J. Anim. Sci. 2005. 83:2175-2181
© 2005 American Society of Animal Science
Supplemental safflower oil affects the fatty acid profile, including conjugated linoleic acid, of lamb
J. A. Boles*,1,
R. W. Kott*,
P. G. Hatfield*,
J. W. Bergman
and
C. R. Flynn
* Animal and Range Sciences Department, Montana State University, Bozeman 59717; and
and
Montana State University Eastern Agricultural Research Station, Sidney 59270
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Abstract
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The objective of this study was to determine whether increasing levels of dietary safflower oil would alter unsaturated fat (especially CLA) and tocopherol content of lamb, animal performance, carcass characteristics, or color stability of lamb muscle tissue. Targhee x Rambouillet wethers (n = 60) were assigned to one of three diets (four pens per treatment with five lambs per pen) in a completely random design. Diets were formulated with supplemental safflower oil at 0 (control), 3, or 6% (as-fed basis) of the diet. Diets containing approximately 80% concentrate and 20% roughage were formulated, on a DM basis, to be isocaloric and isonitrogenous and to meet or exceed NRC requirements for Ca, P, and other nutrients. A subsample of 12 wethers per treatment was selected based on average BW (54 kg) and slaughtered. Carcass data (LM area, fat thickness, and internal fat content) and wholesale cut weight (leg, loin, rack, shoulder, breast, and foreshank), along with fatty acid, tocopherol, and color analysis, were determined on each carcass. The LM and infraspinatus were sampled for fatty acid profile. Increasing safflower oil supplementation from 0 to 3 or 6% increased the proportion of linoleic acid in the diet from 49.93 to 55.32 to 62.38%, respectively, whereas the percentage of oleic acid decreased from 27.94 to 23.80 to 20.73%, respectively. The percentage of oil in the diet did not (P 
0.11) alter the growth and carcass characteristics of lambs, nor did it alter the tocopherol content or color stability of meat. Increasing levels of safflower oil in lamb diets decreased (P < 0.01) the weight percentage of oleic acid in the infraspinatus and LM, and increased linoleic acid (P < 0.01). Oil supplementation increased (P < 0.01) the weight percentage of various isomers of CLA in muscle, with the greatest change in the cis-9,trans-11 isomer. Supplementation of sheep diets with safflower oil, up to 6% of the diet, resulted in increasing levels of unsaturated fatty acids and CLA in the lean tissue, without adversely affecting growth performance, carcass characteristics, or color stability of lamb.
Key Words: Conjugated Linoleic Acid • Fatty Acid Profile • Fat Supplementation • Lamb • Meat Color • Tocopherol
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Introduction
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Altering the fatty acid profile by increasing unsaturated fatty acids in the human diet, especially CLA, has been reported to have potential health benefits (Ip, 1997
; Houseknecht et al., 1998; Park et al., 1999). Other researchers have altered diets of both ruminant (Madron et al., 2002; Kott et al., 2003) and nonruminant (Thiel-Cooper et al., 2001; Wiegand et al., 2002) animals to increase the unsaturated fatty acid and CLA levels of meat, thereby improving potential health benefits of red meat (Pariza and Ha, 1990; Park et al., 1999).
Feeding CLA to nonruminant animals (Thiel-Cooper et al., 2001; Wiegand et al., 2002) results in increasing levels of CLA in muscle and fat tissue; however, the biohydrogenation of unsaturated fatty acids in ruminants decreases the effect of dietary supplementation of PUFA (Scollan et al., 2001). Kott et al. (2003) reported that feeding safflower seeds to lambs increased the unsaturated fatty acids and cis-9,trans-11 CLA concentration in the muscle tissue more than twofold over meat from control lambs. Madron et al. (2002) showed a numeric increase in CLA content when feeding diets containing full-fat soybeans to steers. Bolte et al. (2002) also reported increased levels of PUFA and CLA in muscle and fat from lambs that had been fed diets based on high-oleate and high-linoleate safflower seeds. This finding suggests that increasing levels of CLA precursors fed to ruminant animals could result in increased CLA content, as well as increasing the PUFA in the meat (Bolte et al., 2002; Kott et al., 2003). The increase in the unsaturated fatty acid content can, however, lead to an increase in lipid oxidation in the meat and decreased color stability (Chan et al., 1996; Faustman et al., 1989). Therefore, the objective of this study was to determine whether increasing levels of dietary safflower oil would alter unsaturated fat (especially CLA) and tocopherol content of lamb, animal performance, carcass characteristics, and color stability of lamb muscle tissue.
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Materials and Methods
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This research was carried out in accordance with Montana State University Animal Care and Use Committee guidelines. Targhee x Rambouillet wethers, born in April and May at the Red Bluff Experimental Ranch, grazed native range with their dams until they were weaned in September. After weaning, lambs were returned to range until late October when the feedlot study was initiated.
Lambs, weighing 37.3 ± 0.7 kg, were assigned to one of 12 feedlot pens (five lambs per pen), based on equal pen weight, and pens were assigned randomly to one of three diets (four pens per treatment) in a completely random design. Dietary treatments were a safflower oil (commercial blend; 72.27% linoleic acid)-supplemented diet formulated to contain 0 (control), 3, or 6% oil (as-fed basis). Diets containing approximately 80% concentrate and 20% roughage were formulated (DM basis) to be isocaloric and isonitrogenous and to meet or exceed NRC (1985) requirements for Ca, P, and other nutrients (Table 1). Each diet was pelleted (0.64 cm diameter) by a commercial feed mill and fed as a complete feed. Initially, diets were mixed with alfalfa pellets, and the proportion of experimental diet gradually increased by 10% until a full ration of the experimental diet was achieved over 22 d. The study ended when the BW of all lambs on study averaged 54 kg.
Lambs were allowed ad libitum access to water and offered feed twice daily at approximately 0600 and 1700. Feed bunks were inspected before the 0600 feeding, and the quantity offered was adjusted daily to ensure ad libitum consumption. Feed refusals were removed from the bunks, weighed, and recorded weekly. Random grab samples of the diets were taken weekly throughout the experiment, and samples were combined for analysis of feed. Feed samples were ground to pass a 1-mm screen in a Wiley mill, and analyzed for DM, ether extract, N (AOAC, 2000), ADF, NDF (Van Soest et al., 1991), and fatty acid content. Initial and final lamb BW (at the beginning and end of the finishing period) were taken after an overnight fast.
Following the 83-d feeding period, 12 lambs from each treatment, with an average BW of 54 kg, were selected randomly and transported to a local commercial slaughter facility, where they were slaughtered following normal industry procedures. After a 5-d chilling period at 2°C, carcasses were ribbed between the 12th and 13th ribs to facilitate the measurements of s.c. fat thickness and LM area, and kidney fat (including kidneys) was removed and weighed to calculate actual internal fat percent. Carcasses were separated into the fore- and hindsaddles. The foresaddle was fabricated according to National Association of Meat Purveyors (NAMP) specifications (NAMP, 1988) into the square cut shoulder (NAMP #207), rack (NAMP #204), breast (NAMP #209), and foreshank (NAMP #210). The hindsaddle was processed into the loin (NAMP #232) and leg (trotter removed; NAMP #233A). All primal/subprimal cuts were trimmed and weighed to determine wholesale cut yield.
Two 2.54-cm-thick loin chops were removed from the anterior end of the loin. One chop was placed on a white Styrofoam tray and overwrapped with an oxygen-permeable film (oxygen permeability rate >10,000 cc/ cm), whereas the second chop was deboned, vacuum-packaged, frozen, and stored at –77°C for tocopherol analysis. The remaining portion of the loin, along with the infraspinatus from the shoulder, was removed, vacuum-packaged, frozen at –77°C, and used to analyze fatty acid composition of the lean tissue.
Color Measurement
Color was measured with a Hunter Miniscan (Hunter Associates Laboratories, Reston VA) using A illuminant and a 10° angle of incidence. Lightness (L*), redness (a*), and yellowness (b*) were measured on loin chops after a 20-min bloom time at 4°C, and then daily for 7 d (AMSA, 1991). Samples were displayed on a table in a walk-in cooler under cool white lights. Duplicate measurements were made on each loin chop at a 90° angle to each other, and means of the two values were used for statistical analyses.
Fatty Acid Analysis
Fatty acid methyl esters were prepared by direct esterification with methanolic KOH of muscle tissue and composite feed samples according to the procedures reported by Murrieta et al. (2003). The procedure was optimized to identify the different CLA methyl esters. Cross sections of the LM and infraspinatus, including any residual intermuscular fat, (approximately 5.08 cm) were freeze-dried and ground in an electric coffee grinder. Resulting FAME were then analyzed by GLC using a Hewlett Packard 5890 GLC equipped with a flame ionization detector and an autosampler (Hewlett Packard, Avondale, PA). The column used for the chromatographic separations was a 100 m x 0.25 mm x 0.2 µm film thickness, fused-silica column (SP-2560; Sigma-Aldrich, Co., St. Louis, MO). Helium was used as the carrier gas, with a split ratio of 30:1 and 0.9 mL/ min column flow. Column temperature was programmed to be a constant temperature of 175°C for 65 min. The various fatty acids were identified using standard samples from Nu-Chek-Prep (Elysian, MN), and concentrations were determined using the internal method of Murrieta et al. (2003), with tridecanoic acid (C13:0) methyl ester added before extraction as an internal standard.
Tocopherol Analysis
Tocopherols in LM samples were extracted following the procedures of Katsanidis and Addis (1999). Briefly, 2 g of meat was homogenized in absolute ethanol (8.0 mL) for 30 s with a PowerGen 700 (Fisher Scientific, Pittsburgh, PA) homogenizer. Distilled water (10.0 mL) was added, and the sample was homogenized a second time for 15 s. Finally, 8 mL of hexane was added and the sample homogenized for 15 s. The samples were centrifuged at 655 x g for 10 min. The upper layer (hexane) was collected and analyzed by normal-phase HPLC.
The HPLC consisted of a Beckman Coulter System Gold 126 solvent module and a Beckman Coulter System Gold 168 diode array detector (Beckman Coulter, Fullerton, CA). Twenty microliters of hexane-containing sample was injected onto a 4.6 mm x 25 cm silica column (Ultrasphere Silica, Beckman Coulter, Fullerton, CA). The mobile phase was hexane-isopropanol (99:1) with a flow rate of 1.3 mL/min, and the wavelength was programmed at 295 nm for the vitamin E homologs. Alpha-, gamma-, and delta-tocopherol standards (Fisher Scientific) were used to locate and quantify peaks found in meat extracts.
Statistical Analyses
Data were analyzed as a completely randomized design using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC), with pen as the experimental unit for feedlot data and carcass the experimental unit for carcass, fatty acid, and tocopherol data. The interaction of diet and muscle was analyzed for fatty acid data. Color data were analyzed using a repeated-measures model with diet, day, and the two-way interaction included in the model. The model statement for carcass and tocopherol data included diet, whereas the model statement for fatty acid data included both diet and muscle. Least squares means and planned comparisons were used for mean separation when the model was significant (P < 0.05).
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Results and Discussion
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Increasing safflower oil in the diet decreased the percentage of palmitic, stearic, and oleic acids, whereas percentage of linoleic acid increased in the diets (Table 1
). Linolenic and other minor fatty acids were identified in very low amounts but were not quantified. Neither CLA isomers nor octadecenoic acids (C18:1) was detected in the analysis of the feed samples.
Concentration of oil in diet did not (P 0.11) alter DMI, ADG, G:F, or any measured carcass characteristic of sheep in this study (Table 2). Rizzi et al. (2002) reported an increase in DMI and ADG as the level of ether extract from extruded soybeans and sunflower seeds increased in lab diets. Kott et al. (2003) found no effect of supplemental safflower seeds on DMI, but reported a slight increase in ADG and G:F when diets were supplemented with safflower seeds to supply up to 6% dietary oil. Neither researcher, however, reported an effect of increasing sunflower (Rizzi et al., 2002) or safflower (Kott et al., 2003) seeds in sheep diets on carcass characteristics. Bolte et al. (2002) also failed to detect differences in growth or carcass characteristics when high-oleate or high-linoleate safflower seeds were supplemented in the diet, suggesting that either oil or oilseeds could be fed to sheep at levels that supply up to 6% oil without affecting live animal performance.
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Table 2. Effect of safflower seed supplementation on feedlot performance and carcass characteristics in wether lambs
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Fatty Acid Composition
There was no interaction between diet and muscle location tested for fatty acid composition; thus, only main effects of muscle (Table 3) and diet (Table 4) will be discussed. The LM had a greater proportion of oleic (C18:1 cis-9; P < 0.01), palmitolieic (C16:1; P = 0.07), arachidic (C20:0; P = 0.01, and arachidonic (C20:4; P = 0.04) acids than the infraspinatus (Table 3). Conversely, the weight percentage of palmitic (C16:0; P < 0.01) and myristic (C14:0; P = 0.07) acids were greater in the infraspinatus than in the LM. Adding increasing levels of oil to lamb diets decreased (P < 0.01) the weight percentage of oleic (C19:1cis-9) acid, whereas linoleic acid weight percent increased (P < 0.01) in the lean tissue (Table 3). Conjugated linoleic acid isomers also increased (P < 0.01) in response to oil supplementation, with the largest increase observed in the cis-9,trans-11 isomer. This isomer has been reported to be the most biologically active CLA isomer (Ha et al., 1990). The largest increase (P < 0.01) in CLA isomers was with the addition of 6% safflower oil to the lamb diets compared with meat from animals fed diets containing 0 or 3% safflower oil. Supplementing the diet with 6% safflower oil could result in meat that contains more of the biologically active CLA and improve the health benefits of treated lamb.
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Table 3. Effect of safflower oil supplementation on the fatty acid profile (%, weight basis) and tocopherol concentration in muscle sample
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Table 4. Effect of muscle on the fatty acid profile of extracts from infraspinatus and longissimus muscle (%, weight basis)
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Along with these increases in the PUFA, there were concomitant decreases in palmitic (P < 0.01) and stearic (P = 0.03) acids (Table 4). Other researchers have reported increased unsaturated fatty acids in muscle from lambs fed sunflower or safflower seeds (Bolte et al., 2002; Rizzi et al., 2002; Kott et al., 2003). Madron et al. (2002) also reported increased levels of cis-9,trans-11 CLA, and a decrease in SFA in beef with increasing levels of full-fat extruded soybeans fed to steers. Kott et al. (2003) fed cracked safflower seeds to supply 6% oil to the diet of sheep, and reported an almost twofold increase in CLA, as well as an increase in MUFA and PUFA in both lean and fat tissue.
Tocopherols
Increasing levels of safflower oil in the diet did not (P 0.30) influence tocopherol levels in the muscle tissue (Table 3). Increased 
-tocopherol in meat when diets were supplemented with high levels of tocopherol acetate has been reported previously (Arnold et al., 1992, 1993; Lynch et al., 2000). Yang et al. (2002) reported increased color stability of beef from vitamin E-supplemented, grain-fed steers, but beef from pasture-fed steers had color stability similar to vitamin E-supplemented grain-fed steers, even though measured -tocopherol concentrations were similar. This finding suggests that different antioxidants and/or different tocopherol isomers may be active in beef from pasture-fed steers. Lo et al. (1992) reported that safflower oil has relatively high levels of - (39.6 mg), 
- (17.4 mg), and 
-tocopherol (24.0 mg), suggesting that - and -tocopherol would be the isomers most affected in the muscle by the increased supplemental safflower oil; however, results of the present study do not support this theory.
Color
There was a diet x duration of retail storage interaction (P = 0.05) on Hunter L* values, with chops from sheep fed diets with 6% supplemental safflower oil being consistently darker at all days of storage (Figure 1). Conversely, there was no (P 0.89) diet x duration of retail storage interaction or main effects of diet (P 0.34) on a* and b* values. As expected, the duration of retail storage affected objective color measures, with loin chops becoming darker (lower L* values) and less red (lower a* values) as the time in retail storage increased (results not shown). Differences in L*, a*, and b* values were small, however, and may be of little practical importance. Yang et al. (2002) reported that alterations in fatty acid composition altered color stability; however, the changes in fatty acid composition associated with increased safflower oil supplementation in this study failed to alter color stability of lamb.
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Figure 1. The interactive effect of supplemental safflower oil in the diet and duration of retail storage on L* values (a lower L* value indicates a darker color).
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Implications
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Supplementation of sheep diets with increasing levels of safflower oil (up to 6% of the diet; as-fed basis) increased the level of polyunsaturated fatty acids and conjugated linoleic acids (especially the cis-9,trans-11 isomer) in the lean tissue. The increase of oil in the diet to increase polyunsaturated fatty acid and conjugated linoleic acid isomers did not affect growth or carcass characteristics of the lamb nor did it decrease the color stability of the longissimus muscle during retail storage. Therefore, supplementation with high-linoleic acid safflower oil to modify meat characteristics can be used to produce lamb with a modified fatty acid composition, without detrimental effects on growth, carcass composition, or color stability during retail storage.
1 Correspondence: P.O. Box 172900 (phone: 406-994-7352; fax: 406-994-5589; e-mail: jboles{at}montana.edu).
Received for publication October 17, 2003.
Accepted for publication June 7, 2005.
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Abstract
Introduction
