Dietary CLA supplementation and gender modify fatty acid composition of subcutaneous and intramuscular fat in Iberian × Duroc finishing heavy pigs

The objective of this research was to evaluate the effect of dietary conjugated linoleic acid (CLA) enrichment for finishing heavy fatty pigs on performance, carcass traits and fatty acid composition in subcutaneous and intramuscular fat (IMF), including CLA main isomers concentration. Forty castrated Iberian × Duroc pigs, half males and half females, with 120 (± 2.83) kg live weight were used. Pigs were fed experimental diets containing two levels (0 and 1%) of CLA. No effect of CLA was observed on backfat thickness or IMF concentration. In subcutaneous backfat, dietary CLA increased the C16:0, C18:0, c9,t11-CLA , t10,c12-CLA and saturated fatty acids (SFA) proportion and decreased those C18:1 n-9, C18:2 n-6, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) proportions and MUFA/SFA and C18:1 n-9/C18:0 and C16:1 n-7/C16:0 ratios. In the IMF, dietary CLA increased C16:0, SFA, c9,t11 and t10,c12 proportions and reduced C18:1 n-9 and MUFA, MUFA/SFA and C18:1 n-9/C18:0 ratios, but showed no effect on C18:2 n-6 and PUFA proportion. Subcutaneous backfat fatty acid profile was affected by gender, but no gender effect on intramuscular fatty acid profile was observed. CLA isomer accumulation was lower in heavy pigs compared to the lean genotype probably as a consequence of the higher carcass fat concentration in the former. Additional key words: carcass traits; conjugated linoleic acid; fatty acid profile; heavy pigs.


Introduction
There is a growing interest in the production of high quality dry-cured meat products for niche market, sometimes associated to the production of autochthonous pig breeds. This requires the production of fat pigs slaughtered at heavy weight (140-160 kg), where fat content and composition play a determinant role in the nutritive, sensory and technological quality of the meat (López Bote, 1998).
Fat pigs accumulate large amount of subcutaneous lipids and are extremely inefficient (Serrano et al., 2008). However, any strategy aimed to increase feed efficiency (reducing slaughter weight, crossing with lean pigs, etc.) has proven to reduce also intramuscular fat (IMF), and therefore have been discarded because of its negative effect on dry cured meat products (Carrapiso et al., 2003;Ventanas et al., 2007).
Several studies have reported that dietary CLA administration to growing-finishing pigs reduces subcutaneous fat and improves feed efficiency, without any effect on IMF concentration (Dugan et al., 1997;Joo et al., 2002). This has raised an interest in feeding CLA to pigs (Ostrowska et al., 1999;. Moreover, CLA has also beneficial effects such as anti-carcinogenic (Pariza and Hargraves, 1985), antiatherosclerothic (Lee et al., 1994) and enhance of immune system (Haro et al., 2006), and therefore dietary CLA administration to pigs has proven to be an effective way to concentrate active isomer in pig tissues, thus producing additional benefits to consumers (Pariza et al., 2001;Belury, 2002).
At the moment, most studies with CLA feeding to pigs have been performed on lean (Ostrowska et al., 1999;Martín et al., 2007;Larsen et al., 2009) or moderately fat pigs (Eggert et al., 2001;Fernández-Figares et al., 2008;Cordero et al., 2010) but there is lack of information of the possible effect of CLA on heavy fat pigs.
The objective of this research was to evaluate the effect of dietary CLA enrichment for finishing heavy fat pigs (castrated males and castrated females) on performance, carcass traits, backfat thickness, IMF content, and fatty acid composition including the concentration of CLA isomers in subcutaneous and IMF.

Animals and diets
The routine of animal care and experiment procedures used in this experiment was approved by the University Complutense of Madrid. Forty Iberian × Duroc pigs, half castrated males (CM) and half castrated females (CF), with 120.0 (± 2.83) kg live weight were used. The pigs were randomly distributed and located in eight pens of five animals (CM or CF). Pigs were fed with their respective experimental diets which were formulated to contain either no CLA (0%) or 1% CLA supplementation. The CLA oil supplementation (CLA-60, Loderstar TM , Belgium), contained approximately 60% of CLA isomers (30% c9,t11 and 30% t10,c12) ( Table 1).
All the diets were formulated to provide the same protein and energy levels. Diets were provided ad libitum for 42 days. Before the beginning of the experiment all pigs were subjected to the same feeding and management. Pigs were slaughtered at a local slaughterhouse at 153.2 (± 3.0) kg live weight.
Ingredients, chemical composition and main fatty acids of experimental diets are shown in Table 1. Determination of the compositional analysis of feeds (in triplicate) was carried out according to AOAC (2005). Fatty acids of diets were extracted and quantified by the one-step procedure of Sukhija and Palmquist (1988) from lyophilised samples. Pentadecanoic acid (C15:0) (Sigma-Aldrich, Madrid, Spain) was used as internal standard. Fatty acid methyl esters were analysed by gas chromatography using a Hewlett Packard HP-6890 (Avondale, PA, USA) gas chromatograph equipped with a flame ionization detector and a capillary column (HP-Innowax, 100 m × 0.25 mm i.d. and 0.2 µm polyethylene glycol-film thickness) (Kramer et al., 1998).

Sample collection and chemical analysis
At slaughter, carcass weight, carcass yield, hams, forelegs, loins, chops and bacon weight were recorded. Samples of subcutaneous fat and Longissimus dorsi at the level of the last rib were taken, weighed, vacuumpacked in low-oxygen permeable film, and kept frozen at -20°C for fatty acid analysis. Liver samples (appro-CLA in Iberian × Duroc heavy pigs 963 ximately 30 g) were collected at slaughter, placed in liquid nitrogen and stored at -80°C for enzyme activity analyses. Lipids from subcutaneous backfat and Longissimus dorsi muscle were extracted with a mixture of choloroform/methanol (2:1 v/v) following the procedure described by Folch et al. (1957). Lipid extracts were methylated in the presence of sodium methoxide (Christie, 1982) and analyzed as described for dietary fatty acids.

Enzyme assays
Malic enzyme (ME) and glucose 6-phosphate dehydrogenase (G6PD) activities were determined in liver extracts as described by Álvarez et al. (2000). Weighed quantities of liver tissue sample were homogenised with an Ultra turrax T18basic (IKAw; Labortechnik, Staufen, Germany) in 3 vol. ice-cold buffer [20 mM Tris-HCl, 0.25 M sucrose, 2 mM ethylenediamine-tetraacetate EDTA], pH 7.4 and spun at 20,000 × g for 40 min. G6PD and ME activities were determined using continuous spectrophotometric assays following the NADPH formation at 340 nm. The soluble liver protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as standard. All enzyme assays were conducted at 37°C. Care was taken to ensure that initial rates were being measured in all assays. Control experiments established that the enzyme was stable in the buffer used during the time period of the assay and at the temperature required (Álvarez et al., 1998). All enzyme assays were carried out in triplicate. The enzymatic activity units (IU) defined as µmol substrate converted to product per min at the assay temperature, were expressed per mg liver soluble protein (specific activity). All chemicals were supplied by Amersham Biosciences (Upsala, Sweden) and by Sigma-Aldrich (Madrid, Spain).

Statistical analysis
Data were analyzed using the GLM procedure of SAS (SAS, 2002). The effects of dietary CLA level, gender and the interaction CLA × Gender were studied. The experimental unit was the pen all the analyses done. The data are presented as the means and the standard errors of means. The initial weight was used as covariate for productive variables and the carcass weight (CW) for carcass characteristics. When the covariates were not significant (p > 0.05) they were removed from the model. All differences were considered significant at p < 0.05 and p values between 0.05 and 0.10 were considered a trend.

Results
Dietary CLA and gender had no influence (p > 0.05) on average daily gain (ADG), average daily feed intake (ADFI) and feed conversion efficiency (FCE). The  Dietary CLA had no effect on carcass characteristics ( Table 2). Dietary CLA increased the sum of hams, forelegs and loins weights (H + F + L) (p = 0.10). The forelegs weight (FW) were higher (p = 0.03) in CM than in CF, while the bacon weight (BW) tended (p = 0.05) to be higher in CF than in CM. The interaction dietary CLA × gender showed not effect (p > 0.05) for all carcass traits studied.
The gender had not effect (p > 0.05) on fatty acids profile of IMF (Table 4). However, CM tended (p < 0.10) to have higher C18:0, SFA and c9,t11-CLA proportions and lower MUFA proportion and MUFA/SFA and C18:1 n-9/C18:0 ratios than CF. The interaction dietary CLA level × gender showed not effect for all fatty acid proportion determined.
Dietary CLA, gender and interaction CLA × gender showed not effect on G6PD and ME activity in the liver (Table 5).

Discussion
The productive results observed in the present experiment are in agreement with data from Serrano et al.    (Table 2), which is in disagreement with most previous research in which CLA was included at similar dietary concentration. Thiel-Cooper et al. (1998) in lean pigs fed a diet with 1% of CLA reported a 10% reduction in subcutaneous backfat. Ostrowska et al. (2003) reported a 23% reduction in subcutaneous backfat and an increase 10% of firmness (with 1% of CLA). Wiegand et al. (2001) with a dose of approximately 1% reported a reduction in subcutaneous backfat and an increase in marbling and firmness. Dunshea et al. (1998) andOstrowska et al. (1999) reported up to a 25 to 30% reduction in subcutaneous backfat thickness at higher dietary CLA concentration. However, in other experiments no effect of 1% dietary CLA administration was observed on fat accumulation (Eggert et al., 2001). In this experiment, pigs were slaughtered at higher weights (> 120 kg), thus suggesting that the effectiveness of 1% dietary CLA is lower in fatty pigs than in lean genotypes. According to Ramsay et al. (2001), the influence of CLA on carcass fat and lean deposition seems to be affected by pig phase of growth and genotype, being different in lean than obese or fat pigs. In our experiment the CLA level in the diet (1%) did not affect subcutaneous fat deposition although tended to increase the combined weight of H + F + L.
In the present experiment a 1% dietary CLA inclusion did not affect IMF concentration (Table 2). This is contradictory to some previous research in which IMF increased due to dietary CLA administration with a slaughter weight around 105 kg (Joo et al., 2002;Wiegand et al., 2001) and113 kg (Averette et al., 2002). However, no effect of 1% dietary CLA on IMF concentration have been found in pigs slaughtered at heavy weights (> 120 kg) (Eggert et al., 2001).
The effects of dietary CLA administration on subcutaneous and intramuscular fatty acids are shown in Table 3 and 4 respectively. Dietary CLA increased c9,t11-CLA and t10,c12-CLA, which agrees with the findings of Kramer et al. (1998), Eggert et al. (2001) and Martín et al. (2007). On the other hand, the accumulation of c9,t11-CLA was higher than t10,c12-CLA in subcutaneous and intramuscular fat. This result is also in agreement with data from Eggert et al. (2001), Smith et al. (2002 and Martín et al. (2007) and might suggest that the t10,c12 isomer would be either less efficiently incorporated or have a different metabolism. On the other hand, Larsen et al. (2009) found in lean pigs 1.19 and 0.93% of c9,t11-CLA and t10,c12-CLA respectively in bacon from pigs fed 1% CLA enriched diets for 56 days. However, it is noticeable that despite similar experimental conditions in our study the concentration of both isomers were considerably lower (0.33 and 0.18% for c9,t11-CLA and t10,c12-CLA respectively), than in the study of Larsen et al. (2009). The main distinctive characteristic between these two experiments is the marked adipogenicity of Iberian pigs (López-Bote, 1998).
A possible explanation of the effect of dietary CLA on CLA isomers accumulation observed in our experiment could be the higher carcass fat concentration in Iberian × Duroc heavy pigs compared to lean genotypes. It has been recently reported lack of fat mobilization induced by CLA in obese humans (Desroches et al., 2005), while a similar dose was shown to reduce body fatness either in healthy exercising subjects (Thom et al., 2001) or in postmenopausal healthy women (Raff et al., 2009).
In the present study subcutaneous backfat SFA increased from 40.0% to 44.5% during 42 days of experiment, but Larsen et al. (2009) found that SFA increased from 38.5% to 46% during 56 days, thus suggesting that in finishing heavy pigs 42 days supplementation with CLA is enough to modify backfat SFA. The lowering PUFA concentration of CLA in subcutaneous fat is also a subject of potential interest, since there is a negative relationship between PUFA content and fat consistency. An excessive accumulation of PUFA in pig fat leads to an extension of the processing time of carcass joints due to a low fat consistency and therefore, impaired water migration (López-Bote, 1998), which is of importance in production of quality meat products. The ∆-9 desaturase index, assessed by the MUFA/SFA, C18:1 n-9/C18:0 and C16:1 n-7/C16:0, decreased with the CLA administration, which is in agreement with previous research in which stearoyl coenzyme A desaturase activity in porcine subcutaneous adipose tissue was either measured (Smith et al., 2002) or estimated (Eggert et al., 2001;Martín et al., 2007). According to Park et al. (2000), the t10,c12-CLA isomer of CLA has an inhibitory effect on ∆-9 desaturase activity compared to others CLA isomers, whereas c9,t11-CLA isomer seems to have not effect on fatty acid desaturation.
Regarding the influence of gender on fatty acid composition of subcutaneous backfat, Pieszka et al. (2006) observed that in gilts fed with CLA diets, the SFA content in fat was lower and the content of PUFA, MUFA and PUFA/SFA ratio were higher than in barrows. In agreement with our results, some other experiments also reported that subcutaneous backfat from males is more saturated than that from females (Courboulay and Massabie, 1996;Smithard et al., 1980), although, Serrano et al. (2008 did not observe influence of gender on fatty acid composition of subcutaneous backfat from Duroc × Iberian heavy pigs. In IMF from Longissimus dorsi, Bee (2001), in pigs slaughtered at 105.2 kg live weight, and , in heavy pigs, observed an increase of C16:0 and SFA and a reduction of C18:1 n-9 and MUFA with dietary CLA, whereas on C18:2 n-6, no effect was observed. Eggert et al. (2001) also observed that dietary CLA increased C16:0 and SFA concentration, but did not find effect on C18:2 n-6 and PUFA. It is interesting to note that increase in the C16:0 proportion in IMF is similar or a little bit lower than in subcutaneous backfat. The decreasing effect of CLA on C18:1 n-9, MUFA proportions and ∆-9 desaturase index was of lower magnitude in IMF than in subcutaneous backfat, which suggests lower regulatory effect of ∆-9 desaturase in muscle tissue. In the current experiment this result was likely due to the lower t10,c12-CLA isomer concentration in IMF than in subcutaneous fat.
In this study the relationships found between IMF percentage and C14:0, C16:0 and C18:2 n-6 proportions in IMF is of interest. Previous works have also shown a relationship between SFA and intramuscular fat content (Bee, 2001;Eggert et al., 2001;Ramsay et al., 2001), thus suggesting an indirect mechanism of dietary CLA on IMF content. Future research is needed in this topic.
G6PD and ME are the main enzymes involved in supplying NADPH for the reductive biosynthesis of fatty acids (Mourot et al., 1995), but they also contribute to metabolic pathways other than lipogenesis. Both enzyme activities were unaltered by dietary CLA supplementation. The G6PD activity was higher than ME and seemed to be the main producer of NADPH. This observation agrees with comparative studies in newborns (Le Dividich et al., 1994) and growing pigs (Mourot et al., 1995). Bee (2001) did not observe effect of dietary CLA on malic and fatty acid synthesis enzymes activity in subcutaneous backfat and omental fat in finishing pigs, but the same author (Bee, 2000) found a positive effect of CLA on G6PD and ME activity in piglet adipose tissues. According to Daza et al. (2007), the effect of these enzymes is more marked in young animals or when animals receive different levels of energy in the diet. We did not find any effect of CLA on lipogenic enzymes and this confirm that the increase of SFA and the decrease of MUFA concentrations in both tissues studied is not due to an increased synthesis, but more likely to a down-regulation of ∆-9 desaturase activity. This is in agreement with Bretillon et al. (1999) who reported that both the activity and gene expression of hepatic ∆9-desaturase were inhibited by dietary CLA.
It is concluded that a 1% dietary CLA inclusion produced no effect on carcass traits and IMF content, but affects fatty acids profile of subcutaneous backfat and IMF. Levels of CLA isomers in subcutaneous and IMF were lower than in lean pigs, which suggest that a higher dietary CLA supplementation is needed to achieve the same levels in tissues for obese heavy pig genotypes, thus indicating that a correct combination of CLA concentration and administration time should be defined in each productive circumstance according to pig fatness.
The accumulation of the CLA isomers was lower in heavy pigs compared to the lean genotype probably as a consequence of the higher carcass fat concentration in the former. More studies are needed to understand the underlying mechanism of CLA level in the diet on carcass and fat quality in obese pigs.