Echis ocellatus Venom-Induced Reproductive Pathologies in Rat Model; Roles of Oxidative Stress and Pro-Inflammatory Cytokines

This study reported reproductive pathologies associated with Echis ocellatus venom in animal model. Twenty male Wistar rats with body weight between 180 and 220 g were selected randomly into two groups (n = 10). Rats in group 1 served as the control while rats in group 2 were envenomed with a single intraperitoneal injection of 0.055 mg/kg−1 (LD6.25) of E. ocellatus venom on the first day and a repeated dose on the twenty fifth day. Both control and envenomed rats were monitored for fifty consecutive days. The venom caused a significant (p < 0.05) reduction in sperm motility, count, and volume, with increased sperm anomalies in envenomed rats compared to the control. Likewise, serum concentrations of male reproductive hormones were significantly (p < 0.05) higher in envenomed rats. Increased levels of malondialdehyde were accompanied by a significant (p < 0.05) decrease in reduced glutathione and catalase activity in the epididymis and testis tissues of envenomed rats. The venom enhanced the release of epididymal and testicular tumor necrosis factor-alpha and interleukin1-beta compared to the control. Furthermore, severe pathological defects were noticed in tissues of the testis and epididymis of envenomed rats. This study demonstrated that E. ocellatus venom toxins can induce reproductive dysfunction in male victims of snake envenoming.


Introduction
Reproductive dysfunction is a malady of the reproductive system that has evolved as a serious global public health challenge [1,2]. Reproductive dysfunction resulting from pathological conditions in males is a complex physiological process that is associated with different risks and pathological mechanisms that could directly or indirectly affect male reproductive functions and predispose to the development of infertility [3]. Globally, male reproductive infertility has been a significant health problem since the mid-1950s which is on the rise and seriously becoming a public health concern in this age, attracting the focus of clinicians and researchers in recent decades [1,4].
Researchers have documented several factors that may be liable for the impairment of reproductive function in males, which may be psychological resulting from stress or trauma, pathological due to inflammation or infection, physiological as a result of body constitution venom metalloproteinases (SVMPs) and phospholipase A2, which possesses hemorrhagic, cytotoxic, myotoxic, neurotoxic, cardiotoxic, and anticoagulant effects [24,25]. Based on our findings, there are limited toxicological studies that evaluate the impact of snake venom toxins on the reproductive system combined with a paucity of information regarding the impact of viper venom on reproductive functions. The study on pathological conditions of male reproductive system is essential so as to unravel reproductive inefficiency that may arise in envenomed patients. Accordingly, this current study aimed to broaden scientific knowledge on the pathological effects of viper envenoming on male reproductive physiology by accessing the roles of oxidative stress and elevated pro-inflammatory cytokines in E. ocellatus envenomed male rats.

Clinical Signs of Toxicity
The envenomed rats showed several clinical signs of toxicity, including dizziness, sluggish movements, decrease in food and water consumption, and mild heamorrhage noticed at the site of venom injection. Also, one rat died on the 19th day of the experiment in the envenomed group. However, these clinical signs of toxicity were absent in control rats (Table 1). Body weight gain of the control was significantly (p < 0.05) higher compared to the envenomed rats. Likewise, the testicular weight and testiculo somatic index of envenomed rats significantly (p < 0.05) decreased compared to the control group (Table 2).  Data are represented as mean ± standard error (n = 5). Values in the same column with different superscript are considered significant (p < 0.05).

Epididymal Sperm Parameters
A significant (p < 0.05) decline was noticed in the percentage of motile spermatozoa in envenomed rats compared to the control, resulting in a significant (p < 0.05) increase in the percentage of immotile spermatozoa in envenomed rats (Table 3). Moreover, sperm count and volume showed a significant (p < 0.05) reduction in rats injected with venom when compared to control (Table 3).

Sperm Abnormalities in Envenomed Rats
The venom showed various toxic effects on sperm morphology in the envenomed rats. The percentage of sperm abnormalities in envenomed rats was significantly (p < 0.05) Toxins 2022, 14, 378 4 of 15 higher compared to the control. Higher abnormalities of sperm cells with banana shape, folded sperm, no hook, and wrong-angled hook were noticed in the envenomed rats and values obtained were significantly (p < 0.05) different compared to the control. Unique sperm abnormalities not found in the control but present in envenomed rats are abnormal mid-piece, double tail, and double head (Table 4).

Reproductive Hormone Concentrations
The levels of male sex hormones in the serum of experimental rats are presented in Table 5. There was a significant (p < 0.05) elevation in the concentration of testosterone, follicle stimulating hormones (FSH), and luteinizing hormone (LH) in serum of envenomed rats when compared to the control.

Oxidative Stress Parameters
The results of oxidative stress status of the envenomed rats showed that levels of malondialdehyde (MDA) in testis and epididymis tissues of envenomed rats were significantly (p < 0.05) higher compared to the control ( Figure 1). On the other hand, there was a significant (p < 0.05) reduction in levels of glutathione in testis and epididymis tissues of the envenomed compared to the control ( Figure 2). Moreover, the activity of catalase (CAT) in epididymis tissue of envenomed rats was significantly (p < 0.05) lower compared to the control. However, there was no significant (p < 0.05) difference in the activity of CAT in the testis of control and envenomed rats, but a marked increase in CAT activities was recorded in control when compared to the envenomed rats ( Figure 3) icantly (p < 0.05) higher compared to the control (Figure 1). On the other hand, there was a significant (p < 0.05) reduction in levels of glutathione in testis and epididymis tissues of the envenomed compared to the control ( Figure 2). Moreover, the activity of catalase (CAT) in epididymis tissue of envenomed rats was significantly (p < 0.05) lower compared to the control. However, there was no significant (p < 0.05) difference in the activity of CAT in the testis of control and envenomed rats, but a marked increase in CAT activities was recorded in control when compared to the envenomed rats (  the envenomed compared to the control ( Figure 2). Moreover, the activity of catalase (CAT) in epididymis tissue of envenomed rats was significantly (p < 0.05) lower compared to the control. However, there was no significant (p < 0.05) difference in the activity of CAT in the testis of control and envenomed rats, but a marked increase in CAT activities was recorded in control when compared to the envenomed rats (

Pro-Inflammatory Cytokines Production
The venom significantly (p < 0.05) up-regulated interleukin1-beta (IL-1β) production in epididymis and testis tissues of envenomed rats compared to the control ( Figure 4). Likewise, there was a significant (p < 0.05) elevation of tumor necrosis factor-alpha (TNF-

Pro-Inflammatory Cytokines Production
The venom significantly (p < 0.05) up-regulated interleukin1-beta (IL-1β) production in epididymis and testis tissues of envenomed rats compared to the control ( Figure 4). Likewise, there was a significant (p < 0.05) elevation of tumor necrosis factor-alpha (TNF-α) responses in the epididymis and testis tissues of envenomed rats when compared to the control ( Figure 5).

Pro-Inflammatory Cytokines Production
The venom significantly (p < 0.05) up-regulated interleukin1-beta (IL-1β) production in epididymis and testis tissues of envenomed rats compared to the control ( Figure 4). Likewise, there was a significant (p < 0.05) elevation of tumor necrosis factor-alpha (TNFα) responses in the epididymis and testis tissues of envenomed rats when compared to the control ( Figure 5).

E -I L 1
T  Groups TNF levels (Pg/ML) Figure 5. Tumor necrosis factor-alpha (TNF-α) responses in epididymis and testis tissues after E. ocellatus envenomation. Data are expressed as mean ± standard error, (n = 5). Values with the same superscript are considered not significant (p < 0.05). E-TNF: Epididymis Tumor necrosis factor-alpha, T-TNF: Testis Tumor necrosis factor-alpha.

Histopathology
The slides of testis tissues of control rats showed a closely packed seminiferous tubules with no observable defect, whereas testis tissues of envenomed rats revealed an inflammatory response in the dermis, a distorted germinal epithelium, degeneration of the seminiferous tubules, and tubular atrophy ( Figure 6). Moreover, histological examination of epididymis tissues of envenomed rats revealed inflammatory infiltrate in cauda, atrophy of the tubules, and accentuation of interstitium, while epididymis tissue of the control showed a normal cauda with no observable lesion (Figure 7).

Histopathology
The slides of testis tissues of control rats showed a closely packed seminiferous tubules with no observable defect, whereas testis tissues of envenomed rats revealed an inflammatory response in the dermis, a distorted germinal epithelium, degeneration of the seminiferous tubules, and tubular atrophy ( Figure 6). Moreover, histological examination of epididymis tissues of envenomed rats revealed inflammatory infiltrate in cauda, atrophy of the tubules, and accentuation of interstitium, while epididymis tissue of the control showed a normal cauda with no observable lesion ( Figure 7). pha, T-TNF: Testis Tumor necrosis factor-alpha.

Histopathology
The slides of testis tissues of control rats showed a closely packed seminiferous tubules with no observable defect, whereas testis tissues of envenomed rats revealed an inflammatory response in the dermis, a distorted germinal epithelium, degeneration of the seminiferous tubules, and tubular atrophy ( Figure 6). Moreover, histological examination of epididymis tissues of envenomed rats revealed inflammatory infiltrate in cauda, atrophy of the tubules, and accentuation of interstitium, while epididymis tissue of the control showed a normal cauda with no observable lesion (Figure 7).

Discussion
The venom of E. ocellatus contains numerous toxic enzymes, majorly snake venom metalloproteinases (SVMPs) that interfere with different biological systems of the body, eliciting severe clinical complications in envenomed victims [25]. This study assessed the toxicological effects of E. ocellatus venom on reproductive functions initiated through the induction of oxidative stress (OS) and modulation of pro-inflammatory cytokines in testis and epididymis of rats injected with the venom. Findings from this study revealed that E. ocellatus venom caused a significant reduction in body weight, testicular weight, and organo-somatic index compared to the control, suggesting that the venom may have a direct interaction with the testicular tissues resulting in testicular toxicity. Studies have reported such an observation in the testis of mice after exposure to toxicants [26].
Spermatozoa matures in the epididymis with some required processes, including sperm plasma membrane remodeling, membrane protein reordering, enzyme modification, and nuclear reconfiguration [27]. In this study, E. ocellatus venom caused a significant reduction in the epididymal sperm motility, sperm count, and volume in the envenomed rats which may be due to OS induction in the epididymis as there was a significant increase in levels of OS biomarker; malonaldehyde in testicular and epididymal tissues of the envenomed rat. In addition, the epididymis possesses hormonally sensitive tissues

Discussion
The venom of E. ocellatus contains numerous toxic enzymes, majorly snake venom metalloproteinases (SVMPs) that interfere with different biological systems of the body, eliciting severe clinical complications in envenomed victims [25]. This study assessed the toxicological effects of E. ocellatus venom on reproductive functions initiated through the induction of oxidative stress (OS) and modulation of pro-inflammatory cytokines in testis and epididymis of rats injected with the venom. Findings from this study revealed that E. ocellatus venom caused a significant reduction in body weight, testicular weight, and organo-somatic index compared to the control, suggesting that the venom may have a direct interaction with the testicular tissues resulting in testicular toxicity. Studies have reported such an observation in the testis of mice after exposure to toxicants [26].
Spermatozoa matures in the epididymis with some required processes, including sperm plasma membrane remodeling, membrane protein reordering, enzyme modification, and nuclear reconfiguration [27]. In this study, E. ocellatus venom caused a significant reduction in the epididymal sperm motility, sperm count, and volume in the envenomed rats which may be due to OS induction in the epididymis as there was a significant increase in levels of OS biomarker; malonaldehyde in testicular and epididymal tissues of the envenomed rat. In addition, the epididymis possesses hormonally sensitive tissues that may undergo changes due to effects of toxicants and become senescent [28]. Pasqualotto et al. [28] had suggested that epididymal senescence may lead to a decrease in sperm motility. The resultant increase in immotile sperms with a decline in sperm count and volume post envenomation in rats is a consequence of oxidative damage resulting from enhanced ROS levels in sperm plasma membrane [29]. Normal cell activity must be protected by ceaseless elimination of surplus ROS, and in situations where the antioxidant defence system is overpowered due to a surge in production of free radicals or the endogenous antioxidant capacity is overwhelmed by elevated levels of seminal plasma ROS, the consequence may be detrimental to semen parameters [30], as observed in this study. However, such physiological defects on sperm cells have been reported using a viper venom [12].
In this present study, E. ocellatus venom caused a significant increase in sperm abnormalities and OS factor could be responsible for the induction of sperm anomalies as levels of lipid peroxidation (LPO) significantly increased, resulting in the depletion of glutathione (GSH) and catalase contents in the testis and epididymis of envenomed rats. GSH is a defensive mechanism for sperm cells, eliminating free radicals using its free sulphydryl class through direct interplay with ROS, and sperm plasma membranes are protected from cytotoxic aldehydes released during LPO by the direct reaction of GSH present in the extracellular space [31]. On the other hand, LPO is known to cause the quick depletion of adenosine triphosphate (ATP) in sperm cells, leading to reduction, impaired motility, disruption in acrosome membrane, and depletion in potency to fuse with the ovum [31]. According to Atig et al. [32], defective motility of spermatozoa as a result of instability of the mid-piece is a consequence of LPO of the sperm plasma membrane due to insufficient production of GSH. Therefore, reduction in the antioxidant factors may explain sperm anomalies, such as abnormal mid-piece and decreased sperm motility in the envenomed group, as observed in this study. These factors are known to cause infertility in males, thus substantiating the ability of E. ocellatus venom to induce male reproductive system dysfunction in envenomed victims. Moreover, E. ocellatus venom-induced OS in vital organs of envenomed rats has been reported [33]. However, the mechanism of the venom toxins inducing OS is not yet clear and needs further investigation.
In addition, studies have reported that induction of sperm abnormality by toxicants may be a result of generated hydroxyl radicals (OH-) interacting with DNA in the sperm heads, which may wreak havoc on DNA integrity [34]. The observed venom induced-sperm abnormalities may suggest a genotoxic effect resulting from ROS. It has been established that elevated ROS generation and depleted antioxidant capabilities could result in sperm DNA fragmentation, either by direct or indirect effects, via the stimulation of sperm caspases and endonuclease generation [35,36]. Consequently, apoptosis may occur resulting from DNA fragmentation induced by ROS. Likewise, disruption of the mitochondrial membrane attributed to an upsurge in ROS production could trigger the release of cytochrome-C signaling molecule, which may activate apoptotic caspases and annexin-V phosphatidylserine binding activity [37]. Elevated cytochrome-C release in the seminal plasma of infertile men may result in significant damage to the mitochondria [38].
Li et al. [39] has established that the production of sperm and processes are managed by a complex-regulation of the neuroendocrine hypothalamic-pituitary gonadal axis in concert with local testicular steroids. Reproductive hormones, such as testosterone, LH, and FSH, which are male sex hormones, play a crucial role in spermatogenesis. In this study, it was observed that E. ocellatus venom caused a significant increase in levels of testosterone, FSH, and LH, suggesting that the venom toxins may have an impact on the biological process of steroid hormone production in the testis, thereby interfering with the hypothalamic-pituitary-gonadal axis [39]. A significant elevation of testosterone concentration in the serum of envenomed rats may arise due to an increase in genes that are liable for testosterone biosynthesis [39]. Furthermore, harmful feedback mechanisms on the hypothalamus and pituitary gland may cause a rise in testosterone concentration, leading to Leydig cell dysfunction resulting in spermatogenesis disorder [40]. On the other hand, FSH is an important hormone that plays a crucial role in testicular development combined with sperm production. However, a significant increase in concentration of serum FSH is a dependable baseline for main testicular failure, low sperm count, zero sperm in semen, and genetic inborn error [41]. In this study, significant upregulation of FSH in serum of envenomed rats may indicate damage associated with the germinal cell [42]. This result suggests that the venom could induce male sex hormonal imbalances, thereby disrupting the process of spermatogenesis and ultimately resulting in male infertility. However, the mechanism of the venom inducing hormonal dysregulation is not yet clear.
Cytokines are small proteins secreted by the immune cells, and a moderate amount of these proteins is required to maintain the physiological functions of cells inside the testes [16]. Sertoli or spermatogenic cells produce a cautious amount of inflammatory cytokines, such as TNF-α and IL-1β, during the maturation cycles of the seminiferous epithelium, an indication they play a crucial role in controlling this basic attribute of testicular functions [43]. Detection of cytokines in typical seminiferous tubule cross-sections of the adult testis reflects their temporally regulated synthesis and function. They serve important roles in regulating steroidogenesis and immunoregulation while also contributing to pathophysiology and detrimental effects of inflammatory responses on testicular functions [43,44]. In this present study, E. ocellatus venom upregulated levels of IL1-β and TNF-α in testis and epididymis of the envenomed rats, suggesting that toxins in the venom can modulate pro-inflammatory cytokine release, which corroborated our earlier findings on elevated IL1-β and TNF-α in vital organs following E. ocellatus envenomation [33]. Studies have reported that increase in production of pro-inflammatory mediators, most especially IL1-β and TNF-α, could trigger elevated ROS formation and activate inflammatory processes, which have consequent damaging effects on normal functions of the testicular cells, particularly Sertoli and Leydig cells [45,46]. Furthermore, findings have established that up-regulation in responses of some specific inflammatory cytokines is an indication of initiated testis pathologies and revealed that significant high levels of transcripts encoding pro-inflammatory cytokines, such as IL1-β and TNF-α, are responsible for pathologies in testis cancer samples [47]. Reports from other related studies have detailed that elevated release of IL1-β and TNF-α induces inflammatory lesions, disrupts spermatogenesis [48], or causes other pathophysiological diseases [49].
A well-established report has revealed that tissue damage could occur following alteration in intracellular components, proteins, nucleic acid, and lipids due to an upsurge in ROS production in organ cells [50]. Histopathological examination revealed evidence of gross lesions in the testis tissues and inflammatory response while cauda inflammation, atrophy of tubules, and accentuation of interstitium were noticed in the epidermis of the envenomed rats. These are signs of tissue degeneration and evidence of organ toxicity due to the venom's action on the male reproductive organs of the envenomed rats. This could probably be a prerequisite for the observed sperm anomalies produced by the defective testes. The observed pathologies on reproductive organs of envenomed rats further substantiated our previous reports that E. ocellatus venom is capable of inducing histological alterations on vital organs after envenoming [33]. The reproductive organ defects observed in envenomed rats in this study may have been significantly influenced by the action of the venom toxins through the induction of OS. It should be noted that OS has been implicated in cellular damage and chronic diseases, resulting in malfunctioning of reproductive functions of male organs with harmful effects on male fertility [5].
Finally, studies have established the mechanism underlying OS-induced male reproductive dysfunction and reported that OS alters sperm parameters with damaging effects on their structures and functions through actuated interconnected processes with LPO production of the sperm membrane, spermatozoa intracellular oxidative damage, altered sperm DNA, and apoptotic pathway activation in the germ cells [35], and these represent the underlying mechanisms of action that may be applicable to our findings in this current study.

Conclusions
This current study has demonstrated that E. ocellatus venom has the intrinsic ability to induce reproductive toxicity and disrupt male reproductive functions in envenomed rats. Findings from this study indicate that toxins in E. ocellatus venom have the potential to incapacitate viable sperm cells, disrupt sperm production, and cause damage to the testis, such as injuries to seminiferous tubules, degeneration of Leydig cells, and necrosis of spermatogenic cells, combined with endocrine disruption. Without any doubt, these toxic effects resulting from oxidative stress inflicted on the male reproductive organs could cause various reproductive anomalies in envenomed victims. This assumes significance and is a public health concern considering the rising number of snakebite incidences in rural communities of Africa, particularly in Nigeria. Therefore, findings from this present study will draw more consciousness towards the effective treatment of snakebite envenoming to prevent systemic toxicity that could cause reproductive dysfunction resulting in male infertility, most especially in rural communities where there are challenges in accessing effective antivenom treatment.

Chemicals and Kits
Rat enzyme-Linked Immunosorbent Assay (ELISA) Kits for FSH, LH, and Testosterone assays were purchased from Bio-Inteco Diagnostic, UK Ltd., Ken House, London, UK. Enzyme-linked immunosorbent assay (ELISA) kits manufactured by PeproTech, Inc., London, UK were used for TNF-a and IL-1ß assays. The reagents and chemicals were of good grade and procured from Sigma-Aldrich, St Louis, MO, USA.

Procurement of Snake Venom
A lyophilized sample E. ocellatus venom was procured from herpetarium of the Department of Pharmacology and Toxicology, Amadu Bello University, Zaria, Nigeria. The venom sample was transported at a temperature of 4 • C to the Animal Physiology Laboratory, Department of Zoology, University of Ibadan, Nigeria. The lyophilized venom was stored at 4 • C in the laboratory until use.

Experimental Rats
Twenty male albino Wistar rats weighing between 180 and 220 g used for this study were obtained from the animal breeding unit of the Department of Zoology, University of Ibadan, Nigeria. The rats were kept in well-ventilated, transparent plastic cages at temperature 27 • C and the maximum number of rats in each cages was five. The animals were fed with standard rat pelletized feed and given water ad libitum. The experimental protocols were approved with assigned number UI-ACUREC: 18/0108 by the University of Ibadan-Animal Care and Research Ethics Committee (UI-ACUREC) and their guidelines were strictly followed. All animal experiments complied with the National Research Council's publication on guide for the care and use of laboratory animals [51].

Study Design
The experimental rats were randomly divided into two groups of ten rats (n = 10) each. Rats in group 1 served as the control and were injected with 0.2 mL of saline while rats in group 2 were envenomed with 0.2 mL of E. ocellatus venom.

Envenoming Procedures
Previous studies from our laboratory have shown the lethal dose of E. ocellatus venom to be 0.22 mg/bodyweight [33]. In this current study, the envenomed rats were injected intraperitoneally with 0.055 mg/kg −1 (LD 6.25 ) of E. ocellatus venom to reduce fatalities to the lowest minimum. The rats were envenomed at 8:00 am on day 1 and injected with a repeated dose in the morning on day 25. The duration of the experiment and monitoring of envenomed animals was fifty days to allow the completion of spermatogenesis [52]. The control rats were injected with 0.2 mL of saline water. The experimental animals were monitored twice daily for clinical signs of toxicity, morbidity, and mortality.

Body Weight Determination
Prior to venom injection, the animals were weighed on the first day for their initial body weight and before they were sacrificed as terminal weight. The body weight gain was calculated using the formula:

Body weight gain =
Terminal weight o f mice − Initial weight o f mice Initial weight o f mice × 100

Collection of Blood and Organ Sample
Post-exposure, blood samples were collected from the experimental rats through retro-orbital sinus punctuation using heparinized capillary tubes into plain bottles and centrifuged at 380 g for 10 min to obtain serum for hormonal assays. Rats were thereafter sacrificed through cervical dislocation following guides [53]. The cauda epididymis was surgically removed for sperm parameters analysis and the testes was removed and weighed. A portion of the epididymis and testes tissues was used for biochemical analysis and histological examination. The relative testes weight was determined using the formula: Relative organ weight = Organ weight Termimal body weight × 100

Epididymal Sperm Parameters
The cauda epididymis was placed individually in a Petri dish and minced in normal saline (1 mL) to form sperm suspension, and 10 µL were placed in triplicate on microscopic slides and observed for motility under the light microscope at a magnification of ×400. Sperm motility was assessed by classifying 200 spermatozoa into two categories, motile and immotile spermatozoa. Three sperm classes were categorized as motile spermatozoa: rapid progressive, slow progressive, and nonprogressive spermatozoa [54]. To evaluate the sperm volume, the epididymis was immersed in 5 mL normal saline in a measuring cylinder and the volume displaced was taken as the volume of the epididymis. For the sperm count, a 1:10 dilution from the sperm suspension was made in a Petri dish. Spermatozoa from the left and right cauda were counted using the improved Neubauer hemocytometer. Each sample was counted three times and averaged. The sperm morphology assay was carried out according to Wyrobek et al. [55]. Sperm suspension was mixed with 1% aqueous eosin Y and smeared on microscopic slides. Abnormalities were observed in 250 spermatozoa with four replicates in each mouse at a magnification of ×1000.

Hormonal Assays
The sera obtained were analyzed to determine the concentration of testosterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) using the Enzyme-Linked immunosorbent Assay (ELISA). The ELISA kits used were manufactured by Biocheck, South San Francisco, CA, USA.
5.6. Oxidative Stress Parameters in Testes and Epididymis 5.6.1. Measurement of Catalase (CAT) Activity Catalase activity was determined by measuring the rate of hydrolysis of H 2 O 2 at 240 nm [56]. Briefly, Hydrogen peroxide (8.8 mM) in sodium phosphate buffer (0.1 M, pH 7.0) was added to 0.05 mg protein of tissue samples. The decrease in absorbance was monitored for 3 min and the activity was expressed as l mol H 2 O 2 decomposed/min/mg protein (€−43.6/mM/cm).

Determination of Lipid Peroxidation
Induction of oxidative damage was ascertained by measuring the extent of lipid peroxidation (LPO) in the tissue sample using estimated thiobarbituric acid reactive substances (TBARS) [58]. An aliquot of the sample (1.0 mg protein) was added to tubes containing 1.5 mL of acetic acid (pH 3.5, 20% v/v), SDS (8% w/v, 0.2 mL) and 1.5 mL thiobarbituric acid (0.8% w/v). The mixture was then heated in a boiling water bath for 45 min. The adducts formed were extracted into 1-butanol (3 mL) and the absorbance of TBARS formed was taken at 532 nm.

Preparation of Tissues Samples
Sample of frozen testis and epididymis tissues were homogenized in a 1.5 mL RIPA buffer (25 mM TrisHCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS pH = 7.6) supplemented with Protease inhibitors at 4 • C. The homogenate was incubated on ice for 30 min and then centrifuged at 10,000× g for 30 min at 4 • C. Following centrifugation, the supernatants were transferred to a labelled Eppendorf and stored at −80 • C for cytokine measurement. 5.7.2. Measurement of Tumor Necrosis Factor-Alpha (TNF-α) and Interleukin1-Beta (IL-1β) Production Quantitative measurement of the level of cytokines was performed using Mini Enzyme Linked Immunosorbent Assay (ELISA) Development Kits (Peprotech). Well plates were set up according to the manufacturer's instructions and read using an ELISA plate reader at 405 nm with 650 nm as the correction wavelength. Concentrations (pg/mL) of TNF-α and IL-1β in testis and epididymis were estimated respectively [59].

Histological Evaluation
Histology of the testis and epididymis was assessed using standard laboratory procedures. The testicular and epididymis tissues of the rats were fixed in Bouin's solution, embedded in paraffin, sectioned into 4 µm thicknesses, and placed on microscopic slides. Slides were observed using the light microscope at a magnification of ×400 after staining with haematoxylin and eosin (H & E).

Statistical Analysis
Data were analyzed using Statistical Package for Social Sciences software produced by IBM Corp. Ltd., IBM SPSS, Version 25.0, Armonk, NY, USA. Values were expressed as mean ± standard error of mean (SEM) and analyzed using a t-test to compare the significant (p < 0.05) differences between the control and test group. An independent sample test was used for comparison. Institutional Review Board Statement: All animal experiment in this study was approved with assigned number: UI-ACUREC: 18/0108 by University of Ibadan-Animal Care and Research Ethics Committee (UI-ACUREC) (Approval date: 22 August 2018). The guidelines as provided by the review board were strictly followed.