Meat Science 96 (2014) 541–547

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Flaxseed fed pork: n−3 fatty acid enrichment and contribution todietary recommendations

T.D. Turner a, C. Mapiye a, J.L. Aalhus a, A.D. Beaulieu b, J.F. Patience c, R.T. Zijlstra d, M.E.R. Dugan a,⁎a Agriculture and Agri-Food Canada, Lacombe Research Centre, Lacombe, AB, Canadab Prairie Swine Centre, Inc., Saskatoon, SK, Canadac Iowa State University, Ames, IA, USAd University of Alberta, Edmonton, AB, Canada

⁎ Corresponding author. Tel.: +1 403 782 8125.E-mail address: mike.dugan@AGR.GC.CA (M.E.R. Duga

0309-1740/$ – see front matter. Crown Copyright © 2013http://dx.doi.org/10.1016/j.meatsci.2013.08.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 June 2012Received in revised form 27 June 2013Accepted 13 August 2013

Keywords:Health effectsLong-chain polyunsaturated fatty acidsSaturated fatty acids

The potential to increase n−3 fatty acid (FA) intake via flaxseed fed pork is underestimatedwhen restricted to purelongissimus muscle, whereas a combination of muscle and adipose tissue is typically consumed. Presently, the FAcontent of pigs fed 0%, 5% and 10% dietary flaxseed for 11 weeks was measured in loin, picnic and butt primals(lean muscle with epimysium (L), L plus seam fat (LS), and LS plus 5 mm backfat (LSS)). The n−3 FA contentnecessary for an enrichment claim in Canada (300 mg/100 g serving) was exceeded in L from all primals whenfeeding 5% flaxseed, being 4 fold that of controls (P b 0.001), with further enrichment from inclusion ofassociated adipose tissues (P b 0.001). Increasing flaxseed feeding levels in combination with adipose tissue inclu-sion amplified total long chain n−3 FA (P b 0.05), particularly 20:5n−3 and 22:5n−3. Flaxseed-fed n−3 FAenriched pork can contribute substantially to daily long chain n−3 FA intakes, particularly for societies withtypically low seafood consumption.

Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction

In recent years emphasis has been placed on enriching pork andpork products with n−3 fatty acids due to their positive effects onhuman health (Coats, Sioutis, Buckley, & Howe, 2009; Woods &Fearon, 2009). Increasing n−3 fatty acids in pork by feeding flaxseedto pigs has been demonstrated by several groups as reviewed by Raes,De Smet, and Demeyer (2004). Flaxseed supplementation invokes arapid response, with 18:3n−3 and 20:5n−3 levels increasing notablyin muscle after seven days of feeding (Romans, Johnson, Wulf, Libal, &Costello, 1995). Maximum polyunsaturated fatty acid (PUFA) effectsare reportedly achievable after 40 days of feeding, with most effectsoccurring in the first two weeks (Warnants, Van Oeckel, & Boucque,1999). Feeding flaxseed to pigs to yield enough n−3 fatty acids for en-richment claims, however, presents some challenges.

In Canada, 300 mg n−3 fatty acids per serving are required for anenrichment claim (CFIA & Canada, 2009). Within the EU, n−3 claimscan be made for products containing either 300 mg/serving 18:3n−3or 40 mg/serving combined 20:5n−3, 22:6n−3 (EU, 2010), likewisethe Food Standards Australia and New Zealand have claim levels set at200 mg 18:3n−3 or 30 mg combined 20:5n−3, 22:6n−3 (FSANZ,2013). To date, claims have been made in Canada for chicken eggs(Sim & Cherian, 1994). As opposed to eggs, however, pork is comprisedof a number of tissues with differing fatty acid compositions. Reports on

n).

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the extent different tissues in pork are enriched with n−3 fatty acidsare, however, sparse and relates to a focus on loin muscle instead ofmultiple tissues, and emphasis on comparing effects of feeding flaxseedversus other oil sources with differing fatty acid compositions, such assoybean oil (Lu, Zhang, Yin, Everts, & Li, 2008), sunflower oil (Duran-Montge, Realini, Barroeta, Lizardo, & Esteve-Garcia, 2008) olive oil(Nuernberg et al., 2005), or oil blends (D'Arrigo et al., 2002; Hoz,D'Arrigo, Cambero, & Ordonez, 2004).

Effects of feeding flaxseed on pork fatty acid composition have alsobeen variable across studies, revealing the need to standardise feed pro-cessing andpork production strategies in efforts to consistentlymeet re-quirements for n−3 fatty acid enrichment claims. Variation in n−3fatty acid content when feeding flaxseed can in part be due to differ-ences in feed processing. Recently, we demonstrated optimal n−3fatty acid digestibility can be attained by co-extruding flaxseed withfield peas (50:50) at 135 °C and 400 psi pressure (Htoo, Meng,Patience, Dugan, & Zijlstra, 2008). Even at this, feeding a high level offlaxseed (10%) for an extended period (11 weeks) to pigs was notfound to be sufficient to provide 300 mg total n−3 fatty acids per100 g of longissiumus thoracis trimmed of epimysium and associated ad-ipose tissue (Juarez et al., 2011). However, itwasnoted a combination offat and lean tissue in retail ground pork could provide enough n−3fatty acids to achieve an enrichment claim in Canada.

The present study is an extension of Juarez et al. (2011), and inves-tigates the fatty acid composition of tissue and tissue combinationsfound in various cuts of pork when feeding increasing levels of optimal-ly processed flaxseed. Gender effects on fatty acid composition were

reserved.

Table 1Fatty acid composition (% FAME) of the 0%, 5% and 10% flaxseed diets during the feedingperiod.

Week 1–4; dietaryflaxseed

Week 5–8; dietaryflaxseed

Week 9–11;dietary flaxseed

FAME 0% 5% 10% 0% 5% 10% 0% 5% 10%

16:0 16.7 13.2 9.7 16.6 13.9 9.4 17.9 13.3 10.0c9-16:1 0.7 0.4 0.1 0.9 0.5 0.1 1.0 0.4 0.118:0 5.3 4.0 3.1 5.6 4.7 3.0 6.5 4.4 2.8trans-18:1 0.5 0.3 0.1 0.7 0.3 0.1 0.9 0.4 0.1c9-18:1 21.3 18.2 16.7 24.1 19.4 16.3 22.4 19.4 17.5c11-18:1 1.8 1.5 1.3 1.9 1.3 1.2 1.8 1.5 1.218:2n−6 44.3 35.4 28.0 40.7 31.8 27.9 42.0 30.9 28.418:3n−3 5.4 23.9 38.8 5.1 23.2 38.6 3.7 26.1 36.5c11-20:1 0.7 0.9 0.1 0.8 1.2 1.2 0.7 1.2 1.522:0 0.5 0.4 0.5 0.5 0.8 0.5 0.4 0.4 0.3Minor (b0.5%) 2.7 1.8 1.7 3.1 2.8 1.8 2.8 2.1 1.518:2n−6/18:3n−3 8.1 1.5 0.7 8.0 1.4 0.7 11.3 1.2 0.8

Abbreviation: fatty acid methyl esters, FAME.

542 T.D. Turner et al. / Meat Science 96 (2014) 541–547

also investigated due to barrows having greater rates of endogenous fatsynthesis, whereas gilts have a greater capacity for long-chain (LC,≥20carbon) n−3 fatty acid synthesis (Ntawubizi, Raes, Buys, & De Smet,2009). Effects of feeding flaxseed on levels of 18:3n−3 elongationand desaturation products in porkwere also investigated in anticipationthat separate enrichment or health claims for LC n−3 fatty acids willeventually be possible.

2. Materials and methods

2.1. Animal feeding and housing

The feeding trial was conducted at the Prairie Swine Centre(Saskatoon, SK, Canada) in accordance with the University ofSaskatchewan Committee on Animal Care (protocol # 199970019), in ad-herence with guidelines set by the Canadian Council on Animal Care(CCAC, 1993). The same animals were used as reported in Juarez et al.(2011), and this included a description of diets fed and production perfor-mance. In brief, the trial included 96 pigs with equal numbers of barrowsand gilts (Camborough Plus females by Line C337 sires, PIC, Winnipeg,MB, Canada) with an initial body weight of 48 ± 2 kg (mean ± SD).Four animals were fed per pen, and genders were penned separately.Pigs were blocked, and blocks consisted of one pen of pigs per genderper diet, and blocks were separated by one week to facilitate slaughterand sample collection. Pigs were fed diets containing 0%, 5% or 10% flax-seed. Flaxseed was co-extruded 50:50 with field peas (Linpro®, O & TFarms, Regina, SK, Canada) at 400 psi without added water and a barreltemperature of 135 °C by a single screw to optimise fatty acid digestibility(Htoo et al., 2008). Field peas were fed at equal amounts across diets, anddiet formulations were adjusted at three points to meet the nutrient re-quirements of the pigs (National Research Council, 1998) as reported byJuarez et al. (2011). Feed fatty acids were prepared for analysis using di-rectmethylation (Sukhija & Palmquist, 1988) and fatty acidmethyl esters(FAME) were analysed by gas chromatography (Varian 3800 GC,equipped with a 8100 autosampler, Varian, Walnut Creek, CA, USA) ona 30 m capillary column (SP-2340, i.d. 25 μm, Supelco, Bellefonte, PA,USA) under conditions described by Dugan et al. (2007). Briefly, sampleswere injected using a 20:1 split ratio with the initial column temperatureheld at 50 °C for 30 s, ramped to 170 °C at 25 °C/min, held for 3 min,ramped to 180 °C at 2 °C/min then ramped to 230 °C at 10 °C/min withthe injector and flame ionisation detector held constant at 250 °C. TheFAME were identified by retention time comparison to standards (GLC-463 and GLC-603, Nu-Chek Prep, Inc. Elysian, MN, USA) and integratedusing Varian Star Chromatography Workstation software v6.41 (Varian,Walnut Creek, CA, USA).

2.2. Slaughter and sample collection

After 76 days on test, pigs were transported 6 h to the LacombeResearch Centre (Lacombe, AB, Canada) for slaughter. Pigs arrived inlate afternoon and were held overnight without feed but with freeaccess to water. The following morning, pigs were stunned (1.8 amps,5 s), exsanguinated, scalded (4 min, 63 °C), dehaired, suspended bytheir Achilles tendon, eviscerated, and carcasses were split and chilledovernight in a cooler held at 2 °C with a wind speed of 0.5 m/s. At24 h post-slaughter, one pig closest to the pen average weight wasselected for intensive fatty acid analysis. Carcasses were broken to pri-mals and butt, loin and picnic primals were used for further analyses.These primals were trimmed to 5 mm subcutaneous fat and then dis-sected into lean muscle with epimysium, intermuscular (seam), andsubcutaneous adipose tissue. The fatty acid composition of each leanand fat depot were determined separately for each primal, and thenused to calculate fatty acid amounts in cuts typically seen at retail.These included lean with epimysium (L), lean plus seam fat (LS), andlean plus seam plus 5 mm subcutaneous fat (LSS).

2.3. Tissue FAME preparation and analysis

Tissues were ground three times (3 mm plate, Butcher Boy MeatGrinder Model TCA22, Lasar Manufacturing Co., Los Angeles, CA, USA)then further comminuted (Robot Coupe BX3, Ridgeland, MS, USA) andsubsampled for fatty acid analysis. Muscle lipids were extracted using2:1 chloroform:methanol and a solvent to sample ratio of 20:1 (Folch,Lees, & Stanley, 1957). Muscle lipid extracts were dried using arotoevaporator (P12 Rotavapor, Buchi, Thornhill, ON, Canada) then fur-ther freeze dried (Genesis 25 SQ EL, VirTis Company, Gardiner, NY, USA)overnight to remove traces of water. Adipose tissue subsamples(50 mg) were freeze dried overnight. Muscle and adipose tissue lipidswere disolved in toluene-containing internal standard (10% of lipidweight, C19:0 methyl-ester, Nu-Chek Prep Inc. Elysian, MN, USA) andmethylated with 5% methanolic HCl at 80 °C for 1 h (Kramer et al.,1998). Samples were cooled, hexane added and then 0.88% KCl toexpel hexane containing the FAME. Hexane extracts were dried overanhydrous sodium sulphate and stored at -20 °C until analysed. Whenanalysed, hexane extracts were dried to remove both hexane and tolu-ene and resuspended in hexane to 2 mg lipid/ml and analysed using theGC equipment and conditions as described for the feed analysis.

2.4. Statistical analysis

Data were analysed using the Mixed procedure of SAS© v9.1 soft-ware (SAS, 2003). The FAME data were analysed based on cuts (L, LS,LSS) within primals (loin, picnic, butt) using a two-way ANOVA andthe statisticalmodel included diet (0%, 5% and 10% flaxseed) and gender(gilt, barrow) as main effects together with their interaction. Individualanimal was used as the experimental unit and slaughter date was usedas a random factor. No gender by diet interactions were found for anyfatty acids, therefore, this term was removed from the statisticalmodel. Relationships between the n−3 fatty acid content of subcutane-ous fat and other depots were assessed using the Regression procedureof SAS© v9.1 software (SAS, 2003).

3. Results and discussion

Over the course of the experiment, the percentage of 18:3n−3 intotal FAME for the 0, 5 and 10% flaxseed diets averaged 4.9, 24.4 and38% respectively, and adding 5 and 10% flaxseed to the diet reducedthe n−6/n−3 ratio by 6.8 and 12.4 fold respectively (Table 1). Similarresults were attained by Matthews, Homer, Thies, and Calder (2000)who fed 5 and 10%whole flaxseed, reporting dietary 18:3n−3 contentsof 21% and 33%, respectively. The fatty acid profiles of the present dietswere also similar to those reported in an earlier study by Juarez et al.

543T.D. Turner et al. / Meat Science 96 (2014) 541–547

(2010) when feeding 0, 5 and 10% flaxseed diets. Diet adjustmentsmade to meet the changing dietary requirements of the animals hadminimal effect on the diet fatty acid profiles (Table 1).

Fatty acid composition of cuts from loin, butt and picnic primalsfollowed similar trends when flaxseed was added to the diet; therefore,only fatty acid compositions of cuts from the loin primal have beenpresented and discussed (Tables 2 & 3), except for n−3 fatty acids,where results of cuts across primals are also presented.

3.1. Total FAME

The total FAME content of L, LS and LSS cuts from loinwere unaffectedby the addition of flaxseed to the diet (P N 0.05, Table 2). The fat contentof the loin L cut was 1324 mg/100 g greater in barrows versus gilts(P b 0.001) and the gender difference increased to 3223 mg/100 g inthe LS cut (P b 0.001) due to the greater amount of seam fat in barrows.The gender difference in total FAME content was further widened to5210 mg/100 g in the LSS cut. Barrows are known to be fatter than gilts(Eggert, Grant, & Schinckel, 2007; Juarez et al., 2010; Nuernberg et al.,2005) with greater amounts of seam fat, but adding a constant amountof subcutaneous fat for LSS cuts should have theoretically added thesame amount of fat for both genders. Slight differences in anatomybetween gilts and barrows, or a greater lipid content in barrow subcuta-neous fat may explain the difference. Greater rates of fat synthesis andadipocyte hypertrophy have been noted in barrows (Kouba & Sellier,2011), which could translate into a more mature fat depot with greaterfat content.

3.2. Saturated and monounsaturated fatty acids

Including flaxseed in the diet at 5% or 10% reduced saturated fattyacids (SFA) in loin L, as well as in LS and LSS (P b 0.05). The twomajor SFA, namely 16:0 and 18:0, were lower in loin L, as well as in LSand LSS when flaxseed was included in the diet at 5% with no furtherdecrease when feeding 10% flaxseed (P b 0.05). Increasing dietary flax-seed to 5% also reduced total monounsaturated fatty acids (MUFA) andc9-18:1 in loin L, as well as LS, and LSS (P b 0.01, Table 2), with no fur-ther decrease when feeding 10% flaxseed. Present findings concur withPascual et al. (2007) in that increaseddietary 18:3n−3 content reduced16:0 and c9-16:1 as well as 18:0 and c9-18:1, with effects being mostprominent in muscle. Moreover, suppressive effects of dietary PUFA

Table 2Dietary influence of flax supplemented at 0%, 5% and 10% on the predominant saturated andmoand lean + seam and subcutaneous fat (LSS).

0% Flaxseedz 5% Flaxseed 10% Flaxseed

L Total 6141 5174 5788SFA 2459 a 1892 b 1953

16:0 1523 a 1164 b 118118:0 823 a 635 b 679

MUFA 3018 a 2285 b 2260c9-18:1 2430 a 1842 b 1871

LS Total 11,796 9760 10,684SFA 5001 a 3718 b 3732

16:0 3033 a 2261 b 222818:0 1740 a 1277 b 1326

MUFA 5611 a 4143 b 3991c9-18:1 4627 a 3451 b 3380

LSS Total 22,556 20,343 20,830SFA 9646 a 7725 b 7019

16:0 5774 a 4669 b 415818:0 3434 a 2687 b 2535

MUFA 10,645 a 8610 b 7693c9-18:1 9000 a 7364 b 6699

a–c = differences within a row indicate significance, P b 0.05.z = eight pigs per treatment, sexes equally represented.Abbreviations: saturated fatty acids, SFA; monounsaturated fatty acids, MUFA.

are stronger on tissue c9-18:1 than SFA (Duran-Montge et al., 2008).Lipogenic enzyme activities have not been found to respond to dietaryPUFA in muscle or adipose tissue (Kouba, Enser, Whittington, Nute, &Wood, 2003), however, adipose expression of lipogenic genes, i.e. fattyacid synthase and stearoyl-CoA desaturase, can be reduced by feedingflaxseed compared to low fat or high n−6 diets (Duran-Montge,Theil, Lauridsen, & Esteve-Garcia, 2009). As such, the reduction in SFAfrom feeding 5% flaxseed is likely attributable to dilution by tissuePUFA, whereas the lower level of MUFA may be due to dilution and/orPUFA related suppression of stearoyl-CoA desaturase. Increasing dietaryflaxseed to 10% in the present study did not further decrease SFA orMUFA content, suggesting a maximal effect around 3% PUFA in the diet.

The lower content of 16:0 in pork productsmay be positive, as its con-sumption is linked to an increased risk for cardiovascular disease (CVD)(Hunter, Zhang, & Kris-Etherton, 2010). Conversely, 18:0 and c9-18:1may have neutral or slightly beneficial effects on CVD development(Hunter et al., 2010; Kris-Etherton & Nutrition, 1999). Nonetheless, thecardio-protective effects of displacing 16:0 with 18:0 or c9-18:1 are notas strong as for PUFA (Sanders, 2009).

Barrows had a higher SFA and MUFA content than gilts in loin L, LSand LSS cuts (P b 0.001, Table 2). This matches the higher fat contentof barrows versus gilts (Eggert et al., 2007; Juarez et al., 2010;Nuernberg et al., 2005), and has been attributed to greater rates of en-dogenous fat synthesis and adipocyte hypertrophy in barrows (Kouba& Sellier, 2011).

3.3. Polyunsaturated fatty acids

3.3.1. Total polyunsaturated fatty acidsThe PUFA content of loin L, LS and LSS increased by roughly 1.5 fold

and 2.5 fold when feeding 5% and 10% flaxseed, respectively (P b 0.001,Table 3). These findings are in agreement with Cherian and Sim (1995)who reported the PUFA/SFA ratio increased in relation to flaxseed feed-ing up 17% of the diet before plateauing, however, they found no effectonmuscle SFA. Huang, Zhan, Luo, Liu, and Peng (2008) reported a linearincrease in PUFA and decreased MUFA and SFA proportions in muscleand subcutaneous adipose tissue during flaxseed feeding, however, anincrease in muscle total fat content was also noted. Again, loin cutsfrom barrows had significantly greater total PUFA than gilts (P b 0.05)relating to their greater overall fat content.

nounsaturated fatty acids (mg FA/100 g fresh tissue) of loin lean (L), lean + seam fat (LS)

SE Diet Gilt Barrow SE Sex

278 0.07 5039 6363 227 b0.001b 115 b0.01 1774 2429 94 b0.001b 71 b0.01 1093 1486 58 b0.001b 42 b0.05 597 827 34 b0.001b 148 b0.01 2249 2793 121 b0.01b 123 b0.01 1827 2269 101 b0.01

696 0.14 9135 12,358 568 b0.001b 306 b0.05 3369 4932 250 b0.001b 178 b0.01 2051 2964 145 b0.001b 118 b0.05 1163 1732 96 b0.001b 305 b0.01 3973 5191 249 b0.01b 260 b0.01 3314 4325 213 b0.01

1120 0.36 18,638 23,848 915 b0.001b 517 b0.01 6782 9477 422 b0.001b 307 b0.01 4089 5645 250 b0.001b 195 b0.01 2385 3386 159 b0.001b 512 b0.01 8077 9888 418 b0.01b 448 b0.01 6914 8461 366 b0.01

Table 3Dietary influence of flax supplemented at 0%, 5% and 10% on the polyunsaturated fatty acid profile (mg FA/100 g fresh tissue) of loin lean (L), lean + seam fat (LS) and lean + seam andsubcutaneous fat (LSS).

0% flaxseed 5% flaxseed 10% flaxseed SE Diet Gilt Barrow SE Sex

L PUFA 611 c 937 b 1520 a 44 b0.001 961 1084 36 b0.05PUFA/SFA 0.20 c 0.41 b 0.68 a 0.02 b0.001 0.48 0.38 0.02 b0.01n−6 530 c 596 b 736 a 22 b0.001 595 646 18 0.0618:2n−6 422 c 498 b 653 a 21 b0.001 497 552 17 b0.0520:4n−6 59.8 a 49.5 b 34.3 c 2.2 b0.001 51.9 43.8 1.8 b0.01

n−3 75 c 336 b 779 a 24 b0.001 360 433 20 b0.0518:3n−3 41 c 237 b 615 a 21 b0.001 266 329 17 b0.0520:5n−3 5.3 c 20.4 b 31.9 a 0.9 b0.001 19.1 19.3 0.7 0.9022:5n−3 15.4 c 33.2 b 39.6 a 1.1 b0.001 30.0 28.8 0.9 0.3522:6n−3 6.42 b 9.29 a 8.14 ab 0.77 b0.05 8.55 7.35 0.63 0.19

n−6/n−3 7.19 c 1.78 b 0.95 a 0.20 b0.001 3.41 3.21 0.16 0.39

LS PUFA 1134 c 1844 b 2909 a 132 b0.001 1741 2184 108 b0.01PUFA/SFA 0.19 c 0.43 b 0.71 a 0.02 b0.001 0.49 0.40 0.02 b0.01n−6 981 b 1119 b 1332 a 53 b0.001 1048 1240 43 b0.0118:2n−6 822 c 981 b 1208 a 48 b0.001 911 1096 40 b0.0120:4n−6 65.8 a 52.6 b 36.7 c 2.3 b0.001 55.5 47.9 1.8 b0.01

n−3 142 c 715 b 1569 a 82 b0.001 684 933 67 b0.0518:3n−3 90 c 548 b 1291 a 68 b0.001 537 749 56 b0.0520:5n−3 5.7 c 23.4 b 38.9 a 1.2 b0.001 21.9 23.4 1.0 0.2722:5n−3 20.9 c 48.1 b 59.7 a 2.9 b0.001 41.0 44.8 2.4 0.2622:6n−3 8.7 b 12.4 a 10.6 ab 0.9 b0.05 11.1 10.1 0.7 0.36

n−6/n−3 7.03 a 1.57 b 0.87 c 0.16 b0.001 3.27 3.04 0.13 0.24

LSS PUFA 2223 c 3961 b 6074 a 195 b0.001 3733 4439 159 b0.01PUFA/SFA 0.20 c 0.46 b 0.79 a 0.03 b0.001 0.52 0.44 0.02 b0.05n−6 1921 c 2334 b 2697 a 95 b0.001 2165 2470 78 b0.0518:2n−6 1655 c 2095 b 2472 a 87 b0.001 1930 2218 71 b0.0120:4n−6 75.5 a 59.4 b 42.4 c 2.3 b0.001 62.5 55.8 1.9 b0.05

n−3 277 c 1605 b 3362 a 114 b0.001 1549 1947 93 b0.0118:3n−3 186 c 1269 b 2803 a 95 b0.001 1251 1588 77 b0.0120:5n−3 7.8 c 32.1 b 55.4 a 1.7 b0.001 30.4 33.2 1.4 0.1622:5n−3 31.0 c 80.3 b 102.0 a 4.5 b0.001 66.5 75.7 3.6 0.0922:6n−3 12.8 b 19.4 a 16.2 ab 1.3 b0.01 16.2 16.0 1.0 0.89

n−6/n−3 7.05 c 1.46 b 0.81 a 0.17 b0.001 3.23 2.98 0.14 0.21

a–c = differences within a row indicate significance, P b 0.05.z = eight pigs per treatment, sexes equally represented.Abbreviations: polyunsaturated fatty acids, PUFA; PUFA/SFA = 18:2n−6 + 18:3n−3/14:0 + 16:0 + 18:0.

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3.3.2. n−6 fatty acidsAlthough flaxseed fatty acids contain more than 50% 18:3n−3,

there is also about 20% 18:2n−6. As a consequence, the content of18:2n−6 in loin L, LS and LSS cuts increased in relation to flaxseed feed-ing level (P b 0.001, Table 3). These findings are in agreement withNguyen, Nuijens, Everts, Salden, and Beynen (2003) who found strongcorrelations between dietary PUFA levels and adipose tissue deposition.In contrast, the 20:4n−6 content decreased in relation to flaxseed feed-ing level in loin L as well as LS and LSS cuts (P b 0.001). Interestingly,Cherian and Sim (1995) found no effect on the proportion of 18:2n−6 within muscle, however, the 20:4n−6 proportion decreased with10% flaxseed feeding with no further effects at higher flaxseed feedinglevels. The inverse pattern observed between 18:2n−6 and 20:4n−6in relation to flaxseed feeding illustrates the higher affinity of n−3over n−6 fatty acids for desaturase enzymes along the shared pathwayleading to formation of the LC n−6 and n−3 fatty acids (Sprecher,2000). Overall, increases in 18:2n−6 were greater than reductions in20:4n−6 when feeding flaxseed, leading to an overall increase intotal n−6 fatty acids (P b 0.001 Table 3).

Compared to consumption of 14:0, 16:0 or trans-MUFA, 18:2n−6 isconsidered to have beneficial effects on the prevention of CVD (FAO,2010). Excess n−6 PUFA consumption has, however, also been linkedto pro-inflammatory immune responses, largely in relation to eicosanoidsproduced from 20:4n−6 (Calder, 2006). However, low conversion of es-sential fatty acids to LC n−3 and n−6 fatty acidswithin humans empha-sises the heavy reliance on dietary sources of 20:4n−6 for this activity(Williams & Burdge, 2006). In perspective, this means with increasedrates of flaxseed feeding, the associated increase in 18:2n−6 in loin

cuts coupled with the concomitant reduction in 20:4n−6 would belargely beneficial to human health (Adam, Wolfram, & Zollner, 2003).Likewise, in loin L, the PUFA/SFA ratio increased with flaxseed feeding,which was also evident in the loin LS and LSS cuts (P b 0.001). The min-imum ratio of PUFA/SFA suggested for optimal health is 0.45 (BritishDepartment of Health, 1994). The PUFA/SFA ratio in loin L, LS and LSScuts were below, near and in excess of recommendations when feeding0, 5 and 10% flaxseed, respectively.

Regarding PUFA composition, although the overall n−6 fatty acidlevels increased with flaxseed feeding, the n−6/n−3 ratio in loin L,LS and LS cuts decreased roughly 7 fold from the 0% to the 10% flaxseeddiet (P b 0.001, Table 3). Whereas an n−6/n−3 ratio of 4 is recom-mended to provide an optimal balance between inflammatory vs.anti-inflammatory eicosanoids, the absolute amount of n−6 and n−3fatty acids may be more indicative of health status (Smit, Mozaffarian,& Willett, 2009; Wijendran & Hayes, 2004). Regardless, whereas then−6 fatty acid content increased with flaxseed feeding, the n−3 fattyacid content increased at a greater rate and provided further enhance-ments to the fatty acid profile for human consumption.

The loin L cut from barrows had a trend for more total n−6 fattyacids than gilts, and the difference was significant for both loin LS andLSS cuts (Table 3). This was largely attributable to greater amounts of18:2n−6 across L, LS and LSS cuts in barrows versus gilts (P b 0.05).Barrows, however, had consistently lower amounts of 20:4n−6 acrossloin cuts (P b 0.05)whichmay again relate to differing affinities of n−3and n−6 fatty acids for desaturase enzymes as previously mentioned.Consequently, although loin cuts from barrows have more SFA andtheir consumption by humans may pose more of a risk for developing

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545T.D. Turner et al. / Meat Science 96 (2014) 541–547

CVD in humans, they have less 20:4n−6 which may which may atten-uate pro-inflammatory immune responses. There were no gender dif-ferences in the n−6/n−3 ratio for any loin cuts.

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Fig. 2. The 18:3n−3 content in lean (L), lean + seam (LS) and lean + seam + subcutane-ous (LSS) cuts of loin, picnic and butt primals. Letters within muscle (a–g) indicate statisticaldifference (P b 0.05), error bars indicate standard error of the mean.

3.3.3. n−3 fatty acids

3.3.3.1. Total n−3 and 18:3n−3. Juarez et al. (2011) found longissiumusthoracis (trimmed of epimysium, intermuscular, and subcutaneous fat)from pigs used in the present study had insufficient amounts of totaln−3 fatty acids for an enrichment claim in Canada (300 mg/100 g),even when feeding 10% flaxseed for 11 weeks. Present results indicatethat muscles of the loin with even just the epimysium attached (i.e.the L cut), had sufficient total n−3 fatty acids for an enrichment claimwhen feeding pigs 5% flaxseed for 11 weeks (Table 3), and the samewas true for L cuts from butt and picnic primals (Fig. 1). Feeding 10%flaxseed led to clear additional total n−3 fatty acid increases in L cutsacross primals, and adding adipose tissue in the form of seam and sub-cutaneous fat resulted in excessive amounts of n−3 fatty acids relativeto the amount required for an enrichment claim. Increased risk of oxida-tive instability associated with higher 18:3n−3 may be offset by inclu-sion of vitamin E above recommended requirements, however 18:3n−3 levels up to 12% were not found to affect oxidation or taste for cookedloin (Ahn, Lutz, & Sim, 1996), suggesting packaging or cooking tech-niques may have a more substantial effect. In addition, the inclusion ofadipose tissues in servings of porkwould reduce levels of flaxseed need-ed to be fed to meet requirements for n−3 fatty acid enrichmentclaims, and thus further reduce the potential for undesirable sensoryeffects.

The major n−3 fatty acid found in the loin L was 18:3n−3 and itscontent increased by 4.5 to almost 15 fold when feeding 5 and 10% flax-seed respectively (P b 0.001), with a similar pattern for L cuts from buttand picnic primals (Fig. 2). Further increases in 18:3n−3 were notedfor LS and LSS cuts across primals (Fig. 2). When feeding 10% flaxseed,the 18:3n−3 content in loin L alone was twice the level necessary foran n−3 enrichment claim in Canada (Table 2), and adding seam (LS)and backfat (LSS) when feeding 5 or 10% flaxseed provided excessive18:3n−3 enrichments. Current recommendations call for up to 2% ofdaily energy intake as 18:3n−3 due to associated reductions in coro-nary heart disease (CHD) risk (FAO, 2010; Smit et al., 2009), whichequates to roughly 1300 mg/day based on a 2500 kcal/day intake.Whereas this level can be achieved in pork cuts by feeding flaxseed,health recommendations are based on total diet rather than intakefrom a single source.

Barrows had consistently greater amounts of total n−3 fatty acids inloin L, LS and LSS cuts than gilts (P b 0.05, Table 3). Thiswas attributableto greater amounts of 18:3n−3 (P b 0.05) and again relates to thegreater overall fat content of primal cuts from barrows.

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Fig. 1. Total n−3 fatty acid content in lean (L), lean + seam (LS) and lean +seam + subcutaneous (LSS) cuts of loin, picnic and butt primals. Minimal content requiredfor enrichment labelling in Canada indicated at 300 mg/100 g. Letters within muscle (a–g)indicate statistical difference (P b 0.05), error bars indicate standard error of the mean.

3.3.3.2. Long-chain n−3 fatty acids across primal cuts.A further positive as-pect of tissue 18:3n−3 enrichment in the present study was its elonga-tion, desaturation and deposition in the form of LC n−3 fatty acids.Adding 5 and 10% flaxseed in the diet resulted in roughly a 2.5 to 3-foldincrease in LC n−3 fatty acids in loin L (Table 3), and the same patternwas seen in L cuts from picnic and butt primals (Fig. 3). Likewise, the LCn−3 fatty acid content in loin, butt and picnic roughly doubled from Lto LSS cuts, irrespective of flaxseed feeding level. The LC n−3 fatty acidcontent of the LSS cut from the three primals ranged from130 mg/100 g in loin when feeding 5% flaxseed to 193 mg/100 gin the butt when feeding 10% flaxseed. Based on present findings,the LC n−3 fatty acids in100 g servings of LSS cuts could providefrom 66% to 77% of the daily recommended intakes. In referenceto current actual intakes, a study involving pregnant women inCanada found on average only 141 mg/day LC n−3 fatty acidswere consumed, considerably less than the recommended intake(Denomme, Stark, & Holub, 2005). This is partially a reflectance ofcultural norms and highlights the need for a greater variety of n−3 fatty acid enriched products.

In loin L as well as LS and LSS cuts, increases in LC-n−3 fatty acidswere clearly related to increases in 20:5n−3 and 22:5n−3 when thelevel of flaxseed was increased in the diet (P b 0.001, Table 3). Interest-ingly, the 22:6n−3 content in loin L was highest with the 5% flax-seed diet, with the 10% diet resulting in an intermediate content(P b 0.001). Enser, Richardson, Wood, Gill, and Sheard (2000) reportedan increase in phospholipid 22:6n−3 content when feeding flaxseed,however several studies have reported no increase in 22:6n−3 contentin relation to dietary 18:3n−3 feeding as a consequence of competitionbetween substrates for enzymes (Bee, Jacot, Guex, & Biolley, 2008;Juarez et al., 2010). The Δ6-desaturase enzyme is necessary for the ini-tial desaturation of 18:2n−6 and 18:3n−3, as well as for desaturationof 24:4n−6 and 24:6n−3, however slight preference for 18-carbonPUFA, possibly being further exaggerated in relation to relative

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Fig. 3. Combined LC n−3, including 20:5n−3, 22:5n−3 and 22:6n−3 content in lean(L), lean + seam (LS) and lean + seam + subcutaneous (LSS) cuts of loin, picnic andbutt primals. Letters within muscle (a–g) indicate statistical difference (P b 0.05), errorbars indicate standard error of the mean.

546 T.D. Turner et al. / Meat Science 96 (2014) 541–547

abundances of 18-carbon substrates, limits 22:6n−3 formation(Portolesi, Powell, & Gibson, 2007).

Conversion of 18:3n−3 to its longer chain derivatives is quite low inhumans; hence the greater efficiency of direct dietary consumption ofLC n−3 fatty acid for their accumulation, and the need for readily avail-able dietary sources (Williams & Burdge, 2006). Producing pork withenhanced contents of LC n−3 fatty acids has obvious potential aspigs are reportedly more efficient than humans at elongating anddesaturating 18:3n−3, with more than 33% being converted to20:5n−3, 22:5n−3 or 22:6n−3 (Kloareg, Noblet, & van Milgen,2007). The present findings highlight the LC n−3 fatty acid contribu-tions adipose tissues can make to LS and LSS cuts from primals(Fig. 3). Whereas consumers may prefer to remove subcutaneous fatwhen eating pork, intermuscular fat available in retail cuts is usuallyconsumed; hence feeding flaxseed to pigs provides an opportunity toexploit natural processes through dietary enrichment to increase LCn−3 fatty acids in pork available to consumers.

The benefits of LC n−3 fatty acids for humans have revolved around20:5n−3 and 22:6n−3 with the former as a precursor for anti-inflammatory eicosanoids (Calder, 2006), and the latter more associatedwith cognitive ability in children (Innis, 2007) and slowing of degenera-tive neurological diseases (Dyall &Michael-Titus, 2008). Evidence also in-dicates 20:5n−3 and 22:6n−3 reduce the risk of developing CVD(Albert et al., 2002). Health recommendations for LC n−3 fatty acidshave mainly focused on fish consumption due to high concentrations of20:5n−3 and 22:6n−3, calling for a combined intake of 250 mg/day toreduce the risk of developing CHD (Smit et al., 2009). The greateraccumulation of 22:5n−3 in terrestrial animals, as noted in thepresent study, is often overlooked, but make a considerable contri-bution to daily LC n−3 intake (Howe, Buckley, & Meyer, 2007). Therole of 22:5n−3 in human health is less clear, nevertheless evi-dence suggests stronger inhibition of platelet aggregation than20:5n−3 or 22:6n−3 (Akiba, Murata, Kitatani, & Sato, 2000). Evi-dence also indicates 22:5n−3 reduces the effects of ageing on cog-nitive abilities and can be readily converted to 20:5n−3 in times ofdeficiency (Kaur, Cameron-Smith, Garg, & Sinclair, 2011). In light ofthe functional properties of 22:5n−3 and retro-conversion to20:5n−3, perhaps 22:5n−3 intake should also be included whencalculating total LC n−3 fatty acids as in Australia.

Therewere no differences in loin L, LS or LSS content of LC n−3 fattyacids between barrows and gilts (P N 0.05). Loin results for gilts andbarrows were consistent with picnic and butt results (data notshown). Consequently, although cuts from primals from both gilts andbarrows could attain levels of n−3 fatty acid required for enrichmentclaims, cuts from gilts may be more healthful than barrows due totheir greater relative amounts of LC n−3 fatty acids.

3.3.3.3. n−3 enrichment in further processed products. The presentresults highlight the differences in tissue fat content, fatty acid compo-sition and the potential to enrich the n−3 fatty acid composition ofpork products. Levels of n−3 fatty acids attained in LSS cuts when feed-ing flaxseed far exceeded requirements necessary for an enrichmentclaim, and unlike intermuscular fat, subcutaneous fat would likely betrimmed by the consumer. However, the subcutaneous fat representsa rich source of n−3 fatty acids that could be used to develop processedmeat products or as enriched lard for baked goods, to reach a broaderconsumer base. For example, when lean and adipose tissue are com-bined in a 20% fat grind, the n−3 fatty acid content was 1300 and2800 mg/100 g grind when feeding 5% and 10% flaxseed, respectively(Juarez et al., 2011). Monziols, Bonneau, Davenel, and Kouba (2007)reported differences in adipose tissue development and fatty acid com-position, emphasising the need for further investigations into the fattyacid profile of various retail pork cuts available to consumers. Further-more, expression of theΔ6-desaturase protein, key to subsequent elon-gation and further desaturation to LC fatty acids, was found to be higherin subcutaneous adipose tissue compared to skeletalmuscle, making it a

somewhat potentially richer source (Cánovas, Estany, Tor, Pena, &Doran, 2009). The n−3 fatty acid content of subcutaneous fat inthe present study was strongly correlated between loin and butt(P b 0.001, r = 0.99, slope = 0.92) as well as loin and picnic(P b 0.001, r = 0.99, slope = 0.73), providing a highly uniform prod-uct when feeding 5% and 10% flaxseed supplying up to 68 mg and145 mg n−3 fatty acid per g subcutaneous tissue, respectively. Assum-ing20% added fat in sausage, enrichment levels could be attained in sau-sages made from commercially produced pork (i.e. similar to pork fedthe 0%flaxseed) by replacing 1.5 g fat/100 g sausagewith subcutaneousadipose tissue from 5%flaxseed fed or 1.0 g from 10%flax fed (assuming85% lipid in subcutaneous tissue).

4. Conclusions

Presentfindings show that retail cuts from different primals can eas-ily meet requirements for n−3 fatty acid enrichment when associatedadipose tissue depots are retained with the lean cuts. Moreover, theLC n−3 fatty acid content attained when feeding flaxseed can providea considerable proportion of the daily recommended intake. Feedingstrategies will, therefore, need to be tailored to address end productuses, whether sold as a fresh retail cut, or if excess adipose tissuetrimmed at slaughter is used for enrichment of other processed prod-ucts, including enriched lard.

Acknowledgements

The authors would like to thank the Saskatchewan Agriculture De-velopment Fund and Flax Canada 2015 for project funding. T.D. Turnerand C.Mapiye were supported through funding provided by the AlbertaLivestock and Meat Agency.

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