Infertility is a problem that affects approximately 72.4 million couples worldwide, with 40% of infertility problems occurring in men (1, 2). Infertility can impact a man’s income, psychology, and social relationships, all of which may cause long-term trauma and depression (3, 4). Specifically, infertility in men is often associated negatively with masculinity, which causes a lack of confidence and increased anxiety (5).
Smoking is a known risk factor for many diseases. Indonesia has the highest prevalence of smokers globally, with 33% of fifteen-year-old children being active smokers (6). A cigarette contains tar, carbon monoxide, tobacco specific-nitrosamine, B-a-P (Benzo-a-pyrene), nicotine, and pesticide residue. Nicotine can trigger the production of radical molecules that can damage DNA and the lipid membrane of spermatozoa. The radical superoxide (ROS) and hydrogen peroxide (H₂O₂) can break the plasma membrane of spermatozoa and inhibit ATP production, which is vital for spermatozoa movement (7). As a result, men who smoke tend to have low sperm counts, abnormal spermatozoa motility and morphology, and low testosterone levels, leading to infertility. Furthermore, nicotine can induce the production of pro-inflammatory cytokine-like IL – 1 β, TNF α, and MCP-1 (8–10). It also inhibits Gonadotropin-Releasing Hormone (GnRH) secretion. As a result, the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) do not produce enough testosterone in spermatogenic cells to maintain the spermatogenic process in seminiferous tubules (9, 11, 12).
Temulawak (Curcuma xanthorrhiza Roxb.) is a traditional medicinal plant found easily in Indonesia. Temulawak is well known for its benefits as an anti-inflammatory, antioxidant, diuretic, anti-cancer, anti-hepatotoxic, anti-dysmenorrhea, anti-spasmodic, anti-leucorrhoea, anti-bacterial, and anti-fungal drug (13). The antioxidant effect of temulawak in the binding of free radical molecules has been reported as better than that of vitamin E and beta carotene (9, 14). Moreover, the curcumin compound in temulawak can influence the androgenesis process by increasing testosterone and LH levels. It also increases the FSH level, making Sertoli cells produce higher germinal cells and androgen binding proteins (9). Besides their antioxidant effects, the curcumin and xanthorrhizol in temulawak also work as an anti-inflammatory, which decreases the IL – 1β, IL-6, and TNF α (9, 15). However, the use of temulawak to protect the quality of sperm against the effects of nicotine has not been studied so far. Therefore, this study aims to investigate the effect of temulawak extract on sperm quality of nicotine-induced mice.
 

As shown in Table 1, there was a decrease in the percentage of normal morphology, progressive motility, and sperm counts of the nicotine-treated group compared to those of the control normal (K1 vs K0). Giving temulawak extract to the nicotine-induced mice (groups P1 and P2) increased normal morphology, progressive motility, and sperm counts compared to those of the control nicotine group (K1). The difference in the percentage of normal morphology spermatozoa was not significant. In contrast, the differences in the progressive motility and sperm counts were significant (p < 0.01 and p = 0.007).

Table 1. The sperm characteristics of male mice in experimental groups.

Values are expressed as means ±SD. * : Significant if p<0.05.
 

Post hoc tests were performed to compare the experimental groups. From Table 2, it is clear that administering nicotine lowered the progressive motility of the sperm compared to that of the control normal (p<0.001).  Giving temulawak extract resulted in higher progressive motility compared to that of the nicotine-administered group (p=0.001 and p<0.001). In addition, providing 8 mg/20 g BW temulawak extract (P2) improved the progressive motility, but it did not reveal any significant effect when compared with the P1 group.
Table 3 shows that nicotine results in lower sperm counts than those of the control group (p=0.012). On the other hand, giving temulawak extract improved the sperm counts compared to those of the control normal group (K0). Moreover, providing 8 mg/20 g BW of temulawak extract resulted in significantly higher sperm counts compared to those of the nicotine control group (p=0.019), but there was no significant result when compared to the P1 group.

*: significant, p<0.05
 
Table 3. Comparison of sperm counts between each group

*: significant, p<0.05

Discussion

This experimental study showed that giving temulawak extract, both 4 mg/20 g body weight and 8 mg/20 g body weight, to nicotine–treated mice markedly improves sperm quality. The temulawak could improve sperm quality due to its curcumin and xanthorrhizol compounds, which are known for their antioxidant and anti-inflammatory properties.
Several documents have reported the benefits of curcumin and xanthorrhizol compounds. Curcumin works by lowering the toxicity effect of nicotine in spermatogenic cells by producing more ROS scavenging molecules to bind the free radicals (16–18). Both curcumin and xanthorrhizol inhibit IL – 1β, IL-6, TNF alpha, and MCP 1. Moreover, they also suppress the increase of COX-2 that usually results from the presence of nicotine (9, 15). Additionally, curcumin increases the total antioxidant capacity in seminal plasma, which is closely related to male fertility, to provide a suitable environment for sperm swimming (17). The curcumin also improves the androgenesis in the testis and increases the testosterone and LH levels to improve the spermatogenesis process (9).
In our study, we also observed that giving nicotine by inhalation significantly lowered sperm quality in mice. Results from this study are in line with the previous research. Nicotine significantly reduced the testosterone level, sperm counts, viability and motility of sperm, testis weight, and seminiferous tubules diameters in the nicotine (2.5 mg/kg body weight) treated group compared to those of the control (normal saline) (19). Another study showed that giving nicotine in graded doses (0.5 mg/kg body weight to 4 mg/kg body weight) could lower the percentage of normal morphology of sperm and sperm motility (7). However, the decrease in sperm morphology was not significant, which was perhaps due to an incomplete spermatogenesis cycle. The research was conducted in 28 days, while complete spermatogenesis in mice takes 35 days (20). Moreover, previous research mainly used intraperitoneal injection for the nicotine administration to control the exact dose for each animal, while our research used inhalation. Intraperitoneal injection has a slower absorption time that results in a higher concentration of nicotine inside the body (21).
Our study is in line with previous research. Intraperitoneal (IP) injection of 0.5 ml/kg body weight nicotine for 28 days significantly reduced sperm counts and the spermatozoa’s motility and normal morphology.  Curcumin supplementation (10 mg/kg body weight, 30 mg/kg body weight, and 60 mg/kg body weight) to the nicotine-treated mice increased the sperm counts, motility, and normal morphology significantly (16). Another study showed that giving 4 mg/kg BW of nicotine via IP injection for eight weeks could significantly reduce testis weight, epididymis, seminal vesicles, ventral prostate, sexual hormones, antioxidant, and spermatozoa concentration. A significant increase was also observed in the concentration of cytokine, pro-inflammatory mediators, superoxide anions, lipid peroxide, dead sperm, and abnormal morphology of spermatozoa. However, giving curcumin prevents these degenerative changes caused by nicotine (9).

 

Conclusion

Based on the results of this study, giving temulawak extract improves the sperm parameters in nicotine-treated groups. The most effective improvement was observed in the dose of 8 mg/ 20 g BW.

 

Acknowledgements

None.

 

Conflicts of Interest

The authors declare no conflicts of interest.