ANDROLOGY The Effect of Phytosterols and Fatty Acids of Pistachio (Pistacia vera) Oil on Spermatogenesis and Histological Testis Changes in Wistar Adult Male Rats Soudeh Khanamani Falahati-pour1, Soheila Pourmasumi2,3, Maryam Mohamadi1, Zahra Taghipour4,5, Mohammad Reza Mohammadinasab6, Mojtaba Sajadian7, Fatemeh Ayoobi2, Ali Dini1, Zahra Ahmadi1, Sakineh Khanamani Falahatipour1, Alireza Nazari2,8* Purpose: Oilseeds and their related products are known to have various bioactive and health-promoting ingredi- ents. In this research, we investigated the effects of phytosterols and fatty acids of Pistacia vera on spermatogenesis process and testis histological changes in Wistar male rats for the first time. Materials and Methods: A total number of 64 adult male Wistar rats were divided randomly into eight groups including one control group, and seven test groups. Test groups received phytosterols, fatty acids, and pistachio oil orally for 30 days. Then, LH, FSH, and serum testosterone levels were determined. Also, the spermatogenesis process and changes in testicular tissue in rats were investigated. Results: The results of this research suggest that phytosterols in doses of 10 and 50 mg/kg reduce the spermato- genesis process. Fatty acid in a low dose of 10 mg/kg increases spermatogenesis, but when a high dose of 50 mg/ kg was used, it harmed the spermatogenesis process. When low levels of phytosterols and fatty acids are used simultaneously in dose 5 mg/kg, improvement in spermatogenesis process is observed but when these were used together in the dose of 25 mg/kg, the spermatogenesis process was disrupted. Using pistachio oil alone also im- proved spermatogenesis process. Conclusion: It seems that phytosterols reduce spermatogenesis at high and low doses, while fatty acids increase spermatogenesis when used in low doses and reduce this process when used in high doses. The use of fatty acids extracted from pistachios to treat infertility in men seems hopeful. Keywords: CMA3; male infertility; short abstinence; sperm DNA integrity; TUNEL 1Pistachio Safety Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 2Non-Communicable Diseases Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 3Clinical Research Development Unit (CRDU), Niknafs Hospital, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 4Physiology-Pharmacology Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 5Department of Anatomy, School of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 6Student Research Committee, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 7Department of Clinical Biochemistry, Faculty of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. 8Clinical Research Development Unit (CRDU), Moradi Hospital, Rafsanjan University of Medical Sciences, Rafsanjan, Iran. *Correspondence: Shohada Street, Moradi Hospital, Rafsanjan University of Medical Science, Rafsanjan, Iran. E-mail: Drnazari57@gmail.com Received October 2020 & Accepted July 2021 INTRODUCTION In recent years, one of the problems that human so-cieties have faced is infertility in men(1). Factors involved in male infertility include occupational, en- vironmental, and nutritional factors. Among these fac- tors, diet plays an important role in reproductive health in men(2). Oilseeds are widely used in the human diet; these seeds have many bioactive substances with me- dicinal and biological properties that have been used in the treatment of several diseases. The evidence suggests that ingestion of Oilseeds may impose different cardio- vascular effects thought to be due to their lipid compo- nents, which include unsaturated fatty acids, phytoster- ols and tocopherols(3). Recent research has also shown that dietary intake of edible oil may even have more beneficial effects on total ingested seeds, possibly due to the replacement of carbohydrate diets with unsatu- rated fats or other oil components(4). Pistachios are one of the most important Oilseeds due to their high-fat content. The most important portion of pistachio fat is unsaturated fatty acids, 80% of which are oleic acid and linoleic acid(5). Pistachio oil has chemical compounds that contain sat- urated fatty acids such as myristic acid, palmitic acid, stearic acid and unsaturated oils such as linoleic acid, oleic acid, plant sterols and elements such as selenium, zinc, calcium, potassium, iron, and magnesium(6). Pre- vious studies have shown that compounds in pistachio oil inhibit nitric oxide production. Since this compound can control steroidogenesis, therefore, pistachio oil has been used as a drug for the treatment of related diseases, including increased sexual activity(7). On the other hand, it has been reported that plants containing linoleic acid have been used to treat sexual weakness. Saturated and unsaturated fatty acids, for example palmitic acid, oleic acid, linoleic acid, miristic acid and stearic acid inhib- it 5-alpha-reductase enzyme activity, which causes the conversion of testosterone to di-hydroxy-testosterone Urology Journal/Vol 18 No. 6/ November-December 2021/ pp. 682-687. [DOI: 10.22037/uj.v18i.6605] Vol 18 No 6 November-December 2021 683 (DHT), so inhibiting its activity increases the amount of testosterone in the blood(8). Non-saturated fatty acids can inhibit 5-alpha-reductase enzyme activity, and sat- urated fatty acids increase cholesterol(9). Sterols are a group of natural compounds that are de- rived from hydroxylation of polycyclic isopentanoids and have a structure of 1, 2-cyclopentanophenanthrene. Most plant sterols contain 28 or 29 carbon and have one or two carbon-carbon double bonds in their molecular structure, one of which is dual bonds inside the rings, and the other is on the side chain of the sterol structure. More than 200 different types of plant sterols have been reported in plant species. Five sterols including β-si- tosterol, Δ5-avenasterol, campesterol, stigmasterol and cholesterol have been identified in pistachio oil(10). In recent years, considerable attention has been paid to the study of the effect of different plants on fertility of laboratory mammals. Therefore, this study aimed to investigate the effect of the extracted phytosterols and fatty acids from pistachio (Pistacia vera var. Akbari) oil on spermatogenesis and changes in testicular tissue in adult male Wistar rats and also possibly to predict any benefits or harms of these compounds on fertility. MATERIALS AND METHODS Plant preparation The fruits of Pistacia vera var. Akbari were collected The effect of pistachio oil on spermatogenesis-Falahatipur et al. Body weight (g)a Before treatment After treatment Control (A Group) 203.71 ± 23.26a 234.33 ± 15.00a (B Group) 204.14 ± 30.17a 217.28 ± 33.50a (C Group) 207.57 ± 41.92a 230 ± 38.03a (D Group) 200.57 ± 34.16a 237.67 ± 11.64a (E Group) 202.14 ± 40.58a 221.63 ± 22.83a (F Group) 205.71 ± 44.12a 255.4 ± 17.40a (G Group) 201.86 ± 40.42a 224.33 ± 29.45a (H Group) 203 ± 33.98a 217.5 ± 44.65a P-Value (Kruskal-Wallis) .999 .221 P-Value (post hoc) 1 .276 Table 1. Body weight of mice in different treated group and control before and after treatment Control group (A), and seven test groups (B to H) were treated orally by gavage for a period of 30 days. Test groups received 10 mg/Kg of phytosterols (B group), 50 mg/Kg of phytosterols (C group), 10 mg/Kg of fatty acids (D group), 50 mg/Kg of fatty acids (E group), 5 mg/ Kg of fatty acids plus 5 mg/Kg of phytosterols (F group), 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols (G group) and 50 mg/Kg of pistachio oil (H group). Mean values in a column followed by different letters are significantly different (P < 0.05) Figure 1. Histological sections of the testes of mice in different treated groups and control. Control group (A), and seven test groups (B to H) were treated orally by gavage for a period of 30 days. Test groups received 10 mg/Kg of phytosterols (B), 50 mg/Kg of phytosterols (C), 10 mg/Kg of fatty acids (D), 50 mg/Kg of fatty acids (E), 5 mg/Kg of fatty acids plus 5 mg/Kg of phytosterols (F), 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols (G) and 50 mg/Kg of pistachio oil (H), respectively and (I) demonstrates high magnification photomicrograph obtained from testis of rat showing: Sertoli cells Spermatogonia cells Spermatocyte cells spermatide cells Epithelial thickness Seminiferous diameter Vol 18 No 6 November-December 2021 138 from a garden in Rafsanjan, Iran and approved by a bot- anist at the Vali-e-Asr University of Rafsanjan. Reagents All chemicals and solvents were purchased from Sig- ma-Aldrich (St. Louis, MO) and used without further purification except when mentioned specifically. Pistachio oil preparation The oil of the pistachio kernels was obtained by cold-pressing the dried kernels. The oil was protected from direct sunlight and stored at 4-6 °C. Preparation of phytosterol fraction Unlike fatty acids, phytosterols cannot saponify. There- fore, phytosterols were separated from fatty acids using their reactions with NaOH. To extract the sterols, 0.1 g of the pistachio oil was mixed with 20 mL of 1 M ethanolic NaOH solution and stirred for 12 h at room temperature. Then, 20 mL distilled water and 40 mL diethyl ether were added to the mixture. In this step, the obtained mixture was transferred to a decanter where the sterols were separated from the saponified fatty acids and transferred into the ether phase. After, separating the sterol-rich ether phase, the extraction of the remained sterols from the aqueous phase was us- ing 40mL of excess diethyl ether. The two ether phases were mixed and transferred to a decanter where possible saponifiable components were removed by extracting with 0.5 M ethanolic NaOH solution. The sterol-rich ether phase was finally freeze-dried and the solvent-free sterols were stored at -20 °C until use(11). Preparation of fatty acid fraction To extract the fatty acid fraction, 0.1 g of the pistachio oil was mixed with 20 mL ethanolic NaOH solution (1 M) and stirred for 12 h at room temperature. Then, the mixture along with 20 mL distilled water and 40 mL diethyl ether was transferred to a decanter. The sapon- ified fatty acids were dissolved in water activating the aqueous phase. After separating the aqueous phase, 40 mL of NaOH solution (0.5 M) was added and the ex- traction procedure was carried out again to extract the remaining saponified fatty acids from the ether phase. The separated aqueous phases were mixed and react- ed with 20 mL HCl solution (0.5 M) to convert the saponified fatty acids to free fatty acids. After adding Table 2. The testis weight of mice in different treated groups and control after treatment Testis weight (g)a After treatment Testis weight/BW Control (A Group) 1.35 ± 0.1b 0.005761 ± 0.00052a (B Group) 1.31 ± 0.07b 0.006029 ± 0.0013a (C Group) 1.34 ± 0.15b 0.005826 ± 0.00088a (D Group) 1.31 ± 0.15b 0.005512 ± 0.0007a (E Group) 1.33 ± 0.16b 0.006255 ± 0.0013a (F Group) 1.44 ± 0.17a 0.005638 ± 0.00061a (G Group) 1.16 ± 0.18b 0.005171 ± 0.0015a (H Group) 1.25 ± 0.13b 0.005747 ± 0.0018a P-Value(Kruskal-Wallis) .212 .843 P-Value(post hoc) .119 .815 Control group (A), and seven test groups (B to H) were treated orally by gavage for a period of 30 days. Test groups received 10 mg/Kg of phytosterols (B group), 50 mg/Kg of phytosterols (C group), 10 mg/Kg of fatty acids (D group), 50 mg/Kg of fatty acids (E group), 5 mg/ Kg of fatty acids plus 5 mg/Kg of phytosterols (F group), 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols (G group) and 50 mg/Kg of pistachio oil (H group). Mean values in a column followed by different letters are significantly different (P < 0.05) aData are presented as mean ± SD Figure 2. The number of sertoli cells of mice fed on different concentrations of phytosterols and fatty acids of pistachio oil Andrology 684 The effect of pistachio oil on spermatogenesis-Falahatipur et al. Figure 3. The result of epithelial layer thickness of mice fed on different concentrations of phytosterols and fatty acids of pistachio oil Data are presented as mean ± SD. Bars with same superscript letters are not significantly different whereas those with different superscript letters are significantly different (p < 0.05). Vol 18 No 6 November-December 2021 685 40 mL diethyl ether, the free fatty acids were extracted to the ether phase from the aqueous phase. This proce- dure was repeated again and finally, the separated ether phases were mixed, freeze-dried and stored at -20 °C until use(12). Experimental assays The present study was performed on 64 adult male Wis- tar rats, weighing 200±45 g that were kept in the animal laboratory of Rafsanjan University of Medical Scienc- es. In this experimental study, the animals were housed at room temperature (25 °C), and light was set at 12 h light–dark cycle. They were maintained in plastic cages separately and had free access to food and water. The study protocol was approved by the Ethical Committee of Rafsanjan University of Medical Sciences under the ethical code IR.RUMS.REC.1396.98. They were randomly divided into eight groups (n = 8) including one control group (A), and seven test groups (B to H). Test groups received 10 mg/Kg of phytos- terols (B group), 50 mg/Kg of phytosterols (C group), 10 mg/Kg of fatty acids (D group), 50 mg/Kg of fatty acids (E group), 5 mg/Kg of fatty acids plus 5 mg/Kg of phytosterols (F group), 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols (G group) and 50 mg/Kg of pistachio oil (H group) orally by gavage for 30 days. At the end of the treatment period, the animals were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg) mixture. Then, rats were killed by cervical dislocation and testicular tissues were used for the his- tological analysis. Hormones and biochemical assays Enzyme immunoassay test for serum testosterone, FSH and LH were performed according to manufacturer in- structions. Testosterone, FSH and LH were determined by using kits purchased from AccuBind ELISA Kit, Monobind, USA. Histological analysis For histological investigations, testes were quickly dis- sected and weighed immediately after killing the rats, then divided into small pieces and placed in 10% para- formaldehyde (PH = (7.2) for 72 h for fixation. For each group, testis tissue samples of four rats were selected Table 3. The mean serum testosterone, FSH and LH levels in different treated groups and control after treatment. Parametersa Testosterone (ng/mL) FSH (mlu/dL) LH (mlu/dL) Groups Control (A group) 1.53 ± 0.08c 0.05 ± 0.003a 0.42 ± 0.04a (B group) 0.73 ± 0.05e 0.06 ± 0.008a 0.39 ± 0.02a (C group) 0.68 ± 0.06e 0.07 ± 0.005a 0.44 ± 0.05a (D group) 1.93 ± 0.04b 0.05 ± 0.006a 0.36 ± 0.02a (E group) 1.41 ± 0.7d 0.08 ± 0.009a 0.34 ± 0.01a (F group) 1.45 ± 0.09d 0.06 ± 0.004a 0.45 ± 0.07a (G group) 1.51 ± 0.08c 0.05 ± 0.001a 0.40 ± 0.08a (H group) 2.12 ± 0.12a 0.07 ± 0.002a 0.97 ± 0.02b P-Value(Kruskal-Wallis) .002 P-Value(post hoc) <.001 Control group (A), and seven test groups (B to H) were treated orally by gavage for a period of 30 days. Test groups received 10 mg/Kg of phytosterols (B group), 50 mg/Kg of phytosterols (C group), 10 mg/Kg of fatty acids (D group), 50 mg/Kg of fatty acids (E group), 5 mg/ Kg of fatty acids plus 5 mg/Kg of phytosterols (F group), 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols (G group) and 50 mg/Kg of pistachio oil (H group). Mean values in a column followed by different letters are significantly different (P < 0.05) aData are presented as mean ± SD The effect of pistachio oil on spermatogenesis-Falahatipur et al. Vol 18 No 6 November-December 2021 138 randomly, then ten tissue samples were selected from each testis by a systematic random sampling protocol. Tissue samples were directly dehydrated in a graded series of ethanol, cleared in xylen, impregnated in par- affin wax and embedded in paraffin block. From each sample, thin sections with a thickness of 5 μm were pre- pared by a rotary microtome machine, and finally 5 sec- tions were randomly selected from each sample using the systematic uniform random sampling. All collected sections on slides were stained with hematoxylin-eosin (H & E) and were observed under an optical microscope (E-200, Nikon, Japan). Then the microscopic fields of the slides were photographed randomly with a Nikon camera and the images were transferred to a computer and analyzed with Image tool software. The number of sertoli, spermatogonia, spermatocyte, spermatide cells as well as epithelial thickness and seminiferous tube di- ameter were determined with image tool software and statistically compared in all studied groups. Statistical analysis Normality of the data was tested by Kolmogor- ov-Smirnov method. Results are expressed as Mean ± SD. The difference between groups was compared using one-way ANOVA. Also, post hoc approach was used to compare groups with control. If significant difference was detected, multiple comparisons were made using the Tukey-HSD (α = 0.05). All the statistical analyses were carried out using the SPSS.26 software. A p-value below 0.05 was considered statistically significant. RESULTS This study has attempted to investigate the effect of pistachio (Pistacia vera) oil with phytosterols and fatty acids extracted from it on spermatogenesis and testis histological changes in Wistar male rats and possibly to predict any benefits or harms of these compounds on fertility. Body and testis weight The effects of pistachio oil with phytosterols and fat- ty acids extracted from it were investigated on body weight in male Wistar rats. The mean body weights of mice in all test groups increased but no observed sig- nificant change before and after 30 days’ of treatment (P = .999 and P = .221, respectively). Also, the results of the present study revealed that the body weight in all test groups no had significant value in comparison to the control group (P = .276 and P = 1, respectively) (Table 1). The oral administration of phytosterols and fatty acids extracted from pistachio oil resulted in an increase in testis weight in the F group that the rats received 5 mg/ kg phytosterol + 5 mg/kg fatty acid, but this increase is not significant compared to the control group (P = .119). In addition, the ratio of testis weight to body weight in test groups showed no significant difference in comparison to the control group (P = .815) (Table 2). An increase in the testis weight is related to the number of spermatids and sperm present in the tissue. Serum LH, FSH and testosterone levels Administration of pistachio oil, phytosterols and fatty acids in different concentrations for 30 days had no sig- nificant effect on serum level of FSH hormone in Wis- tar male rats(P = .06). But the serum level of testoster- one was decreased significantly (P = .002) in rats who received phytosterols in 10 mg/Kg and 50 mg/Kg con- centrations (B and C groups, respectively) compared to the control group (P = 0). Also, the serum level of this hormone was increased in D and H groups including rats receiving 10 mg/Kg of fatty acids and 50 mg/Kg of pistachio oil, in comparison to the control, respectively (P = 0). The serum level of LH was unchanged between all test groups and the control group except for the H group that increased after treatment with 50 mg/Kg of pistachio oil (P = .04) (Table 3). Histological examinations Figure 1 shows the tissue sections in all groups under study. According to the results of the histological exam- ination of testes in all groups of study, the mean number of sertoli cells was not significantly changed in all treat- ed groups compared to control group (Figure 2). The epithelial layer thickness in all experimental groups Figure 4. The diameter of seminiferous tubules of mice fed on different concentrations of phytosterols and fatty acids of pistachio oil Data are presented as mean ± SD. Bars with same superscript letters are not significantly different whereas those with different superscript letters are significantly different (p < 0.05). The effect of pistachio oil on spermatogenesis-Falahatipur et al. Vol 18 No 6 November-December 2021 687 was significantly different from the control group (P = 0). According to the results of the present study, the ep- ithelial layer thickness was significantly decreased in B, E and G groups that received 10 mg/Kg of phytosterols, 50 mg/Kg of fatty acids and 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols, respectively. While the epithelial layer thickness in C, D, F and H groups that received 50 mg/Kg of phytosterols, 10 mg/Kg of fatty acids, 5 mg/Kg of fatty acids plus 5 mg/Kg of phytos- terols and 50 mg/Kg of pistachio oil, respectively were significantly increased compared to the control group (Figure 3). The diameter of seminiferous tubules was significantly decreased in the G group while in other groups it did not significantly change (Figure 4). The morphological findings of the study and the sper- matogonia count that used tissue sections as well as the comparison between the mean number of spermatogo- nia showed that there was a significant difference in spermatogonia number, between all test groups and the control group except for the B group that remained un- changed after treatment with 10 mg/Kg of phytosterols. So that the number of spermatogonia was significant- ly decreased in the C group that received 50 mg/kg of phytosterols, while the number of spermatogonia in D, E, F, G and H groups that received 10 mg/kg of fatty acids, 50 mg.kg fatty acids, 5 mg/Kg of fatty acids plus 5 mg/Kg of phytosterols, 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols and 50 mg/Kg of pistachio oil, respectively, were significantly increased compared to control group (Figure 5a). The number of spermatids was significantly decreased in B, C, E and G groups that received 10 mg/Kg of phy- tosterols, 50 mg/Kg of phytosterols, 50 mg/Kg of fatty acids and 25 mg/Kg of fatty acids plus 25 mg/Kg of phytosterols, respectively. While the number of sper- matid in D, F and H groups that received 10 mg/Kg of fatty acids, 5 mg/Kg of fatty acids plus 5 mg/Kg of phytosterols and 50 mg/Kg of pistachio oil, respectively were significantly increased compared to control group (Figure 5b). DISCUSSION Pistacia, a genus of flowering plants from the family Anacardiaceae, contains about twenty species, among them five are more commonly recognized, including P. vera, P. atlantica, P. terebinthus, P. khinjuk, and P. lentiscus. Different parts of Pistacia species have been used in traditional medicine for various aims like aphro- disiac(13). Various types of phytochemical constituents like terpenoids, phenolic compounds, fatty acids, and sterols have also been isolated and identified from dif- ferent parts of Pistacia species(13). Pistacia species have oleaginous fruits considered by several researchers. The oil content in P. vera kernel is about 50–60% (14,15). The main fatty acid in kernel of P. vera is oleic acid(16,17). Other fatty acids identified in these species are linolen- ic, palmitic, palmitoleic, stearic, myristic, eicosanoic, behenic, lignoceric, arachidonic, pentadecanoic, hexa- decanoic, octadecanoic, and margaric acid(14). The most abundant sterol reported in fruits of P. vera is β-sitos- terol followed by Δ5-avenasterol, campesterol and stig- masterol(15). The present study aimed to investigate the effect of the extracted phytosterols and fatty acids from pistachio (Pistacia vera var. Akbari) oil on spermatogenesis and changes in testicular tissue in adult male Wistar rats and also possibly to predict any benefits or harms of these compounds on fertility. In the F group that the rats received 5 mg/kg phytoster- ols + 5 mg/kg fatty acids, the thickness of epithelium layer and also the amount of spermatid and spermatogo- nia increased. In general, spermatogenesis process im- proved. In contrast, in the G group that the rats received 25 mg/kg phytosterol+ 25 mg/kg fatty acid, the thick- ness of the epithelium layer decreased, the diameter of tubules decreased and despite increased spermatogonia cells, the number of spermatids was decreased, result- ing in a general decrease in spermatogenesis process. In the H group that received pistachio oil, the thickness of the epithelium layer, the number of spermatogonia and spermatids and the overall spermatogenesis process increased. Figure 5. The number of spermatogonia (a) and spermatid (b) of mice fed on different concentrationw of phytosterols and fatty acids of pistachio oil, respectively. Data are presented as mean ± SD. Bars with same superscript letters are not significantly different whereas those with different superscript letters are significantly different (p < 0.05). The effect of pistachio oil on spermatogenesis-Falahatipur et al. Vol 18 No 6 November-December 2021 138 Phytosterols extracted from pistachio oil decrease the cholesterol desmolase c activity by reducing the con- version of cholesterol to bergenolone in mitochondria, thereby reducing the synthesis of steroids including tes- tosterone. All phytosterols have anti-androgenic effects and de- crease tissue sensitivity to androgens, besides, andro- gen activity is decreased by inhibiting 5-alpha reductase Inhibition of this enzyme reduces the conversion of tes- tosterone to dihydrotestosterone, an active form of this hormone in tissues(18). In addition, phytosterols can treat benign prostatic hyperplasia (BPH) in the prostate by reducing testosterone and dihydrotestosterone levels. Phytosterols also decrease the level of steroid hormones such as testosterone by lowering cholesterol levels(19). Compounds in pistachio oils such as zinc and linoleic acid can inhibit nitric oxide production. These com- pounds can inhibit steroidogenesis, so pistachio oil probably increases steroidogenic function in Leydig cells through inhibition of the synthesis of nitric oxide and consequently increases the concentration of testos- terone(7). In conclusion, the results of this study suggest that phytosterols in doses 10 and 50 mg/kg reduce spermat- ogenesis process. Fatty acid in a low dose of 10 mg/ kg increases spermatogenesis process, but when a high dose of 50 mg/kg was used, it had a negative effect on spermatogenesis process. When low levels of phytos- terols and fatty acids are used simultaneously in dose 5 mg/kg, improvement in spermatogenesis process is observed but when these were used together in high dose of 25 mg/kg, the spermatogenesis process was dis- rupted. Using pistachio oil alone also improved sper- matogenesis process. It seems that phytosterols reduce spermatogenesis process and fatty acids increase sper- matogenesis when used in low doses and reduce this process when used in high doses. ACKNOWLEDGEMENTS Authors would like to thank Pistachio Safety Research Center, Rafsanjan University of Medical Sciences, Raf- sanjan, Iran for their warm cooperation. This study was financially supported by Rafsanjan University of Med- ical Sciences, Rafsanjan, Iran, under the ethical code of IR.RUMS.REC.1396.98 at Rafsanjan University of Medical Sciences. CONFLICT OF INTEREST The authors report no conflict of interest. REFERENCES 1. Krausz C, Forti G. Clinical aspects of male infertility. In The genetic basis of male infertility, 2000. pp. 1-21: Springer. 2. Bansal AK, Cheema RS. Role of Life Style Factors in Male Reproductive Functions: A Review. Theriogenology Insight-An International Journal of Reproduction in all Animals. 2019: 9: 111-116. 3. Hu FB, Stampfer MJ, Manson JE et al. Frequent nut consumption and risk of coronary heart disease in women: prospective cohort study. Bmj. 1998; 317: 1341-1345. 4. Grundy SM, Florentin L, Nix D et al. Comparison of monounsaturated fatty acids and carbohydrates for reducing raised levels of plasma cholesterol in man. Am J Clin Nutr. 1988; 47: 965-969. 5. Abdolshahi A, Mortazavi S, Naibandi S et al. 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