Effects of vitamin E and flaxseed on rumen-derived fatty acid intermediates in beef intramuscular fat

Meat Science 88 (2011) 434–440

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Meat Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i

Effects of vitamin E and flaxseed on rumen-derived fatty acid intermediates in beef intramuscular fat☆ Manuel Juárez a, Michael E.R. Dugan a,⁎, Jennifer L. Aalhus a, Noelia Aldai a, John A. Basarab b, Vern S. Baron a, Tim A. McAllister c a b c

Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1 Alberta Agriculture and Rural Development, Lacombe Research Centre, 6000 C & E Trail, Lacombe, AB, Canada T4L 1W1 Agriculture and Agri-Food Canada, Research Centre, 5403-1st Ave. S. Lethbridge, AB, Canada T1J 4B1

a r t i c l e

i n f o

Article history: Received 3 September 2010 Received in revised form 24 November 2010 Accepted 21 January 2011 Keywords: Beef Linseed Omega-3 Oxidation α-tocopherol

a b s t r a c t To elucidate the effects of dietary vitamin E with or without flaxseed on beef fatty acid composition, 80 feedlot steers were fed 4 diets: Control-E (451 IU dl-α-tocopheryl acetate/head/day), Control + E (1051 IU dl-αtocopheryl acetate/head/day), Flax-E (10% ground) and Flax + E. Vitamin E had no effect on animal growth or carcass weight (p N 0.05), while flaxseed-fed steers had greater average daily gain (p = 0.007), final live weight (p = 0.005) and heavier carcasses (p = 0.012). Feeding flaxseed increased the total n−3 fatty acid content of beef and this response was further accentuated by the inclusion of high levels of vitamin E in the diet. Feeding flax increased levels of some 18:3n−3 partial hydrogenation products including c15- and t13/ 14-18:1 and several 18:2 isomers (p b 0.001) but decreased t10-18:1 (p b 0.001). Vitamin E enhanced intramuscular levels of 18:3n−3 and its biohydrogenation products leading to greater accumulations of total n−3 fatty acids in lean ground beef. The consequences of increasing the concentrations of partially hydrogenated products on human health have yet to be investigated. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction Increasing consumer demand for healthier food products has led to the development of governmental policies regarding health claims in many developed countries. In this context, omega-3 (n−3) fatty acids are currently the only class of fatty acids with regulatory label claim status for meat and meat products in Canada (≥300 mg n−3 fatty acids per serving) (CFIA, 2003). Dietary intake of n−3 fatty acids has been proven to reduce the incidence of heart disease (Kris-Etherton, Harris, & Appel, 2002; Medeiros et al., 2007; Wilson, 2004) and to play important biological functions in inflammation, brain development, sight and immune function (Connor, 2000). Current market penetration of animal based n−3 fatty acid products is in the monogastric and dairy sectors, and arises primarily from the feeding of flax products to monogastric animals (Haak, Raes, Van Dyck, & De Smet, 2008) or rumen bypass lipids to dairy cattle (Castañeda-Gutiérrez et al., 2007). Several authors have studied the effects of the inclusion of flax products in the diet on beef quality and fatty acid composition (i.e. Drouillard et al., 2002; Raes, De Smet, Balcaen, Claeys, & Demeyer, 2003; Vatansever et al., 2000). However,

☆ Contribution number 1170, Agriculture and Agri-Food Canada, Lacombe Research Centre. ⁎ Corresponding author at: Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, Alberta, Canada T4L 1W1. E-mail address: [email protected] (M.E.R. Dugan).

increasing n−3 fatty acids in beef has been proven quite challenging as the efficiency of transfer of polyunsaturated fatty acids (PUFA) from the diet to tissues is poor in ruminants relative to monogastrics (Haak, De Smet, Fremaut, Van Walleghem, & Raes, 2008; Juárez et al., 2010). The main reason for this inefficient transfer is the hydrogenation of PUFA by the microbial community in the rumen. PUFA are toxic to many rumen bacteria and to cope they isomerize and hydrogenate PUFA leading to variable accumulations of both complete and partial hydrogenation products (McKain, Shingfield, & Wallace, 2010). However, the impact of inclusion of flaxseed in the diet on the accumulation of the full spectrum of 18 carbon partial hydrogenation products is less well defined in beef as compared to dairy cattle (Akraim, Nicot, Juaneda, & Enjalbert, 2007; Kay, Roche, Kolver, Thomson, & Baumgard, 2005; Loor, Ueda, Ferlay, Chilliard, & Doreau, 2004; Pottier et al., 2006). Minimizing ruminal hydrogenation of n−3 fatty acids when flaxseed is included in the diet is a key to increasing n−3 fatty acid levels in beef. Some success has been attained through feed processing and protection (e.g. calcium salts) of n−3 fatty acids (Castañeda-Gutiérrez et al., 2007; Kronberg, Scholljegerdes, Barceló-Coblijn, & Murphy, 2007) and, more recently, vitamin E has been shown to alter ruminal PUFA hydrogenation in dairy (Kay et al., 2005; Pottier et al., 2006) and beef cattle (Juárez et al., 2010). Adding vitamin E to cattle diets has also been demonstrated to prevent discolouration and oxidative rancidity in beef (Lee et al., 2008). It is, therefore, hypothesized that feeding flaxseed to cattle will increase both n−3 fatty acids and their partial hydrogenation products

0309-1740/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.01.023

M. Juárez et al. / Meat Science 88 (2011) 434–440

in beef and these will be further influenced by increasing the vitamin E level in the diet. 2. Materials and methods Eighty feedlot steers were blocked by initial weight and housed in 8 feedlot pens balancing by weight within treatment (2 pens per dietary treatment, 10 animals per pen, and n = 20 animals per dietary treatment) and fed ad libitum. All dietary treatments and experimental procedures were approved by the Animal Care Committee at the Agriculture and Agri-Food Canada — Lacombe Research Centre (AAFC — LRC) and animals were cared for as outlined under the guidelines established by the Canadian Council on Animal Care (CCAC, 1993). Animals in each pen were group fed. Feed was provided at ~2.5% of body weight once daily, which resulted in 10–15% refusals after 18 h and all feeds consumed by 24 h. Access to water was ad libitum and feed bunk space was available to accommodate all animals at any one time (a minimum of 0.7 m/head). Steers (381±7.10 kg) were blocked by weight and assigned to one of the four diets in a 2 × 2 factorial experiment, with two levels of dietary vitamin E, with and without flaxseed (Table 1). The basal control diet supplied 451 IU dl-αtocopheryl acetate/head/day. For the high vitamin E diets, an additional 600 IU dl-α-tocopheryl acetate/head/day was top dressed and mixed with a pitch fork into the basal diet. Thus, the four dietary treatments were: Control-E (451 IU dl-α-tocopheryl acetate/head/day), Control + E (1051 dl-α-tocopheryl acetate IU/head/day), Flax-E (10% ground flax-

Table 1 Composition of total mixed rations used in this experiment.

435

seed substituted for steam-rolled barley) and Flax+ E (10% flaxseed and 1051 IU dl-α-tocopheryl acetate/head/day). Total mixed ration (TMR) samples were collected weekly and stored at −20 °C until pooled (monthly) and composite samples were taken for further analyses (n= 6). Live weights were measured monthly and used to calculate average daily gain (ADG). Backfat was measured monthly by ultrasound (Aloka 500 V with 17 cm 3.5 MHz linear array transducer. Overseas Monitor Corporation Ltd., Richmond, BC, Canada) between the 12th and 13th ribs, over the longissimus thoracis (LT; rib-eye), and animals were slaughtered over 5 slaughter dates when reaching 9–10 mm backfat (4 animals per dietary treatment per slaughter day). Following splitting of the carcass, hot side weights were recorded (trimmed and untrimmed) and an initial (45 min) pH and temperature readings were taken posterior to the grade site (12th rib) in the left LT using a Fisher Scientific Accumet AP72 pH meter (Fisher Scientific, Mississauga, ON, Canada) equipped with an Orion Ingold electrode (Udorf, Switzerland). Hot dressing percentage was calculated as the ratio of hot carcass weight to live weight (dressing percentage = HCW × 100 / slaughter weight). Final pH and temperature measurements were recorded 24 h postmortem after the carcasses had chilled at 2 °C overnight. Final carcass weights (FCW) were recorded to determine shrink losses [shrink losses = (HCW − FCW) / HCW × 100]. Carcasses were knife-ribbed at the grade site (between the 12th and the 13th ribs) and assessed for fat thickness, rib-eye area, estimated lean yield (CFIA, 1992) and marbling (AMSA, 1990) by two certified beef graders. The left LT was collected at this time and one steak from the posterior end (near the grade site) was removed and frozen (−80 °C) for subsequent fatty acid determination.

Diet Control-E

Control + E Flax-E

Flax + E

73.3 – 22.0 1.56 3.14

73.3 – 22.0 1.56 3.14

63.3 10.0 22.0 1.56 3.14

63.3 10.0 22.0 1.56 3.14

Vitamin E (IU head− 1 day− 1) Basal dl-α-tocopheryl acetate 451 Top-dressed dl-α-tocopheryl – acetate Total dl-α-tocopheryl acetate 451

451 –

451 600

451 600

451

1,051

1,051

Diet ingredients, % as fed basis Barley Ground flaxseed Alfalfa brome hay Molasses Feedlot supplement1

Nutrient composition, DM basis2 Dry matter (%) 83.1 Crude protein (%) 12.2 ADF (%) 19.4 NDF (%) 31.7 TDN (%)3 70.5 ME (MJ kg− 1)4 13.7 NEg (MJ kg− 1)4 4.05 NEm (MJ kg− 1)4 6.99 Fatty acid composition (% total 16:0 18:0 c9-18:1 18:2n−6 18:3n−3 Total fatty acids (mg g− 1)2

2.1. Feed analysis Feed samples were analyzed for dry matter, crude protein, acid detergent fiber (AFC) and neutral detergent fiber (NDF) as previously reported by Aldai et al. (2010). Metabolizable energy (ME), net energy for maintenance (NEm) and net energy for gain (NEg) were calculated according to the National Research Council (NRC, 1996). Fatty acid methyl esters (FAMEs) from the finishing TMR were prepared according to Sukhija and Palmquist (1988) and analyzed by using the chromatographic conditions reported by Dugan et al. (2007). Results are shown in Table 1. 2.2. Lipid analysis

(3.80) (0.57) (1.98) (2.36) (2.03) (0.25) (0.06) (0.06)

85.8 12.8 21.3 35.3 68.3 13.4 3.92 6.92

(1.85) (0.85) (2.59) (3.31) (2.65) (0.57) (0.14) (0.14)

83.9 12.0 22.6 36.6 66.8 13.3 3.94 6.88

(2.68) (0.62) (4.42) (5.68) (4.52) (0.33) (0.08) (0.08)

86.1 13.4 19.4 31.6 70.4 13.7 4.05 6.99

(2.72) (0.98) (2.69) (2.21) (2.75) (0.34) (0.09) (0.09)

fatty acids)2 19.6 (0.63) 2.09 (0.18) 20.9 (1.09) 46.9 (0.85) 7.48 (0.31) 2.72 (0.16)

19.7 2.09 20.9 46.9 7.48 2.72

(0.63) (0.18) (1.09) (0.85) (0.31) (0.16)

11.5 3.17 18.6 29.3 35.7 5.76

(0.63) (0.09) (0.13) (1.06) (1.48) (0.47)

11.5 3.17 18.6 29.3 35.7 5.76

(0.63) (0.09) (0.13) (1.06) (1.48) (0.47)

1 Supplement pellets contain the following: 90% dry matter, 32% crude protein, 1.5% crude fat, 7.0% crude fiber; 2.0% sodium, 8.0% calcium, 0.6% phosphorus, 0.57% magnesium, 0.66% sulfur, 1.0% potassium, 7 mg kg− 1 cobalt, 20 mg kg− 1 iodine, 300 mg kg− 1 copper, 950 mg kg− 1 manganese, 1020 mg kg− 1 zinc, 6.0 mg kg− 1 selenium, 240 mg kg− 1 fluorine, 100 000 IU kg− 1 vitamin A and 15 000 IU kg− 1 vitamin D. 2 Mean (Standard Deviation); n = 6. 3 For finishing diets, total digestible nutrients (TDN, %) = 92.2 − 1.12 × ADF, % (Aldai et al., 2010). 4 Metabolizable energy ME and net energy NE values calculated according to NRC (1996).

Subcutaneous fat samples (50 mg) were freeze-dried and directly methylated with sodium methoxide (Cruz-Hernandez et al., 2004). Intramuscular lipids were extracted from the meat samples with 2:1 chloroform methanol using a 20:1 solvent to sample ratio (Folch, Lees, & Stanley, 1957). To derivatize all meat lipid classes, extracts were methylated using 5% methanolic HCl, and to correct for CLA isomerization (Aldai, Murray, Nájera, Troy, & Osoro, 2005), separate methylations with 0.5 N sodium methoxide were conducted. FAMEs were analyzed using the GC and Ag+-HPLC equipment and methods outlined by Cruz-Hernandez et al. (2004). FAMEs were analyzed using GC (acid and basic methylations) and Ag+-HPLC (basic methylation) (Dugan et al., 2007). The trans-18:1 isomers were analyzed using two complementary (150 °C and 175 °C plateau) GC temperature programs (Fig. 1) (Kramer, Hernandez, Cruz-Hernandez, Kraft, & Dugan, 2008). For the identification of fatty acids by GC, the reference standard no. 461 from Nu-Check Prep Inc. (Elysian, MN) was used. All the geometric isomers of linoleic and linolenic acids were prepared by mild iodine isomerization (Cruz-Hernandez et al., 2004). The CLA standards #UC-59 M (Nu-Chek Prep Inc. City, State) was used since it contains all four positional CLA isomers, and additional CLA isomers were obtained from Matreya Inc. (Pleasant Gap, PA) and by using

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9c-18:1

10t-

13t/14t-

11t-

6t/7t/8t-

quality. A high level of vitamin E (2000 IU day− 1) fed for 7, 14 or 28 days had also no effect on animal growth or carcass traits compared with a control diet (Carter et al., 2005). Feeding a high level of flaxseed (11% DM), however, caused a decrease in growth rate and carcass weight of steers, as compared to diets that contained flaxseed at 8% or 5% of dietary DM (Mach et al., 2006). A recent study (Scholljegerdes & Kronberg, 2010) reported an increase in average daily gain and feed efficiency when ground flaxseed was included as a supplement (0.18% of body weight on a dry matter basis/head/day) for steers grazing for ~ 90 days.

12t-

9t-

3.2. Intramuscular fatty acid composition

33.00

33.25

33.50

33.75

34.00

Minutes

Fig. 1. Representative trans-18:1 region partial GLC chromatograms using 175 °C temperature program (control diet: solid line; flaxseed supplementation: dotted line).

iodine isomerization (Cruz-Hernandez et al., 2004). Fatty acid concentrations were reported as percentage of total fatty acids identified. 2.3. Statistical analysis Statistical analyses were conducted using the MIXED procedure of SAS (SAS, 2003). The model included the fixed effects of diet (flax/no flax) and vitamin E (normal/enhanced), the diet × vitamin E interaction and the random effects of kill and pen. When a random effect of pen was found, pen was used as the experimental unit, but when pen was not significant, pen was removed from the model and individual animal was used as the experimental unit. Treatment means were determined using the LSMEANS option and separated using F-test protected LSD (p ≤ 0.05). 3. Results and discussion 3.1. Performance and carcass characteristics Vitamin E level in the diet had no effect on animal growth or carcass weight (p N 0.05; Table 2), but steers fed flaxseed had higher ADG (p = 0.007), final live weight (p = 0.005) and heavier carcasses (p = 0.012). Hot dressing percentage, pH, shrink loss, grade fat, ribeye area, estimated lean yield and marbling were not affected (p N 0.05) by dietary treatments. In previous studies, the inclusion of vitamin E (i.e. Lee et al., 2008; Montgomery et al., 2005; Realini, Duckett, Brito, Dalla Rizza, & De Mattos, 2004) or flaxseed in the diet (i.e. Noci, French, Monahan, & Moloney, 2007; Raes et al., 2004; Scollan et al., 2001) had no effect on animal performance or carcass

Increasing the n−3 fatty acid content in beef can be done by increasing the overall lipid content of meat or by increasing the percentage of n−3 fatty acids in total fatty acids. No difference (pN 0.05) was observed in the total intramuscular fatty acid content among treatments (Table 3). Similar results were reported by Noci et al. (2007) and Scollan et al. (2001) when ~3% flax oil or ~4% whole flaxseed, respectively, were included in grass based (pasture and grass silage, respectively) diets. Feeding flaxseed has been shown to increase intramuscular fat levels of n−3 fatty acids by several authors (i.e. Bartoň, Marounek, Kudrna, Bureš, & Zahrádková, 2007; Choi, Enser, Wood, & Scollan, 2000; Raes, Haak, et al., 2004). In the present experiment, inclusion of flaxseed (p b 0.001) and vitamin E (p b 0.026) was shown to increase the level of total n−3 fatty acids in intramuscular fat (Table 3), with this response being primarily related to higher levels of 18:3n−3 and 20:5n−3. A flaxseed by vitamin E interaction was observed for 18:3n−3, where the highest level was observed when both the high level of vitamin E and flaxseed were included in the diet. Flaxseed and vitamin E had no effect on intramuscular levels of 22:5n−3 and 22:6n−3 (p N 0.05). Increases in long chain fatty acids mainly occur in the phospholipid fraction, and it does not usually include 22:6n−3, although some authors have observed a slight increase when feeding flaxseed (Maddock et al., 2006). The lack of effect on 22:6n−3 may be explained by the competition between 18:3n−3 and the precursor for 22:6n−3 (i.e. 24:5n−3) for the activity of the Δ6 desaturase enzyme (Cameron et al., 2000). Feeding flaxseed reduced the percentage of total n−6 fatty acids (p = 0.046). The decrease in total n−6 was due to a decrease in 20:4n−6 (p b 0.001). Overall, however, the increase in n−3 fatty acids when feeding flax was greater than the reduction in n−6 fatty acids resulting in an increase in total PUFA (p b 0.001) and a reduction in the n−6/n−3 ratio (p b 0.001). Long chain fatty acid synthesis is controlled by a complex enzymatic system, consisting of desaturases and elongases. The competition between the n−6 and n−3 fatty acids for that enzymatic activity and for the incorporation into cell membranes (Brenner, 1989) could explain the decrease in long chain n−6 when n−3 fatty acids were

Table 2 Effects of vitamin E and flaxseed dietary supplementation on animal performance and carcass traits of feedlot steers (n = 80). Diet

Initial live weight (kg) Final live weight (kg) Average daily gain (kg) Hot carcass weight (kg) Hot dressing (%) pH45 Temperature 45 (°C) pH24 Temperature 24 (°C) Shrink loss (%) Grade fat Rib-eye area (cm2) Estimated lean yield (%) Marbling

SEM

Control-E

Control + E

Flax-E

Flax + E

375 562 1.45 328 58.3 6.77 39.69 5.59 4.01 1.14 9.47 83.1 58.3 464

383 555 1.34 326 58.7 6.74 39.94 5.57 4.00 1.20 9.57 83.0 58.5 465

381 578 1.53 338 58.5 6.78 39.65 5.60 3.80 1.13 10.70 83.2 57.4 481

387 591 1.58 342 57.8 6.84 39.39 5.62 4.03 1.12 10.10 84.2 58.3 460

7.077 8.781 0.041 5.444 0.369 0.029 0.252 0.016 0.384 0.041 0.941 2.587 0.925 17.24

P value Vit E

Flax

Vit E Flax

0.471 0.745 0.095 0.899 0.657 0.051 0.084 0.104 0.494 0.395 0.657 0.831 0.294 0.438

0.214 0.005 0.007 0.012 0.300 0.550 0.976 0.956 0.403 0.111 0.094 0.758 0.338 0.613

0.325 0.250 0.078 0.546 0.125 0.114 0.128 0.155 0.362 0.155 0.534 0.776 0.552 0.393

M. Juárez et al. / Meat Science 88 (2011) 434–440

437

Table 3 Effects of vitamin E and flaxseed dietary supplementation on intramuscular fatty acid composition (% total fatty acids) of feedlot steers: polyunsaturated fatty acids (n = 80). Diet

1

−1

Total FA (mg g meat ) ΣPUFA Σn−3 18:3n−3 20:5n−3 22:5n−3 22:6n−3 Σn−6 18:2n−6 20:4n−6 n−6/n−3 Σ18:2 atypical isomers c9, t13-/t8, c12t8, c13c9, t12t9, c12t11, c15c9, c15ΣCLA2 c9, t11c11, t13c12, t14t7, c9t8, c10t9, c11t10, c12t11, c13t12, c14-

SEM

Control-E

Control + E

Flax-E

Flax + E

35.2 6.10 0.95 0.36c 0.13 0.33 0.07 3.88 2.67 0.77 4.11 0.68c 0.16 0.11 0.06 0.03c 0.20c 0.08ab 0.59 0.33 0.01 0.00 0.10a 0.01 0.04 0.01 0.01 0.01

37.4 6.60 1.04 0.35c 0.16 0.41 0.07 4.33 2.83 0.97 4.19 0.64c 0.17 0.11 0.06 0.03c 0.14c 0.09a 0.58 0.36 0.01 0.00 0.07b 0.01 0.04 0.01 0.01 0.01

43.1 7.71 2.05 1.35b 0.19 0.37 0.06 3.31 2.51 0.51 1.61 1.75b 0.58 0.43 0.15 0.05b 0.42b 0.08ab 0.60 0.34 0.01 0.00 0.07b 0.01 0.03 0.01 0.02 0.03

39.0 8.76 2.41 1.60a 0.25 0.41 0.07 3.71 2.83 0.57 1.54 2.00a 0.62 0.48 0.17 0.07a 0.55a 0.07b 0.64 0.34 0.01 0.01 0.08b 0.01 0.04 0.01 0.02 0.05

4.978 0.601 0.142 0.065 0.029 0.051 0.008 0.420 0.268 0.114 0.110 0.082 0.021 0.024 0.006 0.006 0.033 0.006 0.037 0.023 0.001 0.000 0.007 0.001 0.004 0.001 0.001 0.005

P value Vit E

Flax

Vit E Flax

0.762 0.063 0.026 0.015 0.047 0.105 0.397 0.154 0.192 0.116 0.923 0.092 0.267 0.139 0.043 0.058 0.152 0.943 0.604 0.509 0.833 0.132 0.174 0.916 0.942 0.331 0.432 0.029

0.126 b0.001 b0.001 b0.001 0.001 0.661 0.431 0.046 0.688 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 0.184 0.407 0.971 0.895 0.043 0.109 0.038 0.357 0.001 0.001 b0.001

0.315 0.503 0.176 0.007 0.522 0.688 0.931 0.939 0.662 0.384 0.281 0.027 0.513 0.145 0.154 0.005 b0.001 0.020 0.579 0.436 0.747 0.115 0.005 0.723 0.508 0.029 0.671 0.071

1

Total FA: total fatty acids. CLA: conjugated linoleic acid. Different letters indicate statistical difference (P ≤ 0.05).

2

a,b,c

included in the diet. In fact, the enzymes responsible for desaturating and elongating 18:2n−6 and 18:3n−3 to their longer chain metabolites are the same and have been reported to have a higher affinity for n−3 fatty acids (Jump, 2002; Raes, Haak, et al., 2004). A flaxseed by vitamin E interaction was found for total atypical (non-conjugated) 18:2 isomers (p = 0.027), and similar to 18:3n−3, the highest level of atypical 18:2 isomers in intramuscular fat was found when the highest level of vitamin E was included in the flaxseed containing diet (Flax + E). The flaxseed by vitamin E interaction for total atypical 18:2 isomers was mostly related to an interaction found for t11,c15-18:2 (p b 0.001), however, it should be noted that flaxseed by itself significantly increased (p b 0.001) levels of several other atypical 18:2 isomers (c9,t13-/t8,c12-, t8,c13-, c9,t12-18:2). Adding either flaxseed or vitamin E had no effect (p N 0.05) on the level of total CLA. Effects of feeding flaxseed on beef CLA levels have been inconsistent. Some authors have reported that flaxseed has no effect on CLA levels (Raes, De Smet, & Demeyer, 2004; Raes, Haak, et al., 2004), while others found an increase (Bartoň et al., 2007; Mach et al., 2006) in c9,t11-CLA. In the present experiment, feeding flaxseed or vitamin E had no effect on the major CLA isomer (c9,t11-CLA; p N 0.05). Some flaxseed, vitamin E and flaxseed by vitamin E interaction effects were seen for several minor CLA isomers (p b 0.05), but the majority of these were very low in concentration (b0.05%). The effects of feeding flaxseed, vitamin E and their combination on monounsaturated fatty acids (MUFA) were complex (Table 4). Increases in total MUFA (p = 0.011) in the present experiment were related to increases in total trans-18:1 when feeding flaxseed, and a flaxseed by vitamin E interaction indicated adding vitamin E to the control diet (Control + E) decreased total trans-18:1 but actually increased it when included in the flaxseed containing diet (Flax + E; p b 0.001). This interaction was found for t6-t8-, t9 and t10-18:1 (p b 0.05). As previously suggested (Juárez et al., 2010), vitamin E could influence ruminal pathways of PUFA biohydrogenation, acting either as an inhibitor of bacteria producing trans 10–18:1 or as an

electron acceptor for Butyrivibrio fibrisolvens (Pottier et al., 2006). On the other hand, the increased level of total trans-18:1 when feeding flaxseed was mainly attributed to large increases (p b 0.001) in t12-, t13/14-, t15- and t16-18:1, and this was to the point where t13/1418:1 was actually more concentrated than t10- or t11-18:1 (Fig. 2), which are typically the most abundant isomers when feeding concentrate or forage based diets, respectively (Aldai, Dugan, Rolland, & Kramer, 2009; Leheska et al., 2008). The effects of feeding flaxseed and vitamin E on cis-18:1 isomers were equally as complex as the trans-18:1 isomers (Table 4). There was a decrease (p = 0.002) in the level of c9-16:1 when feeding flaxseed, again likely as a result of a reduction in Δ9 desaturase activity. The major cis-18:1 isomer across dietary treatments was c9-18:1. An interaction between flaxseed and vitamin E (p b 0.001) resulted in high levels of vitamin E being associated with an increase in c9-18:1 when it was added to the control diet (Control + E; higher in c9-18:1) and a decrease when it was added to the flaxseed diet (Flax+ E; lower in c9-18:1). Several other cis-18:1 isomers (c12-, c13-, c14- and c16-18:1) were increased with flaxseed (p b 0.05; Fig. 2) and a flaxseed by vitamin E interaction was observed for c15-18:1, which was increased (p = 0.046) when the high level of vitamin E was included with flaxseed (Flax + E). Feeding flaxseed reduced the level of total saturated fatty acids (SFA; pb 0.001) in intramuscular fat (Table 5) and this was mainly attributed to a reduction in 16:0 (pb 0.001), as 14:0 or 18:0 remained unchanged. The reduction in 16:0 may be related to a reduced rate of endogenous fatty acid synthesis, which was inhibited by the oil in the flaxseed diets (Noci et al., 2007). Higher levels of 18:0 might be expected from the complete hydrogenation of 18:3n−3 when flaxseed was included in the diet. However, the lower digestibility of 18:0 in high oil diets (Yang et al., 2009) may have masked hydrogenation outcomes in the rumen, leading to a lack of effect in muscle 18:0 levels (pN 0.05). An interaction between flaxseed and vitamin E for 17:0 (p=0.003) was due to a slight reduction in 17:0 when the high level of vitamin E was added to control (Control +E) diet and a slight increase when it was added to the flaxseed diet (Flax+E).

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Table 4 Effects of vitamin E and flaxseed dietary supplementation on intramuscular fatty acid composition (% total fatty acids) of feedlot steers: monounsaturated fatty acids (n = 80). Diet

ΣMUFA c9-16:1 c9-17:1 ΣTrans-18:1 t6-t8t9t10t11t12t13-/t14t15t16c9-18:1 c11c12c13c14c15c16c17a,b,c,d

SEM

Control-E

Control + E

Flax-E

Flax + E

48.6 3.76 0.91 3.62c 0.26a 0.33a 1.74a 0.64c 0.11 0.27 0.21 0.06c 35.8bc 1.55 0.12 0.47 0.04 0.31c 0.04 0.09ab

49.3 3.68 0.83 2.85d 0.19b 0.25b 1.00b 0.71bc 0.13 0.28 0.21 0.08c 37.5a 1.62 0.14 0.47 0.04 0.24c 0.04 0.08b

50.6 3.40 0.79 4.24b 0.19b 0.25b 0.77b 0.78a 0.39 0.99 0.54 0.32a 36.7ab 1.56 0.21 0.55 0.12 1.10b 0.08 0.09ab

49.6 3.39 0.83 4.76a 0.24a 0.28b 1.04b 0.72ab 0.43 1.14 0.62 0.29b 34.8c 1.53 0.24 0.52 0.13 1.45a 0.10 0.10a

0.460 0.102 0.031 0.178 0.014 0.014 0.121 0.036 0.012 0.046 0.040 0.011 0.544 0.066 0.017 0.025 0.007 0.121 0.007 0.006

P value Vit E

Flax

Vit E Flax

0.690 0.661 0.511 0.475 0.255 0.095 0.061 0.911 0.018 0.069 0.201 0.270 0.827 0.682 0.022 0.592 0.040 0.162 0.062 0.834

0.011 0.002 0.082 b0.001 0.472 0.093 b0.001 0.004 b0.001 b0.001 b0.001 b0.001 0.070 0.320 b0.001 0.004 b0.001 b0.001 b0.001 0.084

0.071 0.671 0.068 b0.001 b0.001 b0.001 b0.001 0.011 0.417 0.137 0.263 0.007 b0.001 0.229 0.699 0.504 0.147 0.046 0.313 0.046

Different letters indicate statistical difference (P ≤ 0.05).

3.3. PUFA hydrogenation The accumulation of PUFA and specifically 18:3n−3 hydrogenation products in intramuscular fat were at variance with what is typically seen when feeding beef cattle either grain or forage-based diets. Forage feeding typically yields increased levels of t11- versus t10-18:1 (Leheska et al., 2008) and, after c9,t11-CLA, the second most abundant CLA isomer is frequently t11,c13-CLA for forage fed and t7, c9-CLA for grain fed beef cattle (Aldai et al., 2009). Under typical conditions in western Canada (i.e. feeding barley based finishing diets), levels of atypical 18:2 isomers in beef are low. Feeding a relatively high level of 18:3n−3 from flaxseed resulted in unusual accumulations of 18:3n−3 partial hydrogenation products with a decided shift away from CLA towards atypical 18:2 isomers, and relatively large shifts toward c15-18:1 and t13/14-18:1 production. These data are in agreement with ruminal concentrations (Loor et al., 2005) and duodenal flows of 18:3n−3 and its biohydrogenation intermediates (Doreau, Laverroux, Normand, Chesneau, & Glasser, 2009; Loor et al., 2004), and with plasma and milk fatty acid responses to increased intake of 18:3n−3 in dairy cows (Kay et al., 2005; Loor, Ferlay, Ollier, Doreau, & Chilliard, 2005; Loor, Ferlay, Ollier, Ueda, et al., 2005). Recent studies (Yang et al., 2009) suggest that total ruminal protozoa and cellulolytic bacteria are reduced, and total proteolytic bacteria are increased with dietary oil (e.g. flax oil) supplementation.

18:2n-6

c15-18:1

c9t13/t8c12-18:2 c9t12/t8c13-18:2 t11c15-18:2 c9c15-18:2

36.0

36.5

37.0

37.5

38.0

38.5 Minutes

Fig. 2. Representative 18:2 region of partial GLC chromatograms using 175 °C temperature program (control diet: solid line; flaxseed supplementation: dotted line).

These changes in the ruminal microbial population and their activity may partially explain the modifications observed in the fatty acid profiles in the present study. Including high levels of vitamin E in the diet resulted in higher levels of 18:3n−3 when flaxseed was included in the diet, which indicates that vitamin E can somehow modify 18:3n−3 hydrogenation. In addition, when either vitamin E or flaxseed was added to the control diet it reduced the accumulation of t10-18:1 in muscle. Although a reduction in t10-18:1 could be positive, as t10-18:1 is associated with increased cardiovascular disease risk in animal models (Bauchart et al., 2007; Hodgson, Wahlqvist, Boxall, & Balazs, 1996; Roy et al., 2007), the cardiovascular consequences of consuming increased levels of t9,c12and c9,t12-18:2 have not been found to be positive (Baylin, Kabagambe, Ascherio, Spiegelman, & Campos, 2003; Lemaitre, King, Mozaffarian, Sootodehnia, & Siscovick, 2006a; Lemaitre et al., 2006b). The health effects of many of the other PUFA partial hydrogenation products, such as c8,t13- or t11,c15-18:2, remain to be investigated. 3.4. n−3 fatty acid enriched beef The Canadian regulatory authority (CFIA, 2003) sets 300 mg 100g− 1 as the minimum required to obtain a source claim for n−3 fatty acids in food products. Despite the increase observed in total n−3 fatty acids, supplementation of the diet with flaxseed, with or without vitamin E, did not provide enough n−3 fatty acid enrichment in lean beef (87.8 ± 5.66 and 82.5 ± 3.43 mg 100 g LT− 1, respectively) to achieve a source claim. To attain the level of n−3 fatty acids in beef required for an enrichment claim in Canada, products with higher fat content will likely be required. In the present experiment, changes in lean beef fatty acid composition were consistent with changes seen in subcutaneous fat (individual fatty acid; data not shown). Similar to LT, feeding flaxseed increased total n−3 fatty acids in subcutaneous fat and the increase was greatest when vitamin E supplementation was included in the flaxseed containing diets (Flax+ E; p = 0.006; Fig. 3). Levels of total n−3 fatty acids in lean (23% fat) ground beef (Fig. 3), if manufactured from LT and subcutaneous fat when feeding flaxseed diets, would not only be much higher than those from the control diets (p b 0.001), but also enough to achieve requirements for n−3 fatty acid source claims. Due to individual animal variation, however, lean ground beef from only 45% of flaxseed fed steers would have met required values compared to 80% for steers fed flaxseed and high vitamin E. Therefore, although individual animal variation is eliminated when meat and fat from different animals are

M. Juárez et al. / Meat Science 88 (2011) 434–440

439

Table 5 Effects of vitamin E and flaxseed dietary supplementation on intramuscular fatty acid composition (% total fatty acids) of feedlot steers: saturated fatty acids (n = 80). Diet

ΣSFA 14:0 16:0 17:0 18:0 a,b

SEM

Control-E

Control+E

Flax-E

Flax+E

43.4 2.58 26.2 0.95a 12.0

42.2 2.34 25.1 0.83b 12.2

39.9 2.41 22.9 0.78b 12.1

39.8 2.47 23.1 0.85b 11.8

0.789 0.159 0.542 0.035 0.263

P value Vit E

Flax

Vit E Flax

0.219 0.379 0.245 0.349 0.716

b0.001 0.809 b0.001 0.021 0.539

0.251 0.138 0.087 0.003 0.284

Different letters indicate statistical difference (P ≤ 0.05).

pooled to industrially produce ground beef, the inclusion of vitamin E in diets would more consistently meet the required levels for n−3 fatty acid enrichment or could possibly reduce levels of dietary flaxseed required to meet n−3 fatty acid enrichments. Moreover, when commercial production of n−3 fatty acid enhanced beef products is being considered, analyses of meat and fat depots typically used for ground beef production will have to be conducted.

4. Conclusions As expected, the inclusion of flaxseed in the diets increased the intramuscular n−3 fatty acid content in feedlot steers. Moreover, instead of increasing t10-18:1, as observed when feeding high concentrate diets or t11-18:1 when feeding high forage diets, feeding a relatively high level of 18:3n−3 from flaxseed mainly resulted in a decrease in t10-18:1 and unusual accumulations of 18:3n−3 partial hydrogenation products (c15- and t13/14-18:1 and 18:2 atypical isomers). On the other hand, vitamin E enhanced intramuscular levels of 18:3n−3 and, in flaxseed supplemented diets it modified the concentrations of biohydrogenation products, leading to greater accumulations of total n−3 fatty acids. Thus, feeding both flaxseed and vitamin E showed positive effects on total n−3 fatty acid levels, however, accumulation of atypical hydrogenation products and their potential bioactivity requires further study.

Acknowledgements Dr. M. Juárez acknowledges the receipt of a NSERC fellowship funded through the AAFC ABIP-FOBI program. Dr. N. Aldai acknowledges the receipt of a research contract from the 7th European Community Program (Marie Curie International Outgoing Fellowship).

400

a

1.4

a

b

350

b

1.2

300

1

250 200

0.8 0.6

c

c

c

c

150

0.4

100

0.2

50

0

mg n-3.100g ground beef -1

% n-3 subcutaneous fat

1.6

0 Control-E Control+E Flax-E

Flax+E Control-EControl+E Flax-E

Flax+E

a,b,c

Different letters indicate statistical difference (P 0.05)

Fig. 3. Effects of vitamin E and flaxseed dietary supplementation on subcutaneous n−3 content (% total fatty acids) and ground beef (23% backfat) of feedlot steers (n = 80).

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