1. Introduction
Cardiovascular diseases (CVDs) have emerged as the predominant public health challenge in China, with incidence and mortality rates showing a concerning upward trajectory alongside rapid economic development and dietary pattern transitions. Current epidemiological data reveal a staggering CVDs mortality rate of one death every 10.5 s, known as the “number one killer” of human health [
1,
2]. This epidemic is strongly associated with modern nutritional imbalances, particularly the combination of excessive saturated fat intake and insufficient consumption of omega-3 polyunsaturated fatty acids (n-3 PUFA)—essential nutrients demonstrated to modulate cholesterol metabolism and attenuate CVDs progression [
3]. One way to increase n-3 PUFA intake without changing consumers’ nutritional behavior is to fortify traditional foods such as meat and meat products with n-3 PUFA.
Emerging evidences highlighted the feasibility of n-3 PUFA enrichment in pork through dietary interventions. Supplementation with 3% linseed oil in swine diet increased the n-3 PUFA content while it decreased the n-6/n-3 ratio from 15 to 2.5 in muscle tissue [
4]. However, dosage optimization is critical, as excessive linseed oil (e.g., 5.0%) produces undesirable flavor and reduces oxidative stability [
5]. Studies on fish oil supplementation have demonstrated dose-dependent effects: while 0.5% supplementation effectively increased long-chain n-3 PUFA (n-3 LC-PUFA), escalating to 8% quadrupled n-3 LC-PUFA content but the meat was softer [
6,
7]. As Zaloga [
8] emphasized, elevated n-3 PUFA deposition heightened susceptibility to lipid oxidation, accelerating rancidity and reduced both sensory quality and product shelf-life.
Selenium (Se), an essential trace element, functions as a critical component of hepatic glutathione peroxidase (GSH-Px) and selenoproteins, mitigating lipid oxidation by scavenging free radicals through GSH-Px-mediated antioxidant pathways [
9]. The biological efficacy of Se supplementation is critically dependent on its chemical form, with organic sources such as selenomethionine (SeMet) exhibiting enhanced bioavailability and tissues deposition compared to inorganic forms (e.g., sodium selenite, SeNa) [
10]. A study demonstrated that dietary 0.3 mg/kg SeMet supplementation in finishing pigs elevated tissue Se deposition, improved oxidative stability, and enhanced meat quality [
11]. Furthermore, Se-enriched (0.25 mg Se/kg) diets of finishing pigs reduced volatile basic nitrogen (TVB-N) and microbial spoilage, effects attributed to its antioxidative properties [
12]. However, it remains unclear whether Se can effectively counteract lipid peroxidation induced by n-3 PUFA and how their combined supplementation affects meat quality. Therefore, this study investigates the interactive effects of n-3 PUFA sources (linseed oil and fish oil) and SeMet on growth performance and meat quality in finishing pigs, providing theoretical and practical insights for producing n-3 PUFA-enriched pork.
2. Materials and Methods
2.1. Animals and Experiments Design
The linseed oil was bought from Defu Oil Co., LTD.(Ya’an, China), and the ALA content was 54%. The fish oil contained 21% DPA + DHA, and was bought from Yikang Natural Flavor Oil Refinery (Ji’an, China). SeMet was provided by Sichuan Xinyimei Biotechnology Co., Ltd. (Mianyang, China); the product is named Selenide ™2000. The main ingredients are L-SeMet and carrier thinner (zeolite powder, medical stone, silica, and rice husk powder). The L-SeMet content is ≥5000 mg/kg, and the actual Se content is ≥2000 mg/kg.
A total of 60 finishing pigs (Duroc × Landrace × Large, female) with an average weight of 73.00 ± 2.99 kg were divided into 5 groups with 12 animals per group, distributed in four finishing housings. To mitigate the environmental influences, each treatment was systematically distributed across three spatial zones (anterior, central, posterior) within the finishing housing, ensuring equally scattered in all housing units. The diets for the 5 groups were as follows: (a) 3% sunflower oil supplement (CON); (b) 3% linseed oil supplement (LO); (c) 3% fish oil supplement (FO); (d) 3% linseed oil supplement with 0.3 mg/kg of SeMet (LSe); (e) 3% fish oil supplement with 0.3 mg/kg of SeMet (FSe) (
Table 1).
The CON group received 3% sunflower oil, selected for its extremely low levels of n-3 PUFA while maintaining comparable saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acid ratios to linseed oil. The sunflower oil in the CON group was substituted with 3% linseed oil and fish oil. SeMet products with an actual Se content of 0.3 mg/kg were added to the LSe and FSe groups. To counteract potential lipid peroxidation from increased oils intake, all diets added 200 IU/kg vitamin E as antioxidant.
The basal diet was formulated according to the NRC [
13], and the diets at the same phase were isoenergetic, isonitrogen, and isofat in all experimental groups. The formulation of the experiment diets and measured values of fatty acid composition are shown in
Table 2 and
Table 3. The feeding experiment was divided into two phases according to body weight (Phase 1, 75–100 kg, and Phase 2, 100–135 kg stage), and lasted 52 days.
2.2. Sample Collection
At the end of the feeding experiment, 6 pigs in each group were selected based on the principle of similar average weight. The selected pigs were slaughtered after a 24 h fasting period. A total of 10 mL of blood from the anterior venous of each pig was collected, left for more than 30 min at 4 °C, centrifuged for 15 min at 3500 r/min, and the upper serum was taken and divided into EP tubes and stored at −20 °C for testing. Muscle samples were taken immediately after slaughter and skin removal. The longissimus thoracis et lumborums (LTL) of the left half of the carcass from the 5th–6th segments of the lumbar spine was taken; cut two muscle blocks of about 50 g from top to bottom, perpendicular to the muscle fibers, and stored at −20 °C for testing. Samples of the LTL and subcutaneous fat were collected, divided into frozen storage tubes, wrapped in tin foil, and stored at −80 °C for testing (
Figure 1).
2.3. Growth Performance Measurement
Daily feed intake was recorded during feeding, and all pigs were weighed on 0, 26, and 52 d of the experiment to calculate the average daily feed intake (ADFI), body weight gain (BWG), average daily gain (ADG), and the ratio of feed intake to body weight gain (F/G).
2.4. Serum Biochemical Indexes
Triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in serum were determined by a fully automatic biochemical analyzer (HITACHI 3100, Tokyo, Japan) according to the instructions of kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at the Institute of Animal Nutrition, Sichuan Agricultural University.
2.5. Meat Quality Measurement
Dissected LTL tissue was used to determine meat quality, considering meat color, pH, marbling score, drip loss, and cooking loss. At 45 min, 24 h, 48 h, and 7d postmortem, meat color and pH were measured. Meat color was measured using the CIE Lab color space system (
L*: lightness;
a*: redness;
b*: yellowness), using a calibrated colorimeter 3nh chromameter (NR60CP, 3nh Co., Ltd., Guangzhou, China) with the following settings: CIE 10° standard observer, D65 illuminant, and Φ8 mm aperture. Before measurement, the instrument was calibrated using a standard whiteboard. Each sample was measured at three different locations and the average was taken as the final result. pH values were determined using a food pH meter (Testo 205, Testo, Schwarzwald, Germany). Each sample was measured at three different locations and the average value was taken [
14]. After the loin-eye blocks were stored at 0–4 °C for 24 h, the marbling scores were subjectively evaluated according to the marbling score map (marbling from 1 to 10, with 1 = no marbling and 10 = overly abundant marbling), and the average of values from different observers was used for each muscle sample.
The LTL samples (100 g) were stripped of the fat and fascia, weighed before cooking, then randomly allocated to a thermostatic water bath at 80 °C and cooked in the same batch to an internal temperature of 70 °C. After cooling to room temperature and weighing again, the percentage of cooking loss was then calculated using the following formula:
For drip loss determination, muscle samples were cut into 5 × 3 × 2.5 cm
3 cuboids along the muscle fibers and weighed at 45 min postmortem, suspended in bags at 4 °C for 24 h, 48 h, and 7 d, and reweighed. The percentage of drip loss was then calculated using the following formula:
2.6. Muscle Chemical Composition
The moisture, crude protein (CP), and intramuscular fat (IMF) content in the LTL tissue was detected by a previous study [
15]. Briefly, about 50 g muscle samples were weighed and placed into a Labconco freeze dryer (model 2.5, Labconco Corp., Kansas, MO, USA) with a temperature of −45 °C and a vacuum of less than 10 μm of Hg. Samples were freeze-dried for 48 h, and then the muscle samples were reweighed to calculate the content of moisture. Dried muscle samples were subsequently pulverized using a hammer mill, then the Kjeldahl nitrogen determination was used to determine the CP content, and the cable extraction method (SOX416, Sox-therm, North Rhine-Westphalia, Germany) was used to determine the IMF content.
2.7. Antioxidant Capacity Measurement
The catalase (CAT), total superoxide dismutase (T-SOD) activity, and the malondialdehyde (MDA) content in serum and muscle were determined according to the instructions of the kit (Nanjing Institute of Biological Engineering, Nanjing, China).
2.8. Amino Acid
The amino acid in the LTL tissue was determined by an automatic amino acid analyzer, as described previously [
16] with minor modifications. The freeze-dried meat was about 150 mg in a hydrolysis tube, and 15 mL of 6.0 mol/L hydrochloric acid was added. The tube was filled with nitrogen, sealed, and hydrolyzed in a 110 °C oven for 24 h. Then, it was cooled and transferred to a 50 mL volumetric bottle, filled with ultra-pure water, and shaken well. Then, we absorbed 200 μL into the amino acid vial and freeze dried it under negative pressure before adding 800 μL of 0.02 mol/L hydrochloric acid for resolution. The 0.22 μm microporous filter membrane was filtered and analyzed with an automatic amino acid analyzer (Hitachi L-8900, Tokyo, Japan). The amino acids were expressed as % of dry matter.
2.9. Fatty Acid Composition Measurement
Lipids in the LTL and subcutaneous fat tissue samples were extracted as previously described [
17] with a minor modification [
18]. Total lipids in the upper layer were collected and determined by a GC 2010 plus model gas chromatogram. Chromatographic conditions: HP-88 (100 m × 0.25 mm × 0.20 μm) was used. The sample volume was 1.0 μL, and the inlet temperature was 240 °C. The detector was FID, and the temperature was 280 °C. We programmed the temperature rise: it was kept at 100 °C for 13 min, then heated up to 180 °C at 10 °C/min for 6 min, then heated up to 192 °C at 1 °C/min for 9 min, then heated up to 230 °C at 3 °C/min for 10 min. The carrier gas was nitrogen, and the flow rate was 1.3 mL/min. The fatty acids in the LTL tissue were expressed as mg/100 g fresh weight, while subcutaneous fat tissue was expressed as g/100 g fresh weight.
2.10. Data Statistics and Analysis
The experimental data were processed using Excel 2021 for preliminary organization, with statistical analyses conducted in SPSS 29.0 (IBM, New York, NY, USA). Normality was verified through Shapiro–Wilk tests, and homogeneity of variance was confirmed via Levene test. The one-way ANOVA was used to analyze the groups supplemented with different oils (CON, LO, and FO). The source of n-3 PUFA and whether SeMet was added (LO, FO, LSe, and FSe) were analyzed by two-factor ANOVA with a general liner model (GLM), and the significance of the main effect and interaction effect were analyzed. If the difference was significant, the least significant difference (LSD) was used for multiple comparison. The results were expressed as mean, and the standard error (SEM) of the mean was used to represent the variability of the values. p < 0.05 was a significant difference, p ≤ 0.01 was an extremely significant difference, and 0.05 ≤ p ≤ 0.1 was a trend.
4. Discussion
n-3PUFA is abundant in unsaturated double bonds, particularly EPA and DHA, and its oxidation stability is suboptimal [
19]. In terms of the primary fatty acid composition of feed, FO was predominantly composed of EPA and DHA, whereas LO primarily contained ALA. EPA and DHA had more unsaturated double bonds than ALA, and FO supplementation induced greater oxidative stress than LO, evidenced by the higher level of the lipid oxidation product MDA and lower T-SOD activity in the FO group. Dietary supplementation of 3% FO increased F/G during the 100–135 kg phase, which might be explained by the fact that FO easily impairs the intestinal antioxidant capacity of finishing pigs, which decreases nutrient utilization efficiency [
20,
21]. Interestingly, 0.3 mg/kg SeMet increased T-SOD activity without significantly affecting growth performance, which was consistent with previous findings [
11], suggesting that SeMet partially eliminated the decrease in antioxidant damage caused by FO. Another study found that a dietary addition of 8% FO resulted in a decrease in ADG and an increase in backfat thickness of finishing pigs [
7]. However, there was no statistically significant difference in BWG or ADG arising from the low supplemental level of FO and diets with identical fat content.
We also noticed a decrease in serum TG level in the LO group, which was in line with recent research [
22]. EPA and DHA reduce serum TG level by activating the transcription factor peroxisome proliferator activating receptor (PPARα) and inhibiting sterol reaction element binding protein 1 (SREBP-1) [
20]. However, we failed to find no significant effect of LO on TG level; this might be explained by the higher SFA level in FO, in addition to EPA and DHA, which mutually restricted the regulation of serum TG level [
23]. In line with Czech et al. [
24], both LO and FO decreased the serum HDL-C level. FO and SeMet co-supplementation elevated serum LDL-C and TC levels. In mammals, SFA can stimulate de novo synthesis of TC, which increases the serum TC level. Combined with the regularity of fatty acid deposition in tissues, serum TC and LDL-C levels increased as a result of higher SFA content in the FO diet [
25].
The purchasing decisions of consumers are also influenced by traditional meat quality indexes when considering n-3 PUFA-enriched pork. Customers notice meat color directly through their senses, showing a preference for meat that is lighter and more red (higher
L* and
a* values) and less yellow (lower
b* value). The meat color stability was reduced due to the presence of double bonds in LO, which is prone to myoglobin and lipid oxidation during storage [
26]. With the addition of SeMet, the
L* value in the LSe group was higher compared to the LO group, indicating that SeMet mitigated the adverse effect of LO on the
L* value of pork. When SeMet and LO were combined, SeMet functioned as an antioxidant to counteract the effect induced by LO, inhibit the cross-linking damage of lipid oxidation byproducts (such as malondialdehyde) to proteins, delay muscle tissue browning, and maintain brightness [
27]. Other studies reported no effect of SeMet on meat color [
27]. Juniper et al. [
28] suggested that once the Se content of tissue exceeded the requirements of antioxidant enzymes, a further increase in tissue Se does not result in noticeable improvement in meat quality. In this study, SeMet had no effect on other meat color indexes, which may possibly be explained by the supplementation period and dose. In addition, theoretically, FO was expected to exhibit higher oxidative stress level compared to LO, a condition that could potentially compromise muscle tissue stability [
29]. However, our findings revealed no significant alterations in meat color parameters, despite the observed differences in oxidative status. Meanwhile, muscle tissue analysis demonstrated higher T-SOD activity in the FO group. In contrast to the findings in serum, the main effect analysis unexpectedly showed that SeMet decreased T-SOD activity in muscle tissue. We postulate that this phenomenon may reflect an adaptive response, where prolonged exposure to lipid oxidation triggers systemic redistribution of T-SOD to muscle tissue as a compensatory antioxidant mechanism. The antioxidative effect of SeMet showed a U-shaped curve, with excess potentially promoting oxidative mechanisms [
30]. This study showed that n-3 PUFA treatment had no significant difference in umami and bitter amino acids content in muscle tissue, whereas SeMet increased Ser content. This observation might be attributed to the fact that the methionine in SeMet regulated the amino acid metabolism of pigs, consequently influencing the amino acid composition of pork. However, amino acid composition of muscle remained unaffected by different source of n-3 PUFA. It was evident that SeMet had the potential to enhance the amino acid favorable for pork flavor [
31].
Our investigation revealed distinct tissue-specific modulation of fatty acid profiles through n-3 PUFA supplementation, which selectively regulated n-3 PUFA composition in both LTL and subcutaneous adipose tissue, with differential effects between lipid sources. LO supplementation mainly increased the ALA and C20:3n-3 content, while FO predominantly increased the EPA and DHA content. These modifications significantly improved the lipid profile, with a reduced n-6/n-3 ratio and an increased PUFA/SFA ratio. These changes could be explained by the competitive enrichment of n-3 PUFA inhibiting the carbon chain extender and desaturase required for n-6 PUFA synthesis [
32]. Notably, we observed significant tissue-specific deposition efficiency, with adipose tissue showing 5.67 times higher n-3 PUFA deposition in the LO group compared to the CON group, 2.85 times higher observed in muscle. This differential deposition pattern aligns with the unique lipid absorb and metabolism of monogastric animals, where dietary PUFA directly deposited into tissue phospholipids and triglycerides the body without hydrogenation, indicating a strong correlation between the fatty acid composition of the diet and the fatty acid composition of the animal body [
33]. The results of fatty acid analysis in various treatments were in agreement with the regularity of fatty acid deposition in various diets. Interestingly, despite comparable dietary C20:3n-3 content across groups, compared with the CON and FO groups, the LO group exhibited 2.9- and 5.7-times increases in muscle and adipose C20:3n-3 content, respectively, which might be explained by the fact that C20:3n-3 is a metabolic intermediate in ALA elongation/desaturation pathways and a direct precursor for EPA and DHA [
34]; thus, C20:3n-3 content in tissues was correlated with ALA content in diets. Our findings demonstrated that while LO supplementation significantly elevated EPA and DHA content in muscle and adipose tissue, which could be because the experimental diet included more lipid supplements, ALA in sunflower oil or LO can still increase DHA content to some extent through fatty acid metabolism. DHA level showed no statistical differentiation between the LO and CON groups. This specific disparity persisted despite higher ALA in the LO diet compared to the sunflower oil-based CON diets. The findings demonstrated that while ALA conversion to long-chain n-3 PUFA in finishing pigs significantly enhanced EPA deposition, its capacity to improve DHA synthesis remained insignificant. This constrained DHA conversion efficiency is strongly correlated with dietary ALA level and the initial body weight. Notably, low-dose LO supplementation (<2%) marginally elevated porcine DHA synthesis [
35], a phenomenon potentially attributable to ALA’s competitive inhibition of Δ6-desaturase activity against C24:5n-3 [
36], the essential precursor for DHA biosynthesis [
37].
As an animal-derived lipid source, FO inherently contains higher SFA and MUFA content. Compared with LO, FO supplementation showed higher SFA, MUFA content, and n-6/n-3 ratio, and lower PUFA, n-3PUFA content, and PUFA/SFA ratio in the adipose tissue, while only the PUFA/SFA ratio was lower in muscle tissue, potentially attributable to inefficient n-3PUFA deposition in the muscle. Additionally, it is worth noting that SeMet enhanced adipose deposition of ALA and C20:3n-3, indicating improved n-3 PUFA deposition capacity. This dual mechanism involves (1) antioxidant protection of SeMet redirecting fatty acids from oxidation towards storage, and (2) synergistic lipid metabolism regulation through combined SeMet-LO action [
38,
39]. Although SeMet potentially elevated DHA synthesis, the degree of unsaturation in FO exceeded the antioxidative capacity of SeMet at a level of 0.3 mg/kg, resulting in non-significant differences. Surprisingly, the efficacy of SeMet in promoting n-3 PUFA deposition was adipose-specific, likely due to the high fatty acid content of adipose tissue stimulating SeMet to regulate lipid metabolism and enhance its antioxidant capabilities [
40].
Data from Chinese National Bureau of Statistics indicate an annual per capita pork consumption of 30.5 kg (≈83.56 g/d) [
41]. Based on standard lean pork composition (55% muscle, 27% fat), this equates to approximately 67% muscle and 33% fat intake [
42]. Referencing Chinese Nutrition Society guidelines [
43], daily recommended intakes for ALA, EPA + DHA, and total n-3 PUFA are 1333.33, 250, and 1111.11 mg, respectively. Our analyses demonstrated that 3% LO-supplemented pork provided 156%, 24%, and 217% of these respective requirements. Given that the conversion efficiency of ALA in the body is about 5–15% [
44], LO-derived pork delivers functionally adequate n-3 PUFA levels to meet recommendations. Comparatively, 3% FO-supplemented pork met 41%, 230% and 106% of daily requirements. While FO pork provided twice the EPA + DHA recommendation, its ALA content remains insufficient. Given that ALA is an essential fatty acid and metabolic precursor, pork produced with 3% LO showed superior nutritional value by meeting n-3 PUFA demand through both direct provision and conversion potential. Consequently, in terms of nutritional value, it surpasses that of pork produced with FO. However, during the production of n-3 PUFA-enriched pork, the supplementation of linseed oil and fish oil may inhibit fat deposition, resulting in lean carcass and reduced marbling, and the energy–protein ratio needs to be adjusted to maintain the growth rate [
45]. However, pork enriched with high levels of n-3 PUFA remains more susceptible to lipid oxidation during storage, necessitating the optimization of packaging conditions. This functional meat product holds significant appeal for health-conscious consumers, particularly those at high risk of cardiovascular disease. Clear and Codex-compliant nutritional labeling (including EPA + DHA content labeling), along with consumer education initiatives emphasizing the importance of optimizing the n-6/n-3 ratio, may enhance consumer purchase intent.