Co-administration of atorvastatin and piperine impairs fertility potential in male Wistar rats through disruption of cholesterol homeostasis and testosterone production

Co-administration of atorvastatin and piperine impairs fertility potential in male Wistar rats through disruption of cholesterol homeostasis and testosterone production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Co-administration of atorvastatin and piperine impairs fertility potential in male Wistar rats through disruption of cholesterol homeostasis and testosterone production Sanjib Ghosh, Sweata Sarkar, Jayanta Mistry, Dr. Maharaj Biswas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4253476/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Atorvastatin is administered to treat hyperlipidemia. The fruit of the black pepper plant contains the alkaloid piperine. Both atorvastatin and piperine are known to have hypolipidemic effects. The goal of the current investigation was to assess the effects of atorvastatin and piperine on reproductive potential in male Wistar rats. For this study, twenty rats were obtained and placed into four groups, each with five rats. Group I served as a control, group II animals are treated with atorvastatin (8 mg/kg BW), group III animals received piperine (10 mg/kg BW) and group IV animals were co-administered with piperine (10 mg/kg BW) and atorvastatin (8 mg/kg BW). All treatments were done by using oral gavage for consecutive 28 days and thereafter assessed for lipid profile, H-E staining, sperm parameter analysis, testosterone level detection using ELISA. Results The results showed that co-administration of atorvastatin and piperine (p < 0.05) significantly reduced weight of reproductive organs, changed histoarchitectural patterns of seminiferous tubules, epididymis, seminal vesicle. Serum and testicular cholesterol and testosterone levels of atorvastatin and piperine co-treated (p < 0.05) groups were found to decrease. Sperm count, motility and viability were decreased significantly in atorvastatin and piperine co-treated animals. Conclusion Results of this study revealed that consumption of piperine (active ingredient of black pepper) along with atorvastatin (lipid-lowering drug) have deleterious effects on reproductive potential of male rat. Toxicology Animal Science Atorvastatin Piperine Cholesterol Testosterone Spermatogenesis Fertility Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Male infertility is a rising concern throughout the globe. Many external and environmental factors are associated with male infertility. Smoking, stress, obesity, and alcohol addiction [ 1 ] are in the frontline causes of men infertility along with pesticide and heavy metal toxicity. Many exogenous and endogenous factors are associated with male infertility. Anatomical abnormalities, endocrinological and genetic defects are the most common endogenous factors causing male infertility. Regular diet patterns, medications, environmental conditions, and life style patterns are contributing as exogenous causes of male infertility [ 2 ]. To evaluate the causes of male infertility exogenous factors are ignored in most of the cases. Statins are lipid-lowering drugs that are used to prevent cardiovascular diseases throughout the world. Statins act as a competitive inhibitor of 3 hydroxy-3-methyl-glutaryl-coenzyme A reductase or HMG-CoA reductase [ 3 ] to reduce the biosynthesis of cholesterol [ 4 ]. Cholesterol is an important component that have direct impacts on male reproductive potential because cholesterol is directly associated with steroidogenesis, spermatogenesis, and fertilization [ 5 ]. Cholesterol is the precursor molecule for testosterone biosynthesis and testosterone is needed for proliferation and differentiation of spermatogonia [ 6 ]. Cholesterol is directly linked to sperm motility and capacitation [ 7 ]. Impaired lipid metabolism is severely related to male infertility [ 8 ]. Atorvastatin (ATR) is one of the most prescribed statin drugs. For a long time, several researchers had claimed that atorvastatin consumption results in compromised fertility potential in males [ 9 , 10 , 11 ]. Cholesterol-lowering agents have long been speculated to have affect steroid production in men through their therapeutic effects on cholesterol [ 12 , 13 ]. Piperine is the main secondary metabolite in black pepper ( Piper nigrum L.). Black pepper is a very popular spice throughout the globe [ 14 ]. Piperine has many pharmacological properties and is also used in medicinal practices mainly in Asian countries for a long time [ 15 ]. Piperine has the potentiality to enhance the bioavailability of various drugs and nutrients in animals and humans [ 16 ]. Piperine is also known to have the lipid-lowering effects [ 17 ]. Mishra and Singh (2009) reported antifertility and antispermatogenic effects of piperine and black pepper in male rats characterized by degenerative changes on seminiferous tubules, epididymis, and sperm parameters [ 18 ]. Ethanol extract of black pepper fruit have seen to decrease the number of primary spermatocyte and sperm motility in experimental rat [ 19 ]. Classical work of Malini et al (1999) also reported antispermatogenic effects of piperine [ 20 ]. Piperine is also reported to initiate apoptosis in testicular tissues [ 21 ]. Interaction between dietary foods, herbs and phytochemicals is a less studied area of research. To develop the efficacy and reaction of pharmaceuticals in various elements of medical practices for different groups of individuals and to generate personalised medication, a great deal of research is needed on the interactions between drugs and diet and herbs. Considering the study and understanding of the interactions between drugs and botanicals, efforts should be made to comprehend the potential therapeutic function of botanicals and to support their safe and appropriate use, as well as to evaluate their toxicity, efficacy, and mechanism of action. Herb–drug interaction (HDI) is a significant clinical issue that can emerge when conventional medications and herbs are used concurrently [ 22 , 23 ]. Thomas et al (2021) studied HDI between ATR and PIP and found increased bioavailability of ATR when co-administered with PIP [ 24 ]. It is claimed for a long time that statin therapy has adverse effects on male fertility and PIP is also known to have antispermatogenic effects. Both ATR and PIP are hypodipidemic agents. Several research works have been done to evaluate antifertility aspects of ATR and PIP. But we have not found any research work to evaluate the reprotoxic effects of ATR and PIP co-administration. In this present study we have targeted to find out the effects of ATR and PIP co-administration on reproductive potential of male rats. Materials and Methods Drugs and chemicals Piperine and Sudan black were purchased from Sigma-Aldrich (Germany). Atorvastatin used in this experiment was purchased from Lupin Ltd. (India). ELISA kit was purchased from Accu-bind, Monobind Inc. (CA, USA). Lipid profile test kit was purchased from Robonik Pvt. Ltd. (India). Eosin, nigrosine, PBS were purchased from SRL (India). All other chemicals and reagents used in this experiment were highly pure and of analytical grade. Laboratory animals The study was conducted on adult healthy male Wistar rats of an average age of 70–80 days and body weight of 150–200 gm. Animals were obtained from Centre for Laboratory Animal Research and Training, West Bengal, India. Standard laboratory conditions like 24-26 0 C room temperature, 50–70% relative humidity and 12hour light/dark cycle were maintained for the animals. Rats were acclimatized in the laboratory conditions for 2 weeks. All experimental procedures performed on the animals were approved by IEAC, University of Kalyani, 892/GO/Re/S/01/CPCSEA. Study design A total of 20 male Wistar rats were taken for the experiment. The experiment was conducted for 28 days. Animals were randomly divided into 4 groups each with 5 rats (n = 5). The first group was control group (C) and individuals of this group received 1 ml distilled water every day. The second group (ATR) rats received atorvastatin at a dose of 8 mg/kg body weight. Third group of animals (PIP) were treated with piperine at a dose of 10 mg/kg body weight every day. Fourth group (ATR + PIP) of rats were co-administered with atorvastatin (8 mg/kg body weight) and piperine (10 mg/kg body weight) every day for consecutive 28 days. Sample collection Soon after sacrificing the animals, blood samples were collected from each animal in heparinized tubes. The serum was centrifuged and preserved at -20 0 C for future use for biochemical analysis. Epididymal spermatozoa were collected in PBS and stored in incubator at 37 0 C for further sperm parameter analysis. After dissecting out the testis, epididymis, seminal vesicle, and prostate glands were cleaned and weighed. Then all the tissues were fixed in Bouin’s solution for further histopathological examinations. Sperm parameters analysis After dissecting out, the epididymis was cleaned and the caudal region were cut into pieces. Small pieces of cauda epididymis were placed in a petridish containing 2 ml of 1X PBS solution and the cauda were pierced with a needle to release the spermatozoa in the media. The petridish then incubated for 10 min at 37 ºC with 5% CO2 to let the sperm to swim out of tubules. The suspension in the petridish then examined to measure sperm motility, sperm concentration, and sperm viability at 400X magnifications [ 25 ]. Ten microliters of the sperm suspension were put on a sterile, previously heated microscope slide and covered with a cover slip to measure the motility of the sperm. Using a microscope with a heated stage, at least ten tiny fields were inspected at higher magnification. After being analysed under a microscope for two to four minutes after being isolated, 200 sperm cells were categorised as progressively motile, non-progressively motile, or immotile, and their proportion of the total sperm count was reported. Sperm that rotated or vibrated in the same spot were regarded as non-progressively motile, but sperm that migrated forward were seen as progressively motile. Eosin-Y (0.05%) was used to measure the viability of the sperm [ 25 ]. After adding 20 µL of sperm suspension to 20 µL of Eosin-Y, the mixture was incubated for two minutes at room temperature. Next, sperm viability was evaluated using a 400 × microscope. Using this technique, cells with a changed plasma membrane turn pink, whereas spermatozoa with an undamaged plasma membrane remain uncolored. The ratio of "unstained /pink spermatozoa" was used to determine viability, and the results were given as a percentage. Using an upgraded Neubauer hemacytometer, the number of sperm was ascertained [ 26 ]. The epididymal sperm suspension was diluted in a 1:5 ratio with PBS medium to examine the sperm concentration. The diluted sperm sample was then fed into the two central grids of a Neubauer’s counting chamber. Under a light microscope with a 100× magnification, the quantity of sperm cells was counted from both chambers and averaged. The placement of the sperm's head determined how many were tallied; only sperm with heads between the row's two inner lines were counted. Hematoxylin-Eosin staining of reproductive organs After cleaning the reproductive organs (Testis, epididymis, seminal vesicle, and prostate gland) were fixed in 10% formalin for histological investigations and subsequently embedded in paraffin. Sections (5–6 µm) were stained with Hematoxylin and Eosin for histopathological assessment. Serum Lipid profile Serum lipid profile including total cholesterol, triglycerides, LDL, VLDL, HDL cholesterol were measured by colorimetric methods. Cholesterol in testicular tissue In a screw-capped centrifuge tube, 0.1 ml of the supernatant was combined with 4.9 ml of ferric chloride solution (50 mg FeCl3 6H2O, dissolved in 100 ml acetic acid) after testicular tissue (50 mg/ml) was homogenised in an ether-alcohol mixture (1:3) and centrifuged at 3000 rpm for 10 min. The mixture was then left to stand for 15 minutes. After centrifuging the mixer for ten minutes at 3000 rpm, 1.5 ml of concentrated H2SO4 was added to 2.5 ml of the clear supernatant, and the mixture was left to develop colour for approximately half an hour at room temperature. Using a spectrophotometer, the colour intensity of the unknown and standard was measured against a blank at 560 nm using the Zlatkis et al. (1953) method. The outcome was given as mg/gm tissue [ 27 ]. Testosterone level in serum and in testicular tissue Serum testosterone concentration was measured by using ELISA testosterone kit according to the manufacturer’s instruction (Accu-bind, Monobind Inc. CA, USA). About 100 mg of testicular tissue was homogenized in 1 ml of PBS buffer (PH – 7.4). Homogenates were centrifuged at 10000 rpm for10 min. [ 28 ] and the resultant supernatant used immediately for determination of testosterone by ELISA Method (AcccuBind™ ELISA). Testicular testosterone content was calculated as ng / ml / mg of tissue. Statistical analysis The statistical analysis was performed using GraphPad Prism statistical software (Graphstats Technologies, USA). Differences among individual groups were determined by one-way ANOVA analysis followed by Tukey’s test to make comparisons. Data are expressed as mean ± SEM (standard error of the mean). A p value of < 0.05 was considered statistically significant. Results Effects on gravimetry of reproductive organs The changes in the weight of reproductive organs of rats in all the treated groups (ATR, PIP, ATR + PIP) along with control group are presented in Fig. 1 and Table 1 . Reduction in testis weight found highly significant in ATR + PIP (p < 0.001) co-treated group, PIP (p < 0.001) group and ATR (p < 0.01) group in comparison with control group (Table 1 ). A significant reduction in the weight of epididymis was also recorded in all the treated groups when compared to control group. Weight of seminal vesicles in all treated groups showed huge reduction (p < 0.001). No any significant changes found in prostate gland weight in ATR and PIP treated groups but ATR + PIP co-treated group showed significant reduction (p < 0.01) in weight. Table 1 Weight changes of reproductive organs CONTROL ATR PIP ATR + PIP Testis weight (gm) 1.226 ± 0.020 1.096 ± 0.028 ** 1.016 ± 0.028 *** 0.856 ± 0.024 *** Epididymis weight (gm) 0.436 ± 0.022 0.350 ± 0.007 ** 0.384 ± 0.014 ** 0.322 ± 0.012 *** Seminal vesicle weight (gm) 0.592 ± 0.015 0.326 ± 0.012 *** 0.468 ± 0.014 *** 0.302 ± 0.018 *** Ventral prostate gland weight (gm) 0.144 ± 0.009 0.136 ± 0.011 ns 0.136 ± 0.009 ns 0.096 ± 0.006 ** Values are expressed as mean ± SEM, n = 5. When p < 0.05 (*), p < 0.01 (**), p < 0.001(***) was significant when compared with control and ns means not significant. Unit in grams (gm). Effects on sperm parameters Sperm count, viability, and sperm motility- these three sperm parameters were evaluated in this experiment (Table 2 and Fig. 2 ). As compared to the control group, the ATR group showed insignificant changes in sperm count and sperm motility parameter but a significant reduction (p < 0.001) in viability of epididymal spermatozoa were seen in this group. PIP group showed a significant decline in sperm count (p < 0.01) and sperm viability (p < 0.001) but no any significant changes found in case of sperm motility compared to control group. A high reduction (p < 0.001) in sperm count and viability were observed in ATR + PIP co-administered group in comparison with control group (Table 2 ). In case of sperm motility changes, ATR + PIP co-treated group showed significant decrease (p < 0.05) in sperm motility when compared to the control group. Table 2 Sperm parameter changes in all the groups CONTROL ATR PIP ATR + PIP Sperm count (X10 6 /ml) 30 ± 1.4 26 ± 1.1 ns 22 ± 0.9 ** 19 ± 1.3 *** Viability (%) 86 ± 0.95 69 ± 2.00 *** 72 ± 1.66 *** 63 ± 1.72 *** Motility (%) 74.4 ± 1.89 66.4 ± 1.69 ns 65.0 ± 2.61 ns 62.6 ± 3.07 * Values are expressed as mean ± SEM, n = 5. Significance levels are denoted as * (p < 0.05); ** (p < 0.01); *** (p < 0.001) and ns for not significant. Effects on histopathology of reproductive organs The histopathological examination of the testicular sections of the animals of control group showed typical histological architecture of seminiferous tubules, spermatogonia cells, Leydig cells, Sertoli cells, and blood vessels. Animals co-administered with ATR + PIP showed mild atrophy of seminiferous tubules associated with mild interstitial oedema. Size of seminiferous tubules lumen increased in all treated groups compared to control group (Fig. 3 ). Epididymis sections from the control group showed normal morphology with compactly arranged tubules. Control group showed high density of sperms in the lumen of cauda epididymis whereas both ATR and ATR + PIP co-treated groups showed a noticeable decrease in epididymal lumen (Fig. 3 ). Histopathologic examination of the seminal vesicle sections of negative control group showed the normal histologic structure with columnar lining epithelium and dense fluid (Fig. 3 ). Co-treatment with ATR + PIP in all groups showed mild hyperplasia in epithelial lining. Ventral prostate from control group showed tubules with normal epithelial height. Epithelial height was decreased in all treated groups. Interstitial oedema was found mostly in ATR + PIP treated group and slightly in ATR and PIP groups compared to control group (Fig. 3 ). ATR + PIP co-treated group showed mild atrophy of prostatic acini associated with slight degeneration of epithelial lining. Effects on serum lipid profile Lipid profile evaluation results showed that all the treatment groups had changed cholesterol and triglyceride levels. A huge decline in cholesterol (p < 0.001) were found in ATR + PIP co-treated group of animals. Both ATR (p < 0.01) and PIP (p < 0.05) treated groups showed significant decrease in serum cholesterol contents. Triglycerides in all the treated groups decreased significantly (p < 0.001). No any observable changes found in HDL levels in treated groups when compared to control group (Table 3 ). Table 3 Changes in lipid profile CONTROL ATR PIP ATR + PIP Cholesterol (mg/dl) 109.4 ± 5.74 82.4 ± 5.52 ** 89.2 ± 2.85 * 64.4 ± 2.60 *** Triglycerides (mg/dl) 118.2 ± 5.15 81.2 ± 2.87 *** 84.8 ± 5.05 *** 56.6 ± 2.50 *** HDL (mg/dl) 33.8 ± 3.86 32.0 ± 2.28 ns 24.2 ± 1.53 ns 23.8 ± 1.85 ns VLDL (mg/dl) 23.6 ± 1.03 16.2 ± 0.58 *** 17.0 ± 0.95 *** 11.4 ± 0.51 *** LDL (mg/dl) 60.8 ± 6.54 33.0 ± 3.94 ** 43.6 ± 4.81 ns 21.4 ± 2.60 *** Changes in lipid profile were recorded in all the groups. Values are expressed as mean ± SEM, n = 5. Significance levels are denoted as * (p < 0.05); ** (p < 0.01); *** (p < 0.001) and ns for not significant. Effects on testicular cholesterol level We evaluated the cholesterol levels in testicular tissues. Results of our experiment exhibited in Fig. 4 . In ATR treated group and ATR + PIP co-administered group, testicular cholesterol levels were found to decrease in a drastic pattern (p < 0.001). A significant decline in cholesterol levels in PIP (p < 0.01) treated group were also seen. Effects on serum and testicular testosterone level The results of serum and testicular testosterone levels are exhibited in Fig. 4 . Treatment with ATR (p < 0.01) and PIP individually resulted in significant decrease in testicular testosterone levels compared to the control group. ATR + PIP co-treated group (p < 0.001) showed drastic decline in testicular testosterone levels. Serum testosterone levels in ATR treated group (p < 0.01), PIP treated group (p < 0.001) and ATR + PIP co-treated group (p < 0.001) were found to have significant decrease when compared to control group. Discussion Serum lipid profile of ATR and PIP treated animals in our study showed changes in total cholesterol, HDL, LDL and TG levels (Table 3 ). A slight change in total cholesterol level found in ATR and PIP treated groups but cholesterol levels in ATR + PIP co-treated animals decreased very much. Vijaykumar et al (2006) also found that PIP treated rats show lowered serum cholesterol levels [ 29 ]. Cholesterol levels in testicular tissues were found to decrease significantly in ATR + PIP co-administered group (Fig. 4 ). The cholesterol levels in serum and testicular tissues found to decline significantly in ATR + PIP co-treated groups. Previous studies regarding effects of ATR and PIP treatment on serum lipid profile support our experimental data [ 29 , 30 ]. For the first time we studied the effects of ATR + PIP co-administration on serum lipid profile and testicular tissue cholesterol levels. The data of our experiment on testosterone levels in serum and testicular tissues are of very much significant (Fig. 4 ). We found drastic reduction in testosterone levels in both serum and testicular tissues. Akdeniz et al (2020) also reported similar changes in serum testosterone levels in ATR administered male rats [ 9 ]. Malini et al (1999) studied effects of piperine on serum testosterone levels and found decrease of testosterone in PIP treated male rats [ 20 ]. The results of this experiment also support the previous studies. Here we found significant decrease in testosterone levels in both serum and testicular tissues of ATR + PIP co-treated animals. Individual treatments with ATR and PIP in terms of cholesterol levels and testosterone levels in serum and testicular tissues showed sufficient similarities with previous studies [ 6 , 9 , 11 , 31 ]. But co-treatment with ATR + PIP revealed there is a clear relationship between cholesterol levels and testosterone levels. In both circulation and tissues, we found reduced levels of cholesterol and testosterone. It implies that co-administration of ATR + PIP affects both cholesterol biosynthesis and testosterone biosynthesis procedures. In this case it seems that PIP could have enhanced the bioavailability of ATR [ 22 , 23 , 24 ] and synergistic effects of both ATR and PIP may have resulted in decreased synthesis of cholesterol that have affected the steroidogenesis in Leydig cells. Weight of reproductive organs, changes in sperm parameters (sperm count, sperm motility, sperm viability) and histopathological aberrations are the main parameters that have immense role in fertility potential of male. In this study we considered the weight changes of testis, epididymis, seminal vesicle, and ventral prostate gland (Fig. 1 and Table 1 ). From the data in Table 1 it is evident that the weight of testis, epididymis and seminal vesicle decreased in all treated groups whereas the weight of ventral prostate gland exhibited no significant weight change in treated groups except ATR + PIP co-treated groups compared to control group. Klinefelter et al (2014) reported that high-dose atorvastatin resulted in low testes weights [ 10 ]. D’cruz and Mathur (2005) studied the changes in weight of epididymis, seminal vesicle and ventral prostate glands in PIP treated rats [ 32 ]. Co-treatment with ATR + PIP has affected the reproductive organs more drastically than other treatment groups. Atrophic and degenerative tissue may be indicated by decreased reproductive organ mass, which is a measure of reproductive toxicity [ 33 ]. Since the mass of differentiated spermatogenic cells determines the weight of the testis, a decrease in the density of mature spermatids and germ cells could be the cause of the testis weight drop. Similarly, decreased sperm production would eventually result in decreased sperm storage, which would lower epididymal weight [ 34 ]. The spermatogenic arrest and regression of seminiferous tubules size in ATR + PIP co-treated rats may be the cause of the testicular weight loss, since seminiferous tubules comprise approximately 90% of the wet weight of a normal rat testis [ 35 ]. ATR + PIP co-treated rats were also found to have low quantities of lipids, which make about 30% of the total testicular weight [ 36 ] and may have also played a role in the testicular weight decrease that was observed. Both the diameters tubular lumen and interstitial space were considerably expanded after ATR + PIP exposure (Fig. 3 ). It also decreased the diameter of seminiferous tubules, the height of the epithelium, and the thickness of tunica albuginea. Exposure to ATR + PIP was also observed to reduce several spermatogonia, primary and secondary spermatocytes, and spermatids. The morphologic indicators of spermatogenic failure include diminished spermatogenic cell numbers and seminiferous tubule atrophy [ 37 ]. Previous studies have shown that PIP inhibited spermatogenesis on male adult albino rats after 30 days of treatment [ 20 , 32 , 38 ]. ATR is also claimed to have anti-spermatogenic effects [ 9 ]. In our study we extended the outcome to that consumption of ATR along with PIP disrupts spermatogenesis. Histopathological sections of Epididymis in control group showing columnar epithelium, cilia, smooth muscle, and spermatozoa. Epididymis of ATR + PIP co-treated groups showing a few ducts with spermatozoa while others are devoid of spermatozoa. The sections of seminal vesicle from control group animals underwent histopathological analysis, which revealed the gland's characteristic histologic structure, which includes columnar lining epithelium and thick fluid. When ATR and PIP were used together, the lining epithelium displayed moderate hyperplasia. This study demonstrates the clear damage to the androgenic dependent accessory reproductive organs, including the prostate gland, seminal vesicle, and epididymis. Androgens have a crucial role in maintaining the accessory gland's ability to secrete [ 39 ]. The changes in sperm parameters were investigated in this experiment. The observed results are depicted in Table 2 and Fig. 2 . Epididymal sperm count, motility and sperm viability adversely affected in ATR + PIP co-treated animals. Previous studies on ATR treatment [ 11 ] and PIP treatment [ 32 ] exhibited similar results. We found drastic decrease in sperm count, motility and sperm viability in ATR + PIP co-treated animals. Atorvastatin treatment have negative impact on sperm parameters that reaches in extreme when the individual intakes piperine orally or with diet. The present study has revealed a decrease in sperm motility, count, and viability. This could be attributed to the lower bioavailability of testosterone, as the epididymis and the fertilising capacity of its contained spermatozoa are dependent on testicular androgens. Conclusion In conclusion, our study shows that co-administration of atorvastatin and piperine decreases serum and testicular cholesterol and testosterone levels. Epididymal sperm count, sperm motility and sperm viability found to decline adversely in ATR + PIP co-treated animals. Weight of testis, epididymis and seminal vesicles were decreased significantly in ATR + PIP co-treated animals. Histopathological studies of seminiferous tubules, cauda epididymis and seminal vesicles exhibited degenerative status. For the first time we evaluated the reprotoxic effects of atorvastatin and piperine co-administration on male rats that resulted in reduced fertility potential due to disruption of cholesterol and testosterone homeostasis. List Of Abbreviations ATR Atorvastatin ELISA Enzyme linked immunoassay HDI Herb Drug Interaction HE Hematoxylin Eosin HMG Co A β-Hydroxy β-methylglutaryl-CoA IEAC Institutional Animal Ethics Committee LDL Low Density Lipoprotein PIP Piperine SEM Standard Error of Mean Declarations Ethics approval and consent to participate This study was performed in accordance with the guidelines for the care and purpose of laboratory animals. All the experiments were carried out in accordance with the recommendations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India (No. 892/GO/ Re/S/01/CPCSEA), with the approval of the Institutional Animal Ethics Committee (IAEC), University of Kalyani. Consent for publication Not applicable. Availability of data and materials Data will be made available on request. Competing of interest The authors declare that they have no conflict of interest. The authors also state that none of the work described in this publication appears to have been influenced by any known competing financial interests or personal ties. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors' contributions SG and MB conceived the idea and designed the study. SG, MB, and SS performed the experiments. SG and MB did the statistical analysis. SG wrote the manuscript. JM and SS checked the manuscript and arranged the references. MB supervised the experiment. All authors read and approved the final version of manuscript. Acknowledgements The authors express their sincere gratitude to the Head of the Department of Zoology, University of Kalyani and the Principal, Rishi Bankim Chandra College for providing all the necessary help and facilities for carrying out this research. Authors’ information Sanjib Ghosh is an Assistant Professor of Molecular Biology at RBC college (affiliated to West Bengal State University, India) and currently involved in research on male infertility at Endocrinology (Reproductive) Laboratory, Department of Zoology, University of Kalyani, India. Dr. Maharaj Biswas is an Assistant Professor of Endocrinology at University of Kalyani, India. Dr. Jayanta Mistry is an Assistant Professor of Endocrinology at Government General Degree College, India. Sweata Sarkar is a researcher at Endocrinology Laboratory, University of Kalyani, India. References Rehman R, Zahid N, Amjad S, Baig M, Gazzaz ZJ (2019) Relationship between smoking habit and sperm parameters among patients attending an infertility clinic. Front Physiol 10:1356. https://doi.org/10.3389/fphys.2019.01356 Saugandhika S, Sapra L, Kumari K, Srivastava RK (2023) High Salt Diet Impairs Male Fertility in Mice via Modulating the Skeletal Homeostasis. 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Indian J Exp Biol 47:706–714 Ekaputri TW, Sari IP, Rizal DM (2018) The Effect of Ethanol Extract of Piper nigrum L. Fruit on Reproductive System in Adult Male Wistar Rats: A Study of FSH, LH, Testosterone Level and Spermatogenic Cells. Indonesian J Pharm 29(3):136 Malini T, Manimaran RR, Arunakaran J, Aruldhas MM, Govindarajulu P (1999) Effects of piperine on testis of albino rats. J Ethnopharmacol 64:219–225. https://doi.org/10.1016/s0378-8741(98)00128-7 D’Cruz SC, Vaithinathan S, Saradha B, Mathur RP (2008) Piperine activates testicular apoptosis in adult rats. J Biochem Mol Toxicol 22:382–388. https://doi.org/10.1002/jbt.20251 Brantley SJ, Argikar AA, Lin YS, Nagar S, Paine MF (2014) Herb drug interactions: Challenges and opportunities for improved predictions. Drug Metab Dispos 42:301–331. https://doi.org/10.1124/dmd.113.055236 Bedada SK, Boga PK (2017) The influence of piperine on the pharmacokinetics of fexofenadine, a P-glycoprotein substrate, in healthy volunteers. Eur J Clin Pharmacol 73:343–349. https://doi.org/10.1007/s00228-016-2173-3 Thomas AB, Choudhary DC, Raje A, Nagrik SS (2021) Pharmacokinetics and Pharmacodynamic Herb-Drug Interaction of Piperine with Atorvastatin in Rats. J Chromatogr Sci 59:371–380. https://doi.org/10.1093/chromsci/bmaa126 World Health Organization (2021) WHO laboratory manual for the examination and processing of human semen Jaâ M (2015) A simple and practical method for rat epididymal sperm count (Rattus norvegicus). Biol Med Nat Prod Chem 4(1):1–3. https://doi.org/10.14421/biomedich.2015.41.1-3 Zlatkis A, Zak B, Boyle AJ (1953) A new method for the direct determination of serum cholesterol. J Lab Clin Med 41:486–492 Li S, Lu DanDan Z Yaling and, Zhang Y (2014) Long-term treatment of hydrogen-rich saline abates testicular oxidative stress induced by nicotine in mice. J Assist Reprod Gen 31(1):109–114. https://doi.org/10.1007/s10815-013-0102-2 Vijayakumar RS, Nalini N (2006) Lipid-lowering efficacy of piperine from Piper nigrum L. in high‐fat diet and antithyroid drug‐induced hypercholesterolemic rats. J Food Biochem 30(4):405–421. https://doi.org/10.1111/j.1745-4514.2006.00074.x Ochiai A, Miyata S, Shimizu M, Inoue J, Sato R (2015) Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins. PLoS ONE 10(10):e0139799. https://doi.org/10.1371/journal.pone.0139799 Esmail M, Kandeil M, El-Zanaty AM, Abdel-Gabbar M (2020) The ameliorative effect of atorvastatin on serum testosterone and testicular oxidant/antioxidant system of HFD-fed male albino rats. https://doi.org/10.12688/f1000research.25926.1 . F1000Res 51300 D'cruz SC, Mathur PP (2005) Effect of piperine on the epididymis of adult male rats. Asian J Androl 7(4):363–368. https://doi.org/10.1111/j.1745-7262.2005.00059.x Michael B, Yano B, Sellers RS, Perry R, Morton D, Roome N, Johnson JK, Schafer K (2007) Evaluation of organ weights for rodent and non-rodent toxicity studies: a review of regulatory guidelines and a survey of current practices. Toxicol Pathol 35:742–750. https://doi.org/10.1080/01926230701595292 Mesbah SF, Shokri S, Karbalay-Doust S, Mirkhani H (2007) The effect of nandrolone decanoate on the body, testis and epididymis weight and semen parameters in adult male rats. Iran J Med Sci 32(2):93–99 Christensen AK, Mason NR (1965) Comparative ability of seminiferous tubules and interstitial tissue of the rat testis to synthesize androgens from progesterone-4 14C in vitro. Endocrinology 76:646–655. https://doi.org/10.1210/endo-76-4-646 Amann RP, Johnson AD, Gomes WR, Vandemark NL (1970) The testis Ma Q, Li Y, Luo M, Guo H, Lin S, Chen J, Du Y, Jiang Z, Gui Y (2017) The expression characteristics of FAM71D and its association with sperm motility. Hum Reprod 32:2178–2187. https://doi.org/10.1093/humrep/dex290 Chen X, Li Y, Ge RS (2018) Diverged effects of piperine on testicular development: Stimulating leydig cell development but inhibiting spermatogenesis in rats. Front Pharmacol 9:333929. https://doi.org/10.3389/fphar.2018.00244 Lina S, Eliza H, Hashida NH, Ibrahim SF, Osman K (2018) Androgen receptor and ultrastructural features of Nigella sativa oil and nicotine-treated male rat reproductive glands. Sains Malays 47(8):1827–1833 Additional Declarations The authors declare no competing interests. Supplementary Files Data.xlsx GA.png Graphical Abstract Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4253476","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290207678,"identity":"87b7a3e2-32f6-49a7-ae2b-6d6be8e5c0f5","order_by":0,"name":"Sanjib Ghosh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sanjib","middleName":"","lastName":"Ghosh","suffix":""},{"id":290207679,"identity":"370b615d-f735-4c8f-b828-e653cecaddb9","order_by":1,"name":"Sweata Sarkar","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sweata","middleName":"","lastName":"Sarkar","suffix":""},{"id":290207680,"identity":"6819f111-740d-48a2-90af-c508fbf8a98b","order_by":2,"name":"Jayanta Mistry","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jayanta","middleName":"","lastName":"Mistry","suffix":""},{"id":290207681,"identity":"197fb014-a277-4b4f-8352-08986a3239dd","order_by":3,"name":"Dr. Maharaj Biswas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDCCAwxsDAkgBg8D4wMQxUeKFmYDEMVGlBYGiBY2CRBNUAvf7dNpDx78OSwv33PGrPJrjp0MGwPzw0c38GiRPJe73SCx7bDhhrM9ZrdltyUDHcZmbJyDR4vBGd5tEokNtxk38POY3ZbcxgzUwsMmTVBLwp/b9vP7ecyKJbfVE6uF7XZiA9BhjB+3HSasRfIML8gv/5M3nDlWLM247TgPGzMBv/ABbXn440+a7fye5I0ff26rtudnb374GJ8WFMDMAyaJVQ4CjD9IUT0KRsEoGAUjBgAAwExJu43GigcAAAAASUVORK5CYII=","orcid":"","institution":"University of Kalyani","correspondingAuthor":true,"prefix":"Dr.","firstName":"Maharaj","middleName":"","lastName":"Biswas","suffix":""}],"badges":[],"createdAt":"2024-04-11 15:59:33","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4253476/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4253476/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54538350,"identity":"0ce3ec11-c4f4-41c6-ae90-c4469fd60e6f","added_by":"auto","created_at":"2024-04-12 03:30:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":738031,"visible":true,"origin":"","legend":"\u003cp\u003eReduction in size and weight of reproductive organs in treated groups. A. Testis, B. Epididymis, C. Ventral Prostate gland, D. Seminal vesicle.\u003c/p\u003e","description":"","filename":"TISSUE.png","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/ff8153b316518c76cc7404d8.png"},{"id":54537708,"identity":"9afc7421-8e57-471f-a2d6-a872b218eddf","added_by":"auto","created_at":"2024-04-12 03:22:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2059238,"visible":true,"origin":"","legend":"\u003cp\u003eEosin-Nigrosin stained sperms.\u003c/p\u003e","description":"","filename":"sperm.png","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/aaf44132c1076682acb488b2.png"},{"id":54538659,"identity":"1398bb50-7e00-4904-b584-4a601da8a0aa","added_by":"auto","created_at":"2024-04-12 03:38:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1283933,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrographs showing histology of testis, epididymis, seminal vesicle, and prostate gland.\u003c/p\u003e","description":"","filename":"histology.png","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/1e4723a1e27f1d4eefaaeede.png"},{"id":54538348,"identity":"b9fb09ab-63ae-4720-ba53-91ab2dec9de5","added_by":"auto","created_at":"2024-04-12 03:30:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45245,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in testicular cholesterol, testicular testosterone, and serum testosterone levels in all the groups. Values are expressed as mean ± SEM, n = 5. Significance levels are denoted as * (p \u0026lt; 0.05); ** (p \u0026lt; 0.01); *** (p \u0026lt; 0.001) and ns for not significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/0492308f975f44e13a9a3fa2.png"},{"id":54539131,"identity":"d3751969-e405-405f-b8d3-fa24fb4d5e44","added_by":"auto","created_at":"2024-04-12 03:54:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4245993,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/03f1fbdb-069d-4888-b227-7ca8a5179610.pdf"},{"id":54537705,"identity":"621a83d8-bb1b-476e-a193-4442175c3616","added_by":"auto","created_at":"2024-04-12 03:22:42","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Data.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/f3cb155a934894db4db2ce48.xlsx"},{"id":54537704,"identity":"0797695a-2609-43dd-a132-b4f19c006d3c","added_by":"auto","created_at":"2024-04-12 03:22:42","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":298439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-4253476/v1/601f987cdb47ed6f53cf38f5.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCo-administration of atorvastatin and piperine impairs fertility potential in male Wistar rats through disruption of cholesterol homeostasis and testosterone production\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eMale infertility is a rising concern throughout the globe. Many external and environmental factors are associated with male infertility. Smoking, stress, obesity, and alcohol addiction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] are in the frontline causes of men infertility along with pesticide and heavy metal toxicity. Many exogenous and endogenous factors are associated with male infertility. Anatomical abnormalities, endocrinological and genetic defects are the most common endogenous factors causing male infertility. Regular diet patterns, medications, environmental conditions, and life style patterns are contributing as exogenous causes of male infertility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To evaluate the causes of male infertility exogenous factors are ignored in most of the cases. Statins are lipid-lowering drugs that are used to prevent cardiovascular diseases throughout the world. Statins act as a competitive inhibitor of 3 hydroxy-3-methyl-glutaryl-coenzyme A reductase or HMG-CoA reductase [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] to reduce the biosynthesis of cholesterol [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cholesterol is an important component that have direct impacts on male reproductive potential because cholesterol is directly associated with steroidogenesis, spermatogenesis, and fertilization [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cholesterol is the precursor molecule for testosterone biosynthesis and testosterone is needed for proliferation and differentiation of spermatogonia [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cholesterol is directly linked to sperm motility and capacitation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Impaired lipid metabolism is severely related to male infertility [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Atorvastatin (ATR) is one of the most prescribed statin drugs. For a long time, several researchers had claimed that atorvastatin consumption results in compromised fertility potential in males [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Cholesterol-lowering agents have long been speculated to have affect steroid production in men through their therapeutic effects on cholesterol [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePiperine is the main secondary metabolite in black pepper (\u003cem\u003ePiper nigrum\u003c/em\u003e L.). Black pepper is a very popular spice throughout the globe [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Piperine has many pharmacological properties and is also used in medicinal practices mainly in Asian countries for a long time [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Piperine has the potentiality to enhance the bioavailability of various drugs and nutrients in animals and humans [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Piperine is also known to have the lipid-lowering effects [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Mishra and Singh (2009) reported antifertility and antispermatogenic effects of piperine and black pepper in male rats characterized by degenerative changes on seminiferous tubules, epididymis, and sperm parameters [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Ethanol extract of black pepper fruit have seen to decrease the number of primary spermatocyte and sperm motility in experimental rat [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Classical work of Malini et al (1999) also reported antispermatogenic effects of piperine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Piperine is also reported to initiate apoptosis in testicular tissues [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInteraction between dietary foods, herbs and phytochemicals is a less studied area of research. To develop the efficacy and reaction of pharmaceuticals in various elements of medical practices for different groups of individuals and to generate personalised medication, a great deal of research is needed on the interactions between drugs and diet and herbs. Considering the study and understanding of the interactions between drugs and botanicals, efforts should be made to comprehend the potential therapeutic function of botanicals and to support their safe and appropriate use, as well as to evaluate their toxicity, efficacy, and mechanism of action. Herb\u0026ndash;drug interaction (HDI) is a significant clinical issue that can emerge when conventional medications and herbs are used concurrently [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thomas et al (2021) studied HDI between ATR and PIP and found increased bioavailability of ATR when co-administered with PIP [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is claimed for a long time that statin therapy has adverse effects on male fertility and PIP is also known to have antispermatogenic effects. Both ATR and PIP are hypodipidemic agents. Several research works have been done to evaluate antifertility aspects of ATR and PIP. But we have not found any research work to evaluate the reprotoxic effects of ATR and PIP co-administration. In this present study we have targeted to find out the effects of ATR and PIP co-administration on reproductive potential of male rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDrugs and chemicals\u003c/h2\u003e \u003cp\u003ePiperine and Sudan black were purchased from Sigma-Aldrich (Germany). Atorvastatin used in this experiment was purchased from Lupin Ltd. (India). ELISA kit was purchased from Accu-bind, Monobind Inc. (CA, USA). Lipid profile test kit was purchased from Robonik Pvt. Ltd. (India). Eosin, nigrosine, PBS were purchased from SRL (India). All other chemicals and reagents used in this experiment were highly pure and of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLaboratory animals\u003c/h2\u003e \u003cp\u003eThe study was conducted on adult healthy male Wistar rats of an average age of 70\u0026ndash;80 days and body weight of 150\u0026ndash;200 gm. Animals were obtained from Centre for Laboratory Animal Research and Training, West Bengal, India. Standard laboratory conditions like 24-26\u003csup\u003e0\u003c/sup\u003eC room temperature, 50\u0026ndash;70% relative humidity and 12hour light/dark cycle were maintained for the animals. Rats were acclimatized in the laboratory conditions for 2 weeks. All experimental procedures performed on the animals were approved by IEAC, University of Kalyani, 892/GO/Re/S/01/CPCSEA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eA total of 20 male Wistar rats were taken for the experiment. The experiment was conducted for 28 days. Animals were randomly divided into 4 groups each with 5 rats (n\u0026thinsp;=\u0026thinsp;5). The first group was control group (C) and individuals of this group received 1 ml distilled water every day. The second group (ATR) rats received atorvastatin at a dose of 8 mg/kg body weight. Third group of animals (PIP) were treated with piperine at a dose of 10 mg/kg body weight every day. Fourth group (ATR\u0026thinsp;+\u0026thinsp;PIP) of rats were co-administered with atorvastatin (8 mg/kg body weight) and piperine (10 mg/kg body weight) every day for consecutive 28 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003eSoon after sacrificing the animals, blood samples were collected from each animal in heparinized tubes. The serum was centrifuged and preserved at -20\u003csup\u003e0\u003c/sup\u003e C for future use for biochemical analysis. Epididymal spermatozoa were collected in PBS and stored in incubator at 37\u003csup\u003e0\u003c/sup\u003e C for further sperm parameter analysis. After dissecting out the testis, epididymis, seminal vesicle, and prostate glands were cleaned and weighed. Then all the tissues were fixed in Bouin\u0026rsquo;s solution for further histopathological examinations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSperm parameters analysis\u003c/h2\u003e \u003cp\u003eAfter dissecting out, the epididymis was cleaned and the caudal region were cut into pieces. Small pieces of cauda epididymis were placed in a petridish containing 2 ml of 1X PBS solution and the cauda were pierced with a needle to release the spermatozoa in the media. The petridish then incubated for 10 min at 37 \u0026ordm;C with 5% CO2 to let the sperm to swim out of tubules. The suspension in the petridish then examined to measure sperm motility, sperm concentration, and sperm viability at 400X magnifications [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTen microliters of the sperm suspension were put on a sterile, previously heated microscope slide and covered with a cover slip to measure the motility of the sperm. Using a microscope with a heated stage, at least ten tiny fields were inspected at higher magnification. After being analysed under a microscope for two to four minutes after being isolated, 200 sperm cells were categorised as progressively motile, non-progressively motile, or immotile, and their proportion of the total sperm count was reported. Sperm that rotated or vibrated in the same spot were regarded as non-progressively motile, but sperm that migrated forward were seen as progressively motile.\u003c/p\u003e \u003cp\u003eEosin-Y (0.05%) was used to measure the viability of the sperm [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. After adding 20 \u0026micro;L of sperm suspension to 20 \u0026micro;L of Eosin-Y, the mixture was incubated for two minutes at room temperature. Next, sperm viability was evaluated using a 400 \u0026times; microscope. Using this technique, cells with a changed plasma membrane turn pink, whereas spermatozoa with an undamaged plasma membrane remain uncolored. The ratio of \"unstained /pink spermatozoa\" was used to determine viability, and the results were given as a percentage.\u003c/p\u003e \u003cp\u003eUsing an upgraded Neubauer hemacytometer, the number of sperm was ascertained [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The epididymal sperm suspension was diluted in a 1:5 ratio with PBS medium to examine the sperm concentration. The diluted sperm sample was then fed into the two central grids of a Neubauer\u0026rsquo;s counting chamber. Under a light microscope with a 100\u0026times; magnification, the quantity of sperm cells was counted from both chambers and averaged. The placement of the sperm's head determined how many were tallied; only sperm with heads between the row's two inner lines were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-Eosin staining of reproductive organs\u003c/h2\u003e \u003cp\u003eAfter cleaning the reproductive organs (Testis, epididymis, seminal vesicle, and prostate gland) were fixed in 10% formalin for histological investigations and subsequently embedded in paraffin. Sections (5\u0026ndash;6 \u0026micro;m) were stained with Hematoxylin and Eosin for histopathological assessment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSerum Lipid profile\u003c/h2\u003e \u003cp\u003eSerum lipid profile including total cholesterol, triglycerides, LDL, VLDL, HDL cholesterol were measured by colorimetric methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCholesterol in testicular tissue\u003c/h2\u003e \u003cp\u003eIn a screw-capped centrifuge tube, 0.1 ml of the supernatant was combined with 4.9 ml of ferric chloride solution (50 mg FeCl3 6H2O, dissolved in 100 ml acetic acid) after testicular tissue (50 mg/ml) was homogenised in an ether-alcohol mixture (1:3) and centrifuged at 3000 rpm for 10 min. The mixture was then left to stand for 15 minutes. After centrifuging the mixer for ten minutes at 3000 rpm, 1.5 ml of concentrated H2SO4 was added to 2.5 ml of the clear supernatant, and the mixture was left to develop colour for approximately half an hour at room temperature. Using a spectrophotometer, the colour intensity of the unknown and standard was measured against a blank at 560 nm using the Zlatkis et al. (1953) method. The outcome was given as mg/gm tissue [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTestosterone level in serum and in testicular tissue\u003c/h2\u003e \u003cp\u003eSerum testosterone concentration was measured by using ELISA testosterone kit according to the manufacturer\u0026rsquo;s instruction (Accu-bind, Monobind Inc. CA, USA). About 100 mg of testicular tissue was homogenized in 1 ml of PBS buffer (PH \u0026ndash; 7.4). Homogenates were centrifuged at 10000 rpm for10 min. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and the resultant supernatant used immediately for determination of testosterone by ELISA Method (AcccuBind\u0026trade; ELISA). Testicular testosterone content was calculated as ng / ml / mg of tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was performed using GraphPad Prism statistical software (Graphstats Technologies, USA). Differences among individual groups were determined by one-way ANOVA analysis followed by Tukey\u0026rsquo;s test to make comparisons. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). A p value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects on gravimetry of reproductive organs\u003c/h2\u003e \u003cp\u003eThe changes in the weight of reproductive organs of rats in all the treated groups (ATR, PIP, ATR\u0026thinsp;+\u0026thinsp;PIP) along with control group are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Reduction in testis weight found highly significant in ATR\u0026thinsp;+\u0026thinsp;PIP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) co-treated group, PIP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) group and ATR (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) group in comparison with control group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A significant reduction in the weight of epididymis was also recorded in all the treated groups when compared to control group. Weight of seminal vesicles in all treated groups showed huge reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No any significant changes found in prostate gland weight in ATR and PIP treated groups but ATR\u0026thinsp;+\u0026thinsp;PIP co-treated group showed significant reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in weight.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eWeight changes of reproductive organs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePIP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATR\u0026thinsp;+\u0026thinsp;PIP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTestis weight (gm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.226\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.096\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.016\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.856\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEpididymis weight (gm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.436\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.350\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.384\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.322\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeminal vesicle weight (gm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.592\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.326\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.468\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.302\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVentral prostate gland weight (gm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.144\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.096\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;5. When p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001(***) was significant when compared with control and ns means not significant. Unit in grams (gm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects on sperm parameters\u003c/h2\u003e \u003cp\u003eSperm count, viability, and sperm motility- these three sperm parameters were evaluated in this experiment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As compared to the control group, the ATR group showed insignificant changes in sperm count and sperm motility parameter but a significant reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in viability of epididymal spermatozoa were seen in this group. PIP group showed a significant decline in sperm count (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and sperm viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but no any significant changes found in case of sperm motility compared to control group. A high reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in sperm count and viability were observed in ATR\u0026thinsp;+\u0026thinsp;PIP co-administered group in comparison with control group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In case of sperm motility changes, ATR\u0026thinsp;+\u0026thinsp;PIP co-treated group showed significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in sperm motility when compared to the control group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSperm parameter changes in all the groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePIP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATR\u0026thinsp;+\u0026thinsp;PIP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSperm count (X10\u003csup\u003e6\u003c/sup\u003e/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eViability (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e74.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e62.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.07\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;5. Significance levels are denoted as * (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); ** (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); *** (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and ns for not significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects on histopathology of reproductive organs\u003c/h2\u003e \u003cp\u003eThe histopathological examination of the testicular sections of the animals of control group showed typical histological architecture of seminiferous tubules, spermatogonia cells, Leydig cells, Sertoli cells, and blood vessels. Animals co-administered with ATR\u0026thinsp;+\u0026thinsp;PIP showed mild atrophy of seminiferous tubules associated with mild interstitial oedema. Size of seminiferous tubules lumen increased in all treated groups compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Epididymis sections from the control group showed normal morphology with compactly arranged tubules. Control group showed high density of sperms in the lumen of cauda epididymis whereas both ATR and ATR\u0026thinsp;+\u0026thinsp;PIP co-treated groups showed a noticeable decrease in epididymal lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Histopathologic examination of the seminal vesicle sections of negative control group showed the normal histologic structure with columnar lining epithelium and dense fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Co-treatment with ATR\u0026thinsp;+\u0026thinsp;PIP in all groups showed mild hyperplasia in epithelial lining. Ventral prostate from control group showed tubules with normal epithelial height. Epithelial height was decreased in all treated groups. Interstitial oedema was found mostly in ATR\u0026thinsp;+\u0026thinsp;PIP treated group and slightly in ATR and PIP groups compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). ATR\u0026thinsp;+\u0026thinsp;PIP co-treated group showed mild atrophy of prostatic acini associated with slight degeneration of epithelial lining.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects on serum lipid profile\u003c/h2\u003e \u003cp\u003eLipid profile evaluation results showed that all the treatment groups had changed cholesterol and triglyceride levels. A huge decline in cholesterol (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were found in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated group of animals. Both ATR (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and PIP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) treated groups showed significant decrease in serum cholesterol contents. Triglycerides in all the treated groups decreased significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No any observable changes found in HDL levels in treated groups when compared to control group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in lipid profile\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePIP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eATR\u0026thinsp;+\u0026thinsp;PIP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholesterol (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e109.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriglycerides (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e118.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e84.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.05\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHDL (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e33.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.28\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVLDL (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDL (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e60.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81\u003csup\u003ens\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.60\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eChanges in lipid profile were recorded in all the groups. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, n\u0026thinsp;=\u0026thinsp;5. Significance levels are denoted as * (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); ** (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01); *** (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and ns for not significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects on testicular cholesterol level\u003c/h2\u003e \u003cp\u003eWe evaluated the cholesterol levels in testicular tissues. Results of our experiment exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In ATR treated group and ATR\u0026thinsp;+\u0026thinsp;PIP co-administered group, testicular cholesterol levels were found to decrease in a drastic pattern (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A significant decline in cholesterol levels in PIP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) treated group were also seen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects on serum and testicular testosterone level\u003c/h2\u003e \u003cp\u003eThe results of serum and testicular testosterone levels are exhibited in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Treatment with ATR (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and PIP individually resulted in significant decrease in testicular testosterone levels compared to the control group. ATR\u0026thinsp;+\u0026thinsp;PIP co-treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) showed drastic decline in testicular testosterone levels. Serum testosterone levels in ATR treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), PIP treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and ATR\u0026thinsp;+\u0026thinsp;PIP co-treated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were found to have significant decrease when compared to control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSerum lipid profile of ATR and PIP treated animals in our study showed changes in total cholesterol, HDL, LDL and TG levels (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A slight change in total cholesterol level found in ATR and PIP treated groups but cholesterol levels in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated animals decreased very much. Vijaykumar et al (2006) also found that PIP treated rats show lowered serum cholesterol levels [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Cholesterol levels in testicular tissues were found to decrease significantly in ATR\u0026thinsp;+\u0026thinsp;PIP co-administered group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The cholesterol levels in serum and testicular tissues found to decline significantly in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated groups. Previous studies regarding effects of ATR and PIP treatment on serum lipid profile support our experimental data [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For the first time we studied the effects of ATR\u0026thinsp;+\u0026thinsp;PIP co-administration on serum lipid profile and testicular tissue cholesterol levels.\u003c/p\u003e \u003cp\u003eThe data of our experiment on testosterone levels in serum and testicular tissues are of very much significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We found drastic reduction in testosterone levels in both serum and testicular tissues. Akdeniz et al (2020) also reported similar changes in serum testosterone levels in ATR administered male rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Malini et al (1999) studied effects of piperine on serum testosterone levels and found decrease of testosterone in PIP treated male rats [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The results of this experiment also support the previous studies. Here we found significant decrease in testosterone levels in both serum and testicular tissues of ATR\u0026thinsp;+\u0026thinsp;PIP co-treated animals. Individual treatments with ATR and PIP in terms of cholesterol levels and testosterone levels in serum and testicular tissues showed sufficient similarities with previous studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. But co-treatment with ATR\u0026thinsp;+\u0026thinsp;PIP revealed there is a clear relationship between cholesterol levels and testosterone levels. In both circulation and tissues, we found reduced levels of cholesterol and testosterone. It implies that co-administration of ATR\u0026thinsp;+\u0026thinsp;PIP affects both cholesterol biosynthesis and testosterone biosynthesis procedures. In this case it seems that PIP could have enhanced the bioavailability of ATR [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and synergistic effects of both ATR and PIP may have resulted in decreased synthesis of cholesterol that have affected the steroidogenesis in Leydig cells.\u003c/p\u003e \u003cp\u003eWeight of reproductive organs, changes in sperm parameters (sperm count, sperm motility, sperm viability) and histopathological aberrations are the main parameters that have immense role in fertility potential of male. In this study we considered the weight changes of testis, epididymis, seminal vesicle, and ventral prostate gland (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). From the data in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e it is evident that the weight of testis, epididymis and seminal vesicle decreased in all treated groups whereas the weight of ventral prostate gland exhibited no significant weight change in treated groups except ATR\u0026thinsp;+\u0026thinsp;PIP co-treated groups compared to control group. Klinefelter et al (2014) reported that high-dose atorvastatin resulted in low testes weights [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. D\u0026rsquo;cruz and Mathur (2005) studied the changes in weight of epididymis, seminal vesicle and ventral prostate glands in PIP treated rats [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Co-treatment with ATR\u0026thinsp;+\u0026thinsp;PIP has affected the reproductive organs more drastically than other treatment groups. Atrophic and degenerative tissue may be indicated by decreased reproductive organ mass, which is a measure of reproductive toxicity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Since the mass of differentiated spermatogenic cells determines the weight of the testis, a decrease in the density of mature spermatids and germ cells could be the cause of the testis weight drop. Similarly, decreased sperm production would eventually result in decreased sperm storage, which would lower epididymal weight [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The spermatogenic arrest and regression of seminiferous tubules size in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated rats may be the cause of the testicular weight loss, since seminiferous tubules comprise approximately 90% of the wet weight of a normal rat testis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. ATR\u0026thinsp;+\u0026thinsp;PIP co-treated rats were also found to have low quantities of lipids, which make about 30% of the total testicular weight [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and may have also played a role in the testicular weight decrease that was observed.\u003c/p\u003e \u003cp\u003eBoth the diameters tubular lumen and interstitial space were considerably expanded after ATR\u0026thinsp;+\u0026thinsp;PIP exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It also decreased the diameter of seminiferous tubules, the height of the epithelium, and the thickness of tunica albuginea. Exposure to ATR\u0026thinsp;+\u0026thinsp;PIP was also observed to reduce several spermatogonia, primary and secondary spermatocytes, and spermatids. The morphologic indicators of spermatogenic failure include diminished spermatogenic cell numbers and seminiferous tubule atrophy [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous studies have shown that PIP inhibited spermatogenesis on male adult albino rats after 30 days of treatment [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. ATR is also claimed to have anti-spermatogenic effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In our study we extended the outcome to that consumption of ATR along with PIP disrupts spermatogenesis. Histopathological sections of Epididymis in control group showing columnar epithelium, cilia, smooth muscle, and spermatozoa. Epididymis of ATR\u0026thinsp;+\u0026thinsp;PIP co-treated groups showing a few ducts with spermatozoa while others are devoid of spermatozoa. The sections of seminal vesicle from control group animals underwent histopathological analysis, which revealed the gland's characteristic histologic structure, which includes columnar lining epithelium and thick fluid. When ATR and PIP were used together, the lining epithelium displayed moderate hyperplasia. This study demonstrates the clear damage to the androgenic dependent accessory reproductive organs, including the prostate gland, seminal vesicle, and epididymis. Androgens have a crucial role in maintaining the accessory gland's ability to secrete [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe changes in sperm parameters were investigated in this experiment. The observed results are depicted in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Epididymal sperm count, motility and sperm viability adversely affected in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated animals. Previous studies on ATR treatment [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and PIP treatment [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] exhibited similar results. We found drastic decrease in sperm count, motility and sperm viability in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated animals. Atorvastatin treatment have negative impact on sperm parameters that reaches in extreme when the individual intakes piperine orally or with diet. The present study has revealed a decrease in sperm motility, count, and viability. This could be attributed to the lower bioavailability of testosterone, as the epididymis and the fertilising capacity of its contained spermatozoa are dependent on testicular androgens.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study shows that co-administration of atorvastatin and piperine decreases serum and testicular cholesterol and testosterone levels. Epididymal sperm count, sperm motility and sperm viability found to decline adversely in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated animals. Weight of testis, epididymis and seminal vesicles were decreased significantly in ATR\u0026thinsp;+\u0026thinsp;PIP co-treated animals. Histopathological studies of seminiferous tubules, cauda epididymis and seminal vesicles exhibited degenerative status. For the first time we evaluated the reprotoxic effects of atorvastatin and piperine co-administration on male rats that resulted in reduced fertility potential due to disruption of cholesterol and testosterone homeostasis.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003eATR Atorvastatin\u003c/p\u003e \u003cp\u003eELISA Enzyme linked immunoassay\u003c/p\u003e \u003cp\u003eHDI Herb Drug Interaction\u003c/p\u003e \u003cp\u003eHE Hematoxylin Eosin\u003c/p\u003e \u003cp\u003eHMG Co A β-Hydroxy β-methylglutaryl-CoA\u003c/p\u003e \u003cp\u003eIEAC Institutional Animal Ethics Committee\u003c/p\u003e \u003cp\u003eLDL Low Density Lipoprotein\u003c/p\u003e \u003cp\u003ePIP Piperine\u003c/p\u003e \u003cp\u003eSEM Standard Error of Mean\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in accordance with the guidelines for the care and purpose of laboratory animals. All the experiments were carried out in accordance with the recommendations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India (No. 892/GO/ Re/S/01/CPCSEA), with the approval of the Institutional Animal Ethics Committee (IAEC), University of Kalyani.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;The authors also state that none of the work described in this publication appears to have been influenced by any known competing financial interests or personal ties.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSG and MB conceived the idea and designed the study. SG, MB, and SS performed the experiments. SG and MB did the statistical analysis. SG wrote the manuscript. JM and SS checked the manuscript and arranged the references. MB supervised the experiment. All authors read and approved the final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to the Head of the Department of Zoology, University of Kalyani and the Principal, Rishi Bankim Chandra College for providing all the necessary help and facilities for carrying out this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSanjib Ghosh is an Assistant Professor of Molecular Biology at RBC college (affiliated to West Bengal State University, India) and currently involved in research on male infertility at Endocrinology (Reproductive) Laboratory, Department of Zoology, University of Kalyani, India. Dr. Maharaj Biswas is an Assistant Professor of Endocrinology at University of Kalyani, India. Dr. Jayanta Mistry is an Assistant Professor of Endocrinology at Government General Degree College, India. Sweata Sarkar is a researcher at Endocrinology Laboratory, University of Kalyani, India.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRehman R, Zahid N, Amjad S, Baig M, Gazzaz ZJ (2019) Relationship between smoking habit and sperm parameters among patients attending an infertility clinic. Front Physiol 10:1356. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2019.01356\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2019.01356\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaugandhika S, Sapra L, Kumari K, Srivastava RK (2023) High Salt Diet Impairs Male Fertility in Mice via Modulating the Skeletal Homeostasis. 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Sains Malays 47(8):1827\u0026ndash;1833\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Kalyani","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Atorvastatin, Piperine, Cholesterol, Testosterone, Spermatogenesis, Fertility","lastPublishedDoi":"10.21203/rs.3.rs-4253476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4253476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAtorvastatin is administered to treat hyperlipidemia. The fruit of the black pepper plant contains the alkaloid piperine. Both atorvastatin and piperine are known to have hypolipidemic effects. The goal of the current investigation was to assess the effects of atorvastatin and piperine on reproductive potential in male Wistar rats. For this study, twenty rats were obtained and placed into four groups, each with five rats. Group I served as a control, group II animals are treated with atorvastatin (8 mg/kg BW), group III animals received piperine (10 mg/kg BW) and group IV animals were co-administered with piperine (10 mg/kg BW) and atorvastatin (8 mg/kg BW). All treatments were done by using oral gavage for consecutive 28 days and thereafter assessed for lipid profile, H-E staining, sperm parameter analysis, testosterone level detection using ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that co-administration of atorvastatin and piperine (p \u0026lt; 0.05) significantly reduced weight of reproductive organs, changed histoarchitectural patterns of seminiferous tubules, epididymis, seminal vesicle. Serum and testicular cholesterol and testosterone levels of atorvastatin and piperine co-treated (p \u0026lt; 0.05) groups were found to decrease. Sperm count, motility and viability were decreased significantly in atorvastatin and piperine co-treated animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults of this study revealed that consumption of piperine (active ingredient of black pepper) along with atorvastatin (lipid-lowering drug) have deleterious effects on reproductive potential of male rat.\u003c/p\u003e","manuscriptTitle":"Co-administration of atorvastatin and piperine impairs fertility potential in male Wistar rats through disruption of cholesterol homeostasis and testosterone production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 03:22:36","doi":"10.21203/rs.3.rs-4253476/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e70b229a-426e-43f0-92fe-db4662ac61bc","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30566661,"name":"Toxicology"},{"id":30566662,"name":"Animal Science"}],"tags":[],"updatedAt":"2024-04-12T03:22:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 03:22:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4253476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4253476","identity":"rs-4253476","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}