Green tea consumption increases sperm concentration and viability in male rats and is safe for reproductive, liver and kidney health

Green tea is a popularly consumed beverage worldwide and contains polyphenols, whose antioxidant activities could improve sperm parameters and fertility thereof. We investigated the effect of green tea on the male rat reproductive system as well as its safety. Male Wistar rats were administered 2 and 5% aqueous extract of green tea for 52 days’ ad libitum, while the control group received tap water. Total polyphenol, flavanol, flavonol and soluble solids significantly increased in a concentration-dependent manner in vitro (P < 0.01). Weights of body, testis, epididymis, prostate gland, seminal vesicles, and liver, serum levels of testosterone, ferric reducing antioxidant power, creatinine, and sperm motility, remained unchanged (P > 0.05). Kidney weight, sperm concentration and vitality, spontaneous acrosome reaction increased (P < 0.05), while alanine transaminase and aspartate transaminase levels decreased (P < 0.05). Catalase, superoxide dismutase, glutathione and lipid peroxidation remained unchanged in the testes, liver and kidney (P > 0.05). Histological sections of testis, epididymis, kidney and liver showed no conspicuous alteration. Diameter and epithelial height of seminiferous tubule decreased, while caudal epididymis epithelial height increased (P < 0.01). Consumption of green tea in the conditions used in the present study seems to be safe and improved sperm parameters. However, subtle structural changes observed in the decreased diameter and epithelial height of the seminiferous tubule and increased acrosome reaction needs further investigation.

and powerful antioxidant 13 . Although, a higher amount of antioxidants, as well as antioxidant potential, are seen in white tea compared to green tea in a dose-dependent manner 1,14 .
Intake of three to four cups of green tea (equivalent to 2 g) three times a day is to be safe for administration in a long term basis 15 . Human intervention and bioavailability studies involving low to moderate doses of green tea preparations or EGCG reported no adverse effect [16][17][18] . However, other studies revealed that administration of high doses of green tea or EGCG resulted in liver and renal toxicity 12,15,19,20 , in which its polyphenol catechins could be the causative agent 20 . High levels of serum aspartate transaminase (AST), alanine transaminase (ALT), and creatinine, respectively indicate liver and renal toxicity or injury as ALT and AST are biomarkers of the liver structure and function, while creatinine is a reliable renal biomarker 21,22 . The harmful effects of green tea have also been attributed to the pro-oxidative property when consumed at high doses 23 . Repeated administration of green tea polyphenols (GTPs) (750 mg/kg) caused a hepatic injury 24 . Oral administration of EGCG (1,500 mg/ kg) increased ALT in plasma and reduced survival rate in CF-1 mice 9 . Mazzanti et al. also showed a link between the intake of green tea supplement and liver damage 25 . Salminen et al. demonstrated that green tea extract (500 and 1,000 mg/kg) potentiated acetaminophen-induced hepatotoxicity in mice 26 . Low doses of GTPs (0.01 and 0.1%) but not the high dose of GTPs (1%) ameliorated dextran sulfate sodium (DSS) increased ALT and AST levels in mice 22 . Administration of high dose GTPs (1%) to male mice caused renal toxicity, as marked by increased serum creatinine level, and increased thiobarbituric acid-reactive substances (TBARS) in both kidney and liver 22 . Protective effects of green tea extract were also demonstrated against nephrotoxic agents in rats [27][28][29] .
The testis performs two crucial functions, that is, spermatogenesis (production of spermatozoa) and steroidogenesis (production of steroid hormones), which are regulated by gonadotropins and locally synthesised factors 30 . The whole spermatogenic cycle takes 52 days in rats 31 . Previous studies have indicated inconsistent results on the exposure of green tea to the male reproductive system. Yassa et al. showed that administration of the aqueous green extract (70 mg/kg/day) for 90 days did not affect the body weight, serum testosterone, and the histological appearance of the seminiferous tubules of the testes was normal 32 . Oral administration of aqueous extract of green tea (70 mg/kg/day) to rats for 63 days did not affect weights of the body and reproductive organs, sperm concentration and viability and diameter of the seminiferous tubule 33 . Oral administration of 1.25% and 5% green tea extract (polyphenone-60) mixed with powdered CE-2 diet to male rats for 2, 4 and 8 weeks' ad libitum decreased weight of the body, testes, and prostate gland, but increased levels of luteinising hormone (LH) and testosterone, with no marked difference in the histological sections of the testis at the highest dose 34 . Consumption of 2.5% and 5% green tea extract (1 ml/100 g body weight) for 26 days resulted in impaired spermatogenesis through the loss of matured and elongated spermatids. Decreased net weight gain and testis weight, sperm count and motility, Δ5 3β-and 17 β-hydroxysteroid dehydrogenase and testosterone levels, with increased follicle-stimulating hormone (FSH) and LH in adult male rats, was also observed 35,36 .
Furthermore, intraperitoneal injection of EGCG (85 mg/kg body weight) for 2-7 days caused acute body weight loss, and reduction in weight of prostate gland and testis as well as the level of testosterone and LH 37 . Administration of lower concentrations of EGCG (2 µM and 20 µM) to human sperm enhanced sperm quality through the increased motility, viability and capacitation. In comparison, administration of a high concentration of EGCG (60 µM) caused a deleterious effect 38 . Supplementation of sperm storage media with tea extract increased sperm viability in a dose-dependent manner 1 . Other studies have shown the ameliorative or cytoprotective property of green tea following the administration of testicular toxicants such as cadmium and acrylamide or by the induction of oxidative stress in animal models 32,33,[39][40][41] . In this study, we report on the effect of green tea in the male rat reproductive system after a complete spermatogenic cycle of 52 days as well as its safety by considering the liver and kidney functions.

Results
chemical analysis of green tea. A significant increase in total polyphenol (P < 0.0001), flavanol content (P < 0.01) and flavonol content (P < 0.0001) were observed in the 5% green tea extract compared to that of 2%. The soluble solid content (P < 0.0001) as well as the ferric reducing power (P < 0.0001) was also higher in the 5% in green tea extract compared to 2% green tea extract) ( Table 1). fluid intake and weight gain. Overall, fluid intake in male rats exposed to green tea (2% or 5%) did not differ to the control group (Table 2; P > 0.05). Bodyweight gain of animals in all groups increased progressively throughout treatment (Fig. 1). Rats treated with green tea showed no change in the weekly weight gain compared to the control (P > 0.05), although the 2% green tea treated group showed a reduced weight gain at week five Serum analysis. Green tea (2% and 5%) did not affect the level of testosterone produced in the male rats (Table 3; P > 0.05). However, a positive trend in the level of testosterone was observed in a dose-dependent manner (ANOVA-trend analysis, repeated measure: P = 0.029). Ferric reducing antioxidant power in serum remained unchanged following the 52 day's treatment (Table 3; P > 0.05). Creatinine remained unchanged in its serum level in the treated rats (Table 3; P > 0.05). The AST level in serum, as shown in Table 3 dropped significantly in rat exposed to 2% and 5% green tea (P < 0.05). Repeated measure ANOVA trend analysis showed an increase in the level of AST in serum (P = 0.0050) at the higher tea concentration used. Serum ALT activity decreased significantly in the treated groups (Table 3; P > 0.05). Although not significant, a trend to a decreased level of   Sperm parameter analysis. Green tea significantly increased sperm concentration (P < 0.05) (Fig. 2a) and sperm vitality control (P < 0.05). The increase in sperm vitality was up to 40% and 54.8% in the 2% and 5% green tea treated groups, respectively (Fig. 2b). Total motility (Fig. 2c) and total progressive motility ( Fig. 2d) remained unchanged (P > 0.05) in both treated groups. Also, green tea caused a significant decline in the percentage of total static spermatozoa (P < 0.05; Fig. 2e). Further to these, sperm velocity parameters, that is, ALH, BCF, LIN, STR, VAP, VCL, VSL, and WOB remained unchanged (P > 0.05; Table 4). following exposure of male rats to 2% and 5% green tea. Lastly, the percentage of acrosome reacted spermatozoa significantly increased (P < 0.05; Fig. 2f).
Histology and morphometric measurements. The histological observation of the testes in the treated rats showed no noticeable distortion in the architecture of the cells compared to the control (Fig. 3). All stages of spermatogenesis were present, and copious spermatozoa in the lumen of the seminiferous tubule in the treated groups, like the control. Furthermore, sections of the epididymis (cauda and caput) also showed no difference in structure compared to the control group (Figs. 4 and 5). On the contrary, the diameter (2% and 5%; P < 0.0001) and epithelial heights (2%, P < 0.05) of seminiferous tubules of the treated groups reduced significantly (Table 5).  www.nature.com/scientificreports/    www.nature.com/scientificreports/ Green tea extract (2%) significantly increased the epithelial height of the cauda epididymis (p < 0.05); however, the epithelial height of the caput epididymis remained unchanged (p > 0.05) ( Table 5). Furthermore, the histological sections of the treated kidney and liver depicted standard architecture compared to the control (Figs. 6 and 7). Table 6 shows the results on lipid peroxidation and antioxidant enzymes in the testes, liver and kidney following treatment with green tea (2% and 5%). No change in the level of MDA in the testes, liver and kidney (P > 0.05; Table 6). Similarly, GSH, CAT and SOD levels in the kidney, liver and testes remained unchanged (P > 0.05).   www.nature.com/scientificreports/

Discussion
The results on the effect of green tea extract or its polyphenols (such as EGCG) on the male reproductive functions and spermatogenesis are conflicting [32][33][34][35]37 . Also, a high dose of green tea extract caused renal toxicity and hepatotoxic effects 12,15,19 . Hence, we aim to report on the overall effect of consumption of green tea ad libitum on the male reproductive system after the completion of one spermatogenic cycle in rats, as well as on the kidney and liver functions. Green tea leaves contain 30-40% polyphenols 2,8 . Similar to previous studies 1, 14 , in vitro analysis of green tea in our study revealed the presence of these polyphenols and further demonstrated a concentration-dependent increase in total polyphenols, flavanol, flavonol, and soluble solids. Further analysis of the antioxidant capacity of green tea in vitro, using FRAP showed a concentration-dependent increase; however, it remained unchanged in the serum of the treated male rats. These were indicating a possible higher antioxidant property with higher concentrations of the extract. Our study however, also showed no change in the serum FRAP after drinking green tea in vivo, which supports the findings of Maxwell & Thorpe 42 . Contrary to our study, consumption of green tea significantly increased plasma FRAP in humans 43 . The possible explanation could be due to the differences in bioavailability of green tea, as bioavailability and transformation of EGCG, the predominant catechin in green tea, differs significantly between humans, mice and rats 44 . Low bioavailability of EGCG was also demonstrated following administration of GTP as the sole source of drinking fluid in rats, with a higher level seen in mice 45 .
The animals consumed green tea extract (2 and 5%) ad libitum, and their average intake was comparable to the control group and corresponds to approximately 40 cups per day in an 80 kg man. The study demonstrated a progressive rise in the weekly weight of animals, with no obvious signs of clinical toxicity or behavioural changes in the rats, indicating the well-being of the animals during the treatment period. Furthermore, body weight gain of the treated rats also remained unchanged. Similar, to our study, administration of green tea (70 mg/kg/day) to rats for 63 days or 90 days did not affect body weight gain 32,33 . However, a transient reduction in body weight was observed at week 5 in the 5% treatment group and may be attributed to taste aversion and reduced food intake. Kao et al. however, reported that EGCG (a predominant green tea polyphenol) reduced body weight in rats 37 . Green tea catechins (especially EGCG) are responsible for the reduced body weight by decreasing the differentiation and proliferation of adipocytes during lipogenesis 46 . Also, a significant rise in weight loss was observed in humans following consumption of a high dose of green tea for 12 weeks. It may be attributed to inhibition of ghrelin secretion, which leads to increased adiponectin levels 47 .
Although our study, however, showed a remarkable rise in kidney weight, the creatinine level remained unchanged, suggesting that green tea did not damage renal function. Another study revealed that high concentrations of green tea catechins (5%) increased the kidney weight, with no histopathologcal observation 48 . Also, l-theanine (a predominant free amino acid found in tea) was shown to increase kidney weight in rats, with no histological correlations 49 . Higher protein or amino acid intake modulates renal hemodynamic by increasing renal blood flow and elevating intraglomerular pressure that results in an increased glomerular filtration rate (GFR) as as well an increased kidney volume and weight 50 . The increased weight in our study may be attributed to the catechins or amino acid (l-theanine) content in green tea and was considered not to be toxicologically significant as there was no obvious histopathological changes in the kidney together with a comparable level of creatinine as the control. Besides, the weight of the liver remained unchanged, with significantly reduced levels of AST and ALT. Both of these findings suggest that consumption of green tea at least in rats had no detrimental effects, as also supported by the histological sections of the treated kidneys and liver. Our study is in corroboration with Bun et al. 6 , as no sign of evidence of distinctive hepatotoxicity was found in green tea treated rats, as this study showed no hepatic functional disorders based on the levels of ALT and AST in serum, as well as histological sections of the liver. The results of the current study are in line with the literature data suggesting hepatoprotective properties of green tea preparations 51-53 . Takami et al. revealed that prolonged intake (90 days) of high concentrations of green tea catechins (5%) brought about hepatotoxic effects in male rats by the increased levels of ALT and AST 48 , which could suggest that the use isolated or pure polyphenols could be detrimental to the functioning of these organs. Furthermore, case-reports have associated the use of concentrated green teabased supplements with liver toxicity in humans 54,55 .
Our study demonstrated that the level of testosterone (which plays a crucial role in spermatogenesis) remained unchanged, even though, a trend to increased values was observed with increasing concentration of green tea. Other studies showed that green tea extract and EGCG produced a substantial drop in testosterone level in Leydig cells in vitro 5,56 , while testosterone production in vivo studies showed a reduction 35,37 , increment 34 or remained unchanged 32 in male rats. A possible explanation for these variations could be due to the use of different forms of the plant (crude extract, or isolated compounds), modes of administration or length of treatment.
Reproductive organ weights are used as indicators of reproductive toxicity 57 , and a reduction of testicular weight is a sensitive parameter for interpretation of male gonadal toxicity 58 . In line with the observed level of testosterone and similar to a previous study 33 , intake of green tea in this study did not alter the weight of testosterone dependent organs (testis, epididymis, seminal vesicles and prostate gland). Also, histological sections of the testis and epididymis revealed no obvious alteration in their structures. Furthermore, sperm quality is also used as an important indicator of chemical-induced toxicity on testis 59 . While a study demonstrated that green tea extract did not affect sperm concentration and viability 33 , our present study showed that green tea significantly increased sperm concentration and sperm vitality, rather, sperm motility and velocity functions were unaffected. However, morphometric measurements in our study showed a significant reduction in the diameter and epithelial height of the seminiferous tubule. In contrast, epithelial heights of cauda epididymis increased, and that of the caput remained unchanged. Contrary to our study, green tea did not affect the diameter of the seminiferous tubule 33 ; however, a reducing trend was observed. Reproductive toxicants are known to reduce the diameter and epithelial height of seminiferous tubule as well as impair spermatogenesis 59 www.nature.com/scientificreports/ an increase in sperm viability, concentration, and epithelial heights of epidiydymis with no dinstinct distortion of the seminiferous tubules or loss of spermatogenic cells. The mechanism of action responsible for these increment is unclear and requires further investigation to understand the impact on the male reproductive functions and fertility outcome. Bioactive compounds in tea such as catechins, caffeine, and l-theanine 62 are shown to possess conflicting effects on the male reproductive system. For instance, low concentrations of caffeine (mainly derived from tea leaves) stimulates lactate (the preferred metabolic substrate for developing germ cells) production (5 and 50 µM), and increases expression of glucose transporters (50 µM; GLUTs) in human Sertoli cells (hSCs) 63 . In another study, administration of caffeine (200 mg/kg body weight) negatively affected the male reproductive functions as demonstrated by the reduced testicular and epidydimal weight, distortion of the histo-architecture of the seminiferous tubule and loss of spermatogenic cells, sperm count, motility and viability with increase abnormal sperm 64,65 . In addition, maternal consumption of caffeine during gestation (26 and 45 mg/kg) and lactation (25 and 35 mg/kg) was shown to also negatively affect male reproductive function in the male offspring rats as indicated by a significant reduction in testicular weight, diameter and epithelial heights of seminiferous tubules, testosterone production and sperm quality 66 . EGCG was shown to have an antiproliferative effect on hSCs (5 and 50 µM), although lactate production was maintained (5 and 50 µM) with an increased consumption of glucose and pyruvate (50 µM). However, it decreased the mitochondrial membrane potential (50 µM) and conversion of pyruvate to alanine (5 and 50 µM), which ensures the regular production of lactate 67 . l-Theanine, on the other hand, increased hSC proliferation, glucose consumption and mitochondrial membrane potential, although lactate production remained unchanged 68 . Also, supplementation of sperm storage media for up to 3 days either with caffeine (71 µg/ml), EGCG (82 µg/ml) or l-theanine (19 µg/ml) had no effect on sperm viability, while the supplementation with a combination of these three compounds significantly increased sperm viability, suggesting a synergestic effect of the components of tea rather than an individual bioactive compound 69 . Hence, a possible explanation for the increased sperm concentration and viability in our study might be attributed to an increased lactate production by the Sertoli cells or increased GLUT protein levels and activities, although further studies are required to validate this.
In order to fertilise an oocyte, capacitated spermatozoa must undergo acrosome reaction, and consists of the release of the hydrolytic enzyme in the secretory vesicle of the sperm acrosome, for the degradation of the zona pellucida of the oocyte. A premature or spontaneous acrosome reaction makes the spermatozoa incompetent to interact with the oocyte and thereby to impair its fertilisation rate 70 . This study revealed a significant increase in spontaneous acrosome reaction, posing a question on the ability of these spermatozoa to fertilise an oocyte. Production of F-actin during capacitation was shown to be crucial in the prevention of spontaneous acrosome reaction 71 . An increase in cyclic adenosine monophosphate (cAMP) results in the activation of protein kinase A (PKA) at the onset of capacitation, which indirectly controls the phosphorylation of protein tyrosine, hyperactivated motility and actin polymerisation 72 . Further to this, calmodulin kinase II (CAMKII) and phospholipase D (PLD) were shown to be major pathways responsible for the polymerisation of actin 71 . Tsirulnikov et al. further showed that PKA protects the sperm from undergoing spontaneous acrosome reaction by inducing actin polymerisation via PLD and CAMKII 73 . The possible explanation for the increased spontaneous acrosome reaction as seen in this study could be due to inhibition of PKA, actin polymerisation or CAMKII and PLD pathways, which needs to be further interrogated. Isotani et al. rather reported that acrosin-disrupted spermatozoa equally pierced the zona pellucida as the wild-type spermatozoa, rather, a delayed dispersal rate of cumulus oophorus cells caused a decrease in the number of fertilised oocyte and litter size 74 . Hence, the effect of green tea or its polyphenolic compounds on fertility rate must be further investigated to understand better the implication of spontaneous acrosome reaction observed in this study.
In comparison to the findings of this study, a previous study showed that black tea increased sperm vitality, motility, the epithelial height of the cauda epididymis, kidney weight, as well as spontaneous acrosome reaction while decreasing the activities of AST and ALT and diameter and epithelial height of the seminiferous tubule 75 , however, an increased creatinine activity was observed. Another study demonstrated the aphrodisiac property of black tea, accompanied by an increase in testosterone production in rats 76 . White tea, which is known to have a higher level of antioxidants compared to green, oolong and black teas 14 , stimulated lactate production in Sertoli cells, which provides nutritional support to the developing germ cells 77 , as well as increased rat sperm viability 1 . White tea was also shown to improve prediabetes-induced reproductive dysfunction through increment in glucose transporters 2 and 3 (GLUT 2 and GLUT 3) protein levels and phosphofructokinase 1 (PFK1) activity, sperm motility, with restoration of testicular lactate content and sperm viability 78 . In another study, white tea improved testiscular antioxidant potential and sperm concentration, and restored sperm quality (motility, morphology and viability) in prediabetic male rats 79 . These are suggestive of possible effects of the antioxidants in these plants on various aspects of male reproductive functions.
Most of the beneficial effects of green tea are attributed to the antioxidant and free-radical scavenging properties, its polyphenols and flavonols 80 . Our study showed that the levels of CAT, GSH, MDA, SOD were unaffected by green tea in the liver, kidney and testes of the treated rats, which indicates the safety of the plant and suggests that a balance between the scavenging activity of the antioxidant and generation of ROS is not compromised, thereby preventing oxidative stress under the tested conditions. Another study showed that testicular CAT and GSH remained unchanged while a significant increase in SOD was noted 33 . Green tea extract was shown to protect testicular tissue against oxidative damage by increasing its antioxidant defence mechanism as demonstrated by the increased SOD and GSH activities with a resultant decrease in lipid peroxidation 81 . Antioxidant therapy is used to eliminate, take up or reduce the formation of ROS. Antioxidant supplements are categorised based on their activities as a preventive antioxidant (e.g. lactoferrin), which prevents the formation of reactive oxygen species (ROS), and scavenging antioxidants (e.g. vitamins C and E), which removes already present ROS 82 . The antioxidants are also classified as enzymatic (natural) oxidants (includes glutathione reductase, catalase, superoxide Scientific RepoRtS | (2020) 10:15269 | https://doi.org/10.1038/s41598-020-72319-6 www.nature.com/scientificreports/ dismutase) and non-enzymatic oxidants, (includes vitamins B, C and E, carotenoid, carnitine, cysteine), which are derived from fruits and vegetables 83 . For instance, intake of a low amount of vitamin C was associated with increased oxidative stress 84 , while high levels of antioxidant (supraphysiological level) resulted in reductive stress, which is as destructive to cells as oxidative stress. For instance, administration of high concentrations of ascorbate was shown to have similar effects by causing oxidative stress 85 . A balance must be attained in the use of medicinal plants with an antioxidant in order to prevent oxidative or reductive stress. This study demonstrates that continuous and prolonged intake of aqueous extract of green tea improved sperm parameters, specifically sperm concentration and vitality, and appears to be safe in the rats. However, subtle structural changes observed in the reduced diameter and epithelial heights of the seminiferous tubule and increased spontaneous acrosome reaction needs further investigation to understand the implications on fertility outcomes. preparation of aqueous extract of green tea. Green tea (Camellia sinensis; Five Roses™) purchased from a retail store in Cape Town, South Africa was infused in freshly boiled tap water for 5 min to final concentrations of 2% and 5% respectively, was filtered through a cheesecloth and Whatman's filter paper (no 4 and 1, respectively) using a vacuum system (Opuwari & Monsees, 2015). chemical analysis of green tea. During the period of treatment, at least three samples of prepared green tea (2% and 5%) were randomly collected at different time points and stored at − 20 °C for further use. In order to determine the quantity of soluble solids in green tea, 1 ml of 2% and 5% green tea (in triplicate) was placed in the dried vials with a known weight. It was then dried overnight at 120 °C, allowed to cool in a desiccator and weighed. The obtained results were expressed as mg/ml of tea.

Methods
Total polyphenols in green tea were determined using the Folin-Ciocalteau (F-C) reagent, with Gallic acid as standard 86 and the result was expressed as mg Gallic acid equivalent (GAE)/ml of tea. In brief, F-C reagent (10%) and sodium bicarbonate (7.5%) was added to the tea extracts and prepared standards (5:4:1). The thoroughly mixed samples were incubated at 37 °C for 2 h and read at 765 nm.
Flavonol content was determined using quercetin as a standard, according to Mazza et al. 87 and expressed as mg Quercetin equivalent/ml of tea. To summarise, 0.1% hydrogen chloride (HCl) in 95% ethanol (50 µl) and 2% HCl (900 µl) was added to blank, standards and diluted samples (50 µl), incubated at 37 °C for 3 min and measured at 360 nm.

Animals. Male
Group one: tap water and served as control.
Group two: 2% green tea. Group three: 5% green tea. The amount of daily intake of fluid and the weekly bodyweight of the animals were recorded. At the end of the experiment (day 52), all rats were euthanized. Carbon dioxide was used to anaesthetize and euthanize the rats as it does not interfere with specific sperm parameters 90 . To avoid dyspnoea and distress, rats were placed in a glass chamber which was then filled slowly with carbon dioxide from a compressed gas cylinder according to AVMA guidelines 91 . When rats lost consciousness, gas flow was increased and maintained until no respiratory movements were observed. Death was confirmed by exsanguination of the heart. Final body and organ weights were noted. Blood obtained through a cardiac puncture, allowed to clot for 30 min and centrifuged (3,000×g, 15 min), was stored at − 80 °C for hormonal and biochemical assays. Left cauda epididymis was excised and used for the determination of sperm concentration, vitality, motility and acrosome reaction. Testis, right epididymis, liver and kidney were fixed in Bouin's solution for histological purposes. Left testis, liver and kidney were frozen at − 80 °C for biochemical assays. www.nature.com/scientificreports/ Serum analysis for testosterone and biochemical assays. The level of testosterone in serum obtained from the male rats was measured with an ELISA kit (EIA 1559, DRG Instruments GmBH, Marburg, Germany), as per the manufacturer's instruction. In brief, enzyme conjugate (200 µl) was added to 25 µl of sample and standard (0, 0.2, 0.5, 1, 2, 6, and 16 ng/ml) well, mixed and incubated at room temperature (RT) for one hour. After that, contents were briskly shaken out and rinsed three times with diluted wash solution (400 µl) and stricken sharply on absorbent paper to remove residual droplet. Substrate solution (200 µl) was added to each well and incubated at RT for 15 min, and the reaction stopped by the addition of stop solution (100 µl). Absorbance was read at 450 nm, and the result expressed as nanogram per millilitre. Serum creatinine was determined, according to Bartels et al. 92 and expressed as mg/ml. In brief, standard solution (0, 15, 30, 60, 90, 120 and 150 mg/l creatinine in dH 2 0) or serum was added to a working solution (0.1 g picric acid in 50 ml dH 2 0 and 50 ml 0.4 NaOH in 4 °C) (1:10), incubated at RT for 15 min and measured at 492 nm.
Antioxidant activities in the serum were measured using FRAP as described earlier 89 and expressed as µM Trolox/ ml of serum.
Lipid peroxidation was assessed according to Yagi 94 by measuring malondialdehyde (MDA), a thiobarbituric acid reacted substances (TBARS) and represented as nanomolar of MDA per milligram of protein. In brief, thiobarbituric acid (TBA) reagent (15% v/v trichloroacetic acid and 0.25 N HCl) was added to each sample (2:1), heated at 95 °C for 15 min and allowed to cool. Samples were centrifuged (1,000×g; 10 min) and measured at 532 nm.
Glutathione (GSH) level was analysed as described by Ellman 95 and expressed as millimolar thiol per milligram of protein. Briefly, Ellman's reagent (3 ml) was added to each sample (20 µl), incubated for 15 min and absorbance read at 412 nm.
Superoxide dismutase (SOD) activity was determined by the method of pyrogallol autoxidation 96 with a slight modification 97 . Briefly, samples were mixed with Tris-EDA-HCl buffer and pyrogallol (1:15:1) and incubated at 25 °C for 10 min. After that, 1 N HCl (50 µl) was added, and SOD activity measured at 440 nm. One unit is the amount of enzyme that inhibited the oxidation of pyrogallol by 50%, and the result represented as units per milligram of protein.
Catalase (CAT) activity was evaluated, according to Aebi 98 . In brief, samples were mixed with PBS and 20 mM H 2 0 2 (1:4:5) and measured at 25 °C within 2 min. A unit of CAT is the activity of the enzyme that catalysed the reduction of 1 µmol of H 2 O 2 per min per milligram of protein.
Analysis of rat spermatozoa. Spermatozoa collected from freshly isolated cauda epididymis by a swim out technique was used to analyse the respective parameters 100 . Sperm diluted with phosphate-buffered saline (PBS) (1:9) was counted with Markler counting chamber (Sefi-Medical Instrument, Haifa, Israel) to determine sperm concentration. Sperm concentration was represented as millions per millilitre (10 6 /ml). Sperm vitality was determined using the fluorescent Duo vital kit (Microptic, Barcelona, Spain) according to the manufacturer's instruction. In brief, a mixture of fluorochrome solution red (1 µl), fluorochrome solution green (1 µl) and sperm sample (5 µl), was placed on a clean slide. The slide was covered with a coverslip and spermatozoa (100) was analysed under a fluorescence microscope. Spermatozoa fluorescing green or red were respectively scored as alive or dead, and the result expressed as percentage alive. Sperm motility was determined by placing sperm suspension (5 µl) in a Leja slide (Leja Products B.V., Nieuw Vennep, Netherlands) and evaluated with Sperm Class Analyzer software (Microptic, Barcelona, Spain). Sperm parameters assessed included total motility, progressive motility, total static, curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity (VAP, µm/s), the amplitude of lateral head displacement (ALH, µm), and linearity (LIN, %), Wobble (WOB, %), beat cross frequency (BCF, Hz) as defined 101 . Acrosome reaction was evaluated according to Larson & Miller 102 . In brief, sperm samples were fixed with 4% p-formaldehyde in PBS (1:5) for 5 min and centrifuged (3,000×g, 3 min). The cells were re-suspended with ammonium acetate (100 µl), smeared on a glass slide, airdried and stained with Coomassie Brilliant Blue G (0.22%) solution for 2 min at RT. Excess dye was removed and covered with a coverslip and observed under a bright-field microscope (1000X). Spermatozoa head stained blue represented spermatozoa with intact acrosome reaction, while those with partial or no stain, spermatozoa undergoing acrosome reaction.
Histology and morphometric measurement. Following the fixing and tissue processing of the testis, epididymis, kidney and liver, hematoxylin & eosin staining was utilised. The diameter and epithelial heights of the seminiferous tubules of the testes, and epithelial heights of the epididymis were conducted according to