Jensen et al. (1998) was used. Two times one gram of meat, was cut into pieces and homogenised in two tubes. 1.2 ml of 20% ascorbic acid solution, 0.6 ml methanol and 1.2 ml of KOH-water (1:1) were added to each tube. After saponification and cooling, retinol and tocopherols were extracted in 2 times 4 ml of hexane. The hexane-vitamin solution was evaporated under nitrogen gas and diluted with the mobile phase. The mobile phase used for retinol and tocopherols
(GC) analyses. The peaks of GC chromatograms were analysed by comparison with authentical standards (GLC-68 A, Nu-Chek Prep, Elysian, USA) as described by Ho¨gberg et al. (2001). MDA, retinol and tocopherols was determined with HPLC using a Merck Hitachi L6200A pump, a FL Lt480 detector and an As-2000 A auto sampler. The HPLC column was a 4.0250 mm RP-18 LiChroCART. Identification and quantification were done by using external standards.
Table 5 Fatty acid composition (least squares mean and standard error) of neutral and polar lipids in intramuscular fat from pigs with different RN genotypea Fatty acid
(a) Neutral lipid +
rn
(n=19)
(b) Polar Lipid RN (n=21)
S.E.
rn+ (n=19)
RN (n=21)
S.E.
P-value
nd Trace 1.31 19.74 0.16 0.46 0.27 7.79 4.18 nd 9.70 2.30 2.57 0.28 29.52 0.28 0.75 nd Trace 0.44 1.01 9.51 0.19 0.76 Trace 1.35 1.82 0.71
nd Trace 1.27 19.47 0.20 0.32 0.28 8.40 3.54 nd 8.81 2.92 2.57 0.28 31.48 0.32 0.79 nd Trace 0.47 0.91 9.18 0.21 0.67 Trace 1.40 1.69 0.60
nd
nd
0.03 0.43 0.02 0.02 0.02 0.02 0.28 nd 0.25 0.07 0.02 0.02 0.39 0.01 0.02 nd
0.93 0.66 0.19 0.001 0.23 0.03 0.11 nd 0.02 0.001 1.00 0.99 0.001 0.05 0.21 nd
0.02 0.02 0.25 0.01 0.24
0.09 0.001 0.37 0.03 0.02c
0.04 0.06 0.34
0.46 0.12 0.03c
IMF 12:0 14:0 Unknown 16:0 16:1 9 tr 16:1 n-7 17:0 18:0 Unknown 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2 9.12tr 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 20:3 n-6 20:4 n-6 20:3 n-3 20:5 n-3 24:0 22:4 n-6 22:5 n-3 22:6 n-3
1.58 Trace 1.35 nd 23.16 0.16 2.98 0.20 12.36 nd 0.23 42.03 3.71 nd 0.14 7.71 Trace 0.64 0.21 0.76 0.33 Trace 0.36 Trace Trace nd Trace Trace Trace
1.85 Trace 1.36 nd 23.78 0.24 3.02 0.20 12.02 nd 0.20 41.63 3.80 nd 0.15 7.36 Trace 0.61 0.21 0.82 0.34 Trace 0.41 Trace Trace nd Trace Trace Trace
SAFA MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans
37.39 49.61 1.24 1.02 0.28 9.63 8.69 0.92 0.52
37.74 49.54 1.38 1.07 0.30 9.31 8.41 0.88 0.59
0.63 0.50 0.06 0.06 0.02 0.55 0.50 0.05 0.04
0.70 0.92b 0.11b 0.51 0.56 0.68 0.69 0.63 0.31
28.06 15.28 15.10 14.13 3.48 46.35 42.12 4.23 0.52
28.42 14.87 15.43 13.60 3.17 47.75 43.78 3.97 0.53
0.49 0.35 0.35 0.31 0.09 0.41 0.46 0.09 0.03
0.61 0.40 0.19 0.24 0.01c 0.07c 0.02c 0.06c 0.90
9.85
9.71
0.17
0.57
10.11
11.18
0.23
0.003c
n-6/n-3 a b c
0.12
P-value
0.02 nd 0.34 0.03 0.10 0.01 0.03 nd 0.02 0.41 0.07 nd 0.01 0.05
0.12 0.75 nd 0.21 0.13 0.76 0.88 0.47 nd 0.29 0.50b 0.39 Nd 0.24 0.59
0.04 0.01 0.02 0.02
0.48 0.94 0.01 0.78
0.02
0.12b
nd
nd
Abbreviations: see Tables 2 and 3. Significant effect of the intramuscular fat content when included as a covariate (P<0.05). Significant interaction between sex and RN genotype. See Table 6.
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extracted according to the method of Børsting, Engberg, Jakobsen, Jensen, and Anderssen (1994) and analysed by HPLC.
consisted of 95% metanol:acetonitrile (1:1) and 5% chloroform with a flow rate of 1.2 ml/min. Tocopherols and retinol were detected with excitation wavelengths of 290 and 344 nm, respectively, and with emission wavelengths of 327 and 472 nm, respectively. For MDA (malondialdehyde) analysis the method of Draper, Squires, Mahmoodi, Agarwal, and Hadley (1993; method 4) was used for preparing the meat samples before HPLC analysis. Two times one gram of meat was homogenised and after preparation 300 ml of MDA/butanol solution was diluted 10 with the mobile phase. HPLC analysis was performed according to the method of O¨hrvall, Tengblad, Ekstrand, Siegbahn, and Vessby (1994; excitation 532 nm and emission 553 nm). A mobile phase consisting of 60% potassium phosphate buffer (50 mM, pH 6.8) and 40% methanol with a flow rate of 0.7 ml/min was used. The analyses of the feed were performed according to the following: the energy analysis was performed using the EFOS method as described by Boisen and Fernandez (1997). The fatty acid composition of the feed was analysed as described above after grinding of the feed. Retinol and tocopherols of the feed 1 and 2 were
2.3. Statistical analysis Statistical evaluation was carried out with Minitab statistical software for Windows 95 and NT, version 12 (Minitab, 1998) on the mean values of the duplicate samples. Production system, sex and RN genotype were regarded as fixed effects of the experiment and were tested by analysis of variance (GLM procedure), and different groups were compared on the basis of leastsquares means. Intramuscular fat content (IMF) was included as covariate in both neutral lipids and polar lipids. In the case of interaction between fixed effects, pair-wise comparisons were made, using Tukey’s method. If a covariate or an interaction failed to reach significance (P>0.05), the term was excluded from the model, except for some of the fatty acids in Table 9. Finally, Pearson overall and partial correlation coefficients were calculated between a-tocopherol, MDA and certain individual and groups of fatty acids of importance in polar
Table 6 Fatty acid composition in polar lipids with interaction between sex and RN genotype (least-square means and pooled standard error)a S.E.
P-valueb
30.69ab 0.63b 0.62a 47.31ab 3.08a 43.50b 3.82b
0.55 0.20 0.05 0.72 0.13 0.65 0.13
0.01 0.01 0.01 0.01 0.01 0.03 0.01
11.57b
0.32
0.05
Sex
Castrates
Genotype
rn+ (n=9)
RN (n=9)
rn+ (n=10)
RN (n=12)
18:2 n-6 20:5 n-3 22:6 n-3 PUFA HUFA n-3 PUFA n-6 PUFA n-3
28.82a 0.71ab 0.57a 44.84a 3.22a 40.83a 4.0ab
32.28b 0.71ab 0.59a 48.18b 3.27a 44.05b 4.13ab
30.23ab 0.80a 0.86b 47.86b 3.74b 43.41b 4.45a
n6/n3
10.35a
10.79ab
9.86a
a b
Gilts
Abbreviations: See Table 2. Values with different letters within a row are significantly different (P<0.05). P-value for the interaction between sex and RN genotype.
Table 7 Vitamin E and retinol content in muscle from indoor pigs, outdoor pigs, castrated male pigs and female pigs (least-square means and pooled standard error) Antioxidant
Production system
Sex
Indoor (n=20)
Outdoor (n=20)
S.E.
P-value
Castrates (n=18)
Female pigs (n=22)
S.E.
P-value
g/100 g meat a-Tocopherol g-Tocopherol Retinol
91.10 5.21 2.86
139.05 3.47 2.44
11.9 0.27 0.22
0.01a 0.001 0.18
97.80 4.25 2.61
132.31 4.42 2.68
11.63 0.26 0.21
0.04a 0.65 0.81
g/g intra muscular fat a-Tocopherol g-Tocopherol Retinol
51.56 2.96 1.61
100.89 2.40 1.72
11.79 0.25 0.16
0.01 0.12 0.65
62.13 2.46 1.58
90.32 2.90 1.74
11.55 0.24 0.16
0.09 0.21 0.48
a
Significant interaction between feed and sex (Table 9).
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and neutral lipids. The partial correlations were calculated after correcting for feed, sex, RN genotype, interactions and IMF content as described earlier.
3. Results In spite of the higher energy content in the feed of the outdoor pigs, as a compensation for higher energy expenditure, the intra muscular fat content in these pigs was lower than in the indoor pigs (Table 3). The intra muscular fat content influenced the monounsaturated (MUFA) and the highly polyunsaturated fatty acids (HUFA) of the neutral lipids (Tables 3, 4, 5). No effect of the intra muscular fat content on the fatty acid composition in the polar lipids was found.
outdoor pigs (7.13 and 0.65, respectively, Table 3). Furthermore, the level of unsaturation was higher in the neutral lipids of the female pig muscles compared with the castrated male pig muscle (Table 4). The differences between the castrated male pigs and the female pigs were seen in HUFA (1.40 and 1.21, respectively) and PUFA n-3 (1.01 and 0.80, respectively). However, there was a production systemsex interaction in the level of C 18:2 n-6 and HUFA n-3 fatty acids. The highest levels of these fatty acids were found in the indoor female pigs (Table 9). In the polar lipids the effect of production system could be seen in PUFA n-3 fatty acids with a higher level in the muscles from the indoor pigs compared with their outdoor counterparts (4.38 and 3.82, respectively, Table 3). 3.2. Fatty acid composition depending on RN genotype
3.1. Fatty acid composition depending on production system and sex The level of n-6 polyunsaturated fatty acids (PUFA n6) and n-3 polyunsaturated fatty acids (PUFA n-3) in the neutral lipid were higher in the muscle of the indoor pigs, (9.97 and 1.15, respectively) compared with the Table 8 Vitamin E and retinol content in muscle from pigs with different assumed RN-genotype (least-square means and pooled standard error) Antioxidant
Genotype rn+ (n=19) RN (n=21) S.E.
g/100 g meat a-Tocopherol g-Tocopherol Retinol g/g intra muscular fat a-Tocopherol g-Tocopherol Retinol
The RN genotype did not affect the levels of any major fatty acids in the neutral lipids. In the polar lipids differences were found. The level of PUFA n-3 fatty acids was higher in non-carrier pigs and in PUFA n-6 fatty acids in the RN-carrier group (Table 5). However, a sexRN genotype interaction was observed in the polar lipids, i.e. the differences between genotypes in PUFA n-3 fatty acids were seen in the castrated male pigs and the differences between genotypes in the PUFA n-6 were found in the female pigs (Table 6).
P-value
112.41 3.83 2.83
117.72 4.85 2.47
12.07 0.77 0.27 0.01 0.22 0.26
80.52 2.57 1.40
71.93 2.79 1.92
11.99 0.63 0.24 0.55 0.16 0.03
3.3. Contents of tocopherols, retinol and lipid oxidation The two production systems resulted in a different content of a-tocopherol. The outdoor pigs had a higher content of a-tocopherol in the muscles compared with the indoor pigs (Table 7). However, the effect of the production system was unevenly distributed within sex. Outdoor female pigs had the overall highest content of a-tocopherol compared with the indoor and outdoor castrated male pigs and the indoor female pigs (Table 9).
Table 9 Content of a-tocopherol, the degree of lipid oxidation and fatty acids of importance of neutral lipids (least-square means and pooled standard error) in muscle from female pigs and castrated male pigs reared indoors and outdoora Sex
Castrates
Rearing conditions
Indoors (n=9)
Outdoor (n=9)
Indoors (n=11)
Outdoor
a-Tocopherol (mg/100 g meat) MDA (ng/g meat) 18:1 n-9 18:2 n-6 18:3 n-3 MUFA HUFA HUFA n-3 PUFA
101.77a 97.07ab 41.84ab 7.65a 0.69a 49.62ab 1.26ab 0.29a 9.60a
102.12a 94.19ab 42.84a 5.65a 0.40c 50.78a 1.14a 0.22a 7.19a
92.30a 142.48a 40.19b 10.06b 0.92b 46.96b 1.57b 0.41b 12.55b
173.29b 65.82b 42.42a 6.77a 0.47c 50.95a 1.27ab 0.25a 8.51a
a b
Female pigs
S.E.
P-valueb
0.17 17.0 0.61 0.65 0.51 0.72 0.09 0.03 0.77
0.06 0.04 0.34 0.34 0.13 0.07 0.38 0.06 0.32
MDA, Malondialdehyde. Other abbreviations: see Table 2. Values with different letters within a row are significantly different (P <0.05). P-value for the interaction sexproduction system.
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Table 10 Overall and partiala correlations between a-tocopherol, MDA and some individual and groups of fatty acids of importance in polar and neutral lipids of pig muscle Overall correlation NL MDA 18:3 n-3 MUFA HUFAn-3 n-6/n-3 MDA
0.35 0.31 0.30 0.35
Partial correlation FL
atocopherol 0.24 0.15 0.06 0.47 0.43
MDA 0.42 0.01 0.08 0.02
NL atocopherol 0.49 0.08 0.12 0.26 0.43
MDA 0.06 0.02 0.10 0.09
PL atocopherol 0.07 0.11 0.29 0.23 0.39
MDA 0.31 0.16 0.15 0.24
atocopherol 0.30 0.06 0.13 0.01 0.39
a After correction for feed, sex, RN genotype, interactions and IMF content as described in Section 2.3. Correlation (r-value) >0.3; P<0.05; n=40 Abbreviations: See Table 2, MDA: malondialdehyde.
The content of g-tocopherol was found to be influenced both by the production system and the RN-genotype. Indoor pigs and carriers of the RN genotype had a higher content of g-tocopherol (Tables 7 and 8). Regarding lipid oxidation, a significant interaction (P=0.043) was seen between production system and sex. Results indicate that there was a higher degree of oxidation in the indoor female pigs in comparison to the outdoor female pigs after storage of the meat (Table 9). Furthermore, a negative global correlation was found between MDA and a-tocopherol (r= 0.43; P=0.005) and there was a positive correlation between MDA and PUFA n-3 fatty acids in both neutral lipids (r=0.35; P=0.02) and polar lipids (r=0.42; P=0.01; Table 10). In addition, a negative correlation was found between a-tocopherol and the fatty acid 18:3 n-3 in the polar lipids (r= 0.49; P=0.001).
4. Discussion The fatty acid composition of the intra muscular fat is affected by several factors, of which diet in general seems to be one of the most important (Nu¨rnberg, Wegner, & Ender, 1998). Increased levels of C22:6 n-3 and C20:5 n-3 in pig muscle have been shown in several earlier studies, in which different amounts of fish oil were included in the diet (Irie & Sakimoto, 1992; Lauridsen et al., 1999; Øverland et al., 1996). In our study, the amounts of C22:6 n-3 and C20:5 n-3 in the feed of the indoor pigs were quite low compared to other studies (Lauridsen et al., 1999; Table 2). However an increased level of these fatty acids in the polar and neutral lipids of the meat in the indoor pigs fed feed 1 was found. We suggest that the found difference in the content of C18:3 n-3 in the diets (Table 2) could be one cause of this effect. Thus, the increased levels of C22:6 n-3 and C20:5 n-3 might be a result of elevated elongation and desaturation of C18:3 n-3. Enser, Richardson, Wood, Gill, and Sheard (2000) have shown that an ele-
vated level of C18:3 n-3, increased the level of C22:6 n-3 and C20:5 n-3 in the polar lipids of pigs. However, it is not well established to which extent C18:3 n-3 is further elongated and desaturated to C22:6 n-3 (Bowen & Clandinin, 2000). It has been shown that the content of polyunsaturated fatty acids in the diet affects the oxidation rate of the final meat product (Leskanich et al., 1997). In our study the MDA content of the meat was influenced by the more unsaturated fatty acid profile in the indoor pigs fed feed 1. We have shown that a correlation between the content HUFA n-3 and MDA as well as between 18:3 n-3 and MDA (Table 10). Thus, we suggest that the oxidative effect can partly be ascribed to the overall increased level of PUFA n-3. Especially C22:6 n-3 and C20:5 n-3 are known to have a high oxidation rate (Cosgrove, Church, & Pryor, 1987). An additional oxidative effect is probably also caused by the higher amount of C18:3 n-3, even if this fatty acid has a lower oxidation rate than the fatty acids mentioned above. The correlation between an oxidative parameter as MDA and an vitamin with antioxidative properties as tocopherol has also been shown in several earlier studies (Corino, Oriani, Pantaleo, Pastorelli, & Salvatori, 1999). In our study the content of a-tocopherol differed between female pigs in the different production systems and there was an overall negative correlation between a-tocopherol and MDA. We suggest this to be an additional factor explaining the difference in susceptibility to oxidation. However, it is intricate to explain the increased level of polyunsaturated fatty acids of the neutral lipids in the female indoor pigs and no increased level in the castrated male indoor pigs (Table 9). One hypothetical explanation might be the possible difference in metabolism between castrated male and female pigs, as suggested in the study of Ho¨gberg et al. (2001). The castration and thereby possible loss of metabolisc functions in connection to puberty might affect the elongation and desaturation activity and rate of C18:2 n-6 and C18:3 n-3 towards their corresponding HUFA in
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castrated male pigs. Thus, the suggested difference in metabolic activity, in addition to the relatively low content of a-tocopherol in the indoor female pig muscle, might contribute to the increased susceptibility of the indoor female pig muscle to post-mortem oxidation, in this study shown as increased MDA content. The level of g-tocopherol in pig muscle was relatively low compared with a-tocopherol in our study. However, in our study the content of g-tocopherol was higher in the indoor pig muscle, probably due to a higher dietary content of g-tocopherol in the feed of the indoor pigs. The dietary influence g-tocopherol has been previously reported by Rey et al. (1998). Ho¨gberg et al. (2001) have shown that grazing increased the level of PUFA n-3 fatty acids in the neutral lipids but not in the polar lipids. In the present study the level of PUFA n-3 fatty acids were increased in both neutral and polar lipid fraction in the indoor pig muscle. One plausible explanation for this effect could be the more pronounced difference in the level of PUFA n-3 fatty acids between the diets of the indoor and the outdoor pigs in our study, compared with the difference between the diets in Ho¨gberg et al. (2001). However, the dietary influence was greater in the neutral lipids compared with the polar lipids, as the level of C18:3 n-3 was doubled in the neutral lipids. Similar results have also been reported in other studies in which the level of C18:3 n-3 was more prone to change in the triacylglycerols than in the phospholipids of pig meat (Leskanich, 1999). Enser et al. (2000) showed that an increased dietary level of C18:2 n-6 resulted in an increased level of this fatty acid in both neutral and polar lipids. We found increased level of C18:2 n-6 only in neutral lipids of the muscles from the indoor pigs fed feed 1, and this effect was more pronounced in the female pigs. Some possible explanations for the increased level of C18:2 n-6 are as follows. (1) Dietary level of C18:2 n-6 was higher in our dietary treatments when compared with other studies (Enser et al., 2000). (2) It is possible that a certain level of C18:2 n-6 is needed and will be included in the membrane while additional C18:2 n-6 will not affect the membrane composition. Thus, the excess of C18:2 n-6 will be stored in the neutral lipids or undergo b-oxidation. Becker and Bruce (1986) have shown that C18:2 n6 was preferentially b-oxidised when rats were fed a high C18:2 n-6 diet. This has also been found in fish (Kiessling & Kiessling, 1993). (3) Another explanation might be that the content of C18:3 n-3 in both diets in our study was lower compared with other studies (Enser et al., 2000). The low content of C18:3 n-3 might result in a demand for this fatty acid causing an incorporation of all C18:3 n-3 available. Fu and Sinclair (2000) suggested a conservation of C18:3 n-3 when fed in low amounts at the expense C18:2 n-6. Thus, it is possible that the unaffected level of C18:2 n-6 in the polar lipids
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in the present study is a consequence of the low dietary level of C18:3 n-3. Pigs carrying the dominant RN allele have a higher muscle glycogen content which will affect muscle quality characteristics of the final meat products (Enfa¨lt et al., 1997). In the present study, we found that the difference in the fatty acid composition between the RN genotypes arose in the polar lipids of M. Longissimus dorsi. Additionally, the difference in fatty acid composition could be seen not only in the PUFA n-3 fatty acids of polar lipids, as mentioned above (Nilze´n et al., 2001) but also in the PUFA n-6 fatty acids of the polar lipids. However, there was a significant interaction between RN genotype and sex in the polar lipids (Table 6). These differences in the level of polyunsaturated fatty acids might indicate the metabolic importance of the PUFA n-3 and PUFA n-6 fatty acids. Clarke (2000) hypothesises that n-3 polyunsaturated fatty acids play an essential role in the maintenance of energy balance and glucose metabolism. Considering suggestions of Clarke (2000), the results of the present study might indicate that polyunsaturated fatty acids can be involved in the mechanism leading to the increased glycogen content of M. Longissimus dorsi in meat of RN carriers. However, the nature of this relationship between glycogen content and polyunsaturated fatty acids needs to be further investigated. In conclusion, both diet and sex influenced the level of oxidation of meat in this study. However, more research is needed to establish whether this difference is due to the balance between antioxidants and polyunsaturation or other metabolic events in the live pig. The fatty acid composition in the diet affected the neutral and polar lipid fraction differently. Finally, the RN genotype was found to influence the fatty acid composition in the polar lipids of pig muscle. Further investigations of the interactions between RN genotype and the fatty acid composition are needed. Two hypotheses can possibly be considered: (1) The difference in membrane fatty acid composition affects the glycogen metabolism in pigs. (2) The RN allele affects the incorporation of fatty acids in membrane lipids. References Andersson, K. (1985). SLU-normen en ny utfodringsnorm till slaktsvin. Konsulentavdelningen, Swedish Univ of Agricultural Sciences, Allma¨nt, 67, 2 1-2:8. Becker, W., & Bruce, A˚. (1986). Retention of linoleic acid in carcass lipids of rats fed different levels of essential fatty acids. Lipids, 21, 121–126. Boisen, S., & Fernandez, J. A. (1997). Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses. Animal Feed Science and Technology, 68, 277–286. Bowen, R. A. R., & Clandinin, M. T. (2000). High dietary 18: 3n-3 increases the 18: 3n-3 but not the 22: 6n-3 content in the whole body, brain, skin, epididymal fat pads, and muscles of suckling rat pups. Lipids, 35, 389–394.
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Børsting, C. F., Engberg, R. M., Jakobsen, K., Jensen, S. K., & Andersen, J. O. (1994). Inclusion of oxidised fish oil in mink diets 1. The influence on nutrient digestibility and fatty-acid accumulation in tissues. Journal of Animal Physiology and Animal Nutrition, 72, 132–145. Clarke, S. D. (2000). Polyunsaturated fatty acid regulation of gene transcription: a mechanism to improve energy balance and insulin resistance. British Journal of Nutrition, 83, 59–66. Corino, C., Oriani, G., Pantaleo, L., Pastorelli, G., & Salbatori, G. (1999). Influence of dietary vitamin E supplementation on ‘‘heavy’’ pig carcass characteristics, meat quality, and vitamin E status. Journal of Animal Science, 77, 1755–1761. Cosgrove, J. P., Church, D. F., & Pryor, W. A. (1987). The kinetics of the autoxidation of polyunsaturated fatty-acids. Lipids, 22, 299–304. Dalrymple, R. H., & Hamm, R. (1973). A method for the extraction of glycogen and metabolites from a single muscle sample. Journal of Food Technology, 8, 439–444. Draper, H. H., Squires, E. J., Mahmoodi, H., Agarwal, J. W. S., & Hadley, M. (1993). A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radical Biology and Medecine, 15, 353–363. Enfa¨lt, A. C., Lundstro¨m, K., Karlsson, A., & Hansson, I. (1997). Estimated frequency of the RN- allele in Swedish Hampshire pigs and comparison of glycolytic potential, carcass composition, and technological meat quality among Swedish Hampshire, Landrace, and Yorkshire pigs. Journal of Animal Science, 75, 2924–2935. Enser, M., Richardson, R. I., Wood, J. D., Gill, B. P., & Sheard, P. R. (2000). Feeding linseed to increase the n-3 PUFA of pork: fatty acid composition of muscle, adipose tissue, liver and sausages. Meat Science, 55, 201–212. Fu, Z., & Sinclair, A. J. (2000). Increased alpha-linolenic acid intake increases tissue alpha-linolenic acid content and apparent oxidation with little effect on tissue docosahexaenoic acid in the guinea pig. Lipids, 35, 395–400. Henderson, R. J., & Tocher, D. R. (1987). The lipid-composition and biochemistry of fresh-water fish. Progress in Lipid Research, 26, 281–347. Hoppe, P. P., Schoner, F. J., & Frigg, M. (1992). Effects of dietary retinol on hepatic retinol storage and on plasma and tissue alphatocopherol in pigs. International Journal for Vitamin and Nutrition Research, 62, 121–129. Ho¨gberg, A., Pickova, J., Dutta, P. C., Babol, J., & Bylund, A. C. (2001). Effect of rearing system on muscle lipids of gilts and castrated males. Meat Science, 58, 223–229. Irie, M., & Sakimoto, M. (1992). Fat characteristics of pigs fed fish oil containing eicosapentaenoic and docosahexaenoic acids. Journal of Animal Science, 70, 470–477. Jensen, C., Lauridsen, C., & Bertelsen, G. (1998). Dietary vitamin E: quality and storage a stability of pork and poultry. Trends in Food Science and Technology, 9, 62–72.
Jensen, S. K., Jensen, C., Jakobsen, K., Engberg, R. M., Andersen, J. O., Lauridsen, C., Sorensen, P., Skibsted, L. H., & Bertelsen, G. (1998). Supplementation of broiler diets with retinol acetate, betacarotene or canthaxanthin: effect on vitamin status and oxidative status of broilers in vivo and on meat stability. Acta Anaesthesiologica Scandinavica, 48, 28–37. Kiessling, K. H., & Kiessling, A. (1993). Selective utilization of fattyacids in rainbow-trout (Oncorhynchus-mykiss walbaum) red muscle mitochondria. Canadian Journal of Zoology, 71, 248–251. Lauridsen, C., Andersen, G., Andersson, M., Danielsen, V., Engberg, R., & Jakobsen, K. (1999). Effect of dietary fish oil supplied to pigs from weaning to 60 kg liveweight on performance, tissue fatty acid composition and palatability of pork when slaughtered at 100 kg liveweight. Journal of Animal and Feed Sciences, 8, 441–456. Leskanich, C. O. (1999). The comparative roles of polyunsaturated fatty acids in pig neonatal development. British Journal of Nutrition, 81, 87–106. Leskanich, C. O., Matthews, K. R., Warkup, C. C., Noble, R. C., & Hazzledine, M. (1997). The effect of dietary oil containing (n-3) fatty acids on the fatty acid, physicochemical, and organoleptic characteristics of pig meat and fat. Journal of Animal Science, 75, 673–683. Lynch, G. L. (1991). Natural occurance and content of vitamin E in feedstuffs. Vitamin E in animal nutrition and management. In (pp. 43–48). Parsippany, NJ: BASF Corporation. Minitab. USA: Minitab Inc. Milan, D., Jeon, J. T., Looft, C., Amarger, V., Robic, A., Thelander, M., Rogel-Gaillard, C., Paul, S., Iannuccelli, N., Rask, L., Ronne, H., Lundstro¨m, K., Reinsch, N., Gellin, J., Kalm, E., Le Roy, P., Chardon, P., & Andersson, L. (2000). A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle. Science, 288, 1248–1251. Nilze´n, V., Babol, J., Lundeheim, N., Enfa¨lt, A., & Lundstro¨m, K. (2001). Free range rearing of pigs with access to pasture grazing— effect on fatty acid composition and lipid oxidation product. Meat Science, 58, 267–275. Nu¨rnberg, K., Wegner, J., & Ender, K. (1998). Factors influencing fat composition in muscle and adipose tissue of farm animals. Livestock production science, 56, 145–156. Rey, A. I., Isabel, B., Cava, R., & Lopez-Bote, C. J. (1998). Dietary acorns provide a source of gamma-tocopherol to pigs raised extensively. Canadian Journal of Animal Science, 78, 441–443. O¨hrvall, M., Tengblad, S., Ekstrand, B., Siegbahn, A., & Vessby, B. (1994). Malondialdehyde concentration in plasma is inversely correlated to the proportion of linoleic-acid in serum-lipoprotein lipids. Atherosclerosis, 108, 103–110. Øverland, M., Taugbøl, O., Haug, A., & Sundstøl, E. (1996). Effect of fish oil on growth performance, carcass characteristics, sensory parameters, and fatty acid composition in pigs. Acta Agriculturae Scandinavica Section A Animal Science, 46, 11–17.
Muscle lipids, vitamins E and A, and lipid oxidation as affected by diet and RN genotype in female and castrated male Hampshire crossbreed pigs A. Ho¨gberga,*, J. Pickovaa, J. Babola, K. Anderssonb, P.C. Duttaa a Department of Food Science, Swedish University of Agricultural Sciences, PO Box 7051, S-75007 Uppsala, Sweden Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, PO Box 7051, S-75007 Uppsala, Sweden
b
Received 4 March 2001; received in revised form 26 May 2001; accepted 25 June 2001
Abstract The objectives of this study were to investigate the polar and neutral lipid fatty acid composition, content of retinol, a-tocopherol, g-tocopherol and level of oxidation of the pig muscle (M. longissimus dorsi). Female and castrated male Hampshire crossbreeds were produced in two systems. One group was raised indoors with a more polyunsaturated diet and the other raised outdoors with a more saturated diet. The level of polyunsaturated fatty acids in muscle was higher in the indoor females compared with the outdoor females, indoor castrated males and outdoor castrated males. The increased level of polyunsaturated fatty acids in the muscle, which was accompanied by a relatively low content of a-tocopherol, increased the susceptibility to lipid oxidation in the form of MDA (malondialdehyde) in the indoor female pigs. Finally, the level of polyunsaturated fatty acids in the polar lipids was affected by the RN genotype, and this difference was dependent on sex. In conclusion, diet has a major effect on the fatty acid composition and oxidation stability in pork muscle, but additional factors such as sex and RN genotype might also contribute. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Malondialdehyde; Pork; Retinol; RN-genotype; Sex; Tocopherol
1. Introduction Fatty acid composition of feed is known to affect the quality of pig meat. Fatty acid composition as well as the oxidation stability of pig muscle, especially the effect of different feeding regimes, have been intensively studied (Irie & Sakimoto, 1992; Lauridsen, Andersen, Andersson, Danielsen, Engberg, & Jakobsen, 1999; Leskanich, Matthews, Warkup, Noble, & Hazzledine, 1997; Øverland, Taugbøl, Haug, & Sundstøl, 1996). However, in most of these studies, total lipids of the muscles have been investigated, not considering a possible difference among lipid fractions. Polyunsaturated fatty acids (PUFA) of polar lipids, especially C20 fatty acids, are important constituents of membranes and they function as precursors in the eicosanoid metabolism, whereas the neutral lipids serve mainly as a depot * Corresponding author. Tel.: +46-18-672011; fax: +46-18673080. E-mail address: [email protected] (A. Ho¨gberg).
of lipids used as energy source (Henderson & Tocher, 1987). Due to different functions, it is likely that different fatty acids are incorporated differently into lipid classes. Thus, it has been shown that the level of C18:3 n-3 was more susceptible to change due to diet in the neutral lipid fraction than in the polar lipid fraction (Ho¨gberg, Pickova, Dutta, Babol, & Bylund, 2001; Leskanich, 1999). Fatty acid composition of the muscle has been shown to be affected by other factors such as sex and RN genotype. In a previous study (Ho¨gberg et al., 2001), we observed that the fatty acid profile of the polar lipids was more polyunsaturated in female pigs compared with castrated male pigs. This indicated a difference in fatty acid metabolism between castrated male pigs and female pigs. However, an interaction between sex and rearing system was also found in the same study (Ho¨gberg et al., 2001). The RN genotype gives an increased content of glycogen in pig muscle (Enfa¨lt, Lundstro¨m, Karlsson, & Hansson, 1997). In addition, Nilze´n, Babol, Lundeheim,
0309-1740/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(01)00153-X
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Enfa¨lt, and Lundstro¨m (2001) have shown that the RN genotype influenced the fatty acid composition in pig muscle. Pigs with the RN allele had a higher level of n-3 polyunsaturated fatty acids in the muscle. Therefore, it is of interest to further study the effects of the RN genotype effects on the fatty acid composition of different lipid classes. Finally, when measuring changes in fatty acid composition, it is important to measure the content of antioxidants, vitamins and the level of oxidation in the meat. Among the antioxidants a-tocopherol has been recognised as one of the most important for preventing oxidation changes in meat (Jensen, Lauridsen, & Bertelsen, 1998). g-Tocopherol is also a potent antioxidant (Lynch, 1991; Rey, Isabel, Cava, & Lopez-Bote, 1998), whereas retinol is known to have an impact on a-tocopherol status resulting in its decrease (Hoppe, Schoner, & Frigg, 1992). The objectives of this study were to investigate how different lipid classes in pig muscle are affected by dietary fatty acids, RN genotype and sex using commonly produced genders, castrated males and female pigs. In addition, the content of retinol, a-tocopherol, g-tocopherol and the level of lipid oxidation were studied.
2. Materials and methods 2.1. Animals and treatments The pigs were raised at the Swedish University of Agricultural Sciences, Experimental Station in Bjertorp, Sweden, from 28 to 107 kg live weight. The growing/ finishing period span from July to November. The animals studied were a subsample of 40 animals selected from a total of 80 crossbreeds [(Swedish LandraceSwedish Yorkshire)(Hampshire)]. The animals were divided in two groups of 40 animals each. In production system 1 (further on called indoors) pigs were raised indoors in five pens, eight animals in each, and fed feed 1 (Tables 1 and 2). In production system 2 (further on called outdoor) pigs were raised outdoors together in one group in an area of about 6000 m2 clay soil overgrown with grass. The outdoor pigs were given feed 2 (Tables 1 and 2). Feed 2 had an Table 1 Analysed nutrient content of diets to the indoor (feed 1) and outdoor pigs (feed 2)
MJ/kg Crude protein (%) Lysine (%) Metionine (%) Treonine (%)
Feed 1
Feed 2
12.0 15.0 0.80 0.22 0.53
12.6 16.8 0.86 0.28 0.60
intentionally higher energy content than feed 1 to compensate for the increased energy expenditure for the outdoor environment. The outdoor pigs had access to huts with straw. All pigs were fed restrictedly in accordance with the standard feeding regimen for growing pigs in Sweden (Andersson, 1985). The daily feed allowances for the outdoor pigs in MJ of ME (metabolisable energy) were 16.5, 19.0, 24.1, 29.0 at 25, 30, 40, 50, 60 kg live weight, respectively, and 34.1 from 60 kg until slaughter. The feed allowances for the indoor pigs were 5% less than Table 2 Fat content, fatty acid composition, vitamin E content and retinol content of diets fed to indoor (feed 1) and outdoor pigs (feed 2)a Feed 1
Feed 2
Fat content (%) 12:0 (mg/100 g) 14:0 Unknown 16:0 16:1 tr 16:1 17:0 Unknown 18:0 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2 tr 18:2 n6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 22:0 22:1 20:5 n-3 24:0 24:1 22:6 n-3
5.3 56.4 43.6 1.4 791.2 3.3 16.8 5.9 0.6 247.0 26.1 1410.0 83.5 7.4 1.7 1541.3 1.4 177.7 17.9 30.8 3.1 10.8 8.5 2.5 7.3 3.6 3.0
3.4 12.1 13.9 0.5 475.1 1.7 9.5 2.9 0.0 80.6 5.6 918.1 44.9 1.8 0.6 1183.1 0.8 88.2 8.0 22.1 1.7 6.7 3.5 nd 4.6 2.1 nd
SAFA (mg/100 g) MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans n-6/n-3
1176.3 1560.7 10.1 4.5 5.5 1729.1 1545.8 183.2 31.1 8.4
601.3 1002.0 2.6 2.5 nd 1273.9 1185.7 88.2 7.9 13.4
19.9 9.0 7.3
25.3 4.9 8.1
a-Tocopherol g-Tocopherol Retinol (mg/g) a
SAFA, saturated fatty acids; MUFA, mono saturated fatty acids; PUFA, polyunsaturated fatty acids; HUFA, highly polyunsaturated fatty acids (18 carbon atoms or more not including 18:2 n-6 and 18:3 n-3); nd, not detectable.
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duction system and taken from the front part of M. Longissimus dorsi (LD) 48 h post-mortem and were frozen and stored at 80 C until analysis. Samples for analysis of MDA were collected from the same muscle and stored at 20 C for 12 months.
for the outdoor pigs. The lysine level was approximately 0.85% of air-dried feed and the total amounts of methionine and threonine were calculated to be 31 and 65% of the dietary lysine level respectively (Table 1). The amount of feed was adjusted every 2nd week depending on the mean weight of each group. The pigs were slaughtered at a commercial abattoir (Scan Farmek, Skara, Sweden). The carcasses were submitted to standard slaughter procedures and divided into joints 2 days after slaughter. Samples were chosen randomly within each sex and RN genotype and pro-
2.2. Analytical procedures The assumed RN genotype of each individual pig was determined with the method of Dalrymple and Hamm (1973), by enzymatic determination of the concentration
Table 3 Intramuscular fat content and fatty acid composition (%, least squares mean and standard error) in neutral and polar lipids of intramuscular fat from indoor (feed 1) and outdoor (feed 2) pigs Fatty acid
(a) Neutral lipid
(b) Polar lipid
Indoor (n=20)
Outdoor (n=20)
S.E.
P-value
IMF 12:0 14:0 Unknown 16:0 16:1 9 tr 16:1 n-7 17:0 18:0 Unknown 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2 9.12tr 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 20:3 n-6 20:4 n-6 20:3 n-3 20:5 n-3 24:0 22:4 n-6 22:5 n-3 22:6 n-3
1.88 Trace 1.34 nd 23.08 0.19 2.76 0.21 12.08 nd 0.24 40.73 3.49 nd 0.14 8.92 Trace 0.82 0.21 0.77 0.38 Trace 0.39 Trace Trace nd Trace Trace Trace
1.55 Trace 1.36 nd 23.86 0.21 3.23 0.19 12.03 nd 0.18 42.93 4.02 nd 0.15 6.16 Trace 0.43 0.22 0.81 0.28 Trace 0.38 Trace Trace nd Trace Trace Trace
0.12
0.05
0.02 nd 0.34 0.03 0.09 0.01 0.31 nd 0.02 0.41 0.07 nd 0.01 0.46
0.65 nd 0.12 0.71 0.001 0.26 0.62 nd 0.06 0.001b,c 0.001 nd 0.13 0.001b
0.04 0.01 0.02 0.02
0.001b 0.76 0.13 0.001
0.02 Trace Trace nd
0.78c Trace Trace Nd
SAFA MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans
37.10 47.84 1.46 1.09 0.35 11.15 9.97 1.15 0.57
38.03 51.31 1.15 1.00 0.23 7.79 7.13 0.65 0.54
0.63 0.50 0.06 0.5 0.01 0.55 0.50 0.05 0.04
0.31 0.001b,c 0.001b,c 0.23 0.001b 0.001b 0.001 0.001 0.59
8.62
10.95
0.17
0.001
n-6/n-3
Indoor (n=20)
Outdoor (n=20)
S.E.
P-value
nd Trace 1.15 19.04 0.15 0.27 0.25 8.69 4.31 nd 9.12 2.59 2.67 0.26 30.34 0.27 0.82 nd Tracea 0.48 0.95 9.30 0.20 0.80 Trace 1.23 1.78 0.79
nd Trace 1.43 20.18 0.20 0.51 0.29 7.50 3.41 nd 9.40 2.64 2.47 0.30 30.67 0.33 0.72 nd Trace 0.44 0.98 9.40 0.20 0.63 Trace 1.52 1.73 0.52
nd
nd
0.34 0.43 0.02 0.02 0.01 0.19 0.28 nd 0.25 0.07 0.19 0.02 0.39 0.01 0.02 nd
0.56 0.07 0.10 0.001 0.01 0.03 0.03 nd 0.43 0.58 0.48 0.21 0.57 0.001 0.003 nd
0.02 0.02 0.25 0.01 0.24
0.07 0.41 0.79 0.46 0.001
0.04 0.06 0.03
0.001 0.56 0.000
28.24 14.90 15.83 13.85 3.56 46.94 42.56 4.38 0.50
28.24 15.26 15.70 13.89 3.10 47.16 43.34 3.82 0.55
0.05 0.35 0.35 0.31 0.09 0.52 0.47 0.09 0.03
1.00 0.47 0.79 0.93 0.002 0.77 0.27 0.00 0.18
9.87
11.41
0.23
0.001
Trace: Identified fatty acids below 0.15% are shown as trace but included in sum of SAFA, PUFA n-6, PUFA n-3, HUFA n-6 and HUFA n-3, respectively. a Abbreviations: see Table 2. b Shown in Table 9 for each sex in the outdoor and indoor rearing system. c Significant effect of the intramuscular fat content when included as a covariate (P <0.05).
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of residual glycogen (including glucose and glucose-6phosphate) in homogenised muscle tissue. Animals with a concentration less than 35 mmol/mg of M. Longissimus dorsi were considered as not carrying the RN allele, while animals with a concentration above 35 mmol/mg were considered as carriers of the RN allele. Since there is some overlap in the distribution of glycogen content between RN and rn+ animals, a diagnostic DNA test for the causative mutation (Milan et al., 2000) was carried out on the four individuals with glycogen concentrations between 30 and 40 mmol to avoid misclassifications.
The intra muscular fat content (IMF) of M. Longissimus dorsi (LD) was analysed with Soxhlet analysis (Soxtec System H+ equipment, Tecator AB, Ho¨gana¨s, Sweden). In the Soxhlet analysis and for all analyses below, meat without connective tissue and subcutanous fat was used. For fatty acid analyses the method was used as described by Ho¨gberg et al. (2001). In brief, 215 g of minced muscle were homogenised and total lipids were separated into neutral lipid and polar lipid fractions. Fatty acid methyl esters (FAME) from both neutral and polar lipids were prepared before gas chromatograph
Table 4 Fatty acid composition (%, least squares mean and standard error) of neutral and polar lipids in intramuscular fat from castrated male pigs and female pigsa Fatty acid
IMF 12:0 14:0 Unknown 16:0 16:1 9 tr 16:1 n-7 17:0 18:0 Unknown 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2tr 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 20:3 n-6 20:4 n-6 20:3 n-3 20:5 n-3 24:0 22:4 n-6 22:5 n-3 22:6 n-3 SAFA MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans n-6/n-3 a b c d
(a) Neutral lipid
(b) Polar lipid
Castrates (n=18)
Female pigs (n=22)
S.E.
1.76 Trace 1.34 nd 23.59 0.17 3.04 0.20 12.48 nd 0.21 42.28 3.80 nd 0.15 6.65 Trace 0.55 0.21 0.80 0.31 Trace 0.35 Trace Trace nd Trace Trace Trace
1.67 Trace 1.36 nd 23.34 0.23 2.95 0.20 11.91 nd 0.21 41.38 3.72 nd 0.15 8.43 Trace 0.70 0.21 0.77 0.36 Trace 0.41 Trace Trace nd Trace Trace Trace
38.00 50.13 1.21 0.96 0.26 8.40 7.58 0.80 0.53
37.13 49.03 1.40 1.14 0.33 10.54 9.52 1.01 0.58
0.61 0.48 0.06 0.05 0.01 0.54 0.49 0.04 0.04
9.77
9.77
0.16
Castrates (n=18)
Female pigs (n=22)
S.E.
P-value
nd Trace 1.29 19.75 0.18 0.43 0.30 7.95 3.82 nd 9.74 2.61 2.64 0.26 30.55 0.31 0.82 nd 0.18 0.45 0.97 8.79 0.19 0.71 Trace 1.37 1.76 0.58
nd Trace 1.30 19.45 0.17 0.35 0.25 8.25 3.90 nd 8.77 2.62 2.50 0.30 30.46 0.30 0.72 nd 0.16 0.46 0.95 9.90 0.21 0.72 Trace 1.38 1.75 0.74
nd
nd
0.33 0.42 0.02 0.02 0.01 0.18 0.28 nd 0.25 0.07 0.19 0.02 0.39 0.01 0.02 nd 0.01 0.02 0.02 0.02 0.01 0.23
0.98 0.63 0.66 0.01 0.001 0.26 0.83 nd 0.01 0.90 0.63 0.25 0.86d 0.45 0.004 nd 0.03 0.78 0.48 0.003 0.13 0.83d
0.32 0.11b,c 0.02b,c 0.03 0.004b 0.01b 0.01 0.002 0.39
28.26 15.75 15.14 13.31 3.24 46.51 42.44 4.07 0.51
0.90
10.57
P-value 0.12
0.02 nd 0.33 0.03 0.09 0.01 0.31 nd 0.20 0.39 0.07 nd 0.01 0.05
0.61 0.52 nd 0.61 0.22 0.50 0.76 0.20 nd 1.00 0.11b,c 0.41 nd 0.86 0.01b
0.04 0.01 0.02 0.02 Trace 0.02 Trace
0.01b 0.80 0.14 0.04 Trace 0.07c Trace
nd
nd
Abbreviations, see Tables 2 and 3. Shown in Table 9 for each sex in the free-range and indoor rearing system. Significant effect of the intramuscular fat content when included as a covariate (P <0.05). Significant interaction between sex and RN genotype. See Table 6.
0.04 0.06 0.03
0.78 0.86 0.002d
28.21 14.40 16.39 14.42 3.41 47.59 43.45 4.14 0.53
0.48 0.35 0.34 0.30 0.09 0.49 0.45 0.09 0.25
0.94 0.01 0.02 0.01 0.21d 0.14d 0.12d 0.60d 0.61
10.72
0.22
0.65d
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For the analysis of retinol and a- and g-tocopherol a modified method by Jensen et al. (1998) was used. Two times one gram of meat, was cut into pieces and homogenised in two tubes. 1.2 ml of 20% ascorbic acid solution, 0.6 ml methanol and 1.2 ml of KOH-water (1:1) were added to each tube. After saponification and cooling, retinol and tocopherols were extracted in 2 times 4 ml of hexane. The hexane-vitamin solution was evaporated under nitrogen gas and diluted with the mobile phase. The mobile phase used for retinol and tocopherols
(GC) analyses. The peaks of GC chromatograms were analysed by comparison with authentical standards (GLC-68 A, Nu-Chek Prep, Elysian, USA) as described by Ho¨gberg et al. (2001). MDA, retinol and tocopherols was determined with HPLC using a Merck Hitachi L6200A pump, a FL Lt480 detector and an As-2000 A auto sampler. The HPLC column was a 4.0250 mm RP-18 LiChroCART. Identification and quantification were done by using external standards.
Table 5 Fatty acid composition (least squares mean and standard error) of neutral and polar lipids in intramuscular fat from pigs with different RN genotypea Fatty acid
(a) Neutral lipid +
rn
(n=19)
(b) Polar Lipid RN (n=21)
S.E.
rn+ (n=19)
RN (n=21)
S.E.
P-value
nd Trace 1.31 19.74 0.16 0.46 0.27 7.79 4.18 nd 9.70 2.30 2.57 0.28 29.52 0.28 0.75 nd Trace 0.44 1.01 9.51 0.19 0.76 Trace 1.35 1.82 0.71
nd Trace 1.27 19.47 0.20 0.32 0.28 8.40 3.54 nd 8.81 2.92 2.57 0.28 31.48 0.32 0.79 nd Trace 0.47 0.91 9.18 0.21 0.67 Trace 1.40 1.69 0.60
nd
nd
0.03 0.43 0.02 0.02 0.02 0.02 0.28 nd 0.25 0.07 0.02 0.02 0.39 0.01 0.02 nd
0.93 0.66 0.19 0.001 0.23 0.03 0.11 nd 0.02 0.001 1.00 0.99 0.001 0.05 0.21 nd
0.02 0.02 0.25 0.01 0.24
0.09 0.001 0.37 0.03 0.02c
0.04 0.06 0.34
0.46 0.12 0.03c
IMF 12:0 14:0 Unknown 16:0 16:1 9 tr 16:1 n-7 17:0 18:0 Unknown 18:1 tr 18:1 n-9 18:1 n-7 18:1 n-5 18:2 9.12tr 18:2 n-6 18:3 n-6 18:3 n-3 20:0 20:1 20:2 n-6 20:3 n-6 20:4 n-6 20:3 n-3 20:5 n-3 24:0 22:4 n-6 22:5 n-3 22:6 n-3
1.58 Trace 1.35 nd 23.16 0.16 2.98 0.20 12.36 nd 0.23 42.03 3.71 nd 0.14 7.71 Trace 0.64 0.21 0.76 0.33 Trace 0.36 Trace Trace nd Trace Trace Trace
1.85 Trace 1.36 nd 23.78 0.24 3.02 0.20 12.02 nd 0.20 41.63 3.80 nd 0.15 7.36 Trace 0.61 0.21 0.82 0.34 Trace 0.41 Trace Trace nd Trace Trace Trace
SAFA MUFA HUFA HUFA n-6 HUFA n-3 PUFA PUFA n-6 PUFA n-3 Trans
37.39 49.61 1.24 1.02 0.28 9.63 8.69 0.92 0.52
37.74 49.54 1.38 1.07 0.30 9.31 8.41 0.88 0.59
0.63 0.50 0.06 0.06 0.02 0.55 0.50 0.05 0.04
0.70 0.92b 0.11b 0.51 0.56 0.68 0.69 0.63 0.31
28.06 15.28 15.10 14.13 3.48 46.35 42.12 4.23 0.52
28.42 14.87 15.43 13.60 3.17 47.75 43.78 3.97 0.53
0.49 0.35 0.35 0.31 0.09 0.41 0.46 0.09 0.03
0.61 0.40 0.19 0.24 0.01c 0.07c 0.02c 0.06c 0.90
9.85
9.71
0.17
0.57
10.11
11.18
0.23
0.003c
n-6/n-3 a b c
0.12
P-value
0.02 nd 0.34 0.03 0.10 0.01 0.03 nd 0.02 0.41 0.07 nd 0.01 0.05
0.12 0.75 nd 0.21 0.13 0.76 0.88 0.47 nd 0.29 0.50b 0.39 Nd 0.24 0.59
0.04 0.01 0.02 0.02
0.48 0.94 0.01 0.78
0.02
0.12b
nd
nd
Abbreviations: see Tables 2 and 3. Significant effect of the intramuscular fat content when included as a covariate (P<0.05). Significant interaction between sex and RN genotype. See Table 6.
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extracted according to the method of Børsting, Engberg, Jakobsen, Jensen, and Anderssen (1994) and analysed by HPLC.
consisted of 95% metanol:acetonitrile (1:1) and 5% chloroform with a flow rate of 1.2 ml/min. Tocopherols and retinol were detected with excitation wavelengths of 290 and 344 nm, respectively, and with emission wavelengths of 327 and 472 nm, respectively. For MDA (malondialdehyde) analysis the method of Draper, Squires, Mahmoodi, Agarwal, and Hadley (1993; method 4) was used for preparing the meat samples before HPLC analysis. Two times one gram of meat was homogenised and after preparation 300 ml of MDA/butanol solution was diluted 10 with the mobile phase. HPLC analysis was performed according to the method of O¨hrvall, Tengblad, Ekstrand, Siegbahn, and Vessby (1994; excitation 532 nm and emission 553 nm). A mobile phase consisting of 60% potassium phosphate buffer (50 mM, pH 6.8) and 40% methanol with a flow rate of 0.7 ml/min was used. The analyses of the feed were performed according to the following: the energy analysis was performed using the EFOS method as described by Boisen and Fernandez (1997). The fatty acid composition of the feed was analysed as described above after grinding of the feed. Retinol and tocopherols of the feed 1 and 2 were
2.3. Statistical analysis Statistical evaluation was carried out with Minitab statistical software for Windows 95 and NT, version 12 (Minitab, 1998) on the mean values of the duplicate samples. Production system, sex and RN genotype were regarded as fixed effects of the experiment and were tested by analysis of variance (GLM procedure), and different groups were compared on the basis of leastsquares means. Intramuscular fat content (IMF) was included as covariate in both neutral lipids and polar lipids. In the case of interaction between fixed effects, pair-wise comparisons were made, using Tukey’s method. If a covariate or an interaction failed to reach significance (P>0.05), the term was excluded from the model, except for some of the fatty acids in Table 9. Finally, Pearson overall and partial correlation coefficients were calculated between a-tocopherol, MDA and certain individual and groups of fatty acids of importance in polar
Table 6 Fatty acid composition in polar lipids with interaction between sex and RN genotype (least-square means and pooled standard error)a S.E.
P-valueb
30.69ab 0.63b 0.62a 47.31ab 3.08a 43.50b 3.82b
0.55 0.20 0.05 0.72 0.13 0.65 0.13
0.01 0.01 0.01 0.01 0.01 0.03 0.01
11.57b
0.32
0.05
Sex
Castrates
Genotype
rn+ (n=9)
RN (n=9)
rn+ (n=10)
RN (n=12)
18:2 n-6 20:5 n-3 22:6 n-3 PUFA HUFA n-3 PUFA n-6 PUFA n-3
28.82a 0.71ab 0.57a 44.84a 3.22a 40.83a 4.0ab
32.28b 0.71ab 0.59a 48.18b 3.27a 44.05b 4.13ab
30.23ab 0.80a 0.86b 47.86b 3.74b 43.41b 4.45a
n6/n3
10.35a
10.79ab
9.86a
a b
Gilts
Abbreviations: See Table 2. Values with different letters within a row are significantly different (P<0.05). P-value for the interaction between sex and RN genotype.
Table 7 Vitamin E and retinol content in muscle from indoor pigs, outdoor pigs, castrated male pigs and female pigs (least-square means and pooled standard error) Antioxidant
Production system
Sex
Indoor (n=20)
Outdoor (n=20)
S.E.
P-value
Castrates (n=18)
Female pigs (n=22)
S.E.
P-value
g/100 g meat a-Tocopherol g-Tocopherol Retinol
91.10 5.21 2.86
139.05 3.47 2.44
11.9 0.27 0.22
0.01a 0.001 0.18
97.80 4.25 2.61
132.31 4.42 2.68
11.63 0.26 0.21
0.04a 0.65 0.81
g/g intra muscular fat a-Tocopherol g-Tocopherol Retinol
51.56 2.96 1.61
100.89 2.40 1.72
11.79 0.25 0.16
0.01 0.12 0.65
62.13 2.46 1.58
90.32 2.90 1.74
11.55 0.24 0.16
0.09 0.21 0.48
a
Significant interaction between feed and sex (Table 9).
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and neutral lipids. The partial correlations were calculated after correcting for feed, sex, RN genotype, interactions and IMF content as described earlier.
3. Results In spite of the higher energy content in the feed of the outdoor pigs, as a compensation for higher energy expenditure, the intra muscular fat content in these pigs was lower than in the indoor pigs (Table 3). The intra muscular fat content influenced the monounsaturated (MUFA) and the highly polyunsaturated fatty acids (HUFA) of the neutral lipids (Tables 3, 4, 5). No effect of the intra muscular fat content on the fatty acid composition in the polar lipids was found.
outdoor pigs (7.13 and 0.65, respectively, Table 3). Furthermore, the level of unsaturation was higher in the neutral lipids of the female pig muscles compared with the castrated male pig muscle (Table 4). The differences between the castrated male pigs and the female pigs were seen in HUFA (1.40 and 1.21, respectively) and PUFA n-3 (1.01 and 0.80, respectively). However, there was a production systemsex interaction in the level of C 18:2 n-6 and HUFA n-3 fatty acids. The highest levels of these fatty acids were found in the indoor female pigs (Table 9). In the polar lipids the effect of production system could be seen in PUFA n-3 fatty acids with a higher level in the muscles from the indoor pigs compared with their outdoor counterparts (4.38 and 3.82, respectively, Table 3). 3.2. Fatty acid composition depending on RN genotype
3.1. Fatty acid composition depending on production system and sex The level of n-6 polyunsaturated fatty acids (PUFA n6) and n-3 polyunsaturated fatty acids (PUFA n-3) in the neutral lipid were higher in the muscle of the indoor pigs, (9.97 and 1.15, respectively) compared with the Table 8 Vitamin E and retinol content in muscle from pigs with different assumed RN-genotype (least-square means and pooled standard error) Antioxidant
Genotype rn+ (n=19) RN (n=21) S.E.
g/100 g meat a-Tocopherol g-Tocopherol Retinol g/g intra muscular fat a-Tocopherol g-Tocopherol Retinol
The RN genotype did not affect the levels of any major fatty acids in the neutral lipids. In the polar lipids differences were found. The level of PUFA n-3 fatty acids was higher in non-carrier pigs and in PUFA n-6 fatty acids in the RN-carrier group (Table 5). However, a sexRN genotype interaction was observed in the polar lipids, i.e. the differences between genotypes in PUFA n-3 fatty acids were seen in the castrated male pigs and the differences between genotypes in the PUFA n-6 were found in the female pigs (Table 6).
P-value
112.41 3.83 2.83
117.72 4.85 2.47
12.07 0.77 0.27 0.01 0.22 0.26
80.52 2.57 1.40
71.93 2.79 1.92
11.99 0.63 0.24 0.55 0.16 0.03
3.3. Contents of tocopherols, retinol and lipid oxidation The two production systems resulted in a different content of a-tocopherol. The outdoor pigs had a higher content of a-tocopherol in the muscles compared with the indoor pigs (Table 7). However, the effect of the production system was unevenly distributed within sex. Outdoor female pigs had the overall highest content of a-tocopherol compared with the indoor and outdoor castrated male pigs and the indoor female pigs (Table 9).
Table 9 Content of a-tocopherol, the degree of lipid oxidation and fatty acids of importance of neutral lipids (least-square means and pooled standard error) in muscle from female pigs and castrated male pigs reared indoors and outdoora Sex
Castrates
Rearing conditions
Indoors (n=9)
Outdoor (n=9)
Indoors (n=11)
Outdoor
a-Tocopherol (mg/100 g meat) MDA (ng/g meat) 18:1 n-9 18:2 n-6 18:3 n-3 MUFA HUFA HUFA n-3 PUFA
101.77a 97.07ab 41.84ab 7.65a 0.69a 49.62ab 1.26ab 0.29a 9.60a
102.12a 94.19ab 42.84a 5.65a 0.40c 50.78a 1.14a 0.22a 7.19a
92.30a 142.48a 40.19b 10.06b 0.92b 46.96b 1.57b 0.41b 12.55b
173.29b 65.82b 42.42a 6.77a 0.47c 50.95a 1.27ab 0.25a 8.51a
a b
Female pigs
S.E.
P-valueb
0.17 17.0 0.61 0.65 0.51 0.72 0.09 0.03 0.77
0.06 0.04 0.34 0.34 0.13 0.07 0.38 0.06 0.32
MDA, Malondialdehyde. Other abbreviations: see Table 2. Values with different letters within a row are significantly different (P <0.05). P-value for the interaction sexproduction system.
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Table 10 Overall and partiala correlations between a-tocopherol, MDA and some individual and groups of fatty acids of importance in polar and neutral lipids of pig muscle Overall correlation NL MDA 18:3 n-3 MUFA HUFAn-3 n-6/n-3 MDA
0.35 0.31 0.30 0.35
Partial correlation FL
atocopherol 0.24 0.15 0.06 0.47 0.43
MDA 0.42 0.01 0.08 0.02
NL atocopherol 0.49 0.08 0.12 0.26 0.43
MDA 0.06 0.02 0.10 0.09
PL atocopherol 0.07 0.11 0.29 0.23 0.39
MDA 0.31 0.16 0.15 0.24
atocopherol 0.30 0.06 0.13 0.01 0.39
a After correction for feed, sex, RN genotype, interactions and IMF content as described in Section 2.3. Correlation (r-value) >0.3; P<0.05; n=40 Abbreviations: See Table 2, MDA: malondialdehyde.
The content of g-tocopherol was found to be influenced both by the production system and the RN-genotype. Indoor pigs and carriers of the RN genotype had a higher content of g-tocopherol (Tables 7 and 8). Regarding lipid oxidation, a significant interaction (P=0.043) was seen between production system and sex. Results indicate that there was a higher degree of oxidation in the indoor female pigs in comparison to the outdoor female pigs after storage of the meat (Table 9). Furthermore, a negative global correlation was found between MDA and a-tocopherol (r= 0.43; P=0.005) and there was a positive correlation between MDA and PUFA n-3 fatty acids in both neutral lipids (r=0.35; P=0.02) and polar lipids (r=0.42; P=0.01; Table 10). In addition, a negative correlation was found between a-tocopherol and the fatty acid 18:3 n-3 in the polar lipids (r= 0.49; P=0.001).
4. Discussion The fatty acid composition of the intra muscular fat is affected by several factors, of which diet in general seems to be one of the most important (Nu¨rnberg, Wegner, & Ender, 1998). Increased levels of C22:6 n-3 and C20:5 n-3 in pig muscle have been shown in several earlier studies, in which different amounts of fish oil were included in the diet (Irie & Sakimoto, 1992; Lauridsen et al., 1999; Øverland et al., 1996). In our study, the amounts of C22:6 n-3 and C20:5 n-3 in the feed of the indoor pigs were quite low compared to other studies (Lauridsen et al., 1999; Table 2). However an increased level of these fatty acids in the polar and neutral lipids of the meat in the indoor pigs fed feed 1 was found. We suggest that the found difference in the content of C18:3 n-3 in the diets (Table 2) could be one cause of this effect. Thus, the increased levels of C22:6 n-3 and C20:5 n-3 might be a result of elevated elongation and desaturation of C18:3 n-3. Enser, Richardson, Wood, Gill, and Sheard (2000) have shown that an ele-
vated level of C18:3 n-3, increased the level of C22:6 n-3 and C20:5 n-3 in the polar lipids of pigs. However, it is not well established to which extent C18:3 n-3 is further elongated and desaturated to C22:6 n-3 (Bowen & Clandinin, 2000). It has been shown that the content of polyunsaturated fatty acids in the diet affects the oxidation rate of the final meat product (Leskanich et al., 1997). In our study the MDA content of the meat was influenced by the more unsaturated fatty acid profile in the indoor pigs fed feed 1. We have shown that a correlation between the content HUFA n-3 and MDA as well as between 18:3 n-3 and MDA (Table 10). Thus, we suggest that the oxidative effect can partly be ascribed to the overall increased level of PUFA n-3. Especially C22:6 n-3 and C20:5 n-3 are known to have a high oxidation rate (Cosgrove, Church, & Pryor, 1987). An additional oxidative effect is probably also caused by the higher amount of C18:3 n-3, even if this fatty acid has a lower oxidation rate than the fatty acids mentioned above. The correlation between an oxidative parameter as MDA and an vitamin with antioxidative properties as tocopherol has also been shown in several earlier studies (Corino, Oriani, Pantaleo, Pastorelli, & Salvatori, 1999). In our study the content of a-tocopherol differed between female pigs in the different production systems and there was an overall negative correlation between a-tocopherol and MDA. We suggest this to be an additional factor explaining the difference in susceptibility to oxidation. However, it is intricate to explain the increased level of polyunsaturated fatty acids of the neutral lipids in the female indoor pigs and no increased level in the castrated male indoor pigs (Table 9). One hypothetical explanation might be the possible difference in metabolism between castrated male and female pigs, as suggested in the study of Ho¨gberg et al. (2001). The castration and thereby possible loss of metabolisc functions in connection to puberty might affect the elongation and desaturation activity and rate of C18:2 n-6 and C18:3 n-3 towards their corresponding HUFA in
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castrated male pigs. Thus, the suggested difference in metabolic activity, in addition to the relatively low content of a-tocopherol in the indoor female pig muscle, might contribute to the increased susceptibility of the indoor female pig muscle to post-mortem oxidation, in this study shown as increased MDA content. The level of g-tocopherol in pig muscle was relatively low compared with a-tocopherol in our study. However, in our study the content of g-tocopherol was higher in the indoor pig muscle, probably due to a higher dietary content of g-tocopherol in the feed of the indoor pigs. The dietary influence g-tocopherol has been previously reported by Rey et al. (1998). Ho¨gberg et al. (2001) have shown that grazing increased the level of PUFA n-3 fatty acids in the neutral lipids but not in the polar lipids. In the present study the level of PUFA n-3 fatty acids were increased in both neutral and polar lipid fraction in the indoor pig muscle. One plausible explanation for this effect could be the more pronounced difference in the level of PUFA n-3 fatty acids between the diets of the indoor and the outdoor pigs in our study, compared with the difference between the diets in Ho¨gberg et al. (2001). However, the dietary influence was greater in the neutral lipids compared with the polar lipids, as the level of C18:3 n-3 was doubled in the neutral lipids. Similar results have also been reported in other studies in which the level of C18:3 n-3 was more prone to change in the triacylglycerols than in the phospholipids of pig meat (Leskanich, 1999). Enser et al. (2000) showed that an increased dietary level of C18:2 n-6 resulted in an increased level of this fatty acid in both neutral and polar lipids. We found increased level of C18:2 n-6 only in neutral lipids of the muscles from the indoor pigs fed feed 1, and this effect was more pronounced in the female pigs. Some possible explanations for the increased level of C18:2 n-6 are as follows. (1) Dietary level of C18:2 n-6 was higher in our dietary treatments when compared with other studies (Enser et al., 2000). (2) It is possible that a certain level of C18:2 n-6 is needed and will be included in the membrane while additional C18:2 n-6 will not affect the membrane composition. Thus, the excess of C18:2 n-6 will be stored in the neutral lipids or undergo b-oxidation. Becker and Bruce (1986) have shown that C18:2 n6 was preferentially b-oxidised when rats were fed a high C18:2 n-6 diet. This has also been found in fish (Kiessling & Kiessling, 1993). (3) Another explanation might be that the content of C18:3 n-3 in both diets in our study was lower compared with other studies (Enser et al., 2000). The low content of C18:3 n-3 might result in a demand for this fatty acid causing an incorporation of all C18:3 n-3 available. Fu and Sinclair (2000) suggested a conservation of C18:3 n-3 when fed in low amounts at the expense C18:2 n-6. Thus, it is possible that the unaffected level of C18:2 n-6 in the polar lipids
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in the present study is a consequence of the low dietary level of C18:3 n-3. Pigs carrying the dominant RN allele have a higher muscle glycogen content which will affect muscle quality characteristics of the final meat products (Enfa¨lt et al., 1997). In the present study, we found that the difference in the fatty acid composition between the RN genotypes arose in the polar lipids of M. Longissimus dorsi. Additionally, the difference in fatty acid composition could be seen not only in the PUFA n-3 fatty acids of polar lipids, as mentioned above (Nilze´n et al., 2001) but also in the PUFA n-6 fatty acids of the polar lipids. However, there was a significant interaction between RN genotype and sex in the polar lipids (Table 6). These differences in the level of polyunsaturated fatty acids might indicate the metabolic importance of the PUFA n-3 and PUFA n-6 fatty acids. Clarke (2000) hypothesises that n-3 polyunsaturated fatty acids play an essential role in the maintenance of energy balance and glucose metabolism. Considering suggestions of Clarke (2000), the results of the present study might indicate that polyunsaturated fatty acids can be involved in the mechanism leading to the increased glycogen content of M. Longissimus dorsi in meat of RN carriers. However, the nature of this relationship between glycogen content and polyunsaturated fatty acids needs to be further investigated. In conclusion, both diet and sex influenced the level of oxidation of meat in this study. However, more research is needed to establish whether this difference is due to the balance between antioxidants and polyunsaturation or other metabolic events in the live pig. The fatty acid composition in the diet affected the neutral and polar lipid fraction differently. Finally, the RN genotype was found to influence the fatty acid composition in the polar lipids of pig muscle. Further investigations of the interactions between RN genotype and the fatty acid composition are needed. Two hypotheses can possibly be considered: (1) The difference in membrane fatty acid composition affects the glycogen metabolism in pigs. (2) The RN allele affects the incorporation of fatty acids in membrane lipids. References Andersson, K. (1985). SLU-normen en ny utfodringsnorm till slaktsvin. Konsulentavdelningen, Swedish Univ of Agricultural Sciences, Allma¨nt, 67, 2 1-2:8. Becker, W., & Bruce, A˚. (1986). Retention of linoleic acid in carcass lipids of rats fed different levels of essential fatty acids. Lipids, 21, 121–126. Boisen, S., & Fernandez, J. A. (1997). Prediction of the total tract digestibility of energy in feedstuffs and pig diets by in vitro analyses. Animal Feed Science and Technology, 68, 277–286. Bowen, R. A. R., & Clandinin, M. T. (2000). High dietary 18: 3n-3 increases the 18: 3n-3 but not the 22: 6n-3 content in the whole body, brain, skin, epididymal fat pads, and muscles of suckling rat pups. Lipids, 35, 389–394.
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