Effect of L‐ascorbic acid supplementation on testicular oxidative stress and endocrine disorders in mature male rats exposed to intensive swimming exercise

Abstract

In order to investigate the ameliorative potential of L‐ascorbic acid on intensive swimming exercise induced testicular oxidative stress, 18 Wistar male rats (age: 3 months, weight: 127.5 ± 5.3 g) were randomly divided into the following groups: (i) control group (CG, n = 6); (ii) experimental group (EG, n = 6); and (iii) supplemented group (SG, n = 6). An exercise protocol of 3 h swimming per day, five days per week was followed for 6 weeks in EG and SG with no exercise in CG. In SG, L‐ascorbic acid was supplied orally at a dose of 25‐mg/kg of bodyweight each day for 6 weeks. A significant decrease (P < 0.05) was noted in paired testicular weights, epididymal sperm count, testicular Δ⁵, 3β‐hydroxyseroid dehydrogenase, 17β‐hydroxyseroid dehydrogenase, plasma levels of testosterone luteinizing hormone, follicle stimulating hormone, prolactin, the numbers of preleptotine spermatocytes, midpachytene spermatocytes and stage 7 spermatids of stage VII seminiferous epithelium cycle in EG when compared with CG. A significant elevation (P < 0.05) in plasma corticosterone and testicular content of malondialdehyde along with a significant reduction (P < 0.05) in glutathione, ascorbic acid, α‐tocopherol, superoxide dismutase, catalase and glutathione‐peroxidase, and glutathione‐S‐transferase were noted in testes of EG compared with CG. No significant change was noted in final bodyweight or numbers of spermatogonia‐A among the groups. Furthermore, L‐ascorbic acid supplementation restored the above parameters to the control level.

Conclusion:  It can be concluded that intensive swimming exercise induced oxidative stress causes dysfunctions in the male reproductive system, which can be protected by L‐ascorbic acid. (Reprod Med Biol 2006; 5: 145–153)

INTRODUCTION

ALTHOUGH EXERCISE HAS some beneficial effects on maintaining disease free health, several studies on humans and animals have indicated that chronic intensive exercise could result in dysfunction in the male reproductive system. ¹ , ² Forced and endurance exercise might be associated with oxidative stress and subsequent neuroendocrine changes. ³ Oxidative stress is a condition associated with an increased rate of cellular damage induced by oxygen and oxygen derived oxidants commonly known as reactive oxygen species (ROS). ROS are reported to damage almost all macromolecules of the cell, including membrane polyunsaturated fatty acids, thus causing impairment of cellular functions. ⁴ Testicular membranes are extremely rich in polyunsaturated fatty acids, therefore, the organ is highly susceptible to oxidative stress. ² However, most of the research findings have indicated that intensive exercise lowers the level of testicular testosterone. ¹ , ² , ⁵ Spermatogenic disorders, suppressed gonadotrophin and androgen production, along with a decrease in antioxidant enzymes and the consequent production of reactive oxygen species (ROS), were evident as the result of chronic intensive swimming exercise. ² , ⁵ , ⁶ Furthermore, the detrimental effect of oxidative stress on testicular activities was elevated with the increasing intensity of exercise. ⁵ Recent studies have drawn increasing attention to the supplementary effect of dietary antioxidants to reduce the detrimental effect of oxidative stress. We have already established that antioxidants, such as vitamin‐E, are able to reduce exercise induced testicular oxidative stress and other reproductive dysfunctions. ⁶ The potential role of dietary L‐ascorbic acid (vitamin C) in reducing the activity of free radical induced oxidative stress has been well established. ⁷ Furthermore, vitamin C plays a significant role against oxidative tissue damage and apoptosis in vital tissues. ⁸ The water‐soluble antioxidant, vitamin C, possibly elevates tissue antioxidant enzyme activities. ⁹ Vitamin C has the capacity to directly reduce peroxyl radicals during exercise. ¹⁰ Exercise‐induced oxidative stress has been reported to result in the loss of vitamin C from plasma and tissues of rats, ¹¹ and the reduced exercise mediated oxidative stress was noted after ascorbic acid supplementation. ¹² Therefore, the aim of the present study was to determine the ameliorating potential of L‐ascorbic acid on testicular steroidgenic and spermatogenic disorders in rats, induced by intensive swimming exercise, in connection to testicular oxidative stress.

METHODS

Animals

THE PRESET EXPERIMENT was carried out using 18 sexually mature male Wistar strain rats that were 3 months of age, weighed 127.5 ± 5.3 g at the beginning of the experiment and were randomly divided into three groups as follows: (i) control group (CG, n = 6); (ii) experimental group (EG, n = 6); and (iii) supplemented group (SG, n = 6). The rats were housed in a temperature controlled room at 25 ± 2°C, with 60% relative humidity and a 12 : 12 light‐dark cycle for 15 days prior to the experiment and, thereafter, during the duration of the experiment. Bodyweight of the animals was maintained among the groups by a proper diet (dietary composition [egg albumin‐420, corn starch‐86, sucrose‐240, cellulose‐40, salt‐80, coconut oil‐70, oligoelements‐16, vitamin B complex‐1] g/kg) and water at libitum. All animals had their bodyweight recorded weekly. The Principles of Laboratory Animal Care (NIH publication no. 85–23, revised 1985) were followed throughout the experimental schedule. The experimental protocol also met the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian Science Academy, New Delhi, India) and the Animal Ethics Committee of the Institute duly approved the experimental protocol.

Experimental design

Exercise protocol and supplementation of ascorbic acid

In EG and SG, an exercise protocol of 3 h of continuous swimming per day, 5 days a week for 6 weeks was used. Whereas, in the CG, no exercise was given. The intensity of exercise used for the present study was based on a previous study by the authors. ⁵ This intensity of exercise caused an optimum level of dysfunction in the male reproductive system. All the rats of EG and SG swam together in a water tank, with a calculated average of 300 cm² of water surface area for each rat and a depth of 60 cm, at a water temperature of 30 ± 1°C. An electric hair dryer was used to dry their bodies immediately on removal from the water. Furthermore, in SG, L‐ascorbic acid (vitamin C) was given orally at a dose of 25‐mg/kg bodyweight per day, 4 h after the cessation of exercise for 6 weeks.

Collection of blood and reproductive organs

Animals of all the groups were killed by light ether anesthesia 24 h after the last day of exercise, in order to avoid the acute effects of exercise. No food was provided for 2 h before the animals were killed. The bodyweights of the animals were recorded. An initial 5 mL of blood was collected from the dorsal aorta using a heparinized syringe with a 21‐gauge needle and allowed to clot for 3 h at 4°C and then centrifuged at 2000 × g for 15 min at room temperature. The plasma samples were separated and stored at −20°C prior to hormone assay. The testes were dissected out and weighed.One testis from each animal was used for histology and the other was used for the study of steroidogenic and scavenger enzyme activities, along with the levels of glutathione, l‐ascorbic acid, α‐tocopherol and lipid peroxidation.

Epididymal sperm count

The epididymal sperm count was measured according to the method described previously. ¹³ Briefly, sperm were collected from an equal length of the caudae from the excised epididymis of each rat by flushing through the vas deferens with the same volume (10 mL) of suspension medium containing 140 mmol NaCl, 0.3 mmol KCl, 0.8 mmol Na₂HPO₄, 0.2 mmol KH₂PO₄ and 1.5 mmol d‐glucose (pH adjusted to 7.3 by adding 0.1 [N] NaOH, E Merck). The collected sample was centrifuged at 100 × g for 2 min and the precipitate part was resuspended in 10 mL of fresh suspension medium. A fraction of suspension (100 µL) was mixed with an equal volume of 1% Trypan blue in the same medium and the number of sperm were counted in four chambers (used for counting of white blood corpuscles) of the hemocytometer slide. At this concentration of Trypan blue (0.5%), the dye was completely rejected by intact sperm, which appeared bright and colorless, but taken up by dead and damaged sperm, which showed blue heads. The sperm number was expressed per ml of suspension.

Quantitative study of spermatogenesis

A quantitative study of spermatogenesis was carried out at stage VII of the spermatogenic cycle according to the method of Clermont and Morgentaler. ¹⁴ The characteristic cellular association present in this stage is spermatogonia‐A (Asg), preleptotine spermatocytes (pLSc), midpachytene spermatocytes (mPSc), stage 7 spermatids (7Sd) and most mature step 19 spermatids (19Sd). The different nuclei of the germ cells (except step 19 spermatids, which cannot be enumerated precisely) were counted.

Radioimmunoassay of testosterone, LH, FSH and prolactine

Radioimmunoassay of testosterone was carried out, ¹⁵ using a double –antibody (¹²⁵I) RIA (ICN, Biochemical, Costa Mesa, CA, USA). Because chromatographic purification of the sample was not carried out, each testosterone value was the sum of testosterone and dihydrotestosterone. The intra‐assay coefficient of variation was 6.5%. The luteinising hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) concentrations in the plasma were measured using a double antibody radioimmunoassay (RIA). The plasma concentrations of LH and FSH were measured according to the standard methods ¹⁶ and the plasma concentrations of PRL were measured following the procedure of Jacobs ¹⁵ with reagents supplied by the Rat Pituitary Distribution Programme and NIDDK (National Institute of Diabetes and Digestive Kidney Diseases, Bethesda, MD, USA). Highly purified rat LH (rLH‐I‐4), rat FSH (rFSH‐I‐8) and rat PRL (rPRL‐I‐6) were iodinated with 1mCi¹²⁵ I (Bhaba Atomic Research Center, Mumbai, India) and freshly prepared chloramine T (Sigma, St Louis, MO, USA). Goat antirabbit γ globulin was used as the second antibody (Indo‐Medicine, Friendswood, TX, USA). NIDDK–rLH‐RP‐3 was used as a standard and NIDDK–anti‐rLH‐S‐5 was used for the LH assay. The limit of the detection of LH was 0.05 ng at 80%. For the FSH, NIDDK–rFSH‐RP‐2 was used as a standard and NIDDK–anti‐rFSH‐S‐11 was used for the assay. The limit of the detection of FSH was 0.04 ng at 98%. NIDDK–rPRL‐RP‐3 was used as a standard and NIDDK–anti‐rPRL‐S‐9 was used for the PRL assay. The least detectable quantity of plasma PRL was 0.40 ng at 90%. All the samples were assayed on the same day to avoid the inter assay variation. Intra assay variation of LH and FSH assay were 3.5%, whereas the intra assay coefficient of variation was 6% in case of PRL assay.

Estimation of plasma concentration of corticosterone

Plasma concentrations of corticosterone were assayed with a rat corticosterone RIA DSL‐80100 kit supplied by Diagnostics System Laboratories (Webster, TX, USA). The rat corticosterone assay is based on the competition between the corticosterone in the sample and ¹²⁵I labeled rat corticosterone tracer for binding to a highly specific rabbit polyclonal antibody of rat corticosterone. After incubation, the separation antigen and antibody complex is achieved by using a double antibody system. The radioactive bound fraction is precipitated by centrifugation and counted in a gamma counter. The assays were carried out following manufacturer's instructions. The corticosterone concentrations were read from a calibration curve (calibrators 1–6 supplied with the kit) and expressed as ng/mL. The minimum detectable concentration was assayed at 2.7 ng/mL. The intra assay coefficient of variation was 2.6%.

Assay of testicular Δ⁵, 3β‐HSD and 17β‐HSD activities

Testicular Δ⁵, 3β‐HSD and 17β‐HSD activities were measured spectrophotometrically at 340 nm according to the method of Talalay ¹⁷ and Jarabak et al., ¹⁸ respectively. One unit of enzyme activity was equivalent to a change in absorbency of 0.001 per min at 340 nm.

Assessment of antioxidant status and lipid peroxidation

Testicular superoxide dismutase (SOD) activity was measured according to the method of Paoletti and Mocali. ¹⁹ One unit of SOD activity was defined as the amount of enzyme required to inhibit the rate of NAD(P)H oxidation by 50% and was expressed as unit/mg of protein. An activity of testicular catalase (CAT) was measured biochemically. ²⁰ One unit of CAT activity was defined as the amount of H₂O₂ consumption per minute. Assay of testicular glutathione peroxidase (GPx) activity was determined by the modified procedure of Paglia and Valentine. ²¹ GPX activity was expressed as nmol NAD(P)H oxidized per min/mg of protein. Testicular glutathione‐S‐transferase (GST) activity was measured spectrophotometrically according to the method of Habig et al. ²² Enzyme activity was expressed as nmol of product formed per min/mg of protein. The level of malondialdehyde (MDA) was expressed as nmol/mg of protein. Testicular glutathione (GSH) was measured following the standard method. ²³ The amount of GSH in the testicular tissue was expressed as nmol/mg of protein. The estimation of ascorbic acid in testicular tissue was carried out following the method of Mitsui and Ohta ²⁴ and expressed as µg/mg of tissue. The testicular α‐tocopherol measurement was described earlier ⁶ and expressed as µg/mg of tissue. Estimation of testicular MDA was carried out by the method described by Ohkawa et al. ²⁵

Statistical analysis

For statistical analysis of the data, analysis of variance (anova) followed by a multiple two‐tailed t‐test was used. anova tests the differences between the variances of two or more groups. We analyzed the data by one‐way anova, which is used to investigate the effects of a single independent variable on a dependent variable. Multiple two‐tailed t‐test with Bonferroni modification was used to find out whether the differences of mean values in each parameter between groups were significant. Differences were considered significant when P < 0.05. Accordingly, the statistical software package, spss, was used.

RESULTS

Growth, reproductive organs weight and epididymal sperm count

THERE WAS NO significant change in bodyweight among the three groups. In contrast, paired testicular weight and epididymal sperm counts had decreased significantly (P < 0.05) in the experimental group when compared with that of the control group. l‐ascorbic acid supplementation restored these parameters to the control group (Table 1).

Table 1.

Testicular weights, quantitative analysis of spermatogenesis at stage VII and epididymal sperm counts in different experimental groups of adult male rats

Treatment	Paired testicular weight (g)	Spermatogenesis at stage VII				Epididymal sperm count (No./mL)
		ASg	pLSc	mPSc	7Sd
CG	2.32 ± 0.14	1.32 ± 0.21	14.32 ± 2.42	18.46 ± 2.32	46.10 ± 4.51	12 110 ± 1240
EG	1.70 ± 0.08*	1.24 ± 0.18	9.24 ± 1.65*	12.10 ± 1.46*	31.20 ± 2.15*	7860 ± 402*
SG	2.18 ± 0.12	1.30 ± 0.24	13.46 ± 2.82	16.62 ± 2.84	44.84 ± 3.84	10 920 ± 840

^* P < 0.05. Each value represents mean ± SEM, n = 6. Asg, spermatogonia‐A; CG, control group; EG, experimental group; mPSc, midpachytene spermatocytes; pLSc, preleptotine spermatocytes; SG , supplemented group; 7Sd, stage 7 spermatids.

Quantitative measurement of spermatogenesis

Quantitative study of spermatogenesis at stage VII showed the detrimental effect of intensive exercise. The numbers of pLSc, mPSc and 7Sd had decreased significantly (P < 0.05) in the experimental rats in comparison with those of in the control group; whereas, no significant difference was noted in the number of Asg among the groups. However, L‐ascorbic acid supplementation restored the numbers of different generations of germ cells at stage VII of the seminiferous epithelium cycle, although the count showed a lower value when compared with controls (Table 1).

Testicular Δ⁵ 3β‐HSD and 17β‐HSD activities

After prolonged intensive swimming exercise, the activities of testicular Δ⁵, 3β‐HSD and 17β‐HSD had significantly decreased (P < 0.05) in the experimental group compared with the control group. However, significant restoration was noted in Δ⁵, 3β‐HSD and 17β‐HSD (P < 0.05) activities in the supplemented group in comparison to the experimental group. Moreover, a difference in Δ⁵, 3β‐HSD and 17β‐HSD existed between the control group and supplemented group (Fig. 1).

Figure 1.

Testicular androgenic key enzyme activities, plasma concentrations of testosterone in different experimental groups of adult male rats. Each value represents mean ± SEM, n = 6. (anova followed by multiple two‐tailed t‐tests with Bonferroni modification, *P < 0.05 as compared with respective control.) (□) CG, control group; (▪) EG, experimental group; () SG, supplemented group.

Plasma testosterone, LH, FSH, PRL and corticosterone concentrations

The levels of plasma testosterone, LH, FSH and PRL were reduced significantly (P < 0.05) in the experimental group when compared with the control group. Furthermore, in the supplemented group, significant elevation was noted in the plasma level of these hormones when compared with the control group, although the level of testosterone and PRL showed a lower value when compared with the controls (1, 2). The concentration of corticosterone was elevated significantly (P ≤ 0.05) in the experimental animals when compared with the control. However, in supplemented animals the concentration of this hormone was significantly restored towards control level, though a higher concentration was also noted in respect to the control animals (Table 2).

Figure 2.

Plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin in different experimental groups of adult male rats. Each value represents mean ± SEM, n = 6. (anova followed by multiple two‐tailed t‐tests with Bonferroni modification, *P < 0.05 as compared with respective control.) (□) CG, control group; (▪) EG, experimental group; () SG, supplemented group.

Table 2.

Testicular malondialdehyde, glutathione, ascorbic acid, α‐tocopherol and plasma concentration of corticosterone in different experimental groups of adult male rats

Treatment	MDA (nmol/mg protein)	GSH (nmol/mg protein)	Ascorbic acid (µg/mg tissue)	α‐tocopherol (µg/mg tissue)	Corticosterone (ng/mL)
CG	3.10 ± 1.76	302.14 ± 14.46	262.18 ± 18.12	146.12 ± 14.34	236 ± 30.18
EG	6.84 ± 1.52*	192.86 ± 10.21*	174.65 ± 15.10*	81.25 ± 10.48*	316 ± 32.10*
SG	3.72 ± 1.02	281.10 ± 12.14	285.26 ± 17.21	132.76 ± 18.21	252 ± 45.14

^* P < 0.05. Each value represents mean ± SEM, n = 6. CG, control group; EG, experimental group; GSH, glutathione; MDA, malondialdehyde; SG, supplemented group.

Testicular antioxidants and lipid peroxidation

Testicular antioxidant scavenger enzymes, such as SOD, CAT, GPx and GST, were found be significantly reduced (P < 0.05) in the experimental group when compared with control group. Lipid peroxidation, as measured by the level of MDA, was elevated significantly (P < 0.05) along with a significant reduction (P < 0.05) in the level of testicular nonenzymatic antioxidants, such as GSH, ascorbic acid and α‐tocopherol, in the experimental group when compared with the control group. Furthermore, L‐ascorbate supplementation restored these parameters to the control level, suggesting the strong ameliorating potential of this vitamin against testicular oxidative stress (Table 2, Fig. 3).

Figure 3.

Testicular activities of testicular catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione‐S‐transferase (GST) in different experimental groups of adult male rats. Each value represents mean ± SEM, n = 6. (anova followed by multiple two‐tailed t‐tests with Bonferroni modification, *P < 0.05 as compared with respective control.) (□) CG, control group; (▪) EG, experimental group; () SG, supplemented group.

DISCUSSION

IN THE PRESENT study, the harmful effect of oxidative stress on the male reproductive system imposed by intensive swimming exercise was evaluated in mature male albino rats. In testicular steroidogenic events Δ⁵, 3β‐HSD and 17β‐HSD play a key regulatory role. ⁵ , ⁶ , ²⁶ The inhibition in steroidogenic enzyme activities after chronic exercise in the experimental rats might be a result of low plasma levels of LH, as this is a prime regulator of testicular steroidogenic enzyme activities. ² , ⁵ , ²⁶ Furthermore, inhibition of testicular steroidogenic enzyme activities in experimental rats after intensive exercise might be the result of the production of ROS in testicular tissue, as mitochondrial and microsomal steroidogenic enzyme activities in testis are reduced in the presence ROS. ¹³ , ²⁷ Moreover, in the present study the increased level of testicular MDA breakdown products of lipid peroxidation by ROS ⁴ was also noted in the experimental rats. ROS can cause cytotoxicity, one of the manifestations of which can be observed through lipid peroxidations. ¹³ , ²⁷ The elevation in testicular ROS in the experimental rats has been supported by the decrease in the activities of testicular SOD, CAT and GST, as these are important scavenger enzymes against free radicals. ²⁷ , ²⁸ GSH is a thiol‐containing tripeptide found in virtually all cells. GSH plays multiple roles in cellular antioxidant defenses. The most important antioxidant function of GSH is to remove hydrogen peroxide and organic peroxides. ²⁸ Furthermore, α‐tocopherol, is an important nonenzymatic antioxidant that scavenges ROS and elevates scavenger enzyme activities. ⁶ Therefore, any decline in the level of GSH indicates the increased production of free radicals, ²⁷ , ²⁸ as found in the present study. Moreover, low plasma levels of testosterone in the experimental rats also strengthen the belief about the inhibitory effect of intensive swimming exercise on testicular steroidogenesis. It is well established that in the course of physical exercise, the increase in oxygen consumption is accompanied by an increased production of ROS and causes oxidative DNA damage. ³

The low levels of testicular Δ⁵, 3β‐HSD and 17β‐HSD has been supported by a decrease in plasma testosterone levels in the present study, as testosterone is the prime androgen in male testis. ²⁹ The inhibition in gonadal steroidogenesis in stressful conditions is consistent with other findings where gonadal steroid synthesis has interfered significantly in mammals. ¹³ In addition, significant inhibition in the testicular somatic index in experimental animals also supported the low level of plasma LH in stressful conditions as testicular growth is purely dependent on the plasma level of LH and FSH. ²⁷ , ²⁹ Indeed, the actual cause for the decrease in plasma LH and FSH levels in the present experiment is not clear, but this could occur as a result of hyperactivation of the hypophysial‐adrenocortical axis. As it has been well established, stressful conditions also stimulate this axis and there is an increased secretion of adrenocorticotrophic hormone (ACTH) and corticosterone. ³⁰ The high levels of ACTH and corticosterone directly suppress testosterone production and secretion by decreasing the testicular LH receptor, and also results in the reduction of spermatogenesis and epididymal sperm count. ³¹ The increased concentration of corticosterone was noted in the experimental animals in the present study. The elevation of plasma levels of corticosterone might suppress the sensitivity of gonadotroph cells to GnRH and, therefore, might prevent gonadotrophin secretion. ³¹ The decrease in the epididymal sperm count in experimental animals is the result of lower concentrations of testosterone, as the sperm production in testis and maturation in the epididymis is controlled by testosterone. ²⁹

In the present study, an intensive exercise‐induced spermatogenic disorder has been reflected by the decrease in number of different generations of germ cells at stage VII in the spermatogenic cycle. Stage VII of the seminiferous epithelial cycle was selected as a quantitative study of spermatogenesis because of the fact that it represents the particular condition of spermatogenesis, as all varieties of germ cells are present at this stage. ³² This inhibition in spermatogenesis might be the result of low levels of gonadotrophins and testosterone, ² which corroborated with the low plasma levels of testosterone, LH and FSH as observed in the present investigation. The concentration of testosterone in the circulation is a function of the amount of testosterone entering (testicular production and secretion) and the amount leaving (metabolic clearance) the blood pool. This process is affected by any change in the physiological state that alters the metabolic turnover of the hormone. ³³ In fact, testosterone secretion is affected mainly by testicular blood flow through the testicular area, because testosterone is lipid soluble and, thus, freely diffusible. Furthermore, the testes apparently have little or no storage capacity for testosterone. Testicular blood flow is a function of the levels of vascular vasoconstriction or vasodilatation. ¹ Therefore, anything that influences vascular tone can affect the rates of testosterone secretion by increasing sympathetic nervous system activity. ¹ It has been observed that endurance exercise reduces the blood flow to the testicles and causes a low level of testosterone secretion, thus, affecting some degree of spermatogenesis, ¹ which closely resembles the decrease of spermatogenesis as found in the present study. However, in the present study, swimming exercise was used in order to minimize the changes in core and testicular temperatures in these animals (compared with running exercise). Hence, the changes that have been observed here in testicular function following the training are less likely to be a result of abnormally high testicular temperatures. In testes, reactive oxygen such as H₂O₂ might have an important role in the initiation of apoptosis with regard to necrosis in germ cells. ¹³ Studies on testicular tissues suggest that oxidative stress in testicular milieu is associated with DNA damage and produces a higher frequency of abnormal sperm, which has a significant effect on male fertility. ³⁴

Besides this, hormonal alterations and the spermatogenic inhibition in experimental rats might be the result of lipidperoxidation. This is indicated by the elevated levels of MDA, which is a product of lipid peroxidation, that have a detrimental effect on spermatogenesis. ² A decrease in the testicular mass in the experimental rats also supports the inhibition of testicular steroidogenesis. The elevation in the levels of MDA in the present study is further strengthened by the decrese in the activities of antioxidant scavenger enzymes such as SOD, CAT and GST as well as the levels of GSH, ascorbic acid and α‐tocopherol in testicular tissues of rats. ²⁷ , ²⁸

The results of the present study indicate that l‐ascorbic acid (vitamin C) has a protective effect on exercise induced testicular disorders. This might be a result of the stimulatory effect of l‐ascorbic acid in testicular steroidogenesis and gametogenesis. ³⁵ Furthermore, vitamin C also facilitates the synthesis and secretion of gonadotrophins from the anterior pituitary. ³⁶ These findings also supported our findings where the plasma concentrations of testosterone, LH and FSH were restored to the control level by l‐ascorbic acid supplementation. However, previous studies have indicated that vitamin C acts as a potent water‐soluble antioxidant by scavenging physiologically relevant reactive oxygen species (ROS) and possibly elevates tissue antioxidant enzyme activities. ⁴ , ⁹ , ³⁷ In addition to scavenging ROS, vitamin C can regenerate other small molecule antioxidants, such as α‐tocopherol, GSH and β‐carotine from their respective radical species. ⁴ Vitamin C has the capacity to directly reduce peroxyl‐radicals during exercise. ¹⁰ Exercise‐induced oxidative stress has been reported to result in the loss of vitamin C from plasma and tissues of rats. ¹¹ Therefore, reduction in tissue vitamin C levels might reduce the antioxidant enzymes activity and increase the suceptability to oxidative damage. During oxidative stress, l‐ascorbic acid is converted to the ascorbyl radical by its reduction of superoxide and other reactive oxygen species. ³⁷ Ascorbic acid is synthesized in the rat liver from glucose in a multistep enzyme mediated pathway, with l‐gulonolactone oxidase catalyzing the terminal reaction. ³⁸ In the present study, it was found that L‐ascorbic acid supplementation lowered the level of MDA and enhanced the antioxidant defense system in testicular tissue of exercised rats. This is strongly supported by other studies where the exercise induced oxidative stress was reduced by this vitamin supplementation. ¹² , ³⁹ Tissue oxidative stress, as indicated by glutathione status, was significantly affected by intensive exercise in the present study. Hence, it seems unlikely that the lower tissue vitamin C levels noted in the experimental group was the result of glutathione deficiency. ⁴ In the present study, it has been found that chronic intensive exercise induced oxidative stress reduced the number of different generations of germ cells, which might lead to infertility. The finding that l‐ascorbic acid prevents oxidative DNA damage of sperm ⁴⁰ was also supported by the present study as significant protection of germ cells degeneration was noted in the supplemented group. Therefore, L‐ascorbate has a strong ameliorating potential against exercise induced reproductive disorders and testicular oxidative stress.

From the present study, it can be concluded that intensive exercise‐induced oxidative stress might be responsible for some cases of low reproductive activities. The overall results of the study indicate that oxidative stress is imposed on male reproductive system by intensive swimming exercise, which might interfere in testicular steroidogenesis and spermatogenesis. The results of the present study suggest that supplementation of l‐ascorbic acid has a significant protective effect against chronic intensive swimming exercise induced oxidative stress and male reproductive dysfunction in rats. In order to extrapolate the results of the present study to male athletes doing such types of intensive exercise (such as an ultramarathon), further studies in this area are an imperative necessity.