Introduction
Beef consumption in China has increased substantially with economic development, driving consumer demand for high-quality marbled beef with superior juiciness, tenderness, and flavor (Benli and Yildiz, 2023; Guo et al., 2022). Intramuscular fat (IMF) deposition is a critical determinant of beef quality and palatability. However, high IMF content raises consumer health concerns due to elevated total fat level. Addressing this challenge requires optimizing fatty acid composition while maintaining IMF level, specifically, increasing functional fatty acids such as conjugated linoleic acid (CLA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) while reducing the n-6/n-3 fatty acid ratio to balance palatability with health benefits. CLA has garnered significant attention for its potential anti-inflammatory effects and metabolic regulatory properties (Koba and Yanagita, 2014). Beef serves as the primary dietary source of CLA for humans, predominantly containing the cis-9, trans-11 CLA isomer (De La Torre et al., 2006). EPA and DHA provide additional health benefits, including anti-inflammatory effects, immune enhancement, and cardiovascular disease prevention (Kousparou et al., 2023; Panda, Varadharaj, and Voruganti, 2022). Enriching these functional fatty acids directly enhances health attributes of beef while improving the overall n-6/n-3 ratio. The internationally recognized optimal n-6/n-3 ratio of 4∶1 positively impacts lipid metabolism and reduces chronic disease risk (Bishehkolaei and Pathak, 2024; Ebrahimi et al., 2017; Gonzalez-Becerra et al., 2023). However, beef cattle industry of China faces significant productivity gaps compared with international standards, primarily due to outdated management practices and inadequate feeding techniques (Gao et al., 2023). This necessitates innovative nutritional solutions for producing high-quality beef that meets evolving consumer demands.
During the late fattening period, IMF content can be effectively increased by optimizing dietary strategies, such as increasing energy level and fatty acid supplementation (Arshad et al., 2018; Lee et al., 2024; Park et al., 2018). However, CLA synthesis and accumulation in ruminant muscle tissue presents greater challenges. Most cis-9, trans-11 CLA in ruminants originates from ruminal bacterial isomerization of linoleic and α-linolenic acids, while endogenous tissue synthesis via stearoyl-CoA desaturase (SCD)-mediated desaturation remains limited (Wang et al., 2020). Oleic acid may facilitate endogenous CLA synthesis by regulating SCD activity (Tibori et al., 2024). Previous studies demonstrate that dietary fatty acid supplementation promotes growth performance and IMF accumulation across livestock species (Bai et al., 2023; Barnes et al., 2012; Wang et al., 2021). While fatty acid supplementation is crucial for beef cattle production, the high cost of pure fatty acids limits its large-scale application in this field. Plant-derived oils, however, can serve as economically viable alternatives due to their high energy density and controllable costs.
Plant-derived oils serve as promising feed additives, enhancing livestock performance through its high energy properties, anti-inflammatory, antioxidant, and rumen metabolism-modulating effects (Dos S. Silva et al., 2019). Soybean oil, the world’s second-largest vegetable oil by production volume, is rich in polyunsaturated fatty acids (linoleic acid) and monounsaturated fatty acids (oleic acid) (Silva et al., 2021). Rapeseed oil, derived from the third most-abundant oilseed crop globally, contains high concentrations of oleic acid, linoleic acid, and α-linolenic acid (Banaś, Piwowar, and Harasym, 2023; Zhang et al., 2021). Both oils possess established nutritional value. While soybean oil improves feed conversion efficiency and modulates fatty acid composition in pork and goat meat (Alencar et al., 2021; Han et al., 2025), and rapeseed oil enhances rumen fermentation, increases EPA and DHA content in lamb muscle, and elevates monounsaturated fatty acids and CLA levels in milk (Altenhofer et al., 2014; Flakemore et al., 2017; Idowu et al., 2025), their specific effects on IMF accumulation and fatty acid profiles in beef cattle remain poorly understood. Their distinct fatty acid profiles may directly influence the patterns of IMF deposition and ultimate fatty acid accumulation. Furthermore, research on their role in promoting endogenous CLA synthesis in beef is lacking, representing such knowledge gaps for developing targeted beef quality improvement strategies. Understanding the molecular mechanisms governing intramuscular lipid metabolism is essential for optimizing dietary strategies. The PI3K/AKT signaling pathway serves as a central regulator of cellular energy metabolism and lipid homeostasis (Han, Lin, and Hu, 2024; Liu et al., 2016). It is involved in IMF deposition in beef cattle (Baldassini et al., 2025), and it may also play a regulatory role in the optimization of fatty acid profiles and the synthesis of CLA.
We hypothesized that dietary supplementation with soybean oil or rapeseed oil would positively influence IMF accumulation and fatty acid composition in beef cattle. This study investigated the effects of 3% soybean oil and rapeseed oil supplementation on IMF accumulation, fatty acid profiles, and underlying molecular mechanisms during the late fattening phase of Woking black cattle.
Materials and Methods
Isolation of bovine skeletal muscle-derived cells
Bovine skeletal muscle-derived cells (BSMC) were isolated following our previously described protocol with minor modifications (Jin et al., 2024). This cell model has demonstrated multipotent differentiation potential, including adipogenic differentiation in vitro, and our team has successfully utilized it in adipogenic induction experiments, further validating its reliability as an in vitro platform for studying the molecular mechanisms of IMF development. Longissimus thoraci muscle samples were obtained from three 30-mo-old Woking black cattle at the Changchun Haoyue Abbatoir. Following collection, samples were immediately placed in phosphate-buffered saline (PBS) containing 0.2% antimicrobial solution (100× gentamicin and amphotericin B, Yuanye, Shanghai, China) and transported to the laboratory on ice. BSMC were isolated via enzymatic digestion using 0.25% collagenase type II (Biosharp, Shanghai, China) and 0.1% dispase II (Yuanye) at 37°C. After filtration and red blood cell lysis, the digestion was terminated by adding Dulbecco’s Modified Eagle Medium (DMEM, HyClone, Logan, Utah) supplemented with 10% fetal bovine serum (Meilun Bio, Dalian, China) and 1% penicillin/streptomycin (P/S, Meilun Bio). Cells were cultured in a humidified incubator at 37°C with 5% CO2. Third-passage BSMC were used for all subsequent experiments.
Adipogenic differentiation and oil treatment
For adipogenic differentiation, BSMC were seeded into 6-well plates containing growth medium at a density of 5.0 × 104 cells per well. When BSMC reached 90% confluence (designated as day 0, D0), cells were treated with adipogenic induction medium consisting of DMEM supplemented with 10% horse serum (HS, Yuanye, Shanghai, China), 10 μg/mL insulin (Sigma-Aldrich, St. Louis, Missouri), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 1 μg/mL rosiglitazone (MCE, Shanghai, China), and 1% penicillin/streptomycin. After 3 d, the medium was replaced with maintenance medium (DMEM containing 10% HS, 10 μg/mL insulin, 1 μg/mL rosiglitazone, and 1% penicillin/streptomycin), which was refreshed every 2 d until day 11 (D11). During the late adipogenic differentiation phase (D7-D11), cells were treated with soybean oil or rapeseed oil (300 μg per well) (Zhong et al., 2020) dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) or vehicle control (DMSO only) to simulate the late fattening period of beef cattle and verify the related mechanisms. Cells were collected from 3 individual cattle to constitute 3 biological replicates. For each treatment condition, 3 parallel experiments were conducted, which served as 3 technical replicates. The detailed experimental design is illustrated in Figure 1A.
Rapeseed oil has a stronger effect than soybean oil in promoting the adipogenic differentiation of BSMC. (A) Cell culture strategy and treatment protocols with soybean oil and rapeseed oil. (B) Effects of different treatments on lipid accumulation in BSMC: Oil Red O staining and TG quantification. (C) Expression of adipogenic differentiation-related proteins in BSMC. Data are presented as mean ± SEM from n ≥ 3 biological replicates. When comparing data between the 2 groups, **P < 0.01 and *P < 0.05. When comparing data among 3 or more groups, P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses, using “B” and “b,” respectively. Abbreviations: BSMC, bovine skeletal muscle-derived cells; C/EBPα, CCAAT/enhancer binding protein α, FABP4, fatty acid-binding protein 4; PPARγ, peroxisome proliferator activated receptor γ; SREBF1, sterol regulatory element binding transcription factor 1.
Oil Red O staining
Oil Red O staining was performed on D11 of adipogenic differentiation. BSMC were washed twice with 1× PBS and then fixed overnight at room temperature with 10% formalin. After washing twice with distilled water (DW), cells were incubated in 60% isopropanol for 5 min. Subsequently, cells were stained with an Oil Red O solution (0.3% Oil Red O in 60% isopropanol, Sigma-Aldrich) for 30 min. Following staining, cells were washed 4 times with DW, observed, and photographed under a microscope.
Triglyceride quantification
At the end of adipogenic differentiation (D11), cells were harvested with 500 μL of isopropanol. Intracellular triglyceride (TG) content was measured using the Amplex Red Triglyceride Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin inhibitor treatment
The PI3K inhibitor ZSTK474 (HY-05847, 10 mM, MedChemExpress, Shanghai, China) was dissolved in DMSO and filtered through a 0.22 μm membrane. Cell cultures were treated with ZSTK474 at a final concentration of 1 μM (Liebscher et al., 2023) starting from D7. The AKT inhibitor GSK2141795 (HY-15965, 10 mM, MedChemExpress) and the mTOR inhibitor rapamycin (HY-10219, 10 mM, MedChemExpress) were prepared using identical methodology, with final working concentrations of 2.5 μM (Jacobsen et al., 2017) and 0.5 μM (Zhou et al., 2023), respectively. Control group cells were treated with vehicle (DMSO) only. The detailed experimental design is illustrated in Figure 1A.
Animal and management
All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (license no. SY202302017) prior to study initiation. The experiment was conducted at a commercial farm operated by Haoyue Group (Haoyue Halal Meat Co., Ltd, Changchun, China). Forty-one Woking black cattle (F2 hybrids of Australian Wagyu and Yanbian Yellow cattle), all of which were steer and aged a mean (SD) of 26 (1) mo with comparable physiological status and body weight, were randomly selected as experimental subjects. The animals were maintained under standardized feeding conditions following the Nutrient Requirements of Beef Cattle and the Technical Specification for Beef Cattle Feeding (National Academies of Sciences et al., 2016). To enhance marbling score, vitamin A administration was strategically implemented during the fattening period by our previous description (Peng, Smith, and Lee, 2021). Specifically, the fattening phase commenced at age 14 mo and continued until slaughter at 30 mo. All cattle were housed in individual steel tethered pens with ad libitum access to water and received formulated diets twice daily. The animals were randomly allocated into control group (n = 7), soybean oil treatment group (n = 16), and rapeseed oil treatment group (n = 18). The treatment group receiving concentrated roughage supplemented with either 3% soybean oil or 3% rapeseed oil, respectively, based on dry matter. Vegetable oil was supplemented throughout the entire late finishing phase of beef cattle, which lasted approximately 4 mo (Figure 2A). That is, vitamin A supply was restricted at the beginning of the fattening phase (age 14 mo), followed by the restoration of vitamin A levels in the feed (Supplementary Table 1) and supplemental vegetable oil feeding during the late fattening phase (age 26 mo). The nutrient composition of feedstuffs during the experimental period is presented in Supplementary Table 1. The results of our determination of the fatty acid composition of the 2 oils used in this experiment are shown in Supplementary Table 2. All cattle underwent standardized pre-slaughter handling and slaughtering procedures in accordance with the national standard for beef cattle slaughtering (GB/T 19477-2018, China). Carcass characteristics, including slaughter weight, carcass weight, dressing percentage, eye muscle area, backfat thickness, rib muscle thickness, and intermuscular fat thickness, were evaluated following protocols outlined in the technical specification for beef cattle performance testing (NY/T 2660-2014, China). Marbling grade assessment was conducted according to the Haoyue Woking black cattle grade assessment standard (Q/HYB0303ND-2017, Changchun, China), which classifies marbling from low to high as A1, A2, A3, A4, and A5. For statistical analysis purposes, these grades were assigned numerical values of 100, 200, 300, 400, and 500, respectively (Figure 2B).
Supplementation of soybean oil or rapeseed oil to the diet of Woking black cattle during the late fattening period can improve the level of marbling accumulation and fatty acid synthesis without disturbing their blood biochemical indicators, with rapeseed oil exhibiting superior efficacy. (A) Feeding strategies for Woking black cattle. (B) Marbling score grading standard established by Haoyue Group. (C) Effects of supplementing soybean oil or rapeseed oil in feed on the proportion of marbling accumulation grades in Woking black cattle. (D) Effects of soybean oil or rapeseed oil supplementation on serum biochemical parameters in Woking black cattle. (E) Effects of soybean oil or rapeseed oil supplementation on adipogenesis-related proteins expression in Woking black cattle. (F) Effects of soybean oil or rapeseed oil supplementation on the expression of fatty acid synthesis-related proteins in Woking black cattle. When comparing data among 3 or more groups, P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses, using “B” and “b,” respectively. Abbreviations: ACACA, acetyl-CoA carboxylase α; C/EBPα, CCAAT/enhancer binding protein α; FABP4, fatty acid-binding protein 4; FASN, fatty acid synthase; GLU, glucose; GOT, glutamic-oxaloacetic transaminase; GPT, glutamic-pyruvic transaminase, HDLC, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol; NEFA, non-esterified fatty acids, PPARγ, peroxisome proliferator activated receptor γ; SCD, stearoyl-CoA desaturase; SREBF1: sterol regulatory element binding transcription factor 1.
Serum and longissimus thoraci muscle sample collection
Immediately following slaughter, longissimus thoraci muscle samples were excised from between the 12th and 13th ribs, sectioned into small pieces, transferred into 5 mL microtubes, and flash-frozen in liquid nitrogen. The frozen samples were transported to the laboratory on dry ice and stored at −80°C. Blood was collected from the heart using sterile disposable syringes and transferred into vacuum collection tubes containing coagulant (KWS, Hebei, China). The tubes were wrapped in aluminum foil to shield them from light and transported to the laboratory in ice boxes. On arrival, blood samples were allowed to stand at room temperature for 1 h before centrifugation (3,500 rpm, rotor radius 12 cm, 15 min, 4°C). The supernatant serum was aspirated, transferred into aluminum foil-wrapped tubes, and stored at −80°C for subsequent analysis. Muscle samples were pulverized in a mortar with liquid nitrogen, transferred to 5 mL microtubes, and stored at −80°C.
Serum biochemical analysis
Serum biochemical analysis examined 6 parameters: glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), glucose (GLU), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and non-esterified fatty acids (NEFA). All analytes were quantified using commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols.
Western blotting
Longissimus thoracis muscle proteins were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer and quantified with a bicinchoninic acid kit (Meilun Bio). Protein samples mixed with loading buffer were denatured at 99°C for 8 min. Proteins (5 μg per well) were separated by SDS-PAGE using gels of appropriate concentration based on target protein size and then transferred to PVDF membranes (Millipore Corp, Billerica, Massachusetts). Membranes were blocked with protein-free rapid blocking buffer (EpiZyme, Shanghai, China) for 15 min and incubated overnight with primary antibodies at 4°C. After 3 10-min washes with tris buffered saline with Tween-20 (TBST) buffer (20 mM Tris-HCl, 137 mM sodium chloride [NaCl], 0.01% Tween 20, pH 7.6), membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. After incubation, the membranes were washed 3 times with TBST buffer for 10 min each. Protein bands were visualized using enhanced chemiluminescence (ECL) substrate (Meilun Bio) on a chemiluminescence imaging system (Tanon, Shanghai, China). Band intensities were quantified using ImageJ software (National Institutes of Health) and normalized to β-actin. Antibody details are presented in Supplementary Table 3.
Analysis of vitamin A in serum
Serum vitamin A analysis was performed as previously described methods (Peng et al., 2017). All procedures were conducted under controlled light conditions to prevent degradation. High-performance liquid chromatography (HPLC)-grade methanol (TEDIA, Fairfield, Ohio), ethanol (Beijing Chemical Industry Group, Beijing, China) and hexane (Tianjin Damao Chemicals Reagent Factory, Tianjin, China) were used. A 0.04% 2.6-di-tert-butyl-4-methylphenol with ethanol (BHT-EtOH) solution was prepared using BHT (Sigma-Aldrich) as a stabilizer. Internal and external standard stock solutions were prepared with retinyl acetate and retinol (Sigma-Aldrich), respectively, and stored in brown glass bottles at −20°C. For sample preparation, 200 μL of thawed serum was combined with 20 μL of internal standard working solution, 200 μL of ultrapure water, and 400 μL of BHT-EtOH solution in a 2 mL centrifuge tube. After vortexing, 400 μL of BHT-hexane solution was added, and the mixture was centrifuged at 3,000 g for 15 min at 4°C. The hexane layer (700 μL) was transferred to a brown centrifuge tube, evaporated under nitrogen (EYELA, Tokyo, Japan), and reconstituted in 500 μL of 95% methanol. The solution was filtered (0.22 μm, PES, SORFA, Beijing, China) before HPLC analysis. Vitamin A content was determined using HPLC (Primaide model, Hitachi, Tokyo, Japan) with a C18 column (Elite, Dalian, Liaoning, China). Chromatographic conditions included a single mobile phase of 95% methanol as mobile phase, 1 mL/min flow rate, 20°C column temperature, and 325 nm detection wavelength. Quantification was performed using external standards (0.1–50.0 μg/mL), with correction by internal standard recovery. All samples were analyzed in duplicate to ensure reliability.
Fatty acid analysis in longissimus thoracis muscle
The fatty acid extraction solution was prepared by mixing trichloromethane (Beijing Chemical Industry Group) with methanol (2:1, v/v). For methylation, a solution containing 14% boron trifluoride-methanol (Sigma-Aldrich), methanol and hexane (4:7:9, v/v/v) was prepared. The longissimus thoracis tissue powder (0.5 g) was combined with 20 mL extraction solution in a 50 mL centrifuge tube, purged with nitrogen, sealed, and shaken (220 rpm) at 4°C overnight. The following day, 8 mL of 0.88% NaCl solution was added, and the mixture was shaken for an additional 2 h before centrifugation (3,000 g, 20°C, 15 min). The lower phase (9 mL) was transferred to a glass tube and evaporated under nitrogen at 55°C. The resulting lipid residue was methylated with 2 mL of methylation solution at 90°C for 1 h under nitrogen with intermittent vortexing. After cooling, 2 mL hexane and 1 mL 0.88% NaCl were added, and the mixture was centrifuged (3,000 g, 20°C, 15 min). The hexane layer was collected and analyzed using a GC7980 gas chromatograph (Techcomp, Shanghai, China) equipped with a silica gel capillary column (100 m × 0.25 mm × 0.20 μm). The temperature program was as follows: 70°C (1 min), increased to 100°C at 5°C/min, held for 2 min, increased to 175°C at 10°C/min, held for 40 min, increased to 225°C at 5°C/min, and held for 40 min. Fatty acids were identified by comparing retention times with standards and quantified as percentage of total fatty acid peak area so as to reflect the compositional pattern or relative distribution characteristics of fatty acids.
Statistical analysis
Statistical analyses were performed using SPSS version 24.0 software (IBM SPSS Statistics, Chicago, Illinois). Data are expressed as mean ± standard error of the mean (SEM). One-way analysis of variance was used to evaluate the differences in slaughter performance, IMF accumulation, Metabolic profile test, muscle fatty acid composition, serum vitamin A concentration, and protein expression in muscles and cells among groups, followed by Tukey’s post hoc multiple comparison test. Statistical extremely significant difference is defined as P < 0.01, and significant difference is defined as P < 0.05, while 0.05 ≤ P < 0.10 indicates a trend toward significance.
Results
Rapeseed oil demonstrates superior efficacy over soybean oil in promoting bovine skeletal muscle-derived cells adipogenic differentiation
To confirm the effects of plant-derived oils on IMF accumulation in beef cattle during the late fattening stage, we treated BSMC at the late differentiation stage with vegetable oils to simulate the treatment conditions of the late fattening period. To verify the efficiency and suitability of BSMC isolated from muscle tissue in this study, we assessed their adipogenic differentiation capacity and the efficacy of treatment with both oils by detecting lipid droplet formation, TG accumulation, and protein expression of key adipogenic transcription factors. Cell culture experiments demonstrated that rapeseed oil significantly promoted intracellular lipid droplet formation and TG accumulation compared to the control group (P < 0.05), while soybean oil showed no significant effect (P > 0.05) (Figure 1B). Consistent with the TG results, rapeseed oil significantly upregulated the expression of fatty acid-binding protein 4 (FABP4) (P < 0.05), whereas soybean oil had no significant regulatory effect (P > 0.05). Furthermore, rapeseed oil specifically enhanced the expression of CCAAT enhancer binding protein α (C/EBPα) and sterol regulatory element binding transcription factor 1 (SREBP1), and its effect was significantly superior to both the control group and soybean oil group (P < 0.05). No significant differences were observed between the control group and soybean oil group (P > 0.05). Additionally, both oils upregulated peroxisome proliferator activated receptor γ (PPARγ) expression (P < 0.05) (Figure 1C). These findings indicate that BSMC possess adipogenic differentiation capacity, and both vegetable oils exert promoting effects on their adipogenic differentiation, with rapeseed oil demonstrating significantly superior efficacy compared to soybean oil.
Soybean oil and rapeseed oil supplementation enhance carcass characteristics and intramuscular fat deposition while maintaining normal metabolic parameters in beef cattle
Based on the promoting effect of the 2 vegetable oils on the adipogenic differentiation of BSMC, we supplemented these 2 vegetable oils in the diet during the late fattening stage to explore their effects in practical application. After slaughter, we examined the effects of dietary oil supplementation on carcass characteristics and meat quality parameters. Supplementation with either soybean oil or rapeseed oil markedly enhanced slaughter performance, with pronounced improvements in IMF accumulation (Table 1). Marbling scores were significantly elevated in both treatment groups compared with the control group (soybean oil, P < 0.01; rapeseed oil, P < 0.01), indicating enhanced IMF accumulation. Given the observed improvements in marbling scores, we next analyzed beef grade distribution to assess practical implications (Figure 2C). The control group showed limited grade advancement, with 100% achieving A1+ but only 57% reaching A2 grade, 29% attaining greater than or equal to A3, and, notably, no animals producing A4 or higher grades. Soybean oil supplementation resulted in substantial grade enhancement, ensuring 100% A2+ achievement, with 56% reaching A3 and 38% achieving A4 grade. In contrast, rapeseed oil demonstrated superior performance, not only securing 100% ≥A3 grades but also yielding the highest proportion of premium beef: 50% achieved A4, and 17% attained the highest A5 grade. Compared with the control group, the A2 grade proportion was significantly higher in the soybean oil and rapeseed oil treatment groups, respectively (P < 0.05). The proportion of A3 grade in the rapeseed oil treatment group was extremely significantly higher than that in the control group (P < 0.01) and significantly higher than that in the soybean oil group (P < 0.05), while there was no significant difference between the soybean oil group and the control group (P > 0.05). There was no significant difference in the proportion of A4 grade between the soybean oil and rapeseed oil treatment groups (P > 0.05). In addition, compared with the control group, rapeseed oil supplementation significantly enhanced intermuscular fat thickness (P < 0.05), but there was no significant difference compared with the soybean oil supplementation group (P > 0.05), and neither was there a significant difference between the soybean oil supplementation group and the control group (P > 0.05). Carcass yield parameters followed a similar pattern of improvement (Table 1). Compared with the control group, the soybean oil supplementation group significantly increased the slaughter weight (P < 0.05), with no significant difference compared with the rapeseed oil supplementation group (P > 0.05). The slaughter weight of the rapeseed oil supplementation group was significantly higher than that of the control group (P < 0.05), while there was no significant difference in slaughter weight between the soybean oil group and either the rapeseed oil group or the control group (P > 0.05). The carcass weight in both oil supplementation groups was significantly higher than that in the control group (P < 0.05). However, there were no significant changes in beef dressing percentage, eye muscle area, backfat thickness, or rib meat thickness (all P > 0.05).
The impact of supplementing soybean oil and rapeseed oil during the late fattening period on marbling score and carcass traits of Woking black cattle.
| Control | Soybean Oil | Rapeseed Oil | SE | P Value | |
|---|---|---|---|---|---|
| Serum vitamin A concentration (IU/dL) | 77.67 | 96.49 | 86.25 | 3.169 | 0.095 |
| Intramuscular fat (marbling score) | 185.71B | 293.75A | 366.67A | 16.524 | <0.001 |
| Slaughter weight (kg) | 649.29b | 730.25a | 722.94a,b | 12.094 | 0.050 |
| Carcass weight (kg) | 384.00b | 435.25a | 435.36a | 7.950 | 0.048 |
| Beef dressing percentage (%) | 58.97 | 59.61 | 60.15 | 0.246 | 0.227 |
| Eye muscle area (cm2) | 50.94 | 53.15 | 55.09 | 1.256 | 0.517 |
| Backfat thickness (cm) | 1.84 | 2.21 | 2.21 | 0.088 | 0.299 |
| Rib thickness (cm) | 5.97 | 6.33 | 6.42 | 0.102 | 0.307 |
| Intermuscular fat thickness (cm) | 3.03b | 3.46a,b | 3.75a | 0.107 | 0.052 |
Soybean oil and rapeseed oil treatment was conducted during late fattening period (27 [1] mo to age 30 mo) in Woking black cattle. Marbling score grade: 500 = A5, 400 = A4, 300 = A3, 200 = A2, 100 = A1. Data are presented as mean ± SEM. P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses.
To evaluate whether these performance benefits compromised animal health, we assessed key metabolic parameters (Table 1 and Figure 2D). Serum biochemical analysis revealed that the oil treatment group had a tendency to increase serum vitamin A concentration (P = 0.095, Table 1), while there was no significant difference between the other treatment groups (P > 0.05, Figure 2D), with liver function markers, glucose metabolism indicators, and lipid profiles remaining within normal physiological ranges. These results indicate that the observed improvements occurred without disrupting metabolic homeostasis. These findings demonstrate that dietary supplementation with soybean oil or rapeseed oil, particularly rapeseed oil, effectively enhances both beef quality and carcass yield while maintaining normal physiological function.
Based on the differences in slaughter performance and beef grades observed in Table 1 and Figure 2C, we further investigated the underlying mechanisms. Western blot analysis was performed to examine the expression of key regulatory proteins involved in adipogenesis and fatty acid synthesis in longissimus thoracis muscle. Compared with the control group, both oil supplements significantly upregulated FABP4 expression, a key regulator of adipogenesis that plays a central role in promoting adipocyte hypertrophy and lipid accumulation (Song et al., 2025) (P < 0.05, Figure 2E). However, rapeseed oil demonstrated a more pronounced effect, significantly enhancing PPARγ expression compared with both control and soybean oil groups (P < 0.05). There was no significant difference in C/EBPα expression levels among the groups (P > 0.05). These findings indicate that, consistent with the cell culture experimental results, rapeseed oil exhibits significantly superior efficacy compared to soybean oil in promoting IMF accumulation in beef cattle.
Soybean oil and rapeseed oil supplementation differentially regulate fatty acid composition in longissimus thoracis muscle of beef cattle during late fattening period
Having confirmed the positive effects of both vegetable oils on IMF accumulation in beef, we next examined fatty acid composition, a key determinant of beef fat healthiness and consumer acceptance. Fatty acid profiling of longissimus thoracis muscle samples revealed distinct compositional changes following dietary oil supplementation (Table 2). Soybean oil supplementation significantly increased concentrations of several fatty acids compared with controls, including elaidic acid (C18:1n9t, P < 0.01), myristic acid (C14:0), linolelaidic acid (C18:2t, n-6), and γ-linolenic acid (C18:3c, n-6) (P < 0.05). A notable trend toward increased linoleic acid (C18:2c, n-6) was also observed (P = 0.056), while cis-11,14,17-eicosatrienoic acid (C20:3c, n-3) content decreased (P < 0.05).
Effects of soybean oil and rapeseed oil on fatty acid profiles in the longissimus thoracis muscle of Woking black cattle.
| Fatty Acid/% | Control | Soybean Oil | Rapeseed Oil | SE | P Value |
|---|---|---|---|---|---|
| Octanoic acid (C8:0) | 0.043 | 0.059 | 0.041 | 0.0073 | 0.065 |
| Decanoic acid (C10:0) | 0.013 | 0.014 | 0.016 | 0.0035 | 0.809 |
| Hendecanoic acid (C11:0) | 0.018 | 0.021 | 0.022 | 0.0025 | 0.666 |
| Lauric acid (C12:0) | 0.018 | 0.021 | 0.019 | 0.0017 | 0.556 |
| Myristic acid (C14:0) | 1.344b | 1.876a | 1.624a,b | 0.1476 | 0.033 |
| Myristoleic acid (C14:1) | 0.220 | 0.305 | 0.276 | 0.0305 | 0.187 |
| Pentadecanoic acid (C15:0) | 0.137 | 0.160 | 0.154 | 0.0175 | 0.566 |
| Cis-10-pentadecanoic acid (C15:1) | 0.038 | 0.037 | 0.034 | 0.0028 | 0.448 |
| Palmitic acid (C16:0) | 35.661 | 37.539 | 36.432 | 1.3481 | 0.336 |
| Palmitoleic acid (C16:1) | 1.435 | 1.805 | 1.684 | 0.1228 | 0.205 |
| Heptadecanoic acid (C17:0) | 0.422 | 0.476 | 0.498 | 0.0505 | 0.434 |
| Cis-10-heptadecenoic acid (C17:1) | 0.285 | 0.330 | 0.345 | 0.0258 | 0.199 |
| Stearic acid (C18:0) | 6.244 | 5.726 | 5.984 | 0.2360 | 0.379 |
| TVA (C18:1,n11t) | 0.568 | 0.735 | 0.842 | 0.1228 | 0.301 |
| Oleic acid (C18:1, n9c) | 50.683 | 47.476 | 48.931 | 1.7492 | 0.125 |
| Elaidic acid (C18:1, n9t) | 0.218B | 0.308A | 0.245B | 0.0152 | <0.001 |
| Linolelaidic acid (C18:2t, n-6) | 0.159b | 0.207a | 0.178b | 0.0086 | 0.001 |
| Linoleic acid (C18:2c,n-6) | 2.091 | 2.584 | 2.259 | 0.1775 | 0.056 |
| Cis-9, trans-11 CLA | 0.066B | 0.053B | 0.091A | 0.0068 | <0.001 |
| Trans-10, cis-12 CLA | 0.006B | 0.006B | 0.010A | 0.0008 | <0.001 |
| γ-linolenic acid (C18:3c,n-6) | 0.003b | 0.006a | 0.005a | 0.0007 | 0.018 |
| α-linolenic acid (C18:3c,n-3) | 0.030b | 0.030b | 0.056a | 0.0085 | 0.008 |
| Arachidic acid (C20:0) | 0.008a | 0.006a,b | 0.005b | 0.0015 | 0.073 |
| Cis-11-eicosenoic acid (C20:1) | 0.038b | 0.047a,b | 0.058a | 0.0042 | 0.003 |
| Cis-11,14-eicosadienoic acid (C20:2c,n-6) | 0.030 | 0.021 | 0.025 | 0.0040 | 0.371 |
| Cis-11,14,17-eicosatrienoic acid (C20:3c,n-3) | 0.003a | 0.002b | 0.002a,b | 0.0005 | 0.060 |
| ARA (C20:4c,n-6) | 0.002 | 0.002 | 0.003 | 0.0004 | 0.201 |
| EPA (C20:5c,n-3) | 0.006B | 0.007B | 0.015A | 0.0018 | <0.001 |
| Heneicosanoic acid (C21:0) | 0.025 | 0.024 | 0.025 | 0.0037 | 0.971 |
| Behenic acid (C22:0) | 0.165 | 0.098 | 0.100 | 0.0280 | 0.081 |
| DHA (C22:6c,n-3) | 0.005b | 0.007b | 0.013a | 0.0016 | 0.001 |
| Tricosanoic acid (C23:0) | 0.001 | 0.002 | 0.003 | 0.0003 | 0.111 |
| Lignoceric acid (C24:0) | 0.004 | 0.004 | 0.004 | 0.0012 | 0.997 |
| Cis-15-tetracosaenoic acid (C24:1) | 0.005b | 0.005b | 0.008a | 0.0010 | 0.011 |
Abbreviations: ARA, arachidonic acid; CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TVA, trans-vaccenic acid.
P values for differences between means are shown. P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”).
Rapeseed oil supplementation produced markedly different fatty acid modifications. This treatment significantly enhanced level of bioactive fatty acids, including cis-9, trans-11 CLA, trans-10, cis-12 CLA, and EPA (C20:5c, n-3) (P < 0.01). Additional increases were observed in γ-linolenic acid (C18:3c, n-6), α-linolenic acid (C18:3c, n-3), cis-11-eicosenoic acid (C20:1), DHA (C22:6c, n-3), and cis-15-tetracosaenoic acid (C24:1) (P < 0.05), while arachidic acid (C20:0) concentrations decreased (P < 0.05). Direct comparison between the 2 oil treatments revealed superior functional fatty acid enrichment with rapeseed oil. Both CLA isomers and EPA content were significantly higher in the rapeseed oil group compared with the soybean oil group (P < 0.01), with α-linolenic acid and DHA also showing significant elevation (P < 0.05). Notably, linoleic acid content was significantly higher in the soybean oil group than in the rapeseed oil group (P < 0.01). In contrast, soybean oil supplementation failed to significantly enhance these functional fatty acid parameters compared with controls.
These differential effects reflect the distinct fatty acid profiles of the supplemented oils (Supplementary Table 2). Soybean oil contained predominantly linoleic acid (65.70%) and oleic acid (13.45%), while rapeseed oil showed higher oleic acid content (63.03%). While soybean oil primarily elevated n-6 fatty acids without affecting n-3 levels, rapeseed oil selectively enhanced n-3 fatty acids and beneficial bioactive compounds, resulting in beef with a significantly more favorable n-6/n-3 ratio. Analysis of overall fatty acid categories confirmed these differential effects (Table 3). Soybean oil supplementation significantly increased total n-6 fatty acids compared with controls (P < 0.05), with a trend toward elevated total polyunsaturated fatty acids (PUFAs) (P = 0.075) but showed no significant changes in n-3 fatty acids or the n-6/n-3 ratio compared with controls. Rapeseed oil supplementation produced more pronounced and nutritionally favorable changes, significantly increasing n-3 fatty acid levels compared with both the control and soybean oil groups (P < 0.05), while simultaneously reducing the n-6/n-3 fatty acid ratio compared with both groups (P < 0.01). In addition, the ratios of c9, t11-CLA/ (trans-vaccenic acid [TVA] + c9, t11-CLA) and c9, t11-CLA/TVA in the rapeseed oil treatment group were significantly higher than those in the soybean oil group (P < 0.05), but there was no significant difference between these 2 groups and the control group (P > 0.05).
Effects of soybean oil and rapeseed oil on the comprehensive profiles of fatty acids in the longissimus thoracis muscle of Woking black cattle.
| Fatty Acid/% | Control | Soybean Oil | Rapeseed Oil | SE | P Value |
|---|---|---|---|---|---|
| SFA | 44.104 | 46.026 | 44.926 | 1.6456 | 0.393 |
| MUFA | 53.490 | 51.048 | 52.422 | 1.6640 | 0.238 |
| PUFA | 2.402 | 2.925 | 2.657 | 0.1809 | 0.075 |
| MUFA/SFA | 1.233 | 1.119 | 1.175 | 0.0898 | 0.301 |
| PUFA/SFA | 0.055 | 0.064 | 0.059 | 0.0051 | 0.257 |
| n-6 fatty acids | 2.291b | 2.827a | 2.481a,b | 0.1786 | 0.043 |
| n-3 fatty acids | 0.045b | 0.046b | 0.086a | 0.0108 | 0.001 |
| n-6 /n-3 fatty acids | 55.620A | 65.810A | 33.412B | 7.5613 | <0.001 |
| C14:1/(C14:0+C14:1) | 0.147 | 0.142 | 0.144 | 0.0142 | 0.956 |
| C16:1/(C16:0+C16:1) | 0.039 | 0.046 | 0.044 | 0.0033 | 0.393 |
| C18:1, n9c/(C18:0+C18:1, n9c) | 0.889 | 0.892 | 0.891 | 0.0056 | 0.905 |
| c9, t11-CLA/(TVA+c9, t11-CLA) | 0.112ab | 0.076b | 0.118a | 0.0134 | 0.020 |
| c9, t11-CLA/TVA | 0.128ab | 0.084b | 0.138a | 0.0170 | 0.021 |
Abbreviations: CLA, conjugated linoleic acid; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TVA, trans-vaccenic acid.
P values for differences between means are shown. P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”).
These results demonstrate that rapeseed oil is significantly superior to soybean oil in optimizing fatty acid profiles and n-6/n-3 ratios while also showing clear advantages in promoting endogenous tissue CLA conversion efficiency. The observed muscle fatty acid modifications suggest that dietary oleic and linoleic acids serve as substrates for enzymatic conversion pathways, leading to enhanced polyunsaturated fatty acid content in beef, particularly γ-linolenic acid, α-linolenic acid, CLA isomers, EPA, and DHA. Notably, rapeseed oil increased CLA content in beef, which may be attributed to its high oleic acid content that upregulates fatty acid enzyme systems and thereby enhances endogenous CLA conversion.
To investigate the underlying mechanisms, we further analyzed lipid synthesis-related proteins in longissimus thoracis muscle (Figure 2F). Western blot analysis revealed that rapeseed oil significantly increased SREBP1 expression compared with both the control and soybean oil groups (P < 0.05). Furthermore, compared with both the control group and soybean oil supplementation group, rapeseed oil substantially increased the expression of fatty acid synthesis enzymes, including acetyl-CoA carboxylase alpha (ACACA), fatty acid synthase (FASN), and SCD (P < 0.05). ACACA, as the rate-limiting enzyme for long-chain fatty acid biosynthesis, primarily promotes de novo fatty acid synthesis while driving adipocyte differentiation and lipid accumulation (Dong et al., 2024; Piórkowska et al., 2020). FASN is a key enzyme that regulates de novo synthesis of long-chain saturated fatty acids and can synthesize precursors of various other fatty acid types (Rowland et al., 2023). SCD influences fat accumulation by participating in adipocyte differentiation and fatty acid desaturation processes, and it promotes CLA synthesis (Du et al., 2022; Yang et al., 2017). The changes in expression of these proteins following rapeseed oil treatment provide a mechanistic basis for the increased CLA content in beef and improved n-3 fatty acid profile observed in Table 3. Compared with the control group, no significant changes in these indices were observed in the soybean oil group (Figure 2F). Dietary supplementation with rapeseed oil reshaped the fatty acid profile of longissimus thoracis muscle in Woking black cattle through regulation of the fatty acid synthase system. In contrast, soybean oil had limited regulatory effects on these enzyme system-related indicators, which further explains the differences in their impacts on beef fat quality in practical applications.
Dietary soybean oil and rapeseed oil regulate adipogenesis and fatty acid synthesis pathways in beef cattle
The activation of adipogenic transcription factors and fatty acid synthase systems is essentially under the precise regulation of upstream signaling pathways. To clarify the upstream regulatory mechanisms, based on the omics data results from our previous study (unpublished data) and changes in phenotypic proteins, we chose to examine the PI3K/AKT/mTOR signaling axis, which plays a central role in regulating lipid metabolism. Western blot analysis revealed that rapeseed oil supplementation significantly upregulated PI3K, AKT, and mTOR, along with their phosphorylated forms (except p-AKT), compared with controls (P < 0.05, Figure 3). In contrast, soybean oil supplementation exhibited no significant effect on these pathway components (P > 0.05). Additionally, rapeseed oil significantly decreased the phosphorylation of β-catenin compared with the control group (P < 0.05), further supporting enhanced adipogenic signaling. Compared with the control group, soybean oil also had no significant effect on phosphorylated β-catenin (P > 0.05). These findings indicate that rapeseed oil can more effectively activate the PI3K/AKT/mTOR signaling axis than soybean oil, thereby upregulating downstream adipogenic transcription factors and the fatty acid synthase system, promote IMF deposition and CLA enrichment. This may explain the differential effects observed in cattle muscle regarding fatty acid composition and overall lipogenic capacity.
Effects of soybean oil and rapeseed oil supplementation during the late fattening period on protein expression in adipogenesis and lipogenesis-related pathways in Woking black cattle. When comparing data among 3 or more groups, P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses, using “B” and “b,” respectively. Abbreviations: AKT, protein kinase B; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase.
Rapeseed oil promotes adipogenesis and lipid metabolism in bovine skeletal muscle-derived cells by activating the PI3K/AKT/mTOR signaling axis
To validate the molecular mechanisms underlying differential oil effects on lipogenesis and lipid metabolism observed in animal experiments, as well as to confirm whether the PI3K/AKT/mTOR signaling axis is specifically activated by rapeseed oil and promotes IMF deposition and endogenous synthesis of CLA in beef cattle, we employed BSMC for functional validation.
Given that TG accumulation and the expression of key adipogenic transcription factors were significantly increased in cell validation assays, whereas soybean oil showed no significant changes in these indices, and the specific activation of PI3K/AKT/mTOR signaling components was observed in rapeseed oil-treated longissimus thoracis muscle (Figure 3), we selected rapeseed oil to further validate the pathway role through pathway-specific inhibition experiments. Treatment with the PI3K inhibitor (ZSTK474) and AKT inhibitor (GSK2141795) significantly reduced the additional lipid accumulation induced by rapeseed oil treatment (P < 0.05, Figures 4 and 5), demonstrating pathway dependency. Next, we detected the signaling axis and its downstream components. In addition, it is known that AKT can inactivate glycogen synthase kinase 3 β (GSK3β) through phosphorylation, thereby maintaining the stability of β-catenin; based on this, we speculate that the downregulation of β-catenin observed in muscle tissue may be related to the activation of the AKT/GSK3β pathway. Western blot analysis revealed that, compared with the control group, rapeseed oil could upregulate the protein expression level of mTOR, as well as upregulated adipogenic transcription factors (C/EBPα, FABP4, PPARγ, and SREBP1) and fatty acid synthesis enzymes (ACACA, FASN, and SCD) (all P < 0.05), and significantly reduced the phosphorylation level of β-catenin (P < 0.05), but it had no significant effect on the protein expression of phosphorylated GSK3β (P > 0.05) (Figures 4 and 5). In addition, compared with the control group, rapeseed oil could upregulate the protein phosphorylation expression level of AKT (P < 0.05) (Figure 5) and significantly upregulated the phosphorylation level of mTOR protein (P < 0.05) (Figure 6).
Inhibition of PI3K (ZSTK474) blocks rapeseed oil-induced adipogenesis in BSMC. Effects of different treatments on lipid accumulation and lipid metabolism in BSMC as indicated by triglyceride quantification and pathway-related protein expression. When comparing data among 3 or more groups, P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses, using “B” and “b,” respectively. Abbreviations: ACACA, acetyl-CoA carboxylase α; BSMC, bovine skeletal muscle-derived cells; C/EBPα, CCAAT/enhancer binding protein α; FABP4, fatty acid-binding protein 4; FASN, fatty acid synthase; GSK3β, glycogen synthase kinase 3 β; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase; PPARγ, peroxisome proliferator activated receptor γ; SCD, stearoyl-CoA desaturase; SREBF1, sterol regulatory element binding transcription factor 1; TG, triglycerides.
Inhibition of AKT (GSK2141795) blocks rapeseed oil-induced adipogenesis and lipogenesis in BSMC. Effects of different treatments on lipid accumulation and lipid metabolism in BSMC as indicated by triglyceride quantification and pathway-related protein expression. When comparing data among 3 or more groups, P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses, using “B” and “b,” respectively. Abbreviations: ACACA, acetyl-CoA carboxylase α; AKT, protein kinase B; BSMC, bovine skeletal muscle-derived cells; C/EBPα, CCAAT/enhancer binding protein α; FABP4, fatty acid-binding protein 4; FASN, fatty acid synthase; GSK3β, glycogen synthase kinase 3 β; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase; PPARγ, peroxisome proliferator activated receptor γ; SCD, stearoyl-CoA desaturase; SREBF1, sterol regulatory element binding transcription factor 1; TG, triglycerides.
Inhibition of mTOR (rapamycin) blocks rapeseed oil-induced adipogenesis and lipogenesis in BSMC. Effects of different treatments on lipid accumulation and lipid metabolism in BSMC as indicated by triglyceride quantification and pathway-related protein expression. When comparing data among 3 or more groups, P < 0.01 was considered as statistically extremely significant difference (marked as “A”), and P < 0.05 was regarded as statistically significant difference (marked as “a”). The same labeling method was used for subsequent analyses, using “B” and “b,” respectively. Abbreviations: ACACA, acetyl-CoA carboxylase α; AKT, protein kinase B; BSMC, bovine skeletal muscle-derived cells; C/EBPα, CCAAT/enhancer binding protein α; FABP4, fatty acid-binding protein 4; FASN, fatty acid synthase; GSK3β, glycogen synthase kinase 3 β; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase; PPARγ, peroxisome proliferator activated receptor γ; mTOR: mechanistic target of rapamycin, FASN, fatty acid synthase, SCD, stearoyl-CoA desaturase; SREBF1, sterol regulatory element binding transcription factor 1; TG, triglycerides.
Pathway inhibition studies revealed hierarchical signaling dynamics that, compared with the control group, ZSTK474 attenuated PI3K phosphorylation (P < 0.05), significantly inhibited the high expression of downstream mTOR protein, adipogenic transcription factors (C/EBPα, FABP4, PPARγ, and SREBP1), and SCD induced by rapeseed oil, and also relieved the inhibitory effect of rapeseed oil on phosphorylated β-catenin (all P < 0.05). Similarly, it had no significant effect on the changes in the expression of phosphorylated GSK3β protein (P > 0.05) (Figure 4). GSK2141795 relieved the promoting effect of rapeseed oil on downstream mTOR protein, fatty acid synthesis enzymes (ACACA, FASN, and SCD), and adipogenic transcription factors (SREBP1, PPARγ) (all P < 0.05). Similarly, it had no significant effect on the changes in the expression of phosphorylated GSK3β and β-catenin protein (P > 0.05) (Figure 5).
In previous animal experiments, we found that supplementary feeding of rapeseed oil could significantly increase the level of mTOR protein (a downstream target of the PI3K/AKT pathway) and its phosphorylation in muscle tissue. Based on this finding, we also conducted corresponding inhibitor-based cell experiments to verify its function in the pathway. The Western blot results showed that rapamycin, as a specific inhibitor of mTOR, could significantly inhibit the upregulation of mTOR protein phosphorylation level induced by rapeseed oil (P < 0.05). In addition, rapamycin also reversed the promoting effect of rapeseed oil on downstream adipogenic transcription factors (including C/EBPα, PPARγ, and SREBP1) (all P < 0.05) (Figure 6). It is worth noting that although mTOR inhibitor treatment did not cause a significant change in intracellular TG content, and the combined use of rapeseed oil and mTOR inhibitor also failed to restore the TG content to the normal level while at the protein level (Figure 6), rapamycin successfully inhibited the promoting effect of rapeseed oil on downstream (SREBP1, C/EBPα, and PPARγ). This finding suggests that the mTOR signaling pathway and the adipogenic transcription factors are connected through an upstream-downstream regulatory mechanism, with mTOR acting as a pivotal core within this pathway. The reasons for the unaffected TG content may be multifaceted: on the one hand rapeseed oil may offset the effect of mTOR inhibition by inhibiting the activation of the β-catenin signaling pathway; on the other hand, the high expression of the PI3K-AKT pathway already activated by rapeseed oil may partially weaken the regulatory effect of the mTOR inhibitor. Altogether, our data indicate that rapeseed oil, by activating the PI3K/AKT/mTOR signaling axis and subsequently activating adipogenic transcription factors and fatty acid synthase systems, not only enhances the quality grade of beef but also optimizes the fatty acid profile of beef and promotes the endogenous synthesis of CLA.
Discussion
Driven by population growth, shifting dietary preferences, and increased health consciousness, beef rich in IMF is highly favored for its tender texture and rich flavor. However, this very characteristic also raises consumers’ concerns about potential health issues such as obesity (Vahmani et al., 2015). Therefore, marbled beef that combines flavor and health attributes should be regarded as the quality pursuit direction of the current beef cattle industry. As a concentrated energy source, vegetable oil can improve the growth performance of ruminants and optimize their rumen microenvironment (Ibrahim et al., 2021). Supplementing oil through the diet is a major method to regulate the IMF content and fatty acid composition of ruminant meat, but the impacts vary significantly due to differences in the fatty acid profiles and active ingredients of different vegetable oils (Adeyemi et al., 2015). Soybean and rapeseed oils, widely consumed for their distinct fatty acid compositions and nutritional value (Chen, Chaudhary, and Mathys, 2022; Zhou et al., 2020). They have been extensively studied in the field of human nutrition and are 2 representative types of vegetable oils.
Currently, the comparative effects of soybean oil and rapeseed oil on IMF accumulation, fatty acid composition, and related molecular mechanisms in beef cattle remain unclear. This study addresses this gap by evaluating how dietary soybean oil or rapeseed oil supplementation during the late fattening phase influences carcass traits, muscle fatty acid profiles, and lipid metabolism signaling pathways in beef cattle.
The present study demonstrates that dietary supplementation with soybean oil and rapeseed oil during late fattening differentially enhances beef quality in Woking black cattle through distinct molecular mechanisms. Our findings reveal that while both oils improve carcass characteristics and IMF accumulation, rapeseed oil exhibits superior efficacy in producing premium-grade beef with enhanced nutritional profiles through coordinated activation of adipogenic signaling pathways. Dietary supplementation with both vegetable oils significantly improved carcass weights and marbling scores without compromising metabolic health, as evidenced by normal serum biochemical parameters. The carcass weight increased significantly, while there was no significant growth in subcutaneous fat. In contrast, there was a significant accumulation of IMF and intermuscular fat, along with a slight increase in eye muscle area and rib meat thickness. This result indicates that the weight gain brought by oil supplementation precisely drives fat accumulation in high-value parts, accompanied by a small amount of muscle growth. The economic implications are substantial, particularly for China’s beef industry, which has long struggled with insufficient high-grade beef production (Gao et al., 2023). While soybean oil supplementation achieved 100% ≥A2 grade beef, rapeseed oil demonstrated superior performance with 100% ≥A3 grade, including 50% A4 and 17% A5 grades. This dramatic improvement from zero A4+ grades in controls to substantial premium production represents a significant advancement in domestic beef quality enhancement. Faced with the dilemma of supply-demand imbalance, China’s beef cattle industry is urgently required to comprehensively improve beef quality through innovative nutritional strategies. In this study, the enhancement of beef quality by 2 types of vegetable oils, especially rapeseed oil, provides a new perspective for China to break through this predicament.
Studies have indicated that mTOR activation upregulates PPARγ and its downstream target genes, utilizing exogenous fatty acids for lipid synthesis (Angela et al., 2016). In this experiment, the significant upregulation of PPARγ and C/EBPα protein expression in muscle and cells should be attributed to oleic acid, which activates the PI3K/AKT cascade to activate mTOR, thereby mediating the expression of PPARγ and downstream proteins and consequently increasing IMF accumulation in beef. In contrast, soybean oil, which is mainly composed of linoleic acid, does not significantly regulate this pathway. Thus, the differential effects observed between the 2 oils stem from their distinct fatty acid compositions and subsequent metabolic outcomes. The soybean oil used in this study was rich in linoleic acid content (65.7%), primarily elevated n-6 PUFAs in muscle tissue, resulting in an n-6/n-3 ratio of 55.62. In contrast, rapeseed oil, rich in oleic acid (63.03%), significantly enhanced beneficial n-3 PUFAs, including EPA, DHA, and α-linolenic acid, achieving a more favorable n-6/n-3 ratio of 33.41. Lower n-6/n-3 ratio in the diet have demonstrated beneficial effects on cardiovascular and lipid metabolic health (Bishehkolaei and Pathak, 2024; Gonzalez-Becerra et al., 2023). This nutritional optimization holds particular significance given the prevalent dietary imbalances in current human consumption patterns. The phosphorylation of AKT can activate mTOR, which in turn promotes the function of SREBP1, cascading regulating the activation of downstream fatty acid synthase systems (Mu et al., 2021). The optimization of fatty acid profiles observed in beef in this study can be attributed to the specific activation of PI3K/AKT by oleic acid.
A particularly noteworthy finding was that rapeseed oil demonstrated superior enhancement of CLA content despite containing lower linoleic acid level than soybean oil. CLA, recognized for its anti-obesity, anti-inflammatory, and anti-cancer properties (He et al., 2024), is primarily synthesized in ruminants through ruminal microbial hydrogenation of linoleic and α-linolenic acids and endogenous tissue conversion via SCD (Gebereyowhans, 2024; Schmid et al., 2006). The synthesis and accumulation of CLA in beef cattle muscle presents significant challenges. The process is susceptible to dietary composition and fluctuations in the rumen environment, while the efficiency of endogenous synthesis pathways is limited by trans-vaccenic acid availability (an intermediate product generated from ruminal biohydrogenation) and the expression and activity of key enzymes such as SCD. However, our results demonstrate that rapeseed oil can activate the PI3K/AKT/mTOR pathway, significantly upregulating SCD expression and activity, thereby efficiently catalyzing the conversion of trans-vaccenic acid to CLA in tissues. This process does not rely on exogenous synthesis by rumen microbes but rather achieves targeted CLA accumulation through the metabolic capacity of muscle tissue itself, compensating for lower substrate availability. The specific activation of the endogenous CLA synthesis pathway by rapeseed oil provides new insights for developing high-CLA beef. In contrast, soybean oil failed to effectively activate SCD, resulting in limited endogenous synthesis capacity. This mechanism may explain why our 3% soybean oil supplementation did not enhance CLA content. However, previous studies using higher supplementation concentrations (5–6%) have successfully observed this effect (Fiorentini et al., 2018; Han et al, 2025), possibly reflecting dosage-dependent effects or disrupted ruminal microbial activity (Griswold et al., 2003).
The lipid metabolism during the late fattening stage of beef cattle is crucial for improving meat quality, with more than one-half of the weight gain in this period being fat (Bai et al., 2024). Therefore, we chose to supplement with oils during this stage. Compared with treatment throughout the entire fattening period, this approach precisely and efficiently increased marbling deposition and saved costs. Additionally, during the fattening process, we adopted a synergistic strategy: Vitamin A is restricted during the fattening period to promote adipogenesis, while in the later stage, vitamin A levels are restored and rapeseed oil is supplemented to specifically activate adipogenic transcription factors and fatty acid synthases. On the basis of increasing adipocyte size, this further enhanced lipid accumulation and metabolism, thereby improving the marbling grade and optimizing the fatty acid composition. Reducing the level of vitamin A in the diet can improve marbling scores during the early fattening period. Note that while reducing dietary vitamin A improves marbling early in fattening, maintaining this restriction later harms health, growth, and marbling itself (Park et al, 2018). In contrast, the findings of this study reveal that supplementing with the 2 types of oils during the late fattening period tends to increase serum vitamin A level, which may be one of the reasons for the observed improvements in carcass weight and marbling scores. This combined fattening strategy thereby serves as an enhancement and refinement of the traditional vitamin A restriction approach.
The core demand of consumers for beef quality has shifted toward balancing flavor and health. The unique value of rapeseed oil lies in its ability to simultaneously enhance both aspects, an advantage that stems from precise regulation of lipid metabolism signaling pathways by the abundant oleic acid in rapeseed oil. This compound can drive adipocyte differentiation and lipid synthesis in beef while specifically enriching functional fatty acids such as CLA and optimizing overall fatty acid composition, thereby achieving simultaneous improvement in both the quantity and quality of beef.
At the molecular level, our findings reveal that rapeseed oil uniquely activated comprehensive lipid metabolism regulatory networks. PI3K/AKT/mTOR signaling pathway represents a key mechanistic insight underlying the effect of rapeseed oil. This pathway serves as a central regulator of lipid metabolism and adipogenesis (Ceccarelli et al., 2022; Chu et al., 2022), with our data showing significant upregulation of pathway components. The expression of the key downstream transcriptional regulators PPARγ and SREBP1 was significantly upregulated. The further downstream lipogenic-related factors, including C/EBPα, FABP4, and lipogenic enzymes including ACACA, FASN, and SCD (Liu et al., 2021; Yang et al., 2020), were significantly upregulated, accompanied by decreased expression of β-catenin, a negative regulator of adipogenesis (De Winter and Nusse, 2021). Therefore, we propose that oleic acid-rich rapeseed oil triggers PI3K phosphorylation via membrane receptors, cascading to activate AKT and mTOR, while relieving the inhibition of adipogenic factors by β-catenin (Choi et al., 2014; Li et al., 2015; Xu et al., 2024), thereby activating a comprehensive adipogenic regulatory network, promoting lipid deposition, and, more importantly, specifically upregulating SCD, thereby driving the endogenous conversion of CLA. This explains its dual advantage of simultaneously improving marbling and nutritional quality. This hypothesis is supported by previous findings that oleic acid can activate PI3K/AKT pathways through free fatty acid receptors (Matoba et al., 2017).
Functional validation using BSMC confirmed the pathway-dependent nature of the effects observed with rapeseed oil treatment. The significant increases in TG accumulation, expression of adipogenic genes, and fatty acid synthase systems could be effectively inhibited to a large extent by specific PI3K, AKT, and mTOR inhibitors, clearly establishing a mechanistic link between dietary oleic acid and the regulation of adipogenic metabolism as well as the enrichment of functional fatty acids, particularly CLA. In addition, it should also be recognized that the bioactive components of vegetable oil is highly complex. Beyond oleic acid, rapeseed oil contains various physiologically active components such as tocopherols and phytosterols, which have reported antioxidant, anti-inflammatory, and lipid metabolism-regulating potential (He et al., 2022) that may indirectly affect adipogenic differentiation and fatty acid metabolic pathways. Therefore, the synergistic or independent effects of other bioactive components must be considered. Future studies should investigate their specific roles in regulating beef fat deposition and fatty acid composition, thereby advancing our understanding of how vegetable oils improve beef quality.
Interestingly, CLA content in longissimus thoracis muscle was significantly increased in the rapeseed oil treatment group, while the content of its precursor, TVA, did not show a significant increase. This phenomenon may be related to efficient TVA conversion by SCD, and future studies can further verify this hypothesis by detecting TVA content in blood samples. Moreover, the observation that β-catenin phosphorylation downregulation occurs independently of the AKT/GSK3β axis indicates alternative regulatory mechanisms that merit future exploration. These results collectively highlight the complexity of adipogenic signaling networks and suggest that multiple pathways may contribute to the observed phenotypic changes. In contrast to rapeseed oil, soybean oil failed to significantly activate the PI3K/AKT/mTOR pathway or key adipogenic transcription factors, indicating distinct mechanisms underlying its moderate effects on IMF accumulation and fatty acid profile. While linoleic acid has been shown to upregulate adipogenic genes in bovine satellite cells (Belal et al., 2019; Belal et al., 2018), the precise regulatory mechanisms and optimal dosage requirements for the effects mediated by soybean oil require further investigation.
Conclusion
In conclusion, dietary rapeseed oil supplementation improved beef quality grade without affecting the physiological health of beef cattle, and rapeseed oil performed better than soybean oil. Rapeseed oil significantly optimized fatty acid profiles by increasing n-3 PUFA and CLA content while reducing the n-6/n-3 ratio, thereby improving beef nutritional value. Mechanistically, rapeseed oil specifically activated the PI3K/AKT/mTOR signaling pathway (Figure 7). In contrast, soybean oil increased n-6 PUFA content but failed to significantly activate this pathway. These results reveal the molecular mechanisms through which rapeseed oil promote endogenous CLA synthesis in beef, and provide new insights for producing beef that balances both quality grade and nutritional value to meet consumer demands.
Molecular alterations and potential regulatory mechanisms underlying rapeseed oil-induced improvement of IMF accumulation and muscle fatty acid profile in Woking black cattle during late fattening phase. Abbreviations: ACACA, acetyl-CoA carboxylase α; AKT, protein kinase B; BSMC, bovine skeletal muscle-derived cells; C/EBPα, CCAAT/enhancer binding protein α; FABP4, fatty acid-binding protein 4; FASN, fatty acid synthase; GSK3β, glycogen synthase kinase 3 β; mTOR, mechanistic target of rapamycin; IMF, intramuscular fat; PI3K, phosphoinositide 3-kinase; PPARγ, peroxisome proliferator activated receptor γ; mTOR: mechanistic target of rapamycin, FASN, fatty acid synthase, SCD, stearoyl-CoA desaturase; SREBF1: sterol regulatory element binding transcription factor 1; TG, triglycerides.
Author Contribution
Bai Han Liu: Writing – Original Draft, Data Curation, Visualization, Formal Analysis, Validation. Cheng Xing Zhang: Validation, Investigation, Resources. Guan Zhu Liu: Validation, Investigation, Resources. Dong Qiao Peng: Conceptualization, Funding acquisition, Supervision, Writing – Original Draft, Writing – Review & Editing. Yong Cheng Jin: Conceptualization, Funding acquisition, Supervision, Writing – Review & Editing. All listed authors have reviewed and approved the manuscript for submission to Meat and Muscle Biology.
Conflict of 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
This work was supported by Jilin Provincial Department of Science and Technology Key R&D Funding Project (20250205026GH), National Natural Science Foundation of China (32302762), and Changchun Science and Technology Key R&D Program Funding Project (21ZGN21). All experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University (license no. SY202302017) prior to study initiation. The datasets analyzed in the current study are available from the corresponding author on request.
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Supplementary Materials
Composition and nutrient content of basal diet (%, DM basis).
| Nutrient contents | Concentrate1 | Forage2 |
|---|---|---|
| Dry matter (DM, %) | 88.51 | 90.43 |
| Crude protein (% DM) | 13.66 | 4.65 |
| Ether extraction (% DM) | 6.36 | 0.60 |
| Crude fiber (% DM) | 5.01 | 32.29 |
| Crude ash (% DM) | 7.47 | 11.98 |
| Acid detergent fiber (% DM) | 11.10 | 51.47 |
| Neutral detergent fiber (% DM) | 25.94 | 66.49 |
| Calcium (% DM) | 1.05 | 0.51 |
| Phosphorus (% DM) | 0.62 | 0.17 |
| Vitamin A (IU/kg) | 1852.87 | 884.93 |
Commercial concentrate product,
Kneaded rice straw
The fatty acid composition profiles of soybean oil and rapeseed oil used in the experiment.
| Fatty acid/% | Soybean Oil | Rapeseed Oil | SE |
|---|---|---|---|
| Octanoic acid (C8:0) | 0.098 | 0.079 | 0.0342 |
| Decanoic acid (C10:0) | 0.021 | 0.020 | 0.0062 |
| Hendecanoic acid(C11:0) | 0.045 | 0.038 | 0.0075 |
| Lauric acid (C12:0) | 0.006 | 0.004 | 0.0004 |
| Myristic acid (C14:0) | 0.031 | 0.023 | 0.0008 |
| Pentadecanoic acid (C15:0) | 0.004 | 0.005 | 0.0004 |
| Cis-10-pentadecanoic acid (C15:1) | 0.029 | 0.022 | 0.0020 |
| Palmitic acid (C16:0) | 11.405 | 4.314 | 0.0562 |
| Palmitoleic acid (C16:1) | 0.034 | 0.064 | 0.0011 |
| Heptadecanoic acid (C17:0) | 0.027 | 0.011 | 0.0003 |
| Cis 10-Heptadecenoic acid (C17:1) | 0.013 | 0.012 | 0.0003 |
| Stearic acid (C18:0) | 1.198 | 0.609 | 0.0060 |
| Oleic acid (C18:1, n9c) | 13.445 | 63.028 | 0.2117 |
| Elaidic acid(C18:1, n9t) | 0.016 | 0.022 | 0.0003 |
| Linolelaidic acid (C18:2t, n-6) | 0.010 | 0.007 | 0.0008 |
| Linoleic acid (C18:2c,n-6) | 65.699 | 25.066 | 0.1626 |
| α-linolenic acid (C18:3c,n-3) | 7.016 | 6.260 | 0.0518 |
| Cis-11-Eicosenoicacid, (C20:1) | 0.899 | 0.415 | 0.0219 |
| Heneicosanoic acid (C21:0) | 0.002 | 0.003 | 0.0004 |
SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids.
SE:Standard Error
Antibody information and dilutions in this study.
| Antibodies Name | Diluted Multiples | Accession Number | Reagent Company |
|---|---|---|---|
| Mouse anti- β-actin polyclonal antibody | 1:5,000 | T0022 | Affinity |
| Rabbit anti- PPARγ polyclonal antibody | 1:5,000 | PAA886Bo01 | Cloud-clone |
| Rabbit anti- C/EBPα polyclonal antibody | 1:2,000 | bs-1630R | Bioss |
| Rabbit anti- FABP4 polyclonal antibody | 1:5,000 | 12802-1-AP | Proteintech |
| Rabbit anti- SCD polyclonal antibody | 1:2,000 | bs-55193R | Bioss |
| Rabbit anti- SREBP1 polyclonal antibody | 1:2,000 | bs-1402R | Bioss |
| Rabbit anti- ACACA polyclonal antibody | 1:8,000 | bs-2745R | Bioss |
| Rabbit anti- FASN polyclonal antibody | 1:5,000 | 10624-2-AP | Proteintech |
| Rabbit anti- PI3K p85 alpha polyclonal antibody | 1:2,000 | AF6241 | Affinity |
| Rabbit anti- Phospho- PI3K p85 alpha (Tyr607) Antibody | 1:2,000 | AF3241 | Affinity |
| Mouse anti- AKT monoclonal antibody | 1:1000 | bsm-33278M | Bioss |
| Mouse anti- phospho- AKT (Ser473) monoclonal antibody | 1:1000 | bsm-33281M | Bioss |
| Rabbit anti-Beta Catenin Polyclonal antibody | 1:5000 | 51067-2-AP | Proteintech |
| Rabbit anti-Phospho-Beta Catenin (Ser675) Polyclonal antibody | 1:2000 | 28853-1-AP | Proteintech |
| Rabbit anti- mTOR polyclonal antibody | 1:2000 | 28273-1-AP | Proteintech |
| Rabbit anti- phospho- mTOR polyclonal antibody | 1:2000 | 67778-1-lg | Proteintech |
| Mouse anti- GSK-3β polyclonal antibody | 1:2000 | bs-0023M | Bioss |
| Rabbit anti phospho- GSK-3 Beta (Ser21+Ser29) polyclonal antibody | 1:1000 | bs-5367R | Bioss |
| Goat anti- rabbit IgG antibody | 1:20,000 | bs-40295G-HRO | Bioss |
| Goat anti- mouse IgG antibody | 1:20,000 | bs-40296G-HRP | Bioss |
