Effects of Magnesium Sulfate Administration on Testicular Ischemia/Reperfusion Injury in Rats

Introduction

Testicular torsion is a urologic emergency that leads to serious infertility ( Parlaktas et al., 2014 ). It can happen in children and young males, thereby requiring urgent diagnosis and treatment ( Celik et al., 2016 ). The duration and degree of twisting of the cord are closely related to the severity of the testicular injury ( Aydıner et al., 2012 ). The testicular torsion is defined as a circulatory failure by testis revolving around the vascular peduncle ( Yulug et al., 2013 ). The main pathophysiologic event in testicular torsion is ischemia followed by reperfusion (Pogorelic et al., 2016). Therefore, testicular torsion detorsion is an ischemia/reperfusion (I/R) injury for the testis ( Asghari et al., 2018a ). Diminished blood flows occur in ipsilateral and contralateral internal spermatic arteries after unilateral torsion. These changes result in testicular function impairment and fertility ( Yulug et al., 2013 ). Interruption in tissue blood supply by I/R leads to cellular and tissue damage ( Koksal et al., 2012 ). Testicular torsion terminates to tissue degeneration and usually requires emergency surgical intervention for the reperfusion of the affected testis ( Celik et al., 2016 ). There are numerous reports on medications and/or interventions utilized to manage this condition. In physical therapy, hyperbaric oxygen (through plasma oxygen transport), hypothermia, and ischemic post-conditioning are being used as surgical techniques against reperfusion injury for the first-line treatment ( Arena et al., 2017 ). Among the medications, agonists of erythropoietin receptors, dexmedetomidine, morphine, and antioxidants, such as dimethylsulfoxide, zinc, vitamin E, melatonin, and plant antioxidant extracts are widely suggested by the clinicians ( Arena et al., 2017 ). The exact pathological mechanisms of testicular torsion causing the injury are not fully elicited; however, the excessive reactive oxygen species (ROS) generation impairs spermatozoal motility ( Eghbali et al., 2010 ). The ROS has a deniable effect on male infertility. Reperfusion injury is related to the elevated of free oxygen radicals which causes cell membrane lipid peroxidation and DNA impairment ( Raju et al., 2011 ). Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen ( Fakouri et al., 2017 ). Magnesium (Mg²⁺) is the second plentiful cation within the cell and has a key role in various physiologic actions, including cell cycle, ATPase activity, channel regulation, and metabolic regulation ( Romani, 2007 ). The Mg²⁺ deficiency is related to oxidative stress in pathologic circumstances (e.g. diabetes, hypertension, atherosclerosis, and neuronal injury) ( Wolf et al., 2009 ). Moreover, it is reported that ROS-mediated DNA damage is related to Mg²⁺ dependent inhibition of cell growth ( Wolf et al., 2009 ). Furthermore, Mg²⁺ has been revealed to prevent lipid peroxidation and reduce neuroprotective effects in I/R injury of the fetal rat brain ( Hasturk et al., 2013 ). In addition, Mg²⁺ protects livestock spermatozoa against freezing and thawing injury ( Pesch et al., 2006 ). The MgSO₄ diminishes superoxide dismutase (SOD) in the bile duct ligation-induced in rats ( Eshraghi et al., 2015 ). The imbalance between ROS and scavenge free antioxidants occurs during oxidative stress ( Agarwal et al., 2009 ). The ROS are crucial requirements of spermatozoa for fertilization, capacitation, motility, and acrosomal reaction ( Hadwan et al., 2014 ). Although there is a report on the correlation between seminal antioxidant and Mg²⁺ levels with sperm viability and motility ( Pesch et al., 2006 ), no studies investigated the effects of MgSO₄ on testicular IR injury in rats. Therefore, this study aimed to evaluate the effects of MgSO₄ injection on testicular IR injury in rats.

Material and Methods

Animals. In total, 50 healthy adult male Wistar rats with a mean weight of 270 g were purchased from the Pasteur Institute and kept in constant room temperature (20±1°C), relative humidity (42±1%), and 12-hour light/ dark cycle. Subsequently, they were randomly divided into five experimental groups of 10 per group. All protocols for animal experiments were approved by the Institutional Animal Ethical Committee, Islamic Azad University, Science and Research Branch, Tehran, Iran (Ethic code: 25876).

Chemicals. The MgSO₄ was purchased from Sigma Chemicals (Poole, Dorset, UK); in addition, the assay kits of malondialdehyde (MDA), SOD, and glutathione biosynthesis (GPx) were obtained from Randox Laboratories (Ltd., Crumlin, Antrim, United Kingdom). The MgSO₄ dosages were selected based on previous reports ( Eshraghi et al., 2015 ; Asghari et al., 2018b ).

Experimental protocol. Anesthesia was accomplished using an intraperitoneal injection of ketamine hydrochloride (60 mg/kg) and xylazine hydrochloride (10 mg/kg) during experimental testicular IR ( Koksal et al., 2012 ). A midline longitudinal incision was made to have access to both testes. Torsion was created by twisting the left testis 720° in a counterclockwise direction and maintained by fixing the testis to the scrotum with a 6-0 nylon suture passing through the tunica albuginea and dartos. The suture was removed 2h post-ischemia, and the left testis was detorted and replaced with scrotum reperfusion continued for 24 h ( Sahin et al., 2005 ). Group 1 was considered as control with no surgery, and group 2 was subjected to 2h of I and 24h of R. Moreover, group 3 was subjected to 2h of I, and after 1 h of I, the rats were injected intraperitoneally with 125 mg/kg MgSO₄ followed by 24h of R. In the similar vein, the rats in group 4 were subjected to 2h of I, and after 1 h of I, they were injected intraperitoneally with 250 mg/kg MgSO₄ followed by 24h of R. Group 5 was also subjected to 2h of I, and after 1 h of I, 500 mg/kg MgSO₄ was administered to the rats intraperitoneally followed by 24h of R. After 2 h of I, the suture was removed and the left testis detorted and replaced into scrotum for 24 h of reperfusion. At the end of the study, the rats were euthanized with pentobarbital (300 mg/kg, IP), the peritoneum was opened, and the left testis was removed. Following that, the testicle was divided into two halves using a sagittal section. The first half of the testicle tissue was fixed in Bouin’s solution, and the second half was stored at -80 °C for the biochemical analysis ( Fakouri et al., 2017 ). The right testis was removed as a control for histological investigations. Diagram 1 describes the entire study protocol.

Diagram 1.

Tissue processing. The tissue was fixed in 7.5 ml saturated picric acid, 2.65 ml glacial acetic acid, and 2.5 mL 7% formaldehyde (Bouin’s solution); additionally, it was post-fixed in 70% alcohol and embedded in paraffin blocks. A 5µm tissue section was obtained, deparaffinized, and stained with Hematoxyline and Eosin. The testicular tissue was evaluated under light microscopy. In the next stage, the testis tissue was fixed at Bouin's solution for complete fixation and processed for paraffin sectioning. A tissue section about 5µm thickness was taken and stained with Hematoxyline and Eosin. The testis sections were graded based on the seminiferous tubule injury according to Johnsen (1970).

Antioxidant activity. The tissue MDA levels were determined by a method based on the reaction with thiobarbituric acid ( Wasowicz et al., 1993 ) and maximum absorption at 532 nm ( Placer et al., 1966 ). The GPx level was measured in the absorbance of 340 nm ( Paglia and Valentine, 1967 ). The total antioxidant status kit was obtained on the basis of suppression in color production which was measured at 600 nm and expressed as mmol/ml ( Paoletti and Mocali, 1990 ; Miller et al., 1993 ).

Statistical analysis. The parametric data were analyzed in SPSS software (version 24) (SPSS, Inc., Chicago, IL, USA) through a one-way analysis of variance and expressed as mean±standard error. In case of heterogeneity occurrence, the groups were separated using Duncan Multiple Range Test. Moreover, the Kruskal-Wallis test was used to compare group medians for histopathological scores. A p-value less than 0.05 was considered statistically significant.

Results

The I/R had the lowest testis damage grade, compared to other groups (P<0.05) (Figure 1).

Figure 1. score of assessing in testis associated with seminiferous tubules injury in experimental I/R rat. Different letters (a-d) indicate significant differences between treatments (P<0.05).

On the other hand, the testis damage grade was the highest in the control group (P>0.05). A significant difference was observed between MgSO₄ treated groups and group 2 regarding testis damage grade (P<0.05). Moreover, no difference was observed among groups 3, 4, and 5 regarding the administration of MgSO₄ at different dosages (P>0.05). According to Table 1, there is a significant increase in the tissue MDA levels in group 2 (P<0.05), whereas the MgSO₄ dosages of 125, 250, and 500 mg/kg (groups 3-5) decrease I/R-induced MDA (P<0.05). Moreover, experimental I/R decreases GPx and SOD activity significantly (P<0.05); however, the injection of the MgSO₄ (125, 250, and 500 mg/kg) elevates SOD and GPx activity significantly (P<0.05). There is no significant difference among the experimental groups in terms of total antioxidant status (P>0.05). Furthermore, the left and right testis sections of group 1 reveal normal seminiferous tubules and spermatogenesis with spermatocytes, Sertoli, and spermatozoa (Figure 2).

Figure 2. Testis section of left testis in control rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (Left). Testis section of right testis in control rats showing normal seminiferous tubules with spermatogonia (black arrow), spermatocyte (black arrow head) and many spermatozoa (white arrow) (Right). H & E: Hematoxylin and Eosin.

Degeneration of seminiferous tubules and loss of spermatogenesis with few spermatocytes were observed in left degenerated testis tubules in group 2. However, there was no significant effect on the right testis (Figure 3). Furthermore, degeneration of seminiferous tubules and loss of spermatogenesis with few spermatocytes were observed on the left testis following the injection of the MgSO₄ (125 mg/kg) (Figure 4). However, no significant effect was observed on the right testis (Figure 4). As can be seen in Figure 5, the IP administration of the MgSO₄ (125 mg/kg) followed by I/R improved testis characteristics with few normal seminiferous tubules and spermatocyte in the testis of the rats in group 2. Moreover, the administration of MgSO₄ (500 mg/kg) improved testis characteristics with normal seminiferous tubules and spermatocyte in the testis of the rats in group 2 (Figure 6).

Figure 3. Testis section of left testis in I/R rats showing degenerated seminiferous tubules (arrow) and loss of spermatogenesis (H&E) (Left) and testis section of right testis in I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (Right). H & E: Hematoxylin and Eosin.

Figure 4. Testis section of left testis in the MgSO₄ (125 mg/kg) followed by I/R rats showing seminiferous tubules (Arrow) with few spermatocyte and interstitial cells (Arrow head) between tubules (Left) and testis section of right testis in the MgSO₄ (125 mg/kg) followed by I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules (Right). H & E: Hematoxylin and Eosin. MgSO₄: magnesium sulfate.

Figure 5. Testis section of left testis in the MgSO₄ (250 mg/kg) followed by I/R rats showing seminiferous tubules (Arrow) with few spermatocyte and interstitial cells (Arrow head) between tubules (Left) and testis section of right testis in the MgSO₄ (250 mg/kg) followed by I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules. H & E: Hematoxylin and Eosin.. MgSO₄: magnesium sulfate.

Figure 6. Testis section of left testis in the MgSO₄ (500 mg/kg) followed by I/R rats showing many normal seminiferous tubules (arrow) (H&E) (Left) with few spermatocyte (Arrow head) and testis section of right testis in the MgSO₄ (500 mg/kg) followed by I/R rats showing normal seminiferous tubules (Arrow) and interstitial cells (Arrow head) between tubules. H & E: Hematoxylin and Eosin. MgSO₄: magnesium sulfate.

Discussion

There is little evidence regarding the effects of MgSO₄ on male infertility and I/R injury. This is the first study that has determined the effects of MgSO₄ on semen MDA, SOD, and GPx in I/R injuries. As observed, MgSO₄ dependently decreased MDA and increased SOD and GPx activities in I/R-induced rats. Moreover, seminiferous tubules degenerated, and loss of spermatogenesis with few spermatocytes were detected in the degenerated testis tubules among I/R rats. In addition, the repeated succession of IR injury in testicular cells caused many biochemical and morphological changes which might lead to lipid peroxidation, protein denaturation, DNA damage, and apoptosis ( Kanter, 2010 ). In the past few years, several anti-inflammatory antioxidants and free-radical scavengers were utilized for the treatment of testicular I/R, which induced male infertility. The MgSO₄ improved testis characteristics in experimental I/R-induced rats. The imbalance between ROS generation and the antioxidants leads to oxidative stress ( Hwang and Lamb, 2012 ). It is reported that more than 60% of male infertility happens via ROS mediated sperm damage ( Agarwal et al., 2014 ).

Additionally, it has been revealed that semen oxidation acts via an increase in ROS levels and a decrease in antioxidant capacity ( Hsieh et al., 2006 ), which leads to infertility ( Masson and Brannigan, 2014 ). The low levels of ROS are critical for normal fertilization, capacitation, hyperactivation, and motility ( Agarwal et al., 2009 ). Spermatozoa have high levels of polyunsaturated fatty acids, which are vulnerable to attacks by ROS ( Ghalehkandi, 2018 ). Seminal plasma is endowed with MDA, SOD, and GPx ( Ghalehkandi, 2018 ). During the oxidation, MDA levels are increased followed by decreases in SOD and GPx status ( Hsieh et al., 2006 ). The ROS reacts with these enzymes via cell membrane lipid oxidative damage in sperm ( Akhondi et al., 2013 ). Magnesium plays a prominent role in the human reproductive system, semen, and fertilization ( Valsa et al., 2012 ). Moreover, seminal plasma has a critical role in the protection of sperms and acts as a buffer for sperm motility. Semen is composed of lipids, ions (e.e., citrate), calcium, Mg²⁺, K+, Na+, zinc, chloride, proteins, and oxidative enzymes that protect the sperm from oxidative stress ( Agarwal et al., 2013 ). Magnesium has been associated with oxidative enzymes and its depletion correlated with reduced antioxidant properties. It is reported that Mg²⁺ has an influence on SOD activity in retinal tissue ( Korkmaz et al., 2013 ). Moreover, Mg²⁺ is required for GPx ( Barbagallo et al., 2010 ). Oxidative stress is responsible for glaucoma pathogenesis and Mg²⁺-induced oxidative enzyme levels can contribute to the progression of glaucoma ( Korkmaz et al., 2013 ). Diminished cellular Mg²⁺ levels alter ATPase function and increase oxidative stress ( Agarwal et al., 2013 ). Magnesium has neuroprotective activity in the brain and spinal cord ischemia ( Yavuz et al., 2013 ). The injection of the Mg²⁺ reduced infarct size after I/R injury via decreasing generation and releasing free oxygen radicals in the left anterior descending artery ( Ravn et al., 1999 ). The Mg²⁺ insufficiency might affect Na+ fluxes and elevated intracellular Na+, which might lead to an increased Ca2+. The severe ionic disturbances have been observed in I/R injury ( Huang et al., 2014 ). One possible mechanism of this effect is that Mg²⁺ may reduce endothelial and neuronal reperfusion injury by minimizing the Mg²⁺ use and lipid peroxidation. Furthermore, Mg²⁺ regulates ATP availability in the reperfusion phase ( Yavuz et al., 2013 ). Eshraghi et al. (2015) reported that Mg²⁺ protected the liver against bile duct ligation-induced in rats. Based on their report, the protective activity of the Mg²⁺mediates by diminishing MDA and increasing SOD and CAT activities ( Eshraghi et al., 2015 ). The cellular Mg²⁺ has different functions in glycolysis, protein synthesis, respiration, and reproduction ( Chandra et al., 2013 ). Testicular plasma and semen contain high levels of Mg 2+ ( Wong et al., 2001 )( The MgSO₄ treatment improved seminiferous tubules with many spermatocytes in the testes of experimental unilateral varicocele established rats ( Asghari et al., 2018b ). Magnesium isoglycyrrhizinate decreased MDA and increased SOD and GPx, which protected I/R-induced hepatic injury ( Huang et al., 2014 ). It is reported that the number of spermatogonia A, preleptotene spermatocytes, mid-pachytene, spermatocytes, and spermatid was increased in Mg²⁺ treated animals. There have also been reports that revealed Mg²⁺ deficiency-induced morphological changes up to 40% in the spermatids ( Chandra et al., 2013 ). Previous reports have mostly investigated the effects of Mg²⁺ on hepatic or cardiac I/R; moreover, they evaluated the hepatic enzymes, as well as inflammatory factors with limited information, existed on the ROS. Accordingly, there were no previous reports on the role of the Mg²⁺ in testicular I/R-injury to be compared with our results. In conclusion, new findings of the current study suggested that treatment with MgSO₄ had beneficial effects on I/R-induced rats. In addition, MgSO₄ improved seminiferous tubules with normal spermatocytes by increasing the oxidative defense system and decreasing ROS generation in I/R-induced rats. Further studies are required to determine the direct cellular and molecular actions of MgSO₄ against I/R injuries in rats and other species.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Conflict of Interest

The authors declare that they have no conflict of interest.

Authors’ Contribution

Study concept and design: Asghari, A., Abedi, G.

Acquisition of data: Moshkelani, S.

Analysis and interpretation of data: Asghari, A., Mortzavi, P.

Drafting of the manuscript: Asghari, A., Moshkelani, S.

Critical revision of the manuscript for important intellectual content: Asghari, A.

Statistical analysis: Jahandideh, A., Asghari, A.

Administrative, technical, and material support: Science and Research Branch Islamic Azad University, Tehran, Iran

References

1. Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male infertility. Reprod Biomed Online. 2014; 29(1):32-58.
2. Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares A, Sabanegh E. Role of oxidative stress in pathogenesis of varicocele and infertility. Urology. 2009; 73(3):461-9.
3. Agarwal R, Iezhitsa I, Awaludin NA, Ahmad Fisol NF, Bakar NS, Agarwal P, et al. Effects of magnesium taurate on the onset and progression of galactose-induced experimental cataract: in vivo and in vitro evaluation. Exp Eye Res. 2013; 110:35-43.
4. Akhondi MM, Mohazzab A, Jeddi-Tehrani M, Sadeghi MR, Eidi A, Khodadadi A, Piravar Z. Propagation of human germ stem cells in long-term culture. Iran J Reprod Med. 2013; 11(7):551-8.
5. Arena S, Iacona R, Antonuccio P, Russo T, Salvo V, Gitto E. Medical perspective in testicular ischemia-reperfusion injury. Exp Ther Med. 2017; 13(5):2115-2122.
6. Asghari A, Akbari G, Beigi AM, Mortazavi P. Tramadol reduces testicular damage of ischemia-reperfusion rats. Anim Reprod. 2018; 13(4):811-9.
7. Asghari A, Akbari G, Galustanian G. Magnesium sulfate protects testis against unilateral varicocele in rat. Anim Reprod. 2018; 14(2):442-51.
8. Aydıner CY, Pul M, İnan M, Bilgi S, Çakır E. Can N-acetylcysteine play a role on preventing tissue damage on experimental testicular torsion. Cum Med J. 2012; 34:462-71.
9. Barbagallo M, Dominguez LJ, Galioto A, Pineo A, Belvedere M. Oral magnesium supplementation improves vascular function in elderly diabetic patients. Magnes Res. 2010; 23(3):131-7.
10. Celik E, Oguzturk H, Sahin N, Turtay MG, Oguz F, Ciftci O. Protective effects of hesperidin in experimental testicular ischemia/reperfusion injury in rats. Arch Med Sci. 2016; 12(5):928-934.
11. Chandra AK, Sengupta P, Goswami H, Sarkar M. Effects of dietary magnesium on testicular histology, steroidogenesis, spermatogenesis and oxidative stress markers in adult rats. Indian J Exp Biol. 2013; 51(1):37-47.
12. Eghbali M, Alavi SS, Asri RS, Khadem AM. Calcium, magnesium and Total Antioxidant Capacity (TAC) in seminal plasma of Water Buffalo (Bubalus bubalis) bulls and their relationships with semen characteristics. Vet Res Forum. 2010; 1: 12-20.
13. Eshraghi T, Eidi A, Mortazavi P, Asghari A, Tavangar SM. Magnesium protects against bile duct ligation-induced liver injury in male Wistar rats. Magnes Res. 2015; 28(1):32-45.
14. Fakouri A, Asghari A, Akbari G, Mortazavi P. Effects of folic acid administration on testicular ischemia/reperfusion injury in rats. Acta Cir Bras. 2017; 32(9):755-766.
15. Ghalehkandi JG. Garlic (Allium sativum) juice protects from semen oxidative stress in male rats exposed to chromium chloride. Anim Reprod. 2018; 11: 526-532.
16. Hadwan MH, Almashhedy LA, Alsalman AR. Study of the effects of oral zinc supplementation on peroxynitrite levels, arginase activity and NO synthase activity in seminal plasma of Iraqi asthenospermic patients. Reprod Biol Endocrinol. 2014; 12:1.
17. Hasturk AE, Harman F, Arca T, Sargon M, Kilinc K, Kaptanoglu E. Neuroprotective effect of magnesium sulfate and dexamethasone on intrauterine ischemia in the fetal rat brain: ultrastructural evaluation. Turk Neurosurg. 2013; 23(5):666-71.
18. Hsieh YY, Chang CC, Lin CS. Seminal malondialdehyde concentration but not glutathione peroxidase activity is negatively correlated with seminal concentration and motility. Int J Biol Sci. 2006; 2(1):23-9.
19. Huang X, Qin J, Lu S. Magnesium isoglycyrrhizinate protects hepatic L02 cells from ischemia/reperfusion induced injury. Int J Clin Exp Pathol. 2014; 7(8):4755-64.
20. Hwang K, Lamb DJ. Molecular mechanisms of antioxidants in male infertility. Male infertility, Springer; 2012 pp. 245-260.
21. Johnsen SG. Testicular biopsy score count--a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970; 1(1):2-25.
22. Kanter M. Protective effects of melatonin on testicular torsion/detorsion-induced ischemia-reperfusion injury in rats. Exp Mol Pathol. 2010; 89(3):314-20.
23. Koksal M, Oğuz E, Baba F, Eren MA, Ciftci H, Demir ME. Effects of melatonin on testis histology, oxidative stress and spermatogenesis after experimental testis ischemia-reperfusion in rats. Eur Rev Med Pharmacol Sci. 2012; 16(5):582-8.
24. Korkmaz Ş, Ekici F, Ali Tufan H, Aydın B. Magnesium: Effect on ocular health as a calcium channel antagonist. J Clin Exper Invest. 2013; 4(2): 244-251.
25. Masson P, Brannigan RE. The varicocele. Urol Clin North Am. 2014; 41: 129-144.
26. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (Lond). 1993; 84(4):407-12.
27. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967; 70(1):158-69.
28. Paoletti F, Mocali A. Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. Methods Enzymol. 1990; 186:209-20 .
29. Parlaktas BS, Atilgan D, Ozyurt H, Gencten Y, Akbas A, Erdemir F. The biochemical effects of ischemia-reperfusion injury in the ipsilateral and contralateral testes of rats and the protective role of melatonin. Asian J Androl. 2014; 16(2):314-8.
30. Pesch S, Bergmann M, Bostedt H. Determination of some enzymes and macro- and microelements in stallion seminal plasma and their correlations to semen quality. Theriogenology. 2006; 66(2):307-13.
31. Placer ZA, Cushman LL, Johnson BC. Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Anal Biochem. 1966; 16(2):359-64.
32. Pogorelić Z, Mustapić K, Jukić M, Todorić J, Mrklić I, Mešštrović J. Management of acute scrotum in children: a 25-year single center experience on 558 pediatric patients. Can J Urol. 2016; 23(6):8594-8601.
33. Raju AB, Challa SR, Akula A, Kiran K, Harinadh GB. Evaluation of oxidant and anti-oxidant balance in experimentally induced testicular injury by ischemia reperfusion in rats. Eur J Gen Med. 2011; 8(2):117-21.
34. Ravn HB, Moeldrup U, Brookes CI, Ilkjaer LB, White P, Chew M. Intravenous magnesium reduces infarct size after ischemia/reperfusion injury combined with a thrombogenic lesion in the left anterior descending artery. Arterioscler Thromb Vasc Biol. 1999; 19(3):569-74.
35. Romani A. Regulation of magnesium homeostasis and transport in mammalian cells. Arch Biochem Biophys. 2007; 458:90-102.
36. Sahin Z, Bayram Z, Celik-Ozenci C, Akkoyunlu G, Seval Y, Erdogru T. Effect of experimental varicocele on the expressions of Notch 1, 2, and 3 in rat testes: an immunohistochemical study. Fertil Steril. 2005 ; 83(1):86-94.
37. Valsa J, Skandhan KP, Khan PS, Sumangala B, Gondalia M. Split ejaculation study: semen parameters and calcium and magnesium in seminal plasma. Cent European J Urol. 2012; 65(4):216-8.
38. Wasowicz W, Nève J, Peretz A. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: importance of extraction pH and influence of sample preservation and storage. Clin Chem. 1993; 39(12):2522-6.
39. Wolf FI, Trapani V, Simonacci M, Boninsegna A, Mazur A, Maier JA. Magnesium deficiency affects mammary epithelial cell proliferation: involvement of oxidative stress. Nutr Cancer. 2009; 61(1):131-6.
40. Wong WY, Flik G, Groenen PM, Swinkels DW, Thomas CM, Copius-Peereboom JH. The impact of calcium, magnesium, zinc, and copper in blood and seminal plasma on semen parameters in men. Reprod Toxicol. 2001; 15(2):131-6.
41. Yavuz Y, Mollaoglu H, Yürümez Y, Ucok K, Duran L, Tünay K. Therapeutic effect of magnesium sulphate on carbon monoxide toxicity-mediated brain lipid peroxidation. Eur Rev Med Pharmacol Sci. 2013; 17 Suppl 1:28-33.
42. Yuluğ E, Türedi S, Alver A, Türedi S, Kahraman C. Effects of resveratrol on methotrexate-induced testicular damage in rats. Sci World J. 2013; 2013: 489659.