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ANIMAL NUTRITION |
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* Animal Nutrition, Management, and Welfare Group, Unitat de Remugants-IRTA (Institut de Recerca i Tecnologia Agroalimentàra), Barcelona, 08193, Spain;
and
Unitat de Química Alimentària-IRTA, Girona, 17118, Spain;
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Unitat de Qualitat de la Canal i Carn-IRTA, Girona, 17118, Spain; and
and
ICREA (Institució Catalana de Recerca i Estudis Avançats), Barcelona, 08010, Spain
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: beef • conjugated linoleic acid • omega-3 • linseed • rumen
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Krauss et al., 2000; Lee and Lip, 2003) and play important biological functions, particularly in inflammation, brain development, sight, and immune function (Connor, 2000). The American Heart Association (Krauss et al., 2000) recommended a reduction in the consumption of SFA and an increase in the consumption of unsaturated FA, mainly n-3, to obtain a ratio of dietary n-6:n-3 FA of 4.0 or less.
The ratio of n-6:n-3 FA can be improved by decreasing n-6 FA consumption, increasing n-3 FA consumption, or both. More recently, Wijendran and Hayes (2004) have described the importance of providing a ratio of n-6:n-3 FA close to 6.0 in human diets but have emphasized that the first consideration when contemplating long-term consumption of FA should be the absolute amounts of n-6 and n-3 consumed, rather than their ratio. In that regard, Wijendran and Hayes (2004) recommended 1.7 g/d of linolenic acid based on the reduction of platelet aggregation in hyperlipidemic subjects reported by Freese et al. (1994).
To enrich beef with n-3 FA, the dietary supply of n-3 FA must escape rumen biohydrogenation (which converts unsaturated FA to SFA) before it can be absorbed in the small intestine and deposited in meat. One strategy to avoid rumen biohydrogenation is to feed whole oilseeds, because the seed coat prevents the access of rumen microorganisms to the unsaturated FA (Aldrich et al., 1997). The n-3 FA content of muscle has been increased in late-maturing breeds of cattle by feeding forage-based diets supplemented with oils or oilseeds rich in cis-9, cis-12, cis-15-18:3 (ALA), EPA, or DHA (Choi et al., 2000; Scollan et al., 2001a; Raes et al., 2004b). However, there are no studies conducted with cattle of early-maturing breeds and less than 12 mo of age.
The objective of this study was to assess the possibility of enriching the concentration of n-3 FA, especially ALA, and to improve the ratio n-6:n-3 FA in meat from young Holstein bulls fed high-concentrate diets using whole linseed.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Fifty-four Holstein bulls were blocked into 3 BW groups (274, 295, and 329 kg) and randomly assigned to 1 of 6 dietary treatments following a 3 x 2 factorial arrangement. The 6 treatments consisted of 3 concentrate lipid levels, 5, 8, and 11% of DM, and 2 lipid sources, whole canola seed and whole linseed. Whole linseed was chosen because it is an oilseed rich in n-3 FA (54.2% ALA) and its seed coat might protect PUFA from rumen biohydrogenation and increase passage of PUFA to the duodenum (Scollan et al., 2001b). Whole canola seed was chosen as a negative control because it is also a seed coat-protected oilseed rich in PUFA but poor in n-3 FA (10.6% ALA).
All concentrate ingredients were ground, with the exception of the oilseeds that were included as whole seeds. The 6 concentrates were isonitrogenous and isocaloric (Table 1) but differed in FA profile, mainly due to differences in 16:0, 18:0, cis-9-18:1, cis-9, cis-12-18:2 (LA), and ALA (Table 2). To ensure that the concentrates were isocaloric, the increase in lipid level was counterbalanced by a decrease in nonfibrous carbohydrates (NFC), mainly by reducing corn meal. Bulls were fed the concentrate in a trough (0.6 x 2.65 m) and were fed barley straw (3.5% CP, 1.6% EE, 70.9% NDF, and 6.1% ash, on a DM basis) in a separate trough (0.6 x 1.2 m), both ad libitum, until reaching the target slaughter weight of 440 kg. Bulls were housed in outdoor paved and partially covered 13.65 x 3.85-m pens (3 bulls/pen) at the IRTA experimental station (Prat de Llobregat, Spain).
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, 4 mL of ruminal liquid were mixed with 1 mL of a solution containing 0.2% (wt/wt) mercuric chloride, 2% (wt/wt) orthophosphoric acid, and 4-methylvaleric acid (internal standard) in distilled water, and frozen until subsequent VFA analysis.
Bulls were transported to the slaughterhouse when they achieved the target slaughter weight of 440 kg. Immediately after slaughter, HCW was recorded, and carcass backfat and conformation were graded according to the EU classification system into 1.2.3.4.5 (EU Regulation No. 1208/81) and into (S)EUROP categories (EU Regulation No. 1208/81, 1026/91), respectively. Dressing percent was calculated from HCW. After 24 h of carcass chilling, carcasses were weighed again, ribbed at the sixth and the eighth ribs, and the resulting rib sample was collected. The pH of the LM at the sixth rib was measured in the center of the LM, and the area of the LM was determined by artificial vision (Pomar et al., 2001). Colorimeter values of LM (L*, a*, and b*) were measured with a Minolta CM-2002 (Minolta Co. Ltd., Osaka, Japan) between the sixth and the seventh ribs. Before reading, ribs were kept in dark and cold conditions (4°C) for 15 min. The LM from the sixth to the eighth ribs was dissected, and a 200-g sample was frozen at –20°C until analysis of fat content and FA composition.
Chemical Analyses
Feed samples were analyzed for DM (24 h at 103°C), ash (4 h at 550°C), CP by the Kjeldahl method (AOAC, 1995), NDF according to Van Soest et al. (1991) using sodium sulfite and alpha-amylase, and fat by Soxhlet with previous acid hydrolysis (AOAC, 1995). The NFC content was calculated as 100% minus the sum of ash, CP, NDF, and fat. Rumen VFA concentration was analyzed with a polyethylene glycol, terephtalic acid-treated capillary column (ID 25 m x 0.25 mm, 0.25-µm film thickness, BP21, SGE, Europe Ltd., Barcelona, Spain) using GLC (CE 5300 HT, Carlo Erba Instruments chromatograph, Milano, Italy) with an initial temperature of 100°C for 1 min, which was increased 8°C/min to 160°C, and was then held at 160°C for 5 min. The injector and flame ionization detector temperatures were 250 and 280°C, respectively. The carrier gas was He at 30 cm/sec, and the injection was performed by split mode at a ratio of 1:30.
The LM sample of 200 g was ground using a food processor (Robot coupe Blixer3, Montceau Les Mines, France), and a subsample of 10 g was used to determine intramuscular fat content by Soxhlet with a previous acid hydrolysis (AOAC, 1995). Intramuscular fat content data were expressed as grams of fat/100 g of muscle. Another subsample of 2 g was used to determine FA composition. Fat was extracted as described by Folch et al. (1957); the 2-g subsample was homogenized in 100 mL of 2:1 (vol:vol) chloroform:methanol. After 24 h, the mixture was filtered and reextracted twice in a separatory funnel. The filtrate was mixed at a ratio of 2.5:1 with 10% NaCl (vol/vol) and 2 mg of internal standard (15:0) to quantify individual FA. After 24 h, the layer-containing lipid in chloroform was decanted and dried in a rotary evaporator at 40°C. Chloroform remaining was evaporated with a N2 stream.
Fatty acids were separated and quantified as FA methyl esters (FAME) prepared by the AOAC (1990) method. The extracted fat was mixed with 1 mL of 1 M KOH and 1 mL of 14% (wt/vol) trifluoride boron in methanol. The sample was methylated by incubation at 100°C for 60 min and, after cooling to room temperature, was extracted with 5 mL of hexane. The FAME in the hexane layer were analyzed by GLC (5890 Series II GC, Hewlett Packard, S.A., Barcelona, Spain). All samples were methylated in duplicate, and 0.2 µL was introduced by split injection into a fused silica capillary column (30 m x ID 0.25 mm, BPX 70; 0.25-µm film thickness, Barcelona, Spain). Helium was the carrier gas at 30 cm/sec. Column temperature was initially 150°C for 1 min, was increased by 4°C/min to 200°C, and was then held at 200°C for 10 min. Individual FAME were identified by retention time with reference to FAME standards (lipid standard: FA methyl ester mixture #189-19 L-9495; Sigma Chemical Co., St. Louis, MO). The cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers were identified with reference to methyl esters of CLA (O-5507, Sigma-Aldrich, St. Louis, MO). The EPA reference (44864) was obtained from Fluka Industriasse (Zurich, Switzerland).
Statistical Analyses
Average daily gain and rumen data were analyzed using mixed-effects ANOVA with repeated measures (SAS Inst. Inc., Cary, NC). The statistical model included block (initial BW), lipid level, lipid source, the interaction between lipid level and lipid source, time, and the interaction of time with lipid level, time with lipid source, and time with lipid source and lipid level as fixed effects, and bull nested within pen as a random effect to account for any potential dependencies between animals within pen. Time was considered a repeated factor, and the interaction of bull with pen nested within the interaction of lipid level and lipid source (the error term) was subjected to 3 variance-covariance structures: compound symmetry, unstructured, and autoregressive order 1. The variance-covariance matrix that yielded the smallest Schwarz’s Bayesian criterion was considered to be the most desirable structure. The linear and quadratic effects of lipid level were evaluated using contrasts. Consumption of concentrate and straw and G:F were analyzed as described above, but pen was the random effect. Carcass quality characteristics, meat fat content, and FA composition were analyzed as described for ADG without the time effect (because there were no repeated measures) and with pen as a random effect.
Because bulls were slaughtered on 5 different days (depending on BW), days on feed were analyzed as a discrete variable using mixed-effects logistic regression analysis with initial BW, lipid level, lipid source, and the interaction between lipid level and lipid source as fixed effects, and pen as a random effect.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Intake and Animal Performance
Daily concentrate intake (7.2 kg of DM/d) and total DMI (8.2 kg of DM/d) were not affected by lipid level or lipid source (Table 3). To our knowledge, there are no studies that report DMI of young bulls consuming a diet with fat levels near 9% of DM (accounting for the consumption of concentrate and straw), but increasing lipid level (3 to 8% DM) does not consistently decrease intake in growing cattle (Chilliard, 1993). Other studies have not reported any negative effects on feed intake when supplementing beef diets with linseed (Choi et al., 2000; Scollan et al., 2001a; Raes et al., 2004b) or canola seed (Hussein et al., 1995) at lower inclusion rates than in our study. From our study, it can be concluded that this high level of dietary fat, when included as whole seed, has no detrimental effect on DMI.
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Average final BW was 443 kg, and it was achieved after 105 ± 4 d of feeding without differences among treatments. Lipid source did not affect ADG in agreement with other studies conducted with finishing beef fed linseed (Choi et al., 2000; Scollan et al., 2001a; Raes et al., 2004b) or canola seed (Rule et al., 1994; Hussein et al., 1995). However, bulls fed the highest lipid level tended (P = 0.07) to grow less (1.27 kg/d) than the bulls fed the low (1.37 kg/d) or the medium (1.42 kg/d) lipid level. The G:F was not affected by treatment.
Ruminal Fermentation
Average rumen pH was greater (P < 0.01) when bulls were fed the high lipid level (6.49) than when they were fed the medium (6.26) or low (6.20) lipid level (Table 4). As expected, total rumen VFA concentration decreased linearly (P < 0.05) with lipid level (Table 4), probably as a consequence of the decrease in NFC content as the lipid level in the concentrates increased (Table 1
). The inverse relationship between NFC and lipid level in the concentrates also could have affected the acetate to propionate ratio in the rumen (Table 4). The acetate to propionate ratio was greater (P < 0.01) in bulls fed the high (2.76) and the medium (2.67) lipid levels than the low (1.99) lipid level. Rumen molar proportion of n-butyrate was lower (P < 0.05) in bulls fed linseed (7.4 mol/100 mol) than in bulls fed canola seed (13.0 mol/ 100 mol).
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. Dietary treatments did not affect HCW (233.3 kg), dressing percent (52.6%), backfat (71% classified as 3, data not shown), conformation (94% classified as 0, data not shown), meat pH (5.53), and LM colorimetric responses (39.1, 13.5, and 4.3, for L*, a*, and b*, respectively). These results are in agreement with those reported by other authors (Choi et al., 2000; Scollan et al., 2001a; Raes et al., 2004b) comparing the effect of linseed supplementation with other fat sources on carcass characteristics. However, cold carcass weight tended (P = 0.10) to decrease in bulls fed the high (224.6 kg) compared with bulls fed the medium (230.6 kg) or the low (230.4 kg) lipid level. Felton and Kerley (2004) observed that HCW tended to decrease as whole raw soybean inclusion level increased from 0 to 24% in steers fed high-concentrate diets; however, in a second experiment this effect was not observed, and the authors concluded that negative performance effects of whole raw soybeans at inclusion levels of 24% seemed to be minimal.
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Fatty Acid Composition of the LM
Fatty acid profile and content of LM are presented in Tables 6 and 7, respectively. Several FA (10:0; cis-10-15:1; trans-10, cis-12-18:2; cis-8, cis-11, cis-14-20:3, cis-13, cis-16-22:2; DHA; 23:0; and cis-15-24:1) were not detectable or detected at <0.01% of total FAME.
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). However, major FA in the concentrates (Table 2) were cis-9-18:1 (22.1 to 49.5%) followed by LA (from 25.8 to 45.8%) and ALA (from 3.5 to 38.4%). Differences between concentrate and LM in 18:0, LA, and ALA proportions suggest that unsaturated dietary FA had undergone extensive biohydrogenation by rumen microorganisms, although 18:0 is also synthesized de novo by elongation of 16:0 (Enser, 1984) or originates from ruminal biohydrogenation of C18 unsaturated FA (Sauvant and Bas, 2001). In our study, it was expected with bulls consuming high-concentrate diets that ruminal pH would be near 5.5, and in consequence rumen biohydrogenation would be reduced (Martin and Jenkins, 2002). However, despite the fact that bulls consumed a high-concentrate diet, the average ruminal pH was >6.0, and consequently extensive rumen biohydrogenation could be expected.
The proportion and content of 8:0 and the content of 12:0 in LM were greater (P 
0.05) in bulls fed linseed than in bulls fed canola seed. The content of 13:0 and 17:0 in LM decreased (P < 0.05) with lipid level. The 16:0 proportion in LM tended (P = 0.09) to be greater in bulls fed the low (23.3%) or the medium (23.2%) lipid level than in those fed the high lipid level (21.9%). In the liver and in the adipose tissue, the major FA synthesized de novo is 16:0 (Enser, 1984), although a portion of 16:0 typically is of dietary origin. In our study, the proportion of de novo FA (8:0, 12:0, 13:0, 14:0, cis-9-14:1, and 16:0) in LM was lower (P < 0.05) in the bulls fed the high lipid level (24.3%) than in those fed the low (26.1%) and the medium (25.9%) lipid levels, suggesting less de novo FA synthesis in the rumen and in the adipose tissue when high levels of fat were included in the diet. In agreement with our study, an apparent reduction in the synthesis of de novo FA 16:0 has been reported when feeding unsaturated lipids to ruminants (AbuGhazaleh et al., 2002; Martin and Jenkins, 2002; Ueda et al., 2003).
The proportion of 18:0 in LM increased linearly (P < 0.05) with lipid level and tended (P = 0.06) to be greater in canola seed (19.6%) than in linseed treatments (18.1%). An interaction between lipid level and lipid source in the percentage of 18:0 was observed (P < 0.01). The percentage of 18:0 in LM of bulls fed canola seed increased linearly with lipid level, whereas in bulls fed linseed the percentage of 18:0 remained constant. The greater percentage of 18:0 in LM of bulls fed canola seed than in those fed linseed could indicate that rumen biohydrogenation might have been greater in bulls fed canola seed than in bulls fed linseed.
The proportion of trans-9, trans-12-18:2 in LM increased quadratically (P < 0.01) with lipid level and was nearly double (P < 0.01) in those bulls fed linseed (0.40%) compared with those fed canola seed (0.22%). Also, the LM trans-9, trans-12-18:2 content increased markedly (P < 0.01) in the bulls fed linseed (6.7 mg/100 g of LM) relative to those fed canola seed (4.0 mg/100 g of LM). The content of cis-9, trans-11-18:2 in LM tended (P = 0.06) to be greater in the bulls fed linseed (6.3 mg/100 g of LM) compared with those fed canola seed (4.9 mg/100 g of LM). The lower content of the biohydrogenation intermediate FA (cis-9, trans-11-CLA and trans-9, trans-12-18:2) further reinforces the hypothesis that biohydrogenation of unsaturated FA to SFA was greater in bulls fed canola seed than in those fed linseed. Therefore, the results from our study indicate that physical treatments such as crushing, bruising, or extrusion are not necessary to ensure rumen microorganism accessibility to unsaturated FA contents of linseed. It is generally assumed that rumen lipolysis and biohydrogenation in low-roughage diets is lower than in high-roughage diets (Gerson et al., 1985; Sackmann et al., 2003), suggesting that the main rumen biohydrogenating bacteria are cellulolytic. In our study, straw intake was greater in bulls fed canola seed than in those fed linseed, which could have contributed to the apparently greater rumen biohydrogenation in bulls fed canola seed than in bulls fed linseed. In addition, recent studies indicate that diets containing seeds rich in LA and ALA stimulate the production of trans11-18:1 and cis-9, trans-11-CLA in the rumen (Chichlowski et al., 2005). Moreover, several authors (Bauman et al., 1999; Beaulieu et al., 2002; Raes et al., 2004a) reported that the major source of cis-9, trans-11-CLA is desaturation of endogenous trans-11-18:1 in the adipose tissue by the enzyme 
9 desaturase. Similar to our study (Table 6), Raes et al. (2004a) feeding Belgian Blue bulls with 17% extruded linseed and 6.80% crushed linseed reported cis-9, trans-11-CLA values of 0.38 g/100 g of total FA.
The content of LA and total n-6 tended (P = 0.06) to be greater in bulls fed linseed (216 and 229 mg/100 g of LM, respectively) than in bulls fed canola seed (174 and 185 mg/100 g of LM, respectively). However, Scollan et al. (2001a) and Raes et al. (2004b) did not observe an increase of n-6 intramuscular content when supplementing 20 or 2.6% linseed, respectively. The differences in LA and total n-6 content in LM observed in our study could be attributed to rumen biohydrogenation of ALA to LA (van de Vossenberg and Joblin, 2003).
Proportions and contents of ALA, EPA, and total n-3 in LM were greater (P < 0.05) in bulls fed linseed than in those fed canola seed, and all increased (P < 0.05) with lipid level. Furthermore, an interaction (P < 0.05) between lipid source and lipid level in the proportion of ALA, EPA, and total n-3 was observed. In bulls fed linseed, the ALA and n-3 proportions and contents in LM increased linearly with lipid level, whereas in bulls fed canola seed they remained constant. These results are in agreement with those reported by other authors (Choi et al., 2000; Scollan et al., 2001a; Raes et al., 2004b).
The ratio of PUFA to SFA tended (P = 0.07) to be greater for linseed-supplemented bulls (0.36) than for those receiving canola seed (0.30). In all treatments, the ratio of PUFA to SFA was around 0.4 and was greater than values reported for feedlot beef cattle (Enser et al., 1996; Rule et al., 2002). The n-6 to n-3 ratio was lower (P < 0.01) in the bulls fed linseed (10.0) than in the bulls fed canola seed (26.0), and decreased (P < 0.05) with dietary lipid level. In bulls fed the high linseed treatment, the n-6 to n-3 ratio was close (6.3) to the 4.0 ratio recommended by Simopoulos (1999) for human health. Furthermore, the amount of ALA in beef increased markedly when linseed was fed, which would help in meeting the daily consumption of ALA recommended by Wijendran and Hayes (2004).
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: alex.bach{at}irta.es
Received for publication November 2, 2005. Accepted for publication June 22, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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