Aerobic exercise improves ejaculatory behaviors and complements dapoxetine treatment by upregulating the BDNF-5-HT duo: a pilot study in rats

Abstract

Accumulating evidence has revealed many clues that regular aerobic exercise benefits brain health and behaviors. The aims of this study were to explore the effect of aerobic exercise on ejaculatory behaviors, as well as to make a preliminary assessment of aerobic exercise as a complementary strategy to dapoxetine treatment in rapid ejaculators. Copulatory tests of rats and a treadmill training protocol were performed in this study. In total, 12 rapid ejaculators were selected on the basis of ejaculation distribution theory and randomly assigned to 4 groups: control (Ctrol) group, aerobic exercise (Ex) group, dapoxetine (Dapo) group, and Ex+Dapo group. We evaluated the changes in ejaculatory parameters in the 4 groups. Variations in biological markers, including serum corticosterone, serotonin (5-HT), and brain-derived neurotrophic factor (BDNF) of the raphe nucleus, were determined by enzyme-linked immunosorbent assay (ELISA). The primary finding of our study was that both aerobic exercise and acute dapoxetine could enhance ejaculation control and prolong ejaculation latency in rapid ejaculator rats. The ejaculation delay effect of aerobic exercise was nearly equivalent to that of acute dapoxetine. In addition, both aerobic exercise and dapoxetine treatment could lead to increased expression of BDNF and 5-HT in the raphe nucleus of rapid ejaculators. Moreover, the two interventions, when applied together, may further upregulate the expression of BDNF-5-HT duo in a complementary manner. This study highlights the positive effects of aerobic exercise on ejaculation control. Regular aerobic exercise might be a promising complementary treatment to dapoxetine in rats.

INTRODUCTION

Performed studies showed that over 30% of adult males suffer from premature ejaculation (PE), which greatly reduces their quality of sexual life.1,2 To date, the exact etiology of PE has not yet been elucidated, and a deficiency of serotonin (5-HT) in the brain is considered the most likely pathogenesis.3,4

Patients with PE are normally treated with selective serotonin reuptake inhibitors (SSRIs) to prolong the intravaginal ejaculatory latency time (IELT), while the effectiveness differs from man to man.5 Dapoxetine, which is taken on demand, is the only in-label SSRI for PE treatment.6 It works by targeting the 5-HT transporter (5-HTT) and increasing the level of 5-HT in the synaptic cleft. Although dapoxetine is the most commonly employed treatment, the current situation is not satisfactory, as approximately 10%–20% of PE patients do not show complete response.7 In addition, it frequently results in several side effects, such as nausea, dyskinesias, and hallucinations.8 Considering these reasons, complementary strategies for drug treatment have received increasing attention in recent years.

The benefits of exercise are widely studied and frequently conveyed by popular media. Metaphorically speaking, exercise is similar to food for the brain throughout the lifespan.9 It can improve performance in many aspects of both humans10–12 and rats.13,14 In addition, the brain displays a variety of molecular, physiological, and structural changes following exercise.15 Therefore, exercise therapy has been recommended as a promising therapeutic strategy. Among various types of exercise, aerobic exercise is a rhythmic and continuous exercise under the condition of adequate oxygen supply. Although there is evidence that oxygenation plays a role in the development of sexual dysfunction,16,17 the effectiveness of aerobic exercise on ejaculatory dysfunction has received less attention. Some physiological mechanisms involved in exercise are also related to the regulation of ejaculation. First, exercise enhances brain function by facilitating monoamine neurotransmitters, especially 5-HT.18,19 Second, exercise can lead to a potentiation of brain-derived neurotrophic factor (BDNF) expression, which plays a crucial role in the neuroplasticity of the brain.20,21 Accumulating evidence in both humans and rats has shown that BDNF is associated with ejaculation control.22–24 Third, exercise is a common method of pelvic floor rehabilitation, which has been a component of PE treatment since 199625 and has been proven effective by many studies.26–28 Thus, it is necessary to further study the effects of aerobic exercise on the regulation of ejaculation.

Copulatory tests of rats have provided us with a classical method to understand the underlying mechanisms of rapid ejaculation. In the present study, rapid ejaculators were selected based on copulatory tests and then received different interventions. A well-designed treadmill exercise protocol was implemented to simulate aerobic exercise. Serotonergic neurons are mainly located in the raphe nucleus of the brainstem.23,29 Therefore, parallel to examining ejaculatory behaviors, the variations in biomarkers, including 5-HT and BDNF, in the raphe nucleus were tested.

The above findings prompted us to focus on the following objectives: (i) to investigate the effect of aerobic exercise on rapid ejaculators; (ii) to explore the combined effect of aerobic exercise and acute dapoxetine on rapid ejaculators; and (iii) to determine whether biomarkers, including 5-HT and BDNF, are affected by different interventions in rapid ejaculators. We hypothesized that aerobic exercise could enhance ejaculation control and have a complementary effect to dapoxetine on delaying ejaculation in rapid ejaculator rats.

MATERIALS AND METHODS

Animals

Experiments were performed in 105 male and 35 female Sprague–Dawley rats (8 weeks, 200 g–250 g). The rats were housed individually in a room with a 12-h day/night light cycle during the entire experimental period. All procedures in this study were approved by the Animal Ethics Committee of Anhui Medical University (Approval No. LLSC20211518).

Selection of rapid ejaculators

Preparations of female rats, including sterilization and estrus, and the protocol of copulatory tests were described in our previous study.23,24 Six copulatory tests were conducted in every male rat, and the first 3 tests (T1–T3) were to obtain stable sexual behaviors. The following parameters were analyzed based on the last 3 tests (T4–T6): mount latency (ML, in s), mount frequency (MF), intromission latency (IL, in s), intromission frequency (IF), ejaculation latency (EL, in s), ejaculation frequency (EF), postejaculatory interval (PEI, in s), and intromission ratio (IR=IF/[MF+IF], %). Eventually, 5 male rats were excluded for no insertion behavior in the copulatory tests. The mean EF values in T4–T6 were normally distributed. According to the ejaculation distribution theory,30,31 10% of male rats with the highest EF were considered rapid ejaculators. Finally, 12 out of 100 male rats belonging to the rapid category were qualified and selected for further interventional experiments. Behavioral parameters at T4–T6 were considered baseline values.

Interventional experimental design

The rapid ejaculators were randomly divided into 4 groups: control (Ctrol) group (n = 3), aerobic exercise (Ex) group (n = 3), dapoxetine (Dapo) group (acute dapoxetine 60 mg kg⁻¹ body weight [bw]; n = 3), and Ex+Dapo group (aerobic exercise plus acute dapoxetine 60 mg kg⁻¹ bw; n = 3). The interventional experiments lasted for another 6 weeks (T1’–T6’). Similarly, parameters at T4’–T6’ were considered endpoint values after different interventions. Moreover, the body weights of the rats were measured pre- and post-intervention. Figure 1 shows a simple flowchart for the experimental design. As running on a treadmill is a skill that the rats should maintain prior to the interventional experiments, rats in the Ex and Ex+Dapo groups were trained on an electric-driven treadmill for a week. The speed was gradually increased from 5 m min⁻¹ to 15 m min⁻¹ with a 0° incline. The training lasted for 1 h every day, and the first and last 5 min were considered the warm-up and recovery periods. A plastic ball instead of a weak electrical shock was placed on the back end of the treadmill as a contact stimulus to keep the rats running.

Figure 1.

A simple flowchart of the implementation plan of the experiment showed (a) the selection of rapid ejaculators, and (b) different interventional group assignments. There are 3 rapid ejaculators in each group: Ctrol group (without intervention treatment), Ex group (6-week aerobic exercise), Dapo group (acute dapoxetine 60 mg kg⁻¹ bw), and Ex+Dapo group (6-week aerobic exercise plus acute dapoxetine 60 mg kg⁻¹ bw). ML: mount latency; MF: mount frequency; IL: intromission latency; IF: intromission frequency; EL: ejaculation latency; EF: ejaculation frequency; PEI: postejaculatory interval; IR: intromission ratio; 5-HT: serotonin; BDNF: brain-derived neurotrophic factor; bw: body weight; W: week; T: test.

After habituation, the rats in the Ex and Ex+Dapo groups regularly ran on the treadmill at a velocity of 15 m min⁻¹ for 60 min, 6 days (Monday to Saturday) a week for 6 weeks, approximately 70% of volume per time oxygen maximum (VO₂ max).32 Here, we used a moderate-intensity treadmill exercise protocol model to reduce lactate accumulation and stress. A sign of stress is the lactate threshold (LT), which can be alternatively regarded as stressful when the treadmill speed continues above the LT (approximately 20 m min⁻¹ in rats).33

At the same time, the rats in the Ctrol and Dapo groups were left on the treadmill for 60 min, 6 days a week for 6 weeks without running, so they could experience stress stimuli similar to those of the training rats. Copulatory tests were always performed every Sunday. The rats in the Dapo and Ex+Dapo groups were orally administered 60 mg kg⁻¹ bw dapoxetine at 3 h before the copulatory tests. This dose was used on the basis of our previous study in rats.34

Preparation of blood and brain samples

Immediately after interventional experiments, 12 rats were anesthetized with pentobarbital (30 mg kg⁻¹ bw, intraperitoneal injection). First, 2 ml blood was taken from the inferior vena cava. Then, the rats were perfused transcardially with normal saline. After death, brains were quickly removed, and raphe nucleus samples were dissected on ice. All samples were stored at −80°C for further analyses.

Quantification of corticosterone, 5-HT, and BDNF

Serum corticosterone was assayed using an ELISA kit (Elabscience, Wuhan, China) and expressed as ng ml⁻¹. For raphe nucleus samples, BDNF and 5-HT concentrations were determined using a BDNF ELISA kit (USCN, Wuhan, China) and a 5-HT ELISA kit (Elabscience) according to the protocol, respectively. The results were expressed as pg ml⁻¹ for BDNF and ng ml⁻¹ for 5-HT. All samples were assayed in triplicate.

Statistical analyses

The results were analyzed using SPSS 17.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). The normal distribution was verified by the Shapiro–Wilk test. Variables with a normal distribution were compared between groups using one-way analysis of variance (ANOVA) followed by a post hoc LSD test. If not, the Kruskal–Wallis H test was used. Paired Student’s t-test was used to assess the differences between body weight before and after interventions. P < 0.05 was considered statistically significant.

RESULTS

Changes in body weight

As mentioned above, the selected rapid ejaculators were divided into 4 interventional groups. All 12 rapid ejaculators successfully completed the interventional experiments without injury or compromised health. Body weight before and after interventions was measured and is shown in Figure 2. There was no significant difference in baseline body weight between groups (P = 0.797). The rats in the Ctrol and Dapo groups gained body weight significantly (P = 0.013 for the Ctrol group and P = 0.012 for the Dapo group). In contrast, rats in the Ex and Ex+Dapo groups maintained their body weight compared to baseline (P = 0.987 for the Ex group and P = 0.778 for the Ex+Dapo group).

Figure 2.

Changes in body weight in different groups pre- and post-intervention. Data are presented as the mean ± standard deviation. ANOVA followed by post hoc LSD test between groups was used. ^**Significant differences versus the Ctrol group, P < 0.01. ^##Significant differences versus the Ex group, P < 0.01. ^&&Significant differences versus the Dapo group, P < 0.01. Paired Student’s t-test was used to assess the differences before and after interventions. ^$Significant differences versus preintervention, P < 0.05. There are 3 rapid ejaculators in each group: Ctrol group (without intervention treatment), Ex group (6-week aerobic exercise), Dapo group (acute dapoxetine 60 mg kg⁻¹ bw), and Ex+Dapo group (6-week aerobic exercise plus acute dapoxetine 60 mg kg⁻¹ bw). ANOVA: one-way analysis of variance; LSD: least-significant difference; bw: body weight.

Ejaculatory behaviors after different interventions

The ejaculatory behaviors before interventions are included in Table 1. There were no significant differences among the 4 groups at baseline (all P > 0.05). Table 2 shows the ejaculatory parameters after another series of 6 weeks of interventional experiments and copulatory tests.

Table 1.

Variations in ejaculatory parameters of rapid ejaculators at baseline

Variable	Ctrol group	Ex group	Dapo group	Ex+Dapo group	aP
ML (s)	290.89±78.84	270.78±124.06	254.78±86.99	294.33±108.99	0.826
MF	13.44±2.70	14.44±3.97	15.78±4.21	13.78±3.56	0.549
IL (s)	298.44±77.78	280.00±117.89	273.00±95.87	311.00±106.86	0.849
IF	17.33±5.15	17.89±3.33	16.89±6.23	15.67±3.16	0.774
EL (s)	660.33±273.41	693.89±205.06	587.33±206.05	708.78±178.40	0.651
EF	3.33±0.50	3.22±0.44	3.44±0.73	3.44±0.73	0.845
PEI (s)	385.67±99.31	399.44±51.82	453.67±77.13	404.11±55.79	0.242
IR (%)	55.82±5.95	55.30±7.83	52.32±8.10	54.85±4.92	0.710

^aANOVA followed by post hoc LSD test among four groups was used. Data are expressed as the mean ± standard deviation. There are 3 rapid ejaculators in each group: Ctrol group (without intervention treatment), Ex group (6-week aerobic exercise), Dapo group (acute dapoxetine 60 mg kg⁻¹ bw), and Ex+Dapo group (6-week aerobic exercise plus acute dapoxetine 60 mg kg⁻¹ bw). ML: mount latency; MF: mount frequency; IL: intromission latency; IF: intromission frequency; EL: ejaculation latency; EF: ejaculation frequency; PEI: postejaculatory interval; IR: intromission ratio; bw: body weight; ANOVA: one-way analysis of variance; LSD: least-significant difference

Table 2.

Variations in ejaculatory parameters of rapid ejaculators after intervention

Variable	Ctrol group	Ex group	Dapo group	Ex+Dapo group	aP
ML (s)	271.89±110.03	156.00±97.41*	339.78±100.05##	318.22±103.29##	0.003
MF	13.89±3.62	18.00±2.50**	18.11±2.93**	19.56±2.51***	0.002
IL (s)	291.33±110.76	164.89±95.56*	353.67±94.36###	335.22±108.07##	0.002
IF	16.78±2.68	23.89±3.82**	22.78±5.14**	30.89±5.37***,#,&	<0.001
EL (s)	667.33±207.48	1016.89±305.50*	1110.33±303.53**	1530.56±403.55***,##,&&	<0.001
EF	3.44±0.53	2.33±0.71**	2.22±0.83***	1.22±0.44***,##,&&	<0.001
PEI (s)	411.11±58.20	392.44±57.51	516.67±94.45**,##	458.00±58.78	0.003
IR (%)	55.05±5.92	56.94±2.93	55.41±4.44	61.09±2.46**,#,&&	0.016

^aANOVA followed by post hoc LSD test among four groups was used. Data are expressed as the mean ± standard deviation. ^*Significant differences versus the Control group, P<0.05; ^**significant differences versus the Control group, P<0.01; ^***significant differences versus the Control group, P<0.001; ^#significant differences versus the Exercise group, P<0.05; ^##significant differences versus the Exercise group, P<0.01; ^###significant differences versus the Exercise group, P<0.001; ^&significant differences versus the Dapoxetine group, P<0.05; ^&&significant differences versus the Dapoxetine group, P<0.01. There are 3 rapid ejaculators in each group: Ctrol group (without intervention treatment), Ex group (6-week aerobic exercise), Dapo group (acute dapoxetine 60 mg kg⁻¹ bw), and Ex+Dapo group (6-week aerobic exercise plus acute dapoxetine 60 mg kg⁻¹ bw). ML: mount latency; MF: mount frequency; IL: intromission latency; IF: intromission frequency; EL: ejaculation latency; EF: ejaculation frequency; PEI: postejaculatory interval; IR: intromission ratio; bw: body weight; ANOVA: one-way analysis of variance; LSD: least-significant difference

For the Ex group, when compared with the Ctrol group, ML and IL were significantly shortened (P = 0.023 for ML and P = 0.013 for IL), while EL was significantly prolonged (P = 0.024). Moreover, MF and IF were significantly greater (P = 0.005 for MF and P = 0.002 for IF), while EF was significantly less than that of the Ctrol group (P = 0.001). No difference was found in PEI and IR (both P > 0.05).

For the Dapo group, there was no significant difference in ML or IL compared with the Ctrol group (both P > 0.05), which was different from the Ex group. Most of the ejaculatory parameters of the Dapo group, including MF, IF, EF, EL, and IR, were comparable to those of the Ex group (all P > 0.05). Of note, PEI of the Dapo group was significantly prolonged when compared with the Ctrol group (P = 0.003) and the Ex group (P = 0.001).

For the Ex+Dapo group, when the two interventions were combined, most of the parameters, including MF, IF, EF, EL, and IR, were further improved compared with those of the Ex or Dapo group alone (all P < 0.05).

The levels of corticosterone, 5-HT, and BDNF

Figure 3 presents the levels of serum corticosterone, 5-HT, and BDNF in the raphe nucleus. There was no significant difference in serum corticosterone among the 4 groups (Figure 3a; all P > 0.05). As shown in Figure 3b and 3c, both BDNF and 5-HT increased significantly in the Ex and Dapo groups compared with the Ctrol group (all P < 0.05). More accurately, the Ex group showed a higher level of BDNF, while the Dapo group had a higher level of 5-HT. Notably, the levels of BDNF and 5-HT were further increased in the Ex+Dapo group compared with the Ex group or Dapo group (all P < 0.05).

Figure 3.

Levels of (a) corticosterone, (b) 5-HT, and (c) BDNF after different interventions. Data are presented as the mean ± standard deviation. ANOVA followed by post hoc LSD test between groups was used. ^*Significant differences versus the Ctrol group, P < 0.05. ^**Significant differences versus the Ctrol group, P < 0.01. ^***Significant differences versus the Ctrol group, P < 0.001. ^##Significant differences versus the Ex group, P < 0.01. ^###Significant differences versus the Ex group, P < 0.001. ^&&Significant differences versus the Dapo group, P < 0.01. ^&&&Significant differences versus the Dapo group, P < 0.001. There are 3 rapid ejaculators in each group: Ctrol group (without intervention treatment), Ex group (6-week aerobic exercise), dapoxetine group (acute dapoxetine 60 mg kg⁻¹ bw), and Ex+Dapo group (6-week aerobic exercise plus acute dapoxetine 60 mg kg⁻¹ bw). NS: not significant; 5-HT: serotonin; BDNF: brain-derived neurotrophic factor; ANOVA: one-way analysis of variance; bw: body weight; LSD: least-significant difference.

DISCUSSION

Our study illustrated that aerobic exercise can improve the control of ejaculation in rapid ejaculators. The delayed effect on ejaculation of aerobic exercise is equivalent to that of acute dapoxetine. Furthermore, our results showed improvement in ejaculation control and overlapping and potentiating upregulation of BDNF and 5-HT in the raphe nucleus when aerobic exercise and dapoxetine were applied together. These findings suggested that aerobic exercise can be used as a complementary strategy to dapoxetine in the treatment of rapid ejaculation.

Unlike humans, the evaluation of ejaculatory function in rats includes a series of behavioral parameters. First, EF and EL are the most direct indicators of ejaculation control. We found that both aerobic exercise and acute dapoxetine could reduce EF and prolong EL. Moreover, ML and IL mainly reflect sexual motivation.35 Intromission requires sufficient penile erection and coordinated activity of the pelvic floor muscles. Due to the benefits of aerobic exercise on erectile function16,36 and pelvic floor muscles,26,37 the ML and IL of the Ex group were significantly shortened in the present study. Increased MF and IF after exercise and dapoxetine treatment indicate that the ability to delay ejaculation was improved to some extent. In addition, IR was increased significantly in the Ex+Dapo group, suggesting that the combination of aerobic exercise and dapoxetine could enhance erectile efficiency and the success rate of intromission.

In recent years, significant progress has been made in understanding the central regulation of ejaculation at the molecular level. Due to methodological and ethical reasons, it is difficult to obtain central 5-HT and BDNF levels after ejaculation in humans. Therefore, the rat model for PE has become the best choice. Our study demonstrated that aerobic exercise, acute dapoxetine, or both could upregulate the expression of BDNF and 5-HT in the raphe nucleus of rapid ejaculators. These findings support the hypothesis that there is a common convergence point at the molecular level for the ejaculation delay effect by these two disparate interventions. Possible signaling pathways for aerobic exercise and dapoxetine leading to a complementary upregulation of BDNF-5-HT are shown in Figure 4.

Figure 4.

Potential mechanism of the complementary effect of aerobic exercise and dapoxetine treatment on rapid ejaculation. The upward arrow (↑) indicates that the parameter has increased, and the downward arrow (↑) indicates that the parameter has decreased. BDNF: brain-derived neurotrophic factor; 5-HT: serotonin; 5-HTT: 5-HT transporter; TPH2: tryptophan hydroxylase 2; EL: ejaculation latency; MF: mount frequency; IF: intromission frequency; EF: ejaculation frequency.

Dapoxetine can raise the level of central 5-HT acutely. The present study confirmed this point of view. In addition, our results showed that dapoxetine treatment can lead to increased expression of BDNF in the raphe nucleus. Similar results have also been found in the field of depression,38 which suggests that upregulating BDNF expression may be another possible mechanism of SSRIs.

Two hypotheses have been suggested to explain the increased 5-HT after aerobic exercise. First, unlike its precursor tryptophan, peripheral 5-HT cannot pass through the blood–brain barrier (BBB).39 The aerobic nature of exercise promotes the release of blood tryptophan from its carrier albumin. Then, the free tryptophan can cross the BBB and consequently lead to higher central 5-HT biosynthesis.40 Another hypothesis emphasizes the neurotrophic effect of BDNF on serotonergic neurons, which increases the synthesis and release of 5-HT. The results of a previous study were consistent with our study, which showed that the expression of BDNF mRNA in the brain increased 2-fold after treadmill exercise.41 Meanwhile, increased BDNF was shown to upregulate the expression of tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme in central 5-HT synthesis.24 Therefore, the effect of ejaculation delay after aerobic exercise could be mainly attributed to the improvement of BDNF. However, the ultimate mechanism of BDNF upregulation is still unclear. It was reported that BDNF was regulated via a cyclic adenosine monophosphate (cAMP) response element binding protein (CREB)-dependent mechanism.42 Moreover, SSRI administration has been found to increase the expression of CREB.43 Thus, we speculate that aerobic exercise and dapoxetine treatment may increase the expression of BDNF through a CREB-dependent mechanism.

In addition to the effects above, exercise is well known to boost mood and ease symptoms of depression.44,45 Due to the negative mood of rapid ejaculators, the benefit to mood induced by exercise could indirectly improve ejaculatory performance. Moreover, regular aerobic exercise helps to form a slim and healthy body image, which is more in line with public esthetics.46 In contrast, physically inactive and highly sedentary lifestyles, such as “couch potatoes”, can lead to weight gain. Discomfort due to poor body image can also lead to rapid ejaculation or even decreased sexual desire.47

A common criticism of the treadmill model is that exercise may cause stress on rats, which may affect the measurement of results. Therefore, we measured the level of serum corticosterone and found no significant difference, indicating that the rats in the 3 interventional groups did not experience greater stress than the Ctrol group. Some efforts were made to minimize the stress in our study. First, sufficient familiarization procedures before treadmill exercise were important to decrease the stress effect. Second, our exercise protocol used well-trained rats that received no electrical shock during running to avoid stimulating stress effects. Finally, the exercise intensity (15 m min⁻¹) was below the lactate steady state previously established, indicating that the training was aerobic. A speed less than 20 m min⁻¹ does not lead to increased lactate or corticosterone.48

Limitations in our study must be acknowledged. First, statistical power was a limitation in our study. The low efficiency of the selection process of rapid ejaculators resulted in this situation. Therefore, the current findings provide suggestions for future studies rather than definite truths. In addition, 5 male rats were excluded due to their low level of sexual activity. Female factors may be an explanation, and replacement of female rats can be considered to solve this problem in future research. Second, the rats were not allowed to drink water during exercise, which may have led to dehydration. A previous study showed that dehydration during exercise can affect cerebral blood flow and the measurement of biomarkers.49 Therefore, sufficient water was available ad libitum before and after exercise to avoid dehydration to the maximum. Finally, there is a lack of evidence in the literature to standardize the aerobic exercise model of rats. Different treadmill exercise protocols limit the homogeneity between studies.

CONCLUSION

Both aerobic exercise and acute dapoxetine could significantly improve the control of ejaculation. Moreover, this study provides preliminary evidence that aerobic exercise may be a promising complementary treatment for PE patients with poor efficacy of dapoxetine. However, since the study was conducted in a limited sample of rats, it should only be considered a pilot study. Further experimental and clinical investigations are necessary to elucidate the mechanisms involved.

AUTHOR CONTRIBUTIONS

YYH and DWP designed the study, carried out the animal study, and drafted the manuscript. QSL participated in helping to perform the copulatory tests. HJ and XSZ participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declared no competing interests.