Effect of Ginseng on Blood Lipid Profile, Testosterone Level and Epididymal Sperm Quality of Aged BALB/C Mice

Muhammad Tayyab Khan1*, Muhammad Irfan-ur-Rehman Khan1, Tehmina Yasmin2, Ghulam Shabbir Khan1 and Amjad Riaz1*

1Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore (54000), Pakistan

2Recep Tayyip Erdgon Hospital, Muzaffargarh 34200, Pakistan

ABSTRACT

It is well known that aging in males leads to male infertility; therefore, the current study determines the impact of ginseng on blood lipid profile, testosterone level, and epididymal sperm quality in aged balb/c mice. Aged male mice (n=48) were assigned to three groups (n=16 in each) i.e., control (CON), high dose ginseng (HDG) and low dose ginseng (LDG). Mice were treated as CON (normal saline), HDG (500 mg/kg/day) and LDG (250 mg/kg/day) for 5 weeks. Daily feed intake, weekly weight, blood lipid profile, testosterone level, epididymal sperm quality, and testicular tissue were evaluated during and 5 weeks after the end of treatment. Results revealed that weight gain and feed intake decreased (P< 0.05) in mice treated with HDG and LDG than CON during and 5 weeks after the treatment. During the treatment, levels of testosterone, cholesterol, and low-density lipoproteins increased (P< 0.05) in mice treated with HDG and LDG than CON; however, cholesterol, triglyceride, and LDL-C were increased in CON than HDG and LDG treated mice after 5 weeks of treatment. Motility and viability (%) of spermatozoa increased (P< 0.05) in HDG and LDG treated mice than those of CON mice whereas sperm concentration was more (P< 0.05) in LDG than HDG and CON treated mice during the treatment. Five weeks after the treatment, motility and viability of spermatozoa were greater (P< 0.05) in HDG and LDG than CON treated mice. In conclusion, Ginseng treatment in aged BALB/c mice enhances the epididymal sperm quality, lipid profile and blood testosterone level during and even five weeks after the end of treatment period.

Article Information

Received 23 March 2023

Revised 25 January 2024

Accepted 13 February 2024

Available online 29 April 2024

(early access)

Published 08 February 2025

Authors’ Contribution

MTK: methadology,writing-original draft. MIRK: supervision,validation. TY: review and editing. GSK: review and validation. AR: conceptualization, financial support and guidance.

Key words

Ginseng, Sperm quality, Lipid profile, Testosterone, BALB/c, Panax ginseng

DOI: https://dx.doi.org/10.17582/journal.pjz/20230323090320

* Corresponding author: [email protected], [email protected]

0030-9923/2025/0002-0557 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

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

Male infertility is the least studied issue worldwide (Agarwal et al., 2015). There are about 48.5 million infertile couples worldwide (Mascarenhas et al., 2012) of which ~40% couples have male origin infertility (Smith et al., 2010; Medicine, 2013). In recent times, age-related infertility is commonly due to socioeconomic and societal reasons as couples wait until the age of 30-35 to begin their families. Using a rodent’s model, researchers have documented that age-related changes occur in both male and female reproductive organs. In male rats, these changes include a decline in the number of germ cells, thinning of seminiferous tubular epithelium, testicular atrophy, sperm motility, and production.Furthermore, the incidences of gene mutations, sperm aneuploidy (Lowe et al., 1995; Walter et al., 1998), fertilization failures, and neonatal deaths (Serre and Robaire, 1998) are greater in aged male rats when mated to healthy young females.

Ginseng (Panax ginseng) is a medicinal plant having aphrodisiac and anti-aging properties (Hu, 1976). The pharmacological actions of ginseng are attributed to one of its ingredients ginsenosides. Red ginseng is known to improve age-related decline in learning and memory (Lee and Oh, 2015). In aged mice, it improves cognition through the regulation of cholinergic and antioxidant activity (Lee et al., 2017). The therapeutic use of ginseng is well documented in frogs, rabbits, rats, and chickens to promote the development of reproductive organs, spermatogenesis, ovulation, female receptivity, and egg-laying (Hong et al., 2002; Rahim, 2014). In addition to its aphrodisiac properties, ginseng is often used to cure diabetes, skin and hair problems, obesity, anxiety, and stress (Cooke, 2019). Several studies emphasize the beneficial effects of ginseng on male fertility in animal models such as mice (Yoshimura et al., 1998), rabbits (Kim et al., 1998), guinea pigs (Kim et al., 1999), and rats (Tsai et al., 2003). These effects of ginseng may not be due to changes in hormonal secretions, but directly effects or its ginsenoside components on the central nervous system and gonadal tissue (Hong et al., 2002; Murphy and LEE, 2002)

There has been no previous study reported in scientific literature to describe the effect of ginseng on blood lipid profile, testosterone level, and sperm quality analysis of aged BALB/c mice. Therefore, the objective of this study was to evaluate the effect of ginseng on blood lipid profile and semen quality in aged BALB/c mice strain.

MATERIALS AND METHODS

Experimental animals and location

The current research was conducted at mice facility of Theriogenology Department, University of Veterinary and Animal Sciences, Lahore. All the procedures performed in the study were approved by Animal Care and Ethical Review Committee of the university. For this study, aged male BALB/c mice (n=24; age: 6 months ± SEM 6 and weight: 40 g ± SEM) were kept under uniform and optimum environmental conditions i.e., 20-26˚C room temperature, and 10:14 h of light and dark cycle. All mice were offered broiler starter ration (National Feeds, Pvt. Ltd., Pakistan) containing 23% crude protein, <5% crude fiber, 6.9% crude fat, and 3000 Kcal/Kg of metabolizable energy. All mice had free access to water ad libitum.

Experimental design, testicualar histology, semen processing, and blood sampling

In this study, aged mice (n=48) were randomly subjected to a high dose of ginseng (HDG; 500mg/kg/day; n=16), a low dose of ginseng (LDG, 250mg/kg/day, n=16), and normal saline (Control, n=16) for 5 weeks. Both ginseng (Vitamin Company, Lahore, Pakistan) and normal saline were administered in a total of 0.2 ml volume through gastric intubation. Weekly bodyweight, feed intake and sexual behaviors of mice were recorded through out the study. Lipid profile and plasma testosterone concentration were analyzed by obtaining blood from heart after killing mice at the end of 5-weeks treatment and then 5-weeks later (n=8 mice per group at each time point). Similarly, both testes were recovered for histological examination after secluding caudal epididymides for semen evaluation.

For histological analyses, testes were fixed (10% formal saline for 24 h), dehydreated in series of alocohol baths, paraffinized into wax blocks. 5 μm thick sections were cut and stained by hematoxylin and eosin method (Bancroft and Stevens, 1996) for examination under the light microscope

For semen analyses, caudal epididymides were sliced open in HTF media containing 4 mg/ml of BSA and incubated in a CO2 incubator (5% CO2 in air at 37ºC) for 10 min, allowing sperm to swim into the medium. To determine the sperm concentration, semen was diluted 100 times with PBS and 10 μl of semen was used to charge the haemocytometer. Heads of the sperms were counted at 400X magnification under a light microscope. To evaluate the sperm motility (%), 5μl of diluted semen was aliquoted on prewarmed glass slide, covered with a coverslip and observed at 400X magnification unser the phase contrast microcope with a heated stage (37°C). At least 3-5 various fiels were observed to estimate the percent motiliy of each sample. To evaluate viability of spermatozoa, 20 μl of the diluted semen was mixed with 0.05% eosin and smeared on glass slide. After air drying the smear at room temperature, at least 200 live (unstained) or dead (stained as pink) spermatozoa were counted under the light microscope at 400X maginification.

Statistical analysis

Effect of ginseng on weekly weight, daily feed intake, serum testosterone, lipid profile and semen parameters were analyzed by one way ANOVA using IBM Statistical Package for Social Sciences (SPSS Version 20). The comparisons among treatments and after withdrawal period were analyzed by paired t-test. A probability level P≤ 0.05 was considered as significant.

RESULTS

The data of the effect of ginseng treatment on daily feed intake, weekly weight gain, testosterone, cholesterol, triglyceride, high density lipoprotein (HDL) and low density lipoprotein (LDL) concentrations in aged male BALB/c mice during treatment has been depicted in Table I. Results reveal that plasma concentration of testosterone was greater (P< 0.05) in HDG group as compared LDG and CON. However, cholesterol concentration was was greater (P< 0.05) in LDG group as compared to LDG and CON.

The effect of ginseng treatment on daily feed intake, weekly weight gain, testosterone, cholesterol, triglyceride, HDL-C and LDL-C concentrations in aged male BALB/c mice 5 weeks after end of treatment period has been presented in Table I. Results reveal that plasma concentration of testosterone was greater (P< 0.05) in HDG group as compared LDG and CON. In contrast, Cholestrol concentration was greater (P< 0.05) in CON group as compared to HDG and LDG.

 

Table I. Effect of ginseng supplementation on weight, feed intake, blood lipid profile and testosterone during 5 weeks and after 5 weeks treatment period.

Variables	Control (n=8)	Low dose (n=8)	High dose (n=8)
During 5 weeks treatment
Feed intake* (g)	23.35± 0.43b	22.13 ± 0.36a	21.78 ± 0.37a
Weight* (g)	40.18 ± 1.64b	29.36 ± 0.7a	30.21 ± 0.77a
Testosterone (ng/dl)	4.98±0.71c	9.55±0.35b	12.99±1.64a
Cholesterol (mg/dl)	149.75±6.17ab	164±4.63b	135.75±8.52a
Triglyceride (mg/dl)	147.75±6.43bc	155±8.93b	116.75±10.42a
HDL-C1 (mg/dl)	19±0.91ac	13.31±1.26a	15.93±0.88ac
LDL-C2 (mg/dl)	93.75±7.04c	118.75±7.46ad	116.75±5.76ab
After 5 weeks treatment
Feed intake (g)	22.40±0.38b	12.97 ± 0.26a	12.76±0.91a
Weight (g)	41.0 ± 1.31b	34.69 ± 0.81a	35.65±0.93a
Testosterone (ng/dl)	4.36 ± 0.81c	29.88 ± 1.50b	116.25±1.49a
Cholesterol (mg/dl)	175 ± 2.34c	134.5 ± 1.32b	89.5±2.21a
Triglyceride (mg/dl)	210 ± 9.30c	68.5 ± 1.70b	116.5±2.10a
HDL-C3 (mg/dl)	60.25±3.88c	83±4.60b	37.31±0.64a
LDL-C4 (mg/dl)	125±6.06c	80.93±3.10b	58.75±2.56a

 

a,b, c Values in the same rows with different superscripts differ significantly (P<0.05). Values are mean ± SEM. No treatment group (CON) representing control group. 3HDL-C: high density lipid cholesterol. 4LDL-C: low density lipid cholesterol.

 

Table II. Effect of ginseng supplementation on semen parameters and testis weight during and after the 5 weeks treatment period.

Parameter	Control (n=8)	Low dose (n=8)	High dose (n=8)
During 5 weeks treatment
Sperm motility (%)	57.73±3.37b	70.2±2.13a	69.8±1.39a
Viability (%)	47.58±2.3b	59.1±2.13a	58.2±2.14a
Concentration (×106/ml)	14.7±3.40c	15.6±3.40b	14.24±2.24a
Testes weight (g)	0.62±0.14ab	0.61±0.12b	0.64±0.17a
After 5 weeks of treatment
Sperm motility (%)	64.2±3.37b	77.8±3.53a	79.8±3.29a
Viability (%)	44.92±23b	55.1±1.45a	56.2±1.14a
Concentration (×106/ml)	13.7±2.40c	15.6±2.40b	18.4±2.24a
Testes weight (g)	0.59±0.16ab	0.56±0.18b	0.60±0.13a

 

a, b, c Values in the same rows with different superscripts differ significantly (P<0.05). Values are mean ± SEM.

 

The effect of ginseng treatment on testes weight, sperm motility percentage, viability and sperm concentration in aged male BALB/c mice during treatment has been illustrated in Table II. Results reveal that sperm motility and viability was greater (P< 0.05) in HDG and LDG groups as compared to CON.

 

The effect of ginseng treatment on testes weight, sperm motility percentage, viability, and sperm concentration in aged male BALB/c mice 5 weeks after the end of treatment period has been shown in Table II. Results reveal that sperm motility and viability was greater (P<0.05) in HDG group as compared both of other groups LDG and CON. The effect of ginseng on testes histological examination has been depicted in Figure 1. The normal appearance of the testicular structure of mice was determined by evaluation of group 1(CON) (Fig. 1C), which had a normal arrangement of germinative cells in the seminiferous tubules. The appearance of group 2 and 3 (Fig. 1) was similar to that of group 1. The germinal epithelial layer appeared nearly normal. In figure black arrow showing seminiferous tubules and blue arrow is showing germinal epithelial cell layer.

DISCUSSION

In the present study, the administration of ginseng for 5 weeks in BALB/c mice decreased daily feed intake, weekly weight gain, serum triglycerides (LDL) whereas increased serum testosterone and HDL levels (Jorge et al., 1998; Lauridsen and Mortensen, 1999). The reduction in triglyceride, LDL-C, and cholesterol might be due to hypolipidemia caused by ginseng administration (Prasad, 1999). Besides, the ginseng due to its antioxidative potential may have decreased cholesterol and LDL-C. Several mechnisms have been described in previous reports for the decline in cholesterol after ginseng treatment. These include extraction of cholesterol through bile acid formation, and accelerated degradation of cholesterol through feces. It seems that all mechanisms of action of ginseng might be involved in the present study. Furthermore, the results of our study are under different studies conducted by Kim et al. (2017) who concluded that long term administration of ginseng in obese women resulted in a significant reduction of cholesterol, LDL-C, and enhanced production of HDL-C. However, the association between ginseng antioxidant property and lipid metabolism were not discussed in previous studies. Controversy still exists in the current study that HDL-C production was enhanced in the low dose treatment group as compared to the control group which supported the previous studies, but it was reduced in high dose group, which is an exception in our study. This discrepancy in results may be attributed to different pre-body weight, dosage variation associated with body weight, and type of ginseng used.

The findings of the current study also revealed that ginseng also had a profound positive effect on serum testosterone, libido, resulting in enhanced plugs formations. The active ingredient in ginseng is ginsenoside which is triterpenoid saponin and structurally resembles with steroid hormones. So, this constituent of ginseng may be associated with the enhanced sexual function, reproductive behavior by activating the steroid receptors (androgen receptors) which are abundantly present in the male reproductive tract, genital organs, and spermatozoa (Solakidi et al., 2005). Similar observations regarding the effects of ginseng have been described by other researchers who explained that ginseng acts on steroid receptors and enhances the production of testosterone and ultimately the libido Matsumoto (Travison et al., 2006). Moreover, behavioral studies have suggested that ginseng effects on the central nervous system (Bhattacharya and Mitra, 1991; Watanabe et al., 1991; Benishin, 1992). It is further suggested that ginseng enhances the spontaneous motor activity which inturns inhibit daily feed intake (Yoshimatsu et al., 1993). For this reason, a decline in the weekly weight gain of mice treated with ginseng was observed in the present study (Aitken and Clarkson, 1987).

In this study semen analysis revealed greater testicular damage in aged mice but that was recovered by ginseng administration. Moreover, improvements in sperm motility, viability, and concentration in response to ginseng administration have been described by previous researchers testing the efficacy of ginseng in fertile and asthenozoospermic men. The success rate of fertilization is directly related to number of motile spermatozoa (Gist et al., 2000). Activities of Na+-K+-ATPase is highly sensitive to ROS which cause depletion of Na+-K+-ATPase and results in loss of motility of spermatozoa. Consequently, the observed protective influence of ginseng against age-related oxidative damage potentially contributes to the restoration of reproductive hormone production.

Additionally, ginseng demonstrated an enhancement in sperm production through its stimulatory effect on testosterone synthesis and the complete mechanisim for antoxident activity of ginseng has been previously described by (Ku et al., 2020). Another plausible mechanism underlying the beneficial effects of ginseng pertains to its conjectured association with the activation of testicular angiogenesis. Ginseng notably elevated the expression levels of two angiogenic factors, VEGF and visfatin. This observation aligns with the understanding that angiogenesis plays a role in the context of spermatogenesis and spermiogenesis, potentially contributing to ginseng’s observed effects on reproductive health (LeCouter and Ferrara, 2002). The effect of ginseng on antioxidation and sexual hormone production significantly contributed to increased sperm production and motility by fostering spermatogenesis (Kilinç et al., 2004). The role of ginseng in minimizing ROS damage to sperm as evident from current study is similar to Chen et al. (2001) who reported that ginseng acts as free radical scavengers (Yun et al., 2016) and improve reproductive hormone receptors expression which are declined by production of ROS (Kim et al., 2017) and avoid further damage to spermatozoa. Some of ginsenosides (Rg3) are well documented possess the many biological properties (Lee et al., 2019). Secondly, decrease in cholesterol and LDL-C which may be attributed towards antioxidative potential of ginseng and thirdly, the reduction in daily feed intake and hence, weekly weight gain possibly caused by the effect of ginseng on central nervous system in enhancing spontaneous motor activity.

It is concluded from the results of current study that treatment with ginseng for 5 weeks improved sperm parameters in aged BALB/c mice, enhances the male fertility during treatment period and even five weeks after the end of treatment period and concluded that ginseng acts as a potent antioxidant in the protection of mice sperm cells against oxidative stress and may be useful to treat male infertility. However, further studies may be planned in the future to study integrating diverse methodologies, including molecular biology, cellular physiology, and pharmacology, will be instrumental in unraveling the intricate mechanisms behind ginseng’s effects on male reproductive health. Such insights hold promise not only for understanding ginseng’s role but also for potential therapeutic applications in addressing fertility issues.

Acknowledgment

The author would like to thank Dr.A. Riaz for support in research.

Funding

Financial support was provided by supervisor Dr. A. Riaz, Chairman Department of Theriogenology thoroughout this study.

IRB approval

IRB approval was granted by the Ethical Review Committee of University of Veterinary and Animal Sciences, Lahore, with Approval No. DR/1320 dated 21/12/2017.

Ethical statement

This study was approved by the university of veterinary and animal Sciences Lahore, local ethical review Committee (Approval No. DR/1320).

Statement of conflict of interest

The author declares that he has no conflict of interests.

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