Introduction
Hyperlipidemia is a critical metabolic disorder characterized by elevated plasma levels of lipids, including triglycerides (TGs), total cholesterol (TC), and proatherogenic low-density lipoprotein (LDL), often coupled with decreased levels of antiatherogenic high-density lipoprotein (HDL)1. Dyslipidemia is the primary risk factor for the initiation and progression of atherosclerosis2,3. Atherosclerosis is a chronic, progressive inflammatory disease characterized by lipid and cholesterol accumulation in the arterial wall, leading to plaque formation and vascular obstruction4.
As the leading cause of heart attacks and strokes, CVDs are responsible for a large percentage of global mortality, underscoring the urgent need for effective preventive strategies5,6.
The pathogenesis of atherosclerosis involves a complex interplay between dysregulated lipid metabolism, chronic inflammation, and endothelial dysfunction. Specifically, apolipoprotein B-containing LDL is particularly atherogenic, as it readily undergoes modifications (e.g., oxidation) and accumulates within the arterial intima, initiating early lesion development7,8. Even moderately elevated LDL levels can significantly exacerbate disease progression, particularly when combined with oxidative stress, which promotes lipid peroxidation and further compromises endothelial integrity1,7,9.
These complex, multifactorial mechanisms emphasize the direct importance of dietary fat quality in the development or prevention of cardiovascular disease10,11.
Dietary fat remains a major energy source12, but the health implications of different types of fats are not uniform. While saturated fatty acids (SFAs) are traditionally viewed as detrimental, recent evidence highlights a nuanced view13,14. The health impact of SFAs is not uniform but depends on factors such as fatty acid chain length, e.g., C:18:0 (stearic acid) has a more favorable effect on LDL-C than does C:16:0 (palmitic acid), and the food matrix15,16.
This complexity underscores the importance of evaluating specific fat sources. For example, animal tallow is a dense source of SFAs but also contains monounsaturated fatty acids (MUFAs), whereas olive oil is consistently cardioprotective because of its high MUFA content and antioxidant compounds17,18.
While pharmacological treatments such as simvastatin are highly effective in lowering LDL cholesterol by inhibiting HMG-CoA reductase and suppressing cholesterol synthesis19–21, dietary modification remains a cornerstone of CVD prevention. Given the limited and inconclusive scientific evidence regarding the atherogenic potential of traditional animal fats (such as tallow) and the scarcity of direct experimental comparisons between these fats and protective oils such as olive oil, a controlled study is essential22–24. The high-cholesterol-fed rat model is highly suitable because it provides a controlled environment to induce dyslipidemia and track key aspects of human lipid metabolism and early atherogenesis for compound screening.
Therefore, this study was designed to evaluate and directly compare the ameliorative effects of traditional dietary fats (sheep and bovine tallow) with those of a cardioprotective oil (olive oil) on hyperlipidemia, oxidative stress, and atherosclerosis progression in a controlled rat model. By including simvastatin as a pharmacological benchmark, this study also aimed to assess the efficacy of dietary interventions against standard clinical treatment. Addressing this critical knowledge gap will provide evidence-based insights into the true metabolic and cardiovascular impact of widely consumed animal fats, determining whether their effects are detrimental, neutral, or potentially beneficial in the context of advanced cardiovascular risk profiles.
Results
Fatty acid analysis of tallow and oil
The fatty acid compositions of bovine tallow, sheep tallow, and olive oil were analyzed via GC/FID. As shown in Table 1; Fig. 1, bovine tallow contained the highest proportion of saturated fatty acids (SFAs) (70.991%), followed by sheep tallow at 40.796%, while olive oil had the lowest SFA content (18.512%).
C18:0 (stearic acid) was the most abundant SFA in all three fat sources, accounting for 34.648%, 7.064%, and 3.535% of the total SFAs in the bovine, sheep, and olive oils, respectively. Notably, C4:0 (butyric acid) was detected exclusively in sheep tallow, and C6:0 (caproic acid) was detected only in bovine tallow.
Olive oil had the highest content of total monounsaturated fatty acids (MUFAs) (67.949%), followed by Sheep Tallow (51.659%) and Bovine Tallow (28.263%). The predominant MUFA in all the samples was C18:1 (oleic acid), which was present at 67.949% in olive oil, 43.737% in Sheep Tallow, and 26.325% in bovine tallow. Among the polyunsaturated fatty acids (PUFAs), olive oil had the highest total content at 12.181%, whereas bovine tallow had the lowest at 1.628%.
The most abundant PUFA was C18:2 t + 1 (trans-linoleic acid), with levels of 11.434%, 1.385%, and 1.353% in olive oil, bovine tallow, and sheep tallow, respectively, as illustrated in Table 1.
Table 1GC/FID analysis of fatty acid compositions in tallow and olive oil.FAsPercentage (%)aSheep tallowBovine tallowOlive oilC4:01.470 ± 0.009––C6:0–0.019 ± 0.002–C8:00.294 ± 0.0020.019 ± 0.001–C10:00.482 ± 0.00310.148 ± 0.003–C11:0–0.310 ± 0.0060.047 ± 0.001C12:00.118 ± 0.0030.515 ± 0.0080.023 ± 0.001C13:01.27 ±0.0071.27 ± 0.003–C14:03.714 ± 0.0033.714 ± 0.007–C14:10.841 ± 0.00210.255 ± 0.0032–C15:01.701 ± 0.0070.362 ± 0.0021–C15:10.226 ± 0.0030.37 ± 0.002–C16:021.768 ± 0.036328.368 ± 1.96414.240 ± 1.029C16:14.271 ± 0.0090.9350 ± 0.0060.937 ± 0.0061C17:02.174 ± 0.00421.276 ± 0.0020.134 ± 0.003C17:12.584 ± 0.0070.254 ± 0.0030.149 ± 0.004C18:07.064 ± 1.08134.648 ± 2.8733.535 ± 0.042C18:143.737 ± 2.82626.325 ± 3.08467.949 ± 3.294C18:2t + 11.353 ± 0.0061.385 ± 0.00511.434 ± 0.85C18:3n62.301 ± 0.0409––C18:3n33.184 ± 0.0340.168 ± 0.0030.630 ± 0.0023C20:0–0.254 ± 0.0010.533 ± 0.001C20:1–0.124 ± 0.0010.272 ± 0.0004C22:2–0.075 ± 0.00050.117 ± 0.0002C23:00.741 ± 0.0020.088 ± 0.0018–(a): Fatty acid percentages were calculated as the ratio of individual peak area to the total peak area of all detected fatty acids. The full details of the fatty acids, IUPAC names, and structures are presented in Fig. 1S-A-C and Tables 1S-A-C.
Lipid profile
Compared with the negative control (NC) group, the atherogenic (ATH) group presented significantly increased serum lipid levels, confirming the induction of hyperlipidemia (Table 2 and S2). The total cholesterol (TC) content increased from 91.4 ± 9.4 mg/dL in the NC group to 157.6 ± 5.51 mg/dL in the ATH group (p < 0.0001), and the triglycerides (TG) content increased from 129.1 ± 6.99 mg/dL to 156.0 ± 3.81 mg/dL (p < 0.0001).
Compared with the ATH group, all treated groups presented significant reductions in TC. The positive control (PC, simvastatin) had the greatest effect, reducing TC to 87.57 ± 4.65 mg/dL (p < 0.01), followed closely by the olive oil (OL) group, at 98.0 ± 8.72 mg/dL (p < 0.0001). The sheep tallow (SHT) group also presented a reduction in TC, at 140.4 ± 2.49 mg/dL, although this reduction was less pronounced (p < 0.05).
A similar pattern was observed for TG levels. The PC group achieved the most substantial reduction, lowering TG to 113.9 ± 6.31 mg/dL (p < 0.0001). Compared with the ATH group, the OL group also had significantly lower TG levels (133.7 ± 7.68 mg/dL) (p < 0.0001).
High-density lipoprotein cholesterol (HDL-C), known for its protective cardiovascular effects, was significantly higher in the sheep tallow (SHT) group (40.14 ± 1.35 mg/dL) than in the ATH group (36.0 ± 1.00 mg/dL), p < 0.001. A modest but statistically significant increase was also observed in the bovine tallow (BT) group (p < 0.01).
Low-density lipoprotein cholesterol (LDL-C) levels were markedly lower in all treated groups than in the ATH group (p < 0.001, p < 0.0001). The most notable reductions were observed in the olive oil (OL) and BT groups (32.69 ± 9.07 and 65.60 ± 9.92 mg/dL), followed by the SHT group. Very low-density lipoprotein cholesterol (VLDL-C) levels were significantly lower in the PC group (p < 0.0001), and to a lesser extent in the OL group (p < 0.01), than in the ATH group.
The atherogenic index (AI) was significantly lower in all treated groups compared to the ATH group (p < 0.001, p < 0.0001), indicating a general improvement in the lipid risk profile. Although all treatment groups significantly improved lipid profiles, the OL group demonstrated relatively greater effects on TC and LDL-C, with reductions comparable to or even greater than those in the simvastatin (PC) group. The sheep tallow (SHT) group consistently showed strong improvements across all lipid parameters, particularly notable reductions in LDL-C and increases in HDL-C.
The bovine tallow (BT) treatment also significantly ameliorated hyperlipidemia, albeit with slightly less pronounced effects than SHT or OL did for some parameters. (Table 2)
Table 2Effect of tallows and olive oil on lipid profile in atherogenic male rat groups.NCATBATHSHTBTOLPCTC(mg/dL)91.43 ± 9.1192.0 ± 3.74157.6 ± 5.51a ****140.4 ± 2.49b*138.4 ± 11.16b*98.0 ± 8.72b ****87.57 ± 4.65b**TG(mg/dL)129.1 ± 6.99175.6 ± 2.3156.0 ± 3.81a ****158.0 ± 4.93165.6 ± 7.91133.7 ± 7.68b****113.9 ± 6.31b****HDL-C (mg/dL)40.14 ± 1. 9535.80 ± 1.336.0 ± 1.00a ***40.14 ± 1.35b***39.71 ± 1.50b**38.57 ± 1.7239.71 ± 0.76b**LDL-C (mg/dL)25.31 ± 9.1121.5 ± 5.2290.40 ± 5.80a ****68.69 ± 11.86b ***65.60 ± 9.92b***32.69 ± 9.07b****86.77 ± 7.61VLDLC (mg/dL)25.83 ± 1.435.12 ± 0.4628.20 ± 0.76a *31.60 ± 0.9933.11 ± 1.5826.74 ± 1.54b**22.77 ± 1.26b****Atherogenic index (AI)1.29 ± 0.34.37 ± 0.33.38 ± 0.22a ****2.49 ± 0.31b ****2.62 ± 0.33b****1.54 ± 0.21b ****2.70 ± 0.19b **Results are expressed as the mean ± SD from 7 rats, with 3 replicates/rat. NC Negative control, ATB Atherogenic Baseline, ATH Atherogenic, SHT Sheep’s tallow, BT Bovine’s tallow, OL Olive oil, PC Positive control (Simvastatin). *= p < 0.05, **= p < 0.01, ***= p < 0.001 and ****= p < 0.0001 a: significant vs.
NC group, b: significant vs. ATH group. The confidence Intervals (CIs) and exact p-values are represented in Table S4
Lipid peroxidation and total antioxidant capacity
Malondialdehyde (MDA), a biomarker of oxidative stress, was significantly elevated (p < 0.0001) in the ATH group compared with the NC group. Treatment with both tallows (SHT and BT) significantly reduced the MDA level (p < 0.05 and p < 0.01, respectively), whereas the OL group presented a more pronounced reduction (p < 0.0001) than both tallow groups did. Notably, the OL group presented an even greater reduction in MDA than the PC (simvastatin) group did (Fig.
1A).
Compared with those in the NC group, total antioxidant (TAO) levels in the serum were lower in the ATH group. Treatment with sheep or bovine tallow significantly elevated TAO levels (p < 0.05 and p < 0.001, respectively), with levels exceeding those of the NC group in both the SHT and BT groups (Fig. 1B).
Fig. 1Effects of tallows and olive oil on serum lipid peroxidation and total antioxidant capacity levels in atherogenic male rats. (A) Malondialdehyde (MDA) content.
(B) Total antioxidant (TAO) content. The data are presented as the means ± SD from 7 rats, with 3 replicates/rat. NC negative control, ATB atherogenic baseline, ATH atherogenic, SHT sheep tallow, BT bovine tallow, OL olive oil, PC positive control (Simvastatin). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ****= p < 0.0001; a: significant vs. the NC group; b: significant vs. the ATH group.
The confidence Intervals (CIs) and exact p-values are represented in Table S3.
C-reactive protein
C-reactive protein (CRP), an acute-phase protein and marker of systemic inflammation, was significantly elevated (p < 0.0001) in the ATH group compared with the NC group. While all the treatment groups showed a reduction in CRP levels, only the OL group exhibited a statistically significant decrease (p < 0.05) compared with the ATH group (Fig. 2).
Fig. 2Effects of tallows and olive oil on serum C-reactive protein (CRP) levels in atherogenic male rats. The data are presented as the means ± SD from 7 rats, with 3 replicates/rat.
NC (negative control), ATB: atherogenic baseline, ATH (atherogenic), SHT (sheep tallow), BT (bovine tallow), OL (olive oil), PC (positive control, Simvastatin). * =p < 0.05, **** = p < 0.0001; a, significant vs. NC group; b, significant vs. ATH group.
The confidence Intervals (CIs) and exact p-values are represented in Table S3.
Histopathological results of the aorta
Histological examination of the aortic cross-sections from the NC group (Fig. 3a) revealed normal architecture, including an open lumen and intact tunica intima, media, and adventitia surrounded by loose connective tissue. The intimal surface appeared continuous with uniform thickness and no signs of bulging or disruption.
In contrast, aortic sections from the ATB and ATH groups revealed marked degenerative atheromatous changes (Fig. 3b,c). These included a thickened tunica intima with fatty streak lesions, subintimal lipid accumulation (foam cells and lipid droplets), and pools of extracellular lipids within both the intima and media layers.
Additional findings included irregular and fragmented elastic fibers, necrotic areas, and pyknotic nuclei (Fig. 3b,c).
Compared with the ATH group, the SHT group presented notable histological improvement, with a reduction in fatty streaks and a thin subintimal lipid layer, along with partial restoration of normal vascular architecture (Fig. 3d). Similar improvements were observed in the BT and OL groups (Fig.
3e,f), although some subintimal lipid accumulation and superficial erosion persisted, and full restoration of normal architecture was incomplete.
The PC group (Fig. 3g) also showed partial amelioration of atheromatous changes, with some recovery of vascular architecture. However, subintimal lipids and foam cells remained, and disorganization of the vascular layers, fragmentation of elastic fibers, and necrotic areas were evident.
Fatty streaks persist along with subintimal lipids, foam cells, superficial erosion, and necrosis. Restoration of normal vascular architecture was minimal.
Fig. 3Microscopy images of aorta cross-sections from atherogenic male rats in different groups showing histological features. (a) negative control (NC); (b) atherogenic baseline (ATB); (c) atherogenic (ATH); (d) sheep tallow (SHT); (e) bovine tallow (BT); (f) olive oil (OL); (g) positive control (PC) (simvastatin).
The aorta sections were excised from the root of the aorta, distal to the aortic valve, to the bifurcation of the aorta. The sections were stained with hematoxylin and eosin (H&E) at 200× magnification. lumen (L), tunica intima (TI), tunica media (TM), tunica adventitia (TA), loose connective tissue (LCT), subintimal lipid (SiL), foam cells (Fo), lipid droplets (Ld), lipid pools (Lp), fragmented fibers (Ff), necrosis (N), pyknotic nuclei (Pn), and superficial erosion (Se).
Morphometric analysis of the aorta
Quantitative assessment revealed a significant (p < 0.0001) reduction in the aortic inner diameter (AID) in the ATH group compared with the NC group. This reduction was significantly reversed in all the treated groups, SHT, BT, OL, and PC, with p-values p < 0.001, p < 0.01, p < 0.0001, and p < 0.01, respectively. A significant (p < 0.0001) increase in the intima + media (I + M) thickness was recorded in the ATH group compared with the NC group.
All the treated groups presented a significant (p < 0.0001) decrease in I + M thickness, reflecting reductions in both the intima and media layers (Fig. 3d–g). Furthermore, the intima/media ratio was significantly (p < 0.0001) greater in the ATH group than in the NC group.
However, no significant changes in these parameters were detected in the treated groups compared with the ATH group (Table 3).
Table 3Evaluating the effects of tallow and olive oil on aorta histology in atherosclerotic male rats.NCATBATHSHTBTOLPCAortic inner diameter (AID) (µM)1147 ± 49.1879.3 ± 17.2909.50 ± 7.1a****1066 ± 23.16b***1031 ± 53.13b **1089 ± 7.20b****1082 ± 52.6b**Intima thickness (µM)4.334 ± 0.7729.47 ± 23.227.77 ± 3.7a****15.74 ± 0.94b****17.15 ± 1.37b ****14.05 ± 0.82b****14.68 ± 0.82b ****Media thickness (µM)120.5 ± 16.4266.3 ± 8.02262.7 ± 5.84a***149.0 ± 4.53b****165.2 ± 3.43b***135.1 ± 9.96b***135,3 ± 1.40b***Intima + media thickness (µM)124.8 ± 16.82295.8 ± 9.12290.5 ± 4.93a***164.7 ± 4.39b****182.3 ± 2.35b****149.2 ± 10.0b****150 ± 2.117b****Intima/media ratio0.036 ± 0.010.11 ± 0.0160.105 ± 0.02a****0.106 ±. 0.01a****0.104 ± 0.01a***0.104 ± 0.01a****0.109 ± 0.01a ****Data are presented as the mean ± SD (n = 7 rats; 3 fields per rat). *= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001; a, vs. NC group; b, vs.
ATH group. NC negative control, ATB atherogenic baseline, ATH atherogenic, SHT sheep tallow, BT bovine tallow, OL olive oil, PC positive control (simvastatin). The inner diameter and wall thickness of the aorta cross-sections were measured at 0°, 90°, 180°, and 270° using an ocular micrometer calibrated with a stage micrometer (40x = 20 µM) and analyzed via ImageJ software.
The intima thickness was measured from the lumen to the internal elastic lamina. The media thickness was measured from the internal to the external elastic lamina.
Histopathological results of heart
Cardiac sections from the NC group presented an intact myocardial architecture with regularly branched muscle fibers, central oval nuclei, and visible striations (Fig. 4A). In contrast, the ATB and ATH groups presented characteristic histopathological alterations, including lipid accumulation (fatty cells and pools), fiber fragmentation, widened intercellular spaces, infiltration by inflammatory cells, and necrotic areas with pyknotic nuclei (Fig.
4B,C).
Treatments with tallow and olive oils in the SHT, BT, and OL groups (Fig. 4D–F) resulted in notable improvements, although residual fatty changes, hemorrhage, congestion, and necrosis persisted. The most pronounced histological recovery was observed in the SHT and OL groups.
Compared with the ATH group, the PC group (Fig. 4G) presented severe cardiac damage, including substantially widened intercellular spaces, extensive necrosis, fatty changes, hemorrhage, and congestion.
Fig. 4Microscopy images of heart cross-sections from atherogenic male rats in different groups. (A) negative control (NC), (B) atherogenic baseline (ATB), atherogenic (ATH), (D) sheep tallow (SHT), (E) bovine tallow (BT), (F) olive oil (OL), and (G) positive control (PC; simvastatin) groups.
The Sect. (5 µM thick) were obtained from the middle of the heart toward the basal part (5 mm), which is the origin of the blood vessels’ exit, and were stained with hematoxylin and eosin (H&E) at 200× magnification. myocardial fibers (MF), regular striations, oval central nuclei (Nu), fatty changes (Fc), hemorrhage (Hr), widened intercellular spaces (Wis), inflammation (Inf), congestion (Co), infiltration (In), and necrosis (N).
Quantitative assessment of cardiac lesions
The results of the quantitative histopathological analysis of key cardiac lesions (fatty changes, wide intercellular spaces, inflammation, infiltration, hemorrhage, congestion, and necrosis) are summarized in Table 4. Compared with the NC group, the ATH group presented a significant (p < 0.0001, p < 0.05) increase in all lesions. Fatty acid changes were significantly reduced in the SHT, BT, OL, and PC treatment groups (p < 0.001, p < 0.001, p < 0.001, and p < 0.0001, respectively).
Inflammation and congestion were also significantly improved in the SHT, BT, OL, and PC groups (p values ranging from p < 0.0001 to p < 0.001 for inflammation and p < 0.0001 and p < 0.01 for congestion in the SHT and OL groups, respectively). However, hemorrhage did not significantly improve in the treated groups, and the PC group even had a significantly worse hemorrhage score (p < 0.001) than the ATH group did. For wide intercellular spaces, only the SHT and OL groups showed significant (p < 0.05, p < 0.0001) improvement.
Compared with the ATH group, the SHT, BT, and OL groups presented significant reductions in necrosis (p < 0.001, p < 0.0001, and p < 0.001, respectively). Conversely, the PC group presented significantly more severe necrosis and widening of the intercellular space than did the ATH group (p < 0.001 and p < 0.05, respectively).
In summary, histological recovery was most evident in the SHT and OL groups. Unexpectedly, the PC group demonstrated the most severe cardiac histopathological changes, which were worse than those in the ATH group (Table 4).
Table 4Evaluating the effects of tallow and Olive oil on heart histology in atherosclerotic male rats.NCATBATHSHTBTOLPCFatty changes+0.10 ± 0.0++++6.10 ± 0.87+++4.63 ± 1.46a****++3.20 ± 0.36b***++3.00 ± 0.05b***++3.07 ± 0.29b***++2.43 ± 0.15b****Infiltration+0.10 ± 0.0++0.20 ± 0.1++0.20 ± 0.10++0.20 ± 0.1++0.17 ± 0.06++0.17 ± 0.06++0.17 ± 0.06Inflammation+0.167 ± 0.06+++0.90 ± 0.1++0.70 ± 0.10a****+0.27 ± 0.12b****++0.50 ± 0.10b***++0.37 ± 0.06b****++0.40 ± 0.10b****Congestion++0.27 ± 0.06++++0.97 ± 0.12++++0.93 ± 0.15a****++0.33 ± 0.15b****+++0.63 ± 0.06+++0.50 ± 0.10b**++++0.80 ± 0.20Hemorrhage+0.30 ± 0.10+++0.67 ± 0.12++0.63 ± 0.06a*++0.43 ± 0.15+++0.93 ± 0.06++0.80 ± 0.20++++1.27 ± 0.06b***Wide intercellular spaces+1.37 ± 0.12++7.90 ± 1.0++7.50 ± 0.10a****++5.67 ± 0.64b*++6.70 ± 0.46+2.40 ± 0.20b****++++21.70 ± 1.15b***Necrosis+0.70 ± 0.10++++17.67 ± 1.27++++17.63 ± 1.03 a****+++13.77 ± 0.86b***+++13.40 ± 0.72b****+++13.87 ± 0.93b***++++20.13 ± 0.65b*Data are presented as the mean ± SD (n = 7 rats; 3 fields per rat). *= p < 0.05, **= p < 0.01, ***= p < 0.001, ****= p < 0.0001; a, vs. NC group; b, vs. ATH group.
Grades are as follows: (-) absent; (+) trace (1–25%); (++) weak (26–50%); (+++) moderate (50–75%); (++++) severe (75–100%). NC negative control, ATB atherogenic baseline, ATH atherogenic, SHT sheep tallow, BT bovine tallow, OL olive oil, PC positive control (simvastatin).
Discussion
This study investigated the potential of sheep tallow, bovine tallow, and olive oil to mitigate hyperlipidemic-induced atherogenesis in rats fed an atherogenic diet. The results demonstrate that olive oil, and to a significant extent, sheep and bovine tallow, exert beneficial effects on lipid profiles, oxidative stress, inflammation, and the progression of atherosclerotic lesions.
The fatty acid analysis of these three different sources of dietary fat revealed distinct compositions for each fat source. Bovine tallow is characterized by a high content of saturated fatty acids, particularly C18:0, whereas olive oil is rich in monounsaturated fatty acids, predominantly C18:1 (oleic acid). Sheep tallow presented an intermediate profile.
These variations in fatty acid composition likely contribute to the differential effects observed on lipid metabolism and atherogenesis.
The atherogenic diet induced significant dyslipidemia, as evidenced by elevated levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), along with reduced high-density lipoprotein cholesterol (HDL-C). Dyslipidemia is a key driver of atherosclerosis, promoting lipid accumulation within arterial walls25. Cholic acid, a component of AGD, enhances cholesterol absorption and inhibits cholesterol-7α-hydroxylase (CYP7A1), thereby reducing cholesterol excretion.
Concurrently, thiouracil-induced hypothyroidism suppresses LDL receptor (LDLR) activity, further contributing to hypercholesterolemia26. Cholesterol metabolism in mammals is regulated by three key mechanisms: (1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis; (2) CYP7A1, which mediates cholesterol catabolism; and (3) LDLR, which facilitates cholesterol uptake via apolipoprotein binding27.
Consistent with previous findings28,29, AGD-fed rats developed pronounced dyslipidemia, particularly in the untreated ATH group, which presented elevated TC, TG, LDL-C, and VLDL-C levels and reduced HDL-C levels compared with those in the NC group. These findings suggest that the diet promoted cholesterol deposition in vascular endothelial cells and elevated the level of circulating lipoproteins30. These findings are consistent with earlier reports31,32.
Although cholesterol and LDL-C levels decreased significantly in most treated groups, they remained elevated compared with those in the NC group. This residual dyslipidemia may result from ongoing disturbances in lipid homeostasis, including dysregulated HMG-CoA reductase activity, impaired cholesterol esterification, and reduced lipoprotein lipase activity33.
The elevated TG and VLDL-C levels in the ATH group compared with those in the control group likely reflect enhanced hepatic VLDL production driven by elevated plasma free fatty acids34. Additionally, oxidized LDL (oxLDL) formation may occur, generating oxysterols that inhibit LDLR activity35,36. All the treatment groups showed improvements in lipid profiles.
The OL group presented the greatest benefit, followed by the SHT, BT, and PC groups. The weakest improvement was observed in the PC (simvastatin) group, suggesting that olive oil and tallow are more effective at ameliorating dyslipidemia. Previous studies also reported that HDL increases in animals fed/treated with 10% sheep tallow as a dietary supplement37.
Olive oil significantly reduced TC, TG, and LDL-C and increased HDL-C, highlighting its potent lipid-modulating capacity. Sheep and bovine tallow also improved lipid profiles, albeit to a lesser extent. Notably, sheep tallow significantly increased HDL-C, a key component of reverse cholesterol transport and a protective factor against atherosclerosis38.
A critical finding was the superior efficacy of sheep tallow in favor of modulating the lipid profile, specifically demonstrating a higher capacity to raise anti-atherogenic HDL-C and reduce pro-atherogenic LDL-C than both OL and BT. This potent effect is likely underpinned by the unique fatty acid signature of the SHT group. Sheep tallow typically contains a greater proportion of C18:0 (stearic acid) than other saturated fatty acids (SFAs) such as C16:0 (palmitic acid)39.
C18:0 is metabolized differently; it is rapidly converted to monounsaturated oleic acid in the liver, leading to a neutral or even beneficial effect on LDL-C levels40. Furthermore, the favorable SFA/MUFA ratio of SHT, a balance between SFAs and beneficial MUFAs, may synergistically promote reverse cholesterol transport and enhance hepatic LDL receptor activity more effectively than the other tested fats do. This unique combination of a metabolically neutral SFA C18:0 and ample MUFAs provides a plausible mechanism for the potent HDL-C-boosting and LDL-C-lowering effects of SHT observed in our model.
Despite concerns regarding the saturated fat content of animal fats, our findings suggest that C18:0, the dominant saturated fatty acid in tallow, may not adversely affect lipid profiles. Rather, since C18:0 is metabolized differently from other saturated fats, it may even reduce LDL-C and support coagulation homeostasis41,42. It is also the predominant saturated fatty acid in sheep meat43.
In addition, C18:1 (oleic acid) in sheep tallow and C12:0 (lauric acid) in bovine tallow may contribute to their positive lipid-modulating effects. C12:0 has been shown to increase HDL-C levels44. While olive oil was more effective at reducing LDL-C, sheep and bovine tallow promoted greater increases in HDL-C.
This aligns with evidence that saturated fats increase both LDL-C and HDL-C, whereas unsaturated fats primarily reduce LDL-C45,46. Saturated fat intake is often associated with increased HDL-C but lower TG levels44. Moreover, HDL-C may also inhibit LDL oxidation, reducing the atherogenesis of oxLDL47.
Compared with those in the controls, the decreased TG and VLDL-C levels observed in the tallow and olive oil-treated groups may be related to their C16:0 content, which is associated with VLDL particles rich in triacylglycerols48.
The atherogenic index (AI), a predictive marker of atherosclerosis, showed the greatest improvement in the OL group. This may be due to the high contents of C18:2 t + 1 (trans-linoleic acid) and C18:1 in olive oil (Table 1). In addition, tocopherols, which are known to modulate lipid metabolism, increase cholesterol efflux and reduce inflammation49.
Oxidative stress and inflammation are central to the pathogenesis of atherosclerosis. In this study, the atherogenic diet induced a significant increase in the level of MDA, a marker of lipid peroxidation, in the ATH group, accompanied by decreased total antioxidant (TAO) levels, indicating increased oxidative stress in parallel with severe hyperlipidemia. This may be attributed to increased reactive oxygen species (ROS) and/or insufficient antioxidant defenses50.
Hypercholesterolemia reduces antioxidant capacity and increases lipid peroxidation, thereby damaging the vascular endothelium51. The significant reductions in the MDA levels in the treated groups reflect the beneficial antioxidant effects of olive oil and tallow. These reductions could help enhance the antioxidant defense system and thereby reduce atherosclerosis52.
The significantly lower MDA levels in the olive oil group than in the simvastatin-treated group can be attributed to the phenolic antioxidants of olive oil, such as oleuropein and hydroxytyrosol53. These compounds act as radical scavengers, preventing LDL oxidation, a key initiating event in atherogenesis, thus providing superior antioxidant protection54.
Antioxidants can inhibit LDL oxidation and reduce serum cholesterol, offering protection against atherosclerosis36. Olive oil effectively reduced both the MDA and CRP levels, further supporting its anti-atherogenic potential. Tallow supplementation also decreased MDA levels and increased total antioxidant capacity, suggesting a protective effect against oxidative stress.
The reduction in oxidative stress and inflammation likely contributed to the observed attenuation of atherosclerotic lesions in the tallow-treated groups55.
CRP, an acute-phase protein and marker of inflammation56, was the lowest in the OL group, followed by the SHT and BT groups. This reduction is correlated with the presence of anti-inflammatory fatty acids such as C18: 2 t + 1 (t-linoleic acid) (LA) and C18: 3n3 (alpha-linolenic acid) (ALA), which are particularly abundant in OLs and SHTs. ALA is known to exert cardioprotective and anti-inflammatory effects57.
LA intake has also been associated with a reduced risk of coronary heart disease58,59. Additionally, tallow is packed with nutrients, including tocopherol, other lipid-soluble vitamins, and linoleic acid, which may contribute to the antioxidant and anti-inflammatory properties that help reduce the effects of inflammation60–62.
Histopathological analysis revealed advanced atheromatous changes in the ATH group, including intimal thickening, lipid accumulation, elastic fiber disruption, necrosis, and architectural distortion, which are hallmarks of type III atherosclerotic lesions3. Similar pathologies have been reported in prior studies31,63. The proliferation of vascular smooth muscle cells (VSMCs) in response to oxLDL and transforming growth factor-beta (TGF-β) may induce aortic wall thickening64–66.
Endothelial dysfunction enhances vascular permeability to cholesterol-rich particles67. Promoting foam cell formation and fatty streak development68.
Treatment with olive oil and tallow led to a notable regression of aortic lesions, with olive oil resulting in the most pronounced improvement. These histological findings, in parallel with the biochemical data, underscore the potential of dietary fats to reverse vascular damage.
Simvastatin treatment produced a striking and unexpected observation in our study: the simvastatin (PC) group exhibited more pronounced cardiac histopathological changes compared with the atherogenic (ATH) group (Table 4). This finding was contrary to our initial findings and the generally reported cardioprotective effects of simvastatin. The simvastatin dose (0.9 mg/kg BW in our study) falls within the range commonly used in rat models of hyperlipidemia and is generally considered well-tolerated69.
However, the 12-week administration, combined with severe metabolic stress induced by the atherogenic diet, may have contributed to the adverse cardiac findings in the PC group. Although simvastatin is generally cardioprotective, some studies suggest that under specific conditions, statins may impair mitochondrial function or exacerbate oxidative stress in cardiac tissue70. Thus, the worsening of cardiac histopathology in our simvastatin group, while unusual, remains biologically plausible and warrants further mechanistic investigation.
While simvastatin effectively improved serum lipid parameters, its direct cardiac effects may be context-dependent and not invariably beneficial, particularly under conditions of advanced metabolic insult. A comprehensive review of the literature indicates that simvastatin generally exerts cardioprotective effects in hyperlipidemic models. However, some studies have reported potential adverse cardiac effects under specific conditions71.
These studies suggest that, under certain circumstances, statins may impair mitochondrial function or promote oxidative stress in cardiac tissue72,73. Taken together, our findings suggest that the observed worsening of cardiac histopathology in the simvastatin group compared with the ATH group is unusual, but biologically plausible under conditions of combined drug exposure and dietary stress. This warrants further mechanistic investigation, including exploration of mitochondrial function, oxidative stress markers, and drug-diet interactions.
While the simvastatin results were anomalous with respect to the heart, its efficacy in lowering serum lipids and improving aortic histopathology still provides a valuable pharmacological benchmark. In contrast, simvastatin further emphasizes the potential therapeutic value of natural compounds (tallow and olive oil), as they consistently improved cardiac function without adverse effects in our model. The differential outcomes of lipid observed among the dietary fat groups may be partly explained by variations in their fatty acid composition.
Sheep tallow, characterized by a relatively higher C:18:0 and favorable SFA/MUFA ratio, has been reported to raise HDL-C and reduce LDL-C more effectively than does bovine tallow. However, olive oil, which is rich in oleic acid and phenolic antioxidants, exerts both lipid-modulating and antioxidant effects. These compositional differences likely contributed to the superior cardioprotective outcomes observed with tallow and olive oil compared with simvastatin.
Overall, the comparative outcomes can be summarized as follows: olive oil showed the most pronounced improvements, followed by sheep tallow, and then bovine tallow, whereas simvastatin improved lipid parameters but was less effective in reversing cardiac injury. These findings suggest that, in the context of severe hyperlipidemia and advanced atherosclerosis, the cardiac effects of simvastatin may be more complex than previously appreciated and warrant further investigation.
Marked histological improvements, including increased luminal diameter and reduced wall thickness, were observed in the SHT, BT, and OL groups. Myocardial injury was also ameliorated, unlike in the PC group, where simvastatin-related toxicity may have limited improvement.
The protective effects of dietary fats can be attributed to their fatty acid composition. C18: (1Oleic acid), which is well known to reduce hepatocellular lipotoxicity74, was found to be the most abundant component in olive oil, followed by sheep and then bovine tallow, which is correlated with the degree of tissue improvement. Moreover, both tallows contain short-chain fatty acids C4:0, C10:0 (capric acid), and C8:0 (caprylic acid), which improve lipid and glucose metabolism75.
C8:0, in particular, reduces lipid accumulation and alleviates atherosclerosis76.
This study has several limitations that should be considered. First, the use of a rat model, while a valuable tool for initial mechanistic investigations, inherently limits the direct translatability of these results to humans owing to significant physiological and metabolic differences in lipid handling, lipoprotein metabolism, and the chronic progression of atherosclerosis between species. Second, the sample size, although adequate for statistical analysis, was modest and included only male rats, which may not reflect potential sex-related biological variability in response to dietary fats.
Third, the 12-week treatment period, while sufficient to observe significant changes in the rat model, may be relatively short for fully assessing the long-term effects of atherosclerosis, a chronic and progressive condition. Longitudinal studies of longer durations would provide a more complete understanding of sustained efficacy and safety. Fourth, even though the study evaluated several key biomarkers related to lipid profiles, oxidative stress, and inflammation, along with histopathological parameters, a more comprehensive mechanistic analysis such as gene expression profiling, RNA-seq, cytokine assays, endothelial function tests, and gut microbiome characterization would yield deeper insights into the pathways involved.
Fifth, a dose-response study for both the tallows and olive oil was not performed; such an analysis could help determine optimal therapeutic concentrations. Finally, although our findings suggest beneficial effects of tallow, the precise molecular and cellular mechanisms by which its specific fatty acid components or other bioactive compounds exert their effects remain unclear. Future studies are recommended to explore all these aspects to identify the particular molecular pathways through which olive oil and tallow exert their anti-atherogenic effects.
Despite these limitations, the present study provides supportive evidence for the potential protective effects of both olive oil and tallow against hyperlipidemic-induced atherogenesis in a rat model, laying the groundwork for future in-depth investigations.
Conclusion
This study systematically evaluated the effects of dietary sheep tallow, bovine tallow, and olive oil in mitigating diet-induced hyperlipidemia and atherosclerosis in a rat model. Our findings demonstrate that both animal-derived tallows and plant-based olive oil exhibit significant therapeutic potential, leading to improved lipid profiles, notable reductions in oxidative stress and inflammation, and amelioration of histopathological damage to both the heart and aorta. While olive oil consistently showed strong beneficial effects, sheep and bovine tallows also demonstrated promising anti-atherogenic properties, particularly in enhancing HDL-C levels and reducing certain cardiac lesions.
These results collectively suggest a potential cardioprotective role for various dietary fats, which appears to be influenced by their specific fatty acid compositions. Importantly, our findings contribute to a more nuanced understanding of animal fats in the context of cardiovascular health, suggesting that a blanket view of their detriment may warrant re-evaluation based on the specific fatty acid profiles rather than generalized classifications. This study lays important groundwork, highlighting the need for further research to elucidate the precise molecular and cellular mechanisms underlying these observed effects and to determine their ultimate relevance and translatability to human health and dietary guidelines.
Methods
Tallow preparation
Fat samples from local sheep and bovines (≥ 12 months old) were obtained from the official abattoir in Dhamar city, Yemen. Internal and external fat samples were collected during slaughter and from areas around the tripe, heart, kidneys, mesentery, and sheep tails. Tallows were extracted via a traditional rendering method.
The fat samples were stored at − 18 °C for 24 h and then cut into small pieces (~ 1 cm³). The pieces were chopped with a knife and minced with a meat grinder (4–5 mm holes). The fatty tissues were placed in a large pan over low heat.
Melted fat was periodically filtered through gauze to remove residual tissue. The purified tallow was poured into glass mason jars for storage. The tallow was orally administered as a liquid via a syringe.
The mixture was gently warmed to a liquid state and incubated at room temperature before being given to the animals.
Olive oil & simvastatin
Commercial Syrian virgin olive oil was purchased from a local store, and the simvastatin used in the study as a positive control was obtained from Shiba Pharma (Sana’a, Yemen). The simvastatin suspension was prepared daily in distilled water, providing a dose of 0.9 mg/kg B.W77.
Determination of fatty acid composition
The fatty acid compositions of sheep tallow, bovine tallow, and olive oil were analyzed via gas chromatography with a flame ionization detector (GC-FID) at the laboratories of the Yemen Standardization, Metrology, and Quality Control Organization.
GC‒FID sample preparation
Fatty acid methyl esters (FAMEs) were prepared by weighing 100 mg of each sample into glass tubes (in triplicate). Each sample was dissolved in 10 mL of n-hexane and mixed with 100 µL of 2 N potassium hydroxide in methanol (11.2 g KOH/100 mL methanol). The tubes were shaken manually for 30 seconds and then centrifuged at 4000 rpm for 5 minutes, after which the clear supernatant was used for analysis.
GC‒FID analysis
The analysis was conducted via a Shimadzu GC‒2010 Plus FID system with a DB‒WAX polyethylene glycol (PEG) capillary column (60 m × 0.25 mm i.d., 0.25 µM film thickness). The column temperature was initially 50 °C, increased at 10 °C/min to 250 °C, held for 25 min, and then returned to the starting temperature within 5 min78. A reference standard (C4–C24 methyl esters; Sigma‒Aldrich, USA) was used to identify individual fatty acids.
The data were recorded and processed via ChemStation software (version 6.0).
Experimental animals
Forty-nine healthy adult male rats (Rattus rattus), weighing 160–200 g, were obtained from the animal house of the Biology Department, Faculty of Science, Sana’a University. The rats were acclimated for two weeks in a controlled environment (18–24 °C, 12 h light/dark cycle, 60% ventilation). Two rats were housed per cage (15 × 20 × 26 cm) with ad libitum access to standard chow and water.
Food and water intake were recorded daily.
This study was conducted according to the Guide for the Care and Use of Laboratory Animals, National Institutes of Health guidelines, 199679, and the protocol was approved by the Committee of Experimental Animal Care, Faculty of Science, Sana’a University (Ethical code: BAHSS 101/2021). The study is reported in accordance with the ARRIVE guidelines.
Preparation of the atherogenic diet
An atherogenic diet (AGD) comprising 4% cholesterol, 1% cholic acid, and 0.5% thiouracil (w/w)64 was used for this study.
Experimental design and treatment
After acclimatization, the rats were randomly assigned to seven groups (n = 7 per group):
Negative Control (NC): Received 1 mL of distilled water (DW) orally for 24 weeks.Atherogenic Baseline (ATB): Received AGD for 12 weeks and was sacrificed, and served as an atherogenic baseline.Atherogenic (ATH): Received AGD for 12 weeks, followed by no treatment (1 mL DW/day) for another 12 weeks.Sheep Tallow (SHT): Received AGD for 12 weeks, followed by 1 mL/kg B.W./day of sheep tallow orally for another 12 weeks.Bovine Tallow (BT): Received AGD for 12 weeks, followed by 1 mL/kg B.W./day of bovine tallow orally for another 12 weeks.Olive Oil (OL): Received AGD for 12 weeks, followed by 1 mL/kg B.W./day of olive oil orally for another 12 weeks.Positive Control (PC): Received AGD for 12 weeks, followed by 0.9 mg/kg B.W./day of simvastatin in DW orally for another 12 weeks.
Blood and tissue sample collection
After overnight fasting, the rats were anesthetized via subcutaneous injection of ketamine/xylazine (50/5 mg/kg). Blood was collected at 08:00 AM from the retro-orbital plexus via a capillary tube. The samples were left to clot at room temperature for 4 h and then centrifuged at 2000 rpm for 15 min.
The serum was stored at − 20 °C until biochemical analysis. The rats were euthanized via cervical decapitation under anesthesia. Hearts and aortae were excised, rinsed in normal saline, fixed in 10% formalin for 24 h, and preserved in 70% ethanol for histopathological examination.
This procedure was performed after 12 weeks for the atherogenic baseline group (ATB), whereas it was performed after 24 weeks for the NC, ATH, SHT, BT, OL, and PC groups.
Lipid profile assay
Lipid profiles (TC, HDL-C, LDL-C, VLDL-C, and TG) were measured via enzymatic colorimetric methods with commercial kits (Spinreact Co., Spain) on a Biochemistry Analyzer RX50V. The protocol was performed according to the manufacturer’s instructions.
The AI was calculated as follows: AI = (TC − HDL-C)/HDL-C.
Lipid peroxidation and total antioxidant capacity
Malondialdehyde (MDA) and total antioxidant capacity (TAOC) were assessed using commercial kits (MOLEQULE-ON, Auckland, New Zealand) and an ELISA reader (Sinothinker) according to the manufacturer’s instructions.
C-reactive protein levels
C-reactive protein (CRP) was measured using a nephelometric immunoassay commercial kit (Hipro Biotechnology Co., Germany) according to the manufacturer’s protocol.
Histopathological analysis
Formalin-fixed heart and aorta tissues were dehydrated in ethanol (80–100%), cleared with xylene, and embedded in paraffin. Section (5 µM) were stained with hematoxylin and eosin80. The aorta was excised from the aortic arch to the bifurcation, and a cross-section was taken from the distal part of the aortic arch for analysis.
Sections of the heart were obtained from the mid-ventricular region for histological examination. The samples were examined at 40x, 200x, and 400x magnifications via an Olympus CX41 light microscope. Images were captured via a digital camera.
Morphometric analysis
The inner diameter and wall thickness of the aorta cross-sections were measured at 0°, 90°, 180°, and 270° using an ocular micrometer calibrated with a stage micrometer (40x = 20 µM) (Fig. 2S) and analyzed via ImageJ software (Version 1.54, Java 1.8.0_345, 64-bit). The intima thickness was measured from the lumen to the internal elastic lamina, and the media thickness was measured from the internal to the external elastic lamina.
The intima + media thickness and the intima/media ratio were calculated, and the mean value of ‘n’ samples/replicates was used for further evaluation/analysis81.
Heart analysis
Quantitative histological assessments based on averages from seven rats per group and 3 microscopic fields (625 µM²) per sample28,82 were conducted.
Statistical analysis
All the data are expressed as the mean ± standard deviation (SD). The Shapiro–Wilk test was applied to assess the normality of all datasets. Since the data conformed to a normal distribution, multiple comparisons among groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.
Statistical analyses were performed using GraphPad Prism software (version 8.0.2.263; San Diego, CA, USA). A probability value of P < 0.05 was considered statistically significant.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1