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The fatty acid composition of lamb liver, muscle and adipose tissues in response to rumen-protected conjugated linoleic acid (CLA) supplementation is tissue dependent Stefano Schiavon, Matteo Bergamaschi, Erika Pellattiero, Alberto Simonetto, and Franco Tagliapietra J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04597 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017
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ABSTRACT
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The tissue-specific response to rumen-protected conjugated linoleic acid supply (rpCLA)
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of liver, two muscles and three adipose tissues of heavy lambs was studied. Twenty-four lambs, 8
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months old, divided into 4 groups of 6, were fed at libitum on a ration supplemented without or
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with a mixture of rpCLA. Silica and hydrogenated soybean oil was the rpCLA coating matrix. The
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lambs were slaughtered at 11 months of age. Tissues were collected and analysed for their FA
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profiles. The dietary rpCLA supplement had no influence on carcass fatness nor on the fat content
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of the liver and tissues, and had little influence on the FA profiles of these tissues. In the adipose
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tissues, rpCLA increased the proportions of saturated FAs, 18:0 and 18:2t10c12, and decreased the
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proportions of monounsaturated FAs in the adipose tissues. In muscles the effects were the
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opposite. The results suggest that ∆9 desaturase activity is inhibited by the rpCLA mixture in
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adipose tissues to a greater extent than in the other tissues.
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KEYWORDS: Adipose tissues; Conjugated linoleic acid; Lamb; Liver; Muscles.
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INTRODUCTION
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The quality of meat is largely influenced by its fat content and fatty acid (FA) profile.1 Meat fat
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content and fatty acid profile are also of importance for the consumer health.2-4 Ruminant products,
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rich in saturated FAs, are not entirely acceptable to consumers.1 Consequently, strategies to
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manipulate the fat content and the FA composition of ruminant meat and milk have been studied.5,6
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Ruminant products are rich in conjugated linoleic acids (CLAs), which have been found to be
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involved in many biological activities. For example, they have anti-carcinogenic, anti-obesity,
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antioxidant and anti-inflammatory properties.7 Lipid, energy and nitrogen metabolism in
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monogastric and ruminants were found to be influenced by small amounts of CLA.7,8 In cattle, to
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prevent rumen hydrogenation, CLA is supplied in rumen-protected forms. Various are the forms of
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protection proposed,9 but few comparisons were completed.10,11
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Administration of a few grams per day of rumen-protected CLA isomers, in particular 18:2c9,t11
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and 18:2t10,c12, to lactating cows and sheep has been found to notably reduced the milk fat
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content, with the short-chain saturated FA content more affected than the longer chain
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polyunsaturated FAs.12-14 CLA mixtures have also been found to reduce body fatness in growing
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animals of many species, resulting from a reduction in body fat deposition rather than mobilization
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of body fat already deposited.15 However, the results are controversial. Schiavon et al.16,17 found
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that long-term rumen-protected CLA supply notably increased the CLA contents in the meat and fat
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tissues of growing double-muscled Piemontese young bulls. However, this treatment had small or
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negligible effects on growth performance, carcass fatness and the lipid content of these tissues.
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Lambs are an interesting model for studying lipid metabolism in growing ruminants. The lamb
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lipid depots and their FA profiles can be altered within a short span of time after weaning. Thus, the
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confounding effects arising from previous lipid depots can be avoided.18 In fact, milk and solid
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feeds differ greatly in the amount and composition of the lipid compounds contained in them, and
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this leads to progressive differentiation of the body fat content and composition during the early
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stages of growth.19 Feeding systems based on grass or, as in the case of indoor diets, on 3 ACS Paragon Plus Environment
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concentrates have been found to be responsible for large differences in the FA contents of lamb
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tissues, particularly n-3 and n-6 polyunsaturated FAs.20 Tissues and organs are known to have
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varying FA compositions,21 reflecting their differing physiological roles and metabolisms,22 and
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they should, therefore, respond differently to rumen-protected CLA supply. A tissue-dependent
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response to rumen-protected CLA would be evidenced by different changes in the FA profiles of
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the different tissues.
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In the present experiment, the tissue-specific response to rumen-protected CLA supplementation
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was studied by analysing the FA profiles of the liver, various muscles, and the subcutaneous and
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internal fat of lambs fed on indoor diets.
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MATERIAL AND METHODS
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Animals were treated in accordance with the Guidelines for the Care and Use of Agricultural
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Animals in Agricultural Research and Teaching.23 This trial was part of a larger project aimed at
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studying the effects of CLA supplementation on the growth performance and carcass quality traits
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of sheep in the Veneto region of north-eastern Italy,24-25 and the FA profiles of lean and fat tissues.
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Experimental Design. Twenty-four heavy lambs (12 males and 12 females) of local
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breeds were assigned to 4 groups of 6 and reared on the University of Padua’s Lucio Toniolo
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experimental farm (Legnaro, Padua, Italy). Two groups (one of males, the other of females) were
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fed at libitum an identical total mixed ration (TMR) supplemented with 20 g/d of a commercial
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rpCLA product (SILA, Noale, Venice, Italy). The rpCLA coating material was based on a silica and
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hydrogenated soybean oil matrix.17 The other two groups (one of males, one of females) were fed
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the same diet but without the rpCLA supplement. The CLA dose provided 1.58, 1.54, 7.76, 1.60,
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and 1.60 g/d of 18:2c9,t11, 18:2t10,c12, 18:0, 16:0 and 18:0cis, respectively.17
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The TMR contained corn grain (400 g/kg DM), corn silage (256 g/kg DM), soybean meal
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(33 g/kg DM), dried sugar beet pulp (113 g/kg DM), wheat bran (70 g/kg DM), wheat straw (66
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g/kg DM), grape seed meal (20 g/kg DM) and a vitamin and mineral mix (35 g/kg DM). At the
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beginning of the experiment the animals were aged 8 months, the ram lambs had a body weight of 4 ACS Paragon Plus Environment
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37.4 ± 6.3 kg, the ewe lambs 37.8 ± 6.7 kg. At the end of the fattening period the animals were aged
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about 11 months and had slaughter weights of 61.1 ± 8.6 kg (ram lambs) and 57.4 ± 8.6 kg (ewe
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lambs).
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Tissue sampling. The liver and the perivisceral and perinephric fats were taken from all
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the lambs immediately after slaughter. Each carcass was divided into two sides and cold stored at 4
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°C. Twenty-four hours after slaughter the right half of each carcass was divided into five cuts (hind
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leg, foreleg and shoulder, ribs-loin, withers, brisket) and each cut was weighed. The ribs-loins were
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vacuum-packed and transported to the laboratory where they were stored at 4 °C in a chilling room
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for 6 days, after which they were divided into ribs and loin. The longissimus thoracis muscle, other
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muscles and the subcutaneous fat of the rib cut were separated from the other components and
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weighed as described by Schiavon et al.26
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Sample preparation. The samples were prepared as reported in detail by Schiavon et al.
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(Grindomix GM200, Retsch, Haan, Düsseldorf, Germany) then stored at -20 °C. The samples were
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then freeze-dried using a CoolSafe 80-90 freeze dryer (Scanvac, Stockholm, Sweden). Each freeze-
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dried sample (liver, 2.21 ± 0.17 g; longissimus thoracis, 2.17 ± 0.10 g; other muscles, 2.20 ± 0.14 g;
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subcutaneous fat, 0.085 ± 0.014 g; perivisceral fat, 0.086 ± 0.021 g; perinephric fat, 0.088 ± 0.013
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g) was subjected to a mild acid-base transesterification/methylation process as described by
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Jenkins.28 Two mL of sodium methoxide (0.5 M in methanol) and 2 mL of toluene containing 2
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mg/mL of methyl 12-tridecenoate as internal standard (# U-35 M, Nu-Chek Prep, Inc., Elysian,
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MN, USA) were added to the dried tissue samples in a culture tube. The samples were incubated in
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a water bath at 50 °C for 10 min, then removed from the bath and cooled for 5 min. After adding 3
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mL of freshly prepared methanolic HCl (1.37 M) the samples were incubated again in a water bath
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at 80 °C for 10 min, then removed from the bath and cooled for 7 min. Next, 5 mL of K2CO3 (0.43
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M) and 2 mL of toluene were added to each tube and these were then vortexed for 30 s and
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centrifuged for 5 min at 400g and 4 °C. The organic phase (upper layer) of the tube was transferred
Briefly, all the tissue samples were ground, mixed and homogenized for 10 s at 4500g
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to a screw-capped tube, to which was added 0.5 g of anhydrous sodium sulphate and 0.5 g of active
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charcoal (Sigma-Aldrich, MO, USA). The solution was vortexed for 5 min and rested for 1 h. After
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centrifugation for 5 min (400g at 4 °C) the clear upper layer containing the FAME was transferred
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to a gas chromatography (GC) vial and stored at -20 °C until GC analysis.
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Fatty acid analysis. Detailed FA profiles were determined using a GC×GC instrument
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(Agilent 7890A, Agilent Technologies, Santa Clara, CA, USA) with two columns in series and
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equipped with a modulator (G3486A CFT, Agilent Technologies), an automatic sampler (7693,
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Agilent Technologies) and a flame ionization detector connected to the chromatography data system
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software (Agilent Chem Station, Agilent Technologies) at DAFNAE, University of Padua (Legnaro,
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Padua, Italy). The operating conditions of the GC apparatus were as follows: first column, 75 m ×
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180 µm (internal diameter) × 0.14 µm film thickness (Supelco, Bellefonte, PA), H2 carrier at a flow
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rate of 0.22 mL/min; second column, 3.5 m × 250 µm (internal diameter) × 0.14 µm film thickness
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(Agilent, Agilent Technologies), H2 carrier at a flow rate of 22 mL/min. Oven temperature
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programme: 50 °C (held for 2 min), increased to 150 °C at 50 °C/min (held for 15 min), then
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increased to 240 °C at 2 °C/min (held for 20 min). Valves were set for a modulation delay of 1 min,
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a modulation period of 2.9 s, and a sample time of 2.77 s. Flame-ionization detector: heater, 250 °C;
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H2 carrier flow, 20 mL/min; air flow, 450 mL/min. The splitless inlet temperature was 270 °C,
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pressure 20.80 MPa, septum purge 3 mL/min and split flow 35.2 mL/min. The resulting two-
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dimensional chromatograms were analysed with the comprehensive GC×GC software (GC Image
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Software, Zoex Corp., Houston, TX, USA) in order to calculate the cone volume of each FA.
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Fatty acid identification and quantification. Fatty acids were identified as reported in
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detail by Schiavon et al.27 Briefly, the cone positions in the chromatogram were compared with the
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cone positions of the FAs in the pure reference standards (#674 and #463; Nu-Chek Prep, Inc.,
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Elysian, MN, USA), 47080-U Bacterial Acid Methyl Esters (BAMEs; Sigma-Aldrich, St. Louis,
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MO, USA), 47085-U Ω3 Menhaden Oil (Supelco, St. Louis, MO, USA), and 5 CLAs: 18:2c9,t11
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(#UC-60M; Nu-Chek Prep, Inc., Elysian, MN, USA), 18:2t10,c12 (#UC-61M; Nu-Chek Prep, Inc., 6 ACS Paragon Plus Environment
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Elysian, MN, USA), 18:2c9,c11 (#1256; Matreya LLC., Pleasant Gap, PA, USA), 18:2t9,t11
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(#1257; Matreya LLC, Pleasant Gap, PA, USA) and 18:2c11,t13 (#1259; Matreya LLC, Pleasant
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Gap, PA, USA). In addition, other FAs were identified by elution order and their position in the
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two-dimensional chromatography grid using GC Image Software (Zoex Corp.) in accordance with
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Vlaeminck et al.29 Some FAs aligned in specific areas of the chromatogram were classified
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according to their main characteristics, length of carbon chain and degree of unsaturation.
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Fatty acid proportions were calculated by dividing the cone volume of each FA by the total
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volume of FAs and were expressed as g/100 g total FAs. In accordance with other experiments by
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our team,30 we assumed a limit of detection for all FAs of 0.015 mg (0.0015 g/g total FAs). All
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values below this limit were taken to be undetectable and considered missing for the statistical
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analyses. The FAs were also summed in groups according to the criteria set out in Table 1.
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Statistical analysis. The statistical analyses were performed with the SAS software
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package (SAS Institute Inc., Cary, NC, USA) using two different models. Meat characteristics were
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analysed with PROC GLM according to the following model:
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yijklmn = µ + CLAi + Sexk + CLA × Sexik + Birthl + Agem + eijklmn,
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where yijklmn is the observed quality trait; µ is the overall intercept of the model; CLAi is the fixed
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effect of the ith CLA treatment (i = 1 to 2); Sexk is the fixed effect of the kth sex (k = 1 to 2); CLA ×
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Sexik is the fixed effect of the ikth CLA × sex interaction; Birthl is the fixed effect of the lth single or
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twin birth (l = 1 to 2); Agem is the linear covariate of the lamb’s age, and eijklmn is the random effect
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of animal. The experimental unit was the animal.
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To evidence the differential response of the tissues to rpCLA supplementation data obtained for
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each individual FA and FA category, expressed as g/100 g total FAs to avoid heteroskedasticity
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among tissues, was performed with the PROC MIXED SAS procedure according to a model:
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yijklmno = µ + CLAi + Sexk + CLA × Sexik + Birthl + Agem + Lamb(CLA × Sex)n:ijk + Tissueo + CLA
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× Tissueio + eijklmno,
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where yijklmno is the observed trait; µ is the overall intercept of the model; CLAi is the fixed effect of
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the ith dietary treatment (i = 1 to 2); Sexk is the fixed effect of the kth sex (k = 1 to 2); CLA × sexik is
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the fixed effect of the ikth diet × sex interaction; Birthl is the fixed effect of the lth birth (l = 1 to 2);
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Agem is the linear covariate of the lamb’s age; Lamb(CLA × Sex)n:ijk is the random effect of the nth
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animal (l = 1 to 24) within diet and sex; Tissueo is the fixed effect of the oth tissue (m = 1 to 6); CLA
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× Tissueio is the fixed effect of the ioth CLA × tissue interaction; and eijklmno is the residual error.
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The random effect of animal was used to test CLA, sex, the CLA × sex interaction, and birth,
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whereas the effects of tissue and the CLA × tissue interaction were tested on the residual.
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Orthogonal contrasts were fitted to test liver vs other tissues, fatty tissues vs muscles, longissumus
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thoracis vs other muscles, subcutaneous fat vs internal (perivisceral plus perinephric) fat, and
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perivisceral fat vs perinephric fat.
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RESULTS AND DISCUSSION
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Effects of gender and CLA on carcass fat and tissue fat content. The lambs used in the
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present experiment were slaughtered at about 60 kg body weight (Table 2). Females differed from
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males in a number of traits, having greater carcass yields, lower percentages of muscle and greater
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percentages of carcass fat content and perinephric fat. The fat contents of the liver and the
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longissimus thoracis muscle of females were greater than males. However, despite a difference of 7
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percentage points between males and females in carcass fat content (P < 0.01), there were only
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small differences between the genders in the average across-tissue FA profiles. Nevertheless, male
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lambs had higher proportions of medium-chain branched FAs, 18:1t11, 18:1c9, ∑18:1others and some
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medium- to long-chain PUFAs (from C16 to C22) than female lambs. The small differences between
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males and females in FA composition, despite different carcass and tissue fat contents, was
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expected, given that few gender differences in intramuscular FA composition, even in carcasses of
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widely different fat levels, have previously been found.31-33 The major differences between males
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and females regarded the branched FAs and some partially hydrogenated FAs, such as 18:1t11,
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which reflect the contribution of the rumen microbial population.34 The results of the present 8 ACS Paragon Plus Environment
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experiment suggest, therefore, that there were some differences between males and females in the
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patterns of rumen fermentation or the feed particle passage rate.
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The dietary CLA mixture did not affect the weight or growth of the lambs, nor body condition
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scores, nor the proportions of fat and lean in the carcass. The rumen-protected CLA supplement did
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not affect the weights of the liver, kidney and perinephric fat, nor the fat content of the liver and
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muscles. It had little or no effect on the large majority of individual and groups of FAs across
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tissues, with some exceptions, these being the greater proportions of 18:0 (P < 0.01), ∑CLA (P
subcutaneous adipose tissue > internal adipose tissues and liver.
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The tissue with the greatest proportion of PUFAs was the liver (23.8 g/100 g total FAs; P
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< 0.01), followed by muscle (9.3 g/100 g total FA) and adipose tissues (3.8 g/100 g total FAs).
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Liver had the lowest proportions of MUFAs (25.9 g/100 g total FA; P < 0.01), and FAs with 8 to 16
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and 18 carbons (P < 0.01), and the highest proportion of very long chain FAs (> 18 carbons; P
liver > muscle, in
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agreement with.48 The predominant isomer formed in the rumen is commonly 18:1t11, but lambs
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fed high-concentrate diets may also have high proportions of 18:1t10 (co-eluted with 18:1t8-10 in
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the present experiment).48 Differences in the proportions of trans FAs in the various tissues would
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therefore suggest differences in the uptake of these FAs from the rumen, but may also reflect
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metabolic differences among the tissues.49
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Tissue-dependent response to CLA supply. The influence of a rumen-protected CLA
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supplement on the fat content and FA profiles of organs and tissues has not been widely studied in
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sheep or lambs.25 However, Terré et al.50 found that the FA profiles of the longissimus dorsi of
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lambs fed a rumen-protected CLA mixture exhibited small variations, except in the content of the
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two CLA isomers provided.
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Sinclair et al.35 reported that CLA supplementation did not alter the weight of the liver nor body
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fat deposition in lactating ewes, and that it had little influence on the FA profile of the longissimus
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thoracis muscle, except for an increase in the two CLA isomers supplied. Wynn et al.51 reported a
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tissue-dependent effect of rumen-protected CLA in sheep and also found that a rumen-protected
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CLA supplement resulted in lower concentrations of monounsaturated fats (palmitoleate and oleate)
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in adipose tissues and liver. These effects on the FA profiles of tissues were consistent with
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inhibition of the ∆9 desaturase enzyme, as reported for pigs by Cordero et al.52
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In the present experiment, the rumen-protected CLA supplement had little influence on the major
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indices of fatness of the lamb carcass, and little or no influence on the fat content of the liver and
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tissues. On average, CLA supplementation increased the proportions of 18:2c9,t11 and 18:2t10,c12
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in the tissues, but had only a small effect on the relative contents of other FAs, although it interacted
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with the tissues for some groups or individual FAs.
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The most notable CLA × (muscle × fat depot) interactions (Figure 1) regarded the proportions of
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SFAs (P < 0.01) and MUFAs (P < 0.01). The CLA mixture resulted in an increase in SFAs and a
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decrease in MUFAs in all the 3 adipose tissues, while the opposite was observed for the muscles,
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which was primarily due to the changes observed in 18:0 and 18:1c9, the main MUFA constituents
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(Figure 2). The greater increase in 18:0, a component of the rpCLA mixture, and the decrease in
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18:1c9 in the adipose tissue might suggest that ∆9 desaturase activity in these tissues was inhibited
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by the CLA mixture more than in other tissues. The liver was the least responsive to the CLA
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supplement in terms of 18:0 and 18:1c9 contents, an interesting finding given the susceptibility of
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the ruminant liver to hepatic steatosis, especially when fed a diet rich in polyunsaturated FAs, such
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as linoleic acid.45
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The CLA × (muscle × fat depot) interaction was also significant for the 18:2t10,c12 isomer (P
