International Journal of Impotence Research (2014), 1–5 © 2014 Macmillan Publishers Limited All rights reserved 0955-9930/14 www.nature.com/ijir
ORIGINAL ARTICLE
Effects of swimming activity on the copulatory behavior of sexually active male rats MZ Allouh Physical activity has long been associated with better sexual function. This study investigated the effects of moderate swimming exercise on the copulatory behavior of sexually potent male rats. Two sets of sexually potent male rats –highly active and moderately active– were identified depending on baseline sexual activity. Each of the two sets of rats was further randomly divided into two groups (swimming and sedentary). There were 16 rats in each of the four study groups (highly active swimming, highly active sedentary, moderately active swimming and moderately active sedentary). The copulatory behavior parameters and serum testosterone levels were measured and compared between the rats of the swimming and sedentary groups following a month long training period in which rats were made to swim for 1 h every alternate day. Swimming significantly improved the sexual performance of highly active rats, as indicated by increased intromission frequency and intromission ratio, compared with the sedentary controls. Swimming improved both sexual desire and performance, as indicated by reduced mount latency and increased intromission ratio, respectively, in swimming moderately active rats compared with the sedentary moderately active controls. Therefore, swimming activity improves the copulatory behavior of both highly active and moderately active male rats. International Journal of Impotence Research advance online publication, 18 December 2014; doi:10.1038/ijir.2014.42
INTRODUCTION Erectile dysfunction (ED), previously known as sexual impotence, is the inability to achieve or maintain a penile erection that is sufficient for sexual satisfaction.1 This problem adversely affects the quality of life, especially, family and social relationships.2,3 The worldwide prevalence of ED in men in 1995 was estimated to exceed 152 million, and this number could reach 322 million by 2025.4 Several risk factors, including, age, smoking, malnutrition, obesity and physical inactivity, have been related to this high incidence of ED.3,5,6 Physical inactivity can have a detrimental effect on normal organ functioning and is an important cause of many chronic diseases.7 In contrast, physical activity is known to have a major role in preventing or delaying many chronic illnesses such as obesity, diabetes and cardiovascular diseases.7–9 In addition, several studies have suggested that an increase in physical activity may improve sexual function.10–12 Swimming is a good alternative to land-based exercises in older patients with conditions such as arthritis,13 who are known to be at a higher risk of ED.14,15 Interestingly, oxygen utilization data suggest that the energy use during swimming in humans is similar to that in rats.16,17 Among the various exercise modalities (treadmill, voluntary wheel running and swimming) used for studying the physiological response to exercise in rat models, swimming has an advantage as it utilizes a natural ability of rats.16 Several studies on animal models have reported benefits of swimming exercise on preventing tumor growth and progression, pregnancy-associated longterm memory impairment and neuropathic pain caused by nerve injury;18–20 however, data on benefits of swimming on ED are scarce. A recent study on rats found that regular exercise may help in improving penile erection by increasing penile neurotransmitter
activity.21 However, to the best of our knowledge, the effects of swimming on male sexual desire and potency have not been assessed using rat models. This study aimed to investigate the direct effects of swimming on the copulatory behavior of adult male rats. The following hypothesis was tested: swimming activity improves the copulatory behavior of both highly active and moderately active male rats. The serum testosterone levels of these animals were also compared. MATERIALS AND METHODS Animals Adult male Sprague–Dawley albino rats (weight: ~ 250 g) bred and raised in the Animal House Unit at Jordan University of Science and Technology (JUST) were used in this study. Approvals for all animal care and experimental procedures were obtained from the Animal Care and Use Committee at JUST and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.22 The rats were maintained under controlled temperature of 21 ± 1 °C with a 12 h light and 12 h darkness schedule (lights on between 0600 and 1800 hours). Food and water were provided ad libitum. Acclimatization time of 2 weeks was provided before beginning the experiments.
Experimental design Rats were subjected to seven pre-experimental mating tests with sexually receptive females to make them sexually experienced. The rats that achieved ejaculation in the last three tests were considered highly active, those that achieved ejaculation in one or two of the last three tests were considered sexually moderately active and those that failed to achieve ejaculation in the last three tests were considered sexually impotent and excluded from the study.23 Both highly active and moderately active rats were randomly divided into two groups: sedentary and swimming. Each group consisted of 16 male rats.
Department of Anatomy, Faculty of Medicine, Jordan University of Science & Technology, Irbid, Jordan. Correspondence: Dr MZ Allouh, Department of Anatomy, Faculty of Medicine, Jordan University of Science & Technology, P.O. Box: 3030, Irbid 22110, Jordan. E-mail: [email protected] Received 9 May 2014; revised 23 July 2014; accepted 18 October 2014
Effect of swimming on copulatory behavior MZ Allouh
2 organs (testes, epididymides, seminal vesicles, prostate and vasa deferentia) were dissected out, cleaned free of fat and weighed. Serum was prepared by centrifugation of the collected blood at 3000 r.p. m. for 30 min and stored at − 40 °C until testosterone assay. Total and free serum testosterone concentrations were determined by electrochemiluminescence immunoassay technology using the Elecsys Testosterone Assay Kits (Roche Diagnostics, Mannheim, Germany) in a fully automated Elecsys 2010 analyzer (Hitachi, Tokyo, Japan).
Female rats of the same strain were also used in this study. Each female was brought into estrus by sequential subcutaneous injections of 50 μg estradiol benzoate (Intervet International B.V., Holland) and 1 mg progesterone (Schering AG, Germany) 48 and 4 h before the mating tests, respectively. The females were screened with non-experimental males, and the ones that showed good sexual receptivity (solicitation and lordosis in response to mounting) were selected for the sexual behavior test.
Swimming exercise Rats in the swimming groups were trained to swim in a 1.5 × 1.0 × 0.8 m rectangular tank half-filled with water at ~ 34–36 °C. Only four to six rats were allowed in the tank simultaneously. The swimming protocol was conducted in two adaptation phases and one training phase. The first adaptation phase extended for 5 days. The rats were placed for 15 min in the swimming tank with a water depth of 0.1 m. The water depth was increased by 0.1 m every day until it reached 0.4 m on the fourth day. On the fifth day, the rats were allowed to rest. The second adaptation phase period followed the first adaptation phase and also extended for 5 days. The rats were allowed to swim for 15 min on the first day of the second adaptation phase. The swimming period was extended by 15 min every day until the rats swam for a total of 1 h. On the fifth day, the rats were allowed to rest. This was followed by the training phase, which consisted of swimming for 1 h per session. The rats were trained on alternate days over a period of 30 days (15 sessions in total). All swimming sessions were supervised to avoid floating and/or clinging of the rats.
Statistical analysis Samples in the two groups (swimming and sedentary) were first subjected to Levene’s test for homogeneity of variance. Data were analyzed using an independent samples t-test. Statistical significance was evaluated at the 0.05 significance level for the two-sided tests.
RESULTS Effects of swimming on the copulatory behavior of highly active rats Highly active rats that were exposed to swimming exercise showed a significant (P o 0.05) reduction in mount frequency and intromission latency compared with sedentary highly active rats (Table 1). Moreover, the number of intromissions significantly (P o 0.05) increased in the sexually highly active rats subjected to swimming than in sedentary rats. Further, a significant (P o0.01) improvement in the intromission ratio was detected in the swimming highly active rats (86%) compared with the sedentary highly active rats (71%). The percentage of ejaculating rats was 100% in both swimming and sedentary groups of sexually highly active rats, within 30 min.
Copulatory behavior test The sexual behavior of male rats was monitored in a sound-attenuated room by two trained observers blinded to the experimental design. Ten male rats from each group were subjected to the mating test. The test was performed 24 h after the last swimming session and during the dark phase of the light/dark cycle. A single male rat was placed in a rectangular Plexiglas observation chamber (0.45 × 0.4 × 0.3 m height) and allowed to acclimate for 5 min. Next, a sexually receptive female was introduced into the chamber. The following parameters of copulatory behavior were measured as described previously:24 (1) mount latency, time from introduction of female until first mount, (2) intromission latency, time from introduction of female until first intromission (vaginal penetration), (3) mount frequency, number of mounts preceding ejaculation, (4) intromission frequency, number of intromissions preceding ejaculation, (5) ejaculation latency, time from first intromission until ejaculation and (6) post-ejaculatory interval, time from ejaculation until next intromission. Further, the following parameters were calculated on the basis of data for parameters 1 − 6: (7) intromission ratio, measure of intromissive success calculated as intromission frequency/(mount frequency+intromission frequency) and (8) inter-intromission interval, average interval between successive intromissions calculated as ejaculation latency/intromission frequency. The percentage of ejaculating rats was calculated based on the ratio of the number of rats that achieved ejaculation within 30 min to the total number of rats assessed in that group. Tests were normally ended after the first post-ejaculatory intromission.
Effects of swimming on the copulatory behavior of moderately active rats The mount latency, mount frequency and intromission latency were significantly (P o 0.01) reduced in the sexually moderately active rats exposed to swimming exercise compared with the sedentary moderately active rats (Table 2). In addition, a greater reduction in the ejaculatory latency was observed in the swimming rats compared with sedentary rats but the difference was not statistically significant (P = 0.07). A significant (P o0.01) improvement in the intromission ratio was detected in the swimming rats (83%) compared with the sedentary rats (61%). The percentage of ejaculating moderately active rats increased significantly (P o 0.05) from 60% in the sedentary group to 100% in the swimming group (Figure 1). Serum testosterone concentrations The total serum testosterone concentrations were significantly higher in the sexually highly active (P o 0.01) and moderately active (P o0.05) male rats exposed to swimming exercise than in their respective sedentary controls (Figure 2a). A similar trend was noted for the free serum testosterone concentrations (Figure 2b).
Testosterone and weight assays Six male rats from each group that were not submitted to mating tests were used to analyze total and free serum testosterone levels, and total body weight, and weight of the internal reproductive organs. The rats were weighed and then killed by ethyl ether. Subsequently, blood was obtained by cardiac puncture and collected into centrifuge tubes. Reproductive
Table 1.
Comparison of the copulatory behavior of highly active male rats by activity group
Group Sedentary Swimming
ML (s)
MF
IL(s)
IF
EL(s)
PEI(s)
IR
III(s)
74.36 ± 11.25 64.00 ± 10.89
4.36 ± 0.54 2.58 ± 0.40*
124.55 ± 14.14 81.92 ± 11.29*
11.00 ± 1.50 16.08 ± 1.60*
545.20 ± 97.41 636.92 ± 76.66
395.70 ± 20.76 389.83 ± 19.81
0.71 ± 0.02 0.86 ± 0.02**
42.38 ± 4.94 39.42 ± 3.14
Abbreviations: ML, mount latency; EL, ejaculatory latency; IF, intromission frequency; IL, intromission latency; IR, intromission ratio; III, inter-intromission interval; MF, mount frequency; PEI, post-ejaculatory interval. Each value represents the mean ± s.e. obtained for ten rats per group. *P o0.05, **Po0.01 compared with the sedentary group (t-test).
International Journal of Impotence Research (2014), 1 – 5
© 2014 Macmillan Publishers Limited
Effect of swimming on copulatory behavior MZ Allouh
3 Table 2.
Comparison of the copulatory behavior of moderately active male rats by activity group
Group Sedentary Swimming
ML (s)
MF
IL (s)
IF
EL (s)
PEI (s)
IR
III (s)
111.45 ± 11.97 64.25 ± 6.01**
8.83 ± 0.75 2.64 ± 0.45**
192.50 ± 21.43 88.27 ± 11.17**
14.08 ± 1.55 12.91 ± 1.44
1095.89 ± 144.33 779.82 ± 96.92
432.89 ± 20.80 398.18 ± 15.78
0.61 ± 0.02 0.83 ± 0.02**
72.37 ± 4.68 61.15 ± 5.13
Abbreviations: EL, ejaculatory latency; IF, intromission frequency; III, inter-intromission interval; IL, intromission latency; IR, intromission ratio; ML, mount latency; MF, mount frequency; PEI, post-ejaculatory interval. Each value represents the mean ± s.e. obtained for ten rats per group. **Po0.01 compared with the sedentary group (t-test).
Figure 1. Percentage of ejaculating moderately active male rats after 30-day swimming activity. *P o0.05 compared with sedentary group (Fisher’s exact test).
Total body weight and weight of internal reproductive organs There were no significant differences in body weights between swimming and sedentary rats in the highly active (313.57 versus 312.14 g) as well as moderately active (314.29 versus 310.00 g) groups (all, P40.05). Similarly weights of reproductive organs were comparable (all, P40.05). DISCUSSION To the best of our knowledge, this is the first study to investigate the influence of swimming activity on the copulatory behavior of adult male rats. Swimming exercise was found to significantly enhance the copulatory behavior of sexually highly active and moderately active male rats. This was accompanied by significant improvements in the total and free serum testosterone concentrations of these rats. The improvement in sexual performance of swimming highly active rats was evidenced by reduced intromission latency and increased intromission frequency. A reduction in intromission latency signifies efficient penile erection and coordinated activity of the penile muscles, while an increase in intromission frequency reflects efficient activation of the ejaculatory reflexes.25 The intromission ratio was also improved in these animals. The intromission ratio signifies the facilitation of erection and penile muscle contraction and is considered an unbiased measurement of potency.24 Moreover, the swimming exercise significantly stimulated sexual desire in moderately active male rats. This was evidenced by reduced mount latency in these rats at the end of the swimming period. Mount latency is a measurement of and inversely proportional to sexual motivation.24 Further, a significant © 2014 Macmillan Publishers Limited
Figure 2. Influence of swimming exercise on total (a) and free (b) serum testosterone levels in sexually highly active and moderately active male rats. Each column represents the mean ± s.e. of six rats/ group. *P o0.05, **P o0.01 compared with sedentary group (t-test).
improvement in sexual performance was evidenced by reduced intromission latency and increased intromission ratio and number of ejaculating animals in the swimming moderately active group compared with the sedentary moderately active group. Copulatory tests, which lasted for 30 min, were completed at the end of the first post ejaculatory interval. The ejaculation frequency (the number of ejaculations in the 30 min copulation test) was beyond the scope of the present study and thus, not measured. However, we believe that the significant increase observed in the percentage of ejaculating rats in the moderately International Journal of Impotence Research (2014), 1 – 5
Effect of swimming on copulatory behavior MZ Allouh
4 active group (60 versus 100% in sedentary and swimming rats, respectively) could be considered as an indication of improvement in ejaculation frequency. There was significant increase in serum testosterone concentrations in the rats subjected to swimming compared with the sedentary rats. Ozbek et al.21 found that regular exercise significantly increased testosterone levels in rats with age-associated decrease in penile neurotransmitter activity. Similarly, in a study on male SAMP8 mice –a senescence-accelerated strain– wherein the mice were maintained as sedentary or subjected to daily 15-min periods of swimming exercise, a significantly greater serum testosterone level was observed in the exercise group.26 A similar trend is observed in humans, with physical activity being associated with higher testosterone levels,27,28 whereas immobilization being associated with lower testosterone levels.29,30 A previous study found no difference in total serum testosterone concentrations between sedentary and swimming rats.31 This could be attributed to the difference in the number and duration of swimming sessions between this study and our study. While rats were allowed to swim for ten sessions (40 min each) in the previous study, they were allowed to swim for 15 sessions (1 h each) in our study. Moreover, any swimming-induced stress, which might be associated with lower serum testosterone concentration, was avoided in our study through the application of two adaptation periods before starting the swimming exercise. The first period focused on adapting the rats to water by incrementally increasing the water depth, and the second period allowed the rats to adapt to the swimming exercise by gradually increasing the period of swimming session to 1 h. In addition, a 48-h resting period was permitted between swimming sessions to allow sufficient recovery in the rats. Ensuring that the moderate swimming test protocol used in our study was designed such that stress levels were controlled was a vital element of our study, as high intensity swimming exercises are known to increase corticosterone/cortisol levels and consequently decrease testosterone levels in both rats32,33 and humans.34 Testosterone is the primary male sex hormone responsible for enhancing sexual desire and improving erectile function.24 Nevertheless, enhanced sexual behavior in male rats subjected to swimming may not be solely attributed to the significant increase in serum testosterone concentrations in these rats compared with the sedentary counterparts. In fact, previous research suggests no relationship between the circulating plasma testosterone levels and sexual behavior (intromission latency, postejaculatory interval and intromission frequency) in intact as well as castrated rats.35 Similarly, in men there is slim evidence of any correlation between serum testosterone and sexual activity when testosterone concentrations are within or above the lower physiological range.36 The effect of swimming on improving sexual activity is in agreement with research that shows that physical activity usually ameliorates sexual activity, when impaired by a sedentary life style. Interestingly, a previous study demonstrated that intermittent swimming exercise is more efficient than continuous swimming exercise in decreasing adiposity in rats fed a high-fat diet.37 In addition, a high body mass index (BMI) and low physical activity have been found to independently increase the risk of ED.38 Though the weights of rats in the present study were comparable between swimming and sedentary groups, the possibility of a decrease in BMI following intermittent swimming exercise resulting in improved copulatory efficacy cannot be entirely ignored. The results of a meta-analysis of populationbased studies suggested protective effect of exercise on ED, with an evident dose–repose relationship.10 In addition, a recent metaanalysis also concluded that lifestyle modification and treatment of cardiovascular risk factors are effective in improving sexual function in men with ED.39 Furthermore, results of the health professionals follow-up study showed that higher physical activity International Journal of Impotence Research (2014), 1 – 5
and lower BMI decrease the risk of ED after adjusting for all relevant factors.40 Several exercise-associated plausible mechanisms like decrease in adiposity and increased cardiovascular performance may support the beneficial effect of swimming on sexual behavior as seen in the present study. In conclusion, this study showed that moderate swimming exercise significantly improved copulation in highly active and moderately active male rats. Swimming was also associated with increase in serum testosterone levels in both these groups. This suggests that swimming activity might be a complementary therapy to restore testosterone levels and enhance male sexual libido and performance. However, future investigations that include different swimming trials are required to verify the efficacy of swimming activity in improving sexual function in humans. CONFLICT OF INTEREST The author declares no conflict of interest.
ACKNOWLEDGMENTS This study was funded by a grant awarded to the author from the Deanship of Research at JUST (174/2012).
REFERENCES 1 NIH. NIH Consensus Conference. Impotence. NIH Consensus Development Panel on Impotence. JAMA 1993; 270: 83–90. 2 Laumann EO, Paik A, Rosen RC. Sexual dysfunction in the United States: prevalence and predictors. JAMA 1999; 281: 537–544. 3 Johannes CB, Araujo AB, Feldman HA, Derby CA, Kleinman KP, McKinlay JB. Incidence of erectile dysfunction in men 40 to 69 years old: longitudinal results from the Massachusetts male aging study. J Urol 2000; 163: 460–463. 4 Ayta IA, McKinlay JB, Krane RJ. The likely worldwide increase in erectile dysfunction between 1995 and 2025 and some possible policy consequences. BJU Int 1999; 84: 50–56. 5 Selvin E, Burnett AL, Platz EA. Prevalence and risk factors for erectile dysfunction in the US. Am J Med 2007; 120: 151–157. 6 Weber MF, Smith DP, O'Connell DL, Patel MI, de Souza PL, Sitas F et al. Risk factors for erectile dysfunction in a cohort of 108 477 Australian men. Med J Aust 2013; 199: 107–111. 7 Booth FW, Roberts CK, Laye MJ. Lack of exercise is a major cause of chronic diseases. Compr Physiol 2012; 2: 1143–1211. 8 Stampfer MJ, Hu FB, Manson JE, Rimm EB, Willett WC. Primary prevention of coronary heart disease in women through diet and lifestyle. N Engl J Med 2000; 343: 16–22. 9 Hu FB, Manson JE, Stampfer MJ, Colditz G, Liu S, Solomon CG et al. Diet, lifestyle, and the risk of type 2 diabetes mellitus in women. N Engl J Med. 2001; 345: 790–797. 10 Cheng JY, Ng EM, Ko JS, Chen RY. Physical activity and erectile dysfunction: metaanalysis of population-based studies. Int J Impot Res 2007; 19: 245–252. 11 Goh VH, Tong TY. The moderating impact of lifestyle factors on sex steroids, sexual activities and aging in Asian men. Asian J Androl 2011; 13: 596–604. 12 Hsiao W, Shrewsberry AB, Moses KA, Johnson TV, Cai AW, Stuhldreher P et al. Exercise is associated with better erectile function in men under 40 as evaluated by the International Index of Erectile Function. J Sex Med 2012; 9: 524–530. 13 Batterham SI, Heywood S, Keating JL. Systematic review and meta-analysis comparing land and aquatic exercise for people with hip or knee arthritis on function, mobility and other health outcomes. BMC Musculoskelet Disord 2011; 12: 123. 14 Keller JJ, Lin HC. A population-based study on the association between rheumatoid arthritis and erectile dysfunction. Ann Rheum Dis 2012; 71: 1102–1103. 15 Corona G, Mannucci E, Mansani R, Petrone L, Bartolini M, Giommi R et al. Aging and pathogenesis of erectile dysfunction. Int J Impot Res 2004; 16: 395–402. 16 Kregel KC (ed). APS Resource Book for the Design of Animal Exercise Protocols. The American Physiological Society: Bethesda, MD, USA, 2006. 17 Jette M, Sidney K, Blumchen G. Metabolic equivalents (METS) in exercise testing, exercise prescription, and evaluation of functional capacity. Clin Cardiol 1990; 13: 555–565.
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5 18 Almeida PW, Gomes-Filho A, Ferreira AJ, Rodrigues CE, Dias-Peixoto MF, Russo RC et al. Swim training suppresses tumor growth in mice. J Appl Physiol 2009; 107: 261–265. 19 Kim K, Chung E, Kim CJ, Lee S. Swimming exercise during pregnancy alleviates pregnancy-associated long-term memory impairment. Physiol Behav 2012; 107: 82–86. 20 Shen J, Fox LE, Cheng J. Swim therapy reduces mechanical allodynia and thermal hyperalgesia induced by chronic constriction nerve injury in rats. Pain Med 2013; 14: 516–525. 21 Ozbek E, Tasci AI, Ilbey YO, Simsek A, Somay A, Metin G. The effect of regular exercise on penile nitric oxide synthase expression in rats. Int J Androl 2010; 33: 623–628. 22 Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals, 8th edn. The National Academies Press: Washington, DC, 2011. 23 Zanoli P, Zavatti M, Montanari C, Baraldi M. Influence of Eurycoma longifolia on the copulatory activity of sexually sluggish and impotent male rats. J Ethnopharmacol 2009; 126: 308–313. 24 Allouh MZ, Khouri NA, Daradka HM, Kaddumi EG. Orchis anatolica root ingestion improves sexual motivation and performance in male rats. J Complement Integr Med 2010; 7, ISSN (Online) 1553–3840. 25 Agmo A. Male rat sexual behavior. Brain Res Brain Res Protoc 1997; 1: 203–209. 26 Zhao X, Bian Y, Sun Y, Li L, Wang L, Zhao C et al. Effects of moderate exercise over different phases on age-related physiological dysfunction in testes of SAMP8 mice. Exp Gerontol 2013; 48: 869–880. 27 Grandys M, Majerczak J, Duda K, Zapart-Bukowska J, Kulpa J, Zoladz JA. Endurance training of moderate intensity increases testosterone concentration in young, healthy men. Int J Sports Med 2009; 30: 489–495. 28 Budde H, Pietrassyk-Kendziorra S, Bohm S, Voelcker-Rehage C. Hormonal responses to physical and cognitive stress in a school setting. Neurosci Lett 2010; 474: 131–134. 29 Dong Q, Salva A, Sottas CM, Niu E, Holmes M, Hardy MP. Rapid glucocorticoid mediation of suppressed testosterone biosynthesis in male mice subjected to immobilization stress. J Androl 2004; 25: 973–981.
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30 Aziz NM, Ragy MM, Gayyed MF. Effect of acute immobilization stress with or without a heme oxygenase inducer on testicular structure and function in male albino rats. J Basic Clin Physiol Pharmacol 2013; 24: 255–262. 31 Allouh MZ. Effect of Ferula hermonis root extract on rat skeletal muscle adaptation to exercise. Exp Biol Med 2011; 236: 1373–1378. 32 Petit-Demouliere B, Chenu F, Bourin M. Forced swimming test in mice: a review of antidepressant activity. Psychopharmacology 2005; 177: 245–255. 33 Kim SH, Kim EK, Choi EJ. High-intensity swimming exercise increases dust mite extract and 1-Chloro-2,4-dinitrobenzene-derived atopic dermatitis in BALB/c mice. Inflammation 2014; 37: 1179–1185. 34 Tyndall GL, Kobe RW, Houmard JA. Cortisol, testosterone, and insulin action during intense swimming training in humans. Eur J Appl Physiol Occup Physiol 1996; 73: 61–65. 35 Damassa DA, Smith ER, Tennent B, Davidson JM. The relationship between circulating testosterone levels and male sexual behavior in rats. Horm Behav 1977; 8: 275–286. 36 Boloña ER, Uraga MV, Haddad RM, Tracz MJ, Sideras K, Kennedy CC et al. Testosterone use in men with sexual dysfunction: a systematic review and meta-analysis of randomized placebo-controlled trials. Mayo Clin Proc 2007; 82: 20–28. 37 Sene-Fiorese M, Duarte FO, Scarmagnani FR, Cheik NC, Manzoni MS, Nonaka KO et al. Efficiency of intermittent exercise on adiposity and fatty liver in rats fed with high-fat diet. Obesity 2008; 16: 2217–2222. 38 Cheng JY, Ng EM. Body mass index, physical activity and erectile dysfunction: an U-shaped relationship from population-based study. Int J Obes 2007; 31: 1571–1578. 39 Gupta BP, Murad MH, Clifton MM, Prokop L, Nehra A, Kopecky SL. The effect of lifestyle modification and cardiovascular risk factor reduction on erectile dysfunction: a systematic review and meta-analysis. Arch Intern Med 2011; 171: 1797–1803. 40 Bacon CG, Mittleman MA, Kawachi I, Giovannucci E, Glasser DB, Rimm EB. Sexual function in men older than 50 years of age: results from the health professionals follow-up study. Ann Intern Med 2003; 139: 161–168.
International Journal of Impotence Research (2014), 1 – 5
ORIGINAL ARTICLE
Effects of swimming activity on the copulatory behavior of sexually active male rats MZ Allouh Physical activity has long been associated with better sexual function. This study investigated the effects of moderate swimming exercise on the copulatory behavior of sexually potent male rats. Two sets of sexually potent male rats –highly active and moderately active– were identified depending on baseline sexual activity. Each of the two sets of rats was further randomly divided into two groups (swimming and sedentary). There were 16 rats in each of the four study groups (highly active swimming, highly active sedentary, moderately active swimming and moderately active sedentary). The copulatory behavior parameters and serum testosterone levels were measured and compared between the rats of the swimming and sedentary groups following a month long training period in which rats were made to swim for 1 h every alternate day. Swimming significantly improved the sexual performance of highly active rats, as indicated by increased intromission frequency and intromission ratio, compared with the sedentary controls. Swimming improved both sexual desire and performance, as indicated by reduced mount latency and increased intromission ratio, respectively, in swimming moderately active rats compared with the sedentary moderately active controls. Therefore, swimming activity improves the copulatory behavior of both highly active and moderately active male rats. International Journal of Impotence Research advance online publication, 18 December 2014; doi:10.1038/ijir.2014.42
INTRODUCTION Erectile dysfunction (ED), previously known as sexual impotence, is the inability to achieve or maintain a penile erection that is sufficient for sexual satisfaction.1 This problem adversely affects the quality of life, especially, family and social relationships.2,3 The worldwide prevalence of ED in men in 1995 was estimated to exceed 152 million, and this number could reach 322 million by 2025.4 Several risk factors, including, age, smoking, malnutrition, obesity and physical inactivity, have been related to this high incidence of ED.3,5,6 Physical inactivity can have a detrimental effect on normal organ functioning and is an important cause of many chronic diseases.7 In contrast, physical activity is known to have a major role in preventing or delaying many chronic illnesses such as obesity, diabetes and cardiovascular diseases.7–9 In addition, several studies have suggested that an increase in physical activity may improve sexual function.10–12 Swimming is a good alternative to land-based exercises in older patients with conditions such as arthritis,13 who are known to be at a higher risk of ED.14,15 Interestingly, oxygen utilization data suggest that the energy use during swimming in humans is similar to that in rats.16,17 Among the various exercise modalities (treadmill, voluntary wheel running and swimming) used for studying the physiological response to exercise in rat models, swimming has an advantage as it utilizes a natural ability of rats.16 Several studies on animal models have reported benefits of swimming exercise on preventing tumor growth and progression, pregnancy-associated longterm memory impairment and neuropathic pain caused by nerve injury;18–20 however, data on benefits of swimming on ED are scarce. A recent study on rats found that regular exercise may help in improving penile erection by increasing penile neurotransmitter
activity.21 However, to the best of our knowledge, the effects of swimming on male sexual desire and potency have not been assessed using rat models. This study aimed to investigate the direct effects of swimming on the copulatory behavior of adult male rats. The following hypothesis was tested: swimming activity improves the copulatory behavior of both highly active and moderately active male rats. The serum testosterone levels of these animals were also compared. MATERIALS AND METHODS Animals Adult male Sprague–Dawley albino rats (weight: ~ 250 g) bred and raised in the Animal House Unit at Jordan University of Science and Technology (JUST) were used in this study. Approvals for all animal care and experimental procedures were obtained from the Animal Care and Use Committee at JUST and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.22 The rats were maintained under controlled temperature of 21 ± 1 °C with a 12 h light and 12 h darkness schedule (lights on between 0600 and 1800 hours). Food and water were provided ad libitum. Acclimatization time of 2 weeks was provided before beginning the experiments.
Experimental design Rats were subjected to seven pre-experimental mating tests with sexually receptive females to make them sexually experienced. The rats that achieved ejaculation in the last three tests were considered highly active, those that achieved ejaculation in one or two of the last three tests were considered sexually moderately active and those that failed to achieve ejaculation in the last three tests were considered sexually impotent and excluded from the study.23 Both highly active and moderately active rats were randomly divided into two groups: sedentary and swimming. Each group consisted of 16 male rats.
Department of Anatomy, Faculty of Medicine, Jordan University of Science & Technology, Irbid, Jordan. Correspondence: Dr MZ Allouh, Department of Anatomy, Faculty of Medicine, Jordan University of Science & Technology, P.O. Box: 3030, Irbid 22110, Jordan. E-mail: [email protected] Received 9 May 2014; revised 23 July 2014; accepted 18 October 2014
Effect of swimming on copulatory behavior MZ Allouh
2 organs (testes, epididymides, seminal vesicles, prostate and vasa deferentia) were dissected out, cleaned free of fat and weighed. Serum was prepared by centrifugation of the collected blood at 3000 r.p. m. for 30 min and stored at − 40 °C until testosterone assay. Total and free serum testosterone concentrations were determined by electrochemiluminescence immunoassay technology using the Elecsys Testosterone Assay Kits (Roche Diagnostics, Mannheim, Germany) in a fully automated Elecsys 2010 analyzer (Hitachi, Tokyo, Japan).
Female rats of the same strain were also used in this study. Each female was brought into estrus by sequential subcutaneous injections of 50 μg estradiol benzoate (Intervet International B.V., Holland) and 1 mg progesterone (Schering AG, Germany) 48 and 4 h before the mating tests, respectively. The females were screened with non-experimental males, and the ones that showed good sexual receptivity (solicitation and lordosis in response to mounting) were selected for the sexual behavior test.
Swimming exercise Rats in the swimming groups were trained to swim in a 1.5 × 1.0 × 0.8 m rectangular tank half-filled with water at ~ 34–36 °C. Only four to six rats were allowed in the tank simultaneously. The swimming protocol was conducted in two adaptation phases and one training phase. The first adaptation phase extended for 5 days. The rats were placed for 15 min in the swimming tank with a water depth of 0.1 m. The water depth was increased by 0.1 m every day until it reached 0.4 m on the fourth day. On the fifth day, the rats were allowed to rest. The second adaptation phase period followed the first adaptation phase and also extended for 5 days. The rats were allowed to swim for 15 min on the first day of the second adaptation phase. The swimming period was extended by 15 min every day until the rats swam for a total of 1 h. On the fifth day, the rats were allowed to rest. This was followed by the training phase, which consisted of swimming for 1 h per session. The rats were trained on alternate days over a period of 30 days (15 sessions in total). All swimming sessions were supervised to avoid floating and/or clinging of the rats.
Statistical analysis Samples in the two groups (swimming and sedentary) were first subjected to Levene’s test for homogeneity of variance. Data were analyzed using an independent samples t-test. Statistical significance was evaluated at the 0.05 significance level for the two-sided tests.
RESULTS Effects of swimming on the copulatory behavior of highly active rats Highly active rats that were exposed to swimming exercise showed a significant (P o 0.05) reduction in mount frequency and intromission latency compared with sedentary highly active rats (Table 1). Moreover, the number of intromissions significantly (P o 0.05) increased in the sexually highly active rats subjected to swimming than in sedentary rats. Further, a significant (P o0.01) improvement in the intromission ratio was detected in the swimming highly active rats (86%) compared with the sedentary highly active rats (71%). The percentage of ejaculating rats was 100% in both swimming and sedentary groups of sexually highly active rats, within 30 min.
Copulatory behavior test The sexual behavior of male rats was monitored in a sound-attenuated room by two trained observers blinded to the experimental design. Ten male rats from each group were subjected to the mating test. The test was performed 24 h after the last swimming session and during the dark phase of the light/dark cycle. A single male rat was placed in a rectangular Plexiglas observation chamber (0.45 × 0.4 × 0.3 m height) and allowed to acclimate for 5 min. Next, a sexually receptive female was introduced into the chamber. The following parameters of copulatory behavior were measured as described previously:24 (1) mount latency, time from introduction of female until first mount, (2) intromission latency, time from introduction of female until first intromission (vaginal penetration), (3) mount frequency, number of mounts preceding ejaculation, (4) intromission frequency, number of intromissions preceding ejaculation, (5) ejaculation latency, time from first intromission until ejaculation and (6) post-ejaculatory interval, time from ejaculation until next intromission. Further, the following parameters were calculated on the basis of data for parameters 1 − 6: (7) intromission ratio, measure of intromissive success calculated as intromission frequency/(mount frequency+intromission frequency) and (8) inter-intromission interval, average interval between successive intromissions calculated as ejaculation latency/intromission frequency. The percentage of ejaculating rats was calculated based on the ratio of the number of rats that achieved ejaculation within 30 min to the total number of rats assessed in that group. Tests were normally ended after the first post-ejaculatory intromission.
Effects of swimming on the copulatory behavior of moderately active rats The mount latency, mount frequency and intromission latency were significantly (P o 0.01) reduced in the sexually moderately active rats exposed to swimming exercise compared with the sedentary moderately active rats (Table 2). In addition, a greater reduction in the ejaculatory latency was observed in the swimming rats compared with sedentary rats but the difference was not statistically significant (P = 0.07). A significant (P o0.01) improvement in the intromission ratio was detected in the swimming rats (83%) compared with the sedentary rats (61%). The percentage of ejaculating moderately active rats increased significantly (P o 0.05) from 60% in the sedentary group to 100% in the swimming group (Figure 1). Serum testosterone concentrations The total serum testosterone concentrations were significantly higher in the sexually highly active (P o 0.01) and moderately active (P o0.05) male rats exposed to swimming exercise than in their respective sedentary controls (Figure 2a). A similar trend was noted for the free serum testosterone concentrations (Figure 2b).
Testosterone and weight assays Six male rats from each group that were not submitted to mating tests were used to analyze total and free serum testosterone levels, and total body weight, and weight of the internal reproductive organs. The rats were weighed and then killed by ethyl ether. Subsequently, blood was obtained by cardiac puncture and collected into centrifuge tubes. Reproductive
Table 1.
Comparison of the copulatory behavior of highly active male rats by activity group
Group Sedentary Swimming
ML (s)
MF
IL(s)
IF
EL(s)
PEI(s)
IR
III(s)
74.36 ± 11.25 64.00 ± 10.89
4.36 ± 0.54 2.58 ± 0.40*
124.55 ± 14.14 81.92 ± 11.29*
11.00 ± 1.50 16.08 ± 1.60*
545.20 ± 97.41 636.92 ± 76.66
395.70 ± 20.76 389.83 ± 19.81
0.71 ± 0.02 0.86 ± 0.02**
42.38 ± 4.94 39.42 ± 3.14
Abbreviations: ML, mount latency; EL, ejaculatory latency; IF, intromission frequency; IL, intromission latency; IR, intromission ratio; III, inter-intromission interval; MF, mount frequency; PEI, post-ejaculatory interval. Each value represents the mean ± s.e. obtained for ten rats per group. *P o0.05, **Po0.01 compared with the sedentary group (t-test).
International Journal of Impotence Research (2014), 1 – 5
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Effect of swimming on copulatory behavior MZ Allouh
3 Table 2.
Comparison of the copulatory behavior of moderately active male rats by activity group
Group Sedentary Swimming
ML (s)
MF
IL (s)
IF
EL (s)
PEI (s)
IR
III (s)
111.45 ± 11.97 64.25 ± 6.01**
8.83 ± 0.75 2.64 ± 0.45**
192.50 ± 21.43 88.27 ± 11.17**
14.08 ± 1.55 12.91 ± 1.44
1095.89 ± 144.33 779.82 ± 96.92
432.89 ± 20.80 398.18 ± 15.78
0.61 ± 0.02 0.83 ± 0.02**
72.37 ± 4.68 61.15 ± 5.13
Abbreviations: EL, ejaculatory latency; IF, intromission frequency; III, inter-intromission interval; IL, intromission latency; IR, intromission ratio; ML, mount latency; MF, mount frequency; PEI, post-ejaculatory interval. Each value represents the mean ± s.e. obtained for ten rats per group. **Po0.01 compared with the sedentary group (t-test).
Figure 1. Percentage of ejaculating moderately active male rats after 30-day swimming activity. *P o0.05 compared with sedentary group (Fisher’s exact test).
Total body weight and weight of internal reproductive organs There were no significant differences in body weights between swimming and sedentary rats in the highly active (313.57 versus 312.14 g) as well as moderately active (314.29 versus 310.00 g) groups (all, P40.05). Similarly weights of reproductive organs were comparable (all, P40.05). DISCUSSION To the best of our knowledge, this is the first study to investigate the influence of swimming activity on the copulatory behavior of adult male rats. Swimming exercise was found to significantly enhance the copulatory behavior of sexually highly active and moderately active male rats. This was accompanied by significant improvements in the total and free serum testosterone concentrations of these rats. The improvement in sexual performance of swimming highly active rats was evidenced by reduced intromission latency and increased intromission frequency. A reduction in intromission latency signifies efficient penile erection and coordinated activity of the penile muscles, while an increase in intromission frequency reflects efficient activation of the ejaculatory reflexes.25 The intromission ratio was also improved in these animals. The intromission ratio signifies the facilitation of erection and penile muscle contraction and is considered an unbiased measurement of potency.24 Moreover, the swimming exercise significantly stimulated sexual desire in moderately active male rats. This was evidenced by reduced mount latency in these rats at the end of the swimming period. Mount latency is a measurement of and inversely proportional to sexual motivation.24 Further, a significant © 2014 Macmillan Publishers Limited
Figure 2. Influence of swimming exercise on total (a) and free (b) serum testosterone levels in sexually highly active and moderately active male rats. Each column represents the mean ± s.e. of six rats/ group. *P o0.05, **P o0.01 compared with sedentary group (t-test).
improvement in sexual performance was evidenced by reduced intromission latency and increased intromission ratio and number of ejaculating animals in the swimming moderately active group compared with the sedentary moderately active group. Copulatory tests, which lasted for 30 min, were completed at the end of the first post ejaculatory interval. The ejaculation frequency (the number of ejaculations in the 30 min copulation test) was beyond the scope of the present study and thus, not measured. However, we believe that the significant increase observed in the percentage of ejaculating rats in the moderately International Journal of Impotence Research (2014), 1 – 5
Effect of swimming on copulatory behavior MZ Allouh
4 active group (60 versus 100% in sedentary and swimming rats, respectively) could be considered as an indication of improvement in ejaculation frequency. There was significant increase in serum testosterone concentrations in the rats subjected to swimming compared with the sedentary rats. Ozbek et al.21 found that regular exercise significantly increased testosterone levels in rats with age-associated decrease in penile neurotransmitter activity. Similarly, in a study on male SAMP8 mice –a senescence-accelerated strain– wherein the mice were maintained as sedentary or subjected to daily 15-min periods of swimming exercise, a significantly greater serum testosterone level was observed in the exercise group.26 A similar trend is observed in humans, with physical activity being associated with higher testosterone levels,27,28 whereas immobilization being associated with lower testosterone levels.29,30 A previous study found no difference in total serum testosterone concentrations between sedentary and swimming rats.31 This could be attributed to the difference in the number and duration of swimming sessions between this study and our study. While rats were allowed to swim for ten sessions (40 min each) in the previous study, they were allowed to swim for 15 sessions (1 h each) in our study. Moreover, any swimming-induced stress, which might be associated with lower serum testosterone concentration, was avoided in our study through the application of two adaptation periods before starting the swimming exercise. The first period focused on adapting the rats to water by incrementally increasing the water depth, and the second period allowed the rats to adapt to the swimming exercise by gradually increasing the period of swimming session to 1 h. In addition, a 48-h resting period was permitted between swimming sessions to allow sufficient recovery in the rats. Ensuring that the moderate swimming test protocol used in our study was designed such that stress levels were controlled was a vital element of our study, as high intensity swimming exercises are known to increase corticosterone/cortisol levels and consequently decrease testosterone levels in both rats32,33 and humans.34 Testosterone is the primary male sex hormone responsible for enhancing sexual desire and improving erectile function.24 Nevertheless, enhanced sexual behavior in male rats subjected to swimming may not be solely attributed to the significant increase in serum testosterone concentrations in these rats compared with the sedentary counterparts. In fact, previous research suggests no relationship between the circulating plasma testosterone levels and sexual behavior (intromission latency, postejaculatory interval and intromission frequency) in intact as well as castrated rats.35 Similarly, in men there is slim evidence of any correlation between serum testosterone and sexual activity when testosterone concentrations are within or above the lower physiological range.36 The effect of swimming on improving sexual activity is in agreement with research that shows that physical activity usually ameliorates sexual activity, when impaired by a sedentary life style. Interestingly, a previous study demonstrated that intermittent swimming exercise is more efficient than continuous swimming exercise in decreasing adiposity in rats fed a high-fat diet.37 In addition, a high body mass index (BMI) and low physical activity have been found to independently increase the risk of ED.38 Though the weights of rats in the present study were comparable between swimming and sedentary groups, the possibility of a decrease in BMI following intermittent swimming exercise resulting in improved copulatory efficacy cannot be entirely ignored. The results of a meta-analysis of populationbased studies suggested protective effect of exercise on ED, with an evident dose–repose relationship.10 In addition, a recent metaanalysis also concluded that lifestyle modification and treatment of cardiovascular risk factors are effective in improving sexual function in men with ED.39 Furthermore, results of the health professionals follow-up study showed that higher physical activity International Journal of Impotence Research (2014), 1 – 5
and lower BMI decrease the risk of ED after adjusting for all relevant factors.40 Several exercise-associated plausible mechanisms like decrease in adiposity and increased cardiovascular performance may support the beneficial effect of swimming on sexual behavior as seen in the present study. In conclusion, this study showed that moderate swimming exercise significantly improved copulation in highly active and moderately active male rats. Swimming was also associated with increase in serum testosterone levels in both these groups. This suggests that swimming activity might be a complementary therapy to restore testosterone levels and enhance male sexual libido and performance. However, future investigations that include different swimming trials are required to verify the efficacy of swimming activity in improving sexual function in humans. CONFLICT OF INTEREST The author declares no conflict of interest.
ACKNOWLEDGMENTS This study was funded by a grant awarded to the author from the Deanship of Research at JUST (174/2012).
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