Andrologia. 2019;00:1–8. wileyonlinelibrary.com/journal/and  | 1© 2019 Blackwell Verlag GmbH

1  | INTRODUC TION

In urological emergencies, testicular torsion following the spermatic cord torsion is a prevalent complication that causes extreme scrotal pain and significant lesions of testes. Testicular torsion usually oc‐curs before 25 years old, although it can be found in all age groups. According to the lasting time and degree of torsion, acute/chronic damages such as hormonal changes, subfertility and infertility are variable. (Gökçe et al., 2010). The golden surgery time for detorsion is within 4–6 hr, and testicular salvage rates at this time is 90%. But

if the detorsion occurs after 12 hr, the surgery success rate reduces to 50%, and it diminishes to 10% after 24 hr (Shokoohi et al., 2018). Therefore, testicular torsion must be treated immediately after diag‐nosis to reduce ipsilateral and contralateral testis injury and to pre‐serve the testis and fertility. The only way to treat a testicular torsion is through surgery; however, blood flow to the ischaemic testicular tissue may induce considerable damage to the testis (Moghimian, Soltani, Abtahi, Adabi, & Jajarmy, 2016; Mogilner et al., 2006).

Reperfusion begins with numerous events, for example, free oxygen radical generation, intracellular calcium accumulation and

Received:9September2019  |  Revised:24September2019  |  Accepted:14October2019DOI:10.1111/and.13479

O R I G I N A L A R T I C L E

The effects of verapamil and heparin co‐administration on sperm parameters and oxidative stress in prevention of testicular torsion/detorsion damage in rats

Farshid Davoodi1  | Abbas Raisi2  | Asghar Rajabzadeh3  | Mohammad Hassan Hablolvarid4 | Amir Zakian2

1Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran2Department of Clinical Sciences, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran3Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran4Razi Vaccine and Serum Research Institute, Agriculture Research, Education andExtensionOrganization(AREEO),Karaj,Iran

CorrespondenceAbbas Raisi, Department of Clinical Sciences, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran.Email: raisi.a@lu.ac.ir

AbstractIn this research, the impacts of combined administration of verapamil and heparin on testiculartorsiondamagewereexamined.Inthisexperimentalstudy,30sexuallyma‐ture male Wistar albino rats were divided into five equal groups haphazardly (n = 6): Group 1 was the sham group. In group 2, a 2‐hr testicular torsion was induced, and thereafter,detorsionwasdone.Ratsingroup3andgroup4experiencedanidenti‐cal surgical procedure like group 2, but verapamil and heparin were administered in 0.3mg/kgand800IU/kgdosesrespectively,andingroup5,acombinationofvera‐pamil and heparin were administered. Intraperitoneal drug injection in all treatment groupswasdone30minbeforetesticulardetorsion.Testiculartorsionsignificantlychanged sperm parameters, oxidative stress biomarkers and Cosentino's histological score compared to the sham group (p < .05). All treatment groups reduced testicular damage by decreasing oxidative stress and improving sperm parameters, but heparin and co‐administration of verapamil and heparin were significantly better than ve‐rapamil injection alone. However, heparin injected group was more effective than other treatment groups (p<.05).Overall,ananticoagulantlikeheparinismoreeffec‐tive than a calcium channel blocker such as verapamil, and it is more likely to reduce testicular torsion injuries.

K E Y W O R D S

heparin, oxidative stress, sperm parameters, torsion/detorsion, verapamil

2  |     DAVOODI et Al.

lipid peroxidation, finally leading to more cellular and tissue dam‐age (Akbas et al., 2005). An increment in the rate of reactive oxygen species(ROS)causesDNAdamage,apoptosisofgermcellsandmi‐tochondrial membrane lipid peroxidation and protein denaturation (Taşkaraetal.,2011).

VerapamilisaCa2+/Na+channelblockerutilisedforcuringhy‐pertension, cardiac arrhythmia and angina pectoris, and can improve theharmfuleffectsofischaemia/reperfusion(I/R)(Meštrovićetal.,2014;Qiu,He,Zhu,&Chen,2017;Uchidaetal.,1994).Also,ithasbeen shown that verapamil has antioxidant features to preserve the testis from the likely damages produced by I/R (Ameli, Hashemi, Moghimian, & Shokoohi, 2018).

Heparin, that is recognised as unfractionated heparin, is a medi‐cation that is used as an anticoagulant to treat heart attacks and un‐stable angina (Boettcher et al., 2016). Previous studies have proved the beneficial impacts of heparin on I/R damage in different body organs such as lungs, heart, liver, pancreas and body systems con‐taining gastrointestinal and urogenital (Gedik et al., 2012; Ghadie etal.,2012;Moccoetal.,2007;Tahaetal.,2009;Warzechaetal.,2012;Yagmurduretal.,2003).Heparincanreducetheaccumulationof protein carbonyl and malondialdehyde in the untreated twisted testicle.Also,heparinhasROS‐scavengingeffectsthatprotecttestisfromischaemicinjury(Mertoğluetal.,2016).

During I/R, one of the reasons that damage occurs is the accu‐mulation of intracellular calcium, and cytosolic Ca2+ increment acti‐vates a kind of protein kinase that is controlled by Ca2+/calmodulin complex (CaMKs) that leads to dysfunction of organs and cellular death (Kalogeris, Baines, Krenz, & Korthuis, 2012). Researchers have shown that blood flow changes during testicular torsion lead to thrombus formation (Chakraborty, Hikim, & Jhunjhunwala, 1985; Rattansingh, Adamson, & Cosgrove, 2009). Coagulation of the mi‐crovessels is a consequence of venous congestion that is caused by the activation of both coagulation pathways (intrinsic and extrinsic), and hypoxia facilitates thrombus propagation (Wakefield, Myers, & Henke, 2008). Heparin's main anticoagulant role is relevant to thrombin inactivation and factor X (factor Xa) activation through an antithrombin (AT)‐dependent mechanism (Hirsh, Anand, Halperin, & Fuster, 2001). Because heparin and verapamil are two important drugs in the anti‐thrombotic and calcium channel blocker groups and are two most commonly used drugs in hospitals, this study set out to investigate the usefulness of verapamil and heparin co‐administra‐tiontodecreasetesticulardamage.Noresearchhasyetcomparedthe protective effects of verapamil and heparin at testicular damage. So this paper attempts to show the effects of verapamil and heparin and also a mixture of both drugs on I/R injury of testes.

2  | MATERIAL S AND METHODS

2.1 | Experimental design

The approval of animal experiments was given by the Animal Ethics Committee (LU.ACRA.2018.14) at the Veterinary Faculty of Lorestan University. Thirty sexually mature male Wistar albino

ratsweighingbetween250and300gwereusedinthisstudy.Therats were maintained in an animal house with a standard condition comprising12‐hrphotoperiod,23±2°Candhumidityof55%–70%,and were provided a standard commercial pellet diet and water ad libitum. Animals were left for 5 days at laboratory condition for ac‐climation and were randomly divided into five groups, with six rats in each group:

1. Group 1 (sham): In this group, the left testis was drawn out and then returned to its ordinary scrotal position. To measure baseline values of all parameters, this group was defined.

2. Group 2 (TD): Torsion was performed, and after 2 hr, detorsion was done.

3. Group3(TDV):Testiculartorsion/detorsionwasinduced,andratsintraperitoneally received verapamil (0.3 mg/kg, VERAHEXAL,HEXAL)(Nesterova,Durnev,&Seredenin,1999).

4. Group 4 (TDH): Testicular torsion/detorsion was induced, and rats intraperitoneally received heparin (800 IU/kg, Alborzdaru) (Mertoğluetal.,2016).

5. Group 5 (TDVH): Testicular torsion/detorsion was induced, and a mixture of verapamil and heparin with doses as mentioned above was intraperitoneally administered.

Druginjectioninalltreatmentgroupswasdone30minbeforetestic‐ular detorsion.

2.2 | Surgical procedure

Surgical operations were carried out in hygienic conditions, and a combination of ketamine 10% (80 mg/kg, Alfasan) and xylazine 2% (10 mg/kg, Alfasan) were injected intraperitoneally to anaesthetise the rats. In the subsequent stage, scrotum was shaved, and to disin‐fect the surgical area, 10% povidone‐iodine solution was used. Then a vertical scrotal incision was performed through the midline, and lefttestiswasdrawnout,andafterwards,aclockwise720°torsionwas performed in the left testis and then stitched to the scrotum with three 5/0 silk nonabsorbable sutures (SUPA, Iran). Two hours after torsion, testicular detorsion was done by untwisting testis. Previous studies have reported different times for testicular ischae‐miaand reperfusion. In somepreviousstudies (Aktaşetal.,2010;Ergur,Kiray,Pekcetin,Bagriyanik,&Erbil,2008;Ozbaletal.,2012),we decided to set 2 hr for torsion time and 2 hr for detorsion time ac‐cording to the mean elimination half‐life of the drugs, and the results are only for a very short reperfusion time. At the end of the surgery, testis was restored to its ordinary scrotal position, and the simple continuous suture was performed by 4–0 nylon suture (SUPA, Iran) to close scrotum and maintained for 2 hr. The right testis was not operated in any of the experimental groups. Two hours after detor‐sion,anoverdoseof thiopental sodium (300mg/kg, i.p.Exipental,Exir, Iran) was injected to sacrifice the rats, and they underwent an orchiectomy surgery. The cauda epididymides were separated for an examination of sperm parameters, and a longitudinal incision was made in the middle of the testicles and divided into two equal

     |  3DAVOODI et Al.

sections.Onepiecewaskeptin10%bufferedformalinsolution,andthesecondpiecewasfrozenat−80°Ctodeterminethebiochemicalparameters.

2.3 | Evaluation of sperm parameters

2.3.1 | Sperm collection

Sperm collection was performed according to the method of Varisli, Uguz, Agca, and Agca (2009). Briefly, the caudal parts of epididy‐mides were cut off and put in petri dishes containing 5 ml RPMI 1640 medium (INOCLON). Then each epididymis was cut at differentpieces using sharp scissors to allow the dispersion of spermatozoa for15minat37°C.Thereafter,evaluationofspermparameterswascarried out according to theWorldHealthOrganization's instruc‐tions(WorldHealthOrganization,2010).

2.3.2 | Sperm motility

For the assessment of sperm motility, an aliquot of epididymis speci‐mens diluted to 5 ml RPMI medium as described above was located on a glass slide with a coverslip on it and observed under a phase‐contrast microscope (Olympus IMT‐2) at a magnification of 400.To obtain an accurate percentage of sperm motile categories, ap‐proximately 200 spermatozoa were assessed for each sample. The motility of sperms was categorised as progressive motility (PR), non‐progressivemotility(NP)andimmotility(IM)basedonWHOspermanalysismethods(WorldHealthOrganization,2010).

2.3.3 | Sperm vitality

AccordingtotheWHOforspermvitalitytestusingeosin,0.9gNaClwas dissolved in 100 ml purified water and then 0.5 g of eosin Y (Merck)wasdissolvedin100mlof0.9%NaCl.Thirtyminutesaftersampling, a mixture of 5 μl of the specimen and 5 μl of eosin solu‐tion previously prepared (0.5%) was placed on slides. The slides were incubatedat37°Cfor30s,andthenthinsmearsofspermsuspen‐sion were provided and examined under the light microscope (CX21, Olympus).Toachieveanacceptablylowsamplingerror,200sperma‐tozoa in each slide were evaluated, and the average percentage of vital spermatozoa was reported. In this method of rapid staining with eosin, live spermatozoa are identified with white or light pink heads, and red or dark pink stained heads indicate dead spermatozoa.

2.3.4 | Sperm count

To examine sperm concentration, the spermatozoa should be fixed onNeubauerhemocytometer(Brand)1mlof35%formalinsolutionand0.5gofsodiumbicarbonate(NaHCO3)wasdissolvedin100mlof purified water to prepare fixative for diluting sperm fluid. A di‐lution rate of 1:5 was prepared by mixing 50 μl of the sample into the 200 μl of fixative solution. To determine sperm concentration, a 10 μl aliquot of the diluted sample was loaded to the hemocytometer

chamber, and 200 spermatozoa were counted under a phase‐con‐trast microscope with a magnification of 400. Then, according to theWHO, the sperm concentration was calculated as sperm permillilitre.

2.3.5 | Sperm morphology

A 10 μl section of the sperm suspension was placed on a microscope slideandsmearedoutwithanotherslide,fixedin70%ethanolandstainedwithhaematoxylinPapanicolaou′ssolution(PadtanTeb).Todetermine the percentage of normal and abnormal forms, nearly 200 spermatozoa for each rat were counted. Abnormal forms of sperma‐tozoa like bent tail, coiled tail, detached head, abnormal head, short tail and cytoplasmic droplet were evaluated in all groups.

2.4 | Biochemical assays

2.4.1 | Tissue preparation for enzyme assay

To examine the oxidative stress damage, testes that were stored in −80°C,were thawed and cut into small pieces. A homogeniser(Cole‐ParmerLabGEN700Homogenizer)wasusedtohomogenisethe samples in saline phosphate buffer (PH = 7.4), and then cen‐trifugation(3,500g,10min,4°C)wasdonetoobtainsupernatants.Supernatant was used to determine total protein and enzymatic activity.

2.4.2 | The evaluation of oxidative stress biomarkers

The concentrations of total protein, malondialdehyde (MDA), total antioxidant capacity (TAC) and the activities of catalase (CAT) and glutathione peroxidase (GPx) in the testes tissue of animals were de‐termined by colorimetrical commercial biochemical kits (Asan).

2.5 | Histopathological evaluations

For haematoxylin and eosin (H&E) staining, testis samples fixed in 10% neutral buffered formalin were set in paraffin wax, and 4‐μm thick cuts were applied from different parts of each testis and stained after deparaffinisation. An expert pathologist unaware of groups and experiments appraised all microscope slides. A light mi‐croscope(CX21,Olympus)with40×,100×,and200×magnificationwas used to estimate testicular histological injury. Histopathological alterations and testicular necrosis were detected by Cosentino's histological grading method (Cosentino, Nishida, Rabinowitz, &Cockett, 1986):

Score I: Normal tissue structure and regular organisation ofgerm cells without tissue necrosis and haemorrhage.Score II: Testicular damage with reduced in order and adhesion between germinal cells and tightly packed seminiferous tubules with slight haemorrhage and necrosis.

4  |     DAVOODI et Al.

Score III: Testicular damage, with sloughing, disorganisation and nuclear pyknosis of germinal cells and decreased differ‐entiation between seminiferous tubule margins and extensive haemorrhage.Score IV: Testicular damage with tightly packed seminiferous tubules and coagulative necrosis in the germ cells and inten‐sive haemorrhage.

2.6 | Statistical analysis

The one‐sample Kolmogorov–Smirnov test was applied to ana‐lyse distributions of the experimental groups. According to Kolmogorov–Smirnov test results, data with normal distribution were reported asmean ± SE. The data which did not have nor‐mal distribution were reported as median and interquartile range. Comparisons between groups with normal distribution were performedby usingone‐wayANOVAwithTukey–Kramer's posthoc test, and between‐group comparisons with non‐normal dis‐tribution were performed by using nonparametric Kruskal–Wallis test. Analyses were carried out by standard commercial statistical packages (Analyse‐it software for Excel, Ver. 4.80.8, Leeds, UK, and MedCalc software for windows, Ver. 14.8.1). Differences were considered significant when p < .05.

3  | RESULTS

Statistical analysis of sperm parameters revealed that the sperm concentration, progressively motile sperm, live sperm, and nor‐mal morphological sperms were significantly decreased in the TD group compared to the Sham group, and nonprogressive mo‐tile sperm and immotile sperm significantly enhanced (p < .05). Significant increment was found in the sperm PR motility in TDH and TDVH groups when compared to the TD group (p < .05), but no significant difference was seen in the TDV group in compar‐isonwith TD group (Figure 1). In NP spermmotility evaluation,TDH and TDVH groups were significantly lower than TD and TDV groups (p < .05), (Figure 1). There was also a significant differ‐encebetweenTDH,TDVHandTDVgroupsinPRandNPmotilesperms (p < .05), (Figure 1). For immotile sperm examination, TDH and TDVH groups were significantly lower than the TDV group (p < .05; Figure 1). The sperm count in the TDH group was signifi‐cantly higher than TD, TDV and TDVH groups (p < .05; Figure 2). No significant difference was seen in sperm vitality parameterbetween treatment groups and the TD group (p> .05;Figures3and 4). Sperm morphology examination in study groups indicated that the percentage of normal spermatozoa in treatment groups was more than the TD group, but it was not significant (p > .05; Table 1). Also, the percentage of abnormal spermatozoa in the TD group was higher than other groups, but a significant difference was not seen (p > .05; Table 1; Figure 5).

The results of testicular tissue MDA, CAT, GPx and TAC levels of all study groups can be compared in Table 2. In the TD group, the

tissue level of MDA was higher than the sham group, and the GPx and CAT levels were lower than the sham group with significant rate of p < .05. In the heparin‐treated group, the GPx and CAT levels

F I G U R E 1   Comparison of sperm motility percentage in different experimentalgroupsofstudy.AlldataareshownasMean±SE. The symbol of ┼ means the significant difference with sham group (p < .05). The asterisk * shows the significant difference with the TD group and the symbol of & means the significant difference with the TDV group (p < .05)

F I G U R E 2   Comparison of sperm concentration in different experimentalgroupsofstudy.AlldataareshownasMean±SE. The symbol of ┼ means the significant difference with sham group (p < .05). The asterisk * shows the significant difference with the TD group and the symbol of # means the significant difference with the TDH group (p < .05)

F I G U R E 3   Comparison of sperm vitality percentage in different experimentalgroupsofstudy.AlldataareshownasMean±SE. The symbol of ┼ means the significant difference with sham group (p < .05)

     |  5DAVOODI et Al.

were significantly higher than the TD group, and MDA significantly decreased in comparison with the TD group (p < .05). In the com‐bined treatment of heparin and verapamil group, there were notable differences in the MDA and GPx levels in comparison with the TD group (p < .05). There was a significant difference in the MDA level between verapamil‐treated group and the TD group (p > .05).

Table3indicatestheCosentino'sscoreanalysis.Histopathologicalexamination in the sham group showed no changes in the testes, and only a few haemorrhages were seen between the seminiferous

tubules (Figure 6a). Unlike the sham group, great histopathological changes were seen in the TD group, and a large amount of coagu‐lative necrosis was observed in different parts of the testis tissue (Figure 6b). All treatments could ameliorate lesions, and all treat‐ment groups had a meaningful difference with the TD group (p < .05; Table3).TheTDHgrouphadasignificantdifferencewithTDVandTDVH groups (p < .05), but no other notable difference was found between other treatment groups (p > .05). Combined administration of verapamil and heparin could improve the testes tissue injury re‐markably in comparison with the TD group, but heparin administra‐tion alone was much better at reducing tissue necrosis (Figure 6e).

4  | DISCUSSION

Testicular ischaemia reperfusion caused by torsion detorsion is one of the main urological complications. Testicular injury followed by torsion detorsion causes severe damage to the reproductive system and results in subfertility or infertility. Previous studies have identi‐fied reperfusion as the major cause of tissue damage (Ünsal et al., 2006). Because the main reasons of testicular injury are the over‐production of reactive oxygen species, calcium accumulation inside mitochondria and increased cellular apoptosis, various drugs can be useful (Arenaetal.,2017).Recentstudieshavedemonstratedthatreperfusion can cause a noticeable increment in the overgeneration of lipidperoxidation,nitricoxideandgermcellapoptosis(Yuluğetal., 2014). The testis is a sensitive tissue to free radical damage. In

F I G U R E 4   Eosin stained slide for evaluation of sperm vitality. Black arrows show dead spermatozoa with red or dark pink heads and white arrows show live spermatozoa with white or pale pink heads

TA B L E 1  Morphologyofcaudaepididymalspermatozoa.DatawithnormaldistributionaredisplayedasMean±SE; non‐normal distribution data are expressed as median and interquartile range

GroupsNormal mor‐phology (%) Bent tail (%) Coiled tail (%)

Distal cytoplasmic droplet (%) Short tail (%) Abnormal head (%)

Sham 82.33±1.4 14.48±0.99 2.35(1.20–3.00) 0.16±0.16 0±0.00 1.01±0.52

TD 71.41±1.28*  19.37±2.15 8.06(3.98–14.24)*  1.24±0.79 0.39±0.25 0±0.00

TDV 74.36±3.16 17.82±2.48 5.78(0.77–7.14) 0.34±0.34 1.33±0.88 1.39±1.04

TDVH 78.82±2.34 14.47±1.83 4.51(3.35–7.12)*  1.43±0.57 1.07±1.07 1.02±0.79

TDH 77.6±2.71 12.82±0.94 6.12(5.16–13.59)*  0±0.00 0.25±0.25 0.83±0.37

*p < .05 compared with the sham group.

F I G U R E 5   Haematoxylin Papanicolaou's solution‐stained spermatozoa with normal and abnormal morphology.(400×)(a)normalspermatozoa; (b) bent tail; (c) coiled tail; (d) abnormal (elongated) head; (e) free head with a very short tail; (f) short tail

(a) (b) (c)

(d) (e) (f)

6  |     DAVOODI et Al.

healthy physical condition, antioxidant enzymes such as glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD)reduce the damage caused by reactive oxygen species (Dogan et al., 2016).

Simsek,Durmus,Yildiz, andOzcelik (2018) in a studyon sur‐gery‐induced intra‐abdominal adhesions indicated the protective roles of heparin and pentoxifylline to prevent adhesions and re‐ducing oxidative stress (Simsek et al., 2018). Boettcher et al. (2016) described the treatment effects of anti‐thrombotic drugs such as alteplase, enoxaparin and heparin and indicated the reduction in testicular damage after using these drugs (Boettcher et al., 2016). Deepa et al. in research on the impacts of heparin on cell protection

demonstrated that heparin reduces oxidative stress (Deepa & Varalakshmi,2003).Inanotherstudyoneffectsofmethylprednis‐oloneandheparinintesticulardamageconductedbyMertoğluetal. (2016), it was shown that heparin could not significantly express theantioxidantenzymesSOD,CATorGSH‐Pxcomparedtocon‐trolsbutdecreasedproteinlevelsofBaxandcaspase‐3significantlyand anti‐apoptotic protein Bcl‐2 increased, and totally heparin had aprotectiveroleinI/Rinjury(Mertoğluetal.,2016).Previousstud‐ies have failed to consider the impact of heparin on sperm parame‐tersattesticularI/Rinjury(Boettcheretal.,2016;Mertoğluetal.,2016). In contrast to theMertoğlu et al. findings, and consistentwith earlier reports, in our study, we demonstrated that heparin can notably increase CAT and GPx activity and decrease MDA lev‐els and leads to reduced oxidative damage. Also, for the first time, we evaluated sperm parameters for heparin administration group and progressive and nonprogressive percentage of sperms, and sperm count was significantly different from the TD group.

Calcium channel blockers are a group of medications that pre‐vent Ca2+ entrance to sensitive cells (Katz, 1986). Also, a lot of drugs in this group, like amlodipine, nifedipine, verapamil and lido‐caine,haveantioxidantproperties(Arenaetal.,2017).Saricaetal.(1999) in a study revealed the useful effects of verapamil adminis‐tration on tubular diameter and histology of contralateral testis. In a research about verapamil's protective effects on testicular tissue conducted by Ameli et al. (2018), it was found that verapamil can

GroupsMDA (µmol/mg‐pr)

CAT (Unit/mg‐pr)

TAC (nmol Trolox equivalent/mg protein)

GPx (Unit/mg‐pr)

sham 0.24(0.17–0.28) 0.42±0.04 0.43±0.05 20.62±1.10

TD 0.98(0.93–0.99)*  0.14±0.02*  0.15±0.02*  8.24±1.48* 

TDV 0.43(0.34–0.49)**  0.16±0.00 0.25±0.01 17.66±1.82

TDVH 0.36(0.30–0.38)** 

0.20±0.02 0.3±0.05 19.96±2.99** 

TDH 0.49(0.41–0.50)**  0.41±0.02**  0.21±0.04 21.01±1.44** 

*p < .05 compared with the sham group. **p < .05 compared with the TD group.

TA B L E 2   MDA, CAT, TAC and GPx activities in testis tissue. Data with normal distribution are displayed as Mean±SE; non‐normal distribution data are expressed as median and interquartile range

TA B L E 3   Results of Cosentino's grading system. All values are displayedasMean±SE

Groups Cosentino's score

sham 1±0.00

TD 3.83±0.16*,** 

TDV 2.5±0.22*,**,*** 

TDVH 2.33±0.21*,**,*** 

TDH 1.33±0.21** 

*p < .05 compared with the sham group. **p < .05 compared with the TD group. ***p < .05 compared with the TDH group.

F I G U R E 6   Haematoxylin and eosin stained slides of seminiferous tubules for histopathologicalevaluation.(H&E100×)Black arrows show necrosis. (a) Sham‐operated group, (b) torsion/detorsion group, (c) torsion/detorsion + verapamil, (d) torsion/detorsion + verapamil and heparin co‐administration, (e) torsion/detorsion + heparin

(a) (b) (c)

(d) (e)

     |  7DAVOODI et Al.

reduce the I/R damage and the sperm parameters assessment in various groups indicated that verapamil cannot remarkably melio‐rate the sperm characteristics; it is perhaps based on verapamil cal‐ciumchannel‐blockingactivitythatperhapsblockedtheCa2+/Na+channels of spermatozoa and could not increase the motility and percentage of normal spermatozoa (Ameli et al., 2018). Also, Juneja et al. (1990) andMoger (1983) have revealed that sperm qualitydiminishes followed by administration of calcium channel blockers likeverapamil(Junejaetal.,1990;Moger,1983).Theseresultsareconsistent with those of our study and suggest that verapamil can ameliorate testicular damage against I/R injury but cannot improve sperm quality.

Oneofouramazingconclusionswasobservedintheco‐admin‐istration of verapamil and heparin. Although the co‐administration of verapamil and heparin was more effective than the injection of verapamil alone, it did not show better results than heparin adminis‐tration alone. The following conclusions can be drawn from the pres‐ent study that the effect of heparin treatment alone and combined administration of heparin and verapamil on the testicular histology, progressively motile sperm, nonprogressive motile sperm, immotile sperm and testes MDA levels were significantly higher than ver‐apamil injection alone, and the heparin treatment group significantly improved the sperm count and degree of tissue necrosis in compar‐ison with combined administration of heparin and verapamil group. TheresultsofthisresearchsupporttheideathatROSformationhasa vital role in I/R injury and verapamil as a calcium channel blocker has a weak shielding effect against the protective effects of heparin as an anti‐thrombotic agent. More research using controlled trials is needed to understand why verapamil could not improve sperm parameters.

ACKNOWLEDG MENT

This paper has been extracted from a DVM thesis of Farshid Davoodi carried out in Lorestan University, and the authors want to appreci‐ate the vice chancellor of research of Lorestan University and Razi Herbal Medicines Research Center of Lorestan Medical University, Khorramabad, Iran.

ORCID

Farshid Davoodi https://orcid.org/0000‐0003‐1420‐4306

Abbas Raisi https://orcid.org/0000‐0001‐8769‐4284

Asghar Rajabzadeh https://orcid.org/0000‐0003‐1590‐2018

Amir Zakian https://orcid.org/0000‐0002‐7535‐4749

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How to cite this article: Davoodi F, Raisi A, Rajabzadeh A, Hablolvarid MH, Zakian A. The effects of verapamil and heparin co‐administration on sperm parameters and oxidative stress in prevention of testicular torsion/detorsion damage in rats. Andrologia.2019;00:e13479.https ://doi.org/10.1111/and.13479