Meat Science 95 (2013) 98–109 Contents lists available at SciVerse ScienceDirect Meat Science journal homepage: www.elsevier.com/locate/meatsci Effects of feeding flaxseed or sunflower-seed in high-forage diets on beef production, quality and fatty acid composition C. Mapiye a, J.L. Aalhus a, T.D. Turner a, D.C. Rolland a, J.A. Basarab b, V.S. Baron a, T.A. McAllister c, H.C. Block d, B. Uttaro a, O. Lopez-Campos a, S.D. Proctor e, M.E.R. Dugan a,⁎ a Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, Alberta T4L 1W1, Canada Alberta Agriculture and Rural Development, Lacombe Research Centre, 6000 C & E Trail, Lacombe, Alberta T4L 1W1, Canada Agriculture and Agri-Food Canada, Lethbridge Research Centre, 1st Avenue South 5403, PO Box 3000, Lethbridge, Alberta T1J 4B1, Canada d Agriculture and Agri-Food Canada, Brandon Research Centre, 18th Street and Grand Valley Road, P.O. Box 1000A, RR3, Brandon, Manitoba R7A 5Y3, Canada e Metabolic and Cardiovascular Diseases (MCVD) Laboratory, Alberta Diabetes and Mazankowski Institutes, Li Ka Shing (LKS) Centre for Health Research Innovation, University of Alberta, Edmonton, Alberta T6G 2E1, Canada, b c a r t i c l e i n f o Article history: Received 7 February 2013 Received in revised form 26 March 2013 Accepted 27 March 2013 Keywords: Beef production Conjugated linoleic acid Grass hay Red clover silage a b s t r a c t Yearling steers were fed 70:30 forage:concentrate diets for 205 d, with either grass hay (GH) or red clover silage (RC) as the forage source, and concentrates containing either sunflower-seed (SS) or flaxseed (FS), each providing 5.4% oil to diets. Feeding diets containing SS versus FS significantly improved growth and carcass attributes (P b 0.05), significantly reduced meat off-flavor intensity (P b 0.05), and significantly increased intramuscular proportions of vaccenic (t11-18:1), rumenic (c9,t11-CLA) and n−6 fatty acids (FA, P b 0.05). Feeding diets containing FS versus SS produced significantly darker and redder meat with greater proportions of atypical dienes (P b 0.05). A significant forage × oilseed type interaction (P b 0.05) was found for n−3 FA, α-linolenic acid, and conjugated linolenic acid, with their greatest intramuscular proportions found when feeding the RC-FS diet. Feeding GH versus RC also significantly improved growth and carcass attributes, sensory tenderness (P b 0.05) and significantly influenced intramuscular FA composition (P b 0.05), but overall, forage effects on FA profiles were limited compared to effects of oilseed. Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved. 1. Introduction Beef lipids typically have high contents of saturated fatty acids (SFA), compared to tissues from monogastric species, due to extensive ruminal biohydrogenation (BH) of polyunsaturated fatty acids (PUFA; Raes, De Smet, & Demeyer, 2004). Efforts have been made to increase amounts of PUFA in beef, particularly omega-3 (n− 3) PUFA and PUFA BH intermediates, which may have health benefits for consumers (Dilzer & Park, 2012; Molendi-Coste, Legry, & Leclercq, 2011). The challenges have been to define appropriate diets and rumen conditions to promote accumulation of PUFA and their BH intermediates in beef. Abbreviations: AD, atypical dienes; ADG, average daily gain; ALA, α-linolenic acid; BCFA, branched-chain fatty acids; BH, biohydrogenation; c, cis; CLA, conjugated linoleic acids; CLNA, conjugated linolenic acids; d, days; DHA, docosahexaenoic acid; DM, dry matter; DMI, dry matter intake; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; FA, fatty acids; FAME, fatty acid methyl esters; GC, gas chromatography; FS, flaxseed; GH, grass hay; LA, linoleic acid; LT, longissimus thoracis; MUFA, monounsaturated fatty acids; n−3, omega-3; n−6, omega-6; PPO, polyphenol oxidase; PUFA, polyunsaturated fatty acids; RC, red clover silage; RA, rumenic acid; SS, sunflower seed; SFA, saturated fatty acids; t, trans; VA, vaccenic acid; vs., versus. ⁎ Corresponding author. Tel.: +1 403 782 8124. E-mail address: duganm@agr.gc.ca (M.E.R. Dugan). To substantially increase PUFA and their BH intermediates in beef, typically an oil or oilseed source of PUFA must be fed while creating rumen conditions conducive to promote PUFA bypass or partial as opposed to complete BH (Jenkins & Bridges, 2007). To this end, our research group has undertaken a series of studies feeding diets containing 10–15% flaxseed (FS), a rich source of α-linolenic acid (18:3n−3, ALA) to cattle and resulting in increased deposition of n−3 PUFA in total muscle FA by 0.7–1.1% (Juárez et al., 2011; Mapiye et al., 2013; Nassu et al., 2011). The type (He et al., 2012; Nassu et al., 2011) and level (Aharoni, Orlov, & Brosh, 2004; Mir et al., 2003) of forage can, however, have overriding effects on accumulation of PUFA BH intermediates in beef. Juárez et al. (2011) fed steers 10% FS in a high (73%) barley grain diet with 22% alfalfa/brome hay, and found limited accumulations of either ALA or its BH intermediates. In this study, the PUFA BH pathway favored accumulation of trans (t)13/t14-18:1 instead of t11-18:1 (vaccenic acid, VA), and atypical dienes (AD, i.e., non-conjugated, non-methylene interrupted dienes) instead of conjugated linoleic acid (CLA). Nassu et al. (2011) fed FS to cull cows in 50:50 forage:concentrate diets with either grass hay (GH) or barley silage as the forage source for an extended period (20 weeks), and found that feeding GH-FS promoted greater accumulations of PUFA BH intermediates, with VA as the major trans mononene. In addition, feeding GH-FS yielded more AD than barley silage-FS, and for both diets, AD exceeded amounts of total CLA and total n−3 PUFA. 0309-1740/$ – see front matter. Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.03.033 99 C. Mapiye et al. / Meat Science 95 (2013) 98–109 Recently, Mapiye et al. (2013) found feeding 15% FS in a 70% forage (red clover silage, RC) diet for 215 d provided even greater accumulations of PUFA BH intermediates in beef lipids with t11-18:1 reaching a high of 9.5% in perirenal fat, cis (c)9,t11-18:2 (rumenic acid, RA) reaching a high of 2.9% in subcutaneous fat, and t11,c15-18:2 reaching a high of 1.9% in perirenal fat. The increased amounts of PUFA BH intermediates were in part attributed to the amount and duration of FS feeding. In addition, the relatively high level of polyphenol oxidase (PPO) activity in RC may have played a role, as PPO reduces rates of PUFA lipolysis and BH in the rumen (Van Ranst, Lee, & Fievez, 2011) thereby enhancing deposition of PUFA and their BH intermediates in tissues (Lee, Evans, Nute, Richardson, & Scollan, 2009). The benefits of feeding RC were, however, not evaluated in comparison with other forage sources such as GH which have demonstrated positive effects on the accumulation of PUFA BH intermediates in beef (Nassu et al., 2011). Feeding sources of linoleic acid (18:2n−6, LA), for example sunflower-seed (SS) or oil, in high-forage diets can also increase PUFA BH intermediates in beef, but these are mostly restricted to VA and RA (Basarab, Mir, et al., 2007; Noci, French, Monahan, & Moloney, 2007). Feeding SS or FS in high forage diets can, therefore, increase VA and RA in beef, and feeding FS can also increase other ALA specific BH intermediates. Consuming VA and RA may have positive health effects for humans (Dilzer & Park, 2012; Jaudszus et al., 2012; Sofi et al., 2010), but effects of many other PUFA BH intermediates have not been evaluated. Consequently, the objectives of the present experiment were to feed steers a high forage diet (either RC or GH) in combination with oilseeds (either FS or SS) for an extended period, and determine which diets would lead to the greatest accumulations of PUFA and their related BH intermediates in beef. In addition, animal performance, carcass traits, meat quality and sensory attributes were evaluated since increasing the degree of FA unsaturation in beef accentuates oxidation of fat, possibly leading to unacceptable changes in shelf-life and eating quality (Wood et al., 2004). 2. Materials and methods Table 1 Nutrient and fatty acid composition of the experimental diets. Dieta Variable GH-FS GH-SS RC-FS RC-SS Diet ingredients (% DM basis) Red clover silage Grass hay Barley straw Sunflower-seed Flaxseed Vitamin/mineral supplementb Barley grain 0.0 70.0 11.5 0.0 14.3 4.2 0.0 0.0 70.0 0.0 18.4 0.0 4.2 7.4 70.0 0.0 11.5 0.0 14.3 4.2 0.0 70.0 0.0 0.0 18.4 0.0 4.2 7.4 Nutrient composition (DM basis) Dry matter (%) Crude protein (%) Crude fat (%) Calcium (%) Phosphorus (%) ADF (%) NDF (%) Digestible energyc (Mcal/kg) 93.1 13.3 6.4 1.1 0.3 44.3 53.2 2.08 93.0 13.4 6.6 1.1 0.3 45.4 57.6 2.02 46.9 14.2 8.2 1.1 0.3 43.0 55.5 2.16 46.9 14.0 8.4 1.2 0.2 44.0 61.6 2.10 Fatty acid (% of total fatty acids) 14:0 16:0 18:0 20:0 22:0 24:0 c9-18:1 c11-18:1 18:2n−6 18:3n−3 0.2 8.6 3.0 0.4 0.7 0.6 11.6 0.8 23.4 50.7 0.2 10.2 4.1 0.5 0.9 0.5 11.3 0.9 66.0 5.3 0.1 7.5 2.9 0.3 0.4 0.4 11.6 0.8 21.4 54.6 0.1 8.4 4.2 0.4 0.8 0.4 11.7 0.7 70.4 2.8 a GH-FS, grass hay + flaxseed; GH-SS, grass hay + sunflower-seed, RC-FS, red clover silage + flaxseed; RC-SS, red clover silage + sunflower-seed. b Vitamin/mineral supplement per kg DM contained 1.86% calcium, 0.93% phosphorous, 0.56% potassium, 0.21% sulfur, 0.33% magnesium 0.92% sodium, 265 ppm iron, 314 ppm manganese, 156 ppm copper, 517 ppm zinc, 10.05 ppm iodine, 5.04 ppm cobalt, 2.98 ppm selenium, 49,722 IU/kg vitamin A, 9944 IU/kg vitamin D3, and 3222 IU/kg vitamin E. c Digestible energy was calculated according to Bull (1981). 2.1. Animals and diets Sixty-four 12-month-old British × Continental crossbred steers with an initial mean body weight of 423.2 ± 5.93 kg were used in the current study conducted at Lacombe Research Centre, Alberta, Canada. Animal care was in compliance with the principles and guidelines established by the Canadian Council on Animal Care (CCAC, 1993). Steers were stratified by weight to four experimental diets, with two pens of eight steers per diet. The four diets were GH-FS, GH-SS, RC-FS and RC-SS. On a dry matter (DM) basis, diets contained 70% forage and either SS (18.4%) or FS (14.3%), with oilseeds added to provide the same amount of oil (5.4%, DM basis; Table 1) to each diet. All diets included 4.2% of a vitamin mineral supplement (Table 1) and in an attempt to equalize the digestible energy of the diets, additional ground barley grain was added to the diets containing SS, and additional barley straw was added to the diets containing FS. Flaxseed was triple rolled, while SS was fed whole. Steers in each pen were group fed to appetite and were all capable of feeding at the feed bunk at the same time (0.8 m of space at the bunk per animal). Steers had free access to fresh, clean water. Feed was provided once daily (feed DM equaling ~2.5% body weight) and the amount adjusted so that 10–15% orts were present after 18 h with all feed being consumed by 24 h. During the study period one animal from the GH-FS treatment was withdrawn due to lameness unrelated to dietary treatment. 2.2. Feed analysis Feed samples were collected weekly and stored at −40 °C, then pooled monthly before determination of DM, minerals, crude fat, crude protein (AOAC, 2006), neutral detergent fiber and acid detergent fiber (Van Soest, Robertson, & Lewis, 1991). Fatty acids from the finishing total mixed ration were extracted and methylated as described by Sukhija and Palmquist (1988) and analyzed according to Dugan et al. (2007). 2.3. Growth measurements and slaughter procedure Individual steer weights were measured monthly and average daily gain (ADG) was calculated by dividing each animal's body weight by days on-test. Animal growth and DM intake (DMI) were recorded from the start of the experiment until the first group of animals were slaughtered due to difficulties in measuring DMI when numbers of animals per pen were reduced. Backfat thickness was measured monthly by a certified ultrasound technician using an Aloka 500 V diagnostic real-time ultrasound with a 17 cm 3.5 MHz linear array transducer (Overseas Monitor Corporation Ltd., Richmond, B.C., Canada) following procedures of Brethour (1992). Steers were slaughtered at the Lacombe Research Centre abattoir over four slaughter dates in November 2011 (two steers/pen/diet/slaughter day) at an average of 205 d on feed corresponding to subcutaneous fat depths of 5–8 mm between the 12th and 13th rib over the right longissimus thoracis (LT) muscle of each animal. On mornings of slaughter, animals were transported 2 km to the Lacombe Research Centre abbatoir. At slaughter, final live weights were recorded and steers were stunned, exsanguinated and dressed in a commercial manner. Following carcass splitting, trimmed side weights were recorded and initial (45 min) pH and temperature were recorded caudal to the grade site on the left LT using a Hanna HI99163 pH meter equipped with a Hanna Smart electrode FC232 for meat (Hanna Instruments, Laval QC, Canada). Upon entry into the cooler, stainless steel thermocouples (10 cm) were placed into the right LT ~2.5 cm anterior to the 100 C. Mapiye et al. / Meat Science 95 (2013) 98–109 12th vertebrae of all animals, and temperatures were recorded every 15 min for 24 h using data temperature loggers (Mark III, MC4000; Sumaq Wholesalers, Toronto, ON, Canada). 2.4. Meat quality Carcass sides were chilled overnight in a cooler set at 2 °C, with an average wind speed of 1.4 m/s. At 24 h post-slaughter, the left and right carcass sides were weighed to determine cooler shrinkage losses. The left carcass sides were knife-ribbed at the Canadian grade site (between the 12th and 13th ribs) and the muscle surface exposed to atmospheric oxygen for 20 min. Full Canadian grade data were collected (grade fat thickness, estimated lean yield, ribeye area and marbling score) along with objective color measurements from three surface locations (CIE L* (brightness), a* (red–green axis), b* (yellow–blue axis) values; Commission Internationale de l'Eclairage, 1978) using the Minolta CR-300 with Spectra QC-300 Software (Minolta Canada Inc., Mississauga, ON, Canada). Color measurements were converted to hue angle [hab = arctan(b*/a*)] and chroma [C*ab = (a*2 + b*2) 0.5]. Grade data, including yield estimation and subjective estimates of marbling were assessed according to the Livestock and Poultry Carcass Grading Regulations (Canadian Food Inspection Agency, 1992) by two certified graders. Temperature and pH of the LT were again measured at 24 h caudal to the grade site. The left LT was dissected from the carcass at 24 h and a 5.0 cm steak removed from the grade site (caudal end of the LT), trimmed of epimysium, subcutaneous and intermuscular fat, comminuted using a Robot Coupe Blixir BX3 (Robot Coupe USA Inc.; Ridgeland, MS, USA), and subsamples were frozen at −80 °C for subsequent FA analyses. The remainder of the muscle was trimmed of all extraneous fat to the epimysium, weighed, vacuum packaged (Multivac AGW; Multivac Inc., Kansas City, MO, USA) and aged for 6 d at 2 °C. After aging, four 2.5 cm thick steaks were removed from the caudal end of the LT. The first steak was pre-weighed onto a polystyrene tray with a dri-loc pad, over-wrapped with oxygen permeable film (8000 cm3·m−2·24 h−1 vitafilm choice wrap; Goodyear Canada Inc., Toronto, ON, Canada) and placed in a fan assisted, horizontal (chest type) retail display case (Hill Refrigeration of Canada Ltd., Barrie, ON, Canada). Steaks were exposed to fluorescent room lighting (GE deluxe cool white; General Electric Canada, Oakville, ON, Canada), supplemented with incandescent lighting directly above the display case (GE clear cool beam 150 W/120 V spaced 91.5 cm apart; General Electric Canada, Oakville, ON, Canada) to provide an intensity of 1076 lx at the meat surface for 12 h·day−1 (Jeremiah & Gibson, 2001). The average temperature in the retail display case was 3.5 °C. Following 0, 2 and 4 days of retail display, three objective color measurements (L*, a* and b*) were collected across the steak and converted to hue angle and chroma as reported previously. Spectral reflectance readings were also collected concurrently and the relative contents of metmyoglobin, myoglobin and oxymyoglobin were calculated as described by Shibata (1966) and Krzywicki (1979). Following the fourth day of objective color measurements, steaks were removed from retail display and final weights were recorded to determine drip-loss. Raw steak weights were recorded on the second steaks and a 10 cm spear point Type T thermocouple probe (Wika Instruments, Edmonton, AB, Canada) was inserted into the mid-point of each steak prior to cooking. Steaks were then cooked, on a Garland grill (Model ED30B, Condon Barr Food Equipment Ltd., Edmonton, AB, Canada) preheated to 210 °C, to an internal temperature of 35.5 °C, turned and cooked to a final temperature of 71 °C. Internal temperatures during cooking were monitored with a Hewlett Packard HP34970A Data Logger (Hewlett Packard Co., Boise ID, USA). Upon removal from the grill, steaks were placed into polyethylene bags, sealed and immediately immersed in an ice/water bath to prevent further cooking. Steaks were then transferred to a 2 °C cooler for 24 h. Final steak weights were recorded and six cores, 1.9 cm in diameter, were removed from each steak parallel to the fiber grain. Peak shear force was determined on each core perpendicular to the fiber grain (TA-XT Plus Texture Analyzer equipped with a WarnerBratzler shear head at a crosshead speed of 200 mm·min−1 and a 30 kg load cell using Texture Exponent 32 Software; Texture Technologies Corp., Hamilton, MA, USA). Shear force was recorded as the average of all six cores per steak. Raw and final steak weights were used to determine cooking losses, and cooking times were also recorded. The third steaks were labeled, individually vacuum packaged, placed into a 2 °C cooler, and aged for 16 d prior to shear force determination as previously described. Fourth steaks were labeled, vacuum packaged, placed into a 2 °C cooler and aged for 16 d. Following aging, steaks were placed into a −35 °C freezer until used for sensory evaluation. The remaining portion of the LT was trimmed of epymysium and comminuted using Robot Coupe Blixir BX3 (Robot Coupe USA Inc., Ridgeland MS, USA) and analyzed for protein, moisture and fat using a CEM rapid analyzer system (Sprint Protein Analyzer Model 558000, Smart Turbo Moisture Analyzer Model 907990, and Smart Trac Fat Analyzer Model 907955; CEM Corporation, Matthews, NC, USA). 2.5. Longissimus thoracis fatty acid analysis Intramuscular lipids were extracted from LT samples using a mixture of chloroform–methanol (2:1, v/v) according to Folch, Lees, and Sloane Stanley (1957). Aliquots of muscle lipids (10 mg) were methylated separately using acid (5% methanolic HCl) and base (0.5 N sodium methoxide) reagents (Kramer, Hernandez, Cruz-Hernandez, Kraft, & Dugan, 2008). Internal standard, 1 ml of 1 mg c10-17:1 methyl ester/ml toluene (standard no. U-42M form Nu-Check Prep Inc., Elysian, MN, USA), was added prior to addition of methylating reagents. Fatty acid methyl esters (FAME) were analyzed using the gas chromatography (GC) and silver-ion high-performance liquid chromatography methods outlined by Cruz-Hernandez et al. (2004), except t-18:1 isomers were analyzed using two complementary GC temperature programs as described by Kramer et al. (2008). For the identification of FAME by GC, the reference standard no. 601 from Nu-Check Prep Inc, Elysian, MN, USA was used. Branched-chain FAME were identified using a GC reference standard BC-Mix1 purchased previously from Applied Science (State College, PA, USA). For CLA isomers, the UC-59M standard from Nu-Chek Prep Inc. was used which contains all four positional CLA isomers. Trans-18:1 CLA isomers and other BH intermediates not included in the standard mixtures were identified by their retention times and elution orders as reported in literature (Cruz-Hernandez et al., 2004; Gómez-Cortés, Bach, Luna, Juárez, & de la Fuente, 2009; Kramer et al., 2008). The FAME were quantified using chromatographic peak area and internal standard based calculations. Only FAME representing more than 0.01% of total FAME were included in tables and figures, except for BH intermediates where all the quantified isomers were reported. 2.6. Sensory analysis Sensory analysis of steaks was conducted using previously described procedures (Aldai et al., 2010; Basarab, Aalhus, et al., 2007). Prior to taste panel assessments, steaks were removed from the freezer and placed in a refrigerator to thaw for 24 h. Fifteen minutes prior to cooking, steaks were removed from the refrigerator and were cooked to a final temperature of 71 °C as described previously for shear force determinations. Each steak was cut into 1.3 cm cubes, avoiding connective tissue and large areas of fat. Eight cubes from each sample were randomly assigned to an eight-member trained sensory panel. Samples were placed in lightly covered glass jars in a circulating water bath (Lindberg/Blue Model WB1120A-1; Kendro Laboratory Products, Asheville, NC, USA) and allowed to equilibrate to 68 °C prior to evaluation. Attribute ratings were electronically collected using Compusense 5, Release 4.6 software (Compusense Inc., Guelph, ON, Canada ) using 8-point descriptive scales for initial and overall tenderness (1 = 101 C. Mapiye et al. / Meat Science 95 (2013) 98–109 extremely tough and 8 = extremely tender), initial and sustainable juiciness (1 = extremely dry and 8 = extremely juicy), beef flavor intensity (1 = extremely bland and 8 = extremely intense), off-flavor intensity (1 = extremely intense off-flavor and 8 = no off-flavor) and amount of connective tissue (1 = abundant and 8 = none detected). Initial tenderness was rated on the first bite through the cut center surface with the incisors; initial juiciness was rated after 3–5 chews with the molars; beef flavor intensity, off-flavor intensity and amount of connective tissue were rated between 10 and 20 chews, and overall tenderness and sustainable juiciness were rated prior to expelling. All panel evaluations were conducted in well-ventilated partitioned booths, under red lighting (124 lx). Distilled water and unsalted soda crackers were provided to purge the palate of residual flavor notes between samples (Larmond, 1977). to SS and final live weights were also lower (P b 0.05) in steers fed RC compared to those fed GH (Table 2). The lower DMI could be partly related to the slightly higher digestible energy of FS diets, but likely relates more to reduced palatability, as FS has been shown to reduce intake when fed at levels above 10% of DM (Kim et al., 2009). Addition of straw to the FS diets to balance digestible energy across diets may have also reduced palatability and DMI due to its lower digestibility (Sarnklong, Coneja, Pellikaan, & Hendriks, 2010). The reasons for the lower final live weight when feeding RC as opposed to GH are less clear, but the higher crude fat content of RC diets, or the quality of silage fed may have had some influence. Levels of fat above 5% have been shown to inhibit ruminal fiber digestion, which can result in increased rumen fill and reduced DMI (Jenkins, 1993). The quality of the silage fed in the current study was, however, not evaluated. 2.7. Statistical analysis 3.3. Carcass traits and longissimus thoracis quality Data were analyzed using the PROC MIXED procedure of SAS (2009). The statistical model used for animal weights and feed intake included oilseed, forage and oilseed × forage interaction as fixed effects, and pen nested within the oilseed × forage interaction as the random effect. Data analysis for carcass traits, meat quality, sensory attributes and muscle FA profiles included the fixed effects of oilseed, forage and oilseed × forage interaction and random effects of slaughter date and pen nested within the oilseed × forage interaction. Meat shear force and retail display data were analyzed with a repeated measures design, with oilseed, forage, day and their interactions as main effects, slaughter date and pen nested within the oilseed × forage interaction as random effects, and day as the repeated factor. Since the random effect of pen nested within the oilseed × forage interaction was not significant, it was removed from carcass traits, meat quality and FA models and individual animal was used as the experimental unit. Treatment means were generated and separated using the LSMEANS and PDIFF options respectively (SAS, 2009). Frequency tables were generated along with Chi-Square tests for yield and quality grade data. The significance threshold for all statistical analyses was set at P b 0.05. At 24 h post-slaughter, feeding diets containing FS reduced (P b 0.05) cold carcass weight, hot dressing percentage and grade fat thickness as compared to feeding SS (Table 3). Feeding diets containing RC vs. GH also reduced cold carcass weight (P b 0.05), but only led to trends for reduced hot dressing percentage (P b 0.07). These findings may be associated with the lower DMI, ADG and final live weights reported for the FS and RC containing diets. Compared to steers fed diets containing SS, steers fed FS had greater (P b 0.05) carcass shrink loss, and steers fed diets containing RC tended (P b 0.07) to have greater carcass shrink loss than steers fed GH. These findings could be related to differences in grade fat thickness. Subcutaneous fat can act as a thermal insulator and reduce water losses during carcass cooling (Bezerra et al., 2012). Type of forage had an effect on ribeye area, with steers fed diets containing GH having larger (P b 0.05) ribeye areas than those fed RC. These results could be explained by differences in DMI, carcass weights and nitrogen utilization efficiency when consuming these two forages (Fraser, Fychan, & Jones, 2000). Diet had no effect on estimated lean yield or marbling score (Table 3). There were no differences in the distribution of yield and quality grades among the dietary treatments (Table 3). Overall, the distribution of yield and quality grades indicates that about 82% and 65% of the carcasses graded Canada 1 and Canada AA, respectively (Table 3). Loin temperature decline by diet data are presented in Fig. 1. Steers fed diets containing SS had higher (P = 0.05) 45 min postslaughter LT temperatures than those fed FS (Table 3), but forage type had no effect on 45 min LT temperature. Types of forage and oilseed had no effect on 24 h muscle temperature (Table 3). Steers fed diets containing RC as opposed GH had higher (P = 0.05) 45 min LT pH. At 24 h, however, an oilseed type × forage type interaction was detected for LT pH, and steers fed the RC-SS diet had the highest pH, the GH-SS the lowest and steers fed the RC-FS and GH-FS diets had intermediate values (P b 0.05; Table 3). Oilseed and forage type affected muscle L* and interacted to influence hue angle (P b 0.05). Feeding diets containing RC or FS resulted in darker (lower L*; P b 0.05) and redder (lower hue angle; P b 0.05) meat compared to feeding GH and SS (Table 3). Slower rates of live weight gain and lighter carcass weights observed for steers fed diets containing RC or FS likely resulted in faster loin cooling rates (Fig. 1) to produce meat with a higher pH and a less 3. Results and discussion 3.1. Nutrient and fatty acid composition of the experimental diets The DM content of GH was double that of RC diets (Table 1). The DM nutrient composition of the experimental diets was similar (Table 1) with RC diets having slightly more crude protein, crude fat and digestible energy than GH diets. Linoleic acid and ALA were the dominant FA in the SS and FS diets, respectively (Table 1). 3.2. Animal performance Over the course of the experiment, steers fed diets containing FS had significantly lower (P b 0.05) DMI than those fed SS, and steers fed diets containing RC consumed less (P b 0.05) feed than those fed GH (P b 0.05; Table 2). As a reflection of these findings, ADG and final live weights were lower (P b 0.05) in steers fed FS containing diets compared Table 2 Effect of forage type and oilseed supplementation on feed intake and growth performance of feedlot steers. Grass hay Variable Dry matter intake, kg DM·d Initial live weight, kg Final live weight, kg Average daily gain, kg·d−1 −1 Red clover s.e.m Flax Sunflower Flax Sunflower 13.0 425 539 0.56 13.6 431 563 0.69 11.2 419 497 0.45 12.9 418 536 0.71 0.37 8.24 9.55 0.08 P-value Oilseed Forage O ∗ F1 0.03 0.77 0.001 0.001 0.03 0.25 0.001 0.22 0.21 0.68 0.44 0.09 Means with different superscripts for a particular animal performance trait are significantly different (P b 0.05); s.e.m, standard error of mean. 1 Oilseed type × forage type interaction. 102 C. Mapiye et al. / Meat Science 95 (2013) 98–109 Table 3 Effect of forage type and oilseed supplementation on carcass and meat quality of feedlot steers. Grass hay Red clover s.e.m P-value Oilseed Forage O ∗ F1 0.05 5.79 0.37 0.01 0.001 0.04 0.07 0.01 0.07 0.62 0.30 0.26 0.67 1.98 1.77 22.0 0.04 0.28 0.43 0.59 0.28 0.04 0.31 0.25 0.87 0.49 0.27 0.31 21.0 4.84 0.89 0.30 0.55 4.76 17.5 1.59 6.35 14.3 4.76 0.87 0.58 0.44 6.73 36.3 5.66ab 1.54 33.3 21.0 21.5b 38.6 6.70 19.9 4.21 73.8 6.71 36.5 5.68a 1.48 33.7 21.1 21.6b 40.1 5.84 20.0 4.26 73.4 0.93 0.05 0.37 0.31 0.04 0.13 0.03 0.66 0.08 0.21 0.88 0.29 0.05 0.70 0.001 0.15 0.01 0.07 0.001 0.53 0.12 0.01 0.86 0.01 0.56 0.34 0.02 0.97 0.20 0.21 0.04 0.77 0.30 0.45 0.97 0.77 Variable Flax Sunflower Flax Sunflower Carcass traits Shrink loss, % Cold carcass weight, kg Hot dressing, % 1.71 304 56.5 1.59 320 56.9 1.76 283 56.8 1.68 310 58.0 Canadian grade data Grade fat, mm Ribeye area, cm2 Estimated lean yield, % Marbling score2 6.35 73.5 56.5 442 7.25 74.3 59.8 456 5.75 68.0 60.3 441 6.81 71.5 59.7 437 Yield grade3, % Canada 1 Canada 2 19.4 4.84 21.0 4.84 21.0 3.23 Quality grade, % Canada A Canada AA Canada AAA 4.76 14.3 4.76 1.59 19.1 4.76 Longissimus thoracis quality measures pH, 45 min Temperature °C, 45 min pH, 24 h Temperature °C, 24 h L*, 24 h Chroma, 24 h Hue angle, 24 h Drip loss, mg·g−1 (average of 6 and 16 d) Shear force, kg (average of 6 and 16 d) Protein, % Fat, % Moisture, % 6.65 36.1 5.64b 1.46 34.0 21.3 21.9b 38.1 5.90 20.4 4.27 72.9 6.67 36.6 5.58c 1.41 35.8 22.7 23.0a 38.4 5.69 20.8 4.36 72.7 0.03 1.17 0.04 0.42 0.52 0.61 0.45 1.86 0.44 0.29 0.86 0.38 a,b Means with different superscripts for a particular carcass or longissimus thoracis quality trait are significantly different (P b 0.05); s.e.m, standard error of mean. Oilseed type × forage type interaction. 2 Marbling score terminology: 100, devoid; 200, practically devoid; 300, trace; 400, slight; 500, small; 600, modest; 700, moderate; 800, slightly abundant; 900, moderately abundant; 1000, abundant; 1100, very abundant. 3 No yield grade done on B4 carcasses. 1 desirable appearance. Młynek and Guliński (2007) obtained similar results and attributed them to variation in muscle fiber type composition with growth rate. Overall, slower growth rates result in higher contents of type I muscle fibers and lower contents of type II muscle fibers (Młynek & Guliński, 2007). Type I muscle fibers are red in color, have high oxidative capacity, and are lower in glycogen content and glycolytic enzyme activity (Klont, Brocks, & Eikelenboom, 1998). Consequently, lactic acid production is reduced, muscle acidification slows down and meat with redder and darker color is produced (Młynek & Guliński, 2007). Diet had no effect on LT drip loss, shear force at 6 or 16 d post-mortem or fat content (Table 3). Longissimus thoracis shear force, however, declined (P b 0.05) from 6.90 kg on day 6 to 5.16 kg on day 16. Slightly higher (P b 0.05) protein content was found when feeding GH vs. RC containing diets (Table 3), and may again be related to differences in DMI and nitrogen utilization efficiency between these two forages (Fraser et al., 2000). The slightly higher (P b 0.05) LT moisture reported for steers fed RC compared to GH containing diets (Table 3) may be partly related to the higher 24 h LT pH. High muscle pH has been reported to elevate moisture content through an increase in water holding capacity (Byrne et al., 2001). 3.4. Steak retail display As expected, all retail steak measurements were affected by time in display except for myoglobin (Table 4). Overall, L* values increased up to day 2 and then decreased to day 4 (P b 0.05), chroma and oxymyoglobin values declined (P b 0.05) whereas hue angle and metmyoglobin values rose (P b 0.05) with increasing time in display. No interactions were found between time in display with either forage type or oilseed type for any retail measurements (P b 0.05). The LT content of oxymyoglobin was influenced by an oilseed × forage type interaction with steaks from steers fed the GH-SS diet having the highest (P b 0.05) values compared to steaks from steers fed other diets (Table 5). Forage type had an effect on L*, chroma, hue angle and the metmyoglobin content of steaks (Table 5). Steaks from steers fed diets containing RC had slightly lower (P b 0.05) L* and chroma values, and higher (P b 0.05) hue angle and metmyoglobin values compared to steaks from steers fed GH. These findings agree with earlier reports by Lee et al. (2009) and Scollan et al. (2006) who attributed the differences to increased PUFA and reduced vitamin E levels in the muscle of steers fed RC. In the current study, vitamin E was fed (135 IU/kg of dietary DM) in excess of the amount known to improve meat shelf-life (Liu, Lanari, & Schaefer, 1995), but muscle levels of vitamin E were not assessed. However, as noted below, the levels of PUFA in the muscle tended (P = 0.10) to be higher in steers fed RC diets than in steers fed GH. Variation in color during retail display observed when feeding these forages may also be partly attributed to the small differences in LT moisture observed in the current experiment. Overall, high moisture content has been reported to reduce the stability of vitamin E in foods (Miquel, Alegrı́a, Barberá, Farré, & Clemente, 2004). 3.5. Longissimus thoracis fatty acid profiles 3.5.1. n − 3 and n − 6 polyunsaturated fatty acids Total LT FA were similar across diets (Table 6), but total PUFA were influenced by oilseed type. Steers fed diets containing SS had 103 C. Mapiye et al. / Meat Science 95 (2013) 98–109 36 34 32 30 28 26 Temperature, °C 24 22 20 18 16 14 12 10 8 6 4 2 0 0 2 4 6 8 10 12 14 16 18 Time, h Red clover silage-Sunflower seed Red clover silage-Flaxseed Grass hay-Sunflower seed Grass hay-Flaxseed Fig. 1. Loin temperature of feedlot steers fed red clover silage or grass hay supplemented with sunflower-seed or flaxseed. higher (P b 0.05) proportions of total PUFA than steers fed diets containing FS. Proportions of total n−6 PUFA and the majority of individual n−6 PUFA were also influenced by oilseed type, with steers fed diets containing SS having greater (P b 0.05) proportions than those fed diets containing FS (Table 6). For all diets, over 75% of n−6 PUFA in the muscle was linoleic acid (LA). The present results show that increasing the supply of LA through feeding SS was more effective in raising the total PUFA in the muscle through an increase in n−6 PUFA than increasing the supply of ALA and n−3 PUFA through feeding FS. These findings could be related to LA's higher proportions in SS containing diets. For most diets, LA also has lower rates of BH as compared to ALA (Shingfield, Bonnet, & Scollan, 2013). Given that SS were fed whole, while FS was triple rolled, rolling may have increased the availability of ALA in the rumen compared to LA provided in whole SS (Doreau, Aurousseau, & Martin, 2009). Feeding RC vs. GH based diets tended (P b 0.10) to increase total PUFA, increased 20:4n−6 (P b 0.05) and may relate to the either greater supply of PUFA in these diets and/or elevated PPO activity in RC (Lee et al., 2009; Van Ranst et al., 2011). Oilseed type × forage type interactions were significant for total n − 3 PUFA and ALA, with steers fed the RC-FS diet having the greatest proportions followed by those fed the GH-FS diet, while steers fed the Table 4 Effect of day on retail measurements of beef from feedlot steers. Variable L* Chroma, % Hue angle, ° Metmyoglobin Myoglobin Oxymyoglobin Day s.e.m 0 2 4 49.2c 10.3a 35.8c 0.23c 0.04 0.73a 50.4a 10.1a 37.4b 0.27b 0.04 0.68b 49.7b 9.73b 38.9a 0.30a 0.04 0.67c P-value 0.22 0.23 0.30 0.01 0.01 0.01 b0.001 0.02 b0.001 b0.001 0.26 b0.001 a,b,c Means with different superscripts for a particular beef retail measurement are significantly different (P b 0.05); s.e.m, standard error of mean. GH-SS and RC-SS diets had the lowest proportions with no difference found between them (P b 0.05; Table 6). Proportions of ALA accounted for about 50% to 70% of the total n−3 PUFA in LT of steers fed diets containing SS and FS, respectively. The observation that steers fed the RC-FS diet had the greatest proportions of total n−3 PUFA and ALA could be associated with their greater dietary supply, a reduction in ruminal lipolysis and BH of ALA from FS through a shift in ruminal microbial population (Huws et al., 2010), or through products of PPO activity, which inhibit lipase or form lipid–phenol matrices (Van Ranst et al., 2011). The effects of RC on n−3 PUFA might also be explained by its increased rumen passage rates (Vanhatalo, Kuoppala, Toivonen, & Shingfield, 2007). The proportion of ALA in LT FA of steers fed the RC-FS diet (1.38%) was slightly lower than that reported by Mapiye et al. (2013, 1.59%) when a similar diet was fed, but is higher than those reported when feeding FS with grain- (0.25%, Kim et al., 2009; 1.35%, Juárez et al., 2011) or forage-based (0.93%, Aharoni et al., 2004; 1.06– 1.22%, Nassu et al., 2011; 1.35%, Noci et al., 2007) diets. The inconsistencies across studies could be a result of differences in the type, physical form and quantity of PUFA and forages fed, levels of plant secondary compounds consumed, time on feed, age at slaughter, breed and gender of the animals assayed. The proportions of long chain n − 3 PUFA were only influenced (P b 0.05) by oilseed type. Steers fed diets containing FS had higher (P b 0.05) proportions of 20:5n−3 (eicosapentaenoic acid, EPA), 22:5n−3 (docosapentaenoic acid, DPA) and 22:6n−3 (docosahexaenoic acid, DHA) compared to steers fed SS (Table 6). Several studies have shown that diets rich in ALA result in increased levels of EPA and DPA in beef with DPA being the most abundant long chain n−3 PUFA while in most cases no effect on DHA level was observed (Mapiye et al., 2013; Nassu et al., 2011; Noci et al., 2007; Raes et al., 2004). Overall, the proportions of long chain n−3 PUFA in the current study are comparable to those reported by Noci et al. (2007) and Nassu et al. (2011), but higher than those reported by Mapiye et al. (2013). Variation in long chain n−3 PUFA across studies could be due to diet-linked differences in rumen metabolism or desaturase and/or elongase activity. Increasing ALA and its long-chain derivatives, especially DHA and EPA in beef 104 C. Mapiye et al. / Meat Science 95 (2013) 98–109 Table 5 Effect of forage type and oilseed type on retail measurements of beef from feedlot steers. Variable L* Chroma, % Hue angle, ° Metmyoglobin Myoglobin Oxymyoglobin a,b Grass hay Red clover silage s.e.m Flax Sunflower Flax Sunflower 50.0 10.1 37.1 0.27 0.04 0.69b 49.9 10.3 37.1 0.26 0.04 0.71a 49.6 9.91 38.0 0.28 0.04 0.68b 49.5 9.82 37.4 0.28 0.04 0.68b 0.23 0.24 0.33 0.01 0.01 0.01 P-value O F O ∗ F1 0.59 0.65 0.32 0.13 0.18 0.03 0.01 0.03 0.04 b0.001 0.11 b0.001 0.77 0.29 0.25 0.15 0.17 0.04 Means with different superscripts for a particular beef retail measurement are significantly different (P b 0.05); s.e.m, standard error of mean. Oilseed type × forage type interaction. 1 Table 6 Effect of forage type and oilseed supplementation on intramuscular fatty acid profiles of feedlot steers. Variable ∑ FA (mg/g muscle) ∑ PUFA ∑ n−6 18:2n−6 20:3n−6 20:4n−6 ∑ n−3 18:3n−3 20:5n−3 22:5n−3 22:6n−3 ∑ CLNA ∑ CLNA c9,t11,t15-18:3 c9,t11,c15-18:3 ∑ AD ∑ CLA ∑ c,t-CLA ∑ t,t-CLA ∑ t-18:1 ∑ c-MUFA c9-14:1 c7-16:1 c9-16:1 c11-16:1 c9-17:1 c9-18:1 c11-18:1 c12-18:1 c13-18:1 c14-18:1 c15-18:1 c9-20:1 c11-20:1 ∑ BCFA iso-15:0 anteiso-15:0 iso-16:0 iso-17:0 anteiso-17:0 iso-18:0 ∑ SFA 14:0 15:0 16:0 17:0 18:0 19:0 20:0 Grass hay Red clover silage s.e.m Flax Sunflower Flax Sunflower 30.6 6.59 4.94 3.72 0.24 0.87 1.66b 1.09b 0.21 0.34 0.02 0.22b 0.22b 0.04 0.17b 2.40 0.76 0.64 0.12 5.85 37.9 0.47 0.16 2.57 0.12 0.52 30.7 1.02 0.83c 0.29 0.08 0.41 0.09 0.10 1.71 0.18 0.20 0.35 0.34 0.51 0.14 45.8 2.32 0.46 24.4 0.85 15.9 0.06 0.06 31.8 6.70 5.85 4.47 0.32 0.92 0.85c 0.49c 0.10 0.24 0.02 0.10c 0.10c 0.02 0.08c 1.52 0.85 0.76 0.08 7.71 36.9 0.49 0.15 2.47 0.12 0.45 29.6 0.91 1.54a 0.27 0.08 0.19 0.06 0.09 1.55 0.17 0.18 0.34 0.31 0.45 0.11 45.6 2.30 0.44 24.1 0.76 16.3 0.05 0.06 28.6 6.99 4.96 3.73 0.24 0.89 2.03a 1.38a 0.26 0.36 0.03 0.27a 0.27a 0.05 0.22a 2.29 0.79 0.67 0.12 5.64 37.9 0.51 0.17 2.80 0.13 0.53 30.6 1.07 0.61d 0.29 0.07 0.38 0.08 0.11 1.65 0.17 0.20 0.35 0.31 0.48 0.14 45.6 2.34 0.48 24.4 0.81 15.8 0.05 0.06 30.5 7.63 6.89 5.17 0.38 1.17 0.74c 0.39c 0.10 0.23 0.02 0.09c 0.09c 0.02 0.08c 1.07 0.86 0.79 0.06 7.53 35.9 0.41 0.16 2.38 0.11 0.47 29.4 0.96 1.07b 0.23 0.07 0.16 0.06 0.10 1.58 0.16 0.19 0.38 0.30 0.45 0.11 46.2 2.22 0.47 23.85 0.78 17.1 0.05 0.07 3.71 0.07 0.59 0.40 0.04 0.15 0.15 0.08 0.03 0.04 0.00 0.02 0.02 0.00 0.01 0.09 0.05 0.05 0.01 0.28 0.63 0.04 0.01 0.12 0.01 0.01 0.62 0.03 0.06 0.01 0.00 0.02 0.00 0.01 0.04 0.00 0.01 0.02 0.01 0.01 0.01 0.70 0.09 0.01 0.42 0.03 0.42 0.00 0.00 P-value Oilseed Forage O ∗ F1 0.48 0.03 b0.001 b0.001 b0.001 0.01 b0.001 b0.001 b0.001 b0.001 0.05 b0.001 b0.001 b0.001 b0.001 b0.001 0.03 0.01 b0.001 b0.001 0.02 0.23 0.33 0.03 0.10 b0.001 0.03 0.001 b0.001 0.001 0.14 b0.001 b0.001 0.18 0.001 0.02 0.10 0.69 0.03 b0.001 b0.001 0.70 0.47 0.19 0.31 0.01 0.04 0.01 0.16 0.43 0.10 0.11 0.15 0.19 0.04 0.21 0.19 0.11 0.73 0.16 0.08 0.08 0.76 0.05 0.001 0.52 0.37 0.21 0.50 0.48 0.66 0.01 0.56 0.54 0.21 0.79 0.13 b0.001 0.09 0.001 0.08 0.83 0.09 0.73 0.02 0.44 0.14 0.04 0.19 0.23 0.79 0.72 0.08 0.66 0.67 0.46 0.01 0.79 0.87 0.50 0.12 0.16 0.08 0.10 0.02 0.01 0.07 0.41 0.52 0.03 0.03 0.41 0.02 0.06 0.76 0.96 0.10 0.96 0.43 0.09 0.65 0.20 0.18 0.69 0.89 0.98 0.01 0.10 0.20 0.97 0.32 0.76 0.20 0.74 0.81 0.23 0.23 0.14 0.93 0.50 0.58 0.99 0.74 0.19 0.27 0.39 0.09 a,b,c Means with different superscripts for a particular fatty acid profile are significantly different (P b 0.05); s.e.m, standard error of mean. ∑ FA, total fatty acids in mg per g of meat; ∑ PUFA, sum of polyunsaturated fatty acids = ∑ n−6 + ∑ n−3; ∑ n−6 = sum of 18:2n−6, 20:3n−6, 20:4n−6; ∑ n−3 sum of 18:3n−3, 20:5n−3, 22:5n−3, 22:6n−3; ∑CLNA, sum of conjugated linolenic acid = c9,t11,t15-, c9,t11,c15-; ∑ AD, atypical dienes = sum of t11,t15-, c9,t13-/t8,c12-, t8,c13-, c9,t12-/c16-18:1, t9,c12-, t11,c15-, c9,c15-, c12,c15-; ∑ CLA, conjugated linoleic acid = sum of t,t-CLA + sum of c,t-CLA; ∑ trans-trans-CLA = sum of t12,t14-, t11,t13-, t10,t12-, t9,t11-, t8,t10-, t7,t9-t6,t8-; ∑ cis-/trans-CLA = sum of c9,t11-, t7,c9-, t11,c13-, t12,c14-, c11,t13-, t10,c12-, t8,c10-, t9,c11-; ∑ t-18:1, sum of trans-18:1 isomers = t6,t7,t8-, t9-, t10-, t11-, t12-, t13,t14-, t15-, t16-; ∑ c-MUFA = sum of c9-14:1, c7-16:1, c9-16:1, c11-16:1, c9-17:1, c9-18:1, c11-18:1, c12-18:1, c13-18:1, c14-18:1, c15-18:1, c9-20:1, c11-20:1; ∑ BCFA, branched chain fatty acids = sum of iso-15:0, anteiso15:0, iso16, iso17:0, anteiso17:0, iso18:0; ∑ SFA = sum of 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0. 1 Oilseed type × forage type interaction; c, cis; t, trans. C. Mapiye et al. / Meat Science 95 (2013) 98–109 remains a priority as they have been reported to have cardiovascular, immune and mental health benefits in humans (Molendi-Coste et al., 2011). In the current study, estimated levels of n−3 PUFA in LT from steers fed SS and FS containing diets would be 0.03 and 0.06 g per 100 g LT, respectively, which would account for 10% and 20% of the levels required to make an n−3 PUFA enrichment claim in Canada (≥0.3 g of n−3 PUFA per serving; CFIA, 2003). However, at retail most lean steak is sold with subcutaneous fat contributing 5 to 15% of the whole steak, and in other species, this fat depot is included as part of the serving and can contribute significantly to the overall n−3 PUFA enrichment level. 3.5.2. Triene and diene biohydrogenation intermediates The proportions of total conjugated linolenic acid (CLNA) and c9, t11,c15-18:3 were affected by oilseed × forage type interactions (P b 0.05; Table 6). Steers fed the RC-FS diet had the highest proportions of total CLNA (Table 6) and c9,t11,c15-18:3 (Fig. 2A) followed by steers fed GH-FS, while RC-SS and GH-SS resulted in the lowest proportions with no difference between them (P b 0.05). These findings are consistent with our previous findings when feeding diets containing FS (Nassu et al., 2011) or its combination with RC (Mapiye et al., 2013). The result that steers fed the RC-FS diet had the highest proportions of LT CLNA would be expected as a similar interaction was found for ALA, and since ALA is first isomerized to CLNA during BH in the rumen (Jenkins, Wallace, Moate, & Mosley, 2008). Red clover silage might have further increased the proportions of CLNA by increasing passage rates (Vanhatalo et al., 2007) or by reducing BH of CLNA (Huws et al., 2010; Van Ranst et al., 2011) or a combination of these mechanisms. The proportions of CLNA in the current study are similar to those reported by Mapiye et al. (2013) but higher than those reported by Nassu et al. (2011). In a large number of cell culture and animal model studies, CLNA isomers have displayed potent anti-inflammatory, immunemodulatory, anti-obesity and anti-carcinogenic activity, along with the ability to improve biomarkers of cardiovascular health (Hennessy, Ross, Devery, & Stanton, 2011). Consequently, their accumulation in beef may be perceived as a positive development from a human health perspective. For AD, there were two clear isomer patterns found related to oilseeds fed and their dominant FA (Fig. 2a). For AD derived from ALA (t8,c13; c9,c15; t11,c15; c12,c15; t11,t15), proportions were mainly increased by feeding FS (P b 0.05). Minor forage effects were, however, noted for c12,c15 (P b 0.05) and t11,t15 (P b 0.05). These results are consistent with earlier reports (Bessa et al., 2007; Mapiye et al., 2013; Nassu et al., 2011) when feeding FS to ruminants. For AD derived from LA (t8,c12; c9,t12; t9,c12), a fairly consistent forage × oilseed type interaction was found (P b 0.05) with the greatest amounts found when feeding the GH-SS diet. This is somewhat inconsistent with CLNA results where feeding RC was found to increase BH intermediates, but Shingfield et al. (2005) has reported higher PUFA transfer efficiency from diet to tissues when hay as compared to silage was provided as a forage source, probably due to alterations in ruminal lipid metabolism and/or forage lipids during preservation. Overall, accumulations of ALA derived AD when feeding FS were greater than when feeding diets containing SS, and this is consistent with previously reported pathways where ALA BH leads mostly through AD vs. CLA, while LA BH leads mostly through CLA (Jenkins et al., 2008; Nassu et al., 2011). The type of forage fed, however, only led to marginal differences in levels of AD. The most concentrated AD isomer found when feeding any diet was t11,c15-18:2, and reached a high of 0.85% when feeding FS containing diets. This is comparable to the ~ 0.9% found when Nassu et al. (2011) fed 15% FS to cull cows in a 50:50 forage-concentrate diet, but lower than 1.65% found by Mapiye et al. (2013) when feeding diets containing 15% FS to steers in a 70% forage diet. A number of AD isomers (t8,c12; t8,c13; t9t12/c9,t13) were clearly enriched by Nassu et al. (2011) and Mapiye et al. (2013) when feeding FS containing diets, but were not preferentially enriched in the present experiment 105 when feeding FS compared to SS containing diets. These isomers may, therefore, be found in common BH pathways for LA (i.e., from SS) and ALA (i.e., from FS). The human health implications of consuming increased amounts of AD in beef fed GH and FS merits investigation as suggested earlier by Nassu et al. (2011). Of particular interest will be if t11,c15-18:2 can be desaturated to c9,t11,c15-18:3 which may have beneficial health properties similar to RA (Hennessy et al., 2011). For CLA, there were again two clear isomer patterns found related to the types of oilseed fed and their dominant FA (Fig. 2b). For CLA isomers with the first double bond at carbon 10 or closer to the carboxyl end, proportions of most isomers (t7,c9; c9,t11 (RA); t10,c12; t10,t12) were elevated (P b 0.05) when feeding diets containing SS as opposed to FS. This likely stems from LA being their common precursor, as demonstrated previously in a number of studies (Bessa et al., 2007; Noci et al., 2007). Of the isomers in this group, a small but significant forage effect was also found for t7,c9-CLA with feeding GH containing diets resulting in slightly higher (P b 0.05) proportions than RC. In addition, small but significant forage × oilseed type interactions (P b 0.05) were found for t8,c10-CLA and t9,c11-CLA. Specifically feeding the RC-SS diet resulted in the greatest proportion of t8, c10-CLA, while feeding GH-SS or RC-SS resulted in the greatest proportions of t9,c11-CLA. For CLA isomers where the first double bond was from carbon 11 or further from the carboxyl end, proportions of most isomers (t11,t13; t12,t14; t12,c14; c12,t14) were elevated (P b 0.05) when feeding FS. This likely resulted from their common precursor being ALA, which was the predominant FA in the diets containing FS, and is consistent with previously outlined pathways (Mapiye et al., 2013; Nassu et al., 2011). Within this group of isomers, a main effect of oilseed type was also found for t11,c13-CLA, with feeding flaxseed increasing its proportions (P b 0.05), but a forage × oilseed type interaction (P b 0.05) indicated its preferential accumulation when feeding the RC-FS diet. Across all diets, RA was the predominant CLA isomer contributing over 60% and 75% of total CLA in LT lipids of steers fed diets containing FS and SS, respectively. This is consistent with other studies feeding diets containing FS and SS to ruminants (Bessa et al., 2007; Noci et al., 2007), and is likely the result from most CLA being further hydrogenated in the rumen, and tissue CLA being synthesized through Δ9-desaturation of VA (Griinari & Bauman, 1999; Jenkins et al., 2008). In the present study, the proportions of RA in the muscle of steers fed SS (0.65–0.69%) are comparable to previous findings when feeding high-forage diets with sunflower oil (0.63%, Basarab, Mir, et al., 2007) or FS (0.7%, Aharoni et al., 2004), but lower than those reported when feeding RC plus FS (Mapiye et al., 2013, 1.41%) or when supplementing grass pastures with FS oil (1.26%) or sunflower oil (1.78%; Noci et al., 2007). Direct comparisons among studies are, however, difficult as for the most part when RA is reported, it is combined with t7,c9-CLA. In the present study, there was no clear advantage in terms of RA accumulation when feeding either GH or RC. 3.5.3. Monounsaturated fatty acids For t-18:1, patterns of isomers found were clearly influenced by both types of oilseed and forage fed (Fig. 2C). For t-18:1 isomers with double bonds from carbon 6 to 12, these were primarily increased (P b 0.05) when feeding diets containing SS compared to FS. Feeding diets containing GH vs. RC also increased these isomers, except for VA, but overall responses to oilseed were greater than forage type. Current findings contradict previous reports by Nassu et al. (2011) and Mapiye et al. (2013) where all t-18:1 isomers were clearly enriched by feeding FS. The relative amounts of t10- and t15-18:1 in particular are lower than those reported in our previous studies (Juárez et al., 2011; Mapiye et al., 2013; Nassu et al., 2011) when feeding diets containing FS. The proportion of t-18:1 isomers with double bonds from carbon 13 to 16 were elevated (P b 0.05) when feeding diets containing GH as opposed RC but these differences were relatively small. Reasons for differences are unclear, but could be partially ascribed to differences in forage-borne 106 C. Mapiye et al. / Meat Science 95 (2013) 98–109 % of total fatty acids A 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 ß a b b b b a c d b ß a b b a b b c c t8,c12 B α,ß a a α ß t8,c13 c9,t12 t9,c12 t9,t12/c9,t13 c9,c15 t11,c15 t11,t15 c12,c15 ß 0.8 % of total fatty acids 0.7 0.6 0.5 0.4 0.3 0.2 α,ß 0.1 a c b bc a b ß α,ß c ba b a ß ß ß α,ß c 0 t7,c9 % of total fatty acids C t8,c10 c9,t11 t9,c11 5 t10,c12 t10,t12 c11,t13 t11,c13 t11,t13 c12,t14 t12,c14 t12,t14 ß 4 3 α 2 α,ß α,ß α,ß α,ß α,ß α 1 0 t6/t7/t8 t9 Grass hay-Flaxseed t10 t11 Grass hay-Sunflower seed t12 t13/t14 Red clover silage-Flaxseed t15 t16 Red clover silage-Sunflower seed Fig. 2. Effect of forage type and oilseed supplementation on atypical dienes (A), conjugated linoleic acid (B) and trans-18:1 isomers (C) in intramuscular fat from feedlot steers. a,b,c,d Means (±standard error) with different superscripts for a particular fatty acid profile are significantly different (P b 0.05). α: Significant forage effect (P b 0.05); ß: Significant oilseed effect (P b 0.05). microbes or alterations in endogenous ruminal microbes (Kong, He, McAlister, Seviour, & Forster, 2010), or disparities in ruminal and duodenal passage rates between these forages (Lee, Harris, Dewhurst, Merry, & Scollan, 2003). Vaccenic acid was the major t-18:1 isomer and constituted over 45% of total t18:1 irrespective of diet fed (Fig. 2C). The proportions of VA found in LT regardless of diet were less than we previously found when feeding FS in a high RC diet (Mapiye et al., 2013; 6.37%) and when supplementing with either sunflower or flaxseed oil on pasture (Noci et al., 2007; 8.56%). Differences between present results and others may, therefore, be related to influences of non-oil components of the diets. Specifically, the SS fed in the present experiment had a relatively low oil content (29.5%) compared to a typical value of 40% (Gunstone & Harwood, 1994). Consequently, the amount of SS that had to be added to the diet to get 5.4% oil was relatively high (18.4%) and the nutritive or forage value of the hull relatively low. Subsequently, to balance the digestible energy content across diets, straw had to be added to the flaxseed containing diets, which would have led to diminished overall quality of forage in FS containing diets. Results of the present study, therefore, dramatically reinforce the importance of the non-oil components of the diet when trying to enrich BH intermediates, particularly VA, in beef. Emphasis on increasing VA in beef relates to its abilities to reduce pro-inflammatory cytokines (Jaudszus et al., 2012; Sofi et al., 2010) and platelet aggregation in humans (Sofi et al., 2010), and substantially reduce plasma triglycerides in animal models (Wang, Jacome-Sosa, & Proctor, 2012). It may also have positive health effects through Δ-9 desaturation to RA, which may reduce risk of cardiovascular disease and cancer, increase bone mass and modulate immune and inflammatory responses (Dilzer & Park, 2012). In the present study, the estimated levels of VA in LT from steers fed FS and SS containing diets would be 0.08 and 0.12 g per 100 g LT, while the RA contents would be 0.01 and 0.02 g per 100 g LT, respectively. Thus, the RA levels in lean steak from steers fed SS would contribute 0.6 to 20% of the estimated dietary CLA intake of 0.1 to 3.0 g/day considered necessary for cancer prevention (Ip, Singh, Thompson, & Scimeca, 1994; Knekt, Järvinen, Seppänen, Pukkala, & Aromaa, 1996; Ritzenthaler et al., 2001). Although the levels of VA required to elicit positive effects on human health remain to be elucidated, up to 30% of VA is converted to RA in humans (Turpeinen et al., 2002) suggesting that VA could be regarded as potential RA. Including VA conversion, LT from steers fed SS could therefore contribute 1.8 to 56% (VA, 1.2 to 36% + RA, 0.6 to 20%) of the dietary CLA intake considered necessary for cancer prevention. The relative risk to human health of consuming the individual t-18:1 isomers other than VA C. Mapiye et al. / Meat Science 95 (2013) 98–109 remains to be elucidated, and thus recommendations to either enrich or deplete these isomers should be reserved until their effects are known. Feeding cattle a combination of oils or oilseeds rich in ALA and LA has been found to result in a synergistic accumulation of VA (AbuGhazaleh, Schingoethe, Hippen, Kalscheur, & Whitlock, 2002) and CLA (Lock & Garnsworthy, 2002) in tissues, which may have additive health effects (Jacome-Sosa et al., 2010). In this respect, it could be worthwhile to evaluate if supplementing high-forage diets with a blend of SS and FS would simultaneously enhance concentrations of VA, RA and n−3 PUFA in beef. Effects of feeding mixtures of SS, FS and sources of long-chain PUFA such as EPA and DHA, which are known potent inhibitors of BH of 18:1 to 18:0 in the rumen (Chow et al., 2004; Maia, Chaudhary, Figueres, & Wallace, 2007) may also be worth investigating. Total c-MUFA and the majority of individual c-MUFA isomers (c9-16:1, c9-17:1, c9-18:1, c11-18:1, c13-18:1, c15-18:1 and c9-20:1) were influenced by oilseed type, with steers fed diets containing FS having greater proportions (P b 0.05) than those fed diets containing SS (Table 6). The observation that feeding FS increased the proportions of total c-MUFA and c9-18:1 in muscle when compared with SS agrees with earlier findings by Jacobs et al. (2011) in cow milk and Bernard, Bonnet, Leroux, Shingfield, and Chilliard (2009) in goat milk when feeding diets rich in LA and ALA. The levels of c-MUFA in ruminant tissues reflect the extent of BH of PUFA to c-MUFA in the rumen and the capacity of PUFA/or its BH intermediates to inhibit stearoyl-CoA desaturase (SCD), which converts 18:0 to c-MUFA in tissues (Nakamura & Nara, 2004). The current findings, therefore, confirm that ALA has a higher level of BH in the rumen than LA (Doreau & Ferlay, 1994) and/or indicates that ALA is less effective in down-regulating SCD activity than LA as suggested earlier by Jacobs et al. (2011). The lower total c-MUFA in SS steers may also relate to displacement of c-MUFA due to increased total t-18:1 observed in the current study. A forage × oilseed type effect was noted for c12-18:1, with steers fed GH-SS having the largest proportions, followed by steers fed RC-SS, GH-FS and RC-FS, respectively (P b 0.05; Table 6). The proportion of a few minor c-MUFA isomers (c7-16:1 and c14-18:1) were significantly influenced (P b 0.05) by forage type (Table 6), with steers fed GH having more (P b 0.05) c7-16:1 and less (P b 0.05) c14-18:1 (Table 6) than those fed RC. These findings may also be partially ascribed to dissimilarities in forage-borne microbes, changes in endogenous ruminal microbes (Kong et al., 2010) or differences in ruminal and duodenal flow rates between these forages (Lee et al., 2003). Oleic acid (c9-18:1) was the most abundant c-MUFA in muscle making up over 65% of total c-MUFA when feeding all diets (Table 6). Overall, c-MUFA including oleic acid are considered to be beneficial for human health by reducing inflammation and blood coagulation factors (Williamson, Foster, Stanner, & Buttriss, 2005), but the effects of the minor c-MUFA on human health are not known and warrant further research. For example, serum triglyceride levels of c11-18:1 have now been associated with markers of insulin resistance in men (Zulyniak et al., 2012). 3.5.4. Saturated fatty acids The proportions of total branched chain FA (BCFA), iso-15:0, iso-17:0, anteiso-17, iso-18:0 and some odd-chain SFA (17:0 and 19:0) were affected by oilseed type, with steers fed FS containing diets having higher (P b 0.05) proportions than those fed SS (Table 6). Overall, the reduction of BCFA and odd-chain SFA in ruminant products is related to a direct inhibition of PUFA on microbial FA synthesis (Vlaeminck, Fievez, Cabrita, Fonseca, & Dewhurst, 2006). Present results, therefore, further suggest that ALA has lower inhibitory effects on rumen microbial FA synthesis than LA. The proportions of iso-15:0, and iso-17:0 were also influenced (P b 0.05) by forage type with steers fed GH having higher (P b 0.05) proportions than those fed RC (Table 6). These findings are consistent with earlier reports by Vlaeminck et al. (2006) who reported that grass-based diets promote the growth of cellulolytic bacteria rich in iso-FA. The variation between these two diets might also be linked to 107 dietary differences in leucine, which is deaminated and decarboxylated to yield isovaleryl-CoA that serve as a substrate for microbial synthesis of iso-15 and iso-17 (Vlaeminck et al., 2006). A slight increase in 19:0 was also found when feeding GH as opposed to RC (P b 0.05). Enriching BCFA in beef could be of interest because of their potential to reduce cancer (Wongtangtintharn, Oku, Iwasaki, & Toda, 2004) and necrotizing enterocolitis (Ran-Ressler et al., 2011) in humans. Total SFA and proportions of 14:0, 15:0, 16:0 and 20:0 were not influenced by diet (Table 6). The amount of 18:0 was significantly increased (P b 0.05) when feeding SS (Table 6). These results could again suggest that ALA and its BH intermediates were less effective at down-regulating SCD activity than LA. Alternatively, differences in 18:0 might also relate to changes in endogenous FA synthesis, but lack of differences in 16:0, the major endogenous FA synthesis product indicates that endogenous synthesis may not have been differentially affected by diet. 3.6. Sensory attributes Initial (P = 0.04) and overall tenderness (P = 0.05) scores were higher for LT steaks from steers fed GH compared to RC (Table 7). These results may relate to lower LT pH at 24 h observed for the former steers. Overall, the improved tenderness observed as pH falls below 6.0 has been attributed to higher protease activity (Yu & Lee, 1986), increased sarcomere length (Purchas & Aungsupakorn, 1993) and greater calcium-induced weakening of myofibrillar protein structures (Takahashi, Kim, & Yano, 1987). As compared to FS, feeding SS containing diets resulted in steaks with higher (P b 0.05) ratings for flavor intensity, off-flavor intensity and lower (P b 0.05) ratings for initial juiciness (Table 7). These findings may be linked to the differences in intramuscular proportions of n−3 and n−6 PUFA observed for steers fed these diets. Overall, thermally-induced oxidation of PUFA produces volatile compounds which may contribute to desirable or undesirable meat flavor depending on type, amounts and proportions in meat (Elmore, Mottram, Enser, & Wood, 1999). The finding that steaks from steers fed SS had more desirable flavors and less intense off-flavors may also be partly related to some positive influences of increased barley grain intake on beef flavor (Purchas & Davies, 1974). Overall, although some differences in sensory attributes were noted in the present study, absolute differences were all less than one sensory panel unit, and these differences would not likely be detectible by the average consumer. 4. Conclusions Feeding SS compared to FS in high-forage diets improved beef production, enhanced sensory qualities and enriched levels of VA (up to 0.55-fold), RA (up to 0.50-fold) and n − 6 PUFA in beef. On the other hand, feeding FS compared to SS in high-forage diets resulted in enrichments of n−3 PUFA, CLNA and AD. However, feeding FS in high-forage diets also resulted in lower growth rates and smaller carcasses, which interacted with faster cooling rates to produce meat with less desirable appearance. Overall, differences in FA profiles, meat and sensory quality were more influenced by oilseed than forage type. The influence of the non-oil fraction of the diets, however, may have resulted in lower PUFA BH intermediates compared to other studies. To consistently increase proportions of BH intermediates in beef, careful attention must be paid to the non-oil fraction of the diet, and feeding combinations of SS, FS and long-chain PUFA sources should be further investigated. The choice of which oil or oilseed combinations to supplement in diets will, however, ultimately depend on which produces the healthiest FA profile. Extensive bioactivity testing of many previously uncharacterized BH intermediates will, therefore, also be required. 108 C. Mapiye et al. / Meat Science 95 (2013) 98–109 Table 7 Effect of forage type and oilseed type on sensory of beef from feedlot steers. Variables Initial tenderness Initial juiciness Flavor intensity Off-flavor intensity Amount of connective tissue Overall tenderness Sustainable juiciness Grass hay Red clover silage Flax Sunflower Flax Sunflower 6.50 5.99 5.01 7.09 8.24 6.52 5.59 6.30 5.59 5.27 7.96 8.26 6.53 5.38 5.98 6.01 4.82 6.96 8.09 6.06 5.65 6.16 5.79 5.05 7.63 8.24 6.42 5.57 s.e.m 0.20 0.18 0.18 0.28 0.26 0.24 0.17 Oilseed Forage F ∗ O1 0.91 0.001 0.10 0.001 0.21 0.11 0.08 0.04 0.13 0.16 0.26 0.21 0.05 0.20 0.09 0.21 0.87 0.39 0.35 0.08 0.30 a,b,c Means with different superscripts for a particular sensory trait are significantly different (P b 0.05); s.e.m, standard error of mean. Oilseed type × forage type interaction; 8-point descriptive scales (1 = extremely tough, extremely dry, extremely bland flavor, extremely intense off-flavor, abundant connective tissue; and 8 = extremely tender, extremely juicy, extremely intense flavor, no off-flavor, no connective tissue detected). 1 Acknowledgments This research was funded by the Alberta Meat and Livestock Agency (ALMA). Drs. C. Mapiye and T.D. Turner acknowledge the receipt of NSERC Fellowships funded through ALMA. Dr. S.D. Proctor holds a New Investigator Award from the Heart and Stroke Foundation of Canada. Special thanks are extended to staff at the Lacombe Research Centre (LRC) Beef Unit of AAFC for animal care, animal management and sample collection. The slaughter and processing of the cattle by the LRC abattoir staff is gratefully acknowledged. Contributions of the meat grading and quality staff at the LRC to the results are appreciated. Ms. I.L. 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