Clinical Phytoscience

International Journal of Phytomedicine and Phytotherapy

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Pleurotus tuber-regium mushrooms in the diet of rats ameliorates reproductive and testicular injury caused by carbon tetrachloride

  • Kenneth Obinna Okolo1,
  • Orish Ebere Orisakwe2Email author and
  • Iyeopu Minakiri Siminialayi3
Clinical PhytoscienceInternational Journal of Phytomedicine and Phytotherapy20173:14

https://doi.org/10.1186/s40816-017-0051-x

Received: 21 April 2017

Accepted: 16 June 2017

Published: 15 July 2017

Abstract

Background

The incidence of male infertility arising from male sexual dysfunction is high especially in the sub saharan Africa. African foods may hold promise to reverse this trend. The aim of this study therefore is to evaluate the improvement of the reproductive and testicular injuries mediated by CCl4 by the use of a wild edible mushroom, P. tuber-regium.

Methods

Sixty rats were divide into six groups. Group I received 3 mL/kg olive oil by intraperitoneal injections twice weekly. Group II received 3 mL/kg (30% in olive oil) injected twice weekly i.p, Groups III, IV and V received 100 mg, 200 mg and 500 mg wild edible P. tuber-regium (33.3% in feed) daily in addition to 3 mL/kg CCl4 in oil injected twice weekly i.p. Group VI received 500 mg P. tuber-regium (33.3% in feed) daily. After 13 weeks, the animals were sacrificed and testes weighed.

Testicular counts and viability were evaluated. Serum levels of FSH, LH, testosterone, estrogen and prolactin were assayed. Malondialdehyde (MDA), ascorbic acid, α-tocopherol, superoxide dismutase (SOD), catalase, total glutathione and peroxidase were determined in testis homogenate. Also, histopathological examinations of the testes were performed.

Results

Administration of CCl4 to rats significantly (p < 0.01) increased the relative testicular mass in treated group when compared to control group. Also, CCl4 administration decreased significantly (p < 0.01) the levels of sperm motility, epidydymal and testicular sperm count and viability ratio in the CCl4 group when compared to the control group. Exposure to CCl4 decreased significantly the levels of FSH, LH and testosterone when compared to the control while increasing the levels of estrogen, prolactin and MDA when compared to the control. The levels of ascorbic acid, α-tocopherol, SOD, catalase, total glutathione and peroxidase decreased significantly (p < 0.01) in treated groups when compared to control group. These changes were reversed by diets containing Pleurotus tuber-regium mushrooms in a dose-dependent manner. Photomicrographs also showed that P. tuber-regium prevented the edema, spermatogenic distortions and maturation arrest observed in the CCl4 only group.

Conclusion

P. tuber-regium is effective in protecting the testes from the free radical injuries mediated by CCl4.

Keywords

P. Tuber-regium CCl4 MushroomSperm analysisHormonesAntioxidantsHistopathology

Background

The global incidence of male sexual dysfunction (MSD) is alarming with estimates put at over 30% [1]. In sub Saharan Africa, there is lack of data on the true incidence of MSD but researchers believed that it is higher than 60% [2]. MSD is a syndrome with many diseases that usually but not always involve problems of sperm concentration, morphology and motility, hormonal imbalance that could result from low levels of testosterone, aging process, and drugs like antidepressants or environmental chemicals and cigarettes [35]. Carbon tetrachloride is a well-known industrial solvent that had been banned for its toxicological concerns and is known to be a toxicant to the liver, kidney, lungs, testis etc. [6]. Metabolism of carbon tetrachloride produces free radicals (CCL3 and CCl3O2 ) that bind to poly unsaturated free acids (PUFA) of sperm membranes to generate alkoxy and peroxy radicals that can perturb signal transduction mechanisms and promote infertility [7]. These radicals are unstable; the pathological consequences in the testis of these very reactive agents may include reduced sperm counts, disrupted hormone levels, impaired enzyme activities, and necrosis [8]. Also, these radicals react with molecular structures of the body’s defense system like the sulfhydryl groups of glutathione and protein thiols altering their structure and leading to loss of activity [9]. The mammalian body is endowed with protective mechanisms against the destructive effects of free radical damage [10]. Normal protective mechanisms may be overwhelmed in cases of excessive free radical production; cellular damage will ensue in these situations unless additional free radical scavenging capabilities are available through dietary constituents or other exogenous sources. Abundant evidence confirms the ability of antioxidants to treat or prevent CCl4-induced testes injury [5, 7]. Several natural products, especially medicinal plants and mushrooms, have been shown to possess antioxidant properties [11, 12].

Mushrooms, such as Pleurotus tuber-regium, are often used as foods and as folk medicines [13]. Medicinal mushrooms are known to contain several phytoactive constituents which are mainly secondary metabolites like flavonoids, tannins, alkaloids, phenols etc. Pleurotus tuber-regium is an edible mushroom belonging to the family Pleurotaceae. In addition to its nutritive properties, it had been used as an anti-inflammatory, antioxidant, antiviral, antipyretic and hepatoprotective [14, 15].

The present study has evaluated the potential of Pleurotus tuber-regium to protect the reproductive functions of the testes from free radical injury mediated by carbon tetrachloride.

Methods

Collection of fungi

Fresh fruiting bodies of P.tuber-regium were collected from a forest close to University of Nigeria, Nsukka. They were identified by a taxonomist at the International Center for Ethnomedicine and Drug Development (INTERCEDD) Nsukka, Nigeria. The mushrooms were air dried, pulverized and stored in airtight containers until use. The voucher specimen for the same is conserved under the reference number INTERCEDD/050.

Experimental Animals

Male Sprague Dawley rats with average weight of 170–180 g were obtained from the Animal House of University of Port Harcourt, Rivers State Nigeria. The animals were maintained under standard laboratory conditions at ambient temperature of 25 °C ± 15%, with darkness cycle of 12 h. They were allowed access to standard commercial pellet diet and water ad libitum. All the experimental studies conformed to the guidance for care and use of animals in experimental studies of the University of Port Harcourt Ethical protocols.

Acute toxicity test

Various doses of P. tuber-regium were administered to male Sprague Dawley rats per oral (50–5000 mg kg−1 b.w). The animals were observed for gross behavior, neural and autonomic toxicity as described on OECD guidelines [16]. There was no mortality or toxic signs recorded in this period even up to 5000 mg/kg dose.

Experimental Design

The animals were divided into six (6) groups (n = 10). They were treated as follows;
  • Group I Control (received olive oil 3 mL/kg i.p. twice weekly for 13 weeks in addition to feed and water).

  • Group II Carbon tetrachloride treated (received 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly for 13 weeks) [5] modified.

  • Group III Rats were treated with 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly in addition to 100 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks [17].

  • Group IV Rats were treated with 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly in addition to 200 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks.

  • Group V Rats were treated with 3 mL/kg i.p. of 30% CCl4 in olive oil twice weekly in addition to 500 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks.

  • Group VI Rats received olive oil 3 mL/kg i.p. twice weekly in addition to 500 mg/kg BW of Pleurotus tuber-regium (33.3% in feed) for 13 weeks.

Measurement of organ weights

At the end of the 13th week post treatment, the animals were sacrificed under ether anesthesia and the right testes removed, carefully trimmed of fats and rinsed with ice cold saline before being weighed using a digital weighing balance. The relative organ weights were determined as follows;

Relative organ weight (ROW) = Organ weight/Animal weight x 100.

Part of the testes were homogenized in phosphate buffered saline for lipid peroxidation and antioxidant studies while part were preserved in 10% formalin for histological evaluations.

Collection of Blood samples

At the end of the 13th week post treatment, blood samples were withdrawn from the medial canthus of the eye using micro hematocrit tubes into a clean test tube [18]. The blood was allowed to coagulate for 30–60 min, then centrifuged at 1800 x g relative centrifugation force (RCF) for 30 min to effect separation of serum from blood cells. The serum was stored below 0 °C in 1.5 mL tubes until used for hormonal analyses.

Sperm analysis

Sperm motility was determined by macerating the epididymis in mortar and pestle and mixing it with 1 ml of phosphate-buffered saline (37 °C) in order to release the sperm cells, and a drop of the suspension was quickly placed on a glass microscope slide, covered with a glass slip and examined using a light microscope at × 40 magnification. At least 200 sperm cells were counted and the number of motile sperm cells reported as a percentage of the total cells [19]. Sperm viability was assessed microscopically from smears that were prepared by mixing equal volumes of eosin-nigrosin stain with epididymal suspensions, incubation for 30 s, distributing the stained suspensions on glass slides, and drying in air. After 30 s, a drop of the mixture was placed on a glass slide and spread out to make a smear, and this was allowed to air-dry. The smear was examined under x100 oil immersion using a light microscope. Live sperm were left unstained whereas dead sperm stained pink or red. At least 100 spermatozoa were evaluated and the sperm vitality recorded as Live to Dead sperm ratio [20]. The caudal epididymal sperm reserves and testicular counts were determined using the standard hemocytometric method [19].

Lipid peroxidation and antioxidant assays

At the end of the 13th week post treatment, one gram of testis tissue was homogenized in 9 ml ice cold Phosphate buffered saline (pH 7.4) to make 10% homogenates. The homogenates were centrifuged at 1800 x g RCF for 15 min at 4 °C and the supernatants were used to determine the levels of malondialdehyde (MDA), ascorbic acid, alpha tocopherol, and total glutathione as well as superoxide dismutase, catalase, and peroxidase enzyme activities. Lipid peroxidation was quantified as malondialdehyde [21]. Ascorbic acid was assayed colorimetrically using the 2,4-dinitrophenylhydrazine method [22]. Alpha tocopherol was analysed colorimetrically with 2, 4, 6 – tripridyl - s – triazin and FeCl3 after extracting with absolute ethanol and xylene as described by Martinek [23]. Superoxide dismutase (SOD), catalase, total glutathione and peroxidase were assessed using a commercial kit (Biovision, Mountain View, CA, USA) obtained from a local representative and assayed according to the manufacturer’s protocol.

Serum analysis of hormones

Serum analysis of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, estrogen and prolactin were estimated using commercial kits (Biocheck Inc, 323 Vintage Park Dr, Foster City, CA 94404) obtained through a local representative and assayed according to the manufacturer’s protocol.

Histological studies

Portions of the testis from all animals were sliced and dehydrated with a range of concentrations (70%, 80%, 90% and absolute) of alcohol and then cleared with xylene (twice) before embedding in paraffin. The embedded tissue blocks were section with Shandon AS 325 rotary microtomes into approximately 5 μm thick section and slides were prepared with the sections. The tissues were stained with Ehrlich’s haematoxylin and eosin blue.

Statistical analysis

All statistical analyses were done on GraphPad Prism version 5.02 (www.graphpad.com/scientific-software/prism). Data were expressed as mean ± standard deviation (SD). ANOVA test was used to analyze the difference among various treatments with least significance difference (LSD) at 0.05 followed by Bonferroni’s posttest.

Results

Effect of P. tuber-regium on the average kidney weight of CCl4 treated animals

Figure 1 shows the effect of P. tuber-regium treatment on testes of animals administered CCl4 for 13 weeks. Administration of CCl4 to the rats resulted in a significant (p < 0.05) increase in relative weight of the testis 0.92 ± 0.15 in the control groups when compared to the relative weight 1.2 ± 0.24 of the treated group 1.2 ± 0.24. P. tuber-regium in the diet resulted in a dose-dependent reversal of the increases in testicular weight caused by CCl4 treatments.
Fig. 1

Effect of P. tuber-regium on relative weight of testes of animals treated with CCl4 for 13 weeks (n = 10). ***: significantly different from control at p < 0.001. $$$: significantly different from CCl4 only at p < 0.001

Effect of P. tuber-regium on sperm analysis

Administration of CCl4 to rats significantly (p < 0.05) decreased the levels of motile sperms (74.0 ± 1.50%), epididymal reserve (46.0 ± 2.10 x 106), testicular count (449 ± 37 x 106) and viability (46.33 ± 4.62) in the control group when compared to the CCl4 only treatment group values (1.70 ± 1.1%, 10.0 ± 2.1 x 106, 180 ± 13.0 x 106 and 6.23 ± 0.30 respectively). Treatment with P. tuber-regium restored the levels of these biomarkers to different degrees when compared to the control group. However, treatment with P. tuber-regium alone did not alter the levels of these markers significantly (p < 0.05) when compared to the control group (Fig. 2).
Fig. 2

Effect of P. tuber-regium treatment on sperm parameters of animals treated with CCl4 for 13 weeks (n = 10). ***, * significantly different from control at p < 0.001and p < 0.05. $$$, $$, $ significantly different from CCl4 only at P < 0.001

Effect of P. tuber-regium on reproductive hormones

The effects of P. tuber-regium on follicle stimulating hormone, luteinizing hormone, testosterone, estrogen and prolactin are shown in Fig. 3. 13 weeks administration of CCl4 significantly decreased (p < 0.05) the hormonal levels of FSH, LH and testosterone (0.93 ± 0.04 mIU/mL, 0.53 ± 0.02 mIU/mL and 0.25 ± 0.04 ng/mL) when compared to the control group (3.53 ± 0.40 mIU/mL, 2.78 ± 0.30 mIU/mL and 4.48 ± 0.60 ng/mL respectively). Alterations of these hormones were significantly reversed (p < 0.05) by the treatment with P. tuber-regium at different doses in a dose dependent manner. However, there is no significant difference between the negative control treated and the control. Also, administration of CCl4 increased significantly (p < 0.05) the levels of estrogen and prolactin from 4.70 ± 0.56 pg/mL and 0.15 ± 0.06 ng/mL in control group to 14.5 ± 2.2 pg/mL and 1.33 ± 0.12 ng/mL in CCl4 only group respectively. These alterations were reversed with P. tuber-regium treatment.
Fig. 3

Effect of P. tuber-regium treatment on hormonal profile of animals treated with CCl4 for 13 week (n = 10). ***, ** significantly different from control at p < 0.001, p < 0.01 probability levels. $$$, $$, $ significantly different from CCl4 only at p < 0.001, p < 0.01, p < 0.05 probability levels

Effect of P. tuber-regium on lipid peroxidation and antioxidant vitamins

The effects of P. tuber-regium on the levels of malondialdehyde, ascorbic acid, and alpha tocopherol in the testis are shown in Fig. 4. Administration of CCl4 significantly (p < 0.05) increased the level of MDA in the testes from 0.68 ± 0.08 μmol/L in control group to 1.60 ± 0.30 μmol/L in the treated group. Ascorbic acid and alpha tocopherol levels decreased from 0.65 ± 0.04 mg/dL and 3.50 ± 0.03 μg/mL in the control group to 0.31 ± 0.03 mg/dL and 0.69 ± 0.01 μg/mL respectively. P. tuber-regium administration decreased the levels of MDA and increased the levels of ascorbic acid and alpha tocopherol significantly in a dose dependent manner in the groups that took this mushroom. Also, the levels of MDA, ascorbic acid and α-tocopherol in the negative control treated is comparable to control.
Fig. 4

Effect of P. tuber-regium on lipid peroxidation and antioxidant vitamins of animals treatment with CCl4 for 13 weeks (n = 10). *** Significantly different from control at p < 0.001. $$$, $$, $ significantly different from CCl4 only at P < 0.001, P < 0.01, P < 0.05

Effect of P. tuber-regium on antioxidant enzymes

The effect of P. tuber-regium treatment on the antioxidant enzymes in the testis is shown in Fig. 5. There was a significant (p < 0.001) decrease on all the antioxidant enzymes assayed; SOD decreased from 10.30 ± 0.40 U/mL in control group to 2.28 ± 0.03 U/mL in the treated group, catalase from 93.90 ± 4.0 mU/mL to 31.40 ± 1.83 mU/mL, total glutathione from 1.52 ± 0.07 μg/mL to 0.47 ± 0.04 μg/mL and peroxidase from 11.20 ± 0.40 mU/mL to 4.57 ± 0.30 mU/mL respectively. Co-administration of CCl4 with P.tuber-regium resulted in improvement of the aforementioned antioxidant enzymes in a dose dependent manner. However, treatment of P. tuber-regium to negative control produced no significant difference in antioxidants levels when compared to the control.
Fig. 5

Effect of P. tuber-regium on antioxidant enzymes of animals treated with CCl4 for 13 weeks (n = 10). ***, **, * significantly different from control at p < 0.001, p < 0.01, and p < 0.05 probability level. $$$, $$, $ significantly different from CCl4 only group at p < 0.001, p < 0.01, P < 0.05 probability level

Histological studies of the Testes

The histoarchitecture of the testes at different treatments are presented in Fig. 6. The histological studies of the testes showed well preserved testicular architecture (Fig. 6a). Administration of CCl4 resulted in scanty spermatogenic cells, maturation arrest (MA) and edema (Fig. 6b). Treatment with P. tuber–regium improved these observed pathologies in a dose dependent manner (Fig. 6c, d and e). Micrographs of P. tuber-regium only group shows well preserved architecture which is comparable to control group (Fig. 6f).
Fig. 6

Photomicrograph showing histopathological changes in the testes of rats (magnification H & E X 400). a Control testis showing normal histology. b CCl4 only group showing scanty spermatogenic cells and maturation arrest (MA) (c) CCl4 + 100 mg P.t. group showing seminiferous tubules without full population of spermatozoa (d) CCl4 + 200 mg P.t. group showing seminiferous tubules without full population of spermatozoa. e CCl4 + 500 mg P.t. group showing normal testis. f 500 mg P.t. only group with normal histology

Discussion

Oxidative stress resulting from increased generation of free radicals or depletion of antioxidant defense systems is well known to produce toxic effects in animal models [4, 6]. Carbon tetrachloride, a toxic industrial solvent and environmental contaminant, exerts its destructive effects by the generation of free radicals. [24]. CCl4 is bio transformed by phase 1 metabolism of the P-450 system to generate trichloromethyl radical (CCl3 ) and peroxy trichloromethyl radicals (CCl3OO2 ) [25]. These free radicals have the ability to bind poly unsaturated fatty acids (PUFA) abundant in sperm cell membranes to generate alkoxy (R) and peroxy radicals (ROO) that in turn will generate lipid peroxides that are very reactive with potential to alter enzymes and cause necrosis [5, 26]. The resultant effects of these free radicals is the depletion of the antioxidant capacity in vivo, caused by decreasing the levels of strategic antioxidant enzymes and vitamins, leading ultimately to oxidative stress [27].

In this study, we tested the hypothesis that P. tuber-regium will protect and reduce lipid peroxidation in the testes of CCl4 intoxicated Sprague Dawley rats. P. tuber-regium possesses active phytochemicals that, when present in the diet of rats, can supply exogenous antioxidant capacity to ameliorate oxidative stress [13]. Oxidative stress appears to be a major contributor in male infertility and development of effective antioxidants to combat it will be a giant stride in the prevention and management of male sexual dysfunction [28, 29]. Administration of CCl4 increased significantly both the absolute and relative organ weights of the testes. This increase may be due to increase in lipid peroxidation induced by the highly reactive metabolites of CCl4 that may possibly activate the inflammatory pathway leading to edema [5, 30]. CCl4 metabolites react with poly unsaturated fatty acids and form covalent adducts with lipids and proteins that lead to lipid peroxide formation and destruction of cell membranes and consequently testis injury [31]. Amelioration of this effect was seen in the groups that were treated with P. tuber-regium suggesting its abilities to mop up free radicals generated by CCl4 [32]. The protective effects of P. tuber-regium against oxidative stress in rat testis that we observed in this study are consistent with those reported for medicinal plants with known antioxidant properties, such as Launaea procumbens [5].

CCl4 induced significant reduction in the sperm motility (%), count and vitality when compared to the control group. Theses alterations in the sperm parameters were significantly restored to different degree in the groups of animals treated with P. tuber-regium. Other reports on the intoxication of animals with CCl4 recorded similar reductions [26, 33]. Alterations in sperm parameters may result from low levels of reproductive hormones secondary to the effect of CCl4 in the gonadal-pituitary axis or direct toxic effects to the sperm cells [4].

Effects of CCl4 administration on FSH, LH and testosterone showed significant decrease in the CCl4 group compared to the control group. Reductions in the FSH and LH may be due to the distortions in the hypothalamic-pituitary axis leading to testicular dysfunction as indicated by the results of sperm analysis [26, 34]. The decrease in testosterone level may be as a result of low levels of FSH and LH since its activity is under their direct influence. Also, decrease in testosterone level could be due to direct toxicity of CCl4 on the sertoli cells [35]. The levels of estrogen and prolactin increased significantly in the CCl4 only treatment group when compared with those of the control group. In the male reproductive system, prolactin and estrogens promote infertility in males by antagonizing the effects of testosterone [36]. These observed hormonal alterations were reversed by the treatment with P. tuber-regium.

Antioxidant enzymes may have a major role in preventing male infertility due to oxidative stress since CCl4 and reactive oxygen species are associated with impaired antioxidant enzyme status which determine in part the body’s antioxidant capacity [26]. Administration of CCl4 decreased the levels of ascorbic acid, α-tocopherol, SOD, catalase, total glutathione and peroxidase while increasing the level of lipid peroxidation as measured by MDA. Reduction in the levels of antioxidant enzymes and vitamins is inversely related to the MDA suggesting the involvement of oxidative stress in the levels of these enzymes. Decreased glutathione levels may be due toxic effects of CCl4 on NADP (H) enzyme which is the limiting factor in its production [37]. Catalase is known to convert peroxides especially hydrogen peroxide to water and in the circumstance of its lipid peroxides, this enzyme will be consumed leading to its low levels [38]. Treatment with P. tuber-regium improved the levels of the antioxidant enzymes and vitamins further leading credence to the antioxidant properties of this mushroom. Plants with polyphenolics like flavonoids, phenolic compounds, glucans are known to be effective in anti-oxidation. This they do by scavenging hydroxyl and superoxide radicals, chelate metal ions or exert synergistic effects in conjunction with other antioxidant metabolites [7].

Histological studies revealed that CCl4 administration caused degeneration of the testicular structure and germ cells with scanty spermatogenic cells, maturation arrest, and edema. The histological changes in the testis caused by CCl4 were largely reversed by the presence of P. tuber-regium in the diet. This is in agreement with other researchers that used CCl4 model of oxidative stress [26] and points to the involvement of oxidative stress in male infertility and the possible potential benefits natural products like mushrooms may play in the management of this ailment.

Conclusion

P. tuber-regium may be useful in the management of male reproductive hormonal dysfunction possibly secondary to oxidative stress.

Declarations

Authors’ contributions

KO: Carried out the bench work analysed data and write up, OO: Designed the study, analysed data and write up and IS: Design. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Madonna University
(2)
Toxicology Unit, Faculty of Pharmacy, University of Port Harcourt
(3)
Department of Pharmacology, Faculty of Basic Medical Sciences, College of Health Sciences, University of Port Harcourt

References

  1. Laumann EO, Paik A, Rosen RC. Sexual dysfunction in the United States: prevalence and predictors. JAMA. 1999;281:537–44.View ArticlePubMedGoogle Scholar
  2. Ramlachan P, Campbell MM. An integrative treatment model for patients with sexual dysfunctions. SAMJ. 2014;104:440–5.View ArticleGoogle Scholar
  3. Khaki A. Protective effect of quercetin against necrosis and apoptosis induced by experimental ischemia and reperfusion in rat liver. Afr J Pharm Pharmacol. 2010;4:022–6.Google Scholar
  4. Khaki A, Ouladsahebmadarek E, Javadi L, Farzadi L, Fathiazad F, Nouri M. Anti-oxidative effects of citro flavonoids on spermatogenesis in rat. Afr J Pharm Pharmacol. 2011;5:721–5.View ArticleGoogle Scholar
  5. Khan RA. Protective effects of launaea procumbens on rat testis damage by CCl 4. Lipids Health Dis. 2012;11:1.View ArticleGoogle Scholar
  6. Adewole S, Salako A, Doherty O, Naicker T. Effect of melatonin on carbon tetrachloride-induced kidney injury in wistar rats. Afr J Biomed Res. 2007;10:2.Google Scholar
  7. Al-Olayan EM, El-Khadragy MF, Metwally DM, Moneim AEA. Protective effects of pomegranate (punica granatum) juice on testes against carbon tetrachloride intoxication in rats. BMC Complement Altern Med. 2014;14:164.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Sikka SC, Rajasekaran MAHA, Hellstrom WJ. Role of oxidative stress and antioxidants in male infertility. J Androl. 1995;16:464–8.PubMedGoogle Scholar
  9. Cemek M, Aymelek F, Buyukokuroglu ME, Karaca T, Buyukben A, Yilmaz F. Protective potential of royal jelly against carbon tetrachloride induced-toxicity and changes in the serum sialic acid levels. Food Chem Toxicol. 2010;48:2827–32.View ArticlePubMedGoogle Scholar
  10. Sies H. Strategies of antioxidant defense. FEBS J. 1993;215:213–9.View ArticleGoogle Scholar
  11. Mosquera OM, Correa YM, Buitrago DC, Nio J. Antioxidant activity of twenty five plants from Colombian biodiversity. Mem Inst Oswaldo Cruz. 2007;102:631–4.View ArticlePubMedGoogle Scholar
  12. Mohamed EM, Farghaly FA. Bioactive compounds of fresh and dried pleurotus ostreatus mushroom. Int J Biotechnol Wellness Ind. 2014;3:4.View ArticleGoogle Scholar
  13. Dandapat S, Sinha MP, Kumar M, Jaggi Y. Hepatoprotective efficacy of medicinal mushroom pleurotus tuber-regium. Environ Exp Biol. 2015;13:103–8.Google Scholar
  14. Ikewuchi CC, Ikewuchi JC. Chemical profile of pleurotus tuberregium (Fr) sings sclerotia. Pacific J Sci Technol. 2008;10:295–9.Google Scholar
  15. Patel Y, Naraian R, Singh VK. Medicinal properties of pleurotus species (oyster mushroom): a review. World J Fungal Plant Biol. 2012;3:1–12.Google Scholar
  16. Oecd (1994). OECD Guidelines for the Testing of Chemicals. Organization for Economic.Google Scholar
  17. Okolo KO, Siminialayi IM, Orisakwe OE. Carbon tetrachloride induced hepatorenal toxicity in rats: possible protective effects of wild pleurotus tuber-regium. Clinical Phytoscience. 2017;3:2.View ArticleGoogle Scholar
  18. Stone SH. Method for obtaining venous blood from the orbital sinus of the rat or mouse. Science. 1954;119:100.View ArticlePubMedGoogle Scholar
  19. Amann RP, Almquist JO. Reproductive capacity of dairy bulls. I. Technique for direct measurement of gonadal and extra-gonadal sperm reserves. J Dairy Sci. 1961;44:1537–43.View ArticleGoogle Scholar
  20. Lasley J.F, Easely G.T, McKenzie F.F. A staining method for the differentiation of live and dead spermatozoa. I. Applicability to the staining of ram spermatozoa. The Anatomical Record. 1942:82(2):167-174.Google Scholar
  21. Todorova I, Simeonova G, Kyuchukova D, Dinev D, Gadjeva V. Reference values of oxidative stress parameters (MDA, SOD, CAT) in dogs and cats. Comp Clin Pathol. 2005;13:190–4.View ArticleGoogle Scholar
  22. Omaye ST, Turnbull JD, Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods Enzymol. 1979;62:3–11.View ArticlePubMedGoogle Scholar
  23. Martinek RG. Method for the determination of vitamin E (total tocopherols) in serum. Clin Chem. 1964;10:1078–86.PubMedGoogle Scholar
  24. Towner RA, Reinke LA, Janzen EG, Yamashiro S. In vivo magnetic resonance imaging study of kupffer cell involvement in CCl4-induced hepatotoxicity in rats. Can J Physiol Pharmacol. 1994;72:441–6.View ArticlePubMedGoogle Scholar
  25. Bruckner JV, Ramanathan R, Lee KM, Muralidhara S. Mechanisms of circadian rhythmicity of carbon tetrachloride hepatotoxicity. J Pharmacol Exp Ther. 2002;300:273–81.View ArticlePubMedGoogle Scholar
  26. Khan MR, Ahmed D. Protective effects of digera muricata (L.) mart. On testis against oxidative stress of carbon tetrachloride in rat. Food Chem Toxicol. 2009;47:1393–9.View ArticlePubMedGoogle Scholar
  27. Meister A. Glutathione, ascorbate, and cellular protection. Cancer Res. 1994;54:1969s–75s.PubMedGoogle Scholar
  28. Gin I. Antioxidant activity of food constituents: an overview. Arch Toxicol. 2012;86:345–91.View ArticleGoogle Scholar
  29. Halliwell B, Gutteridge JM. Free radicals in biology and medicine. USA: Oxford University Press; 2015.View ArticleGoogle Scholar
  30. Aitken RJ, Smith TB, Jobling MS, Baker MA, De Iuliis GN. Oxidative stress and male reproductive health. Asian J Androl. 2014;16:31.View ArticlePubMedGoogle Scholar
  31. Raja S, Ahamed KN, Kumar V, Mukherjee K, Bandyopadhyay A, Mukherjee PK. Antioxidant effect of cytisus scoparius against carbon tetrachloride treated liver injury in rats. J Ethnopharmacol. 2007;109:41–7.View ArticlePubMedGoogle Scholar
  32. Jayakumar T, Ramesh E, Geraldine P. Antioxidant activity of the oyster mushroom, pleurotus ostreatus, on CCl 4-induced liver injury in rats. Food Chem Toxicol. 2006;44:1989–96.View ArticlePubMedGoogle Scholar
  33. Abraham P, Wilfred G, Cathrine SP. Oxidative damage to the lipids and proteins of the lungs, testis and kidney of rats during carbon tetrachloride intoxication. Clin Chim Acta. 1999;289:177–9.View ArticlePubMedGoogle Scholar
  34. Latif R, Lodhi GM, Aslam M. Effects of amlodipine on serum testosterone, testicular weight and gonado-somatic index in adult rats. J Ayub Med Coll Abbottabad. 2008;20:8–10.PubMedGoogle Scholar
  35. McVey MJ, Cooke GM, Curran IH, Chan HM, Kubow S, Lok E, et al. An investigation of the effects of methylmercury in rats fed different dietary fats and proteins: testicular steroidogenic enzymes and serum testosterone levels. Food Chem Toxicol. 2008;46:270–9.View ArticlePubMedGoogle Scholar
  36. Ostby J, Cooper RL, Kelce WR, Gray Jr LE. The estrogenic and antiandrogenic pesticide methoxychlor alters the reproductive tract and behavior without affecting pituitary size or LH and prolactin secretion in male rats. Toxicol Ind Health. 1999;15:37–47.View ArticlePubMedGoogle Scholar
  37. Ozgoví Ŝ, HeÖmínek J, Gut I. Different antioxidant effects of polyphenols on lipid peroxidation and hydroxyl radicals in the NADPH-, Fe-ascorbate-and Fe-microsomal systems. Biochem Pharmacol. 2003;66:1127–37.View ArticleGoogle Scholar
  38. Yoshida Y, Itoh N, Hayakawa M, Piga R, Cynshi O, Jishage K i, et al. Lipid peroxidation induced by carbon tetrachloride and its inhibition by antioxidant as evaluated by an oxidative stress marker, HODE. Toxicol Appl Pharmacol. 2005;208:87–97.View ArticlePubMedGoogle Scholar

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