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Anatomy & Physiology: Current Research
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Effect of Propolis on the Reproductive Toxicity of Deltamethrinin Male Albino Rats

Wael M. Al-Amoudi*

Biology Department, Faculty of Applied Sciences, Umm Al-Qura University, Makkah, KSA

*Corresponding Author:
Wael M. Al-Amoudi
Biology Department, Faculty of Applied Sciences
Umm Al-Qura University, Makkah, KSA
Tel: +49 511 856 73 81
E-mail: [email protected]

Received date: August 14, 2015; Accepted date: August 21, 2015; Published date: August 31, 2015

Citation: Al-Amoudi WM (2015) Effect of Propolis on the Reproductive Toxicity of Deltamethrinin Male Albino Rats. Anat Physiol S5:004. doi: 10.4172/2161-0940.S5-004

Copyright: © 2015 Amoudi WM. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The present study was undertaken to assess the effects of the pyrethroid insecticide, deltamethrin on reproduction of male albino rats and the possible ameliorative role of propolis. Treating rats with deltamethrin (1/10 LD50) brought about marked reduction in sperm counts, increase in sperm head abnormalities and a decrease in serum testosterone and L.H concentration. Histological examination of the testes revealed severe degenerative changes in seminiferous tubules with reduction of germ cells. Moreover, total carbohydrates and proteins diminished in the testicular tissue. Animals treated with deltamethrin and propolis showed an improvement in the testicular damage, increase in sperm count, increase in testosterone and L.H, and decrease in sperm head abnormalities. Testicular carbohydrates and proteins increased. It is supposed in this work that propolis ameliorated deltamethrin-induced reproductive toxicity in male rats and this might due to the antioxidant activity of its flavonoids compounds.

Keywords

Deltamethrin; Propolis; Male reproduction; Rats; Antioxidants

Introduction

Synthetic pyrethroids account for more than 30% of insecticide use worldwide in household and agricultural application. On the other hand, exposure to these chemicals was accompaniedwith several toxicities. Deltamethrin (S)-alpha-cyano-3-phenoxybenzyl-(1R,cis)2,2-dimethyl-3-(2,2-dibromvinyl)-cyclopropanecarboxylate is a pyrethroid used as an insecticide in crop production and public health programme [1]. Exposure to this insecticide was found accompanying with many toxic effects. Abdel-Khalik et al. [2] reported that treating pregnant rats from day 6 to day 15 of pregnancy with deltamethrin brought about hindrance of development, hypoplasia of the lungs, enlargement of the renal pelvis and increment in placental weight. Administration of deltamethrin caused DNA fragmentation and necrotic changes in thymocytes [3]. It also caused an immunosuppressive effect [4] and inhibited the mitotic index and increased the frequency of chromosomal aberrations in the bone marrow of rats [5]. Tos-Luty et al. reported that that deltamethrin administered intragastrically to mice led to degenerative changes in liver and kidney as well as hematological alterations. Conceptive harmfulness and endocrine interruption, impacts identified with deltamethrin introduction have been accounted for [6,7]. As of late, a wide mixed bag of cell reinforcements and different eating methodologies have been tried to counterbalance the oxidative anxiety instigated by xenobiotic. Propolis (honey bee paste) is a resinous hive item gathered by bumble bees from different plant sources.Propolis has been mainly used as home remedies and a personal product [8]. Raw propolis typically contains more than 300 components including phenolic aldehydes, polyphenol, sequiterpene quinines coumarins, steroids, amino acids and inorganic compounds [9]. Proplis has been found to have an extensive variety of such as antibacterial [10], anti-in?ammatory [11] anticarcinogenic [12], antioxidative [13], hepatoprotective effects [14] and immunomodulatory [15]. The present study expects to assess the protective impact of propolis against the regenerative poisonous quality of deltamethrin in male albino rats.

Materials and Methods

Male albino Wister rats weighing (155 ± 5g) were used in the present study. The animals were kept under controlled hygienic conditions and maintained at a temperature of 25 ± 2°C relative humidity of 50 ± 5% and photoperiod at 12-h dark/ light. They were fed on rodent pellets which composed of wheat bran 10%, soya bean powder 44%, net protein 22%, fats 4.7%, fibers 3.3%, fish meal, and molasses, salts (sodium chloride, calcium carbonate, and calcium phosphate). Water was allowed ad libitum during the experiment period. Rats were allowed to acclimatize to the laboratory environment for one week before start of the experiment. All the experiments were done in compliance with the guide for the care and use of laboratory animals. The animals were divided into 4 groups:

Group 1 (control group): Animals (10 rats) were orally administrated with corn oil.

Group 2: These animals (10 rats) were orally given propolis at a dose of 100 mg/kg b.w.3 times/ week for 6 weeks.

Group 3: Animal of this group (20 rats) were orally given deltamethrin at a dose level of 1/10 LD50 (0.6 mg/kg body weight) [16] in corn oil, 3 days weekly for 6 weeks.

Group 4: Animals (20 rats) were orally administrated with deltamethrin and propolis, 3 times /week for 6 weeks.

Histological and Histochemical Study

Ten animals from each group were dissected and their testes were removed. For histological preparations, the testes were fixed in Bouin’s fluid, dehydrated, cleared and embedded in paraffin wax. Five-micrometer thick sections were prepared and stained with Ehrlich’s haematoxylin and eosin. For histochemical study specimens were fixed in Carnoy’s fluid. Periodic acid Schiff’s reaction [17] was used for demonstration of polysaccharides. Total proteins were detected using the mercury bromophenol blue method [18]. The diameter of seminiferous tubules and height of the germinal epithelium were measured in the normal spermatogenic cells on the inner surface of the basement membrane through the most advanced cell types lining the lumen of the tubules.

Sperm Study (count and abnormalities)

Sperm suspensions were prepared from cauda epididymies of rats by mincing the cauda in 2 ml of phosphate buffered physiological saline. The resulting suspension was pipetted and filtered through an eighty micrometer stainless steel mesh to remove the tissue fragments. An hour after the sperm diffusion in saline solution, sperm count was performed in hemocytometer chamber (Nubauer Bright Line Improved, 0.10 mm). A fraction of each suspension was then mixed (10:1) with 1% eosin Y in distilled water and 30 minute later smears were made, allowed top dry in air, and were mounted under a cover-slip with DPX For each suspension 1000 sperm were examined at 400-fold magnifications by light microscopy.

Biochemical Assays

For biochemical assays determination, blood samples were collected from the inferior vena cava and then centrifuged. Sera were obtained by centrifugation of the blood sample and stored at –20°C. Testosterone and LH were determined using radioimmunoassay kits supplied by Diagnostic Products Corp. (Los Angeles, CA, USA) according to [19].

Statistical analysis

Data were expressed as mean ± Standard deviation (SD). The significance of differences among group's means was evaluated by using one-way ANOVA at P<0.05. SPSS program (Version 18) for windows software was used.

Results

Histological and Morphometrical Results

Examination of testes of control animals or those treated with propolis for 6 weeks showed normal seminiferous tubules with normal size. Each tubule is lined with germ cells (spermatogonia, primary and secondary spermatoocytes, spermatids) and mature spermatozoa that occupy the center of the tubule.

The tubules are surrounded by the interstitial tissue containing Leydig cells (Figure 1a). Sections of testes of rats treated with deltamethrin showed many histopathological alterations. In these specimens, the seminiferous tubules were disorganized and appeared with irregular basement membrane. The germ cells were scattered in the lumens of the tubules and were detached from the basal lamina (Figure 1b). Interstitial tissue were degenerated and showed blood hemorrhage (Figure 1c). The blood vessels were congested (Figure 2a).

anatomy-physiology-Section-testis-control-rat

Figure 1a: Section in testis of a control rat, SC: spermatocytes, S; sperms, it: interstitial tissue, (X400).

anatomy-physiology-Testis-deltamethrin-treated

Figure 1b: Testis of deltamethrin-treated rat showing detachment of spermatogenic cells (arrows), (X 200).

anatomy-physiology-Interstitial-hemorrhage

Figure 1c: Interstitial hemorrhage (H), (X200).

anatomy-physiology-Congested-blood-vessel

Figure 2a: Congested blood vessel (C), (X200).

Most of the tubules showed few germ cells with pyknotic nuclei and the spermatozoa were degenerated and accumulated in the center of the lumen (Figure 2b).

anatomy-physiology-Reduction-germ-cells

Figure 2b: Reduction of germ cells, (X 200).

Examination of testis of rats treated with deltamethrin and propolis revealed that it recovered about its ordinary structure and stamped reclamation of the typical picture of seminiferous tubules was accomplished. Data in Figures 3 and 4 showed that treating rats with deltamethrin caused significant decrease (p< 0.05) in the diameter of the tubules and their epithelial heights. Animals treated with deltamethrin and propolis showed significant increase in the diameter of seminiferous tubules and their epithelial heights in comparison with deltamethrin group. No significant changes were recorded in the diameter of the tubules or the epithelial height in rats treated with propolis compared with control animals.

anatomy-physiology-seminiferous-tubules

Figure 3: Mean diameter of seminiferous tubules in control and treated groups.

anatomy-physiology-Mean-epithelial-height

Figure 4: Mean epithelial height of seminiferous tubules in control and treated groups.

Histochemical Results

PAS-positive materials appeared in tunica albuginea as well as in the intertubular connective tissue of testes of control rats and in those given propolis. The spermatogenic cells exhibited weak reaction while the sperms showed strong reaction (Figure 5a).

anatomy-physiology-Section-testis-control

Figure 5a: Section of testis a control rat showing PAS positive.

Testes of rats treated with deltamethrin revealed a decrease of PAS-positive materials. In these specimens, tunica albuginea, the boundaries of the seminiferous tubules as well as the intertubular connective tissue had weak PAS-positive materials (Figure 5b). An increase in polysaccharides contents were illustrated in testes of animals treated with deltamethrin and propolis (Figure 5c).

anatomy-physiology-testis-rat-treated-deltamethrin

Figure 5b: Section of testis of a rat treated with deltamethrin showing weak PAS reaction.

anatomy-physiology-PAS-inclusions-testis-rat

Figure 5c: A restoration of PASinclusions in testis of a rat given deltamethrin+ propolis (PAS X 400).

Total proteins appeared in the testicular tissues of control rats as deeply stained granules inside the nuclei and cytoplasm of all spermatogenic cells. The tunica albuginea, intertubular connective tissue as well as the boundaries of seminiferous tubules showed strong reaction (Figure 6a). Animals treated with deltamethrin showed a decrease in the protein content of the spermatogenic cells and most cells were degenerated and contained diffused proteins (Figure 6b). Animals treated with deltamethrin and propolis showed that a large number of germ cells restored their protein content but it still had a diffused appearance (Figure 6c).

anatomy-physiology-Normal-protein-content

Figure 6a: Normal protein content in the testis of a control rat.

anatomy-physiology-Reduction-protein-content

Figure 6b: Reduction of the protein content in the germ cells after treatment with deltamethrin.

anatomy-physiology-content-germ-cells

Figure 6c: Restoration of total protein content in most germ cells of a rat treated with deltamethrin+ propolis(Bromophenol blue X 400).

Number of Sperms and Sperm Head Abnormalities

Data in Table 1 shows that the number of sperms was decreased significantly in animals treated with deltamethrin while animals given deltamethrin and propolis showed an increase in the number of sperms. The abnormal heads of the sperm had many shapes. It have banana, without hook, amorphous or hummer shape. Treatment with deltamethrin induced significant increase (P<0.05) in sperm abnormalities. Combined treatment with deltamethrin and propolis significantly reduced the number of sperm head abnormalities.

Animal groups Mean number of sperms Mean number of sperm head abnormalities
Control 987.5 ± 2.5 10.2 ±1.4
Propolis 981.6 ± 2.9 11.8 ± 2.1
Deltamethrin 544 ± 6.6* 78.4± 6.6*
Deltamethrin+ Propolis 705 ± 5.5 37.5 ± 4.5
(*). Significant at p<0.05 compared with controls.

Table 1: Effect of deltamethrin and/or propolison number of sperms and sperm head abnormalities.

Biochemical Results

Figure 7 showed the means of serum testosterone levels in different groups. There was no statistical difference in testosterone level between control and propolis treated groups. Testosterone level significantly (P< 0.05) decreased in sera of rats treated with deltamethrin when compared to the control group. When animals treated with both detamethrin and propolis, significant elevation in testosterone level was observed as compared to deltamethrin- treated rats.

anatomy-physiology-testosterone-control

Figure 7: Change in testosterone in control and treated groups. Deltamethrin group showed significant decrease compared with control group.

Figure 8 showed that rats treated with deltamethrin showed significant reduction in serum LH level (P< 0.05) when compared to control group. Treating animals with detamethrin and propolis revealed elevation in serum LH level when compared to detamethrin-treated group.

anatomy-physiology-Deltamethrin-group-showed

Figure 8: Change in L.H in control and treated groups. Deltamethrin group showed significant decrease compared with control group.

Discussion

The environmental contamination of insecticides as a result of their extensive use has become a serious problem. This stimulated the scientists to study its biological effects. The present results showed that deltamethin caused histological and histochemicalalterations in the testis. Moreover, a decrease in number of sperms and increase in the number of sperm head abnormalities was observed in the treated animals. Biochemical results revealed decrease in testosterone and L.H. Similarly, [20] reported that deltamethrin decreased sperm cell concentrations and sperm motility, and caused many histological alterations in testes of rats. Also noted declines in sperm concentrations, rate of live cells and sperm motility in male rats treated with deltamethrin [21]. reported that subcutaneous deltamethrin treatment created a capture of spermatogenesis and a noteworthy diminishing in F.S.H, L.H. what's more testosterone in rats [22]. Mulled over the impacts of deltamethrin, dimethoate and mixture of the two pesticides on male propagation in mice. An altogether diminished sperm tally, motility and reasonability and fundamentally expanded percent morphologically unusual spermatozoa contrasted and the controls was noted in the gathering presented to deltamethrin and the group exposed to the mixture of dimethoate + deltamethrin [23]. Assessed the adverse effects of deltamethrin on reproductive organs and fertility in male rats and evaluated the protective role of a combination of vitamin E and selenium in alleviating the detrimental effect of deltamethrinon male fertility. They found that that treatment with deltamethrin alone caused statistically significant reductions in epididymal sperm count, motility, and viability, and serum levels of testosterone [24]. Treated mice daily from gestation day 3-21 by oral gavage with deltamethrin, dimethoate or admixture of the two pesticides. Their results showed that a significant reduction in testis weights, epididymal sperm count, motility, and viability in adulthood was noted in deltamethrin-treated mice.

A reduction of total carbohydrates and total proteins was observed in testis of deltamethrin-treated rats. Similar results were obtained by some investigators in testes of animals exposed to different insecticides [25]. Reported that chlorpyrifos brought about marked reduction in testicular glycogen. A reduction in testicular glycogen was found in rats treated with Malathion [26]. A highly significant decrease in glycogen, Fructose, Cholesterol and Sialic acid occurred in testes of mice treated with Cyfluthrin [27] and [28]reported that carbofuran caused reduction of carbohydrates and proteins in testes of mice [29]. Speculated that protein synthesis in spermatogenic cells is dependent upon glucose and a decrease in the glycogen content could affect protein synthesis and thus subsequently inhibit spermatogenesis.

Recently, it has been observed that the oxidative damage to testicular cells can result in testicular dysfunction leading to male infertility [30]. Oxidative stress occurs when the production of Reactive Oxygen Species (ROS) exceeds the body’s natural antioxidant defense mechanisms, causing damage to macromolecules such as DNA, proteins and lipids [31]. It was reported that deltamethrin exert its toxic actions through formation of free radicals [32]. Thus, deltamethrin may induce oxidative stress and resulted in the observed testicular toxicity in albino rats.

Deltamethrin and propolis in combination improved the histological and histochemical alterations induced by deltamethrin. Testosterone, L.H. and sperm count increased while the sperm head abnormalities decreased. In agreement with these results, [33] reported that propolis diminished dead and irregular sperm and TBARS, and expanded testosterone, GSH, 17-ketosteroid reductase, CAT and GST in rats treated with AlCl3.They included that the checked injuries impelled by AlCl3 in seminiferous tubules were improved [34]. Reported that chlorpyrifos and profenofoscaused histopathological alterations in testis of male rats like spermatogenic arrest and edema and degeneration of spermatids, while administration of both propolis and ginseng ameliorate these reproductive toxicity and increased testosterone [35]. Showed that propolis ameliorates nonylphenol-induced testicular alterations and decrease of testosterone and L.H. in albino rats. Treating rats with propolis was found to cause a critical lessening in histological, immunohistochemical, and morphometric changes actuated by cisplatin in the testis [36].

The pharmacological impact of propolis was ascribed to its cell reinforcement action. In this worry, propolis was found to balance cancer prevention agent catalysts and lessening lipid peroxidation forms in plasma, liver, lungs, and cerebrum of mice in a measurement and tissue-subordinate way [37]. Russo et al. reported that propolis has free radical scavenging activity that protects sperm membranefrom the deleterious action of oxidative attack and reduces thiobarbituric acid reactive substances (TBARS) formation [38] demonstrates the scavenging effect of propolis on free radicals produced by liver in response to 4-tert-OP toxicity [39]. Reported a diminishing in superoxide anion radicals and restraint of the lipid peroxidation in rats getting propolis extricate [40] showed that Brazilian propolis have antioxidant effect, scavenging ability on free radicals and inhibitory effect on the superoxide radical mediated contractile activity of guinea pig isolated trachea.

Many investigators recorded that the protective effect of propolis against reproductive toxicity was attributed to its antioxidant activity [33]. Propolis was found to alleviate testicular oxidative stress off AlCl3 [33], nonylphenol [35], profenofos [22], and chlorpyrifos [20], Propolis contains high content of phenolic compounds such as flavonoids which are able to scavenge free radicals [15]. It is supposed in this work that propolis ameliorated deltamethrin-induced reproductive toxicity in male rats and this might due to the antioxidant activity of its flavonoids compounds.

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