Manipulation of the n-3 polyunsaturated fatty acid content of muscle and adipose tissue in lambs 1

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1 Manipulation of the n-3 polyunsaturated fatty acid content of muscle and adipose tissue in lambs 1 S. L. Cooper*, L. A. Sinclair* 2, R. G. Wilkinson*, K. G. Hallett, M. Enser, and J. D. Wood *ASRC, School of Agriculture, Harper Adams University College, Edgmond, Newport, Shropshire, TF10 8NB, United Kingdom and Division of Farm Animal Science, School of Veterinary Science, University of Bristol, Bristol BS40 5DU, United Kingdom ABSTRACT: Fifty Suffolk-crossbred wether lambs, with an initial live weight of 29 ± 2.1 kg, were allocated to one of five concentrate-based diets formulated to have a similar fatty acid content (60 g/kg DM), but containing either linseed oil (high in 18:3n 3); fish oil (high in 20:5n 3 and 22:6n 3); protected linseed and soybean (PLS; high in 18:2n 6 and 18:3n 3); fish oil and marine algae (fish/algae; high in 20:5n 3 and 22:6n 3); or PLS and algae (PLS/algae; high in 18:3n 3 and 22:6n 3). Lambs were slaughtered when they reached 40 kg. Growth performance and intake were similar (P > 0.35) among treatments. By contrast, gain:feed was higher (P < 0.05) in lambs fed the fish oil compared with the linseed oil or PLS/algae diets. Total fatty acid concentration (mg/100 g) in the neutral lipid of the longissimus muscle was not affected by treatment (P > 0.87) but was least (P < 0.05) in the phospholipid fraction in lambs fed the linseed oil diet. Lambs fed either diet containing marine algae contained the highest (P < 0.05) percentage of 22:6n 3 in the phospholipid (mean of 5.2%), 2.8-fold higher than in sheep fed the fish oil diet. In lambs fed the fish/algae diet, the percentage of 20:5n 3 was highest (P < 0.05), contributing some 8.7, 0.8, and 0.5% of the total fatty acids in the muscle phospholipid, neutral lipids, and adipose tissue, respectively. The percentage of 18:3n 3 in the phospholipid fraction of the LM was highest (P < 0.05) in lambs fed the linseed oil diet (6.9%), a value double that of sheep fed the PLS diet. By contrast, lambs fed the PLS diet had twice the percentage of 18:3n 3 in the muscle neutral lipids (3.8%) than those offered the linseed oil diet, and 5.5-fold greater than lambs fed the fish/algae treatment (P < 0.05), an effect that was similar in the adipose tissue. The percentage of 18:2n 6 was highest (P < 0.05) in lambs fed the PLS diet, where it contributed 33.7, 10.1, and 11.2% in the muscle phospholipid, neutral lipids, and adipose tissue, respectively. The highest (P < 0.05) muscle PUFA-to-saturated fatty acid (P:S) ratio was obtained in lambs fed the PLS diet (0.57), followed by the PLS/algae diet (0.46), and those fed the fish oil or linseed oil diets had the lowest ratios (0.19 and 0.26, respectively). The favorable P:S ratio of lambs fed the PLS/algae diet, in conjunction with the increased levels of 20:5n 3 and 22:6n 3, enhanced the nutritional qualities of lamb to more closely resemble what is recommended for the human diet. Key Words: Adipose Tissue, Lambs, Marine Algae, Oils, Phospholipids 2004 American Society of Animal Science. All rights reserved. J. Anim. Sci : Introduction In concentrate- and forage-finished lambs, dietary PUFA are extensively biohydrogenated in the rumen, resulting in the absorption of predominately saturated fatty acids at the small intestine (Doreau and Ferlay, 1994; Wachira et al., 2000). Sheep meat is, therefore, 1 The authors would like to acknowledge the Dept. for Environment, Food, and Rural Affairs; ABNA Ltd.; Roche Products Ltd.; Tesco Stores Ltd.; and Pedigree Pet Foods for funding this work and acknowledge DHA Nutrition, a subsidiary of BioProgress plc, for the supply of the marine algae. 2 Correspondence phone: ; fax: ; lsinclair@harper-adams.ac.uk. Received July 29, Accepted January 26, characterized as having a high saturated fatty acid content and a low PUFA-to-saturated fatty acid (P:S) ratio (Enser et al., 1996). Recently, research has been directed at improving the n 3 PUFA content of sheep meat by feeding diets high in α-linolenic acid (18:3n 3), and, in particular, the longer chain eicosapentaenoic (20:5n 3) and docosahexaenoic (22:6n 3) acids (Wachira et al., 2002; Ponnampalam et al., 2001b). These fatty acids have been associated with a reduction in the thrombotic tendency of blood and associated with lower coronary heart disease in humans (Department of Health, 1994; Sanderson et al., 2002). Results from several studies have shown that muscle and adipose tissue levels of n 3 PUFA can be increased substantially, al- 1461

2 1462 Cooper et al. Table 1. Raw materials and the chemical and fatty acid content of diets differing in their fatty acid source Linseed Fish Fish oil/ PLS/ Item oil oil PLS a algae algae Ingredients, g/kg Wheat straw Soya hulls Barley Sugar beet pulp Soybean meal Linseed oil 43 Fish oil Algae PLS a Molasses Mineral and vitamins Ammonium chloride Sodium chloride Chemical composition, g/kg DM DM, g/kg OM CP NDF Fatty acid composition, g/kg DM 12:0 lauric :0 myristic :0 palmitic :1n 9 palmitoleic b :0 stearic :1n 9 oleic b :2n 6 linoleic b :3n 3 α-linolenic b :5n 3 eicosapentaenoic b :6n 3 docosahexaenoic b Σ n 6 fatty acids c Σ n 3 fatty acids d Total fatty acids a Protected linseed and soybean. Algae = marine algae. b n 9 = omega-9 fatty acid; n 6 = omega-6 fatty acid; and n 3 = omega-3 fatty acid. c Σ n 6 fatty acids = 18:2n :3n :4n :4n 6. d Σ n 3 fatty acids = 18:3n :5n :5n :6n 3. though the P:S ratio was little affected (Wachira et al., 2002; Ponnampalam et al., 2001b; Demirel, 2000). The hypothesis to be tested in the current experiment was that the n 3 fatty acid profile and P:S ratio of lamb could be nutritionally manipulated to more closely meet the values considered as optimal for the human diet. To this effect, linseed oil was used because it is high in 18:3n 3, whereas fish oil was used because it is a rich source of the longer chain n 3 fatty acids. A protected source of linoleic acid (18:2n 6) and 18:3n 3 was used to improve the P:S ratio while maintaining the n 6/n 3 ratio similar to that reported in lambs when grassfinished. Finally, marine algae were used as they are particularly high in 22:6n 3 (Givens et al., 2000) and the level of biohydrogenation has been shown to be lower than that in fish oil (Cooper, 2002). Materials and Methods The experiment described in this paper was conducted in accordance with the requirements of the Animals (Scientific Procedures) Act Animals, Diets, and Experimental Procedure Fifty Suffolk-cross wether lambs, with an initial live weight of 29 kg ± 2.1 kg were fed one of five concentratebased diets formulated to be isoenergetic and isonitrogenous, as well as a similar fatty acid content (approximately 60 g/kg DM) from different fat sources. The linseed oil diet contained linseed oil, whereas the fish oil diet contained Scandinavian crude, unrefined fish oil (both supplied by Trouw UK Ltd., Cheshire, U.K.). The fish/algae diet contained marine algae (from a dinoflagellate of the class Dinophyceae; Martek Biosciences Corp., Columbia, MD) and fish oil in equal proportions on an oil basis. The diet containing the protected linseed and soybean supplement (PLS) contained linseed, sunflower seed, and soybean encapsulated in formaldehyde-treated protein (CSIRO, Blacktown, NSW, Australia). The PLS/algae diet contained equal proportions on an oil basis of PLS and algae. The formulation of the five diets is presented in Table 1. Lambs were stratified according to live weight and randomly allocated to one of the five treatments. The

3 PUFA content of lamb 1463 animals were housed in individual, raised-floor pens, with continuous access to water. Feed was offered daily at 0900 at the rate of 110% of ad libitum intake calculated by weighing-back refusals three times each week. Feed samples were taken weekly and stored at 20 C before analysis. Live weight was recorded weekly at At approximately half their estimated mature live weight (40 kg), the lambs were slaughtered and the carcasses scored for conformation and fat score using a 15-point scale (1 = low to 15 = high; Fisher et al., 2000). The carcasses were then weighed (hot carcass weight), and the ph of the LM was recorded. Then, carcasses were hung by the hind legs for 24 h at 1 C before being reweighed (cold carcass weight) and the 24-h LM ph recorded. After slaughter, samples from the LM were dissected from the sixth to eighth ribs, inclusive and trimmed of all visible fat. Subcutaneous adipose tissue samples (full thickness, mm) were dissected from the cold carcass above the position of the LM sample, vacuum-packaged, and stored at 20 C before subsequent analysis. Chemical Analyses Feed samples were bulked and analyzed for DM, OM (AOAC, 1990), N (Kjeltec 1035 Analyser; Foss UK Ltd., Cheshire, U.K.), and NDF (Van Soest et al., 1991). Fatty acid content of samples of muscle, adipose tissue, and feed samples were determined as described by Wachira et al. (2002). Samples of the LM were blended in a food processor, and the lipids extracted from duplicate 10- g samples using chloroform-methanol (2:1, vol/vol; Folch et al., 1957). Lipids were then loaded onto a silicic acid solid-phase extraction (50 mg/10 ml; Isolute; Jones Chromatography, Hengoed, U.K.) column, and the neutral lipids were extracted using 2 10 ml chloroform, followed by 10 ml of methanol to remove phospholipids. After addition of the fatty acid standard (heneicosanoic acid methyl ester; Sigma, Poole, Dorset, U.K.), the solvents were removed under N 2 and the lipids hydrolyzed with 2 M KOH in methanol-water (1:1, vol/vol) containing 1 g of hydroquinone per liter (as an antioxidant) at 60 C for 1 h. After dilution with water and removal of nonsaponifiable compounds by three extractions with light petroleum ether (boiling point 40 to 60 C), the hydrolyzate was acidified and the fatty acids extracted into light petroleum ether. After neutralizing and drying with solid NaHCO 3 and anhydrous sodium sulfate, the fatty acids were methylated with a solution of diazomethane in diethyl ether and their composition determined by GLC. Samples were injected in the split mode (70:1) onto a 50 m 0.25 mm i.d. CP-Sil 88 WCOT for fatty acid methyl esters (Catalog No. 7488; Chrompak Ltd., Welwyn Garden City, Herts, U.K.), with He as the carrier gas. The output from the flame ionization detector was quantified using a computing integrator (Spectra Physics 4270; Darmstadt, Germany), and linearity of the system was tested using saturated (fatty acid methyl ester 4) and monounsaturated (fatty acid methyl ester 5) methyl ester quantitative standards (Thames Restek UK Ltd., Berks, U.K.). A sample of subcutaneous adipose tissue was blended in a food processor. Lipid was extracted by homogenizing duplicate 1-g samples in chloroform containing 100 mg of 2,6-di-tert-butyl-p-cresol per liter (antioxidant) and then adding anhydrous sodium sulfate to remove water. After filtration, samples were hydrolyzed, and the fatty acids processed and analyzed as previously described. Feed fatty acid samples were attained after the addition of 21:0 methyl ester as the internal standard by direct hydrolysis in 5 M KOH in aqueous methanol (1:1, vol/vol) for 3 h at 60 C, and then another 1 h at 60 C after acidification to ph 1.0 with sulfuric acid. A standard of mixed isomers of CLA methyl esters was obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). Samples were run with and without the internal standard, and, at the column loadings used, only one significant peak corresponding to a peak in the CLA standard was detected, which was identified as cis-9, trans-11 CLA (Enser et al., 1999). Sample concentrations of other isomers were low and did not allow for quantification. Diazomethane has been shown not to affect CLA isomer composition (Kramer et al., 1997), and, in the current study, the recovery of cis-9, trans- 11 CLA indicated there was no loss or isomerization. Fatty acid results are presented as grams per kilogram of DM for the feeds and as milligrams per 100 g of fresh tissue or percentages of the total fatty acids. The trans 18:1 fatty acids are reported as a single value due to incomplete resolution on the GLC column used. The content (mg/100 g tissue) of each individual fatty acid in the LM was determined by combining the content (percentage of each fatty acid multiplied by the total fatty acid content; mg/100 g tissue) in the phospholipid and neutral lipid fractions. Statistical Analyses Each lamb was considered as the experimental unit, and live weight gain was determined by regression analysis of live weight (kilograms) on time (days). All data were subjected to ANOVA as a randomized block design using the model y ij =b i +t j +e ij, where b = blocks and t = treatments. Analysis was conducted using Genstat 5 (VSN Int. Ltd., Oxford, U.K.), and the results presented as treatment means with a SEM. When significant treatment effects were detected, means were separated using LSD, with P < 0.05 being considered as statistically significant. Results Diets and Animal Performance Dietary CP content averaged 188 g/kg of DM, with the highest level in the linseed oil diet and the lowest in the PLS diet (Table 1). The highest concentration of NDF was in the PLS/algae diet, a value 22% higher than

4 1464 Cooper et al. Table 2. Performance and carcass characteristics of lambs fed diets differing in their fatty acid source Overall Linseed Fish Fish oil/ PLS/ treatment Item oil oil PLS a algae algae SEM significance Initial live wt, kg Slaughter wt, kg DM intake, kg/d ADG, kg/d G:F c d cd cd c Carcass measurements Hot wt, kg Cold wt, kg Muscle ph 45 min 6.48 c 6.47 c 6.52 c 6.70 d 6.58 cd Muscle ph 24 h Conformation score b 8.70 d 7.50 c 8.70 d 8.25 cd 8.00 cd Fat score b a PLS = protected linseed and soybean. Algae = marine algae. b Conformation and fat scored on a 1 (low) to 15 (high) scale. c,d Within a row, means without a common superscript letter differ (P < 0.05). the fish/algae diet, which had the lowest concentration. Total fatty acid content averaged 62.5 g/kg DM, with the linseed oil diet having the highest value and the PLS/algae diet the lowest. The fatty acid composition of the diets reflected their fat source, with the fish/algae diet having the highest content of 22:6n 3 (15% of the total fatty acids), a value nearly twofold higher than that in the PLS/algae diet, which was, in turn, higher than in the fish oil diet. The fish oil diet contained the highest concentration of 20:5n 3 (6% of the total fatty acids), a value nearly twofold greater than that in the fish/algae diet, whereas the PLS, PLS/algae, and linseed oil diets had undetectable levels. The highest concentration of 18:3n 3 was in the linseed oil diet, which was some 3.1-fold greater than that in the PLS diet, with the PLS/algae diet having a value intermediate to that in the PLS and the fish oil or fish/algae diets. The concentration of 18:2n 6 was greatest in the PLS diet (44% of the total fatty acid content), and lowest in the fish/algae diet (14%). The percentage of total n 3 fatty acids was highest in the linseed oil diet where they contributed 47% of the total fatty acids. By contrast, total n 6 fatty acids levels were highest in the PLS, followed by the PLS/algae diet, and lowest in the fish/ algae diet. Growth performance and intake were not (P > 0.35) affected by treatment, although lambs fed the fish oil diet had a higher (P < 0.05) G:F than those fed the linseed oil or PLS/algae diets (Table 2). There was no effect (P > 0.82) of treatment on hot or cold carcass weights, whereas LM ph was highest (P < 0.05) in lambs fed the fish/algae diet at 45 min after slaughter, but not at 24 h. Conformation score was lower (P < 0.05) in lambs fed the fish oil diet than those fed the linseed oil or PLS diets; however, there was no (P > 0.74) effect on carcass fat score. Phospholipids Total fatty acid content of the phospholipid was lower (P < 0.05) in lambs fed the linseed oil diet than those fed the PLS, fish/algae, or PLS/algae diets (Table 3). Lambs fed either diet containing the marine algae had the highest (P < 0.05) percentage of 22:6n 3, with values some 2.8- to 8.3-fold higher (P < 0.05) than in lambs fed the fish oil, linseed oil, or PLS diets. The percentage of 20:5n 3 was also highest (P < 0.05) in lambs fed the fish/algae diet and lowest (P < 0.05) in lambs fed the PLS diet. By contrast, 20:4n 6 was 2.5-fold higher (P < 0.05) in lambs fed the PLS diet compared with those fed the fish/algae diet, which had the lowest value. The percentage of 18:3n 3 in lambs fed the linseed oil diet was 2.3-fold greater (P < 0.05) than that of lambs fed either the PLS or PLS/algae diets, which had similar values. The concentration of 18:2n 6 was highest (P < 0.05) in lambs fed either diet containing PLS, with values approximately twice that of lambs fed any of the other diets. The content of trans 18:1 was greatest (P < 0.05) in lambs fed the PLS/algae diet, in which it contributed 3.9% of the total fatty acids, whereas the percentage of cis-9, trans-11 CLA was greatest (P < 0.05) in lambs fed the linseed oil diet and lowest (P < 0.05) in lambs fed the PLS, fish/algae, or PLS/algae diets. Lambs fed either the linseed oil or fish oil diets had the highest percentage of 18:1n 9 at approximately 17.7%, a value 1.7-fold higher (P < 0.05) than in lambs fed either the PLS or PLS/algae diets. By contrast, lambs fed the PLS diet had a higher (P < 0.05) percentage of 18:0 than those fed the fish oil diet, whereas the highest percentages of 16:1n 9 (P < 0.05) and 16:0 (P < 0.05) were observed in lambs fed diets containing fish oil. Total n 3 fatty acids in the phospholipid were highest in lambs fed the fish/algae diet and twofold higher (P < 0.05) than in lambs fed the PLS diet. By

5 PUFA content of lamb 1465 Table 3. Phospholipid composition (% of total fatty acids) of the longissimus muscle of lambs fed diets differing in their fatty acid source Overall Linseed Fish Fish oil/ PLS/ treatment Item oil oil PLS a algae algae SEM significance 14:0 myristic 0.29 fg 0.44 h 0.26 f 0.58 i 0.39 gh :0 palmitic 11.9 f 15.9 i 12.8 g 15.4 i 14.1 h :1n 9 palmitoleic b 0.98 g 1.95 h 0.66 f 1.74 h 0.86 fg :0 stearic 12.0 g 8.33 f 12.3 g 8.53 f 8.45 f trans 18: g 2.84 h 1.06 f 2.89 h 3.89 i :1n 9 oleic b 17.1 h 18.3 h 9.71 f 14.4 g 10.7 f :2n 6 linoleic b 17.0 g 11.5 f 33.7 i 13.2 f 27.7 h CLA c 0.57 h 0.36 g 0.18 f 0.26 f 0.26 f :3n 3 α-linolenic b 6.91 h 1.73 f 3.05 g 1.42 f 3.04 g :4n 6 arachidonic b 4.47 h 2.56 f 5.52 i 2.24 f 3.23 g :5n 3 eicosapentaenoic b 3.84 g 5.77 i 2.59 f 8.73 j 4.81 h :5n 3 docosapentaenoic b 3.28 h 3.04 gh 2.82 g 3.10 gh 2.19 f :6n 3 docosahexaenoic b 1.12 f 1.93 g 0.64 f 5.34 h 5.35 h Σ n 6 fatty acids d 22.4 g 14.6 f 40.2 i 15.9 f 31.7 h Σ n 3 fatty acids e 15.2 h 12.5 g 9.1 f 18.6 i 15.4 h Total fatty acids, mg/100 g of fresh tissue 604 f 656 fg 701 g 716 g 716 g a PLS = protected linseed and soybean. Algae = marine algae. b n 9 = omega-9 fatty acid; n 6 = omega-6 fatty acid; n 3 = omega-3 fatty acid. c CLA = cis-9, trans-11 conjugated linoleic acid. d Σ n 6 fatty acids = 18:2n :3n :4n :4n 6. e Σ n 3 fatty acids = 18:3n :5n :5n :6n 3. f,g,h,i,j Within a row, means without a common superscript letter differ (P < 0.05). contrast, total n 6 fatty acids were highest (P < 0.05) in lambs fed the PLS diet. Muscle Neutral Lipids and Adipose Tissue Total muscle neutral lipids in the LM averaged 3,027 mg/100 g, and was not (P > 0.87) affected by dietary treatment (Table 4). The content of long-chain n 3 PUFA 20:5n 3 and 22:6n 3 was generally low across all the dietary treatments, with lambs fed the fish/algae diet having the highest (P < 0.05) percent. The content of 20:4n 6 was also low, but was highest (P < 0.05) in lambs fed the PLS, fish/algae, or PLS/algae diets and lowest (P < 0.05) in the LM of those fed the linseed Table 4. Neutral lipid composition (% of total fatty acids) of the longissimus muscle of lambs fed diets differing in their fatty acid source Overall Linseed Fish Fish oil/ PLS/ treatment Item oil oil PLS a algae algae SEM significance 14:0 myristic 2.66 f 3.24 g 2.44 f 3.35 g 2.74 f :0 palmitic 22.6 f 26.9 h 21.9 f 26.5 h 24.9 g :1n 9 oleic b 1.80 f 2.43 g 1.71 f 2.40 g 1.81 f :0 stearic 18.7 g 14.7 f 16.4 f 14.7 f 15.1 f trans 18: g 5.21 g 3.17 f 5.38 g 5.86 g :1n 9 oleic b 34.7 h 30.7 f 33.8 gh 29.9 f 31.9 fg :2n 6 linoleic b 2.11 f 1.51 f 10.1 h 1.96 f 6.15 g CLA c 1.20 g 0.82 f 0.79 f 0.87 f 0.95 f :3n 3 α-linolenic b 1.79 g 1.51 g 3.83 i 0.70 f 2.38 h :4n 6 arachidonic b 0.04 f 0.07 g 0.12 h 0.10 h 0.10 h :5n 3 eicosapentaenoic b 0.04 f 0.33 g 0.09 f 0.77 h 0.32 g :5n 3 docosapentaenoic b 0.11 f 0.41 g 0.16 f 0.78 h 0.36 g :6n 3 docosahexaenoic b 0.02 f 0.33 g 0.03 f 1.88 i 1.50 h Σ n 6 fatty acids d 2.15 f 1.61 f 10.2 h 2.09 f 6.28 g Σ n 3 fatty acids e 1.96 f 1.66 f 4.10 g 4.12 g 4.56 g Total fatty acids, mg/100 g of fresh tissue 2,797 3,051 3,115 2,956 3, a PLS = protected linseed and soybean. Algae = marine algae. b n 9 = omega-9 fatty acid; n 6 = omega-6 fatty acid; n 3 = omega-3 fatty acid. c CLA = cis-9, trans-11 conjugated linoleic acid. d n 6 fatty acids = 18:2n :3n :4n :4n 6. e Fatty acids = 18:3n :5n :5n :6n 3. f,g,h,i Within a row, means without a common superscript letter differ (P < 0.05).

6 1466 Cooper et al. Table 5. Subcutaneous adipose tissue fatty acid composition (% of total fatty acids) of lambs fed diets differing in their fatty acid source Overall Linseed Fish Fish oil/ PLS/ treatment Item oil oil PLS a algae algae SEM significance 14:0 myristic 3.26 f 4.39 g 3.38 f 4.59 g 3.55 f :0 palmitic 19.1 f 23.7 h 18.8 f 24.1 h 22.0 g :1n 9 palmitoleic b 1.83 f 2.77 g 1.90 f 2.78 g 2.01 f :0 stearic 20.3 h 14.9 f 18.2 g 15.7 f 15.6 f trans 18: i 7.41 gh 4.81 f 6.73 g 8.12 h :1n 9 oleic b 24.1 g 21.8 f 27.7 h 24.7 g 25.0 g :2n 6 linoelic b 2.59 f 1.78 f 11.2 h 2.11 f 7.31 g CLA c 1.30 g 0.94 f 0.97 f 1.00 f 1.06 f :3n 3 α-linolenic b 2.23 g 0.63 f 4.10 i 0.64 f 2.68 h :4n 6 arachidonic b 0.03 f 0.04 f 0.09 h 0.04 f 0.06 g :5n 3 eicosapentaenoic b 0.04 f 0.29 g 0.05 f 0.54 h 0.27 g :5n 3 docosapentaenoic b 0.11 f 0.44 g 0.13 f 0.72 h 0.39 g :6n 3 docosahexaenoic b 0.01 f 0.32 g 0.01 f 1.46 h 1.57 h Σ n 6 fatty acids d 2.63 f 1.85 f h 2.16 f 7.37 g Σ n 3 fatty acids e 2.39 g 1.68 f 4.30 i 3.36 h 4.91 i Total fatty acids, mg/100 g of fresh tissue 84,088 gh 82,516 fg 86,159 h 81,143 f 84,018 gh a PLS = protected linseed and soybean. Algae = marine algae. b n 9 = omega-9 fatty acid; n 6 = omega-6 fatty acid; n 3 = omega-3 fatty acid. c CLA = cis-9, trans-11 conjugated linoleic acid. d Σ n 6 fatty acids = 18:2n :3n :4n :4n 6. e Σ n 3 fatty acids = 18:3n :5n :5n :6n 3. f,g,h,i Within a row, means without a common superscript letter differ (P < 0.05). oil diet. Lambs fed the PLS diet had a 2.1-fold higher percentage of 18:3n 3 than those fed the linseed oil diet, and some 5.5-fold the level of those fed the fish/ algae diet (P < 0.05), which had the lowest value. The content of 18:2n 6 in LM neutral lipids reflected dietary supply, with the highest (P < 0.05) level in lambs fed the PLS diet, a value nearly fivefold greater (P < 0.05) than in lambs fed the linseed oil diet; lambs fed the PLS/algae diet had intermediate values. Concentration of trans 18:1 was higher (P < 0.05) in lambs fed the PLS/ algae than the PLS diet, whereas the concentration of cis-9, trans-11 CLA was highest (P < 0.05) in lambs fed the linseed oil diet. Lambs fed the linseed oil diet also had a higher (P < 0.05) percentage of 18:0 and 18:1n 9 than those fed either of the diets containing fish oil. By contrast, the percentage of 16:0 and 16:1n 9 was greatest (P < 0.05) in lambs fed diets containing fish oil, and lowest (P < 0.05) in lambs fed the linseed oil or PLS diets. Similar to 16:0, the percentage of 14:0 was highest (P < 0.05) in lambs fed either fish oil diet, where it contributed approximately 3.3% of the total fatty acids. Total n 3 fatty acids in the neutral lipid of the LM was highest (P < 0.05) in lambs fed either the PLS, fish/algae, or PLS/algae diets and lowest (P < 0.05) in lambs fed either the linseed oil or fish oil. Total n 6 fatty acids reflected dietary source, with the highest (P < 0.05) percent in lambs fed the PLS diet and the lowest (P < 0.05) in lambs fed the fish oil diet. Total fatty acid content in the subcutaneous adipose tissue was higher (P < 0.05) in lambs fed the PLS diet than in those fed the fish/algae diet (Table 5). The content of 20:5n 3 and 22:6n 3 in the adipose tissue was generally low across treatments; however, lambs fed diets containing marine algae had the highest (P < 0.05) concentration of 22:6n 3, contributing 1.5% of the total fatty acids. Subcutaneous adipose tissue of lambs fed the PLS diet contained nearly twofold (P < 0.05) the percentage of 18:3n 3 than those fed the linseed oil diet, and over a sixfold greater (P < 0.05) level than lambs fed the fish oil or fish/algae diets. Lambs fed either the linseed oil or PLS/algae diets had a higher (P < 0.05) percentage of trans 18:1 than those offered the PLS diet, whereas cis-9, trans-11 CLA was elevated only in lambs fed the linseed oil diet (P < 0.05). The highest (P < 0.05) percentage of 18:1n 9 was observed in lambs fed the PLS diet, whereas levels of 18:0 were highest (P < 0.05) in lambs fed the linseed oil diet. By contrast, lambs fed either diet containing fish oil had higher (P < 0.05) percentages of 16:0 and 16:1n 9 compared with lambs fed either the PLS or linseed oil diets. Total n 3 fatty acids were higher (P < 0.05) in lambs fed either diet containing PLS compared with those fed the fish oil diet, which had the lowest value. Similar to the neutral lipids of the LM, total n 6 fatty acids were highest (P < 0.05) in lambs fed the PLS diet and lowest (P < 0.05) in those fed the fish oil diet. Nutritional Indices There was no effect (P > 0.03) of dietary treatment on the saturated or monounsaturated fatty acid content of the LM (phospholipid and neutral lipid combined); however, there was an effect of diet on the PUFA content. Lambs fed the PLS or PLS/algae diets had the highest (P < 0.05) concentration of PUFA, some 2.5-fold higher than in lambs fed the linseed or fish oil diets,

7 PUFA content of lamb 1467 Table 6. Fatty acid classes (mg/100 g fresh meat) and ratios of the longissimus muscle (phospholipid and neutral lipid combined) in lambs fed diets differing in their fatty acid source Overall Linseed Fish Fish oil/ PLS/ treatment Item oil oil PLS a algae algae SEM significance SFA b 1,388 1,528 1,446 1,490 1, MUFA c 1,186 1,256 1,231 1,168 1, PUFA d 333 hi 275 h 784 j 427 i 686 j n 6 PUFA e 191 h 143 h 595 j 174 h 428 i n 3 PUFA f 142 h 132 h 189 i 254 j 257 j P:S g 0.26 hi 0.19 h 0.57 k 0.30 i 0.46 j :2 to 18:3 ratio 1.84 h 4.17 i 3.97 i 5.30 j 4.11 i n 6 ton 3 ratio 1.37 j 1.10 i 3.15 l 0.68 h 1.70 k a PLS = protected linseed and soybean. Algae = marine algae. b SFA = saturated fatty acids (14:0 + 16:0 + 18:0). c MUFA = monounsaturated fatty acids (16:1n :1n :1n :1n 9). d PUFA = 18:2n :3n :3n :4n :5n :4n :5n :6n 3. e n 6 PUFA = 18:2n :3n :4n :4n 6. f n 3 PUFA = 18:3n :5n :5n :6n 3. g P:S = total PUFA-to-saturated fatty acid ratio. h,i,j,k,l Within a row, means without a common superscript letter differ (P < 0.05). which had similar values (Table 6). The highest P:S ratio was recorded in lambs fed the PLS diet, which was higher (P < 0.05) than in lambs fed the PLS/algae diet. The lowest (P < 0.05) P:S ratio was recorded in lambs fed the fish or linseed oil diets, followed by the fish/algae diet. The lowest (P < 0.05) ratio of 18:2n 6 to 18:3n 3 was observed in lambs fed the linseed oil, and highest (P < 0.05) in lambs fed the fish/algae diet. In contrast, the lowest (P < 0.05) total n 6:n 3 ratio was observed in lambs fed the fish/algae diet, whereas the highest (P < 0.05) value was observed in lambs fed the PLS diet. Animal Performance Discussion In the current study, there was no effect of the oil supplements on intake or growth performance. These results are in agreement with other studies that have compared linseed with fish oil in sheep (Demirel, 2000) and growing cattle (Scollan et al., 2001). In contrast, Wachira et al. (2002) reported that feeding fish oil depressed the intake and performance of lambs, an effect that was associated with a reduction in microbial growth and efficiency in the rumen (Wachira et al., 2000). Relatively few studies have been conducted to evaluate the effects of feeding marine algae to ruminants; yet, in dairy sheep (Papadopoulos et al. 2002) and cows (Offer et al., 2001), inclusion of marine algae had little effect on DM intake or milk production. Carcass fat levels in the current study were not affected by treatment, although, in other studies, fish oil was reported to increase fat levels (Demirel, 2000; Wachira et al., 2002). By contrast, Ponnampalam et al. (2001b) reported that fat levels were lowest in lambs fed fish oil, and attributed this effect to a reduced diet-induced increase in plasma insulin concentrations. In the current study, feeding fish oil reduced carcass conformation score and it would appear, therefore, that the fish oil used had more of an effect on carcass protein deposition than overall fat levels. Phospholipid Compared with diets containing fish oil or marine algae, the provision of the linseed oil or PLS diets resulted in a lower percentage of 20:5n 3 and 22:6n 3 in the phospholipid fraction of the LM. This low rate of conversion of 18:3n 3 to long-chain PUFA compared with preformed sources, has been reported elsewhere (Scollan et al., 2001; Demirel, 2000) and emphasizes the dietary requirement for these fatty acids to effectively manipulate carcass long-chain n 3 PUFA. The highest inclusion of 22:6n 3 was observed in lambs fed fish oil or PLS along with marine algae, almost 2.5 times the level of that in lambs fed the fish oil diet, and higher than that reported by Ashes et al. (1992) when formaldehyde-protected fish oil was fed. The detected increase in 22:6n 3 by feeding marine algae is also greater than that reported in the breast meat of broiler chickens (Mooney et al., 1998). The high concentration of 22:6n 3 achieved by feeding marine algae in this study can be accounted for by its greater dietary concentration and lower level of biohydrogenation compared with fish oil (60 vs. 86% respectively; Cooper, 2002). Despite the absence of 20:5n 3 in the PLS/algae diet, levels of this fatty acid observed in muscle phospholipid and neutral lipid fractions were similar to those in lambs fed the fish oil diet, with which dietary concentrations were highest. Chain elongation and desaturation from 18:3n 3 may provide one possible explanation. However, the lowest phospholipid levels of 20:5n 3 and 22:6n 3 were observed in lambs fed the linseed oil or

8 1468 Cooper et al. PLS diets, in which the dietary supply of 18:3n 3 was greatest. The very low levels of 20:4n 3 (data not presented) in lambs fed the PLS/algae diet also indicates little evidence of inhibition of 5 -desaturase activity (Brenner, 1989). An alternative explanation of the results obtained here is the retroconversion of 22:6n 3 to 20:5n 3 that has been reported in both rats and humans (Sprecher et al., 1995). Ashes et al. (1992) reported that an additional supply of 20:5n 3 and 22:6n 3 substituted primarily for 18:1n 9 and/or 18:2n 6 in the muscle phospholipid. In the current study, the effects are less clear; additional long-chain n 3 PUFA substituted primarily for 20:4n 6, 18:2n 6, and 18:0 in lambs fed the PLS/algae compared with the PLS diet. However, 20:5n 3 and 22:6n 3 substituted primarily for 18:1n 9 in lambs fed the fish/algae compared with the fish oil diet. The phospholipid 18:2n 6 content in lambs fed the PLS diet was particularly high, whereas 18:1n 9 levels were higher in lambs fed the fish oil diet. It would seem, therefore, that under dietary conditions of high n 6 PUFA the provision of long-chain n 3 PUFA replaced 18:2n 6 in the phospholipid, whereas under conditions of lower n 6 PUFA 18:1n 9 was replaced. In the current study, lambs fed the linseed oil diet had a content of 18:3n 3 in the phospholipid of 6.9%, which is higher than the 5.1% reported in lambs fed formaldehyde-protected linseed (Demirel, 2000). However, lambs fed either the PLS or PLS/algae diets had a lower phospholipid content of 18:3n 3. The failure of the PLS diet to enhance phospholipid 18:3n 3 levels above that of unprotected linseed oil can be attributed to the greater supply of 18:2n 6 in the PLS diet, as it is well established that 18:2n 6 is preferentially incorporated into the phospholipid fraction (Marmer et al., 1984; Enser et al., 1996). Indeed, 18:2n 6 levels in lambs fed the PLS diet accounted for over one-third of the total fatty acids, a value substantially higher than in other studies that have supplemented lambs with diets high in n 6 fatty acids (Ponnampalam et al., 2001a). The phospholipid content of palmitic (16:0) and stearic (18:0) acids reflected their dietary concentration, with 16:0 being higher in the fish oil and marine algae diets and 18:0 being greater in the linseed oil and PLS diets. These results are in contrast with those of Demirel (2000), who reported little difference in the muscle phospholipid content of 16:0 in lambs fed either protected linseed or a combination of fish oil and linseed, but they are in agreement with those of Scollan et al. (2001), who reported an increase in 16:0 in beef muscle when fed fish oil alone. There seems to be little consistency between the reported effects of long-chain PUFA on 18:0 levels. This was attributed, by Demirel (2000), to the different fatty acid composition of individual phospholipids, with an increase of 20:5n 3 phosphatidyl ethanolamine increasing 18:0, whereas phosphatidyl choline would raise 16:0 because 18:0/20:5n 3 and 16:0/20:5n 3 are the major 20:5n 3 containing molecular species in sheep muscle (Scott et al., 1993). Neutral Lipids and Adipose Tissue The low content of long-chain n 3 PUFA in the neutral lipids of the LM and adipose tissue are consistent with a number of other reports (Ponnampalam et al., 2001a,b; Wachira et al., 2002) and reflect the low incorporation of long-chain fatty acids in the triacylglycerol fraction, as well as the low content of phospholipid in the adipose tissue (Ashes et al., 1992; Enser et al., 1996). Despite the dietary content of 18:3n 3 in the PLS diet being approximately one-third of that in the linseed oil diet, LM neutral lipid and adipose tissue levels were double in lambs fed the PLS diet, indicating a sixfold benefit to protection. The concentration of 18:3n 3 in the adipose tissue of lambs fed the PLS diet at 4.1% is in excess of the 3% that is considered to be the maximum acceptable to maintain good meat-eating qualities (Sheard et al., 2000). However, the combination of the PLS with marine algae resulted in an adipose tissue concentration of 2.7%, which is within the limits deemed satisfactory. In contrast to that reported previously (Enser et al., 1996; Wachira et al., 2002), there was little evidence of a preferential deposition of 18:3n 3 over 18:2n 6 in the neutral lipids, with the dietary ratio of 18:2n 6 to 18:3n 3 in the PLS diet being 2.8, and that in the neutral lipids of the LM and adipose tissue being 2.6 and 2.7, respectively. In the current experiment, the LM neutral lipid and adipose tissue content of cis 9, trans 11 CLA was highest in lambs fed the linseed oil diet, with no difference between any of the other four treatments. The content of CLA can be affected by impaired biohydrogenation of 18:2n 6 in the rumen, or increased production of trans 11 18:1, from which CLA can be produced in tissues (Griinari et al., 2000; Santora et al., 2000). However, the flow of cis 9, trans 11 CLA at the duodenum has been shown to be small compared with trans 18:1, indicating the importance of tissue desaturation for enhanced content of CLA in growing ruminants (Duckett et al., 2002). Indeed, the higher content of trans 18:1 and cis 9, trans 11 CLA in lambs fed the linseed oil diet does suggest a precursor-product relationship. Compared with lambs fed the PLS diet, there was a higher neutral lipid content of trans 18:1 in lambs fed any of the diets that were supplemented with longchain n 3 PUFA. Despite this, lambs supplemented with these longer chain fatty acids had a content of cis 9, trans 11 CLA similar to those offered the PLS diet. This apparent impairment of 9 -desaturase activity by long-chain n 3 PUFA is in accordance with what we have found previously (Wachira et al., 2002). Nutritional Indices There are three factors that are generally considered to be important when judging the nutritional value of

9 PUFA content of lamb 1469 fatty foods: 1) total fat content, 2) the P:S ratio, and 3) the n 6:n 3 ratio. In the current study, the total fat content of muscle tissue with the visible fat removed was below 5 g/100 g, a value generally considered to characterize a low fat food (Food Advisory Committee, 1990). On this basis, muscle tissue from lambs fed any of the dietary treatments would be considered as low fat. However, it has to be accepted that if the visible fat is not removed then the fat content of lamb chops can increase to 30%, with values as high as 51% fat being reported (Enser et al., 1996). The inclusion of linseed oil and fish oil in the current study resulted in a P:S ratio of 0.26 and 0.19, respectively. These values are comparable to that reported for lambs finished on grass (Enser et al., 1998), supplemented with fish meal, canola meal or soybean meal (Ponnampalam et al., 2001a), and in our previous work using fish oil (Wachira et al., 2002; Demirel, 2000). Feeding the PLS diet improved the P:S ratio above the recommended value of 0.45 for the human diet as a whole, whereas the combination of PLS and marine algae also resulted in a beneficial ratio of Feeding protected lipid supplements has been reported to improve the P:S ratio to values similar to those reported in the present experiment, although the majority of these studies have focused on the effects on n 6 fatty acids (Scott et al., 1971). The lowest ratio of 18:2n 6 to 18:3n 3 was 1.8, which was observed in lambs fed the linseed oil diet, a value comparable to that reported previously for lambs fed whole linseed (Wachira et al., 2002) but higher than that of lambs fed protected linseed (Demirel, 2000). It was the intention in the current experiment to maintain the balance of 18:2n 6 to 18:3n 3 in meat from lambs fed the PLS or PLS/algae diets at approximately 1.5 to 2.0, the value reported for lambs finished on grass (Enser et al., 1996; Enser et al., 1998). The resultant value of approximately 4.0 reflects the greater dietary content of 18:2n 6 but may also be attributed to the relatively high uptake into the LM neutral lipid fraction of 18:2n 6, or the oxidation of longer-chain PUFA, such as 18:3n 3 (Enser, 1984). The lowest n 6:n 3 ratio of 0.68 was obtained in lambs fed the fish/algae diet and reflects the greater dietary concentration and tissue uptake of long-chain n 3 PUFA in lambs on this diet. In humans, it has been shown that increasing the dietary supply of 18:3n 3, compared with 18:2n 6, has little beneficial effect on coronary heart disease (Sanderson et al., 2002). By contrast, the beneficial effects of longchain n 3 PUFA are well established, and it is recommended that the daily intake of these fatty acids be increased to 200 mg/d (Department of Health, 1994), or higher. A 100-g serving of lamb, the amount assumed to be an average portion (MAFF, 1994), from sheep fed the linseed or PLS diets would supply 32 and 26 mg per serving of 20:5n 3 and 22:6n 3, respectively. This value increases to 71 mg in lambs fed the fish oil treatment; however, the largest supply of the long-chain n 3 fatty acids would be provided from lambs fed the PLS/ algae or fish/algae diets, where a 100-g serving would supply 132 and 179 mg, respectively. Further research is required to determine the eating quality of lamb having these fatty acid profiles. Implications In lambs fed concentrate-based diets, the fatty acid content of the meat can be improved to more closely resemble what is recommended for the human diet as a whole. This alteration in the essential fatty acid profile of sheep meat provides an alternative for nutritionand health-conscious meat eaters. Feeding an unprotected source of α-linolenic acid may not be an effective means of improving the polyunsaturated fatty acid content of sheep meat or increasing the tissue content of eicosapentaenoic and docosahexaenoic acids, with a preformed source of these fatty acids being the preferred option. The combination of marine algae and a rumen-protected source of linseed produced a nutritionally favorable polyunsaturated-to-saturated ratio, with increased levels of eicosapentaenoic and docosahexaenoic acid and an omega-6 to omega-3 ratio similar to lambs finished on grass. Literature Cited AOAC Official Methods of Analysis. 15th ed. Assoc. Offic. Anal. Chem., Arlington, VA. Ashes, J. R., B. D. Siebert, S. K. Gulati, A. Z. Cuthbertson, and T. W. Scott Incorporation of n 3 fatty acids of fish oil into tissue and serum lipids of ruminants. Lipids 27: Brenner, R. R Factors influencing fatty acid chain elongation and desaturation. Pages in The Role of Fats in Human Nutrition. A. J. 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