Reproductive and Developmental Medicine

: 2019  |  Volume : 3  |  Issue : 4  |  Page : 222--229

Effects of carbophos on rat testopathy following resveratrol administration: An experimental study

Cyrus Jalili1, Shiva Roshankhah2, Azita Faramarzi2, Mohammad Reza Salahshoor2,  
1 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran

Correspondence Address:
Mohammad Reza Salahshoor
Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah


Objective: Carbophos (CAR) is an organophosphate that is most capable of producing free radicals and inducing alteration in some of the male reproductive parameters. Resveratrol (RES) is an herbal polyphenol and exerts beneficial antioxidant effects. This study aimed to evaluate the effects of RES against CAR-induced alteration in reproductive parameters of male rats. Methods: In this experimental study, 48 male rats were randomly assigned to eight groups: Group 1: Normal control (saline) group; Group 2: CAR control (250 mg/kg) group; Groups 3–5: 2, 8, and 20 mg/kg RES, respectively, groups; Groups 6–8: CAR plus 2, 8, and 20 mg/kg RES, respectively, groups. The agents were administered intraperitoneally and by gavage daily for 65 days. Sperm parameters, testis malondialdehyde (MDA) levels, total antioxidant capacity, testosterone levels, and germinal layer height were evaluated and compared among these groups. Results: No significant alterations were observed in RES groups (with different doses) compared to the normal control group (P > 0.05). The levels of all parameters except MDA (which decreased) significantly increased in the CAR plus RES (with different doses) groups compared to the CAR control group (P < 0.01). Conclusions: RES attenuates the toxic effect of CAR on some of the male reproductive parameters.

How to cite this article:
Jalili C, Roshankhah S, Faramarzi A, Salahshoor MR. Effects of carbophos on rat testopathy following resveratrol administration: An experimental study.Reprod Dev Med 2019;3:222-229

How to cite this URL:
Jalili C, Roshankhah S, Faramarzi A, Salahshoor MR. Effects of carbophos on rat testopathy following resveratrol administration: An experimental study. Reprod Dev Med [serial online] 2019 [cited 2021 Jan 17 ];3:222-229
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Full Text


Human infertility is a highly complex disorder of the reproductive system that is influenced by many factors such as parents' age, maternal conditions, smoking, alcohol and coffee consumption, socioeconomic status, genetics, hormonal imbalance, and pesticide exposure.[1] Swan et al. demonstrated that pesticides might increase the risk of male infertility.[2] Occupational exposure to pesticides and their detrimental effects on male infertility can cause delayed pregnancy with no contraceptive use, miscarriage, stillbirth, reduced birth weight, and growth disorders.[3] Carbophos (CAR) is an organophosphate and has the appearance of a yellow to dark brown oil.[4] This toxin is extensively used in agricultural fields and gardens to kill pests. The administration of organophosphates has been shown to result in reduced weight of sex organs, reduced sperm motility, increased morphological abnormality, and increased sperms death.[5] Fortunato et al. showed that CAR can induce the production of free radicals and oxidative stress and can also increase the activity of antioxidant enzymes.[6] In physiological conditions, there is a balance between the elimination and production of free radicals in the body of living organisms. An imbalance in these processes causes oxidative stress, which may result in serious cell damage if the stress is intense or prolonged.[7] Antioxidant enzymes are responsible for the detoxification of free radicals. Catalase and superoxide dismutase are the key enzymes of the antioxidant system; furthermore, glutathione and thiol are the most commonly found nonenzymatic intracellular antioxidants.[8] Organophosphates can alter the antioxidant system of cells, cause membrane lipid peroxidation, and induce cell membrane damage via the production of free radicals.[9] Increased lipid peroxidation and free radical production as a result of organophosphate metabolism have been proposed as the main mechanisms involved in the impairment of cell functions and body tissue damage.[10] Organophosphates have alkylating agents. These agents have negative effect on spermatogenesis, sperm protamine phosphorylation, and sperm chromatin structure resulting in poor sperm viability, motility, and morphology.[11],[12] Resveratrol (RES) is a plant-derived polyphenolic phytoalexin that is produced by stilbene synthase enzyme in response to environmental stresses.[13] RES exists in at least 72 plant species and fruits, especially grape skin (50–100 μg/l wet weight).[14] RES inhibits inflammation through direct inhibition of cyclooxygenase (COX)-1 and COX-2 activity [15] and can reduce prostaglandin E2 and reactive oxygen species (ROS) production from the lipopolysaccharides of activated microglial cells via suppressing nuclear factor kappa β and IKβ kinase activity.[16] In addition to the potential antioxidant activity, RES also activates a number of cellular antioxidative enzymes, thus exerting the inhibitory effects on free radical activity. The antioxidant ability of this polyphenol is dependent on the properties of its polyphenolic hydroxyl groups.[17] A review of the literature shows that no study has evaluated the effects of RES against CAR-induced oxidative stress that alters the reproductive parameters of male rats. Therefore, this study aimed to determine these effects.


Preparation of chemicals and measuring kits

CAR and RES were bought from Merck (Germany). Ether, formalin, sodium acetate, ferric chloride, iron sulfate, hematoxylin and eosin, and zinc sulfate powder were purchased from Sigma (USA). For biochemical analysis, the Commercial Pars Azmun colorimetric kits (Pars Azmun, Tehran, Iran) were obtained from Pars Azmoon (I.R. Iran). All other buffer additives and solvents were obtained from Merck (Germany). For preparing 2 mg/kg body weight of RES, 1.5 mg of RES was dissolved in 1.2 mL of normal saline (0.9%). This solution was passed through a 0.45-mm pore size filter (Lida Manufacturing, Kenosha, Wis. USA) and each rat received 200 mL of this solution intraperitonealy. The other doses were made similarly and immediately injected into animals after being prepared. For preparing gavage of 27 mg/kg CAR (C10H19O6PS2), 40.5 mg of CAR was dissolved in 6 mL of saline, and each rat received 1 mL of this solution by using gavages (These values were calculated for one group and each group had six rats).


This experimental study was conducted on 48 male Wistar rats (8 weeks, weighing 220–250 g) at the Kermanshah University of Medical Sciences. All animals were treated in accordance with the guidelines of National Institute of Health for the Care and Use of Laboratory Animals approved by Research Deputy at the Kermanshah University of Medical Sciences; the guidelines are based on the World Medical Association Declaration of Helsinki (Ethics proposal number; IR.KUMS.REC.1397.306). One week before the experiment, the rats were adapted and maintained on a regular diet and water ad libitum with a 12:12 h light/dark cycle at a temperature of 23°C ± 2°C with a relative humidity of 50% ± 5%, in the animal room of the medical school of the Kermanshah University of Medical Sciences.[18]

Study groups and treatment of animals

The animals were randomly divided into eight groups (n = 6). The Group 1 was the normal control group received normal saline (via an intraperitoneal injection) equivalent to the amount of other agents administered to the experimental groups. The Group 2 received CAR at a dose of 250 mg/kg (1/50 LD50) body weight per day (single dose) by gavage (normal saline was used as the solvent for CAR). The Groups 3–5 received 2, 8, and 20 mg/kg RES, respectively, intraperitoneally daily for 65 days at 10 a.m. The Groups 6–8 received a single dose (250 mg/kg) of CAR by gavage to induce alteration in reproductive parameters and then received 2, 8, and 20 mg/kg of RES, respectively, intraperitoneally daily for 65 days at 10 a.m. Start day of RES administration was the day of CAR administration. Furthermore, the latest groups were administrated CAR by gavage and RES by intraperitoneally. All sample collections were done on the day of 66.[13],[19]

Animal dissection and sampling

At the end of the treatment period, all rats were deeply anesthetized with an intraperitoneal injection of ketamine HCl (100 mg/kg) and xylazine (10 mg/kg). Blood samples were collected from the heart without cutting the chest. The samples were kept in an incubator at 37°C for 20 min and then centrifuged at 255 g for 15 min. The serum was isolated, and part of it was kept at −70°C for evaluating total antioxidant capacity (TAC) as well as nitrite oxide and testosterone levels. Then, the chest and abdomen of the animals were cut. The epididymis tail was isolated from the testes and placed in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 12/5% fetal bovine serum culture medium. The left testis was removed from the abdominal cavity and fixed in a 10% formalin solution for histological and morphometric examinations; the right testis was used for malondialdehyde (MDA) level estimations with respect to group assignment.[20]

Sperm cell collection

Both cauda epididymides from each animal were crushed and conserved in a warmed petri dish containing 10 mL of Hank's balanced salt solution at 37°C. The spermatozoa were allowed to disperse into the buffer. After 15 min, the cauda was removed and the suspension was slightly shaken to normalize it. Then, the samples were observed under a light microscope at a magnification of ×400.[18]

Progressive motility

To evaluate progressive motility, four degrees of sperm motility were studied on the basis of the World Health Organization (WHO) methods. Class A: Progressive motility. Progressive motility of the sperm cells from each sample was examined under an optical microscope with a magnification of ×40 in 10 fields of view. For this purpose, one drop of the sperm cells suspension was placed on the chamber and the motile and immotile sperm cells were analyzed by microscope with magnification ×40. In all experimental and control groups, sperm parameters assessment was done by two qualified expert to minimize observer subjectivity.[13]

Sperm viability

Eosin staining was used to identify living sperm cells from dead sperm cells. The basis of this staining is the absorption of stain by the membranes of dead cells and its disposal by the membranes of living cells. At the end of the given time, approximately 20 μL of the medium containing semen fluid was collected from each dish and then mixed with an equal volume of eosin stain solution (approximately 20 μL). After approximately 2–5 min, part of the mixture was poured onto a Neubauer's slide culture. The living sperm cells appeared to lack stain and the dead sperm cells appeared pink. The prepared slide culture was examined at ×40 magnification. At least 100 sperm cells were counted from each random sample from the 10 fields of view, and the percentage of live sperm cells was calculated.[20]

Sperm cell morphology

Normal sperm cell morphology was assessed through the examination of sperm smears from the right cauda epididymis. An aliquot of the sample was used to prepare the smears to appraise the malformations in the spermatozoa. Eosin/nigrosin stain was used to guesstimate the normal spermatozoa morphology. One drop of eosin stain was added to the suspension and mixed slightly. The slides were then observed under a light microscope at ×400 magnification. A total of 400 spermatozoa were studied on each respective slide (4,000 cells in each group) to evaluate the abnormalities in the head and tail.[13]

Sperm calculation

To analyze the quantity of sperm cells, 400 μL of the sperm suspension was diluted with a formaldehyde fixative (10% formalin in phosphate buffered saline) (Sigma; USA). Approximately, 15 μL of the diluted solution was removed and added into a hemocytometer using a Pasteur pipette. The hemocytometer was placed in a petri dish with dampened filter paper and allowed to stand for 10 min. The stable sperm cells were counted and assessed for per 250 small squares of the hemocytometer under a ×40 objective. The amount of sperm per mm 3= (number of sperm counted × dilution)/(number of squares ×4) in a million/mL.[20]

Tissue preparation and staining for the evaluation of germinal layer seminiferous tubules

The nonparenchymal tissues (fat, fascia, and vessels) of the removed left testis were dissected, and paraffin-embedded blocks were prepared using an automatic tissue processor. This procedure included fixation with 10% formal saline (for 72 h), washing thoroughly under running water, dehydrating with increasing concentrations of ethanol (50%, 60%, 70%, 80%, 90%, and 100%, 3 min for each step, with the 100% ethanol step repeated three times), clearing with xylene (three times, 10 min each time), and embedding in soft paraffin (three times, 15 min each time). At this stage, 5-μm coronal histological thin sections were cut from paraffin-embedded blocks using a microtome instrument (Leica RM 2125, Leica Microsystems Nussloch GmbH, Germany); five sections per animal were chosen. For the unification of the section selection, the fourth tissue section was selected as the first and the twenty-fourth tissue section was selected as the last specimen. Of the 20 tissue samples, 4 samples with a 5-fold interval were selected for histological examination. Finally, the routine protocol for hematoxylin and eosin staining was implemented. At the end of tissue processing, the stained sections were mounted using entalan glue and assessed under Olympus BX-51T-32E01 research microscope connected to a DP12 Camera with 3.34-million pixel resolution and Olysia Bio software (Olympus Optical Co. LTD, Tokyo, Japan).[18]

Testosterone measurement

The collected blood was centrifuged at 23°C for 15 min at 5,000 g to separate the serum, which was then kept in a deep freezer (−18°C). The serum testosterone level was examined through ELISA (Abcam 108666, USA).[13]

Testis malondialdehyde measurement

MDA levels in right testis tissue were evaluated as an index of lipid peroxidation. For this purpose, samples were homogenized using a homogenization buffer containing 1.15% KCl solution and then centrifuged at 1,500 g for 10 min. Then, the homogenate samples were added to a reaction mixture containing sodium dodecyl sulfate, acetic acid (pH 3.5), thiobarbituric acid, and distilled water. Following boiling the mixture for 1 h at 95°C and centrifuging it at 3,000 g for 10 min, the absorbency of the supernatant was measured using spectrophotometry at 550-nm wavelength.[21]

Testis total antioxidant capacity estimation

To measure TAC, an acquisition kit (Cat No: TAC-96A) ZellBio GmbH-Germany whose acts based on the oxidation colorimetry resuscitation was purchased. The kit contains one ready-to-use reagent, a Triton X-100, a dye powder, a reaction suspension solution, standard, and a 96-well microplate. In this assay, the level of TAC indicated the level of existing. This comparison is based on the selection of ascorbic acid as the standard for TAC measurement. The kit's sensitivity was equal to 0.1 mmol/L and final absorbance was read at 490 nm, and unit conversion was performed.[21]

Statistical analysis

After extracting the information, the Kolmogorov–Smirnov test was performed to confirm the normality of the data distribution. Data were analyzed using SPSS software for windows (version 20, SPSS Inc., Chicago, IL, USA) using one-way analysis of variance, followed by Tukey's post hoc test. P < 0.05 was considered statistically significant. Variables were presented as mean ± standard error of the mean.


Progressive sperm motility and viability

No significant variations were detected in the RES groups (with different doses) compared to the normal control group (P > 0.05). Moreover, a significant increase in sperm cell viability and progressive motility was observed in the CAR plus RES groups (with different doses) compared to the CAR control group (P < 0.01) [Table 1].{Table 1}

Sperm cell count and normal morphology

No significant deviations were observed in the RES groups (with different doses) compared to the normal control group (P > 0.05). Moreover, a significant enhancement in the sperm cell count and normal morphology were observed in the CAR plus RES groups (with different doses) in comparison with the CAR control group (P < 0.01) [Figure 1] and [Table 1].{Figure 1}

Height of germinal layer in seminiferous tubule

No significant differences were observed in the RES groups compared to the normal control group (P > 0.05). Moreover, a significant improvement in the height of germinal layer in seminiferous tubule was observed in all the CAR plus RES groups in comparison with that in the CAR control group (P < 0.001) [Figure 2] and [Figure 3].{Figure 2}{Figure 3}


No significant alterations were detected in the RES groups (with different doses) compared to the normal control group (P > 0.05). Moreover, a significant increase in testosterone levels was observed in all CAR plus RES groups (with different doses) compared to those in the CAR control group (P < 0.01) [Figure 4].{Figure 4}

Malondialdehyde levels

No significant alteration was observed in the RES groups compared to the normal control group (P > 0.05). Moreover, a significant decrease in MDA levels was observed in all the RES plus CAR groups (with different doses) compared to those in the CAR control group (P < 0. 01) [Figure 5].{Figure 5}

Total antioxidant capacity levels

No significant alteration was observed in the RES groups (with different doses) compared to the normal control group (P > 0.05). Moreover, a significant increase in TAC levels was observed in the RES plus CAR groups (with different doses) compared to those in the CAR control group (P < 0.01) [Figure 6].{Figure 6}


The WHO has reported that 20,000 people die annually due to pesticide poisoning and an additional 3,000,000 people suffer from nonlethal pesticide poisoning; this number has been increasing, with annually more than 700,000 people experiencing chronic effects of contact with pesticides.[22] Organophosphates have the most pronounced effects on the immune and reproductive systems; they can negatively affect male fertility through mechanisms such as direct impairment of cell function and interference with biological processes.[23] Our results suggest that the CAR administration has adverse and destructive effects on testis histology and sperm parameters, oxidant–antioxidant imbalance, and testosterone levels. On the other hand, RES, a natural flavonoid, appears to relieve the detrimental effects of CAR on male reproductive parameters. RES also helps recover cell damage, as indicated by decreased MDA levels and histological evaluation, and decreases oxidation rate, as indicated by increased TAC levels. These data also show that RES reduces lipid peroxidation (indicated by decreased MDA levels) and increases antioxidant capacity (indicated by increased TAC levels) in testis tissues thus, reducing oxidative stress. Consistent with these findings, many studies have demonstrated the antioxidant properties of RES;[13],[15] it apparently prevents lipid peroxidation induced by tert-butyl hydroperoxide in sperm cells. RES is also a lipophilic molecule that inhibits lipid peroxidation via the Fenton reaction.[24] Thus, it appears that RES's antioxidant properties reduced MDA and increased TAC levels in the treatment groups of the present study by inhibiting the production of ROS. Our results also indicated the recovery effect of RES on some male reproductive parameters as well as its effect of decreasing oxidative stress that was evident from a decline in MDA levels. Because sperms lose a large amount of their cytoplasm during spermatogenesis (lack of antioxidant systems), they seem to have a higher sensitivity to elevated ROS than somatic cells.[13] The first outcome of a ROS attack on membrane structures can be cellular peroxidation in the cell membrane and organelles.[4] Use of antioxidants such as RES to eliminate toxic materials and free radicals from the cell surroundings can inhibit lipid peroxidation, thereby maintaining the biochemical structure of cells.[25] The findings of Nahid et al. are in line with the results of the present study; they reported that CAR (malathion) administration significantly reduced catalase and serum total antioxidant levels, increased lipid peroxidation as well as MDA and spermatogenesis damage, decreased germinal epithelium height, and reduced the number of primary spermatocytes in male rats compared with those in controls.[26] Our results showed that the values of all sperm parameters in the CAR control group were significantly reduced compared with those in the normal control group. In the RES and CAR plus RES groups, a significant increase in the values of all sperm cell parameters was observed compared with those in the CAR control group. Spermatogenesis is a highly complex process that may be negatively influenced by numerous factors, leading to infertility and reduced fertility.[20] One such factor is oxidative stress induced by ROS accumulation due to oxidant–antioxidant imbalance.[7] ROS can affect DNA and RNA synthesis in the sperm cell and inhibit mitochondrial functioning.[18] CAR-induced oxidative stress disrupts the cell division and sperm differentiation to such an extent that a numerous spermatogonia relying on the basement membrane are impaired and the number of primary and secondary spermatocytes, spermatids, and mature sperms is reduced.[5] Aitken and Curry findings corroborate those of the present study; they reported that oxidative stress disrupts spermatogenesis and leads to the formation of defective gametes with remodeled chromatin that is vulnerable to the attack of free radicals, resulting in a reduction in the number of spermatogonia, spermatocytes, spermatids, and spermatozoa.[27] The reduced number of sperms in the CAR group might be due to the direct increase in oxidative stress-induced lipid peroxidation, which might have altered the natural properties of the membrane and consequently resulted in the loss of sperms transmitted to and present in the epididymis.[5] On the other hand, high ROS levels result in mitochondrial impairment and consequent release of proapoptotic proteins in the intermembrane space, activation of caspases, reduction of ATP synthesis, increased release of ROS, increased concentration of intracellular calcium, and release of calcium from mitochondria into cytosol, leading to the activation of apoptosis process.[28] The findings of Selmi et al. are in agreement with our study results, suggesting that oral administration of malathion (CAR) significantly decreases the testis and body weight; sperm count, motility, and viability; and normal sperm morphology and increases sperm DNA damage compared with the control group.[5] Elevated free radicals can lead to the impairment of Sertoli cell function and destruction of cytoplasmic bridges via loss of epithelial cells, thereby decreasing sperm count and causing sperm cell deformity.[29] RES seems to have inhibitory effects on free radicals, possess antioxidant properties, and increase the number of antioxidative enzymes. The antioxidant ability of this polyphenol depends on the properties of its polyphenolic hydroxyl groups.[13] RES can exert its effects via a mechanism that is involved in the expression of oxidative phosphorylation genes and in mitochondrial biogenesis.[30] RES is also able to stabilize the blood–testis barrier and protect sperm DNA against the free radical-induced oxidative stress.[13] Revel et al. have reported that RES can inhibit apoptosis induction and DNA damage against benzo[a]pyrene-induced oxidative stress in sperm cells, confirming the results of the present study.[31] Our data demonstrated a significant decrease in serum testosterone levels and seminiferous tubule diameter in the CAR control group compared with those in the normal control group. Moreover, RES significantly elevated testosterone levels and germinal layer of seminiferous tubule height in all groups receiving CAR plus RES compared with the CAR control group. Organophosphates can disrupt the expression of steroidogenic acute regulatory protein (StAR), which is a determinant of the biosynthesis of steroids such as testosterone; organophosphates directly affect steroidogenesis in Leydig cells by disrupting the expression of StAR.[32] The results of Maliji et al. confirmed the findings of the current research in that administration of diazinon 5 days per week for 1 month significantly elevates interleukin-1 and reduces testosterone levels in rats.[33] Besides, it seems that organophosphates increase adrenocorticotropic hormone and cortisol levels. Increased ACTH and cortisol levels can inhibit the activity of hypothalamic–pituitary–gonadal axis, thereby disrupting spermatogenesis.[34] Considering its potent antioxidant properties, RES exerts positive effects on hypothalamic–pituitary–gonadal axis, testosterone levels, spermatogenesis, and sperm motility. Furthermore, RES can reduce apoptosis in germinal cells.[35] Apparently, elevation in ROS levels due to CAR administration increases lipid peroxidation, which in turn induces atrophy of the germinal layer of seminiferous tubules.[36] Salahshoor et al. have shown a reduction in the epithelial volume of seminiferous tubules due to oxidative stress, which is consistent with our study findings.[20] RES seems to protect lipids against peroxidation and prevent testicular oxidative stress and plays a role in the production of testicular steroids.[13] The findings of Erthal et al. are also consistent with our findings, suggesting that oxidative stress causes atrophy of the germinal layer of seminiferous tubules compared with that in the control group and that RES improves the germinal layer of seminiferous tubule height, reduces GSH and MDA levels, and elevates testosterone levels in groups exposed to oxidative stress.[37] Our data showed that CAR-induced alterations in reproductive parameters in male rats could be reduced by plant antioxidants such as RES. Considering the antioxidant properties of RES, we suggest that it alleviates the alteration in male reproductive parameters caused by CAR-induced toxicity.

This study demonstrates that CAR can cause alterations in some male reproductive parameters and that RES has an antioxidant and protective effect. RES improves the quality of some spermatozoa and helps recover the normal sperm morphology; sperm cell viability, motility, and count; germinal layer of seminiferous tubule height; and TAC levels and reduces testis MDA levels. RES may be valuable for the treatment of infertility in men or for enhancement of male fertility. The antioxidant properties of RES could be the main reason for its positive effects on reproductive parameters. Future studies are required to explain its exact mechanism of action.

Financial support and sponsorship

This work was conducted in partial fulfillment of the requirements of an MD degree and was financially supported by the Research Council of the Kermanshah University of Medical Sciences, Kermanshah, I.R. Iran (Grant No. 1397.306).

Conflicts of interest

There are no conflicts of interest.


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