The Journal of Basic & Applied Zoology (2016) 76, 52–59 H O S T E D BY The Egyptian German Society for Zoology The Journal of Basic & Applied Zoology www.egsz.org www.sciencedirect.com Protective effect of curcumin and vitamin C each alone and in combination on cisplatin-induced sperm abnormalities in male albino rats Sabha Elsayed Elballat Zoology Department, Faculty of Science, Zagazig University, Egypt Received July 2016; revised 11 November 2016; accepted 13 November 2016 Available online 17 January 2017 KEYWORDS Cisplatin; Curcumin; Vitamin C; Spermatogenesis; Epididymal sperm morphology Abstract Infertility is a major concern for young men of reproductive age under-going chemotherapy Unfortunately, chemotherapeutic treatment for neoplastic diseases commonly impairs fertility either temporally or permanently In general, chemotherapeutic agents in cancer treatments target all rapidly growing healthy cells, such as those of reproductive system in addition to tumor tissues and it therefore results in impairment of spermatogenesis leading to abnormalities in sperm morphology in cancer survivors Consequently, antioxidants have been shown to protect nonmalignant cells and organs against damage by chemotherapeutic agents Hence the present study was designed to evaluate the possible ameliorative role of curcumin or vitamin C alone and their combination in alleviating the toxicity of cisplatin on sperm morphology when given to normal albino rats The results of the present investigation concluded that the combination between curcumin and vitamin C in cisplatin treatment afforded the best ameliorative effect on cisplatin induced sperm shape abnormalities This may be due to the synergistic effect between curcumin and vitamin C as both of them have antioxidant properties which in turn lead to repairing of sperm abnormalities Ó 2017 The Egyptian German Society for Zoology Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Cisplatin (cis-diamminedichloroplatinum-II, CIS), one of the most effective and widely prescribed anticancer drugs, is still used in the treatment of many types of solid tumors including testicular cancer (Ahmed et al., 2011) It has been proven highly effective in curing testicular cancer in combination with E-mail addresses: sabhael@yahoo.com, Sabhael@Zu.edu.eg Peer review under responsibility of The Egyptian German Society for Zoology other drugs even at an advanced stage of the disease (Peckham et al., 1983) Cisplatin (CIS) kills cancer cells by forming covalent adducts with the cellular DNA molecules and thereby terminating the vital processes like replication and transcription and inducing apoptosis (Johnson et al., 1998) The cytotoxic action of the drug is often thought to be associated with its ability to bind DNA to form cisplatin-DNA adducts (Goldstein and Mayor, 1983) Zahra et al (2014) concluded that percentage of normally shaped sperm was decreased in mice administered cisplatin at a single dose Also, cisplatin decreased sperm motility and http://dx.doi.org/10.1016/j.jobaz.2016.11.001 2090-9896 Ó 2017 The Egyptian German Society for Zoology Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Protective effect of curcumin and vitamin C on cisplatin-induced sperm abnormalities increased anomalous spermatozoa in rats (Aldemir et al., 2014) Cisplatin toxicity is occurred by increased generation of reactive oxygen species (ROS) and decreased formation of antioxidants (Chirino and Pedraza-Chaverri, 2009) The production of ROS depends on the dose of cisplatin and the duration of treatment (Brozovic et al., 2010) Therefore, it has become essential to supplement with antioxidants to reduce the toxicity created by anti-cancer drugs (Mohamed et al., 2011) Antioxidants such as vitamin C reduces many cisplatinassociated side effects Additionally, numerous natural products of plant origin protect against drug-induced toxicity (Antunes et al., 2001) Vitamin C or ascorbic acid (L, 3-ketothreohexuronic acid lactone) is a water soluble vitamin with antioxidant properties Ascorbic acid is an active reducing agent involved in different biological effects (Henson et al., 1991) The treatment with a mixture of ascorbic acid and cisplatin should be very useful to ameliorate cisplatin-induced toxicities including nephrotoxicity, hepatotoxicity and testes/sperms anomalies (Amenla and Surya, 2015) Azu et al (2011) concluded that herbal plant extracts with protective effects against cisplatin -induced reproductive toxicities are due to the presence of antioxidant agents Curcumin is a major yellow pigment in turmeric ground rhizome of Curcuma longa (commonly known as turmeric) which is used widely as a spice and coloring agent in many foods for example curry, mustard and potato chips as well as beautifying agents and medications (Joe et al., 2004) Curcumin has been claimed to be a potential antiinflammatory agent with phyto-nutrient and bio-protective properties (Aggarwal and Sung, 2009) It has also shown to alleviate various forms of male reproductive disorders in experimental animals and thus to enhance fertility (Khorsandi et al., 2013) Curcumin was shown to be a potent scavenger of a variety of reactive oxygen species (Goel et al., 2008) So, it has been shown to have strong antioxidant activity and decreases oxidative stress (Igbal et al., 2003) Omur and Coyan (2016) demonstrated that curcumin, methionine and ellagic acid have protective effects on ram sperm parameters In addition, Ehab et al (2014) recorded that the treatment of rats with curcumin caused protective effect against cisplatin-induced testicular disorders The sperm abnormality significantly decreased in curcumin treated groups compared with cisplatin treated groups Materials and methods Animals Forty five (45) adult male albino rats (Rattus norvegicus) were selected for the present study Their weights ranged from 200 to 210 g each The animals were housed in an air conditioned animal house facility at 25 °C, under a controlled 12 h light/dark cycle The rats were reared on a standard pellet diet and tap water ad libitum 53 Test chemicals Cisplatin was purchased from the local pharmacy shop in Egypt The drug was dissolved in 0.9% normal saline and was injected intraperitoneally in a dose level of (0.4 mg/kg b wt) according to the literature Pratibha et al (2006) VitacidC (Vitamin C) was purchased from the local pharmacy shop in Egypt The vitamin was dissolved in distilled water and was given orally in a dose level of (100 mg/kg b wt) according to literature Rana and Ahmed (2012) Curcuma longa extract (Curcumin) was purchased from National Bio Lab (medical laboratory) 15El Nour St, Floor 1, Dokki, Giza Egypt Curcumin was suspended in 0.05% gum acacia solution then it was given orally at dose level of (20 mg/kg b.wt) according to literature Xu et al (2007) Experimental design The study was performed on 45 mature male rats (Rattus norvegicus), divided into main groups; each group was consisted of rats as following: i Water control group: Animals received distilled water orally daily for 60 successive days ii Saline control group: Animals were injected intraperitoneally with physiological saline (0.9% sodium chloride) daily for 60 successive days iii Acacia control group: Animals orally received 0.05% gum acacia solution daily for 60 successive days using metallic stomach tube iv Curcumin treated group: Animals received curcumin orally at a dose of (20 mg/kg) suspended in 0.05% gum acacia solution daily for 60 successive days using metallic stomach tube v Vitamin C treated group: Animals were orally administered vitamin C at a dose of (100 mg/kg) dissolved in distilled water daily for 60 successive days using metallic stomach tube vi Cisplatin treated group: Animals were injected intraperitoneally with cisplatin at a dose of (0.4 mg/kg) dissolved in physiological saline daily for 60 successive days vii Cisplatin + curcumin treated group: Animals received curcumin (20 mg/kg) orally and after 20 animals were injected intraperitoneally with cisplatin at a dose of (0.4 mg/kg) dissolved in physiological saline daily for 60 successive days viii Cisplatin + vitamin C treated group: Animals received vitamin C (100 mg/kg) orally and after 20 animals were injected intraperitoneally with cisplatin at a dose of (0.4 mg/kg) dissolved in physiological saline daily for 60 successive days ix Cisplatin + curcumin + vitamin C treated group: Animals received curcumin (20 mg/kg) as well as vitamin C (100 mg/kg) orally and after 20 animals were injected intraperitoneally with cisplatin at a dose of (0.4 mg/kg) dissolved in physiological saline daily for 60 successive days Table and Fig shows that the frequency of abnormally shaped sperms in the testes of male albino rat significantly increased (p < 0.05) after the injection of cisplatin at a dose of (0.4 mg/kg) daily for 60 successive days The percentage of total deformed sperms reached (7.58%) compared to (1.8%) in the saline control group (see Fig 2) On the contrast, a non significant changes in the frequency of total deformed sperms were recorded in the curcumin treated group and vitamin C treated group when compared with control groups 17.4 ± 0.67e 18.00 ± 0.54de 18.00 ± 0.31de 19.80 ± 0.37cde 19.40 ± 0.24cde 75.80 ± 2.26a 21.40 ± 0.50c 25.00 ± 0.89b 20.60 ± 0.24 cd 1.74 1.80 1.80 1.98 1.94 7.58 2.14 2.50 2.06 Mean ± SE No % 87 90 90 99 97 379 107 125 103 0.00 ± 0.00 0.00 ± 0.00a 0.00 ± 0.00a 0.20 ± 0.20a 0.20 ± 0.20a 0.40 ± 0.24a 0.00 ± 0.00a 0.20 ± 0.20a 0.00 ± 0.00a 16.80 ± 0.48 17.00 ± 0.54d 17.00 ± 0.31d 19.00 ± 0.70bcd 18.00 ± 0.63 cd 69.20 ± 2.41a 20.20 ± 0.37bc 21.20 ± 0.48b 19.60 ± 0.24bcd 0 1 0.00 0.00 0.00 0.02 0.02 0.04 0.00 0.02 0.00 a Mean ± SE % No Deformed head and tail 0.60 ± 0.24 1.00 ± 0.00c 1.00 ± 0.31c 0.60 ± 0.60c 1.20 ± 0.37c 6.20 ± 0.48a 1.20 ± 0.20c 3.60 ± 0.97b 1.00 ± 0.00c 84 85 85 95 90 346 101 106 98 1.68 1.70 1.70 1.90 1.80 6.92 2.02 2.12 1.96 d 0.06 0.10 0.10 0.06 0.12 0.62 0.12 0.36 0.10 5 31 18 5000 5000 5000 5000 5000 5000 5000 5000 5000 Mean ± SE Means within the same column in each category carrying different letters are significant at (p 0.05) using Duncan’s multiple range test, where the highest mean value has symbol (a) and decreasing in value were assigned alphabetically Similar letters are non significant on the statistical level Results 5 5 5 5 Data were collected, arranged and reported as mean ± standard error of mean (S.E.M) of nine groups (each group was considered as one experimental unit), summarized and then analyzed using the computer program SPSS/version (15.0) The statistical method was one way analysis of variance ANOVA test (F-test) and if significant differences between means were found, Duncan’s multiple range test (whose significant level was defined as (P < 0.05) was used according to Snedecor and Cochran (1982) to estimate the effect of different treated groups Water control Saline control Acacia control Curcumin Vitamin C Cisplatin Cisplatin + curcumin Cisplatin + vitamin C Cisplatin + curcumin + vitamin C Statistical analysis c The sperm smears were obtained from the caudae epididymis of the testes of adult control and treated males The caudae epididymis were cut into small pieces in cc saline solution Sperm smears were obtained from the resulting suspension They were stained by Feulgen nuclear stain Approximately 1000 sperm cells were microscopically examined for each rat A binocular microscope with Â10 eyepieces and Â100 oil immersion objective lenses were used for this study Abnormally shaped sperm cells were recorded randomly and microphotographs were taken whenever necessary No % Collection of epididymal sperm smears Deformed tail One gm of light green is dissolved in 100 ml of methyl alcohol Mean ± SE ii- Light green No % One gm of basic fuchsin is dissolved in 200 ml of distilled water and boiled The solution is shacked for and is allowed to cool to 50 °C then 20 ml N-hydrochloric acid are added After cooling to 25 °C, g of sodium metabisulfite is added The solution is stored in dark overnight then, g of activated charcoal is added and shacked for The solution is filtered and the filtrate is stored at °C in a dark bottle Deformed head i- Schiff’s reagent Total deformed sperms The reagents used in the present work for the investigation of sperm’s abnormalities from the caudae epididymis of the testes of rats were prepared as follows: No of rats No of examined sperms 1000/rat Deformed sperms Reagents Groups Estimation of the frequency of abnormally shaped sperm cells S.E Elballat Table Comparison between the frequencies of deformed sperms from the testes of male rats treated with cisplatin (0.4 mg/kg b.wt), curcumin (20 mg/kg b.wt), vitamin C (100 mg/ kg b.wt.) or their combinations 54 Protective effect of curcumin and vitamin C on cisplatin-induced sperm abnormalities 55 7.58 w ater control saline control curcum in vitam inC 0.02 0.1 0.36 1.96 2.06 2.14 2.12 2.02 0.12 0.02 0.62 cisplatin 0.04 1.98 1.8 0.12 0.02 1.8 1.9 1.7 Acasia control 0.06 0.1 0.06 0.1 1.7 1.68 1.8 1.74 1.94 2.5 6.92 Percentage (%) Total deform ed sperm cells Deform ed head and tail regions Deform ed tail region Deform ed head region cisplatin cisplatin cisplatin +curcum in +vitam inC +curcum in +vitam inC Groups Fig Comparison between the frequencies of deformed sperms from the testes of male rats treated with cisplatin (0.4 mg/kg b.wt.), curcumin (20 mg/kg b.wt.), Vitamin C (100 mg/kg b.wt.) or their combinations Fig Normal sperms from control male albino rat Meanwhile, cisplatin plus curcumin (2.14%) and cisplatin plus vitamin C (2.50%) treated groups elucidate significant (p < 0.05) decrease in total deformed sperms as compared with cisplatin treated group (7.58%) Cisplatin plus curcumin and cisplatin plus vitamin C treated groups also afforded significant increase in the total deformed sperms as compared with normal control groups, but the effect was much less intense compared to the cisplatin treated group Moreover, the combination of cisplatin with curcumin and vitamin C elucidates non significant changes (2.06%) in total deformed sperms compared with saline and acacia control groups (1.8%) The sperm shape abnormalities involved either the head or the tail and/or head and tail regions In the present investigation, the percentage of deformed head region from testis of male rats was significantly increased in the cisplatin treated group (0.62%) compared with all normal control groups The head region abnormalities presented in various forms, sometimes the acrosome showed up with unusual curvature, it might be sharply curved or abnormally straight, in some cases the head region had an irregular shape In addition, sperms without head and head breaks were scored (Fig 3) Cisplatin plus vitamin C elucidates significant decrease in total deformed head region (0.36%) compared with the cisplatin treated group while they afforded significant increase in total deformed head region compared with normal control groups On the other hand, cisplatin plus curcumin and cisplatin plus curcumin and vitamin C groups elucidate non significant effects in the total deformed head region compared with all normal control groups In the tail region, the injection of cisplatin to male rats elucidates significant increase in total deformed tail region with percentage reached (6.92%) compared with all control groups The tail abnormalities appeared in the form of sperm without tail or shortening in tail region (Fig 4) While, treatment with the combination of cisplatin plus vitamin C (2.12%) and cisplatin plus curcumin (2.02%) afforded significant decrease as compared with the cisplatin treated group Moreover, the results revealed that the treatment of male rats with the combination of cisplatin plus curcumin and vitamin C exhibited non significant change (1.96%) in total deformed tail region compared with all control groups On the contrary, cisplatin administration to male rats afforded non significant changes in the percentage of total deformed head and tail regions when given alone and in its combination with either curcumin or vitamin C or/and their combination It is worth mentioning that the combination between curcumin and vitamin C in cisplatin treatment afforded the best ameliorative effect on cisplatin induced sperm shape abnormalities This may be due to the synergistic effect between curcumin and vitamin C as both of them have antioxidant 56 S.E Elballat Fig Different sperms from testes of male albino rats injected intraperitoneally with cisplatin (0.4 mg/kg) showing head region abnormalities as following: A = Sperm without head, B = head with irregular shape, C = straight acrosome, D = sharply curved acrosome, E = head break Fig Different sperms from testes of male albino rats injected intraperitoneally with cisplatin (0.4 mg/kg) showing tail region abnormalities as following: F = Sperm without tail, G = shortening in tail Protective effect of curcumin and vitamin C on cisplatin-induced sperm abnormalities properties which in turn lead to repairing of sperm abnormalities Discussion The present work was an attempt to evaluate the side effects of cisplatin (anticancer drug) alone and in its combination with either curcumin or vitamin C or/and their combination as it is well known that curcumin and vitamin C have been reported to be effective antioxidant, therefore, the present study aimed to elucidate the possible ameliorative role of curcumin and vitamin C in alleviating the toxicity of cisplatin on sperm morphology when given to normal male albino rats In this study, after the end of the experiment, all rats were sacrificed and sperm smears obtained from semen of caudal epididymis of each rat The results demonstrated that the frequency of abnormally shaped sperms in the testes of male albino rat significantly increased after the chronic injection of cisplatin 0.4 mg/kg i.p for 60 successive days These results in full agreement with those of Atesßßs ahin et al (2006) who concluded that the harmful effects caused by cisplatin treatment include a decrease in sperm count, alteration in sperm motility and an increase in abnormal sperm ratios These toxic effects may indicate that cisplatin induces the DNA damage in germ cells leading to abnormalities in sperm morphology Also, Mohammadnejad et al (2012) added that cisplatin has been considered as a potent mutagen causing induction of abnormal male germ cells In our opinion, spermiotoxicity followed by cisplatin treatment is generally ascribed to oxidative stress mediated by increased free radical generation and depletion of antioxidants These results were strongly supported by Zahra et al (2014) who concluded that the percentage of normally shaped sperm was decreased in mice administered cisplatin at a single dose In addition, cisplatin toxicity is occurred by increased generation of reactive oxygen species (ROS) and decreased formation of antioxidants (Chirino and Pedraza-Chaverri, 2009) Also, Makker et al (2009) explained that the oxidative stress is harmful to sperm function and a significant factor in the etiology of male infertility The oxidative stress impairs male fertility by changing the cell function like sperm motility, increase in DNA damage by induction of gene mutations, DNA denaturation, base pair oxidation and DNA fragmentation The acute exposure to antineoplastic agents like cisplatin has shown an increase in the frequency of germ-cell apoptosis in experimental animals It can also lead to decreased reproductive organ weights, azoospermia and degenerated spermatogenic cells (Amin et al., 2008; Turk et al., 2008) In the present study, the percentage of deformed tail region increased progressively than that of the head region The abnormalities in tail region appeared in most cases as a head without tail or tail with abnormal length and this result was significant So, the motility of sperm may be affected and then affect on fertilization process These data are in full agreement with data reported by Aldemir et al (2014) who studied that cisplatin decreased sperm motility and increased anomalous spermatozoa in rats Also, Amr et al (2012) explained that cisplatin significantly decreased the weights of testes and epididymis in addition to the caudal sperm count and motility compared with the con- 57 trol group Cisplatin adversely damages testicular tissue and significantly reduces sperm production through increasing oxidative stress and inducing apoptosis Ahmet et al (2006) and (Amr and AlaaEldin, 2006) supported that both short- and long-term cisplatin treatment decreased sperm concentration, sperm motility and increased abnormal sperm ratio compared with the control Cisplatin administration altered clearly the testicular function In the present investigation, the percentage of deformed head region from testes of male rats was significantly increased in the cisplatin treated group The abnormality of the head region may refer to the sensitivity of the component of head region (DNA) to this anticancer drug and these data indicated the genotoxicity of cisplatin drug on germ cell and cause risk on future generations These results are in agreement with those explained by Florea and Buăsselberg (2011) who demonstrated that the capacity of cisplatin to interact with DNA to form cisplatinDNA adducts with inter- and intra-strand nuclear DNA cross-links is proposed to be the principle mechanism underlying its cytotoxic effect Likewise, the main cytotoxic activity of cisplatin to destroy malignant tumors, is to intercalate the DNA backbone of rapidly growing cells and interfere with cell division (Victoria et al., 2007) However, its dose related side effects, chiefly production of reactive oxygen species and induction of cell apoptosis could adversely damage non target tissues that are not the goal of treatment (Jordan and CarmoFonseca, 2000) Regarding the effect of vitamin C, in the present study, non significant changes in the frequency of total deformed sperms were recorded in the vitamin C treated group when compared with control group Moreover, the pretreatment of the cisplatin treated group with vitamin C at a dose of 100 mg/kg orally for 60 successive days succeeded in ameliorating the toxic effect of cisplatin on the sperm morphology from 7.58% to 2.50% This may be explained as vitamin C as antioxidant repairs cisplatin-associated side effects on sperm morphology by lowering oxidative stress caused by cisplatin These results were supported by Amenla and Surya (2015) who explained that treatment with a mixture of vitamin C and cisplatin significantly decreased the cisplatin-induced sperm abnormalities in the hosts Ascorbic acid’s ameliorating effects to be likely mediated via inhibition of free radicals generation and/or free radical scavenging activity Steinberger and Steinberger (1966) added that ascorbic acid has long been known to participate in spermatogenesis process of rodents and ameliorate oxidative stress related to testicular impairments in animals Also, dietary ascorbic acid was found to protect against endogenous oxidative DNA damage in human sperms Thus, ascorbic acid pre-treatment may be thought to enhance the defense system during stress to combat tissue destruction and safeguard cellular structure and function thereby, ameliorating cisplatin-induced testicular toxicity as observed for kidney and liver in the hosts (Fraga et al., 1991) In addition, Wyrobek et al (1983) investigated that cytotoxic drugs depress spermatogenesis in mammals by causing the death of the developing germ cells in the seminiferous tubules Antioxidants such as vitamin C reduce many cisplatin-associated side effects (Antunes et al., 2001) The previous response was also observed with curcumin, where the frequency of total deformed sperms non significantly changed in the curcumin treated group when compared with 58 control group Also, the combination of curcumin + cisplatin as pretreatment with curcumin significantly decreased sperm abnormalities from 7.58% in the cisplatin treated group to 2.14% These results were in full agreement with Ehab et al (2014) who concluded that the treatment of rats with curcumin caused protective effects against cisplatin-induced testicular disorders The sperm abnormality significantly decreased in curcumin treated groups compared with cisplatin treated group Moreover, curcumin reduced all oxidative stress and improving anti-oxidant defense system and prevented all toxic effects of cisplatin Co-administration of curcumin attenuated the adverse effects of cisplatin in rats by reducing the elevated level of NO (Ilbey et al., 2009) In our opinion, curcumin may exert its protective actions against cisplatin-induced sperm toxicity in rats possibly through its antioxidant mechanisms This result is strongly supported by Goel et al (2008) who concluded that, curcumin was shown to be a potent scavenger of a variety of reactive oxygen species including hydroxyl radicals, nitrogen dioxide radicals and superoxide radicals Ali and Adel, 2013 recorded that, one of the possible ameliorative mechanisms of curcumin on sperm morphology, sperm count, sperm motility and sperm viability in adult male Wister rats is to scavenge the free radicals and thereby act as good antioxidants Curcumin has been shown to have strong antioxidant activity and decrease oxidative stress (Igbal et al., 2003) It is worth mentioning that the combination between curcumin and vitamin C in cisplatin treatment afforded the best ameliorative effect on cisplatin induced sperm shape abnormalities This may be due to the synergistic effect between curcumin and vitamin C as both of them have antioxidant properties which in turn lead to repairing of sperm abnormalities This result is supported by Silici et al (2009) who explained that, the sperm damage induced with cisplatin has been reported to be associated with oxidative stress Thus, the combination of curcumin delivery together with a potent antioxidant may be the appropriate approach to reduce the toxic side effect of cisplatin In addition, Contri et al (2011) recorded a positive correlation between sperm parameters and total antioxidant capacity in seminal plasma References Aggarwal, B.B., Sung, B., 2009 Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets Trends Pharmacol Sci 30 (2), 85–94 Ahmed, E.A., Omar, H.M., Elghaffar, S.K., Ragb, S.M., Nasser, A Y., 2011 The antioxidant activity of Vitamin C, DPPD and lcysteine against Cisplatin-induced testicular oxidative damage in rats Food Chem Toxicol 49 (5), 1115–1121 Ahmet, A., Engin, S ß , Affari, T., Ali, O., Seval, Y., Abdurrauf, Y., Oăzguăr, B., 2006 Chemoprotective effect of melatonin against cisplatin-induced testicular toxicity in rats J Pineal Res 41 (1), 21– 27 Aldemir, M., Okulu, E., Koăsemehmetoglu, K., Ener, K., Topal, F., Evirgen, O., Guărleyik, E., Avcı, A., 2014 Evaluation of the protective effect of quercetin against cisplatin-induced renal and testis tissue damage and sperm parameters in rats Andrologia 46 (10), 1089–1097 Ali, S., Adel, S., 2013 Effect of curcumin on rat sperm morphology after the freeze-thawing process Vet Res Forum (3), 185–189 S.E Elballat Amenla, L., Surya, B., 2015 Biochemical changes associated with ascorbic acid-cisplatin combination therapeutic efficacy and protective effect on cisplatin-induced toxicity in tumor-bearing mice Toxicol Rep http://dx.doi.org/10.1016/j.toxrep.2015.01.017 Amin, A., Hamza, A.A., Kambal, A., Daoud, S., 2008 Herbal extracts counteract cisplatin-mediated cell death in rat testis Asian J Androl 10 (2), 291–297 Amr, A., AlaaEldin, A.H., 2006 Effects of Roselle and Ginger on cisplatin-induced reproductive toxicity in rats Asian J Androl (5), 607–612 Amr, A., Christeena, A., Alaaeldin, A.H., Abdalla, A., Shaikha, B.A., Saina, S.H., Sayel, D., 2012 A standardized extract of ginkgo biloba neutralizes cisplatin-mediated reproductive toxicity in rats J Biomed Biotechnol 2012 (2012), 11 http://dx.doi.org/10.1155/ 2012/362049 362049 Antunes, L.M., Darin, J.D., Bianchi, N.d., 2001 Effects of the antioxidants curcumin or selenium on cisplatin-induced nephrotoxicity and lipid peroxidation in rats Pharmacol Res 43, 145– 150 Atesßßs ahin, A., Sahna, E., Turk, G., Ceribasßi, A.O., Yilmaz, S., Yuce, A., Bulmus, O., 2006 Chemoprotective effect of melatonin against cisplatin-induced testicular toxicity in rats J Pineal Res 41, 21–27 Azu, O.O., Duru, F.I., Osinubi, A.A., 2011 Long-term treatment with Kigelia africana fruit extract ameliorates the testicular toxicity following cisplatin administration in male Sprague-Dawley rats J Med Plants Res (3), 388–397 Brozovic, A., Ambriovic’-Ristov, A., Osmak, M., 2010 The relationship between cisplatin-induced reactive oxygen species, glutathione, and BCL-2 and resistance to cisplatin Crit Rev Toxicol 40 (4), 347–359 Chirino, Y.I., Pedraza-Chaverri, J., 2009 Role of oxidative and nitrosative stress in cisplatin-induced nephrotoxicity Exp Toxicol Pathol 61 (3), 223–242 Contri, A., De Amicis, I., Molinari, A., 2011 Effect of dietary antioxidant supplementation on fresh semen quality in stallion Theriogenology 75, 1319–1326 Ehab, T., Ezar, H., Ahmed, M., Aya, A.H., 2014 Abrogation by curcumin on testicular toxicity induced by cisplatin in rats J Cancer Res Treat (3), 6468 Florea, A.M., Buăsselberg, D., 2011 Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects Cancers (Basel) 3, 1351–1371 Fraga, C.G., Motchnik, P.A., Shigenaga, M.K., Helbock, H.J., Jacob, R.A., Ames, B.N., 1991 Ascorbic acid protects against endogenous oxidative DNA damage in human sperm Proc Natl Acad Sci U S.A 88, 11003–11006 Goel, A., Jhurani, S., Aggarwal, B.B., 2008 Multi-targeted therapy by curcumin: how spicy is it? J Nat Prod 70 (2), 143–146 Goldstein, R.S., Mayor, G.H., 1983 The nephrotoxicity of cisplatin Life Sci 32, 685–690 Henson, D.E., Block, G., Levine, M., 1991 Ascorbic acid: biological functions and relation to cancer J Natl Cancer Inst 83, 547– 550 Igbal, M., Sharma, S.D., Okazaki, Y., 2003 Dietary supplementation of curcumin enhances antioxidant and phase II metabolizing enzymes in ddY male mice: possible role in protection against chemical carcinogenesis and toxicity Pharmacol Toxicol 92, 33– 38 Ilbey, Y.O., Ozbek, E., Simsek, A., Otunctemur, A., Cekmen, M., Somay, A., 2009 Potential chemoprotective effect of melatonin in cyclophosphamide and cisplatin-induced testicular damage in rats Fertil Steril 92 (3), 1124–1132 Joe, B., Vijaykumar, M., Lokesh, B.R., 2004 Biological properties of curcumin-cellular and molecular mechanisms of action Crit Rev Food Sci Nutr 44, 97–111 Johnson, S.W., Ferry, K.V., Hamilton, T.C., 1998 Recent insights into platinum drug resistance in cancer Drug Resist Updates (4), 243–254 Protective effect of curcumin and vitamin C on cisplatin-induced sperm abnormalities Jordan, P., Carmo-Fonseca, M., 2000 Molecular mechanisms involved in cisplatin cytotoxicity Cell Mol Life Sci 57 (8–9), 1229–1235 Khorsandi, L., Mirhoseini, M., Mohamadpour, M., Orazizadeh, M., Khaghani, S., 2013 Effect of curcumin on dexamethasone-induced testicular toxicity in mice Pharm Biol 51 (2), 206–212 Makker, K., Agarwal, A., Sharma, R., 2009 Oxidative stress and male infertility Indian J Med Res 129 (4), 357–367 Mohamed, J., Wei, W.L., Husin, N.N., Alwahaibi, N.Y., Budin, S.B., 2011 Selenium supplementation reduced oxidative stress in diethylnitrosamine-induced hepatocellular carcinoma in rats Pak J Biol Sci 14, 1055–1060 Mohammadnejad, D., Abedelahi, A., Soleimani-rad, J., Mohamadiroshandeh, A., Rashtbar, M., Azami, A., 2012 Degenerative effect of Cisplatin on testicular germinal epithelium Adv Pharm Bull 2, 173–177 Omur, A., Coyan, K., 2016 Protective effects of the antioxidants curcumin, ellagic acid and methionine on motility, mitochondrial transmembrane potential, plasma membrane and acrosome integrity in freeze-thawed Merino ram sperm Vet Med 61 (1), 10–16 Peckham, M.J., Barrett, A., Liew, K.H., 1983 The treatment of metastatic germ-cell testicular tumours with bleomycin, etoposide and cis-platin (BEP) Br J Cancer 47 (5), 613–619 Pratibha, R., Sameer, R., Padmanabh, V.R., Dayanand, A.B., Chitra, Y.D., 2006 Enzymatic studies of cisplatin induced oxidative stress in hepatic tissue of rats Eur J Pharmacol 532 (3), 290–293 Rana, M.A., Ahmed, H.A., 2012 Effect of vitamin C on the hepatotoxicity induced by cisplatin in rats J Sci 23 (2), 23–33 59 Silici, S., Ekmekcioglu, O., Eraslan, G., Demirtas, A., 2009 Antioxidative effect of royal jelly in cisplatin-induced testes damage Urology 74 (3), 545–551 Snedecor, G.W., Cochran, W.G., 1982 Statistical Methods, eight ed Ames Iowa State University Steinberger, A., Steinberger, E., 1966 Stimulatory effect of vitamins and glutamine on the differentiation of germ cells in rat testes organ culture grown in chemically defined media Exp Cell Res 44, 429– 435 Turk, G., Atessahin, A., Sonmez, M., C¸eribas, A.O., Yuce, A., 2008 Improvement of cisplatin-induced injuries to sperm quality, the oxidant-antioxidant system, and the histologic structure of the rat testis by ellagic acid Fertil Steril 89, 1474–1481 Victoria, C., Miguel, A., Fuertes, J.C., Carlos, A., Celia, Q., Jose, M P., 2007 Biochemical mechanisms of cisplatin cytotoxicity Anticancer Agents Med Chem (1), 3–18 Wyrobek, A., Gordon, L., Bukhart, J.G., Francis, M.W., Kapp, R.W., Letz, G., Malling, H.V., Topham, J.C., Whorton, M.D., 1983 An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals Mutat Res 115, 1–72 Xu, M., Deng, B., Chow, Y.L., Zhao, Z.Z., Hu, B., 2007 Effects of curcumin in treatment of pulmonary fibrosis: a comparison with hydrocortisone J Ethenopharmacol 112, 292–299 Zahra, K., Shahrbanoo, O., Ali, G., Mozafar, K., 2014 Protective effect of Tribulus terrestris hydroalcoholic extract against cisplatininduced cytotoxicity on sperm parameters in male mice Int J Morphol 32 (2), 551–557