a variety of stress factors ( i.e. , exposure to microorganisms , hyperthermia , heavy metals , prolonged immobilization , swimming stress , and noise ) are known to inhibit male reproductive functions . noise is one of the major problem today that enhances the apoptosis of the testicular germ cells . this environmental stress may cause increase in the number of free radicals ( i.e. , hydrogen peroxide [ h2o2 ] and reactive oxygen species [ ros ] ) that can cause apoptosis in the spermatogenic cell lineage to decrease the rate of normal sperms . many of these may lack specificity , have low efficiency , or have potentially severe side effects on the treated men . it is generally believed that the antioxidants prevent infertility in men via scavenging of free radicals . honey is full of enzymatic and non - enzymatic antioxidants such as catalase , ascorbic acid , flavonoids , and alkaloids . it acts against oxidative stress and prevents oxygen contact with non - saturated fatty acids and also lipoprotein oxidation . in addition , honey is rich in fructose , glucose , minerals , magnesium , potassium , calcium , sodium chloride , sulfur , ferrous , phosphate , and vitamins c , b6 , b5 , b3 , b2 , and b1 . the efficacy of honey on the fertility of males of different species was also tested in some studies . generally , it was suggested that honey can affect the spermatogenesis process and sperm fertility in males . vitamin e is another antioxidant that presents in cell membranes , it neutralizes h2o2 and prevents cell membrane damage . indeed , -tocopherol is the most biologically active form of vitamin e and is the most abundant form in the body . although the absorption processes of all forms of vitamin e are same , the alpha form is found more in the organs and blood . food supplemental prescriptions that contain vitamin e may contribute to the functional improvement of spermatozoa . also , vitamin e decreases lipid peroxidation produced in oxidative stress and enhances the motility and fertilization power of sperms . to our knowledge , there has been no research regarding the effects of both honey and vitamin e on the testes of rat exposed to noise stress . therefore , this study suggests that treatment by antioxidants like honey and vitamin e may compensate the oxidative damages as a result of noise stress on the germinative and somatic cells of testis parenchyma . honey was obtained from a beekeeping center in uremia , iran and prepared as a 5% solution . mature wistar male rats ( n = 24 , 200  15 g ) were obtained from the laboratory animal reproduction and breeding center , ahvaz , iran . the cages of groups 1 - 3 were placed in a woody and acoustic room ( dimensions of 3  4  3 m ) and then , routinely , exposed to 90 - 130 db voice with 300 - 350 hz frequency of noise emitted by a white noise generator from 7:00 pm to 7:00 am for 50 days . it is worth mentioning that the timer was set in a way that the sound - generating ( speech ) device was turned on for 1 h and turned off for 15 - 60 min and then turned on again . group 1 was exposed to voice along with feeding of 5% honey solution by gavage method . group 2 was exposed to voice waves along with receiving of vitamin e ( 75 mg / ml ) by gavage method and group 3 was exposed to the voice without any complementary material . after 50 days that equals with the duration of one spermatogenesis cycle of rat , testes of rats of all groups were removed and then testicular cells of them were cultured in dulbecco 's modified eagle medium ( dmem ) medium with 10% fetal bovine serum ( fbs ) and penicillin / streptomycin . for this purpose , briefly after separating the capsule under stereo microscope , seminiferous tubules were digested in two steps . first , seminiferous tubules were placed in tubes contains 1 mg / ml collagenase type iv and 200 - 700 g / ml dnasei for 15 min in 37c incubator with gently pipetting action . next , the tubes were centrifuged ( 100g for 5 min ) , the supernatant was discarded , and the cells were resuspend in 1 ml trypsin - ethylenediamminetetraacetate ( edta ; sigma ) and 200 g / ml dnasei for 5 min in 37c incubator . the cells were washed in pbs solution and incubated in fixing buffer ( 1% paraformaldehyde solution ) . next , the cells were centrifuged ( 300g for 5 min ) , washed twice in pbs , resuspended in solution containing dna - labeling , and then incubated ( 37c ) for 1 hour . subsequently , the supernatant was discarded and the cells pre - coated with an antibody - anti brdu solution ( at room temperature for 5 min ) . the cells were then incubated in propidium iodide / rnse staining buffer according to the manufacturer 's instructions . finally , after removing of propidium iodide / rnse staining , the cells were washed again with pbs twice , and assayed for apoptosis by flow cytometry and tanell kit ( invitrogen , ca , usa ) according to the manufacturer 's directions . the significance of differences among different groups was assessed by one - way analysis of variance ( anova ) . honey was obtained from a beekeeping center in uremia , iran and prepared as a 5% solution . mature wistar male rats ( n = 24 , 200  15 g ) were obtained from the laboratory animal reproduction and breeding center , ahvaz , iran . the cages of groups 1 - 3 were placed in a woody and acoustic room ( dimensions of 3  4  3 m ) and then , routinely , exposed to 90 - 130 db voice with 300 - 350 hz frequency of noise emitted by a white noise generator from 7:00 pm to 7:00 am for 50 days . it is worth mentioning that the timer was set in a way that the sound - generating ( speech ) device was turned on for 1 h and turned off for 15 - 60 min and then turned on again . group 1 was exposed to voice along with feeding of 5% honey solution by gavage method . group 2 was exposed to voice waves along with receiving of vitamin e ( 75 mg / ml ) by gavage method and group 3 was exposed to the voice without any complementary material . after 50 days that equals with the duration of one spermatogenesis cycle of rat , testes of rats of all groups were removed and then testicular cells of them were cultured in dulbecco 's modified eagle medium ( dmem ) medium with 10% fetal bovine serum ( fbs ) and penicillin / streptomycin . for this purpose , briefly after separating the capsule under stereo microscope , seminiferous tubules were digested in two steps . first , seminiferous tubules were placed in tubes contains 1 mg / ml collagenase type iv and 200 - 700 g / ml dnasei for 15 min in 37c incubator with gently pipetting action . next , the tubes were centrifuged ( 100g for 5 min ) , the supernatant was discarded , and the cells were resuspend in 1 ml trypsin - ethylenediamminetetraacetate ( edta ; sigma ) and 200 g / ml dnasei for 5 min in 37c incubator . the cells were washed in pbs solution and incubated in fixing buffer ( 1% paraformaldehyde solution ) . next , the cells were centrifuged ( 300g for 5 min ) , washed twice in pbs , resuspended in solution containing dna - labeling , and then incubated ( 37c ) for 1 hour . subsequently , the supernatant was discarded and the cells pre - coated with an antibody - anti brdu solution ( at room temperature for 5 min ) . the cells were then incubated in propidium iodide / rnse staining buffer according to the manufacturer 's instructions . finally , after removing of propidium iodide / rnse staining , the cells were washed again with pbs twice , and assayed for apoptosis by flow cytometry and tanell kit ( invitrogen , ca , usa ) according to the manufacturer 's directions . the significance of differences among different groups was assessed by one - way analysis of variance ( anova ) . in the control group , the mean percentage of apoptotic , necrotic , and live cells were 0.9  0.1 , 0.24  0.01 , and 0.98.41  0.06 , respectively . the level percentage of apoptotic and necrotic cells in the noise group ( 89.31  0.5 and 9.88  0.3 , respectively ) was increased as compared to the control group ( p = 0.003 and p = 0.001 , respectively ) . also , the mean percentage of viability of cells was decreased when compared with that in the control group ( p = 0.003 ) . however , in the case of group pre - treated with honey , the mean percentage of apoptotic and necrotic cells ( 1.89  0.7 and 0.26  0.01 , respectively ) decreased when compared with that in the noise group ( p = 0.002 and p = 0.002 , respectively ) . the level of apoptotic and necrotic cells in groups pre - treated with vitamin e ( 3.42  0.6 and 0.43  0.08 , respectively ) was decreased when compared with that in the noise group ( p = 0.01 and p = 0.01 , respectively ) . it is noteworthy that honey increased the live cells percentage more than did vitamin e ( 97.1%  0.03 vs. 95.56  0.07 , respectively , p < 0.05 ) , but the necrotic cells level was similar between both honey and vitamin e treated groups ( p < 0.05 ) [ figure 1 ] . cells were analyzed for green fluorescence ( fitc ) and for red fluorescence ( pi ) by flow cytometry . percentages of viable , apoptotic and necrotic testicular parenchyma cells were determined by the dot plot of fl1 ( in the x axis ) to fl3 ( in the y axis ) . lower left indicates viability percent and lower right and upper right indicate percentage of apoptosis and necrosis respectively . the comparisons between cell cycle / apoptotic responses of honey , vitamin , noise stress and control groups are showed . in the present study , it was suggested that the noise stress may have negative influences on germinative and somatic cells of testes parenchyma by increasing apoptotic and necrotic cells . considering the current results , some studies confirmed that , in generally , stress can injure testicular cells . yazawa et al . , reported the effect of immobilization stress on the apoptotic rate of the germ cells as well . in normal situation , apoptosis process occurs permanently for maintaining the tissue homeostasis during spermatogenesis . however , high occurrence of apoptosis can cause negative effects in male genital system . apoptotic activity is dependent on the expression of some apoptosis - related genes and proteins ( i.e. , caspase , apaf-1 , nf - kb , p53 , and death receptors ) and also some anti - apoptosis - related genes and proteins ( i.e. , bcl-2 ) . furthermore , some studies have shown that there is a relationship between apoptosis - related proteins and genes and male infertility . in addition , some studies determined whether there is a relationship between steroid hormones or testicular cells and the expression patterns of apoptosis - related proteins and genes . the results of abovementioned studies showed that reduction or inhibition of gonadotropin as well as steroid hormones secretion can lead to apoptosis of testicular cells . therefore , it seems that every factor that can cause , reduce , or even inhibit gonadotropin and sex hormones secretion , normally can lead to induction of apoptosis in germinative and somatic cells of testes parenchyma . therefore , noise stress may disrupt steroid hormones concentration and neuroendocrine gonadal axis in turn is result in increased rate of cellular damage by expressing , as mentioned for testicular tissue , apoptosis - related proteins and genes in testicular cells . in accordance with the above suggestion , chandralekha et al . , reported that the testosterone serum level in rats under noise stress ( 100 db ) was reduced . also , more structural changes in the testis tissue was observed following the noise stress . in addition , it was reported that apoptosis ratio is negatively correlated with normal morphology and motility of sperm and conversely positively correlated with sperm tail defects . the results in this study also show that the necrosis in cells increased following noise stress . consequently , it seems that noise stress can directly lead to induce necrosis in cells population and destruct germ cells of the testicular tissue . alternatively , this study reported that honey and vitamin e decreased apoptosis and necrosis in cells by noise stress and thereby increased cell viability . in fact , focusing on the above suggestions , the only thing that can be concluded from this finding is that vitamin e and especially honey , being rich in enzymatic and nonenzymatic antioxidants , work by neutralizing the expression patterns of apoptosis - related proteins and genes ( may be due to destructive effects of noise stress ) or by regulation of anti - apoptotic patterns . abdul - ghani et al . , claimed that honey enhances the spermatogenesis process without any disruption in sexual hormone concentrations . however , asiyah et al . , reported that honey has no more positive influence on the sex hormones concentration in male . in general , each factor that affects the function of interstitial space cells ( leydig and myoid cells ) as well as epithelium germinal cells ( sertoli and germ cells ) , may consequently lead to suffer a harmful effect on the neuroendocrine gonadal axis and , in turn , on the spermatogenic cell lineage . the above information suggests that honey and vitamin e , because of their effect on both neuroendocrine gonadal axis and testicular cells , may create conditions that modifies or decreases the process of apoptosis and necrosis in the cells . the second assumption is that honey and vitamin e , because of enhanced steroid hormones level to sertoli cells , can have positive effect on the nutrition of germ cells . mahanem et al . , as well as syazana reported that if appropriate dose of honey is applied to rats , the spermatogenic cells lineage and sperm rate increases . therefore , considering the above results and the results of this study , it is suggested that honey and vitamin e can be useful for enhancing the longevity of cells that suffer from deleterious factors such as noise pollution . the findings of this study may specify a novel natural therapeutic approach based on modulation of the apoptotic process induced by pathogenesis stress , i.e. , noise and enhancing spermatogenesis through honey and vitamin e as possible future antioxidant in fertile men .
aims : a variety of stress factors are known to inhibit male reproductive functions . 
 so this study was conducted in order to investigate the effects of honey and vitamin e on the germinative and somatic cells of testes of rats exposed to noise stress.materials and methods : mature male wistar rats ( n = 24 ) were randomly grouped as follows : group 1 ( honey + noise stress ) , 2 ( vitamin e + noise stress ) , 3 ( noise stress , ) and 4 as the control group . in groups 1 , 2 , and 3 , rats were exposed to noise stress . in groups 1 and 2 
 , rats also were given honey and vitamin e , respectively , orally for 50 days . 
 after that , the germinative and somatic cells of testes parenchyma were isolated by digesting the whole testes by a standard method . 
 next , viability , apoptosis , and necrosis of the cells were evaluated by tunel kit and flow cytometry.results:the rates of apoptosis and necrosis of the testicular cells were increased ( p = 0.003 and p = 0.001 , respectively ) , but viability of these cells decreased in testes of rats exposed to noise stress ( p = 0.003 ) . 
 however , administration of honey and vitamin e were significantly helpful in keeping the cells of testis parenchyma alive , which suffers from noise pollution ( p < 0.05 and p < 0.05 , respectively).conclusions : noise stress has negative influences on the cells of testicular tissue by increasing apoptotic and necrotic cells . 
 however , the associated enhancement in healthy cells suggests that honey and vitamin e have positive influences on the testis parenchyma .