Abstract
Cigarette smoking is associated with sexual dysfunction and impaired fertility in males. The aim of this study was to determine the potential protective effect of honey against the toxic effect of cigarette smoke (CS) on sexual behavior and fertility of male rats. Thirty-two adult Sprague-Dawley rats were randomly divided into four groups (8 rats/group) as control, honey (H), CS and H plus CS (H + CS) groups. Rats in control and CS groups received oral administration of distilled water daily while rats in H and H + CS groups received honey (1.2 g/kg body weight/day) by oral gavage. Rats in CS and H + CS groups were also exposed to CS for 8 min 3 times/day. From 10 to 13 weeks of treatment, each male rat was cohabited with 3 untreated female rats for sexual behavioral and reproductive performance studies. Honey significantly increased the percentages of rats achieving intromission and ejaculation as well as increased mating and fertility indexes of male rats exposed to CS. Thus, honey has a protective effect against CS-induced impaired sexual behavior and fertility in male rats.
Introduction
The decline in male reproductive health and fertility for the past 30 years has been linked to environmental toxicants or xenobiotics (Saradha and Mathur, 2006; Sikka and Wang, 2008). One of the toxicants that have been reported to have detrimental effects on male reproductive health is cigarette smoke (CS). Cigarette smoking has been shown to be associated with the abnormalities in male reproductive function such as decreased sperm count (Richthoff et al., 2008) and motility (Gaur et al., 2007) as well as erectile dysfunction (ED) (Shiri et al., 2004) and early pregnancy loss (Venners et al., 2004) in human studies. It is also reported that smokers of more than 20 cigarettes daily had a greater risk of ED compared to nonsmokers (Lam et al., 2006). In experimental studies, rodents exposed to CS have been shown to have reduced fertilizing potential of sperm (Yamamoto et al., 1998), blastocyst capacity for implantation (Kapawa et al., 2004) and produce oxidative stress in the penile tissue (Bivalacqua et al., 2009).
Honey is a natural product of honey bees and traditionally used as a sweetener or food and as a medicine such as for ulcer and wound treatments (Bogdanov, 2010). It is also traditionally consumed among the Malays for enhancement of fertility (Pengarang, 2008) and possesses antioxidant properties (Beretta et al., 2007; Mohamed et al., 2010). Recently, honey supplementation at a dose of 1.2 g/kg/day has been shown to reduce the impaired testicular function in terms of testosterone and sperm productions in rats exposed to CS for 13 weeks (Mohamed et al., 2011). However, to date, whether honey supplementation might protect against impaired sexual behavior and fertility induced by CS is not known. Therefore, the aim of this study was to determine the potential protective effect of honey against the toxic effect of CS on sexual behavior and fertility of male rats.
Materials and methods
Honey used in this study (locally known as Tualang Honey) was supplied by Federal Agricultural Marketing Authority, Malaysia. It is a wild multifloral honey collected from beehives built on a tall tree, Koompassia excelsa (locally named as ‘Tualang’ tree) that grows in the rain forest of Kedah, Malaysia. This honey was filtered to remove solid particles and concentrated (about 20% w/v water) by oven drying at 40°C by the supplier. Commercially available cigarettes (Benson & Hedges, British American Tobacco Bhd., Malaysia) containing about 1.4 mg nicotine and 15 mg tar per cigarette were used for all CS exposures. All chemicals used in this study were of analytical grade.
Thirty-two male Sprague-Dawley rats aged 10 weeks (270–320 g) were obtained from Laboratory Animal Research Unit of Health Campus, Universiti Sains Malaysia (USM), Malaysia. Rats were individually housed in a polycarbonate cage and maintained on a 12-h light/dark cycle at 20–24°C. They were provided rodent chow pellets (Gold Coin Sdn. Bhd., Kuala Lumpur, Malaysia) and water ad libitum and acclimatized to the environment for 2 weeks prior to the experiment. This study protocol was approved by the Animal Ethics Committee, USM (PPSG/07(A)/044/[2007][32]) and the animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals by National Institute of Health. Animals were randomly divided into control, honey (H), CS and H plus CS (H + CS) groups (8 rats/group). Each rat in control and CS groups received oral administration of distilled water (0.5 mL/day) while each rat in H and H + CS groups received honey (1.2 g/kg body weight/day) by oral gavage between 08:00 h and 08:30 h daily. This dose was worked out relative to the local human consumption of honey which is 0.2 g/kg body weight daily. Honey at the dose of 1.2 g/kg body weight was freshly diluted with distilled water to prepare 0.5 mL of diluted honey for each rat. Then, 0.5 mL of the diluted honey was immediately mixed and administered to each rat by oral gavage. All rats in CS and H + CS groups were also exposed to CS for 8 min 3 times/day (at 08:30 h, 12:30 h and 16:30 h) using a whole body smoke exposure chamber (45 × 25 × 20 cm with 2 compartments). For each CS exposure, the smoke produced from 10 burning cigarettes in 1 compartment was continuously ventilated by 2 air pumps to another compartment where the rats were exposed to the smoke (Cesar-Neto et al., 2003). The total exposure to CS for each rat was 24 min/day. Based on our preliminary study, this method of exposure could produce serum cotinine levels ranging from 1.58 to 3.73 ng/mL in male rats (Mohamed and Sulaiman, 2009). These levels correspond to the serum cotinine level in humans who smoke more than 20 cigarettes/ day (more than 0.26 ng/mL; Barua et al., 2002). The rats in control and H groups were subjected to the similar condition but exposed to room air. All treatments were given to all male rats for 13 weeks.
From 8 to 13 weeks of treatments, all male rats were maintained in a reversed light/dark cycle (with light on from 12:00 h to 24:00 h) at room temperature of 22–24°C. Meanwhile, after 10 weeks of treatment, each male rat was cohabited for 3 h daily (from 09:00 h to 12:00 h) for up to 2 weeks with 2 untreated female rats (8–10 weeks old, 180–220 g). All female rats with two regular estrus cycles were used in this study. Vaginal smear was performed daily at the end of cohabitation period. Female rats with sperm-positive vaginal smear were considered being successfully mated, separated from male rats and considered as Day 0 of gestation (Kuriyama et al., 2005). In the following week, each male rat was paired with 1 untreated female rat in estrus phase to assess the male sexual behavior for 20 min (between 09:00 h and 12:00 h) as reported earlier (Agmo, 1997; Chahoud and Faqi, 1998; Faqi et al., 2004). It was recorded under dim white light illumination using a video camcorder (HDD Handycam DCR-SR42, Sony Corporation, Japan). The following parameters were recorded and calculated: (a) presence of precoital exploratory activities such as sniffing, nose-to-nose contact and genital exploring; (b) presence of mount; (c) presence of intromission; (d) presence of ejaculation (e) presence of two or more ejaculations; (f) mount and intromission latencies (the time interval between the introduction of female and the first mount or intromission by the male); (g) ejaculatory latency (the time interval between the first intromission and the first ejaculation) and (h) mount and intromission frequencies (the number of mounts or intromissions until the first ejaculation).
All 24 female rats paired with the male rats (3:1) in this study were used for male fertility or reproductive performance assessment (Kuriyama et al., 2005). For all sperm-positive female rats, weekly body weight was taken and the signs of pregnancy such as enlargements of nipples and abdomen were observed. About half of the pregnant female rats (dams) (from the first two and third mating groups) were killed on Day 21 of gestation by overexposure to diethyl ether. The gravid uterus was excised for further analysis including gravid uterine weight, fetal weights and numbers of implantations, resorptions, corpora lutea and fetuses (live and dead). The remaining half of pregnant dams (from the first two and third mating groups) were allowed normal delivery and killed on Day 7 of postdelivery. The uterus was excised and soaked in 10% ammonium sulfate to assess the number of implantation sites. The pups were examined for any gross external abnormalities, weighed daily and killed on Day 7 of life. Female rats with sperm-positive vaginal smear but did not show any pregnancy signs were killed on the next 24 days after mating. The following parameters were evaluated for male reproductive performance: (a) mating index ([number of sperm-positive females/total number of females cohabited]× 100); (b) fertility index ([number of cohabited females becoming pregnant/total number of females cohabited] × 100); (c) pregnancy index ([number of females with live born pups/number of pregnant females] × 100); (d) gravid uterine weight at Day 21 of pregnancy; (e) percentage of preimplantation loss ([number of corpora lutea − total implantation)/number of corpora lutea] × 100); (f) percentage of resorption or postimplantation loss ([total implantation − number of live pups at term]/total implantation × 100); (g) gestational length; (h) litter size/dam; (i) birth weight of the pup; (j) presence of congenital abnormality; (k) pup weight at Day 7 of postnatal life and (l) 7-day survival index of the pup ([number of live pups at day 7/number of live pups delivered] × 100).
Statistical analyses were carried out using the Statistical Package for Social Science (SPSS) version 12.0.1. Numerical data were analyzed by Kruskal-Wallis test followed by Mann-Whitney U test and presented as median (interquartile range). Categorical data were analyzed using Pearson chi-square or Fisher’s exact test and presented as percentage. A value of p < 0.05 was considered to be statistically significant.
Results
The findings on the sexual behavior of male rats are presented in Table 1. Rats in all groups had shown the presence of precoital exploratory activities and mount. However, the percentages of rats from CS group having intromission (25%) and ejaculation (25%) were significantly lower compared with control (100%) and H groups (100%). In H + CS group, the percentage of rats having intromission (75%) was significantly higher than CS group but not significantly different with control and H groups. Meanwhile, the percentage of rats having ejaculation in H + CS group (62.50%) was slightly higher than CS group but slightly lower than control and H groups. However, the differences were statistically insignificant. No significant differences were also observed for other parameters such as the percentage of rats having two or more ejaculations as well as for the latency and frequency of mount, intromission and ejaculation among all the groups.
Effect of honey on sexual behavior of male rats in all experimental groups a
CS: cigarette smoke; H: honey; H + CS: honey plus CS; NS: not significant.
a n = 8 per group.
b Fisher’s exact test.
c p < 0.05 compared with control group.
d p < 0.05 compared with H group.
e p < 0.05 compared with CS group.
f Data are presented as median (interquartile range).
g Kruskal-Wallis test.
The findings on the reproductive performance of male rats are shown in Table 2. Mating and fertility indexes for rats from H group were similar with control group. However, rats from CS group had significantly lower mating and fertility indexes compared with those from control and H groups. In H + CS group, both mating and fertility indexes were significantly higher than CS group but not significantly different with control and H groups. Meanwhile, pregnancy index was similar among all the groups. Pups in CS group had significantly lower birth weight compared to control and H groups. Although the birth weight of the pups in H + CS group was slightly higher than CS group and slightly lower than control and H groups, the differences were not statistically significant. Other parameters such as gravid uterine weight, pre- and postimplantation loss, gestational length, pup’s weight on Day 7 of life and 7-day survival index were similar among all the experimental groups. Furthermore, none of the pups had gross congenital abnormality.
Effect of honey on reproductive performance of male rats in all experimental groups a
CS: cigarette smoke; H: honey; H + CS: honey plus CS; NS: not significant.
a n = 8 males and 24 females per group.
b p < 0.05 compared to control group.
c p < 0.05 compared to H group.
d p < 0.05 compared to CS group.
e Fisher’s exact test.
f Pearson chi-square test.
g Data are presented as median (interquartile range).
h Kruskal-Wallis test followed by Mann-Whitney U test.
Discussion
CS has been reported to have detrimental effects on male sexual function and fertility in human and experimental studies (Shiri et al., 2004; Venners et al., 2004; Yamamoto et al., 1998). CS also induced oxidative stress in the penis of mice exposed to CS (Bivalacqua et al., 2009). Honey is traditionally consumed for enhancement of male fertility (Pengarang, 2008) and possesses antioxidant properties (Beretta et al., 2007; Mohamed et al., 2010). This study was designed to determine the protective effect of honey against the toxic effect of CS on sexual behavior and fertility of male rats.
In this study, all rats from all groups showed precoital exploratory activities and had mounting activity with similar mount latency and frequency suggesting the presence of sexual motivation or libido (Meisel and Sachs, 1994). These observations might also suggest that honey, CS or both did not produce any detrimental effect on sexual motivation or libido in male rats. Following the sexual motivation, intromission may occur which requires penile erection and a sustained penile erection is needed for ejaculation (Meisel and Sachs, 1994). An inability to achieve or maintain penile erection sufficient for satisfactory sexual performance is defined as ED (NIH, 1993). In this study, CS exposure reduced the ability of rats to achieve and maintain penile erection as shown by reduced percentages of rats achieving both intromission and ejaculation in CS group compared to control and H groups. These findings might suggest that CS could cause ED in rats, which is consistent with previous findings among smokers (Shiri et al., 2004). Interestingly, supplementation of honey improved the erectile function as the percentages of rats achieving intromission and ejaculation in H + CS group were significantly increased.
Reproductive performance of male rats was also assessed in this study in order to evaluate the ability of male rats to successfully mate female rats and subsequently produce viable offspring (OECD, 2004). The similar findings on reproductive performance of rats from H group might suggest that honey supplementation for 13 weeks does not produce any adverse effect on the reproductive performance of male rats. However, CS significantly reduced the mating and fertility indexes of male rats from CS group, and these indexes were significantly increased in rats from H + CS group. Hence, this finding might suggest that honey supplementation might improve the mating ability and fertility by partly improving the erectile function of male rats. It has been suggested that CS reduces fertility of male rats by reducing the fertilizing potential of the sperm (Yamamoto et al., 1998). Consequently, it is also possible that the improved fertility could be attributed to the improved fertilizing potential of the sperm as in our previous findings the percentages of abnormal and motile sperm were significantly improved with the supplementation of honey in male rats exposed to CS (Mohamed et al., 2011).
Although no gross congenital abnormalities were observed, CS exposure produced smaller pups as shown by lower birth weight in CS group as compared with control and H groups. This finding is supported with another study that showed the newborns of nonsmoking mothers whose fathers smoke more than 20 cigarettes/day had lower birth weight, suggesting that fetal growth may be adversely affected by CS (Martinez et al., 1994). However, supplementation of honey showed improvement in the birth weight of pups, indicating that honey might reduce the adverse effect of CS on the fetal growth in rats. The reason for this observation remains unclear. Studies have been shown that smokers significantly had higher percentage of sperm with DNA fragmentation (Sepaniak et al., 2006). Therefore, it is possible that honey might reduce the sperm DNA damage. This in turn might improve fetal growth which needs further study.
Normal level of testosterone is one of the important factors for the ability to achieve and maintain penile erection as testosterone therapy has been shown to improve ED in both human and animal studies (Baba et al., 2000; Rhoden and Morgentaler, 2010). In our earlier observations, testosterone level was significantly increased with the supplementation of honey in male rats exposed to CS for 13 weeks (Mohamed et al., 2011). Thus, it is plausible to suggest that the improved erectile function found in this study could be partly attributed to the improved testosterone level in rats from H + CS group. Experimental studies in rodents showed that CS significantly decreased penile nitric oxide synthase (NOS) and endothelial NOS activities as well as decreased penile neuronal NOS content. These findings might in turn reduce NO level, leading to impaired endothelium-dependent vasodilation and neuronal control of penile erection (Bivalacqua et al., 2009; Xie et al., 1997). The decreased penile NOS activity has been suggested to be due to increased oxidative stress as the levels of superoxide anion and nitrotyrosine, a specific marker of the toxic radical peroxynitrite, were significantly increased in the penis of mice exposed to CS (Bivalacqua et al., 2009). Honey has been reported to have antioxidants such as phenolic compounds as well as possesses antiradical and antioxidant properties (Beretta et al., 2007; Mohamed et al., 2010). Therefore, it is possible that the effect of honey on the increased percentages of rats achieving intromission and ejaculation when exposed to CS in this study could also be partly mediated by its counteraction on oxidative stress within penile tissues via its antioxidant property which needs further study.
In conclusions, our results demonstrated that honey at the dose of 1.2 g/kg body weight daily significantly increased the percentages of rats achieving intromission and ejaculation as well as increased mating and fertility indexes of male rats exposed to CS. Therefore, honey is suggested to have a protective effect on CS-induced impaired sexual behavior and fertility in male rats. However, further studies are needed to identify the components of honey responsible for these effects as well as to investigate the potential use of honey as a natural product in protecting human male sexual function and fertility from toxicants such as CS.
Footnotes
Acknowledgment
The authors are grateful to Federal Agricultural Marketing Authority, Malaysia for supplying the honey.
Funding
This research was funded by USM, Malaysia (304/PPSP/6131586).