Research Article

Effects of Probiotics on Cardiovascular Disease Markers in Male Rats

Walaa Sabri Raheem1, Ahmed M. Amshawee1, Mohanad Salam Hussein2, Islam Isam Kamel Alazzawi3, Omar Ala Dawod Almashaykhi4, Ahmed Flayyih Hasan5,6*

1Department of Radiology Techniques, College of Health and Medical Technologies, University of Hilla, Iraq; 2National Center of Hematology, Mustansiriyah University, Baghdad, Iraq; 3college of Pharmacy, Alfarahidi University, Baghdad, Iraq; 4College of Dentistry, Al-Farabi University, Baghdad, Iraq; 5Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq; 6Department of Medical Laboratory Techniques, College of Health and Medical Technology, Al-Farabi University, Baghdad, Iraq.

Received | October 10, 2025; Accepted | January 04, 2026; Published | April 02, 2026

*Correspondence | Ahmed Flayyih Hasan, Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq; Email: [email protected]

Citation | Raheem WS, Amshawee AM, Hussein MS, Alazzawi IIK, Almashaykhi OAD, Hasan AF (2026). Effects of probiotics on cardiovascular disease markers in male rats. J. Anim. Health Prod. 14(2): 527-533.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.2.527.533

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Obesity is defined as an excessive accumulation of adipose tissue relative to height and is commonly accompanied by chronic low-grade inflammation of the gastrointestinal tract, In recent years, obesity has been recognized as a chronic disease and is frequently associated with the development of several comorbid conditions, including type 2 diabetes mellitus, cardiovascular disease (CVD), certain cancers, and musculoskeletal disorders (Gadde et al., 2018). Body mass and the development of obesity are influenced by a complex interplay of genetic, epigenetic, and lifestyle factors operating within a broad physical and metabolic environment (Alankooshi et al., 2023).

The global rise in obesity parallels an increasing incidence of cardiovascular disease, which remains the leading cause of mortality worldwide. Although the etiology of CVD is multifactorial and not fully understood, obesity is a well-established and significant risk factor that contributes substantially to the development of heart disease. Lifestyle and metabolic status both closely linked to gut health, play central roles in the pathogenesis of obesity. Accordingly, the gut microbiota has gained considerable attention for its role in maintaining host physiological and metabolic homeostasis (Eslami et al., 2026).

Probiotics are believed to exert their beneficial effects by modulating the composition and functional activity of the gut microbiota, thereby influencing multiple host metabolic pathways (Fan et al., 2023). Disruption of the normal host–microbial balance, known as microbiota dysbiosis, has been associated with increased susceptibility to various diseases (Qiu et al., 2018). Alterations in microbial composition or the production of community-derived metabolites and genotoxins may directly or indirectly activate signaling pathways that contribute to the pathophysiological processes underlying cardiovascular diseases. Consequently, these microbiome-associated factors represent promising targets for therapeutic intervention (Tang and hazen, 2017).

The gut microbiota functions as a metabolic bioreactor, fermenting dietary components into bioactive metabolites such as trimethylamine (TMA), secondary bile acids, and short-chain fatty acids (SCFAs) (Tang and hazen, 2017). These bacterial metabolites play critical roles in digestion, energy harvest, and maintenance of gut barrier integrity. After entering systemic circulation, they further influence key physiological processes in distant organs, including glucose metabolism in the pancreas, lipid metabolism in the liver, and cognitive functions in the brain (Schugar et al., 2017).

Among these metabolites, TMA is produced by gut bacteria through the metabolism of dietary choline, betaine, and L-carnitine (Zhu et al., 2018). Specific microbial enzymes, such as choline trimethylamine lyase, catalyze the conversion of TMA-containing nutrients abundant in cholesterol- and fat-rich diets into TMA. Because mammals lack TMA lyase enzymes, the gut microbiota utilizes these nutrients as carbon sources, releasing TMA as a metabolic by-product.

Lactic acid bacteria and Bifidobacterium species are among the trillions of microorganisms inhabiting the gut that have been extensively studied for their beneficial roles in regulating gut microbial balance, metabolic activity, and immune function. Importantly, these probiotic bacteria have been shown to reduce traditional cardiovascular risk factors, highlighting their potential role in the prevention and management of obesity-related cardiovascular disease (Zhou et al., 2025). Therefore, this study aimed to investigate the impact of probiotic supplementation on selected physiological, hormonal and biochemical parameters in rats fed a high-fat diet.

Materials and Methods

Animals and experimental groups

Thirty eight-weeks old male Wistar rats were obtained from the National Laboratory Animal Center, Taipei, Taiwan. Each animal was housed individually under controlled environmental conditions (temperature: 20 ± 2 °C; relative humidity: 55 ± 5%) and maintained on a 12-h light/12-h dark cycle, with lights on from 08:00 to 20:00 h. During an eight-week acclimatization period, rats were provided ad libitum access to standard chow pellets (AIN-76; Young Li Trading Co. Ltd., Taipei, Taiwan) and water.

Following acclimatization, the animals were randomly assigned into experimental groups: normal control (TC); high-fat diet containing 15.47% butter powder (TF); high-fat diet supplemented with probiotic yogurt (78 mg/kg body weight/day; 4.18 × 10⁵ CFU/mL; TP). Daily feed intake was measured.

Analyses

At the end of the experimental period (8 weeks), five rats from each group were randomly selected, weighed, and euthanized. The liver was excised and weighed, and blood samples were collected by cardiac puncture under anesthesia. Liver enzyme activities, including alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), as well as lipid profile parameters, total cholesterol (TCH), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were measured using the method described by Reitman and Frankel (1957).

Serum hormone concentrations were determined using radioimmunoassay (RIA) kits. Testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were quantified using commercially available kits (DiaSorin, Stillwater, MN, USA). The assay sensitivities were 0.05 ng/mL for testosterone and 1 mIU/mL for LH.

Statistical analysis

Data were analyzed using a completely randomized design (CRD). The experiment followed a simple design, and data from the experimental groups were analyzed individually. Mean differences among groups were compared using the least significant difference (LSD) test at a significance level of p ≤ 0.05. All statistical analyses were performed using GenStat software (version 2011) (Al-Kafaji et al., 2018).

Results and Discussion

At the end of the experimental period, the animals were fasted for 12 hours and then anesthetized prior to euthanasia. Blood samples were collected by cardiac puncture, and serum was separated for biochemical analysis.

Lipid profile

In Figure 1A, the data for LDL show a clear and statistically significant trend (p < 0.05). The control group (TC) recorded an LDL level of 19.38 mg/dL, which increased significantly to 31.28 mg/dL in the high-fat diet group (TF). In contrast, supplementation with probiotic-prepared yogurt (TP) resulted in a marked reduction in LDL levels to 9.8 mg/dL. This finding strongly supports the hypothesis that probiotics can positively modulate lipid metabolism and reduce harmful cholesterol levels. These results are consistent with previous studies demonstrating that specific probiotic strains reduce intestinal cholesterol absorption, modulate bile salt metabolism, and promote the production of short-chain fatty acids (SCFAs) that regulate hepatic cholesterol synthesis (da Silva Pontes et al., 2021; Rahman et al., 2022).

As shown in Figure 1B, HDL levels were significantly reduced in the high-fat diet group (TF), reaching 15.1 mg/dL, compared with 30.0 mg/dL in the control group (TC) (p < 0.05). Conversely, rats receiving probiotic yogurt in combination with a high-fat diet (TP) exhibited a significant increase in HDL levels to 35.0 mg/dL, exceeding even the control group (p < 0.05). The concurrent decrease in LDL and increase in HDL levels highlights the regulatory role of probiotics in improving lipid metabolism. These findings align with those reported by Yuanyue et al. (2024), who observed reduced LDL and elevated HDL levels in probiotic-treated mice.

 

In Figure 1C and 1D, TG and TCH levels in the high-fat diet group (TF) were 31.4 mg/dL and 77.7 mg/dL, respectively, showing a significant increase compared with the control group (11.4 mg/dL and 60.0 mg/dL, respectively; p < 0.05). In contrast, the probiotic-treated group (TP) showed significantly reduced TG and TCH levels (11.2 mg/dL and 56.0 mg/dL, respectively), which were comparable to or slightly lower than those of the control group. This improvement is attributed to the beneficial effects of probiotics on lipid metabolism, including reduced intestinal cholesterol absorption, regulation of bile acid secretion, and enhanced enzymatic activity involved in fat metabolism (Asgary et al., 2021). Additionally, Yuanyue et al. (2024) reported increased production of SCFAs such as acetic, propionic, and butyric acids in probiotic-treated mice, further supporting the lipid-lowering effects observed in this study.

Liver enzymes

Figure 2 illustrates the effects of different dietary treatments on liver enzyme activities, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP).

As shown in Figure 2A, ALT activity increased markedly from 98 U/L in the control group (TC) to 192 U/L in the high-fat diet group (TF), indicating significant hepatic stress (p < 0.05). In contrast, rats receiving probiotic supplementation with the high-fat diet (TP) exhibited a substantial reduction in ALT levels to 84 U/L, approaching control values.

Similarly, Figure 2B demonstrates that AST levels rose from 32.3 U/L in the control group to 46 U/L in the high-fat diet group, while probiotic supplementation significantly reduced AST activity to 30 U/L (p < 0.05).

 

In Figure 2C, ALP activity was elevated in the high-fat diet group compared with the control group and showed a marked reduction in the probiotic-treated group, indicating improved liver function following probiotic administration.

The elevated liver enzyme activities observed in the high-fat diet group (TF) can be attributed to excessive lipid accumulation in the liver, which promotes oxidative stress, inflammation, and disruption of gut microbiota. These alterations increase intestinal permeability and facilitate the translocation of bacterial endotoxins to the liver via the portal circulation, ultimately leading to hepatocellular injury. Serum levels of ALT and AST are well-established biomarkers of liver damage, and their elevation reflects hepatocyte membrane disruption (Ding et al., 2023).

Conversely, the reduction in liver enzyme activities observed in the probiotic-treated group (TP) highlights the hepatoprotective effects of probiotics. These beneficial effects are primarily mediated through modulation of the gut microbiome, enhancement of intestinal barrier integrity, reduction of oxidative stress and inflammation, and regulation of lipid metabolism via the production of short-chain fatty acids (SCFAs), particularly butyrate, These findings are consistent with previous animal studies demonstrating that probiotic supplementation reduces hepatic lipid accumulation, improves liver enzyme profiles (ALT and AST), and attenuates inflammation and oxidative stress in high-fat diet-induced fatty liver models (Li et al., 2013).

Body weight and organ weight

Table 1 presents the effects of different dietary treatments on body weight gain and liver weight relative to body weight in the experimental animals. Rats fed a high-fat diet with distilled water for eight weeks (TF group) showed a significant increase in body weight compared with the control group (TC). The mean body weight of the TF group at the end of the experiment reached 250.5 g, whereas the control group fed a standard diet and distilled water recorded an average body weight of 237.0 g. In contrast, the probiotic-treated group (TP), which received a high-fat diet supplemented with 4 mL of yogurt, did not differ significantly from the control group, with an average final body weight of 235.5 g. Feed consumption rates did not differ significantly among all treatments.

 

Table 1: Effect of probiotic feeding on liver weight, body weight, feed consumption, and liver-to-body weight ratio of Wistar rats.

Treatment*	Liver weight / body weight (%)	Liver weight (g)	Body weight (g)	Feed consumption rate (g/day)
TC	3. 69٠	7.40±0.20	٢٣7.0±٢٠.٧	١٩٧.٥±3.4
TF	4.56٠	٩.٦٦±0.20	٢٥٠.٥±٢٠.٧	١٩٨.٥±3.4
TP	٣.080	0٧.2±0.20	٢٣5.٥±٢٠.٧	١٨٩.٩±3.4
LSD (0.05)	1.48	٢.46	٩.20	0.09

 

* TC: normal control; TF: high-fat diet; TP: high-fat diet supplemented with probiotic yogurt; LSD: least significant difference.

 

Regarding liver weight and the liver-to-body weight ratio, the TF group differed significantly from the control group. Rats in the TF group exhibited a liver weight of 9.66 ± 0.2 g, corresponding to 4.56% of body weight, compared with 7.40 ± 0.2 g and 3.69% in the control group. Diets high in fat, particularly saturated and trans fats, promote hepatic lipid accumulation, a condition known as hepatic steatosis. This accumulation triggers a cascade of cellular events, including oxidative stress and inflammation, ultimately leading to liver damage (Rector et al., 2011).

In contrast, the TP group showed a significant reduction in liver weight compared with the TF group, with a liver weight of 7.26 ± 0.2 g, representing 3.08% of body weight. This improvement is attributed to probiotic supplementation, which enhances hepatic lipid metabolism and reduces fat accumulation in the liver. Probiotics may exert these effects through the production of SCFAs, such as butyrate, which have well-documented beneficial metabolic properties (Li et al., 2013). These findings are consistent with previous studies reporting that probiotics influence body weight regulation and fat accumulation by modulating gut microbiota composition, improving insulin sensitivity, reducing caloric absorption, and attenuating systemic inflammation associated with obesity and non-alcoholic fatty liver disease (León-Aguilera et al., 2022; Zhang et al., 2022).

Male hormones

Table 2 illustrates the effects of dietary treatments on male reproductive hormones, which play a critical role in the initiation and maintenance of testicular function, including androgen synthesis and spermatogenesis. These processes are tightly regulated by the hypothalamic–pituitary–gonadal (HPG) axis, a complex network of endocrine, paracrine, and autocrine signaling pathways that coordinate reproductive function (Baile et al., 1983; Shankar et al., 2021). The results exhibited that FSH, LH and serum testosterone levels were significantly increased (p < 0.05) in the high-fat diet group, whereas probiotic supplementation resulted in a significant reduction in these enzymes (p < 0.05).

 

Table 2: Effect of probiotic feeding on the male hormones of Wistar rats.

Treatment*	Testosterone (ng/ml)	FSH (ng/ml)	LH (ng/ml)
TC	5.12±1.45b	3.26±1.34b	4.34±0.10b
TF	10.54±0.24a	12.30±1.23a	13.25±0.21a
TP	3.11±0.65c	2.91±0.47c	3.76±0.10c

 

The values for each treatment group are given as means ±SD. abc Significant differences were observed between mean values within a column that did not share a common superscript letter (p<0.05). * TC: normal control; TF: high-fat diet; TP: high-fat diet supplemented with probiotic yogurt.

 

The present study demonstrated that the high-fat diet treatment (TF) adversely affected the hypothalamic–pituitary–testicular axis in experimental rats. This disruption was evidenced by significantly elevated circulating levels of FSH and LH and serum testosterone levels. Elevated hormonal levels indicate Leydig cell dysfunction and compromised endocrine regulation. Furthermore, the increased FSH levels observed in the TF group suggest dysfunction of the seminiferous epithelium and impaired spermatogenic activity (Lin et al., 2022; Kim et al., 2021).

Conclusions

The results of the present study demonstrate that consumption of a high-fat diet induces significant metabolic, hepatic, and hormonal disturbances in male rats, as evidenced by dyslipidemia, increased liver enzyme activities, elevated liver-to-body weight ratio, and disruption of the hypothalamic–pituitary–gonadal axis. Rats fed a high-fat diet exhibited increased levels of LDL, total cholesterol, triglycerides, and liver enzymes (ALT, AST, and ALP), along with reduced HDL and testosterone levels and elevated FSH and LH concentrations, indicating impaired lipid metabolism, hepatic dysfunction, and testicular endocrine imbalance.

Importantly, supplementation with probiotic-prepared yogurt containing lactic acid bacteria and Bifidobacterium infantis markedly attenuated these adverse effects. Probiotic administration significantly improved lipid profiles by reducing LDL, total cholesterol, and triglycerides while increasing HDL levels. It also restored liver enzyme activities toward normal values, reduced hepatic enlargement, and improved liver-to-body weight ratios. Furthermore, probiotic treatment ameliorated hormonal disturbances by increasing testosterone levels and normalizing FSH and LH concentrations, suggesting recovery of hypothalamic–pituitary–testicular axis function.

These beneficial effects are likely mediated through modulation of the gut microbiota, enhancement of intestinal barrier integrity, reduction of systemic inflammation, and improved lipid and energy metabolism. Collectively, the findings highlight the potential of probiotic-based dietary interventions as an effective strategy for mitigating obesity-related metabolic, hepatic, and reproductive dysfunctions. The study supports the development of functional foods enriched with probiotics as a promising approach for reducing the risk of cardiovascular and metabolic disorders associated with high-fat diets.

Acknowledgments

No institution provided support for the article; all support was provided by the authors.

Novelty Statement

This study demonstrates that probiotic supplementation with a specific yogurt formulation can improve lipid metabolism, liver function, and male reproductive hormones in rats fed a high-fat diet. It provides new evidence for the role of gut microbiota in preventing obesity-related metabolic and reproductive disorders, supporting the development of functional foods for metabolic and cardiovascular health.

Author’s Contribution

Walaa Sabri Raheem and Ahmed M. Amshawee: Study design.

Omar Ala Dawod Almashaykhi, Ahmed Flayyih Hasan

and Omar Jamal Ibrahim: Methodology.

Mohanad Salam Hussein and, Islam Isam Kamel Alazzawi: Corresponding author and manuscript editing, writing.

Funding

No institution provided support for the manuscript.

Ethical approval

Approval for sample collection and study procedures was obtained from the Biotechnology Research Center, Al-Nahrain University, in September 2024.

Generative AI and AI-assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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