# Genotype and dietary inclusion of omega-3 fatty acids and fiber influence metabolism of subcutaneous adipose tissue and systemic metabolic outcomes in growing pigs

Florence Gondret, Kamil Sierżant, Bénédicte Lebret

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Florence Gondret, Kamil Sierżant, Bénédicte Lebret. Genotype and dietary inclusion of omega-3 fatty acids and fiber influence metabolism of subcutaneous adipose tissue and systemic metabolic outcomes in growing pigs. Meat Science, 2025, 229, pp.109911. 10.1016/j.meatsci.2025.109911. hal-05168351

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https://hal.inrae.fr/hal-05168351v1

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# Genotype and dietary inclusion of omega-3 fatty acids and fiber influence metabolism of subcutaneous adipose tissue and systemic metabolic outcomes in growing pigs

F. Gondret<sup>a,*</sup>, K. Sierżant<sup>b</sup>, B. Lebret<sup>a</sup>

## ARTICLE INFO

Keywords:

Subcutaneous fat

Lipogenesis

Lipid oxidation

Plasma metabolites

Linseed

Faba bean

## ABSTRACT

Adipose tissue properties are considered as determinants of meat quality, but also play roles in animal robustness. From 33 kg to 116 kg on average, a total of 60 Duroc (D) or Pietrain (P) crossbred female pigs were individually fed either a conventional (C) or an experimental (R) feeding strategy with a diet rich in omega 3 fatty acids and crude fibers (n = 15 pigs per group). The D pigs had a lower feed efficiency and a higher proportion of subcutaneous adipose tissue (SCAT) at slaughter than P pigs (P < 0.01). SCAT in D pigs had lower levels of polyunsaturated fatty acids (PUFA; P < 0.001) and higher levels of peroxidized lipids (P < 0.05) than SCAT in P pigs. In plasma, reactive oxygen metabolites to total antioxidant capacity was higher (P < 0.01) in D pigs than in P pigs. Compared to C, feeding the R diet resulted in a marked decrease in the n-6:n-3 PUFA ratio in SCAT, which was even more pronounced in D pigs than in P pigs (P < 0.05). The R feeding strategy increased beta-hydroxyacyl-coenzyme A dehydrogenase activity (P < 0.05) acting in fatty acid oxidation, and SCAT susceptibility to peroxidation. This also led to a reduction in plasma phospholipid concentrations and oxidative stress in the D pigs (P < 0.05). Thus, the nutritional value of fat products for humans and the systemic metabolic outcomes for pigs could be improved with an appropriate feeding strategy.

## 1. Introduction

Pigs are a common source of both lean and fatty meat products for the human population. In the last decades, selecting pigs to improve efficiency of lean meat production for the economic benefit of farmers and retailers and to meet nutritional concerns (healthy food) has led to a deterioration of some pork quality traits and consumer acceptance (Schwab et al., 2006). This is partly due to the reduced fat content of the meat, which influences pork eating properties, especially texture, one of the most important quality attributes for consumers (Fernandez et al., 1999). More recently, the societal image of pig farming systems has become as important, or even more important, than the intrinsic qualities of pork (Candek-Potokar et al., 2024). This includes among others, the origin of feed (confidence in domestic production, environmental consciousness leading to decreasing or avoiding importation of protein resources) and breeds (incorporating diversity into livestock systems). In this context, Lebret et al. (2023) have shown that feeding Duroc

crossbred pigs (a genotype with a high capacity for intramuscular fat deposition) with local plant resources rich in *n*-3 polyunsaturated fatty acids (PUFA) and fiber (grain legumes as a domestic protein source alternative to imported soya), can jointly improve the intrinsic (sensory, nutritional and technological) and extrinsic (societal image) quality attributes of pork. However, the effects of genotype and feeding strategy combinations on the biology of adipose tissue (anabolic and catabolic metabolic properties), and the consequences for pig systemic metabolic outcomes have been neglected.

Subcutaneous adipose tissue (SCAT) is the preferred site for the storage of energy in the form of triglycerides during overfeeding and to restore it when pigs face productive, reproductive and environmental challenges (Louveau et al., 2016). Therefore, the anabolic and catabolic pathways that orchestrate the deposition and release of fatty acids (FA) are important to study for their impact on fat composition traits. They may also be involved in the adaptation (robustness) of pigs to different challenges (feeding, environmental temperature, disease, etc.). Stored

<footnote><sup>a</sup> PEGASE, INRAE, Institut Agro, 35590 Saint-Gilles, France</footnote>

<footnote><sup>b</sup> Department of Animal Nutrition and Feed Science, The Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Chełmońskiego St. 38C, 51-630 Wrocław, Poland</footnote>

<footnote>* Corresponding author at: UMR PEGASE, INRAE, 16 Le Clos, 35590 Saint-Gilles, France. E-mail address: Florence.gondret@inrae.fr (F. Gondret).</footnote>
FA largely reflects the composition of the diet received by the pig (Ovilo et al., 2014), so feeding pigs an n-3 PUFA-enriched diet can alter the typically high omega-6 (n-6) to omega-3 (n-3) ratio in their lean and adipose tissues to a more favorable value for human nutrition (Dugan et al., 2015). However, the reactions with reactive oxygen species (ROS) need to be considered not only for the preservation of the shelf life of animal products (Amaral et al., 2018), but also for their propensity to induce prolonged oxidative stress in living animals. Indeed, it has been suggested that redox imbalance may facilitate or prevent adaptation in farm animals at critical stages of their lives (such as the pre-slaughter period) or when faced with specific challenges (Durand et al., 2022). Oxidative phenomena in SCAT may be particularly important in a situation where PUFA levels are elevated (Franco et al., 2006). Activation of SCAT antioxidant enzymes which have a critical role in detoxifying ROS in the tissue, is also important in the determination of systemic oxidative stress in pigs (Sierżant et al., 2019).

This study aims to investigate the metabolic pathways and FA composition of SCAT, as well as the blood metabolites and systemic redox status, in two pig genotypes that differ in their fat deposition capacity and were fed diets with different levels of n-3 PUFA and fiber. This study is based on the experimental animal design set up by Lebret et al. (2023), who examined the effects of these genetic and feeding strategies on pork lean meat quality. This study aimed to provide new insights into how to improve the nutritional value of adipose tissue for human consumption and how to manage systemic metabolic outcomes for the pig.

## 2. Material and methods

The experiment was performed at the INRAE experimental pig facilities (UE3P, 35590 Saint-Gilles, France; doi.org/10.15454/1.5573932732039927E12) in compliance with the EU directive 802,010/63/EU for animal experiments and the French legislation. The experiment was approved by the local Committee on Ethics in animal experimentation (CREEA-07) and was authorized by the French Ministry of Higher Education, Research and Innovation (Authorization: APAFIS#19388-2,019,022,208,529,883 v2 delivered on 18 May 2019).

### 2.1. Experimental design

The experimental design was recently published in a companion paper examining the combination of pig genotype and feeding strategies on various quality properties of lean meat (Lebret et al., 2023). It is only briefly summarized here. Crossbred Large White × Landrace sows (INRAE herd, UE3P, Saint-Gilles, France) were inseminated with semen from either Duroc boars or from Pietrain NN boars. The Duroc boars came from a herd of males selected by a breeding company (Genesis herd, 22400 Lamballe, France) for growth rate, feed intake and intramuscular fat content. The Pietrain NN (non carriers of the hal mutation at the RYR1 gene) boars originated from a herd of males selected by a breeding company (Nucleus, 35650 Le Rheu, France) for growth rate, feed efficiency and pork technological quality. A total of 60 crossbred females, issued from 7 litters of Duroc (D; n = 30 pigs) and 9 litters of Pietrain (P; n = 30 pigs), were used for the feeding trial. In each genotype, 15 pairs of female littermates were divided into two groups with similar average and standard deviation of body weight (BW) and fed individually one of two feeding strategies from 33.7 kg ± 2.0 kg BW to approximately 116 kg BW. All pigs were housed in individual pens (85 × 265 cm) in the same room during the entire trial period. The feeding strategies were either a conventional diet (C) based on imported soybean, rapeseed and hulled sunflower oilseed meals used as the protein sources, or a shift in feed resources in the diet formula (R) with extruded linseed (Valorex, France) as the source of n-3 PUFA and extruded faba bean (French origin) as the main local protein source. The experiment was based on a 2 × 2 factorial design with 4 experimental groups (n = 15 pigs per group), namely DR, DC, PR and PC. In each group, the

nutritional value of the diet, i.e. the ratio of digestible lysine (LysD) to net energy (NE), was adjusted weekly, in order to control the pig's growth rate by dietary protein supply. Thus, the LysD:NE ratio (g/MJ) varied progressively along the experimental period from 1 to 0.70 in the DC and DR groups, and from 0.95 to 0.68 in the PC and PR groups. Pig's requirements along growth were determined for each genotype based on their growth potential, targeted feed intake and assumptions using the INRAPorc software (van Milgen et al., 2008). The Lys requirements were calculated considering an expected protein deposition as 170 g per kg of expected growth, and majored by 10 % to account for individual variability within the group (van Milgen et al., 2008). During the experiment, pig's requirements were calculated weekly for each group, according to the average per group of individual BWs, that were determined every two weeks.

For all pig groups, the diet was prepared each week by mixing the correct ratio of two R and C formulae, respectively, containing either high and low protein and Lys levels. The Table 1 shows the composition and nutritional value of the C and R high and low-protein formulae. Altogether, the R formulae contained more crude fiber, and much more α-linolenic acid (C18:3 n-3, ALA) which is the precursor of the omega-3 (n-3) family of FA, so that the n-6:n-3 PUFA ratio was 8.7 times lower in the R than in the C formulae. The R and C formulae contained the same amount of mineral-vitamin blend, but the R formulae also contained 0.2 % of plant-derived antioxidants to prevent PUFA oxidation and the amount of vitamin E in the R formulae was three times that in the C formulae. All pigs had free access to feed and water. Individual feed consumption was measured weekly (feed offered minus refusals) and average daily feed intake, average daily gain and feed efficiency were calculated per pig over the experimental period.

### 2.2. Blood collection and adipose tissue sampling

Pigs were slaughtered at the INRAE experimental slaughterhouse (UE3P, 35590 Saint-Gilles, France) on three different days, with the same number of pigs from each experimental group on each day. All pigs were fasted for 24 h prior to slaughter. Pigs were killed by exsanguination after electronarcosis. At exsanguination, jugular venous blood was collected on heparinized Vacutainer tubes (9 mL), immediately centrifuged (2500 xg, 4 °C, 10 min), and plasma was aliquoted and stored at -76 °C until analysis. Thirty minutes after slaughter, samples of dorsal SCAT were taken from the right side of each carcass at the level of the last rib, cut into small pieces, immediately frozen in liquid nitrogen and stored at -76 °C until analysis of lipogenic, oxidative and antioxidant enzymatic activities. The carcasses were weighed and their lean meat content was determined using the Lean Fat Sensor (CGM device, Fives Syleps, Lorient, France) based on measurements of muscle (M2) and backfat (G2) depths and the equation developed by Daumas et al. (2010). This equation was lean meat content (%) = $62.19-0.729 \times G2 + 0.144 \times M2$. After carcass chilling at 4 °C for 24 h, the entire backfat (dorsal SCAT) was dissected from the left side of the carcass, weighed, and expressed relative to the chilled carcass weight as a surrogate for body adiposity. On the same day, a 100 g sample of SCAT (encompassing the whole tissue thickness) was taken at the level of the second last rib, vacuum packaged and stored at -20 °C until analysis of lipid content and fatty acids (FA) profile.

### 2.3. Lipid and fatty acid composition

Lipid content was determined on SCAT samples after chloroform-methanol (2:1 v/v) extraction (Folch et al., 1957). The results were expressed in grams of lipids per 100 g of wet tissue weight. Fatty acid (FA) profiles were determined after methylation with boron trifluoride-methanol according to the procedure developed by Morrison and Smith (1964). The FA methyl esters were saponified with a methanolic sodium hydroxide solution in the presence of heptadecanoic (C17:0) acid (H3500, Sigma Aldrich) as the internal FA standard. They were then
<table><caption>**Table 1**
Composition and nutritional value of the formula used in the R (relocation of feed resources) and Control (C) feeding strategies.</caption><thead><tr><th rowspan="2"></th><th colspan="2">R</th><th colspan="2">C</th></tr><tr><th>High protein</th><th>Low protein</th><th>High protein</th><th>Low protein</th></tr></thead><tbody><tr><td>Ingredients, % (as fed basis)</td><td></td><td></td><td></td><td></td></tr><tr><td>Wheat</td><td>17.41</td><td>21.03</td><td>22.13</td><td>20.98</td></tr><tr><td>Maize</td><td>17.41</td><td>21.03</td><td>22.13</td><td>20.98</td></tr><tr><td>Barley</td><td>17.41</td><td>21.03</td><td>22.13</td><td>20.98</td></tr><tr><td>Wheat bran</td><td>3.22</td><td>18.63</td><td>4.00</td><td>19.92</td></tr><tr><td>Dehydrated beet pulp</td><td>1.61</td><td>9.32</td><td>2.00</td><td>9.96</td></tr><tr><td>Soybean meal 48</td><td>4.75</td><td>–</td><td>14.28</td><td>0.94</td></tr><tr><td>Rapeseed meal</td><td>–</td><td>–</td><td>3.10</td><td>–</td></tr><tr><td>Shelled sunflower meal</td><td>–</td><td>–</td><td>3.10</td><td>–</td></tr><tr><td>Extruded linseed</td><td>3.52</td><td>3.52</td><td>–</td><td>–</td></tr><tr><td>Faba bean<sup>1</sup></td><td>29.28</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Cane molasses</td><td>2.00</td><td>2.00</td><td>2.00</td><td>2.00</td></tr><tr><td>Rapeseed oil</td><td>–</td><td>0.50</td><td>–</td><td>–</td></tr><tr><td>Sunflower oil</td><td>–</td><td>–</td><td>1.81</td><td>1.50</td></tr><tr><td>L-Lysine HCl</td><td>0.23</td><td>0.27</td><td>0.43</td><td>0.28</td></tr><tr><td>Mineral-vitamin mix<sup>2</sup></td><td>0.50</td><td>0.50</td><td>0.50</td><td>0.50</td></tr><tr><td>Antioxidants (Sevefarm®)<sup>3</sup></td><td>0.20</td><td>0.20</td><td>–</td><td>–</td></tr><tr><td>Analyzed chemical composition, %<sup>4</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>Dry matter</td><td>88.39</td><td>87.92</td><td>87.36</td><td>87.64</td></tr><tr><td>Crude protein</td><td>17.60</td><td>12.11</td><td>16.32</td><td>12.00</td></tr><tr><td>Crude fat</td><td>3.24</td><td>3.59</td><td>3.73</td><td>3.86</td></tr><tr><td>Crude fiber</td><td>4.23</td><td>4.44</td><td>3.87</td><td>3.87</td></tr><tr><td>Starch</td><td>42.18</td><td>41.39</td><td>40.75</td><td>41.71</td></tr><tr><td>Ash</td><td>4.96</td><td>4.96</td><td>4.84</td><td>4.74</td></tr><tr><td>Gross energy, MJ/kg</td><td>16.21</td><td>15.90</td><td>16.43</td><td>16.08</td></tr></tbody></table>

<table><caption>Fatty acid (FA) composition, % of identified FA<sup>5</sup></caption><tbody><tr><td>C10:0</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C12:0</td><td>0.02</td><td>0.03</td><td>0.06</td><td>0.03</td></tr><tr><td>C14:0</td><td>0.11</td><td>0.11</td><td>0.12</td><td>0.12</td></tr><tr><td>C14:1</td><td>0.03</td><td>0.04</td><td>0.02</td><td>0.05</td></tr><tr><td>C15:0</td><td>0.07</td><td>0.08</td><td>0.06</td><td>0.08</td></tr><tr><td>C16:0</td><td>11.74</td><td>12.22</td><td>11.00</td><td>12.30</td></tr><tr><td>C16:1 n-9</td><td>0.05</td><td>0.07</td><td>0.05</td><td>0.06</td></tr><tr><td>C16:1 n-7</td><td>0.14</td><td>0.17</td><td>0.14</td><td>0.15</td></tr><tr><td>C18:0</td><td>2.82</td><td>2.01</td><td>2.53</td><td>2.25</td></tr><tr><td>C18:1 n-9</td><td>20.90</td><td>25.30</td><td>27.48</td><td>25.71</td></tr><tr><td>C18:1 n-7</td><td>0.85</td><td>1.13</td><td>1.00</td><td>0.79</td></tr><tr><td>C18:2 n-6 t</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C18:2 n-6c</td><td>43.22</td><td>42.20</td><td>53.91</td><td>54.42</td></tr><tr><td>C18:3 n-6</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C18:3 n-3 (ALA)<sup>6</sup></td><td>18.90</td><td>15.26</td><td>2.30</td><td>2.66</td></tr><tr><td>C18:4 n-3</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C20:0</td><td>0.37</td><td>0.30</td><td>0.31</td><td>0.27</td></tr><tr><td>C20:1 n-9</td><td>0.33</td><td>0.58</td><td>0.31</td><td>0.40</td></tr><tr><td>C20:2</td><td>0.00</td><td>0.10</td><td>0.00</td><td>0.04</td></tr><tr><td>C20:3 n-6</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C20:3 n-3</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C20:4 n-6</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C20:4 n-3</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C20:5 n-3</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C22:0</td><td>0.24</td><td>0.22</td><td>0.44</td><td>0.41</td></tr><tr><td>C22:1</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C22:1 n-9</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C22:4 n-6</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C22:5 n-6</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C22:5 n-3</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C22:6 n-3</td><td>0.00</td><td>0.00</td><td>0.00</td><td>0.00</td></tr><tr><td>C24:0</td><td>0.21</td><td>0.17</td><td>0.28</td><td>0.28</td></tr></tbody></table>

<table><caption>**Table 1 (continued)**</caption><caption>Families of FA (sum: Σ), % of identified FA</caption><tbody><tr><td>Σ Saturated (SFA)</td><td>15.58</td><td>15.15</td><td>14.79</td><td>15.74</td></tr><tr><td>Σ Monounsaturated (MUFA)</td><td>22.29</td><td>27.29</td><td>29.00</td><td>27.14</td></tr><tr><td>Σ Polyunsaturated (PUFA)</td><td>62.12</td><td>57.56</td><td>56.21</td><td>57.12</td></tr><tr><td>Σ n-6</td><td>43.22</td><td>42.20</td><td>53.91</td><td>54.42</td></tr><tr><td>Σ n-3</td><td>18.90</td><td>15.26</td><td>2.30</td><td>2.66</td></tr><tr><td>Σ n-6 / Σ n-3 PUFA</td><td>2.29</td><td>2.77</td><td>23.55</td><td>20.48</td></tr><tr><td>C18:2 n-6:C18:3 n-3</td><td>2.29</td><td>2.77</td><td>23.55</td><td>20.48</td></tr></tbody></table>

<table><thead><tr><th rowspan="2"></th><th colspan="2">R</th><th colspan="2">C</th></tr><tr><th>High protein</th><th>Low protein</th><th>High protein</th><th>Low protein</th></tr></thead><tbody><tr><td>Calculated composition<sup>7</sup></td><td></td><td></td><td></td><td></td></tr><tr><td>Digestible lysine (LysD), %</td><td>0.97</td><td>0.52</td><td>0.97</td><td>0.52</td></tr><tr><td>Net energy (NE), MJ/kg</td><td>9.71</td><td>9.35</td><td>9.67</td><td>9.21</td></tr><tr><td>LysD:NE, g/MJ</td><td>1.00</td><td>0.55</td><td>1.00</td><td>0.57</td></tr><tr><td>Vitamin E, mg/100 g</td><td>60</td><td>60</td><td>20</td><td>20</td></tr></tbody></table>

¹ 90 % faba beans +10 % soy beans extruded together.

² Contains 4000 mg/kg vitamin E.

³ Contains 20,000 mg/kg vitamin E, 50000 mg/kg vitamin C, and aromatic substances.

⁴ Analyzed as described by Lebret et al. (2023) and expressed relative to fresh feed for dry matter, and to standardized dry matter of 88.0 % for all other components within each diet.

⁵ Fatty acid (FA) composition of diets analyzed by gas chromatography after chloroform-methanol extraction of lipids as described for FA contents of subcutaneous fat.

⁶ ALA: Alpha linolenic acid.

⁷ Calculated values as described by Furbeyre et al. (2020) and expressed on a fresh feed basis, except vitamin E that was calculated from its concentration in the ingredients of each diet.

methylated with boron trifluoride (140 g BF3 per liter of methanol; reagent was purchased from Sigma). FA methyl esters were recovered with a solution made with pentane (purchased from Carlo Erba) and distilled water. They were then analyzed via gas chromatography (Agilent Technologies 7890 A, Santa Clara, CA, USA) equipped with an injector, a capillary column (30 m × 0.25 mm internal diameter) filled with a stationary phase containing 50 % cyanopropylphenyl and 50 % dimethylpolysiloxane (Agilent Technologies), and a flame ionization detector (280 °C). The temperature treatment began at 150 °C and increased 4 °C per minute up to 220 °C, where it was maintained for 10 min. Injector and flame-ionization detector temperatures were kept constant at 220 °C and 280 °C, respectively. Hydrogen was the carrier gas. Retention times and peaks were determined using the chromatography software Agilent ChemStation. Retention times and peak areas were determined for all samples. The identities of the peaks were determined by comparison with the retention times of standard FA methyl esters. The amount of each FA was calculated as a function of the internal standard.

The FA composition of SCAT lipids was expressed as the percentage of the identified FA (g/100 g lipids). To facilitate comparison with the dietary requirements for human nutrition, the FA profile was also expressed as amount of each FA (mg/100 g of tissue).

## 2.4. Lipogenic enzymes

Samples were homogenized in ice-cold 0.25 M sucrose solution containing EDTA (1 mM) and dithiothreitol (DTT; 1 mM). The mixtures were ultra-centrifuged at 100,000 xg for 1 h and at 4 °C. The resulting supernatants containing cytosolic proteins were collected. Activities of fatty acid synthase (FASN), which synthesizes palmitate as the primary end product of de novo FA synthesis from glucose, and of malic enzyme (ME1) and glucose-6-phosphate dehydrogenase (G6PDH), which generate NADPH for de novo FA synthesis, were determined by spectrometry at 340 nm absorbance after the decrease (FASN) or appearance (ME1, G6PDH) of NADPH, as previously described (Castellano et al., 2015). The volumes of supernatants and reagents were adjusted to be suitable for measurements on a clinical chemistry analyzer (Konelab 20i, Thermo Fisher Scientific, Courtaboeuf, France). Cytosolic protein content was measured (Bradford, 1976) to calculate specific enzyme activities (units/min and per mg protein).
## 2.5. Oxidative and antioxidant enzymes

Samples were homogenized in phosphate buffer (pH 7.4) containing 2 mM EDTA. Mixtures were sonicated (60 s, 50 Hz; Bioblock scientific, Illkirch, France), centrifuged (at 1500 kg, 4 °C) during 13 min, and the resulting supernatants were stored on ice. The activities of the mitochondrial citrate synthase (CS), which converts pyruvate to citrate in the tricarboxylic acid cycle, and of L-3-hydroxyacyl coenzyme A dehydrogenase (β-HAD), which is involved in FA beta oxidation, were immediately determined by spectrometry (KoneLab 20i analyzer), at 30 °C at 340 nm absorbance for β-HAD and 405 nm for CS, respectively, as previously described (Sierżant et al., 2019).

To evaluate the endogenous antioxidant capacity of SCAT, samples were homogenized in 0.8 mL ice-cold sucrose buffer (0.5 M) containing 0.05 M Tris-HCl and 1 mM EDTA (pH 7.4). The homogenates were centrifuged at 10,000 kg for 30 min at 4 °C, and the supernatant was collected. Enzyme activity levels were measured spectrophotometrically, as previously described (Castellano et al., 2015; Sierżant et al., 2019). Briefly, the copper-zinc superoxide dismutase (SOD) activity, which is responsible for the elimination of cytotoxic reactive oxygen generated in mitochondria, was measured at 450 nm by inhibiting the xanthine/xanthine oxidase-mediated oxidation of cytochrome c using a specific kit (19,160 SOD, Sigma Aldrich, France) and a multimode microplate reader (Varioskan™, Thermofisher, Franklin, MA). The catalase (CAT) activity, which detoxifies H₂O₂ to water and oxygen, was measured spectrophotometrically (Uvikon Bio-Tek device, Secomam, Alès, France) at 240 nm at 25 °C. The activities of glutathione peroxidase (GPX), which converts H₂O₂ to water by forming oxidized glutathione (GSSG) from reduced glutathione (GSH), and of glutathione reductase (GRX), which catalyzes the reduction of GSSG and the regeneration of GSH, were evaluated at 37 °C by following the NADPH decrease at 340 nm absorbance (KoneLab 20i clinical analyzer). The protein content in each homogenate was then measured (Bradford, 1976) to calculate the specific enzyme activities (units/min and per mg protein).

## 2.6. Lipid peroxidation

We assessed the intensity of lipid peroxidation in SCAT samples in the basal state as well as along a forced oxidation kinetics induced by iron trichloride and sodium ascorbate. The malondialdehyde (MDA) as the product of lipid peroxidation at the initial time (T0) and the MDA-thiobarbituric acid reactive substances (TBARS) contents produced under these acidic conditions after 75, 150, 225 and 300 min of incubation time (T) at 37 °C were measured. As previously described (Sierżant et al., 2019), the method is based on the formation due to the presence of TBARS of a pink complex with strong absorption at 532–535 nm monitored on a spectrophotometer (Uvikon Bio-Tek device, Secomam, Alès, France). Values were expressed as mg of MDA per kg of SCAT.

## 2.7. Systemic oxidative stress

The concentrations of determinants of reactive oxygen metabolites (dROM) were measured in plasma samples (250 µL) as a surrogate of ROS, by using a dROM test (Diacron, Grosseto, Italy) on the Konelab analyzer. Total antioxidant capacity in plasma was estimated by the FRAP method, which is based on the reduction of 2,4,6-tripyridyl-s-triazine (TPTZ) with ferric chloride hexahydrate which produced blue iron complexes with an intense blue color. Final absorbance was measured at 595 nm after 30 min incubation at 37 °C in the dark using the microplate reader, as previously described (Sierżant et al., 2019). The plasma oxidative stress index (OSI) was calculated as the ratio of dROM to FRAP (dROM:FRAP).

## 2.8. Plasma metabolites

Plasma glucose, triglycerides (TAG), phospholipids (PPL) and non-esterified fatty acids (FFA) concentrations were determined in duplicate using dedicated kits on the Konelab 20i. The suppliers were Bio-Mérieux (Marcy l'Etoile, France) for the colorimetric kits used to assess glucose (Glucose RTU) and TAG (Triglyceride Enzymatique PAP 150), Wako Chemicals GmbH (Neuss, Germany) for FFA (NEFA-HR2 kit) and Sobioda (Montbonnot Saint Martin, France) for PPL (W1306170). Intra-assay coefficients of variation for the measurements were less than 5%.

## 2.9. Statistical analyses

Statistical analyses were performed using SAS software v9.4 (SAS Institute, Cary NC, New-York) with the pig as the observational unit. For all traits, MIXED procedures with genotype (G), feeding strategy (F) and the interaction between genotype and feeding (G x F) as the main effects, were used to analyze data. For TBARS, the model included also the time effect (T) and a repeated statement to indicate that data from the same animal were correlated during the forced oxidation kinetics; for that, the RANDOM statement was used to specify that pig is the random effect term along with the covariance structure (TYPE = CS). For MDA and TBARS concentrations, data were log-transformed to fit a normal distribution before statistical analyses. In all analyses, *P* ≤ 0.05 was considered significant, and 0.05 < *P* ≤ 0.10 was discussed as a trend. Data are presented by the least square means (Lsmeans) for the main effects and pooled standard error of the mean (SEM). When significant interaction effects were observed, charts also illustrate the results for the four experimental groups (PR, PC, DR and DC).

Principal component analysis (PCA) was performed to provide an overview of the correlation matrix for metabolic traits and FA profile in SCAT and systemic metabolic outcomes. The PCA transforms the original (mean-centered) observations into a new set of variables using the eigenvectors and eigenvalues computed from a covariance matrix of the original variables. Each data point was projected onto principal components (dimensions) defined as the eigenvectors each obtained by the linear combination of the original variables that maximized the variance of the projected data. In doing so, the PCA provides a graphical way to facilitate the interpretation of the relationships between the different traits. The PCA was performed using the PRINCOMP procedure of SAS. The three first components of the PCA, called dim_i_with *i* = 1 to 3, were considered.

# 3. Results

## 3.1. Growth performance and carcass traits

Regarding growth performance, results recently published (Lebret et al., 2023) indicated that genotype and feeding strategy influenced growth without any significant interactions between these factors (Table 2). The D pigs tended to eat more on a daily basis (*P* = 0.07), but had a lower feed efficiency than the P pigs (*P* < 0.001); this resulted in a lower average growth rate and a lower slaughter BW for the D pigs (*P* < 0.001). The effect of diet on growth traits was of lesser magnitude. Pigs fed the R diet had a higher average daily feed intake and a better feed efficiency (*P* < 0.05), resulting in a higher growth rate and a higher final BW than pigs fed the C diet (*P* < 0.01). There was however a trend (*P* = 0.053) towards a G x F interaction for final BW, so that the effect of the R diet tended to be more pronounced in the D pigs than in the P pigs (data not shown). The D pigs had a lower carcass lean meat content (*P* < 0.001) and a higher proportion of SCAT (*P* = 0.003) than P pigs, irrespective of the feeding strategy. The diet had no influence on SCAT proportion (*P* > 0.10).
<caption>**Table 2**</caption>

Growth performance and carcass traits in Duroc (D) and Pietrain (P) crossbred pigs fed relocated feed resources (R) or a control (C) diet (Lebret et al., 2023).

<table><thead><tr><th rowspan="2"></th><th colspan="2">Genotype</th><th colspan="2">Feeding</th><th rowspan="2">SEM</th><th colspan="3">Probability<sup>1</sup></th></tr><tr><th>P</th><th>D</th><th>R</th><th>C</th><th>G</th><th>F</th><th>GxF</th></tr></thead><tbody><tr><td>Final BW, kg<sup>2</sup></td><td>120</td><td>113</td><td>119</td><td>114</td><td>0.9</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td><strong>0.053</strong></td></tr><tr><td>ADG, g</td><td>1036</td><td>943</td><td>1020</td><td>959</td><td>14</td><td><strong>&lt;0.001</strong></td><td><strong>0.003</strong></td><td>0.14</td></tr><tr><td>ADFI, g</td><td>2729</td><td>2803</td><td>2812</td><td>2720</td><td>29</td><td><strong>0.072</strong></td><td><strong>0.03</strong></td><td>0.14</td></tr><tr><td>FE</td><td>0.38</td><td>0.34</td><td>0.36</td><td>0.35</td><td>0.003</td><td><strong>&lt;0.001</strong></td><td><strong>0.02</strong></td><td>0.41</td></tr><tr><td>Lean meat content, %</td><td>61.7</td><td>60.4</td><td>61.0</td><td>61.0</td><td>0.2</td><td><strong>&lt;0.001</strong></td><td>0.85</td><td>0.26</td></tr><tr><td>SCAT, %</td><td>5.90</td><td>6.49</td><td>6.24</td><td>6.16</td><td>0.13</td><td><strong>0.003</strong></td><td>0.65</td><td>0.25</td></tr></tbody></table>

## 3.2. Lipid composition in SCAT

The lipid content of SCAT did not differ according to pig genotype or

diet, but the FA composition of total lipids in SCAT was significantly affected by these factors (Table 3). The proportion of saturated fatty acids (SFA) in SCAT lipids was higher in D pigs than in P pigs (P =

<caption>**Table 3**</caption>

Lipid content and fatty acid composition of subcutaneous adipose tissue in Pietrain (P) and Duroc (D) crossbred pigs fed relocated feed resources (R) or a control (C) diet.

<table><thead><tr><th rowspan="2"></th><th colspan="2">Genotype</th><th colspan="2">Feeding</th><th rowspan="2">SEM</th><th colspan="3">Probability<sup>1</sup></th></tr><tr><th>P</th><th>D</th><th>R</th><th>C</th><th>G</th><th>F</th><th>GxF</th></tr></thead><tbody><tr><td>Lipids, %</td><td>73.29</td><td>72.27</td><td>72.76</td><td>72.80</td><td>0.76</td><td>0.35</td><td>0.96</td><td>0.56</td></tr><tr><td>Individual fatty acids, % lipids</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C10:0</td><td>0.07</td><td>0.07</td><td>0.07</td><td>0.07</td><td>0.001</td><td><strong>0.08</strong></td><td>0.21</td><td>0.28</td></tr><tr><td>C12:0</td><td>0.07</td><td>0.07</td><td>0.07</td><td>0.07</td><td>0.002</td><td><strong>0.01</strong></td><td>0.34</td><td>0.62</td></tr><tr><td>C14:0</td><td>1.17</td><td>1.13</td><td>1.15</td><td>1.14</td><td>0.01</td><td><strong>0.07</strong></td><td>0.65</td><td>0.93</td></tr><tr><td>C14:1</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.003</td><td>0.88</td><td>0.46</td><td>0.35</td></tr><tr><td>C15:0</td><td>0.06</td><td>0.06</td><td>0.06</td><td>0.06</td><td>0.002</td><td><strong>0.04</strong></td><td>0.11</td><td>0.28</td></tr><tr><td>C16:0</td><td>22.3</td><td>22.8</td><td>22.7</td><td>22.3</td><td>0.17</td><td><strong>0.04</strong></td><td>0.10</td><td>0.45</td></tr><tr><td>C16:1 n-9</td><td>0.42</td><td>0.40</td><td>0.40</td><td>0.42</td><td>0.01</td><td><strong>0.04</strong></td><td><strong>0.04</strong></td><td><strong>0.06</strong></td></tr><tr><td>C16:1 n-7</td><td>1.58</td><td>1.57</td><td>1.64</td><td>1.51</td><td>0.03</td><td>0.94</td><td><strong>0.01</strong></td><td>0.44</td></tr><tr><td>C18:0</td><td>13.3</td><td>14.4</td><td>13.9</td><td>13.8</td><td>0.20</td><td><strong>&lt;0.001</strong></td><td>0.60</td><td>0.30</td></tr><tr><td>C18:1 n-9</td><td>36.0</td><td>36.4</td><td>36.0</td><td>36.3</td><td>0.25</td><td>0.27</td><td>0.46</td><td>0.68</td></tr><tr><td>C18:1 n-7</td><td>2.04</td><td>2.07</td><td>2.11</td><td>2.00</td><td>0.02</td><td>0.44</td><td><strong>0.003</strong></td><td>0.54</td></tr><tr><td>C18:2 n-6 t</td><td>0.10</td><td>0.10</td><td>0.10</td><td>0.10</td><td>0.008</td><td>0.89</td><td>0.93</td><td>0.14</td></tr><tr><td>C18:2 n-6c</td><td>17.5</td><td>16.0</td><td>15.0</td><td>18.5</td><td>0.30</td><td><strong>0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.34</td></tr><tr><td>C18:3 n-6</td><td>0.06</td><td>0.05</td><td>0.05</td><td>0.07</td><td>0.003</td><td>0.06</td><td><strong>&lt;0.001</strong></td><td>0.58</td></tr><tr><td>C18:3 n-3</td><td>2.57</td><td>2.21</td><td>3.82</td><td>0.96</td><td>0.05</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.29</td></tr><tr><td>C18:4 n-3</td><td>0.07</td><td>0.08</td><td>0.07</td><td>0.08</td><td>0.003</td><td><strong>0.03</strong></td><td><strong>0.08</strong></td><td>0.64</td></tr><tr><td>C20:0</td><td>0.24</td><td>0.26</td><td>0.25</td><td>0.25</td><td>0.007</td><td>0.07</td><td>0.98</td><td>0.26</td></tr><tr><td>C20:1 n-9</td><td>0.67</td><td>0.67</td><td>0.67</td><td>0.68</td><td>0.02</td><td>0.99</td><td>0.72</td><td>0.68</td></tr><tr><td>C20:2</td><td>0.73</td><td>0.68</td><td>0.63</td><td>0.78</td><td>0.02</td><td>0.08</td><td><strong>&lt;0.001</strong></td><td>0.06</td></tr><tr><td>C20:3 n-6</td><td>0.09</td><td>0.09</td><td>0.08</td><td>0.11</td><td>0.004</td><td>0.48</td><td><strong>&lt;0.001</strong></td><td>0.18</td></tr><tr><td>C20:3 n-3</td><td>0.33</td><td>0.30</td><td>0.49</td><td>0.14</td><td>0.009</td><td><strong>0.04</strong></td><td><strong>&lt;0.001</strong></td><td>0.51</td></tr><tr><td>C20:4 n-6</td><td>0.21</td><td>0.18</td><td>0.15</td><td>0.25</td><td>0.008</td><td><strong>0.04</strong></td><td><strong>&lt;0.001</strong></td><td>0.52</td></tr><tr><td>C20:4 n-3</td><td>0.03</td><td>0.02</td><td>0.03</td><td>0.02</td><td>0.002</td><td>0.23</td><td><strong>&lt;0.001</strong></td><td>0.76</td></tr><tr><td>C20:5 n-3</td><td>0.05</td><td>0.04</td><td>0.06</td><td>0.03</td><td>0.002</td><td>0.22</td><td><strong>&lt;0.001</strong></td><td>0.69</td></tr><tr><td>C22:0</td><td>0.02</td><td>0.02</td><td>0.02</td><td>0.02</td><td>0.003</td><td>0.87</td><td>0.75</td><td>0.12</td></tr><tr><td>C22:1 n-11</td><td>0.03</td><td>0.02</td><td>0.03</td><td>0.02</td><td>0.003</td><td>0.20</td><td>0.43</td><td>0.44</td></tr><tr><td>C22:1 n-9</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.01</td><td>0.003</td><td>0.98</td><td>0.53</td><td>0.11</td></tr><tr><td>C22:4 n-6</td><td>0.07</td><td>0.06</td><td>0.05</td><td>0.09</td><td>0.003</td><td><strong>0.01</strong></td><td><strong>&lt;0.001</strong></td><td>0.72</td></tr><tr><td>C22:5 n-6</td><td>0.09</td><td>0.09</td><td>0.09</td><td>0.09</td><td>0.01</td><td>0.85</td><td>0.71</td><td>0.90</td></tr><tr><td>C22:5 n-3</td><td>0.14</td><td>0.13</td><td>0.19</td><td>0.08</td><td>0.006</td><td>0.12</td><td><strong>&lt;0.001</strong></td><td>0.27</td></tr><tr><td>C22:6 n-3</td><td>0.05</td><td>0.05</td><td>0.05</td><td>0.05</td><td>0.004</td><td>0.79</td><td>0.33</td><td>0.22</td></tr><tr><td>Contribution of families of fatty acids (Σ: sum), % lipids<sup>2</sup></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Σ SFA</td><td>37.2</td><td>38.8</td><td>38.3</td><td>37.7</td><td>0.34</td><td><strong>0.001</strong></td><td>0.25</td><td>0.31</td></tr><tr><td>Σ MUFA</td><td>40.7</td><td>41.1</td><td>40.9</td><td>40.9</td><td>0.28</td><td>0.32</td><td>0.87</td><td>0.87</td></tr><tr><td>Σ PUFA</td><td>22.1</td><td>20.1</td><td>20.8</td><td>21.4</td><td>0.36</td><td><strong>&lt;0.001</strong></td><td>0.34</td><td>0.28</td></tr><tr><td>Σ n-6</td><td>18.1</td><td>16.5</td><td>15.5</td><td>19.2</td><td>0.30</td><td><strong>0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.34</td></tr><tr><td>Σ n-3</td><td>3.2</td><td>2.8</td><td>4.7</td><td>1.4</td><td>0.07</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.31</td></tr><tr><td>Σ PUFA: Σ SFA</td><td>0.60</td><td>0.52</td><td>0.55</td><td>0.57</td><td>0.01</td><td><strong>&lt;0.001</strong></td><td>0.30</td><td>0.27</td></tr><tr><td>Σ n-6: Σ n-3</td><td>8.3</td><td>9.3</td><td>3.3</td><td>14.3</td><td>0.16</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td></tr><tr><td>C18:2 n-6: C18:3 n-3</td><td>10.8</td><td>12.8</td><td>4.0</td><td>19.6</td><td>0.16</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td></tr></tbody></table>

<footnote n="1">¹ P values of effects of genotype (G), feeding regimen (F) and their interaction (G x F). Values are shown as least square means for the main effects of genotype or feeding strategy (n = 30 pigs per genotype, and per feeding strategy) together with pooled SEM obtained from MIXED models applied to raw data.</footnote>

<footnote n="2">² Abbreviation used: BW: body weight; ADG: average daily gain; ADFI: average daily feed intake; FE: feed efficiency; SCAT: subcutaneous adipose tissue, calculated as relative percentage of the cold right carcass side.</footnote>

<footnote n="1">¹ Probability values of effects of genotype (G), feeding regimen (F) and their interaction (G x F). Values are shown as least square means for the main effects of genotype or feeding strategy (n = 30 pigs per genotype, and per feeding strategy) together with pooled SEM obtained from MIXED models applied to raw data.</footnote>

<footnote n="2">² Abbreviations used: SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.</footnote>
0.001), and this was mainly due to the higher proportions of C16:0 (palmitic acid) and C18:0 (stearic acid) in the lipids of D pigs compared to P pigs (P < 0.05). Conversely, PUFA were less represented in SCAT lipids of D pigs (P < 0.001). This was due to lower proportions of the *n*-6 family of FA (P = 0.001), including linoleic acid (LA, C18:2 *n*-6) and C20:4 *n*-6 (P < 0.05), and of the *n*-3 family of FA (P < 0.001), including mainly linolenic acid (ALA, C18:3 *n*-3) and C20:3 *n*-3 (P < 0.05). The proportions of eicosapentaenoic acid (EPA; C20:5 *n*-3) and docosahexaenoic acid (DHA; C22:6 *n*-3) in SCAT lipids did not differ between genotypes. Proportion of MUFA did not differ between D and P pigs.

Feeding the R or C diet did not change the relative proportions of SFA, MUFA and PUFA in SCAT. However, the feeding strategy specifically affected the proportions of *n*-6 and of *n*-3 PUFA (*P* < 0.001) and the *n*-6:*n*-3 PUFA ratio which was four times lower in pigs fed the R diet compared to the C diet (*P* < 0.001). This was because of the lower levels of LA (C18:2 *n*-6) and its derivatives C20:3 *n*-6, C20:4 *n*-6 and C22:4 *n*-6 (*P* < 0.001), and of the higher levels of ALA (C18:3 *n*-3) and its derivatives C20:3 *n*-3, C20:4 *n*-3, EPA (C20:5 *n*-3) and C22:5 *n*-3 (*P* <

0.001) in SCAT of pigs fed the R diet. Diet did not affect the relative abundance of C22:6 *n*-3 (DHA) in SCAT lipids. The decrease in the ratios of LA:ALA and of *n*-6:*n*-3 PUFA due to the R diet was more pronounced (G x F; *P* < 0.001) in D pigs than in P pigs (Fig. 1).

When the concentrations of FA were expressed per mg in 100 g of adipose tissue for easier comparison with the dietary requirements of the human population (Table 4), most of the effects or trends for the combinations of genotype and feeding strategies were the same as found when FA were expressed in percentage of identified FA, especially for MUFA, *n*-6 PUFA including LA, *n*-3 PUFA including ALA, EPA and DHA, and *n*-6:*n*-3 and PUFA:SFA ratios. The main differences were for the genotype by feeding strategy interaction effects, that were significant for SFA and more specifically for C16:0 and C18:0 (Fig. 2), with higher amounts in DR pigs than in the other three groups (G x F, *P* < 0.05).

### 3.3. Enzyme activities

The activities of the lipogenic enzymes were affected by the pig

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Ratio of *n*-6 to *n*-3 families of polyunsaturated fatty acids in subcutaneous adipose tissue from Duroc (D) and Pietrain (P) crossbred pigs fed a relocated (R) or control (C) diet.</figcaption></figure>

The fatty acids (FA) composition was determined by gas chromatography as detailed in material and methods. Effects of genotype (G), feeding strategy (F) and their interactions G x F were tested using a MIXED model. Interaction effects were shown (*P* < 0.001) for Σ *n*-6:Σ *n*-3 FA and for C18:2 *n*-6:C18:3 *n*-3. Data are means ± standard errors of the means for the four experimental groups (n = 15 pigs per group). a, b, c: means sharing a common letter did not differ (*P* > 0.05).
<table><caption>**Table 4**
Fatty acid amounts in subcutaneous adipose tissue of Duroc (D) and Pietrain (P) crossbred pigs fed relocated feed resources (R) or a control (C) diet.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Genotype</th><th colspan="2">Feeding</th><th rowspan="2">SEM</th><th colspan="3">Probability<sup>1</sup></th></tr><tr><th>P</th><th>D</th><th>R</th><th>C</th><th>G</th><th>F</th><th>GxF</th></tr></thead><tbody><tr><td colspan="9">Individual fatty acids, mg/100 g tissue</td></tr><tr><td>C10:0</td><td>50.6</td><td>47.1</td><td>49.7</td><td>48.0</td><td>1.18</td><td><strong>0.04</strong></td><td>0.30</td><td><strong>0.06</strong></td></tr><tr><td>C12:0</td><td>47.4</td><td>43.6</td><td>46.0</td><td>44.9</td><td>0.9</td><td><strong>0.01</strong></td><td>0.42</td><td>0.11</td></tr><tr><td>C14:0</td><td>798</td><td>752</td><td>780</td><td>770</td><td>14.8</td><td><strong>0.03</strong></td><td>0.64</td><td>0.15</td></tr><tr><td>C14:1</td><td>8.2</td><td>8.1</td><td>8.4</td><td>8.0</td><td>0.4</td><td>0.90</td><td>0.55</td><td>0.24</td></tr><tr><td>C15:0</td><td>38.3</td><td>40.8</td><td>38.1</td><td>41.0</td><td>1.2</td><td>0.15</td><td>0.11</td><td>0.68</td></tr><tr><td>C16:0</td><td>15,273</td><td>15,187</td><td>15,430</td><td>15,030</td><td>243</td><td>0.80</td><td>0.25</td><td><strong>0.048</strong></td></tr><tr><td>C16:1 n-9</td><td>289</td><td>264</td><td>267</td><td>285</td><td>6.9</td><td><strong>0.01</strong></td><td>0.07</td><td>0.39</td></tr><tr><td>C16:1 n-7</td><td>1078</td><td>1048</td><td>1109</td><td>1017</td><td>25</td><td>0.42</td><td><strong>0.01</strong></td><td>0.76</td></tr><tr><td>C18:0</td><td>9091</td><td>9597</td><td>9450</td><td>9238</td><td>181</td><td><strong>0.053</strong></td><td>0.41</td><td><strong>0.03</strong></td></tr><tr><td>C18:1 n-9</td><td>24,664</td><td>24,215</td><td>24,475</td><td>24,404</td><td>396</td><td>0.43</td><td>0.90</td><td>0.08</td></tr><tr><td>C18:1 n-7</td><td>1400</td><td>1379</td><td>1430</td><td>1350</td><td>25</td><td>0.54</td><td><strong>0.02</strong></td><td>0.35</td></tr><tr><td>C18:2 n-6 t</td><td>67.1</td><td>65.1</td><td>65.9</td><td>66.3</td><td>5.2</td><td>0.79</td><td>0.95</td><td><strong>0.08</strong></td></tr><tr><td>C18:2 n-6c</td><td>12,027</td><td>10,588</td><td>10,139</td><td>12,476</td><td>249</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.64</td></tr><tr><td>C18:3 n-6</td><td>44.2</td><td>36.6</td><td>31.3</td><td>49.5</td><td>2.3</td><td><strong>0.02</strong></td><td><strong>&lt;0.001</strong></td><td>0.99</td></tr><tr><td>C18:3 n-3</td><td>1744</td><td>1495</td><td>2587</td><td>652</td><td>43</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.68</td></tr><tr><td>C18:4 n-3</td><td>49.9</td><td>54.7</td><td>50.0</td><td>54.6</td><td>2.0</td><td>0.10</td><td>0.11</td><td>0.90</td></tr><tr><td>C20:0</td><td>163</td><td>172</td><td>169</td><td>166</td><td>5.4</td><td>0.27</td><td>0.72</td><td><strong>0.06</strong></td></tr><tr><td>C20:1 n-9</td><td>462</td><td>447</td><td>455</td><td>454</td><td>13</td><td>0.42</td><td>0.98</td><td>0.54</td></tr><tr><td>C20:2</td><td>498</td><td>454</td><td>427</td><td>525</td><td>11</td><td><strong>0.006</strong></td><td><strong>&lt;0.001</strong></td><td>0.54</td></tr><tr><td>C20:3 n-6</td><td>64.7</td><td>59.5</td><td>50.9</td><td>73.4</td><td>2.7</td><td>0.17</td><td><strong>&lt;0.001</strong></td><td>0.56</td></tr><tr><td>C20:3 n-3</td><td>224</td><td>204</td><td>334</td><td>93.9</td><td>7.3</td><td><strong>0.05</strong></td><td><strong>&lt;0.001</strong></td><td>0.33</td></tr><tr><td>C20:4 n-6</td><td>143</td><td>122</td><td>98.4</td><td>166</td><td>6.1</td><td><strong>0.02</strong></td><td><strong>&lt;0.001</strong></td><td>0.89</td></tr><tr><td>C20:4 n-3</td><td>18.6</td><td>15.8</td><td>22.8</td><td>11.5</td><td>1.7</td><td>0.24</td><td><strong>&lt;0.001</strong></td><td>0.94</td></tr><tr><td>C20:5 n-3</td><td>31.9</td><td>28.5</td><td>43.0</td><td>17.4</td><td>1.7</td><td>0.16</td><td><strong>&lt;0.001</strong></td><td>0.90</td></tr><tr><td>C22:0</td><td>14.8</td><td>14.0</td><td>14.0</td><td>14.9</td><td>1.9</td><td>0.77</td><td>0.73</td><td>0.10</td></tr><tr><td>C22:1 n-11</td><td>18.0</td><td>14.2</td><td>17.1</td><td>15.2</td><td>1.9</td><td>0.16</td><td>0.48</td><td>0.34</td></tr><tr><td>C22:1 n-9</td><td>6.6</td><td>6.1</td><td>7.0</td><td>5.7</td><td>1.9</td><td>0.87</td><td>0.61</td><td><strong>0.09</strong></td></tr><tr><td>C22:4 n-6</td><td>50.2</td><td>41.0</td><td>32.6</td><td>58.6</td><td>2.1</td><td><strong>0.003</strong></td><td><strong>&lt;0.001</strong></td><td>0.76</td></tr><tr><td>C22:5 n-6</td><td>58.4</td><td>59.6</td><td>57.6</td><td>60.4</td><td>6.9</td><td>0.91</td><td>0.78</td><td>0.96</td></tr><tr><td>C22:5 n-3</td><td>97.4</td><td>87.1</td><td>131</td><td>53.4</td><td>4.1</td><td><strong>0.08</strong></td><td><strong>&lt;0.001</strong></td><td>0.49</td></tr><tr><td>C22:6 n-3</td><td>34.1</td><td>33.3</td><td>35.3</td><td>32.1</td><td>2.8</td><td>0.85</td><td>0.41</td><td>0.34</td></tr><tr><td colspan="9">Contribution of families of fatty acids (Σ: sum), %<sup>2</sup></td></tr><tr><td>Σ SFA</td><td>25,476</td><td>25,854</td><td>25,977</td><td>25,353</td><td>422</td><td>0.53</td><td>0.30</td><td><strong>0.03</strong></td></tr><tr><td>Σ MUFA</td><td>27,926</td><td>27,381</td><td>27,768</td><td>27,539</td><td>443</td><td>0.39</td><td>0.72</td><td>0.10</td></tr><tr><td>Σ PUFA</td><td>15,153</td><td>13,344</td><td>14,107</td><td>14,390</td><td>295</td><td><strong>&lt;0.001</strong></td><td>0.50</td><td>0.73</td></tr><tr><td>Σ n-6</td><td>12,455</td><td>10,972</td><td>10,476</td><td>12,950</td><td>254</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.62</td></tr><tr><td>Σ n-3</td><td>2200</td><td>1918</td><td>3203</td><td>915</td><td>52</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td>0.75</td></tr><tr><td>Σ PUFA: Σ SFA</td><td>0.60</td><td>0.52</td><td>0.55</td><td>0.57</td><td>0.01</td><td><strong>&lt;0.001</strong></td><td>0.30</td><td>0.27</td></tr><tr><td>Σ n-6:Σ n-3</td><td>8.3</td><td>9.3</td><td>3.3</td><td>14.3</td><td>0.6</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td></tr><tr><td>C18:2 n-6:C18:3 n-3</td><td>10.8</td><td>12.8</td><td>4.0</td><td>19.6</td><td>0.8</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.001</strong></td></tr></tbody></table>

genotype, with higher activity levels of FASN (P < 0.05) and ME1 (P < 0.001) in the SCAT of D pigs than of P pigs, whereas the activity level of G6PDH did not differ between P and D pigs (Table 5). The diet did not affect activities of the lipogenic enzymes. On the contrary, mitochondrial enzyme activities were influenced by both genotype and diet. The CS activity in SCAT was higher in D pigs than in P pigs regardless of diet (P < 0.01), and HAD activity was higher when pigs were fed the R diet as compared with the C diet (P < 0.05) regardless of genotype. Among the antioxidant enzymes studied, only the activity of GPX was different between the genotypes of pigs, with D pigs having a lower activity of GPX in the SCAT than P pigs (P < 0.001). The activities of the four antioxidant enzymes studied were not affected by diet.

### 3.4. Lipid peroxidation

Pig genotype influenced TBARS (i.e. MDA) level, a widely used marker of lipid peroxidation, when measured in SCAT. The D pigs had higher levels of TBARS at T0 than P pigs (P < 0.001), and there was no difference between pigs fed the R or C diets. The levels of TBARS formed as secondary oxidation products of lipids in SCAT increased with time (T: P < 0.001) during the forced oxidation kinetics (Fig. 3) in the four

experimental groups. The levels of TBARS did not differ between the D and P pigs from 75 min of incubation onwards. Regardless of genotype, the time-related increase in TBARS levels was greater in the SCAT of pigs fed the R diet (P < 0.001) and lasted longer than for pigs fed with the C diet (feeding x time, P < 0.001). Indeed, the TBARS levels continuously increased up to 225 min for pigs fed the R diet, whereas the oxidation level reached it maximum after 150 min in SCAT of pigs fed the C diet.

### 3.5. Systemic metabolic outcomes

Plasma glucose concentrations were lower (P < 0.001) in the D pigs than in the P pigs (Table 6). Diet tended to have opposite effects on glycaemia in the two genotypes (G x F, P < 0.10). Compared to pigs fed the C diet, the R diet tended to decrease circulating glucose concentrations in D pigs but to increase them in P pigs: the values of plasma glucose concentrations for the four experimental groups were 5.04 mmol/L for DR pigs and 5.58 mmol/L for DC pigs, 6.03 mmol/L for PR pigs and 5.95 mmol/L for PC pigs, respectively. Circulating concentrations of free fatty acids (FFA) and triglycerides (TAG) were not affected by genotype and diet. As shown in Fig. 4, circulating concentrations of phospholipids (PPL) were reduced by the R vs. C diet in D but not P pigs

<footnote n="1">¹ P values of effects of genotype (G), feeding regimen (F) and their interaction (G x F). Values are shown as least square means for the main effects of genotype or feeding strategy (n = 30 pigs per genotype, and per feeding strategy) together with pooled SEM obtained from MIXED models applied to raw data.</footnote>

<footnote n="2">² Abbreviations used: MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Amounts of saturated fatty acids in subcutaneous adipose tissue of Duroc (D) and Pietrain (P) crossbred pigs fed relocated feed resources (R) or a control (C) diet.</figcaption></figure>

The fatty acids (FA) composition was determined by gas chromatography as detailed in material and methods. Effects of genotype (G), feeding strategy (F) and their interactions G x F were tested using a MIXED model. Interaction effects were shown (*P* < 0.001) for C16:0, C18:0 and the sum of saturated fatty acids (SFA). Data are means ± standard errors of the means for the four experimental groups (n = 15 pigs per group). a, b, c: means sharing a common letter did not differ (*P* > 0.05).
<table><caption>**Table 5**
Enzyme activities in subcutaneous adipose tissue of Duroc (D) and Pietrain (P) crossbred pigs fed relocated feed resources (R) or a control (C) diet.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Genotype</th><th colspan="2">Feeding</th><th rowspan="2">SEM</th><th colspan="3">Probability<sup>1</sup></th></tr><tr><th>P</th><th>D</th><th>R</th><th>C</th><th>G</th><th>F</th><th>GxF</th></tr></thead><tbody><tr><td>FASN<sup>2</sup></td><td>46.4</td><td>57.4</td><td>52.9</td><td>50.9</td><td>3.3</td><td><strong>0.02</strong></td><td>0.68</td><td>0.77</td></tr><tr><td>ME1</td><td>1093</td><td>1463</td><td>1325</td><td>1232</td><td>54</td><td><strong>&lt;0.001</strong></td><td>0.24</td><td>0.15</td></tr><tr><td>G6PDH</td><td>562</td><td>527</td><td>549</td><td>541</td><td>22</td><td>0.27</td><td>0.82</td><td>0.41</td></tr><tr><td>CS</td><td>0.45</td><td>0.51</td><td>0.49</td><td>0.46</td><td>0.01</td><td><strong>0.003</strong></td><td>0.15</td><td>0.19</td></tr><tr><td>HAD</td><td>0.40</td><td>0.40</td><td>0.42</td><td>0.37</td><td>0.01</td><td>0.82</td><td><strong>0.03</strong></td><td>0.20</td></tr><tr><td>GPX</td><td>95.0</td><td>66.2</td><td>79.4</td><td>81.9</td><td>5.7</td><td><strong>&lt;0.001</strong></td><td>0.76</td><td>0.48</td></tr><tr><td>GRX</td><td>15.8</td><td>15.6</td><td>16.3</td><td>14.9</td><td>1.3</td><td>0.91</td><td>0.44</td><td>0.94</td></tr><tr><td>SOD</td><td>42.3</td><td>41.2</td><td>43.1</td><td>40.4</td><td>3.3</td><td>0.82</td><td>0.57</td><td>0.90</td></tr><tr><td>CAT</td><td>280</td><td>270</td><td>272</td><td>278</td><td>6.3</td><td>0.29</td><td>0.52</td><td>0.53</td></tr></tbody></table>

¹ P values of effects of genotype (G), feeding regimen (F) and their interaction (G x F). Values are shown as least square means for the main effects of genotype or feeding strategy (n = 30 pigs per genotype, and per feeding strategy) together with pooled SEM obtained from MIXED models applied to raw data.

² Abbreviations used: FASN: fatty acid synthase; ME1: malic enzyme; G6PDH: glucose-6-phosphate dehydrogenase; CS: citrate synthase; HAD: beta-hydroxyacyl-coenzyme A dehydrogenase; GPX: glutathione peroxidase; GRX: glutathione reductase; SOD: superoxide dismutase; CAT: catalase. FAS, ME1, G6PDH, GRX, GPX, SOD and CAT are expressed in nmol/min and per mg proteins. CS and HAD are expressed in µmol/min and per mg proteins.

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Thiobarbituric reactive substances (TBARS) in subcutaneous adipose tissue from Duroc (D) and Pietrain (P) crossbred pigs fed a relocated (R) or control (C) diet.</figcaption></figure>

Thiobarbituric reactive substances (TBARS) were measured in subcutaneous adipose tissue at different time points along the forced chemical oxidation kinetics and expressed as malondialdehyde (MDA) concentration (mg/kg tissue) for the four experimental groups (n = 15 pigs per group). Effects of time (T), genotype (G), feeding strategy (F) and their interactions were tested using a MIXED model for repeated measures data. Values were first log-transformed to fit a normal distribution. For simplicity, statistics are reported only for paired comparisons between the consecutive times in each feeding strategy (R diet: dot line; C diet: solid line). ***: *P* < 0.001, **: *P* < 0.01, *t*: *P* < 0.10. Data are means ± standard errors of the means.

(G x F, *P* < 0.01).

The concentrations of determinants of reactive oxygen metabolites tended to differ between pig genotypes (*P* = 0.08), with higher dROM levels in the plasma of the D pigs than of the P pigs (Table 6). Plasma FRAP was lower in the D pigs than in the P pigs (*P* < 0.05). Feeding pigs with the R diet did not alter plasma dROM concentrations but this was associated with higher plasma FRAP levels when compared to the situation with C diet (*P* < 0.05). Consequently, as shown in Fig. 4, the oxidative stress index (OSI) was higher in the D pigs fed the C diet compared to the three other experimental groups (G x F, *P* < 0.05).

### 3.6. Correlated variation patterns of metabolic and composition traits

The PCA analysis was used to obtain an integrated view of the correlated changes between biochemical and metabolic traits in SCAT

and plasma indicators. The first three axes explained 44 % of the variance. The Fig. 5 shows the projection of the variables onto the correlation circle around dim 1 and dim 2. In the lower left quarter, C16:0, C18:0, C20:0 and the sum of all SFA were grouped together with the activity levels of FASN, ME1, HAD, SOD and GRX enzymes. This group of variables was contrasted with the *n*-6 family of PUFA which was represented by C18:2 *n*-6, C20:4 *n*-6, C22:4 *n*-6 and C20:2 in the upper right quarter. The *n*-3 family of PUFA was represented by C18:3 *n*-3, C20:3 *n*-3, C20:4 *n*-3, C20:5 *n*-3 and C22:5 *n*-3 in the upper left quarter; they contrasted with *n*-6:*n*-3 ratio, and especially the ratio of C18:2 *n*-6 (LA) to C18:3 *n*-3 (ALA), in the lower right quarter. The dim 3 (data not shown) opposed MUFA (C18:1 and C20:1 *n*-9) to short and medium chain FA (C10:0, C12:0 and C14:0).
<table><caption>**Table 6**

Circulating concentrations of metabolites and plasma redox status in Duroc (D) and Pietrain (P) crossbred pigs fed relocated feed resources (R) or a control (C) diet.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Genotype</th><th colspan="2">Feeding</th><th rowspan="2">SEM</th><th colspan="3">Probability<sup>1</sup></th></tr><tr><th>P</th><th>D</th><th>R</th><th>C</th><th>G</th><th>F</th><th>GxF</th></tr></thead><tbody><tr><td>Glucose, mmol/L</td><td>6.0</td><td>5.3</td><td>5.3</td><td>5.8</td><td>0.1</td><td><strong>&lt;0.001</strong></td><td>0.19</td><td><strong>0.08</strong></td></tr><tr><td>FFA<sup>2</sup>, µmol/L</td><td>1007</td><td>1152</td><td>1057</td><td>1101</td><td>85</td><td>0.23</td><td>0.72</td><td>0.59</td></tr><tr><td>TAG, mg/L</td><td>1022</td><td>1137</td><td>999</td><td>1159</td><td>74</td><td>0.28</td><td>0.13</td><td>0.19</td></tr><tr><td>PPL, mg/L</td><td>1146</td><td>1229</td><td>1168</td><td>1211</td><td>31</td><td><strong>0.06</strong></td><td>0.29</td><td><strong>0.006</strong></td></tr><tr><td>dROM, Carru</td><td>989</td><td>1065</td><td>1018</td><td>1037</td><td>30</td><td><strong>0.08</strong></td><td>0.66</td><td>0.13</td></tr><tr><td>FRAP, mg/mL</td><td>64.7</td><td>61.7</td><td>64.8</td><td>61.5</td><td>1.0</td><td><strong>0.04</strong></td><td><strong>0.02</strong></td><td>0.47</td></tr><tr><td>OSI</td><td>15.3</td><td>17.4</td><td>15.7</td><td>17.0</td><td>0.5</td><td><strong>0.005</strong></td><td><strong>0.07</strong></td><td>0.04</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Circulating concentrations of phospholipids and oxidative stress index in plasma of Duroc (D) and Pietrain (P) crossbred pigs fed relocated feed resources (R) or a control (C) diet.</figcaption></figure>

The oxidative stress index (OSI) was calculated as the ratio of the concentrations of determinants of reactive oxygen metabolites (dROM) to total antioxidant capacity estimated by the FRAP. Effects of genotype (G), feeding strategy (F) and their interactions G x F were tested using a MIXED model. Data are means ± standard errors of the means for the four experimental groups (n = 15 pigs per group). a, b, c: means sharing a common letter did not differ (P > 0.05).

<footnote n="1">¹ P values of effects of genotype (G), feeding regimen (F) and their interaction (G x F). Values are shown as least square means for the main effects of genotype or feeding strategy (n = 30 pigs per genotype, and per feeding strategy) together with pooled SEM obtained from MIXED models applied to raw data.</footnote>

<footnote n="2">² Abbreviations used: FFA: free fatty acids; PPL: phospholipids; TAG: triacylglycerides; dROM: reactive oxygen metabolites; FRAP: ferric reducing ability of plasma. Systemic reactive oxygen metabolites (dROM) were expressed as Carratelli Unit (1 CARRU = 0.08 mg H2O2/100 mL). Ferric reducing ability of plasma (FRAP), a measure of antioxidant power, was expressed as Trolox equivalents mg per mL. The oxidative stress index (OSI) was calculated as the ratio of dROM to FRAP.</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Correlation patterns between biochemical traits in subcutaneous adipose tissue and plasma.

Principal component analysis (PCA) was performed to provide an overview of the correlation matrix and to facilitate graphical interpretation of the relationships between the biochemical traits measured in subcutaneous adipose tissue and plasma of pigs. The relative proportions of each fatty acid (% of identified FA) and their families (PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids) were load together with specific activities (U/mg proteins) of the different enzymes, plasma metabolites and oxidative stress index (OSI). After mean centering and unit variance scaling, the data set was summarized by the first two principal components (dim 1, dim 2) obtained by a linear combination of the original variables that maximized the variance of the projected data. The figure shows the score plot of the first two principal components mapping variables. Positively correlated variables are grouped together, while variables in diagonally opposite quadrants are negatively correlated. Abbreviations used for variables can be found in the various tables.</figcaption></figure>

**4. Discussion**

The metabolic properties of SCAT were influenced by both pig genotype and feeding strategy in this study. The greater adiposity of the Duroc compared to the Pietrain crosses was explained by the greater lipogenic capacity of the D pigs. This was illustrated by their lower glycemia which suggests a greater glucose uptake by the SCAT, and by higher activities of citrate synthase (CS) which converts glucose carbons into the mitochondrial tricarboxylic acid cycle, and of two regulatory lipogenic enzymes (FASN and ME1) in SCAT of D pigs compared to P pigs. The variations in FASN and ME1 activities were closely related to variations in the proportions of SFA in SCAT lipids, namely palmitic acid and its elongation derivatives stearic acid and arachidic acid (C20:0) FA. This correlated pattern was only partly expected, considering that Marriott et al. (2013) found a positive relationship between FASN protein expression and SFA amount in the subcutaneous adipose tissue of Large White × Pietrain pigs but not of Duroc × Pietrain pigs. Finding lower proportion (% of total FA) and amount of PUFA (mg / 100 g adipose tissue) in SCAT of D pigs compared to P pigs is inconsistent with Alonso et al. (2009) who reported that SCAT of D x (Landrace x Large White) pigs tended to have higher proportion and amounts of *n*-3 PUFA than P x (Landrace x Large White) pigs. These differences are probably

due to differences in selection intensity for feed efficiency and leanness between the Duroc sire lines used between studies. Indeed, genetically leaner pigs may be less able to synthesize FA while having a greater mobilization of FA, at least when lean pigs were compared with autochthonous fat breeds (Poklukar et al., 2020 for a review). In our study, the variations in the activities of the lipogenic enzymes were negatively correlated with the proportion of PUFA and the ratio of PUFA to SFA. This is likely due to the “dilution effect” of dietary essential PUFA among endogenously synthesized saturated FA (Wood et al., 2008). In both genotypes and compared with the C, feeding the R diet rich in *n*-3 FA and fiber did not alter the activities of lipogenic enzymes in SCAT. The higher content in *n*-3 PUFA in the R diet as compared to the C diet was likely due to the inclusion of extruded linseed and rapeseed oil, as faba bean does not provide much more *n*-3 PUFA than soybean (Scerra et al., 2011). Therefore, our results agree with Kouba et al. (2003) who found no effects of *n*-3 enriched diet on acetyl-CoA carboxylase, ME1 and G6PDH activities in SCAT of Duroc crossbred gilts. Similarly, Guillevic et al. (2009) reported that a linseed diet rich in *n*-3 PUFA did not affect FASN activity, but they found a reduced ME1 activity in SCAT of pigs fed a linseed diet compared to a sunflower based diet. A goal for human nutrition is the reduction of *n*-6:*n*-3 PUFA ratio in both fat and lean tissues as animal-source foods. We observed that the
SCAT of D pigs had a slightly less favorable *n-6:n-3* ratio compared to P pigs, when fed the C diet. This is in contrast to Lebret et al. (2023) who reported no effect of genotype on the *n-6:n-3* ratio in the *Longissimus* muscle of the same pigs. However, when the pigs were fed an R diet, this disadvantage of the D pigs was corrected. On a metabolic point of view, this suggests that the deposition of dietary ALA as compared to LA was more efficient in D pigs than in P pigs. Consistent with other studies (Arterburn et al., 2006), very small amounts of EPA and docosahexaenoic acid (DHA; C22:6 *n-3*), two essential PUFA for human nutrition, were found in SCAT of the pigs. Proportions and amounts of these FA did not differ between D and P genotypes. As ALA was stored at lower levels in the SCAT of D pigs than in Pi pigs, this suggests that the enzymatic conversion of ALA into EPA was more efficient in D pigs. We also revealed closer correlations between variations in ALA and EPA proportions than between ALA and DHA proportions in SCAT of the pigs. This result was expected according to a meta-analysis conducted on the effect of dietary linseed on the *n-3* PUFA contents in pig adipose tissue (Corino et al., 2014). This suggests a competition for the fate of 24-carbon precursors of long-chain *n-3* PUFA when dietary ALA concentrations are increased (Sprecher, 2000).

The autonomous capacity of SCAT to oxidize fatty acids in growing pigs, in whichabolism exceeds catabolism, has been little studied. Contrary to CS activity (discussed above), the activity of β-HAD which catalyzes the penultimate step in the fatty acid β-oxidation pathway, did not differ between D and P genotypes. This is in contrast to the situation observed in the *Longissimus* muscle of the same pigs by Lebret et al. (2023) who reported a lower activity of β-HAD in D pigs compared to P pigs. Feeding the R diet was associated with an increase in β-HAD activity in SCAT when compared to pigs fed the C diet, whereas Guillevic et al. (2009) observed no effect of a linseed-based diet on β-HAD activity in porcine adipose tissues. In our study, the activity of β-HAD in SCAT was also positively associated with the proportions of ALA and of the *n-3* family of PUFA. These findings are consistent with the view that a diet rich in ALA may increase the rate of ALA oxidation as observed in healthy human subjects (Vermunt et al., 2000). In addition, oxidative phosphorylation may be an important pathway to remodel adipocytes in response to environmental stimulation such as nutrient intake (Qian et al., 2020). The diet did not alter the activities of the antioxidant enzymes catalase (CAT), SOD, GPX, and GRX in SCAT of the pigs. Similarly, in rats, antioxidant enzymes are unresponsive to dietary ALA levels (Couëdelo et al., 2022). However, the formation of peroxidized lipids (TBARS) under forced chemical oxidation, which mimics an oxidative challenge, was much higher and persisted longer in pigs fed the R diet compared to the C diet. This finding is consistent with the view that FA oxidation controlled by β-HAD activity is required for diet-induced oxidative stress and would potentiate oxidative-induced inflammation (Lee et al., 2015). The levels of vitamin E and plant antioxidants added to the R diet were thus likely not sufficient to counterbalance an increased oxidation susceptibility, due to the higher *n-3* PUFA content in SCAT and to enhanced β-HAD activity in SCAT adipose cells. For the D pigs, we show specific metabolic outcomes when fed the R diet. Circulating concentrations of phospholipids were reduced when D pigs were fed the R diet compared to the C diet. There was no significant reduction in circulating TAG concentrations when D pigs were fed the R diet, whereas other studies reported a trend for a linear decrease in plasma TAG concentrations in D x (Landrace x Yorkshire) with decreasing *n-6:n-3* ratio (17 to 5) of diets with linseed and fish oil (Sun et al., 2020). It is difficult to directly compare our results with other studies, as effects of dietary *n-3* FA on pig's serum lipids depend on the FA profile of the control diet and their actual level of *n-3* FA intake (Harris, 1997). Moreover, in our study, the R diet also contained faba beans, which may have lipid-lowering effects (Fernández-Quintela et al., 1998; Madar & Stark, 2002). This may also contribute to the reduction in plasma phospholipids concentrations in the D pigs fed the R diet. We observed a lower plasma glucose concentration in D pigs fed the R diet compared to the C diet. This agrees with the reduction of plasma glucose

concentrations observed in rats fed faba beans (Fernández-Quintela et al., 1998). Finally, the disadvantage of D pigs having more reactive oxygen metabolites (dROM) and lower plasma antioxidant potential (FRAP) than P pigs when fed the conventional C diet, was suppressed by feeding them an R diet. In fact, the R feeding strategy reduced systemic oxidative stress index (OSI) of D pigs to the level of P pigs. This may be related to the inclusion of faba beans, described a good source of antioxidants (Madar & Stark, 2002). Indeed, the faba bean is a rich source of polyphenol compounds including condensed tannins with high antioxidant potential (Rybiński et al., 2019); however, the preprocessing and dehulling techniques may affect the bioactivity of these components (Furbeyre et al., 2020; Salvador-Reyes et al., 2024). Overall, the response of the two pig genotypes to the feeding strategy in terms of plasma metabolic outcomes is consistent with the idea that ‘fatter’ genotypes may be more susceptible to dietary factors than ‘lean’ ones (Cameron et al., 2000).

## 5. Conclusion

Genotype and locally produced feeds are two opportunities to diversify pig farming systems and their products. We showed that adipose tissue biology was highly dependent on pig genetics which modulated lipogenesis and PUFA:SFA ratio, and on diet which modulated *n-6:n-3* PUFA ratio and oxidative and peroxidative activities. Interaction effects between genotype and diet were specifically revealed on the ratio of *n-6:n-3* fatty acids in subcutaneous adipose tissue but also on systemic metabolic outcomes such as glycemia, circulating phospholipids and oxidative stress index. These findings may provide new directions for the simultaneous improvement of the nutritional quality of pork fat as food for humans and the systemic metabolic outcomes of pigs for harmonious growth and adaptation to environmental challenges. Further studies involving a larger number of pigs in different contexts and with different feeding levels are needed before stronger recommendations can be proposed.

## Consent form

The work follows the rules of ethics in animal experimentation (ARRIVE guidelines and national formal agreement for the experimentation based on ethics/welfare committee's evaluation) and the ethics in publishing. No data on human are included in this manuscript.

## Human data

No data on human experimentations are included in this manuscript.

## CRediT authorship contribution statement

**F. Gondret:** Writing – review & editing, Writing – original draft, Supervision, Formal analysis, Data curation, Conceptualization. **K. Sierzant:** Writing – review & editing, Validation, Investigation, Formal analysis, Data curation. **B. Lebret:** Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization.

## Funding

This work is part of the ROC+ Regional Project and the animal design was supported by Région Bretagne and Rennes Métropole (France). The funding sources were not involved in the conduct of the research, interpretation of data, or writing of the manuscript. The research was supported by INRAE and a French Government Scholarship (Campus France) for the internship stay of Kamil Sierzant at INRAE PEGASE.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors are grateful to Etienne Labussière (UMR PEGASE) for his expertise in diet formulation and Isabelle Louveau (UMR PEGASE) for helpful discussions during the conception of the study. The authors would also like to thank Sophie Daré, Nathalie Bonhomme and Sandrine Tacher (UMR PEGASE) for their expert technical assistance.

Data availability

Data will be made available on request.

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