J. Anim Sci. 2008. 86:3033-3044. doi:10.2527/jas.2007-0533
© 2008 American Society of Animal Science
Effect of feeding high-temperature, microtime-treated diets with different lipid sources on conjugated linoleic acid formation in finishing Hanwoo steers1
C. X. Xu*,
,2,
Y. K. Oh
,2,
H. G. Lee
,
T. G. Kim#,
Z. H. Li*,
J. L. Yin*,
Y. C. Jin*,
H. Jin,
Y. J. Kim||,
K. H. Kim,
J. M. Yeo¶ and
Y. J. Choi*,3
* Laboratory of Animal Cell Biotechnology, School of Agricultural Biotechnology, and
and
Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 130-712, Korea;
and
National Institute of Animal Science, RDA, Suwon 441-706, Korea;
and
School of Bio-Resources, PNU-Special Animal Biotechnology Center, Pusan National University, Gyeongnam 627-706, Korea;
and
# Agribrands Purina Korea Inc., Seongnam 463-808, Korea;
and
|| Department of Food Science & Biotechnology, Korea University, Chochiwon, Chungnam 339-700, Korea; and
¶ Korea National Agricultural College, RDA, Suwon, 441-706, Korea
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Abstract
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The present study was conducted to examine the effects of different plant oils or plant oil mixtures and high-temperature, microtime processing (HTMT) on the CLA content in Hanwoo steers. Experiment 1, consisting of 3 in vitro trials, was conducted to determine how the biohydrogenation of C18 fatty acids and CLA production were affected by fat sources (tallow, soybean oil, linseed oil, or mixtures of soybean oil and linseed oil) or HTMT treatment in the rumen fluid. The results showed that HTMT was capable of protecting unsaturated fatty acids from biohydrogenation by ruminal bacteria. The HTMT-treated diet containing 4% linseed oil (LU) and a supplement containing 2% linseed oil and 1% soybean oil treated with HTMT + 1% soybean oil (L2S1U+S1) produced an increased quantity of trans-11 C18:1 and cis-9, trans-11 CLA, and a reduced quantity of trans-10, cis-12 CLA. Based on these results, in vivo studies (Exp. 2) were conducted with LU and L2S1U+S1. These 2 treatments increased the content of cis-9, trans-11 CLA in LM compared with the control diet. The content of trans-10, cis-12 CLA in subcutaneous fat was also increased in the L2S1U+S1 treatment compared with other treatments. The subcutaneous fat thickness in the LU treatment was decreased compared with the L2S1U+S1 treatment. The LU treatment significantly decreased fatty acid synthase expression but simultaneously increased leptin expression. In this report, we showed that diets containing LU and L2S1U+S1 were capable of increasing CLA in the intramuscular fat of beef.
Key Words: beef • conjugated linoleic acid • high-temperature microtime treatment • trans-11 C18:1
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INTRODUCTION
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Conjugated linoleic acid is a mixture of positional and geometric isomers of linoleic acid with conjugated double bonds. It occurs naturally, mainly in foods of ruminant origin, and has shown a variety of biological activities in animal studies, including anticancer, antidiabetes, and antiobesity properties (Ha et al., 1987
; Chin et al., 1992; Houseknecht et al., 1998; Blankson et al., 2000). If CLA also has these potential health effects in humans, an increase in the CLA content of beef could enhance its nutritive and therapeutic values.
In ruminant fat, the major isomer of CLA is cis-9, trans-11 CLA, and this can be derived from biohydrogenation (BH) of linoleic acid by rumen microbes. In addition, trans-11 C18:1, an intermediate product of the BH of linoleic or linolenic acid, is important for increasing the cis-9, trans-11 CLA content because it can be transformed to cis-9, trans-11 CLA in ruminant tissues (Bauman et al., 2000). The cis-9, trans-11 CLA content in ruminant fat can be affected by various factors, with the main factor being dietary fat source (Bauman et al., 2000). Feeding soybean oil increased the cis-9, trans-11 CLA content in milk (Dhiman et al., 2000), but it did not increase it in beef (Beaulieu et al., 2002). Processing methods of plant seeds that contain increased quantities of PUFA could also affect the CLA content of ruminant fat (Chouinard et al., 2001). Our previous study demonstrated that 40.2% of the dietary fat was in the bound form with starch in a diet treated with high-temperature, microtime processing (HTMT), and feeding the HTMT-treated diet containing extruded soybeans increased the CLA content in beef (Xu et al., 2006). However, the influence of a high dietary concentrate with plant oil mixtures on the CLA content of beef has not yet been studied. In this study, we examined the effects of different plant oils or plant oil mixtures and the HTMT processing method on CLA content in Hanwoo steers.
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MATERIALS AND METHODS
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All animal-based procedures were in accordance with the "Guidelines for the Care and Use of Experimental Animals of Seoul National University," which was formulated from the "Declaration of Helsinki and Guiding Principles in the Care and Use of Animals."
Exp. 1
Experiment 1, which consisted of 3 in vitro trials, was conducted to determine how CLA production and the BH of C18 fatty acids were affected by fat sources and HTMT treatment in the rumen fluid. A supplement containing 1.8 g of ground concentrate and 0.2 g of ground alfalfa hay was used as the energy and N sources for the inoculum, and 4% (wt/wt) of each fat source was mixed with the supplement. For the HTMT treatment, the supplement mixed with each fat source was heat-treated at 125 to 170°C for 3 to 4 s with a Universal Pellet Cooker (Wenger Manufacturing Inc., Sabetha, KS). The fatty acid compositions of the supplement mixed with each fat source are shown in Table 1.
Experimental Design.
In trial 1 we examined the effects of soybean oil supplementation and HTMT treatment on the production of CLA and trans-11 C18:1 production and on C18 fatty acid BH. The treatments were 1) a supplement containing 4% tallow (T), 2) a supplement containing 4% tallow treated with HTMT (TU), 3) a supplement containing 4% soybean oil (S), and 4) a supplement containing 4% soybean oil treated with HTMT (SU).
Trial 2 was designed to determine the effects of linseed oil supplementation and HTMT treatment on the production of CLA and trans-11 C18:1 and on C18 fatty acid BH. The treatments were 1) T, 2) TU, 3) a supplement containing 4% linseed oil (L), and 4) a supplement containing 4% linseed oil treated with HTMT (LU).
Trial 3 examined the effects of plant oil combinations and HTMT treatment on the production of CLA and trans-11 C18:1 and on C18 fatty acid BH. The oil combination treatments were chosen on the basis of the results obtained from trials 1 and 2. The treatments were 1) LU, 2) S, 3) a supplement containing 3% linseed oil treated with HTMT + 1% (wt/wt) soybean oil (L3U+S1), and 4) a supplement containing 2% linseed oil and 1% soybean oil treated with HTMT plus 1% (wt/wt) soybean oil (L2S1U+S1).
Sampling and Analysis.
Rumen contents were collected 3 h after the morning feeding from a ruminally cannulated Holstein cow fed 4 kg of concentrate and 0.5 kg of alfalfa hay on a DM basis twice daily. The collected rumen contents were strained through 4 layers of cheesecloth under a flux of CO2 to remove the feed particles. Strained rumen fluid was mixed with prewarmed McDougall’s (1948) artificial saliva at the ratio of 1:1 under flushing with CO2. The mixed solution (200 mL) and the supplement were added to a 500-mL conical flask. Incubation times were 3, 6, 12, and 24 h. Three replicates per treatment were incubated. After each incubation time, the solution was freeze-dried and lipids were extracted (Folch et al., 1957). Methylation of the lipids followed the method of Lepage and Roy (1986), and their compositions were determined by GLC (CP-3800, Varian Inc., Palo Alto, CA). Fatty acid methyl esters were separated on a fused-silica capillary column (100 m x 0.25 mm i.d. x 0.20 µm thickness, SPTM-2560, Supelco, Bellefonte, PA) with a 1 mL/min N2 flow. The oven temperature was programmed at 100°C for 2 min, followed by 175°C for 30 min and then 220°C for 20 min. The injector and detector temperatures were maintained at 250°C.
Statistical Analysis.
In trials 1 and 2, data were analyzed as a factorial 2-way arrangement of treatments by using the MIXED procedure (SAS Inst. Inc., Cary, NC). In trial 3, the results obtained were subjected to least squares ANOVA according to the GLM procedure of SAS, and significances were compared by the Student-Newman-Keuls test. Differences were considered significant at P < 0.05.
Exp. 2
Experiment 2, which consisted of 2 in vivo trials, was conducted to determine the effects of dietary fat sources on trans-11 C18:1 and CLA content in the rumen, plasma, and body fat of Hanwoo steers (Korean native cattle). Because trans-10, cis-12 CLA can decrease the intramuscular fat content in beef (Xu et al., 2006), we selected 2 diets from Exp. 1 that produced an increased quantity of cis-9, trans-11 CLA and trans-11 C18:1 and a reduced quantity of trans-10, cis-12 CLA.
Animals and Diets of Trial 1.
Three Hanwoo steers (519 ± 5 kg) fitted with permanent rumen cannulas were used. The animals were fed a diet of 1.8% of BW daily (DM basis), and the concentrate-to-hay ratio in the diet was 9:1. All diets were formulated to meet NRC (1996) nutrient requirements. The chemical composition and fatty acid composition of the concentrate are presented in Tables 2 and 3. The trial was conducted in a 3 x 3 Latin square with 3 dietary treatments and three 7-d periods. The treatments were 1) T (control), 2) LU, and 3) L2S1U+S1.
Sampling and Analysis of Trial 1.
Rumen fluid (100 mL) was collected from 5 different sites in the rumen 10 min before feeding, and at 1, 3, 6, and 9 h after feeding on the last day of each period, and the collected rumen fluid was strained through 4 layers of cheesecloth. The collected solution was freeze-dried and stored at –85°C until analyzed. Samples of blood were taken from a jugular vein [10 mL using heparinized vacutainers (Becton Dickinson, Franklin Lakes, NJ)] 30 min before feeding and at 3, 6, and 9 h after feeding on the last day of each period. The blood was centrifuged immediately at 1,200 x g for 10 min and separated into plasma, and then stored at –85°C until analyzed.
Animals and Diets of Trial 2.
Twenty-seven Hanwoo steers (26 mo old) were randomly divided into 3 groups of 9 animals based on BW (644 ± 12 kg), subcutaneous fat thickness (12.1 ± 0.53 mm), and marbling score (5 ± 0.4). The subcutaneous fat thickness and marbling score were measured by ultrasonography. The diets and the 3 treatments were the same as those used in trial 1. The feeding trial lasted for 2 mo. Steers had free access to concentrate, water, and a mineral block (Tithebarn, Winsford, UK) during the experimental period. In the first month of the trial, steers were fed 0.5 kg of alfalfa hay per steer daily.
Carcass Characteristics and Fatty Acid Analysis.
For determining carcass characteristics and fatty acid composition, LM and subcutaneous fat tissue samples were collected from the 13th rib interface of the left side at 24 h after slaughter. The analysis of carcass characteristics followed the method of the Korean Animal Products Grading Service (http://www.apgs.co.kr/english/html/grades/grade.asp; last accessed Mar. 24, 2007). Samples of LM and subcutaneous fat tissues were ground after freeze-drying and mixed well to obtain subsamples to determine the fatty acid composition.
Adipose Tissue Sampling and Real-Time PCR Analysis.
Samples of adipose tissue were collected from the 13th rib within 5 min after slaughter to analyze the levels of mRNA expression of enzymes associated with lipid metabolism. The collected adipose tissue was snap-frozen in liquid N2 and stored at –85°C until analysis. Total RNA was isolated from adipose tissue using RNeasy Lipid Tissue Mini Kits (Qiagen Co., Hilden, Germany). The real time-PCR technique was used to measure mRNA expression levels for peroxisome proliferator-activated receptor (PPAR) β , PPAR
, fatty acid synthase (FAS), adipocyte fatty acid-binding protein (aP2), lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), perilipin, and leptin. β-Actin was used as an invariant control. The reactions were begun at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The primer sequences for real-time PCR are shown in Table 4.
Statistical Analysis.
Data were analyzed by using the GLM procedure (SAS Inst. Inc.), and significances were compared by the Student-Newman-Keuls test. Differences were considered significant at P < 0.05.
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RESULTS
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Effects of Soybean Oil Supplementation and HTMT Treatment on In Vitro CLA Production and C18 Fatty Acid BH
The composition of C18 fatty acids and production of CLA were affected by soybean oil supplementation and HTMT treatment (Table 5). At all incubation times, trans-11 C18:1; C18:2n-6; cis-9, trans-11 CLA; trans-10, cis-12 CLA; and C18:3n-3 fatty acid proportions were significantly greater for the soybean oil treatment than for the tallow treatment. The content of C18:2n-6 was greater for SU than for S at 6 and 12 h of incubation, and C18:3n-3 was greater for SU than for S at 3 and 6 h of incubation. However, cis-9, trans-11 CLA was less for SU than for S at 3 h of incubation. The content of trans-10, cis-12 CLA in SU was increased compared with S at 12 and 24 h of incubation.
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Table 5. Effect of soybean oil supplementation and high-temperature, microtime (HTMT) processing treatment on C18 fatty acid composition (% of total fatty acids) at different sampling times in culture solution (Exp. 1)
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Effects of Linseed Oil Supplementation and HTMT Treatment on In Vitro CLA Production and C18 Fatty Acid BH
At all incubation times, the contents of cis-9, trans-11 CLA and trans-11 C18:1 with linseed oil supplementation were significantly greater compared with tallow supplementation. The proportion of C18:1 with linseed oil supplementation was greater at 6 h, and the proportion of C18:2n-6 was greater at all incubation times than with tallow supplementation.
The proportions of C18:2n-6 and trans-11 C18:1 were greater in LU than in L at 6 and 12 h of incubation, respectively. The proportion of C18:3n-3 was greater in LU than in L at the 3- and 6-h incubation times. However, cis-9, trans-11 CLA was less with LU at 3 h (Table 6).
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Table 6. Effect of linseed oil supplementation and high-temperature, microtime processing (HTMT) treatment on C18 fatty acid composition (% of total fatty acids) in culture solution (Exp. 1)
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Effects of Mixtures of Soybean Oil and Linseed Oil with HTMT Treatment on In Vitro Ruminal CLA and Trans-11 C18:1 Production
From each of the above 2 trials, we selected 1 supplement that produced a reduced quantity of trans-10, cis-12 CLA and an increased quantity of cis-9, trans-11 CLA and trans-11 C18:1. We then compared the production of CLA and trans-11 C18:1 from these 2 supplements with that from their mixtures (Table 7). Our results showed that the content of trans-11 C18:1 was less for S than for the other treatments at 6, 12, and 24 h of incubation, but the content of trans-10, cis-12 CLA was significantly greater for S than for the other treatments at all incubation times. The content of trans-11 C18:1 tended to be greater for LU than for the other treatments at all incubation times, except for the 3-h incubation. Although there were no significant differences between L2S1U+S1 and L3U+S1 in the content of trans-11 C18:1, the content of cis-9, trans-11 CLA was significantly greater in L2S1U+S1 than in L3U+S1 at 6 and 12 h of incubation.
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Table 7. Effects of mixtures of soybean oil and linseed oil, and high-temperature, microtime processing (HTMT) treatment on the production of trans-11 C18 fatty acid and CLA in culture solution (% of total fatty acids; Exp. 1)1
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Proportions of CLA and Trans-11 C18:1 Fatty Acid in the Rumen Fluid of Hanwoo Steers
From the results of the third in vitro trial, we selected 2 diets that produced a reduced amount of trans-10, cis-12 CLA and increased amounts of cis-9, trans-11 CLA and trans-11 C18:1 for in vivo experiments. Compared with the control treatment, the L2S1U+S1 treatment significantly increased the content of trans-11 C18:1 at all sampling times after feeding, but there were no significant differences in the content of trans-11 C18:1 between L2S1U+S1 and LU, except at 9 h after feeding. The content of cis-9, trans-11 CLA in L2S1U+S1 was significantly increased compared with other treatments at 1 and 9 h after feeding. Although the content of trans-10, cis-12 CLA was significantly greater for L2S1U+S1 than for the other treatments at 3 h after feeding, no significant difference was found afterward (Table 8).
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Table 8. Effects of mixtures of soybean oil and linseed oil, and high-temperature, microtime processing (HTMT) treatment on the production of trans-11 C18 fatty acid and CLA (% of total fatty acids) in the rumen fluid of Hanwoo steers (Exp. 2)1
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Proportions of CLA and Trans-11 C18:1 in Plasma
The plasma content of trans-11 C18:1 was greater for the L2S1U+S1 and LU treatments than for the control treatment at 6 and 9 h after feeding, but no significant difference was observed between the plant oil treatments (Table 9). The trans-10, cis-12 CLA isomer was detected only in the 2 plant oil treatments at 9 h after feeding, and the content of trans-10, cis-12 CLA in L2S1U+S1 was significantly greater than that in LU.
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Table 9. Composition (% of total fatty acids) of CLA and trans-11 C18:1 fatty acid in plasma of Hanwoo steers (Exp. 2)1
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Carcass Characteristics
No significant differences were observed between the treatments in carcass weight, quality grade, firmness, maturity, meat color, and marbling score (Table 10). However, the subcutaneous fat thickness of Hanwoo steers was significantly less for LU than for L2S1U+S1.
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Table 10. Effects of mixtures of soybean oil and linseed oil, and high-temperature, microtime processing (HTMT) treatment on carcass characteristics in Hanwoo steers (Exp. 2)1
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Contents of CLA and Other Fatty Acids in Fat Tissues
The 2 plant oil treatments increased the content of cis-9, trans-11 CLA in LM and subcutaneous fat tissues compared with the control group (Tables 11 and 12). The content of trans-10, cis-12 CLA in both LM and subcutaneous fat tissues was significantly greater for L2S1U+S1 than for the other treatments. The content of trans-11 C18:1 in subcutaneous fat was significantly increased by linseed oil supplementation compared with the control group, but the difference was not found in LM. The contents of palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2n-6) were not significantly affected by plant oil supplementation in any of the tissues examined. However, the content of linolenic acid (C18:3n-3) was significantly increased by the LU treatment in subcutaneous fat and LM compared with the control group. In addition, the increased linolenic acid content caused the total PUFA content in subcutaneous fat to increase. Feeding both oil-supplemented diets also caused significant increases of MUFA in muscle compared with the control group.
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Table 11. Effects of mixtures of soybean oil and linseed oil, and high-temperature, microtime processing (HTMT) treatment on fatty acid composition of subcutaneous fat in Hanwoo steers (g/100 g of fatty acid; Exp. 2)1
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Table 12. Effects of mixtures of soybean oil and linseed oil, and high-temperature, microtime processing (HTMT) treatment on fatty acid composition of LM in Hanwoo steers (g/100 g of fatty acid; Exp. 2)1
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Effects of Fat Sources on mRNA Expression of Enzymes Associated with Lipid Metabolism in Adipose Tissue
There were no significant differences between the treatments in mRNA expression of PPARβ , PPAR, LPL, aP2, perilipin, and HSL, with the exception that leptin expression was significantly increased and FAS expression was significantly decreased by linseed oil supplementation (Figure 1).
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Figure 1. Effects of mixtures of soybean oil and linseed oil, and high-temperature, microtime processing (HTMT) treatment on mRNA gene expression of enzymes associated with lipid metabolism in Hanwoo steers (Exp. 2). Values are mean ± SEM of 7 steers, a,bWithin each item, means not bearing a common letter differ (P < 0.05). LU = 4% linseed oil + HTMT; L2S1U+S1 = 1% soybean oil + HTMT-treated supplement containing 2% linseed oil + 1% soybean oil. PPAR = peroxisome proliferator-activated receptor; FAS = fatty acid synthase; aP2 = adipocyte fatty acid-binding protein; LPL = lipoprotein lipase; HSL = hormone-sensitive lipase.
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DISCUSSION
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In Vitro Ruminal Fatty Acid Composition
Alterations in the PUFA concentration of rumen fluid are directly related to the fatty acid composition of the diet. Consistent with this, our results showed that the concentrations of PUFA in rumen fluid were affected by different fat sources. Supplementation of plant oils increased the content of cis-9, trans-11 CLA compared with tallow supplementation. This may be related to the content of linoleic acid in the plant oils, because cis-9, trans-11 CLA is an intermediate product of linoleic acid BH in the rumen (Kim et al., 2000) and the content was dose-dependent on the supplemented linoleic acid concentration in the rumen (Dhiman et al., 2000). In our study, the linoleic acid content of plant oils, especially soybean oil, was greater than that of tallow.
Trans-11 C18:1 is also an intermediate product from the BH of unsaturated fatty acids in the rumen, and it is an important precursor to the biosynthesis of cis-9, trans-11 CLA in tissue (Griinari et al., 2000). In the rumen, linoleic acid and linolenic acid are the major sources for production of trans-11 C18:1 (Kellens et al., 1986). In our in vitro studies, we also observed increased quantities of trans-11 C18:1 production in the soybean oil and linseed oil treatments.
To increase the content of CLA in ruminant fat, plant oil has been added to ruminant diets (Bauman et al., 2000). However, most of the unsaturated fatty acids in plant oil have been quickly hydrogenated to saturated fatty acids by ruminal bacteria (Beam et al., 2000). Therefore, it is necessary to regulate the rate of hydrogenation of unsaturated fatty acids by rumen bacteria to increase CLA biosynthesis more efficiently; partial inhibition of the BH of linoleic acid can increase cis-9, trans-11 CLA production (Kim, 2003). To protect the unsaturated fatty acids from BH by ruminal bacteria, the most commonly used method is heat treatment. In this study, we used the HTMT method, and the in vitro results showed that the HTMT treatment partially protected the major unsaturated fatty acid of plant oils from BH.
The content of trans-10, cis-12 CLA was significantly increased in the SU treatment at 12 and 24 h of incubation compared with the S treatment. This result might be related to the decreased ruminal pH (data not shown). The trans-10, cis-12 CLA isomer is an intermediate product of linoleic acid BH in the rumen (Kim et al., 2002), and the ruminal pH is an important factor in trans-10, cis-12 CLA biosynthesis in the rumen. Feeding a high-concentrate, low-fiber diet causes low ruminal pH (Griinari et al., 1998), and the rumen concentration of trans-10, cis-12 CLA is dose-dependent on soybean oil concentration in the diet of steers (Beaulieu et al., 2002). However, the content of cis-9, trans-11 CLA was less for SU and LU than for S and L at 3 h of incubation. This result could be related to the rate of linoleic acid release. In the non-HTMT-treated group in which the plant oil was added normally, linoleic acid released into the rumen fluid more quickly and the ruminal pH was nearly neutral (data not shown), thus producing increased concentrations of cis-9, trans-11 CLA.
In linseed oil, the major unsaturated fatty acid was C18:3n-3. The content of C18:3n-3 was greater in the HTMT treatment than in the non-HTMT treatment, and the content of trans-11 C18:1 was increased in the LU treatment compared with the L treatment at 12 h of incubation. The result might be related to the contents of linoleic acid and linolenic acid, because trans-11 C18:1 was an intermediate in the BH of linoleic acid and linolenic acid in the rumen (Harfoot and Hazlewood, 1988).
Between the soybean oil and linseed oil treatments, the production of trans-11 C18:1 was greater for the linseed oil treatment than for the soybean oil treatment. These results may have been caused by the ruminal pH changes. As described above, feeding an increased quantity of concentrate causes low ruminal pH, and the altered rumen environment induces a change in the rumen trans-11 C18:1 profile (Griinari et al., 1998). In this situation, trans-10 C18:1 becomes the predominant trans-11 C18:1 isomer in milk fat. However, feeding an increased quantity of concentrate with linseed oil supplementation increased trans-11 C18:1 in the duodenal flow of cows more than did feeding a reduced quantity of concentrate with linseed oil supplementation (Loor et al., 2004). Supplementation of linseed oil and soybean oil significantly increased the content of milk CLA (Bauman et al., 2000). In this study, we mixed linseed oil and soybean oil at a ratio of 3:1 or 2:2 to examine the effects of mixtures of linseed and soybean oils on CLA production. The linseed oil and soybean oil combinations and their HTMT treatments increased the production of cis-9, trans-11 CLA and trans-11 C18:1. In the oil combination treatments, linoleic acid content was less than in the soybean oil treatment, but the amount of cis-9, trans-11 CLA was similar among the treatments.
In Vivo Experiment
To examine the effects of HTMT treatment and plant oil supplementation on the CLA content in beef, 2 experimental diets were selected on the basis of the results obtained from the in vitro trials. These were LU and L2S1U+S1, which produced increased quantities of trans-11 C18:1 and cis-9, trans-11 CLA, and a reduced quantity of trans-10, cis-12 CLA.
In the in situ trial, we observed results similar to those in the in vitro trial. In general, the plasma fatty acid composition reflected those found in the rumen. The pattern of plasma concentrations of CLA and trans-11 C18:1 was similar to that of the rumen fluid. In steers fed plant oil-supplemented diets, the plasma proportions of trans-11 C18:1 and CLA increased at 6 and 9 h after feeding compared with those in steers fed the control diet.
For cows, feeding soybean oil has been found to increase the cis-9, trans-11 CLA content in milk (Dhiman et al., 2000). However, Beaulieu et al. (2002) reported that for beef, feeding a high-concentrate diet supplemented with soybean oil did not increase cis-9, trans-11 CLA but did increase trans-10, cis-12 CLA. Efforts to enhance the CLA content of beef have focused on improving the output of cis-9, trans-11 CLA from ruminal BH, but in most studies little CLA was derived from the rumen (Duckett et al., 2002). Most of the cis-9, trans-11 CLA was formed in tissue from trans-11 C18:1 (Griinari et al., 2000). The lack of increase in cis-9, trans-11 CLA in muscle tissues with oils rich in linoleic acid could have been due to the low production of trans-11 C18:1 in the rumen (Hristov et al., 2005). Griinari et al. (1998) reported that feeding a high-concentrate diet resulted in a low pH and that trans-10 C18:1 became the predominant trans-11 C18:1 isomer. Therefore, in a high-concentrate feeding system, an increased quantity of linoleic acid supplementation is not the best method for increasing cis-9, trans-11 CLA. Loor et al. (2004) reported that trans-11 C18:1 was increased more efficiently in a high-concentrate feeding system than in a low-concentrate feeding system when the diet was supplemented with linseed oil. In this study, we also observed similar results. Trans-11 C18:1 was increased in subcutaneous fat by feeding a diet containing 4% linseed oil compared with the control diet. In addition, the content of cis-9, trans-11 CLA was significantly increased in both subcutaneous fat and LM by feeding a diet containing linseed oil compared with feeding the control diet. Increasing the content of trans-11 C18:1 in beef is also important, because nonruminants have 
9-desaturase and may synthesize cis-9, trans-11 CLA by using trans-11 C18:1. In fact, Miller et al. (2003) reported that treatment of human mammary (MCF-7) and colon (SW 480) cancer cell lines with 20 mg/mL of trans-11 C18:1 decreased the cell growth, and that trans-11 C18:1 was converted to cis-9, trans-11 CLA.
Feeding an HTMT-treated diet containing linseed oil or oil mixtures did not affect growth performance and carcass characteristics except for subcutaneous fat thickness. Others have reported variable performance responses to supplemental fat. Engle et al. (2000) observed decreases in marbling score, dressing percentage, yield grade, and quality grade when Angus steers were fed a high-concentrate diet supplemented with 4% soybean oil. However, Andrae et al. (2000) reported an increased marbling score and quality grade when Angus steers were fed a diet containing increased quantities of oil corn, although other quality variables were unaffected. The effect of dietary oil on subcutaneous fat thickness is variable, and the differences may be partly due to breed differences. Mir et al. (2002) reported that feeding diets with 6% sunflower oil (high in linoleic acid content) increased the subcutaneous fat thickness in Wagyu x Limousin and Limousin cattle, but decreased it in Wagyu cattle. In this study, subcutaneous fat thickness tended to be greater in Hanwoo steers fed a diet containing an oil mixture (high linoleic acid) than in those fed the control diet (P < 0.06). However, supplementation with linseed oil caused significant decreases in subcutaneous fat thickness compared with the oil mixture. Bernard et al. (2005) reported that feeding formaldehyde-treated linseed oil increased the C18:3n-3 content in goat milk, and LPL activity tended to decrease in the mammary gland of goats. In other animals, similar results have been observed. In chickens, dietary linseed oil produced less abdominal fat deposition (Crespo and Esteve-Garcia, 2002). In rats, dietary linolenic acid decreased FAS activity (Ikeda et al., 1998) and increased lipolysis (Larking and Nye, 1975).
To explain the effect of linseed oil supplementation on the decrease in subcutaneous fat thickness, we selected several enzymes associated with lipid metabolism and examined their mRNA expression in subcutaneous fat tissue. Peroxisome proliferator-activated receptor β and PPAR stimulate adipocyte differentiation, and aP2, LPL, FAS are key enzymes in adipocyte lipogenesis. In this study, oil supplementation did not affect PPARβ , PPAR, aP2, and LPL expression, but FAS expression was less for the linseed oil treatment than for the other treatments. Kim et al. (2003) observed that an increased quantity of linolenic acid supplementation significantly reduced mRNA expression and the activity of FAS in rats. Ikeda et al. (1998) also observed similar results in rats, and they reported that linolenic acid inhibited FAS activity more efficiently than did linoleic acid.
We also examined other proteins associated with lipolysis, including HSL, perilipin, and leptin. Hormone-sensitive lipase is a key enzyme in adipocyte lipolysis that stimulates lipolysis. In contrast, perilipin coats the surface of the triglyceride drop, protecting triglyceride lysis from HSL (Holm, 2003). Leptin also has an important role in the regulation of body fat mass. It is a hormone secreted from adipose tissue, and it induces adipocyte apoptosis (Della-Fera et al., 2001). In this study, expression of HSL and perilipin was not affected by linseed oil supplementation, but leptin expression was significantly increased by linseed oil supplementation.
In summary, the HTMT treatment partially protected the major unsaturated fatty acids from BH by rumen microbes, and the HTMT-treated diet containing linseed oil or the plant oil mixture increased the proportion of cis-9, trans-11 CLA in the rumen, blood, and body fat compared with the control diet in Hanwoo steers. Similar to other reports dealing with feeding systems using an increased quantity of concentrates, trans-10, cis-12 CLA in the rumen, blood, and body fat was increased more efficiently by soybean oil supplementation, and trans-11 C18:1 was greater with linseed oil supplementation than with the control diet. Oil supplementation did not affect most carcass characteristics. Subcutaneous fat thickness was decreased by feeding a supplement containing 4% linseed oil, partially through increasing leptin expression and decreasing FAS expression in subcutaneous fat tissue.
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Footnotes
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1 This study was supported by the National Institute of Animal Science (NIAS), Korea, and in part by the Brain Korea 21 program. 
2 These authors contributed equally to this work.
3 Corresponding author: cyjcow{at}snu.ac.kr
Received for publication August 21, 2007.
Accepted for publication June 2, 2008.
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Abstract
INTRODUCTION
