Impact of Four Week Swimming Exercise with Alpha-Tocopherol Supplementation on Fertility Potential in Healthy Rats Abolfazl Kalantari, Abbas Saremi,* Nader Shavandi, Ali Foroutan Nia Purpose: The aim of this study was to evaluate the effect of 4 week intensive swimming exercise and alpha-to- copherol supplementation on testicular oxidative stress and spermatogenesis in rats. Materials and Methods: 40 male rats were randomly assigned to Control (C), Sham (S), Exercise (E) and Exer- cise + supplement (ES) groups. Exercise training performed for 4 weeks (1session/day, 6days/week). Each session included 180 minutes of swimming. In ES group, alpha-ocopherol was injected at a dose of 50 mg/kg/day. 48 hours after last training session, all rats were killed and gonads of them were removed from their body for histological and biochemical assays. All statistical analysis was performed by SPSS 16. P values less than 0.05 were considered as statistically significant. Results: Total testicular antioxidant capacity increased significantly in E (P = .003) and ES groups (P = .001) whereas there was no significant difference between C and E group in testicle Malondialdehyde (a lipid peroxida- tion marker) level (P = .999) and spermatogenesis quality (P = .381). Testicle Malondialdehyde level decreased (P = .009) and spermatogenesis quality was improved significantly in ES group (P = .001). Conclusion: Alpha-tocopherol supplementation is effective in order to improve spermatogenesis process in ath- letes who exercise with high intensity. Key words: alpha-tocopherol; fertility; free radical; spermatogenesis; swimming Department of exercise physiology, Faculty of Sport Science, Arak University, Arak, Iran. *Correspondence: Department of exercise physiology, Faculty of Sport Science, Arak University, Arak, Iran. Tel: +98 086 32777400. Fax: +98 086 32760104. E-mail: a-saremi@araku.ac.ir. Received April 2017 & Accepted June 2017 INTRODUCTION Nowadays, male infertility is one of the most im-portant medical challenges. Several factors are related to increased risk of infertility such as: reproduc- tive diseases, cancers, genetic anomalies, long-term use of some medications, and oxidative stress.(1-3) Oxidative stress is the result of Reactive Oxygen Species (ROS) or Reactive Nitrogen Species (RNS) overproduction or body antioxidant defense weakness. It has negative effects on sperm quality and motility and also causes sperm DNA damage and lipid peroxidation of sperm membrane.(2) Doing exercise training with high intensi- ty is one of the conditions which can lead to oxidative stress occurrence. It has been reported that intensive ex- ercise develops free radical production and, simultane- ously, reduced spermatogenesis quality and male fertili- ty.(4) Since it is estimated that semen ROS level is higher than normal range in approximately 25% of infertile men,(5) oxidative stress due to intensive exercise can be suggested as a main reason for reduced fertility in high trained men. Some antioxidants like alpha-tocopherol (vitamin E) are strong ROS and RNS detergents and using them may have positive effects on male reproduc- tive function.(6) So in current study, it was assumed that vitamin E intake during intensive training may prevent negative effects of exercise on spermatogenesis process via antioxidant defense improvement. MATERIALS AND METHODS 40 Sperague Dawley rats were selected as subjects. General specifications of the rats are given in Table 1. They were housed at temperature 22 ± 2°c and 12-hour light/dark cycle with full access to food and water, in animal lab of Arak University of Medical Sciences. The subjects were randomly classified into four experimen- tal groups: 1. Control group (C): Rats of this group did not perform any exercise and no substance was injected to them. 2. Exercise group (E): During research, rats of this group were performing aerobic swimming exercise at 10 A.M 3. Exercise + Supplement group (ES): During research, rats of this group were performing aerobic swimming exercise at 10 A.M, after intraperitoneal (I.P.) injection of 50mg/kg vitamin E. 4. Sham group (S): In order to investigate stress of injection and floatation in water, rats of this group were put in water at 10 A.M (15min/day) after injec- tion of normal saline solution. Water depth was 10 cm, therefore they could not swim in it. Health deputy approved codes for working with labo- ratory animals were respected at all stages of the ex- periment. SEXUAL DYSFUNCTION AND ANDROLOGY Vol 14 No 05 September-October 2017 5023 pieces was submersed in Bohen liquid for 24 hours. Af- ter fixation, tissue preparation was done by the follow- ing method: Fixed tissue samples were put in Xylene and then in Paraffin for transparency. So paraffin blocks including samples were produced. 12 sections with 5 micron thickness were prepared from each block. Tis- sue slices were investigated by microscope after paint- ing with Hematoxyline and Eosin (H & E) method and spermatogenesis was scored based on modified Johnsen score.(12) In this classification system, spermatogenesis is rated from1 (only sertoli cell existence in seminifer- ous tubules) to10 (normal condition). Then scores are classified into three groups: Scores of 1–3: poor spermatogenesis Scores of 4–7: moderate spermatogenesis Scores of 8–10: good spermatogenesis. For each sample, all tissue sections were investigated by microscope and an overall score was calculated (Ta- ble 3). Statistical analysis Data from histological and biochemical assessment was collected and Kolmogorov - Smirnov test was applied to check the normality of data distribution. Compari- sons were carried out using one way analysis of vari- ance (ANOVA) followed by post hoc Tukey test. All statistical analysis was performed by SPSS 16. P values less than 0.05 were considered as statistically signifi- cant. RESULTS There was no significant difference between C and S groups in FRAP level (P = .986), MDA level (P = .990) and Spermatogenesis quality (P = .882). Also, there was no significant difference in MDA level of tissue samples between E and C groups (P = .999) But FRAP level of E group was significantly more than C group (P = .003). In ES group, FRAP level were significant- ly higher (P = .001) and MDA level was significantly lower (P = .009) than C group. Spermatogenesis quality of ES group was significantly higher than C group (P = .001) but difference between E and C group was not significant (P = .381). DISCUSSION Large numbers of athletes, especially professional ath- letes, have heavy exercise programs. These programs impose great stresses on their body and consequently, body homeostasis is disturbed. This condition may mistune some organs or systems, for example repro- ductive system. In some studies, defect in male sexual hormones, sperm parameters and reproductive system function have been reported following intensive exer- cises.(13,14) Etiology of these disorders are not clear ex- actly but oxidative stress has been introduced as one Aerobic exercise program Exercise program was aerobic swimming (1 session/ day, 6 days/week) that was performed in two stages: Stage1 (one week preparation): During this stage, rats of E and ES groups swam in a pool (150*50*50 cm) containing water with 32°c temperature . Swimming duration was 60 minutes in first session which was in- creased gradually (20min/session) until reached up to 160 minutes. Stage 2 (main exercise): During this stage, the rats swam in the pool for 4 weeks. Exercise duration was 180 minutes in each session. This program is a mode of intense aerobic training.(7) Providing gonad tissue samples 48 hours after last training session, all rats were anes- thetized with I.P. injection of ketamine (70mg/kg) com- bined with Xylazine (4mg/kg). Then gonads of each rat were removed from its body and one of them was put in Bohen solution for histological assay and the other one was put in an ice container for biochemical analyzing. Evaluation of testicular biochemical parameters Lipid peroxidation of testicles One of the most important productions of lipid perox- idation is Malondialdehyde (MDA).(8) Therefore, its level was determined in tissue samples with Ohkawa method. Results were expressed as nmol/Gkw (Table 2). Details of this procedure have been explained com- pletely in previous studies.(9) Ferric Reducing Antioxidant Power (FRAP) as- say The FRAP test uses antioxidants as reductants. In sam- ples, reductants reduce ferric tripyridyl-triazine com- plex (Fe3+- TPTZ) in stoichiometric excess to a blue ferrous form (Fe2+) with an increase in absorbance at 593 nm.(10) The absorbance values were read at 593 nm immediately and 4 min later using a spectrophotometer UV-1018 (Table 2). Details of this procedure have been explained completely in previous studies.(11) Histological assay Left gonad of each rat was divided into two equal pieces with a longitudinal cutting. In order to fix, each of these Swimming exercise and fertility-Kalantari et al. Table 1. General characteristics of rats. GROUP QUANTITY AGE (month) WEIGHTª (gr) C 10 3-4 250.7 (± 5) S 10 3-4 250.4 (± 5.4) E 10 3-4 250.6 (± 5.3) ES 10 3-4 250.5 (± 5.1) Abbreviations: C, control group; S, sham group; E, exercise group; ES, supplement group. ª Weight of rats is presented as Mean (±SD) GROUP FRAP (µM) P value a MDA(nmol/Gkw) P value b C .76 (± .02) _ .11 (± .01) _ S .76 (± .03) .986 .1 (± .01) .990 E .81 (± .03) .003 .11 (± .01) .999 ES .87 (± .02) .001 .09 (± .01) .009 Abbreviations: C, control group; S, sham group; E, exercise group; ES, supplement group. ª P value for FRAP compared with control group b P value for MDA compared with control group Table 2. Mean (±SD) of biochemical assessment data. GROUP SPERMATOGENESIS SCORE P value a C 8.6 (± .33) _ S 8.5 (± .33) .882 E 8.4 (± .35) .381 ES 9.4 (± .15) .001 Abbreviations: C, control group; S, sham group; E, exercise group; ES, supplement group. ª P value for spermatogenesis score compared with control group Table 3. Mean (±SD) of Spermatogenesis scores. Sexual Dysfunction and Andrology 5024 of the possible mechanisms.(15) Oxidative stress occurs due to overproduction of free radicals or body disabil- ity for decrease their destructive effects optimally.(2) It’s estimated that in normal metabolism state of the body, within oxygen consumption, 2-5 percent of elec- trons are used in free radical generation. Since oxygen consumption increases up to 20 times during intense aerobic exercises, free radical production is developed vigorously in these practices too.(16) Furthermore, exer- cise trainings can lead to overproduction of free radicals through some other pathways such as: energy charge deficit in skeletal muscles, elevated catecholamine se- cretion and oxidation, Nitric-oxide production, visceral ischemia, released Fe union from red blood cells, blood flow reduction and reflux in cells and etc.(17,18) Our body confronts with these free radicals by its antioxidant en- zymes like Superoxide dismutase (SOD), Catalase and Glutathione-S-Transferase (GST). Suitable recovery after exercise sessions increases generation of these en- zymes and therefore elevates body antioxidant capacity in athletes. But following high intensity exercise ses- sions which are repeated over and over, free radical pro- duction may overcome body antioxidant power and ox- idative stress may occur.(19) This condition can impose some destructive effects on spermatogenesis process because testicular and sperm cell membrane are very vulnerable to attack by free radicals since they are rich in polyunsaturated fatty acids. Sperm motility can be af- fected by increased lipid peroxidation and altered mem- brane function. Also, epididymis is the site of sperm capacitation and maturation and also has an essential role in sperm motility. Oxidative stress results harmful effects in the Epididymis. Furthermore, high concen- tration of free radicals causes sperm DNA damage and reduces spermatogenesis quality.(20) However, results of current study showed that 4 weeks of intense aerobic exercise had no destructive effect on spermatogenesis quality in male rats. Our results do not match with some previous studies.(14,21-23) This contradiction is perhaps pertaining to duration of research and shows that body antioxidant system, at least in short term training pro- grams, is probably able to frustrate harmful effects of free radical overproduction. If period of our study was longer, harmful effects might be created. Existence of antioxidants like Selenium, vitamin C and alpha-tocoferol (vitamin E) in diet, enhances body an- tioxidant capacity. Vitamin E is a powerful antioxidant that because of its lipophilic characteristic can locate itself in the interior of cell membrane, react with fatty acid peroxyl radicals and terminate the chain reactions. Therefore it is able to alleviate wrecking functions of free radicals on male reproductive performance.(24) In the current study it was observed that testicular anti- oxidant capacity was elevated, testicle MDA level was decreased and simultaneously, spermatogenesis quality was improved significantly in ES group. These findings confirm beneficial effect of vitamin E supplementation on spermatogenesis process in male athletes and are supported by some previous studies.(25-27) However, it must be noted that free radicals also have some bene- ficial effects on male reproductive system. They have important roles in several processes like sperm matu- ration, Capacitation and the hyper activation. They also control Acrosome reaction and sperm-oocyte fusion.(28) Therefore dose of vitamin E or other antioxidants for supplementation should be selected accurately because over suppressing free radicals may weaken their advan- tageous efficacy. CONCLUSIONS Alpha-tocopherol supplementation is useful to attenuate destructive effects of free radicals on spermatogenesis process and improves male fertility potential in athletes who have intensive exercise programs. ACKNOWLEDGEMENTS We thank to Arak University of Medical Sciences for cooperating with us during laboratory stages of this re- search. CONFLICT OF INTEREST The authors report no conflict of interest. REFERENCES 1. Auger J, Eustache F, Andersen A, et al. Sperm morphological defects related to environment, lifestyle and medical history of 1001 male partners of pregnant women from four European cities. Human reproduction. 2001;16:2710-7. 2. Bansal AK, Bilaspuri G. Impacts of oxidative stress and antioxidants on semen functions. Veterinary Medicine International. 2010;2011. 3. Jungwirth A, Giwercman A, Tournaye H, et al. European Association of Urology guidelines on Male Infertility: the 2012 update. European urology. 2012;62:324-32. 4. Tartibian B, Maleki BH. 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