Effects of conjugated linoleic acid supplementation on growth , carcass characteristics and fatty acid profiles of muscle and fat in growing-finishing pigs *

This study investigated the effects of dietary supplementation of conjugated linoleic acid (CLA) on the growth performance, carcass characteristics, muscle quality and fatty acid compositions in the skeletal muscle and backfat of growing-finishing pigs from 39.5 to 108.0 kg. Forty-eight pigs were randomly allotted to four diets containing 0 (control), 0.5, 1.0 or 1.5% of CLA mixture (35.2% cis-9, trans-11 and 37.5% trans-10, cis-12) for 12 weeks. The results showed that dietary CLA supplementation did not affect the animal’s growth performance and carcass characteristics, whereas significantly increased dry matter content of the Longissimus dorsi muscle (P<0.05). The CLA supplementation also increased the content of saturated fatty acids, but decreased (P<0.05) the content of unsaturated fatty acids (UFA) in both the backfat and Longissimus dorsi muscle. The CLA concentrations in the Longissimus dorsi muscle and backfat were increased linearly by the CLA supplementation in a dose-dependent manner. However, CLA cis-9, trans-11 and CLA trans10, cis-12 showed different deposition efficiencies in pigs. * Supported by Hunan Science and Technology Major Projects, Grant No. 2009FJ1005, and the Knowledge Innovation Program (KIP) Important Projects of The Chinese Academy of Science, Grants No. KSCX-YW-N-49 and KZCX2-YW-T07 4 Corresponding author: e-mail: zltan@isa.ac.cn 5 The authors wish it to be known that, the first two authors should be regarded as joint First Authors Journal of Animal and Feed Sciences, 20, 2011, 171–185 172 CLA PIG PERFORMANCE AND CARCASS CHARACTERISTICS


INTRODUCTION
Conjugated linoleic acid (CLA) refers to a group of positional and geometric (cis or trans) isomers of linoleic acid.It has been initially isolated and identified as an anticarcinogenic agent from fried ground beef (Ha et al., 1987).In recent years, CLA has been reported to have a wide range of biologically beneficial effects, including anticarcinogenic, antiobesity, antiatherogenic and immunomodulatory functions, and lean body mass promotion (Azain, 2003;Pariza, 2004;Bhattacharya et al., 2006).
Food products derived from ruminants are the richest natural sources of CLA (Tanaka, 2005).Researchers have demonstrated that dietary alterations of both ruminant and non-ruminant animals could increase the unsaturated fatty acid and CLA levels of meat, thereby improve its potential health benefits (Boles et al., 2005).The only plausible way to increase muscle CLA content effectively would be to provide chemically synthesized CLA as a dietary supplement in pigs (Joo et al., 2002).Thus, feeding pigs with CLA has been suggested as a potential strategy for obtaining meat products enriched with CLA (Schmid et al., 2006).
An important issue is the effect of dietary CLA on growth traits and fatty acid profiles.However, the results on this issue are inconsistent.Some researchers have shown the improvement in growth rate and carcass traits of pigs when fed diets enriched in CLA (Thiel-Cooper et al., 2001;Weber et al., 2006), but other studies did not find any significant responses (Ramsay et al., 2001;Tischendorf et al., 2002;Corino et al., 2003).On the other hand, few studies on the effects of dietary CLA on growth performance and fatty acids compositions have been conducted in pigs throughout grow-finish phase.
Thus, the present study was therefore designed to evaluate the influence of dietary levels of CLA supplementation on growth rate, carcass characteristics and fatty acid profiles in skeletal muscle and subcutaneous fat, and to determinate the optimum dietary CLA dose for growing-finishing pigs.

Animals and diets
The use of the animals and the experimental procedure was approved by the Animal Welfare Committee of the Institute of Subtropical Agriculture, the Chinese Academy of Sciences.
Forty-eight Landrace × Large White pigs with the initial weight of 39.5 ± 1.7 kg were used for the study and randomly assigned to four dietary treatments in 12 animals with each.The animals were housed in individual pens, four piglets per pen (two barrows and two gilts in each pen), so each treatment had triplicates.Fresh water was accessed ad libitum (nipple drinkers).
The basal diet was composed to meet the nutrient requirements according the NRC (1998) standard and was purchased from a commercial feed company (Liuhe Ltd., Qingdao, China).Chemical composition and nutrient contents of the basal diet are shown in Table 1.The four diets were prepared by the basal diet supplemented with four levels of CLA (0.0, 0.5, 1.0 and 1.5%) (Institute of Geographic Sciences and Natural Resources Research, CAS, China).The CLA compound contained 72.[7][8][9].The animals were fed twice at 08.00 and 18.00.The whole experimental period lasted for 13 weeks, consisting of one week of adaptation and 12 weeks of feeding period.Individual liveweight of pigs was recorded at the end of the experiment and used to calculate daily weight gain (ADG) over the experimental period.Pigs were slaughtered when their average weight reached 108 kg.

Sample collection and chemical analysis
At the end of the experiment, two animals (one barrow and one gilt) from each pen were randomly selected for slaughter, thus there were six animals slaughtered for each treatment.Pigs were electrically stunned and followed exsanguinations.The carcass was scalded, for easy removal of hair, and then eviscerated.Hot carcass weight was recorded.The dressing percentage was calculated as hot carcass weight as a percentage of the final liveweight.
The left loin starting at the third from last thoracic vertebra and extending back to 30 cm was removed from the carcass within 0.5 h post slaughter, and taken to the laboratory immediately.The M. longissimus dorsi sample was taken to measure chemical composition: dry matter, ash, crude protein and crude fat (ether extract); the analyses were performed according to AOAC (2000) methods.
Samples of subcutaneous backfat and Longissimus dorsi muscle of about 10 cm length ended at the third from last thoracic vertebra on the left side of the carcass were taken, frozen in liquid N 2 , and stored at -80°C for fatty acid analysis.On the analysis the samples of the muscle (about 2 g) and backfat (about 1 g) were hemogenated.Then 10 ml of extraction solution (chloroform:methanol, 1:1, vol/vol) and 3 ml distilled water were added and the extraction was allowed for 2 h at room temperature.After vacuum filtration, the residue was rinsed with 10 ml of chloroform and then filtered again.The filtered solution was pooled and centrifuged at 5500 g for 10 min.The upper layer (methanol and water layer) was removed and the bottom layer (chloroform layer containing lipid extracts) was sucked into a test tube.The tube was placed in a water bath at 37°C for evaporating chloroform under nitrogen stream.Then the extract was stored at -40°C until analysis of fatty acids.
Fatty acids in the extract were analysed as fatty acid methyl esters (FAME) by gas chromatography.Briefly, 2 mg of the extract was placed in a glass vial, and then 1.5 ml hexane, 100 µl methyl acetate and 100 µl sodium methoxide were added in order, and thoroughly mixed.The mixture was kept for 20 min at 20°C, and then 10 min at -20°C in a refrigerator.Afterward, 60 µl of oxalic acid was added quickly, and the vial was vortexed.After centrifuging at 5500 g for 10 min the supernatant was added with Na 2 SO 4 to absorb moisture and stored for gas chromatography analysis.
The FAMEs was analysed by using an Agilent 6890N gas chromatographer, equipped with a flame ionization detector (Agilent Technologies, Palo Alto, CA, USA).A CP-Sil 88 fused silica open tubular capillary column (100 m×0.25 nm) (Chrompack, Bridgewater, NJ, USA) was used.The oven temperature started at 45°C for 4 min, thereafter, it was raised to 175°C at 13°C /min, held at 175°C for 27 min and then increased to 215°C at 4°C/min and then held at 215°C for 35 min.
The injector and detector temperatures were set at 250°C.Carrier gas was hydrogen at a flow rate of 30 ml/min.Identification of individual FAMEs was accomplished by the retention times of an authentic standard.Four specific isomers of CLA (cis-9, trans-11; trans-10, cis-12; cis-9, cis-11; and trans-9, trans-11; Matreya LLC, USA) were identified.The concentration of individual fatty acids was quantitated according the peak area, and expressed as proportion to total fatty acids detected.

Statistical analysis
Statistical analysis of experimental data was performed by using the GLM procedure of SAS (1999).Dietary treatment was treated as a factor.For growth rate of pigs, there were three replicates (i.e. three pens).As for carcass traits and fatty acids, there were six replicates which came from six pigs slaughtered.Significant level of the treatment was set P<0.05, and significance of differences between the treatment means was tested using Duncan's test.A contrast analysis was also done to compare the linear (L) or quadratic (Q) response to CLA inclusion levels.

RESULTS AND DISCUSSION
Animal's growth performance, carcass traits and chemical composition of Longissimus dorsi muscle are shown in Table 2.The average feed intake and ADG were not affected by the inclusion of CLA (P>0.05).Similarly, Ramsay et al. (2001), Tischendorf et al. (2002) and Weber et al. (2006) found average daily gain of pigs was not influenced by feeding CLA.On the contrary, Thiel-Cooper et al. ( 2001) reported a linear increase in average daily gain in response to the level of CLA in the diet.The hot carcass weight and dressing percentage were not affected by dietary CLA level (P>0.05),which is in agreement with the results of Cordero et al. (2010).There were also no significant effects (P>0.05) of dietary CLA on the content of crude protein, ether extract and ash in the Longissimus dorsi muscle, but the dietary supplementation of CLA increased (P<0.05)dry matter content in L. dorsi muscle.Similar results are found by Migdal et al. (2004) where a significant increase in dry matter content in meat occurred in pigs fed 2% CLA.
In this study, there was a numerically higher intramuscular fat content in the Longissimus dorsi muscle due to dietary CLA supplementation.Higher intramuscular fat content in L. dorsi has also been observed with dietary CLA supplementation (Joo et al., 2002;Martin et al., 2008).However, Bee (2001), Weber et al. (2006) and Corino et al. (2008) reported that CLA supplementation to pigs did not affect intramuscular fat content in pork.Although no significant influence of dietary CLA supplementation on intramuscular fat content was observed, the higher intramuscular fat content appeared to contribute to higher dry matter content in the L. dorsi muscle of the CLA groups.
The effects of dietary CLA on growth performance of the animals, carcass and meat quality were not completely consistent with other studies.This might be due to the differences in the genetics, the isomeric composition of CLA, the concentration and duration of supplementation of CLA, or CLA × genetic population interaction (Mersmann, 2002;Corino et al., 2003).
The amounts of CLAcc (cis8, and total CLA in the backfat linearly increased (P<0.05) with the dietary CLA level.The concentration of total CLA in the backfat (3.11%) of 1.5% CLA group was more than two times (P<0.05) of the control group (1.06%).
The overall effects of dietary CLA supplementation on fatty acid composition in the backfat were similar to that in Longissimus dorsi muscle.The CLA supplementation resulted in greater SFA contents in the backfat, but lesser MUFA and UFA contents (P<0.05),therefore higher SFA/UFA (P<0.05)ratios than those of the control group.Adding CLA to pig diets increased belly fat firmness associated with an increased ratio of SFA:UFA (Eggert et al., 2001).Soft bellies can cause some difficulty during processing, decrease appearance and shelf-life of retail products (NPPC, 2000).The increase of SFA:UFA ratio could be of certain practical significance from a pork processing point of view (White et al., 2008).
Furthermore, there were some differences in the responses of MUFA and polyunsaturated fatty acids (PUFA) between the backfat and the muscle to CLA supplementation, where MUFA exhibited a decreasing tendency (P=0.055) in the backfat with the increment of dietary CLA, and this phenomenon was not observed in the muscle.Meanwhile, we observed that PUFA content in the Longissimus dorsi muscle of the CLA groups was lower than that of the control group.Interestingly, unlike the L. dorsi muscle, the 1 and 1.5% CLA supplementation had numerically higher PUFA content in the backfat compared with the control group.Therefore the decrease of UFA in the backfat and muscle might be mainly attributed to the changes in MUFA and PUFA.Above results also showed that the effects of dietary CLA supplementation on MUFA and PUFA contents differed between the fat and the muscle.It might be ascribed to the differences of lipid metabolism between the two tissues.Further research is needed to explain the mechanism on an increase of PUFA content in backfat of pigs fed CLA.
Previous experiments observed that an inclusion of CLA in the diet increased CLA cis-9, trans-11, CLA trans-10, cis-12, and SFA contents and decreased MUFA and PUFA in both backfat and muscle of pigs (Martin et al., 2007;Cordero et al., 2010).In this experiment it was not observed that the decreases of MUFA proportion in the muscle and PUFA proportion in the backfat with CLA supplementation.This is not in agreement with the previous study by Martin et al. (2007).
The effect of the decrease in UFA, especially MUFA, might be related to the inhibition of ∆9 desaturase activity and mRNA expression by CLA.The ∆9 desaturase index has been used as an estimator of ∆9 desaturase enzyme activity (Korniluk et al., 2007).In this study, the ∆9 desaturase index, defined as ratio of (C16:1cis-9 + C18:1cis-9) to (C16:1cis-9 + C18:1cis-9 + C16:0 + C18:0), numerically decreased with dietary CLA supplementation in both the Longissimus dorsi muscle and backfat, and the ∆9 desaturase index for the backfat decreased linearly with the dietary CLA levels (P = 0.021).However, the linear relationships differed between the backfat and the muscle, with regression coefficients of -0.0892 and -0.0187 (Figure 1), respectively, for the backfat and the muscle, illustrating a reason for different changes in MUFA between the two tissues.For the modified fatty acid profiles in adipose and muscular tissues, the direct substitution effect of dietary source on fatty acids in the body is one likely reason.Moreover the modulation of gene expression and activity of lipogenic enzymes and ∆9 desaturase by CLA supplementation are likely another explanation.It has been demonstrated that CLA reduced acetyl-CoA carboxylase activity in the liver and adipose tissues of rabbits (Corino et al., 2002).Corino et al. (2003) also reported that CLA supplementation significantly reduced the acetyl-CoA carboxylase activity in adipose tissue of pigs.Several studies have shown that dietary CLA supplementation inhibited the expression and activity of hepatic ∆9 desaturase (Lee et al., 1998;Choi et al., 2000;Shang et al., 2005).These results suggest that CLA has an inhibitory effect on ∆9 desaturase activity.Therefore, the reduction in MUFA content was a reflection of decreased C18:1 content and increased abundance of saturated fatty acids in the tissues of pigs fed CLA, and might be a direct result of the decrease in the desaturase activity (Corino et al., 2003).
The amounts of  in the Longissimus dorsi muscle and backfat showed linear increases with dietary CLA levels.Even so, the mean concentration of CLA in the backfat (2.30%) was about three times of that in the L. dorsi muscle (0.75%) for the CLA groups.
Moreover, though CLA-isomers, cis-9, trans-11 and trans-10, cis-12 had similar contents in the diet, and CLA cis-9, trans-11 was slightly lower than CLA trans-10, cis-12, a preferential incorporation of CLA cis-9, trans-11 was observed in this study.This phenomenon was also found by Thiel-Cooper et al. (2001) and Lauridsen et al. (2005).As shown in Figure 2, the responses of CLA isomers differed between the subcutaneous backfat and the Longissimus dorsi muscle.The concentrations of CLA isomers were lower in the Longissimus dorsi muscle than in the backfat.And CLA cis-9, trans-11 concentration was higher than CLA trans-10, cis-12 isomer in both the tissues.Both the isomers showed linear responses to dietary CLA supplementation.The ratios (R trans-10, cis-12/cis-9, trans-11 ) of CLA cis-9, trans-11 to CLA trans-10, cis-12 in the subcutaneous backfat and Longissimus dorsi muscle of pigs fed the CLA diets were lower than the R trans-10, cis-12/cis-9,trans-11 in the CLA compound that was added to the diets (i.e.0.25-0.41vs 1.07).Czauderna et al. (2004) also found the ratio of CLA cis-9, trans-11 to CLA trans-10, cis-12 in the liver of rats fed the diets enriched with CLA isomer mixture was lower compared with the ratio of these two isomers in the CLA isomer mixture (i.e.0.712-0.833vs 1.024).The accumulation rates of CLA cis-9, trans-11 and CLA trans-10, cis-12 differed between backfat and Longissimus dorsi muscle in piglets fed 1% CLA oil (Weber et al., 2006).As shown in Figures 3 and 4 there were linear relationships between CLA trans-10, cis-12 and CLA cis-9, trans-11 content in the L. dorsi muscle and the subcutaneous backfat.The deposition rate for CLA trans-10, cis-12 was only 42.1% of that for CLA cis-9, trans-11 in the L. dorsi muscle (slope = 0.421), and 55.8% for the subcutaneous backfat (slope = 0.5577).The results of linear responses of CLA trans-10, cis-12 and CLA cis-9, trans-11 might have a value in CLA application.The CLA trans-10, cis-12 and CLA cis-9, trans-11, these two CLA isomers, have been shown to exhibit a wide range of physiological properties in cell culture and biomedical studies with a potential to improve long-term human health.In the nutritional management practice by feeding CLA to pigs in order to improve CLA content in pork products, a high level of supplementation (about 1 time of CLA cis-9, trans-11) of CLA trans-10, cis-12 should be considered, if we wish to have the same enrichment of both CLA trans-10, cis-12 and CLA cis-9, trans-11 in porcine meat and fat.
The reason for the difference between CLA cis-9, trans-11 and trans-10, cis-12 content in tissues might be their dissimilar deposition efficiencies in pig.Czauderna et al. (2004) reckoned that CLA trans-10, cis-12 isomer is more efficiently driven through β-oxidation in cells of the muscles, kidneys, adipose tissue or liver of rats than their homologues.As for pigs, the varied deposition efficiencies of CLAisomers are possibly attributed to their variations in absorption, transportation and catabolism (β-oxidation, etc.), or with competitive inhibition of CLA cis-9, trans-11 on CLA trans-10, cis-12 incorporation.Further study however, is needed to identify factors and precise molecular mechanisms of deposition and metabolism of CLA cis-9, trans-11 or CLA trans-10, cis-12.

CONCLUSIONS
The supplementation of diets with conjugated linoleic acid (CLA) did not notably affect growth performance, carcass characteristics, while it increased dry matter content in the Longissimus dorsi muscle in growing-finishing pigs.Furthermore, dietary CLA supplementation modified the fatty acid profiles in both the Longissimus dorsi muscle and backfat by increasing the concentrations of saturated fatty acids and decreasing the concentrations of unsaturated fatty acids, so the ratio of saturated fatty acids to unsaturated fatty acid increased.The CLA contents in the muscle and backfat increased in a CLA dose-dependent manner.The CLA cis-9, trans-11 and CLA trans-10, cis-12 displayed different deposition efficiencies in the muscle and backfat.Further studies should be undertaken to define the deposition mechanism of CLA isomers in pig tissues, and to determine the optimum CLA concentration in feed and time of feeding to achieve high CLA concentration in pig meat in a more economic way.

Table 1 .
Ingredients and chemical composition of the basal diet

Table 2 .
Effects of dietary CLA supplementation on average daily gain, carcass characteristics, chemical composition of Longissimus dorsi muscle a,b means within a row with different superscripts are different (P<0.05)

Table 3 .
Effects of dietary CLA supplementation on fatty acid profiles in Longissimus dorsi muscle

Table 4 .
Effects of dietary CLA supplementation on fatty acid profiles in backfat