Green Synthesized Selenium Nanoparticles Mitigate Cyclophosphamide-Induced Reproductive Toxicity in Male Wistar Rats

Green Synthesized Selenium Nanoparticles Mitigate Cyclophosphamide-Induced Reproductive Toxicity in Male Wistar Rats | 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 Green Synthesized Selenium Nanoparticles Mitigate Cyclophosphamide-Induced Reproductive Toxicity in Male Wistar Rats Bhakti S. Aiwale, Ranika Maurya, Saba Naqvi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6759007/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 8 You are reading this latest preprint version Abstract Cyclophosphamide (CP), a widely used alkylating chemotherapeutic and immunosuppressive agent, is associated with significant reproductive toxicity in male patients, primarily through oxidative stress and inflammatory damage to testicular tissue. This study examines the protective effects of green-synthesized selenium nanoparticles (SeNPs) from Azadirachta indica leaf extract against CP-induced reproductive toxicity in male Wistar rats. SeNPs were characterized using FTIR, SEM, EDX, TEM, XRD, and dynamic light scattering (DLS); XRD confirmed crystalline SeNPs, with elemental analysis (EDX) revealing 0.57% selenium content. TEM and SEM imaging indicated average particle sizes of 72.32 ± 5.00 nm and 190.2 ± 2.0 nm, respectively. CP administration (15 mg/kg/week, i.p.) induced significant reductions in enzymatic antioxidants and serum hormone levels, alongside abnormal spermatogenesis and histopathology. SeNPs (0.2 mg/kg/day, oral) restored antioxidant enzyme activity, normalized testosterone and gonadotropin levels, improved sperm quality, and ameliorated testicular histoarchitecture. Moreover, SeNPs reduced pro-inflammatory markers, suggesting an anti-inflammatory mechanism. These findings highlight the potential of SeNPs as a pharmacological intervention to mitigate CP-induced reproductive toxicity, with implications for preserving fertility during chemotherapy. cyclophosphamide (CP) cyclophosphamide toxicity testicular toxicity Selenium nanoparticles (SeNPs) serum hormonal level oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cyclophosphamide (CP), a widely used antineoplastic and immunosuppressive agent, is particularly detrimental to the reproductive system. The primary mechanism of CP involves disrupting cell division, growth, and function by cross-linking DNA strands. This mechanism is responsible for both the therapeutic effects and toxic properties of CP, particularly in rapidly proliferating tissues like the reproductive system. Studies have demonstrated that CP treatment causes biochemical and structural disruptions in the testes and epididymis of male rats [ 1 ]. Additionally, CP exposure often leads to oligospermia and azoospermia, as evidenced by experimental animal studies. Testicular injury induced by CP is associated with aberrant gonadotropin secretion and reduced testosterone levels in male patients. Given the significant impact of chemotherapy-induced infertility on quality of life, it is crucial to develop strategies for protecting germ cells during and after chemotherapy [ 2 ]. Nanotechnology has garnered significant attention globally due to its biocompatible properties and potential to enhance the safety and efficacy of treatments. Among various nanoparticles, selenium nanoparticles (SeNPs) have emerged as a promising candidate, owing to their potent antioxidant properties. Studies have demonstrated that nanoparticulate selenium formulations offer superior bioavailability and targeted delivery compared to conventional selenium supplements, thereby enhancing their therapeutic potential [ 3 ]. The protective effects of SeNPs against various forms of toxicity, including reproductive toxicity, have been increasingly explored in preclinical studies. These nanoparticles have shown efficacy in neutralizing reactive oxygen species (ROS), reducing lipid peroxidation, and enhancing the function of the body's natural antioxidant enzymes [ 4 ]. Given their antioxidant properties, SeNPs present a promising intervention to counteract the oxidative damage induced by cyclophosphamide in male reproductive organs. Green synthesis offers several benefits over chemical method, including being non-toxic [ 5 ], pollution-free [ 6 ], economical and more sustainable [ 7 ]. The phytoconstituents present in the plant extract imparts a wide range of therapeutic effects due to their diverse biological activities It also provide characteristic size and shape to the nanoparticles. The nanoparticles synthesised from plant extract are often have superior biocompatibility in In-vitro and In-vivo experiments. In this study we have synthesized selenium nanoparticles by Azadirachta indica leaves extract, an ecofriendly green approach. After the synthesis and characterization of nanoparticles the nanoparticles were investigated in mitigating cyclophosphamide (CP)-induced reproductive toxicity in male rats. The dose of CP, 15mg/kg weekly [ 8 – 10 ]and SeNP, 0.2mg/kg/day [ 3 , 11 ] was decided based on previous research. Various parameters, including sperm count, motility, morphology, and antioxidant enzyme activities, hormonal parameters were evaluated to elucidate the protective mechanisms of SeNPs and explore their potential as a therapeutic strategy to safeguard male fertility during chemotherapy [ 12 ]. 2. Materials and methods 2.1 Chemical Reagents Sodium selenite (214885-5G), cyclophosphamide (PHR1404-1G), were procured from Sigma-Aldrich chemicals., St. Louis (USA). The serum total protein kit and the serum albumin kit were procured from Accurex Biomedical Private Limited, Thane, Maharashtra. All ELISA kits such as FSH (Cat: ELK1315), Rat testosterone (Cat: ELK8314), Rat LH (Luteinising hormone) Cat: ELK2367), General Estradiol Cat: ELK1208, Rat T3 (triiodothyronine) Cat: ELK1339, Rat T4 Cat: ELK1204, TNF-α (Cat: ELK1396), and caspase 3 (Cat: ELK1396), were obtained from ELK Biotechnology Wuhan. The ELISA kit Rat IL-6 (Cat. #IT17833) was purchased from G-bioscience, while all other chemicals were purchased from Hi media and Merck. 2.2 Green Synthesis of selenium nanoparticles 2.2.1 Plant source The fresh green leaves of Azadirachta indica were collected from botanical garden at NIPER Raebareli – transit campus Lucknow. The identification of collected leaves is done at the research laboratory in NIPER facility. 2.2.2 Preparation of aqueous extract After collection the leaves were cleaned with water and dried. The dried leaves were then ground and extraction was done with water for 1 hour. The extract was concentrated and filtered through a crude cellulose filter paper and then Whatman #1 filter paper. The resulting filtrate was stored at 2–4 ͦ C until required. 2.2.3 Synthesis of selenium nanoparticle Selenium nanoparticles (SeNPs) were synthesized using a green synthesis method, as described by [ 13 ], [ 14 ], with slight modifications. For SeNP synthesis, sodium selenite was added dropwise to the neem extract under continuous stirring on a magnetic stirrer. A red colloidal solution of SeNPs formed instantaneously. After completion of the reaction, the solution was centrifuged at 9000 rpm for 30 minutes. The supernatant was discarded, and the pellet was resuspended in milli-Q water and washed 3–5 times. The pellet was stored in a -20°C deep freezer and lyophilized for further use. 2.3 Characterization of selenium nanoparticles 2.3.1 Dynamic light scattering (DLS) The particle size, zeta potential and polydispersity index (PDI) of the biomolecule-functionalized SeNPs were ascertained by dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments Ltd., UK). 2.3.2 Infrared spectroscopy using Fourier transform (FT-IR) The FTIR spectrophotometer (ECO-ATR, ALPHA, Bruker, Germany) was used to examine the FTIR spectral analysis of biosynthesized SeNPs. 2.3.3 X-ray diffraction (XRD) analysis At 40 keV, the crystalline characterization was carried out at IIT-Roorkee, India (Grazing Incidence-X-Ray Diffractometer (GI-XRD)), in the range of 20~ # 2q # 80. PowderX software was used to design the crystals' lattice constant. 2.3.4 Microscopic analysis via SEM and TEM A Jeol JSM-IT200 scanning electron microscope (Japan) was used to examine the surface morphology of biosynthesized SeNPs. The sample preparation was done on clean electric stub, the stub was covered with a drop of the sample, which was then let to dry. The dried sample was prepared for imaging by mounting it using double adhesive tape on an aluminum stub. The microscope had an 8 mm working distance and an accelerating voltage of 10 kV. Energy-dispersive X-ray spectroscopy (EDX) was used to analyze the elemental content present in the nanoparticles. The transmission electron microscopy (TEM) was performed at Jamia Hamdard. It was used to analyze the size and shape of the SeNPs. To prepare the samples for examination, a single drop of aqueous biosynthesized SeNPs was placed on a copper-coated grid. A Jeol JEM-2100 electron microscope (Japan) was used to record a TEM micrograph. 2.4 Animals and experimental study design The experiment was performed on male Wistar rats having body weight of 120–140 grams. The rats were procured from CSIR-IITR, Lucknow. Prior to the experimentation the protocol was approved by the IAEC (Institutional Animal Ethics Committee) members of NIPER-Raebareli (Approval No. NIPER/RBL/IAEC/96/AUG 2022). The rats were kept under standard conditions and feed with standard pellet diet and had unrestricted access to water. Housing condition of animals included a temperature of 20 ± 2°C, relative humidity of 50 ± 10%, and a 12-hour light/dark cycle. After one-week of acclimatization period, the animals were randomly assigned to five groups, each comprising six rats. The groups were designated as follows: Group 1 (Control): normal saline. Group 2 (CP): cyclophosphamide only (15 mg/kg/Week). Group 3 (NaSe): sodium selenite (0.2 mg/kg/day). Group 4 (SeNPs): selenium nanoparticles (0.2 mg/kg/day). Group 5 (CP + SeNPs): cyclophosphamide (15 mg/kg/week) and SeNP (0.2 mg/kg/day). All study treatments were administered orally to the animals over a 28-day period. The care and use of the animals were conducted in accordance with CPCSEA guidelines. Tissue Processing Upon completion of the study, rats were anesthetized with urethane (5mg/kg) via intraperitoneal injection. Blood samples were then collected by retro-orbital sinus puncture. The blood samples were divided into two groups: one was transferred into non-EDTA microcentrifuge tubes for serum collection, while the other was transferred into EDTA-coated microcentrifuge tubes for whole blood collection. For serum separation, the non-EDTA blood samples were allowed to clot for 30 minutes at room temperature, followed by centrifugation at 10,000xg for 10 minutes at 4°C. The resulting serum was stored at -80°C for subsequent serum analysis and hormonal determination. The anticoagulated blood samples (EDTA-coated tubes) were stored at 2–4°C for hematological estimation.[ 10 ]. Following blood collection, the animals were humanely euthanized by cervical dislocation. The testes and epididymides were carefully excised, washed with ice-cold phosphate-buffered saline (PBS), and divided into two portions. One portion was fixed in 10% formalin for histopathological examination. The other portion was stored at -80°C for biochemical assays. Tissue homogenates were prepared by homogenizing the testicular tissue in a 10-fold volume of ice-cold PBS buffer (0.01 M, pH 7.4) using a tissue homogenizer. The homogenates were then centrifuged at 10,000xg for 10 minutes at 4°C. The resulting supernatants were collected as tissue homogenates and stored at -80°C for further analysis [ 15 , 16 ]. 2.5 Sperm collection and sperm parameters One epididymis was placed in a Petri dish containing Hanks' Balanced Salt Solution (HBSS) medium. The caudal portion of the epididymis was gently incised at a few sites using a scalpel, taking care to minimize exposure to air. The tissue was then dispersed in the medium for 5–15 minutes, allowing the sperm to be released. Following this incubation period, the tissue was removed, and the sperm were collected for further analysis [ 17 ]. 2.6 Sperm count The sperm-containing HBSS medium (1 ml) was centrifuged at 1000 rpm for 3 minutes. The resulting supernatant was collected and used for sperm counting. Sperm count was estimated using a hemocytometer, and the results were expressed as millions of sperm per milliliter (ml)[ 18 ]. 2.7 Sperm motility Sperm motility was assessed by placing a 10 µl aliquot of the sperm suspension on a microscope slide, which was then covered with a coverslip. For each animal, at least five microscopic fields were examined, with a minimum of 200 sperm evaluated per field. The percentage of motile sperm was calculated by determining the proportion of total sperm that exhibited movement [ 19 ]. 2.8 Sperm morphology Sperm morphology was evaluated by preparing thin smears of semen on clean, grease-free microscope slides. The slides were air-dried overnight and then stained with a 1% eosin-Y dye solution. Using a light microscope equipped with a 40× objective lens, the stained sperm cells were examined for any morphological abnormalities. Sperm with irregularities, such as amorphous or irregularly shaped heads, missing or malformed acrosomes, double heads, coiled tails, or other deviations from normal morphology, were identified and counted [ 20 ]. 2.8 JC-1 microplate assay The JC-1 microplate assay is a widely used method for assessing mitochondrial membrane potential (ΔΨm\Delta \Psi_mΔΨm​), a critical indicator of mitochondrial health and function. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, which is reflected in a shift in its fluorescence emission. For the fluorescence microplate analysis, a 200 µL aliquot of the sample (25 x 10^6 cells/mL) were placed undiluted into every well (doublet) in a 96-well plate. Fluorescence was measured using a microplate reader equipped with a 485 nm excitation filter, with the gain set to 100% for all assays. Initially, a 595 nm emission filter was used to detect total orange fluorescence, which corresponds to JC-1 aggregates. Subsequently, a 535 nm emission filter was employed to detect all green fluorescence, which corresponds to JC-1 monomers. Each well's total fluorescence was corrected by subtracting the blank fluorescence from HBSS containing 2 pM JC-1 dye only. The mean of total and blank corrected fluorescence values were then calculated for both treatment and control groups [ 21 ]. 2.9 Hematological parameter Blood samples were isolated from the retro-orbital plexus to assess hematological parameters. Key metrics, including RBC count, WBC count, hemoglobin concentration (HBT), hematocrit percentage (HCT), granulocyte, lymphocyte, and monocyte counts, were measured using a Mindray BC-5130 automated hematoanalyzer following transfer of the blood into EDTA-treated microcentrifuge tubes[ 22 , 23 ]. 2.10 Serum biochemical analysis We assessed serum total protein content, albumin levels, and the albumin/globulin (A/G) ratio to evaluate the overall health and physiological status of the organism. The A/G ratio serves as a crucial marker for immune function and inflammation, whereas total protein and albumin levels can indicate liver dysfunction or malnutrition. Serum biochemical indices were measured using an Accurex colorimetric assay kit (Accurex Biomedical Pvt. Ltd., Mumbai, India), following the manufacturer's protocols (Accurex Biomedical Pvt. Ltd., Thane, India). Specifically, the biuret method was employed to determine serum total protein levels, and the Bromocresol Green (BCG) method was used to estimate serum albumin levels [ 24 , 25 ]. 2.11 TBARS Assay The estimation of lipid peroxidation was performed by measuring malondialdehyde (MDA), a well-known lipid peroxidation marker. Tissue homogenate was treated with acetic acid, sodium dodecylsulphate (SDS), followed by the addition of thiobarbituric acid. The mixture was then heated at 95°C for 1hr and centrifuged at 12,000 g for 10 minutes. The resulting pink supernatant was measured at an absorbance of 532 nm. MDA concentration was determined using a standard curve generated with tetraethoxypropane (TEP) and expressed as nmol/mg of protein [ 26 ]. 2.12 Nitrate assay Using the Griess reagent, the nitric oxide (NO) level was determined. The diazotization process mediated by Gries reagent, which spectrophotometrically identifies nitrite generated by spontaneous oxidation of NO under physiological circumstances, is the principle at play here. Here, sulfanilic acid reacts with nitrite in an acidic solution to quantitatively produce a diazonium salt. Following this, the diazonium salt is combined with N-(1-naphthyl) ethylenediamine to create an azo dye, which can be measured spectrophotometrically at the absorbance at 548 nm [ 27 ]. 2.13 Glutathione assay The concentration of glutathione (GSH) in the testis was determined using a colorimetric method. Tissue homogenates were treated with an equal volume of 5% sulphosalicylic acid, vortexed, and incubated in an ice bath for 30 minutes to precipitate proteins. The resulting supernatant was then used to measure GSH levels using Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) solution. The results were expressed as µM GSH/mg protein [ 28 , 29 ]. 2.14 Catalase assay To determine the catalase activity in the testis, a modified version of the method described by Sinha et al. was employed. The assay involved adding 250 µl of the sample to 250 µl of hydrogen peroxide, followed by incubation at 37°C for 1 minute. The reaction was terminated by adding 1 ml of a solution containing potassium dichromate and acetic acid in a 1:3 ratio, and the mixture was then placed in a boiling water bath at 95°C for 10 minutes. After cooling, 300 µl of each solution was loaded into well plates, and the absorbance was measured at 570 nm[ 30 ]. 2.15 Superoxide Dismutase (SOD) To determine the antioxidant enzyme SOD, The Sodium pyrophosphate was added to the supernatant, and then phenazine methosulphate, nitro blue tetrazolium, nicotinamide adenine dinucleotide, and distilled water was added in a test tube. The mixture was incubated at 30°C for 90 s. The reaction was ceased by adding glacial acetic acid within 90 s and the violet color complex formed was determined at the absorbance of 560nm [ 28 ]. 2.16 Measurement of serum proteins The blood serum was used to estimate the level of serum total protein, albumin, globulin and albumin/globulin ratio by using Elisa kits procured from ELK biotechnology. Assay was performed as per manufacturer’s protocol. 2.17 Measurement of serum hormonal levels The serum was used for estimation of level of Follicular stimulating hormone (FSH), Testosterone, Luteinizing hormone (LH), Estradiol (E2), T3 (triiodothyronine) and T4 (Thyroxine). These hormones level was measured by ELISA which were procured from ELK biotechnology. Assay was performed according to manufacturer’s protocol[ 23 , 31 ]. 2.18 Measurement of inflammatory biomarkers Testis homogenate was used to measure inflammatory biomarkers. Testis inflammatory mediators TNF-α and IL-6 were measured by ELISA. ELISA kits were procured from ELK biotechnology. The assays were performed as per manufacturer’s instruction[ 25 ]. 2.19 Estimation of apoptotic biomarker Caspase-3 Testis homogenate was used to measure apoptosis biomarker. Testis apoptosis biomarker Caspase-3 was measured by ELISA. ELISA kits were procured from ELK biotechnology. The assay was performed as per manufacturer’s instruction[ 15 ]. 2.20 Histology of testis and epididymis of rat Testis and epididymis collected in the formalin were processed in ascending concentrations of ethanol such as 70%, 80%, 90%, and 3 times in 100%, two times changed with xylene, and three times changed with paraffin (60C) for 40 min per each solution. Finally, the samples were sectioned at a thickness of 5 µm and these sections were mounted on the coated slides. After that deparaffinization is done in xylene and rehydration is done in graded ethanol ranging from 100–50%, followed with hematoxylin and Eosin (H&E) staining. Further, gross cellular damage was observed by using light microscope[ 19 , 32 ]. 2.21 Statistical analysis The data was presented as the Mean ± Standard Error (SE). Graph Pad Prism 8 software was utilized to analyze the statistical differences using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. The level of statistical significance was set at *p < 0.05. Results Characterization of Selenium Nanoparticles (SeNPs) Zeta Potential and Size Using dynamic light scattering (DLS), the average particle size, polydispersity index (PDI), and zeta potential of the selenium nanoparticles were ascertained. The results revealed the formation of selenium nanoparticles with an average size of 100.2 nm, as depicted in Fig. 2 (a) and the polydispersity index was found to be 0.024, signifying a homogeneous size population of nanoparticles in the aqueous medium. The zeta potential of the synthesized nanoparticles was − 4.75 mV, as shown in Fig. 2 (b). Fourier Transform Infrared (FTIR) and XRD Analysis The FTIR analysis was conducted to identify the functional groups present on the selenium nanoparticles. The spectra covered the range from 400 cm⁻¹. The FTIR data of selenium nanoparticles shown in Fig. 2 (c) shows the peak at 3300 cm-1 = Hydroxyl group of phenol, 1655cm-1 = Amide group, 588 cm-1 = Se-O interaction. The X-ray diffraction (XRD) pattern of the synthesized selenium nanoparticles is depicted in Fig. 2 (d). The analysis revealed prominent diffraction peaks at 2θ values of 23.62°, 29.81°, 41.42°, 43.74°, 45.45°, 51.80°, 55.71°, 61.32°, and 65.28°. The most intense peak was observed at 29.81°, indicating a high degree of crystallinity. SEM, EDX, and TEM Analysis of Selenium Nanoparticles Energy-dispersive X-ray (EDX) analysis (Fig. 1 e) confirmed the presence of selenium, sodium, carbon, and oxygen. The elemental composition of Se was found to be 0.057 ± 0.03. The size and shape of the selenium nanoparticles were analyzed using Scanning electron microscopy (SEM) (Fig. 1 f), revealing an average size of 190.2 nm. Transmission electron microscopy (TEM) (Fig. 1 g) analysis showed that the nanoparticles were not aggregated, and their surface morphology was consistent with the size found using the zeta-sizer. In vivo toxicity assessment of Cyclophosphamide Effect of treatment on body and organ (testis) weight The body weight of rats in each group was monitored throughout the study, and organ weight (testis) was measured at the end of the study. The results are shown in Fig. 2 (a) & (b). Cyclophosphamide (CP) treatment (Group 2) caused a slight decrease in body weight compared to the control group (Group 1). In contrast, rats treated with selenium nanoparticles (SeNPs, Group 4) exhibited a gradual and consistent increase in body weight throughout the study, suggesting the non-toxic nature of SeNPs. In Group 5, where animals received both CP and SeNPs, the body weight also increased steadily, although the increment was slightly lower than in Group 4. However, compared to the CP-only group, the SeNP treatment significantly mitigated CP's toxic effects, resulting in a notable improvement in body weight. Similarly, Group 3 (sodium selenite) showed a trend towards increased body weight compared to the CP group, but the protective effect was less pronounced than that observed with SeNPs. Cyclophosphamide treatment also caused a significant reduction in testis weight compared to the control group (***P < 0.004 vs. Control), indicating CP-induced testicular toxicity. This reduction was significantly reversed by SeNPs treatment in Group 5 (#P < 0.011, ###P < 0.0005 vs. CP), suggesting their protective effect. While sodium selenite in Group 3 showed a mild improvement in testis weight compared to the CP group, it was less effective than SeNPs. Overall, the findings demonstrate that SeNPs not only counteract CP-induced testicular toxicity but also improve body weight, highlighting their potential as a protective agent against CP-induced systemic toxicity. The data was expressed as mean ± SEM (n = 5). The statistical analysis included Tukey's multiple comparison tests after a one-way ANOVA. The significance level was set at **p < 0.01, ***p < 0.001 in comparison to the control. And # p < 0.05, ## p < 0.01, ### p < 0.001 vs CP. Cyclophosphamide's Influence on Sperm Characteristics, and Mitochondrial Function The effects of green synthesized selenium nanoparticles (SeNPs) on cyclophosphamide (CP)-induced reproductive toxicity were evaluated (Fig. 3 ). The CP-treated group (Group 2) exhibited a significant reduction in sperm count compared to the control group (Group 1, p < 0.0002). Notably, treatment with SeNPs (Group 5) significantly improved sperm count compared to the CP group (p < 0.0005), indicating a reversal of CP-induced toxicity. Sperm motility was significantly impaired in the CP group compared to the control group (p < 0.0002). SeNP treatment (Group 5) restored sperm motility to near-control levels (p < 0.0001 vs. CP), demonstrating its protective potential. The JC-1 assay was carried out to assess mitochondrial membrane potential (MMP) of the sperm as MMP is a critical parameter for assessing sperm function and quality. As we know Mitochondria in sperm provide the energy (ATP) necessary for motility and capacitation. Sperm with high mitochondrial membrane potential are considered more viable, motile, and healthy. Whereas decreased membrane potential​ may indicate poor sperm quality. In our study, we noticed a significant reduction in mitochondrial membrane potential in the sperm of CP-treated rats (Group 2) compared to the control group. Treatment with SeNPs (Group 5) markedly restored mitochondrial membrane potential compared to the CP group. Sperm morphology analysis showed that CP treatment led to a higher incidence of abnormalities, including hooked heads, coiled tails, and detached hooks. These abnormalities were significantly reduced in the SeNP-treated group, with morphology comparable to the control group. These findings indicate that SeNPs effectively mitigate CP-induced reproductive toxicity, as evidenced by improved sperm parameters, restored mitochondrial function, and reduced sperm morphological abnormalities. Statistical significance was noted as ***p < 0.0004 vs. control and ###p < 0.001 vs. CP. Effect of cyclophosphamide on hematological parameters of rat blood The effects of cyclophosphamide (CP) on haematological parameters were assessed by measuring white blood cells (WBC), red blood cells (RBC), eosinophils (Eos), neutrophils (neu), monocytes (Mon), lymphocytes (Lym), hemoglobin (HGB), and hematocrit percentage (HCT%) and protective effect of SeNP were evaluated (Fig .4). Cyclophosphamide treatment (Group 2) caused a significant reduction in all measured parameters compared to the control group (Group 1), reflecting the hematotoxic effects of CP. Specifically, WBC counts were significantly decreased (**p < 0.014 vs. Control), as were RBC counts (***p < 0.00051 vs. Control), eosinophils (**p < 0.0021 vs. Control), neutrophils (***p < 0.0002 vs. Control), and monocytes (*p < 0.03 vs. Control). Lymphocyte counts (**p < 0.0053 vs. Control), HGB levels (**p < 0.0027 vs. Control), and HCT% (***p < 0.0001 vs. Control) were also markedly reduced in the CP group compared to the control group. Treatment with SeNPs (Group 5) significantly reversed these CP-induced alterations. WBC counts in the SeNP-treated group showed a marked recovery (#p < 0.02 vs. CP), indicating restoration of immune function. RBC counts were significantly improved compared to the CP group (#p < 0.01, ###p < 0.0008 vs. CP), suggesting enhanced erythropoiesis. Eosinophils, neutrophils, and monocyte counts demonstrated substantial recovery with SeNP treatment (#p < 0.031 for eosinophils, #p < 0.03 for neutrophils, and #p < 0.03 for monocytes vs. CP), reflecting improved immune and inflammatory responses. Lymphocyte counts in the SeNP group were significantly higher compared to the CP group (##p < 0.0023, ##p < 0.0093 vs. CP), highlighting the mitigation of CP-induced lymphotoxicity. HGB levels (#p < 0.0130 vs. CP) and HCT% (###p < 0.0003, ###p < 0.0007 vs. CP) were also significantly restored in the SeNP-treated group, indicating recovery of oxygen-carrying capacity and blood volume integrity. These findings demonstrate that SeNPs effectively counteracted the hematotoxic effects of CP by improving all evaluated hematological parameters. Statistical significance was observed for all comparisons, indicating the robust protective potential of SeNPs. Estimation of serum total protein content, albumin and albumin/globulin (A/G) ratio The effects of cyclophosphamide (CP) on serum total protein, albumin, and albumin/globulin (A/G) ratio were evaluated to assess the impact of treatments on protein metabolism (Fig. 5 ). Cyclophosphamide treatment (Group 2) significantly reduced serum total protein, albumin levels, and the albumin-to-globulin (A/G) ratio compared to the control group (Group 1). Specifically, total protein levels were markedly decreased in the CP group (***p < 0.0005 vs. Control), reflecting impaired protein synthesis. Similarly, albumin levels (**p < 0.009 vs. Control) and the A/G ratio (**p < 0.0067 vs. Control) were significantly lower in the CP group, indicating disruptions in hepatic function and protein homeostasis. Treatment with selenium nanoparticles (Group 5) significantly restored these parameters. Total protein levels were significantly improved in the SeNP group compared to the CP group (##p < 0.0099, ##p < 0.028 vs. CP), highlighting a recovery in overall protein synthesis. Albumin levels also showed a significant recovery (#p < 0.0342 vs. CP), suggesting an improvement in liver function. Furthermore, the A/G ratio was significantly enhanced in the SeNP-treated group compared to the CP group (#p < 0.01, ##p < 0.02 vs. CP), reflecting a restored balance between albumin and globulin levels. These results indicate that SeNP treatment effectively deciphers the adverse effects of CP on serum protein metabolism, with statistically significant improvements in total protein, albumin levels, and A/G ratio. Estimation of oxidant (MDA, nitrite level) and Antioxidant parameters (GSH, Catalase, SOD, Gpx activity) The impact of cyclophosphamide (CP), sodium selenite (NaSe), selenium nanoparticles (SeNPs), and CP combined with SeNPs (CP + SeNP) on oxidative stress was evaluated through the measurement of lipid peroxidation (TBARS), nitrate levels, and antioxidant parameters including catalase, superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) activity (Fig. 6 ). Cyclophosphamide treatment (Group 2) significantly elevated TBARS and nitrate levels, indicating increased oxidative stress, while reducing the activities of catalase, SOD, GPx, and GSH levels compared to the control group (Group 1). Specifically, TBARS levels were significantly increased in the CP group (*p < 0.0308, **p < 0.0065 vs. Control), while nitrate levels were markedly higher (***p < 0.0003 vs. Control), reflecting an enhanced production of reactive oxygen and nitrogen species. Antioxidant parameters, including catalase (***p < 0.001 vs. Control), SOD (***p < 0.0005 vs. Control), GSH (*p < 0.01, ***p < 0.001 vs. Control), and GPx (**p < 0.001 vs. Control), were significantly diminished, underscoring a compromised antioxidant defense system. Treatment with selenium nanoparticles (Group 5) significantly ameliorated oxidative stress by reducing TBARS levels (#p < 0.02, #p < 0.01 vs. CP) and nitrate levels (###p < 0.0008, ###p < 0.006 vs. CP). Antioxidant defense markers showed significant recovery in the SeNP-treated group compared to the CP group. Catalase activity was significantly restored (###p < 0.0006, ###p < 0.0003 vs. CP), as were SOD (##p < 0.002, ##p < 0.007 vs. CP), GSH (##p < 0.007, ##p < 0.006 vs. CP), and GPx activity (#p < 0.01, ##p < 0.0090 vs. CP). These improvements highlight the capacity of SeNPs to counteract CP-induced oxidative damage and enhance endogenous antioxidant defenses. Overall, the results demonstrate that SeNP treatment significantly alleviates CP-induced oxidative stress, restoring a balance between pro-oxidant and antioxidant systems, with statistically significant improvements across all parameters. Measurement of serum hormonal levels The serum hormonal levels of follicle-stimulating hormone (FSH), testosterone, luteinizing hormone (LH), estradiol, triiodothyronine (T3), and thyroxine (T4) were evaluated in different treatment groups. The results are expressed as mean ± SEM (n = 5) and analysed using one-way ANOVA followed by Tukey's multiple comparison tests, as shown in Fig. 7 (a)–(f). FSH levels in the CP group (Group 2) showed a slight increase compared to the control (Group 1); however, this difference was not statistically significant across the groups. Testosterone levels were significantly decreased in the CP-treated group compared to the control (**p < 0.005), indicating CP-induced testicular toxicity. Treatment with SeNPs (Group 4) and the CP + SeNP combination (Group 5) significantly restored testosterone levels compared to the CP group (#p < 0.012, ##p < 0.0385). Sodium selenite (Group 3) also showed an improvement, although the effect was less pronounced than SeNPs. LH levels followed a similar trend, with significant reduction in the CP group compared to the control (**p < 0.0038, *p < 0.0244). Both SeNP (Group 4) and CP + SeNP (Group 5) treatments significantly increased LH levels compared to the CP group (#p < 0.0457), highlighting the protective potential of SeNPs. Estradiol levels were significantly increased in the CP group compared to the control (**p < 0.0022), reflecting hormonal imbalance caused by CP. This elevation was effectively normalized in Group 5, while sodium selenite group doesn’t have any significant changes. The estradiol level was slightly higher in SeNP group compared to cyclophosphamide (#p < 0.0104 vs. CP) indication beneficial effect of low dose of selenium on estradiol hormone. Triiodothyronine (T3) levels were severely reduced in the CP group compared to the control (***p < 0.0001, **p < 0.0022). Treatment with SeNPs and CP + SeNPs significantly restored T3 levels compared to the CP group (###p < 0.0009, ###p < 0.0006), with SeNPs showing the most pronounced effect. Similarly, thyroxine (T4) levels were significantly decreased in the CP group compared to the control (****p < 0.0001), and treatment with SeNPs significantly improved T4 levels (#p < 0.0415, ###p < 0.0004 vs. CP). Overall, SeNPs effectively mitigated CP-induced alterations in serum hormonal levels, highlighting their potential to restore hormonal homeostasis disrupted by CP toxicity. Measurement of inflammatory and Apoptosis markers The levels of inflammatory markers, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), as well as the apoptosis marker caspase-3, were assessed across treatment groups (Fig. 8 a-c). TNF-α levels were significantly elevated in the CP-treated group (Group 2) compared to the control (**p < 0.0024), indicating CP-induced inflammation. In contrast, treatment with SeNPs (Group 5) significantly reduced TNF-α levels compared to the CP group (##p < 0.051, ##p < 0.0099), demonstrating a potent anti-inflammatory effect. Similarly, IL-6 levels were significantly increased in the CP group compared to the control (**p < 0.006), reflecting a pro-inflammatory response. Treatment with SeNPs (Group 5) effectively reduced IL-6 levels compared to the CP group (#p < 0.02), highlighting the anti-inflammatory potential of SeNPs. Caspase-3 activity, a marker of apoptosis, was markedly elevated in the CP group compared to the control (***p < 0.0001). Treatment with SeNPs (Group 5) significantly reduced caspase-3 activity compared to the CP group (###p < 0.0002), demonstrating its role in mitigating CP-induced apoptotic damage. Notably, Group 3 (Sodium Selenite) and Group 4 (SeNPs only) showed lower caspase-3 levels compared to the CP group (#p < 0.02, ##p < 0.01). These findings collectively suggest that SeNPs effectively attenuate CP-induced inflammation and apoptosis, thereby reducing oxidative damage and tissue injury. Histopathological investigation of rat testis and epididymis Hematoxylin and Eosin (H&E) staining was performed to evaluate histological alterations in the epididymis and testis, as shown in Fig. 9 (a) and (b). Sections were examined under a light microscope to identify structural and cellular changes. In the seminiferous tubules of the testis, the cyclophosphamide (CP)-treated group (Group 2) displayed significant histopathological abnormalities, including severe degeneration of spermatocytes and Sertoli cells, perivascular cell degradation, disturbed Leydig cells, and a marked loss of mature spermatids, compared to the control group (Group 1). In contrast, the selenium nanoparticles (SeNPs) group (Group 4) and the CP + SeNPs-treated group (Group 5) exhibited normal seminiferous structure, indicating retained testicular physiology and reduced CP-induced damage. The histology of the cauda epididymis in the control group showed well-organized luminal epithelial cell layers with abundant sperm. In contrast, the CP-treated group exhibited a significant reduction in sperm count and disrupted epithelial cell integrity. Treatment with SeNPs and the combination of CP + SeNPs effectively preserved the normal architecture of principal cells and luminal structure, demonstrating the protective effects of SeNPs against CP-induced reproductive toxicity. Discussion Cyclophosphamide (CP), a widely used alkylating agent in chemotherapy, is known to cause severe reproductive toxicity in males, posing challenges to long-term reproductive health. The mechanisms of CP-induced toxicity include germ cell damage, oxidative stress, hormonal disruptions, and depletion of spermatogonial stem cells, collectively impairing spermatogenesis and testosterone production. These findings highlight the urgent need for protective strategies against CP-induced reproductive dysfunction. In this study, green selenium nanoparticles (SeNPs) were synthesized and extensively characterized to evaluate their protective role against CP-induced reproductive toxicity. The synthesized SeNPs demonstrated desirable physicochemical properties, including a particle size of 100.2 nm and a zeta potential of -4.75 mV, as confirmed by dynamic light scattering (DLS). Spectroscopic and microscopic techniques further validated the nanoparticles' composition and morphology. FTIR analysis revealed characteristic peaks of SeNPs, while XRD data confirmed their crystalline nature. TEM and SEM images showed spherical, monodisperse particles, and EDX analysis identified selenium as a major component. These findings confirm the suitability of SeNPs for biological applications, enabling their evaluation in vivo. In CP-treated rats, significant reductions in body weight gain and testis weight were observed, indicating systemic and organ-specific toxicity. SeNP treatment mitigated these effects, suggesting a protective role in preserving testicular integrity. Sperm morphology assessment revealed severe abnormalities, including hooked and damaged sperm in the CP group, while SeNP treatment restored normal morphology. Similarly, mitochondrial potential, measured using the JC-1 assay, was significantly improved in SeNP-treated groups, further supporting the nanoparticles' protective effects. CP exposure adversely affected blood parameters, with reductions in hematocrit and hemoglobin levels. Serum biochemical analysis revealed a decrease in total protein, albumin, and the albumin-to-globulin (A/G) ratio, indicating systemic inflammation and metabolic dysregulation. SeNP administration reversed these alterations, restoring blood parameters closer to control levels. Oxidative stress, a key contributor to CP toxicity, was evident from elevated malondialdehyde (MDA) and nitrate levels in CP-treated rats. This was accompanied by a reduction in antioxidant defenses, as indicated by decreased levels of superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPX). Treatment with SeNPs significantly reduced oxidative stress markers and restored antioxidant enzyme levels, demonstrating their potential as a potent antioxidant therapy. Hormonal disruptions were also prominent in CP-treated rats, with significant reductions in thyroid hormones (T3, T4) and thyroid-stimulating hormone (TSH), alongside minor, non-significant increases in follicle-stimulating hormone (FSH). These alterations can severely impair spermatogenesis and overall reproductive health. SeNP treatment partially reversed these hormonal imbalances, underscoring their role in preserving endocrine function. Inflammatory and apoptotic markers, including TNF-α, IL-6, and caspase-3, were markedly elevated in CP-treated groups, indicating inflammation and testicular cell death. Notably, SeNP treatment significantly reduced these markers, demonstrating their anti-inflammatory and anti-apoptotic potential. Histopathological analysis corroborated the biochemical findings, revealing profound testicular damage in CP-treated rats, including distorted seminiferous tubules, germ cell exfoliation, and disrupted germinal epithelium. Conversely, SeNP-treated groups exhibited near-normal testicular morphology, with preserved seminiferous tubules and reduced interstitial space. Conclusion This study demonstrates the protective effects of green SeNPs against CP-induced reproductive toxicity through their antioxidant, anti-inflammatory, and anti-apoptotic properties. By mitigating oxidative stress, preserving hormonal balance, and reducing inflammatory responses, SeNPs offer a promising therapeutic strategy to safeguard male reproductive health during chemotherapy. 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Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 07 Jul, 2025 Reviews received at journal 30 Jun, 2025 Reviewers agreed at journal 30 Jun, 2025 Reviewers agreed at journal 29 Jun, 2025 Reviewers invited by journal 03 Jun, 2025 Editor assigned by journal 30 May, 2025 Submission checks completed at journal 30 May, 2025 First submitted to journal 27 May, 2025 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-6759007","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465943021,"identity":"50afa796-b052-40c5-809e-4f1ca0cc679f","order_by":0,"name":"Bhakti S. Aiwale","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research (NIPER-R), Lucknow (UP)","correspondingAuthor":false,"prefix":"","firstName":"Bhakti","middleName":"S.","lastName":"Aiwale","suffix":""},{"id":465943022,"identity":"aad2f788-a0f0-458f-884d-d60d56c4c046","order_by":1,"name":"Ranika Maurya","email":"","orcid":"","institution":"National Institute of Pharmaceutical Education and Research (NIPER-R), Lucknow (UP)","correspondingAuthor":false,"prefix":"","firstName":"Ranika","middleName":"","lastName":"Maurya","suffix":""},{"id":465943023,"identity":"000adc69-c677-4ab4-a771-1244973ab447","order_by":2,"name":"Saba Naqvi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYFACxgYILcHA+ICB4QBRWhobGBLAWpgNiNQCsgaihU2CKC0Gtw+3P/j4w8aef3bzs2qemjty/AzMDx/dwKflXGJj44yEtMQZd46Z3eY59sxYsoHN2DgHjxazM4yNzTwJhxMYbiQAtbAdTtxwgIdNmqCWPwmH7eVvpH8r5vlHrBaGhMOMG27kmDHzthGhxR6oZWZPWlrixjtniiXn9h02lmwm4BfJHvYHH37Y2NjL3W7f+OHNt8Ny/OzNDx/j04ICmHhAJDOxykGA8QcpqkfBKBgFo2DEAACYD1NwaxvRbwAAAABJRU5ErkJggg==","orcid":"","institution":"National Institute of Pharmaceutical Education and Research (NIPER-R), Lucknow (UP)","correspondingAuthor":true,"prefix":"","firstName":"Saba","middleName":"","lastName":"Naqvi","suffix":""}],"badges":[],"createdAt":"2025-05-27 11:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6759007/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6759007/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-025-04929-8","type":"published","date":"2025-12-26T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84215194,"identity":"8a4eaf33-92b5-4dcd-b7d4-f93c20459e89","added_by":"auto","created_at":"2025-06-09 10:37:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1536068,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of selenium nanoparticles: (a) DLS showing size distribution of selenium nanoparticles, (b) Zeta potential analysis indicating surface charge, (c) Fourier-transform infrared (FTIR) spectroscopy identifying functional groups on the nanoparticle surface, (d) X-ray diffraction (XRD) pattern confirming crystalline structure, (e) Energy-dispersive X-ray (EDX) spectroscopy detailing elemental composition, (f) Scanning electron microscopy (SEM) showing nanoparticle surface topology, (g)Transmission electron microscopy (TEM) revealing nanoparticle morphology.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/c50903ca1b4cb0ef0b24c6d4.jpg"},{"id":84214341,"identity":"67c531c9-02ff-4eca-90a7-b844d0a0aa87","added_by":"auto","created_at":"2025-06-09 10:29:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":450910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cyclophosphamide on the (a)body weight, and (b)Organ (testis) weight of rat.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data was expressed as mean ± SEM (n=5). The statistical analysis included Tukey's multiple comparison tests after a one-way ANOVA. The significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/7018c1a1e37c1e6b5a02a5eb.jpg"},{"id":84215193,"identity":"465e1347-1aef-4d5f-834a-94c927a8493a","added_by":"auto","created_at":"2025-06-09 10:37:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":841346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cyclophosphamide on various reproductive parameters of rat: (a)Sperm motility (b)Sperm count, (e) Mitochondrial membrane potential in the sperm.\u003c/strong\u003e The data were expressed as mean ± SEM (n=5). The statistical analysis included Tukey's multiple comparison tests after a one-way ANOVA. The significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/b9eca4400e9c3dc4946d666c.jpg"},{"id":84214337,"identity":"dfce32e5-cd00-4535-83b1-a006613367e8","added_by":"auto","created_at":"2025-06-09 10:29:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":102629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of cyclophosphamide on hematological parameters\u003c/strong\u003e. \u003cstrong\u003e(a) WBC (*10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e3 \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/µL), (b) RBC (*10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/uL), (c) Eos (*10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/L), (d) Neu (*10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/L)) monocytes (e) Mon (*10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/L) and (f) Lym (*10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e/L) counts, (g) HGB (g/dL) and (h) HCT (%).\u003c/strong\u003e \u0026nbsp;Data are presented as mean ± SEM (n=5). Statistical analysis was performed using One-way ANOVA followed by Tukey’s multiple comparison test to determine the significance of data. The significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/d7f41acbbd24ecc950b58caf.jpg"},{"id":84215195,"identity":"9563c784-239c-4e83-81f7-b3e7f0ef72f7","added_by":"auto","created_at":"2025-06-09 10:37:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":540534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of cyclophosphamide on various protein metabolism. (a)Total serum protein (b) Albumin (c)A/G in rat.\u003c/strong\u003e All values are expressed as mean ± SEM (n=5). One-way ANOVA was used in the statistical analysis, followed by Tukey's multiple comparison tests. The statistical significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/3b2f3f5db2f630a458f5d366.jpg"},{"id":84214343,"identity":"00361acb-7ca0-48e6-bd85-3a6d399dee9f","added_by":"auto","created_at":"2025-06-09 10:29:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":930071,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of cyclophosphamide (CP), on pro-oxidant and antioxidant parameters (a) TBARS, (b) nitrate, (c) catalase, (d) superoxide dismutase (SOD), (e) glutathione (GSH), and (f) glutathione peroxidase (GPx) activity. The data is presented as the mean ± standard error of the mean (SEM) for n=5 samples. One-way ANOVA was used for statistical analysis, followed by Tukey's multiple comparison tests. The statistical significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/0ed0f786f693ef50d9418ce4.jpg"},{"id":84212826,"identity":"3ff59a54-27c4-4560-a0b9-b856bc014c7e","added_by":"auto","created_at":"2025-06-09 10:21:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":788124,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of cyclophosphamide, sodium selenite, selenium nanoparticles (SeNPs), and combined treatment CP+ SeNPs on (a) level of serum follicle stimulating hormone (FSH), (b) Level of serum testosterone (c) Luteinizing Hormone (LH), (d) Estradiol (e) Triiodothyronine T3 (f) Thyroxine T4. The data is presented as the mean ± standard error of the mean (SEM) for n=5 samples. One-way ANOVA was used for statistical analysis, followed by Tukey's multiple comparison tests. The statistical significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/380b42afea7a1bfef38485fb.jpg"},{"id":84212829,"identity":"53d3f485-7467-48e7-9334-ab4a84c2eeab","added_by":"auto","created_at":"2025-06-09 10:21:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":53965,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of cyclophosphamide (CP), sodium selenite, selenium nanoparticles (SeNPs), and the combination of CP and SeNPs on inflammatory biomarkers (TNF-α and IL-6) and the apoptotic marker caspase-3. The data is presented as the mean ± standard error of the mean (SEM) for n=5 samples. One-way ANOVA was used for statistical analysis, followed by Tukey's multiple comparison tests. The statistical significance level was set at **p\u0026lt;0.01, ***p\u0026lt;0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003ep\u0026lt;0.001 vs CP.\u003c/p\u003e","description":"","filename":"Fig8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/5bc868dfe32c497b1d0d7cb6.jpg"},{"id":84214344,"identity":"1ddb0a61-4c40-4633-a6d5-353de80b4e32","added_by":"auto","created_at":"2025-06-09 10:29:11","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":205020,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative photomicrographs of a histological section of (a) epididymis of rat and (b) Testis tissue, stained with hematoxylin and eosin (H\u0026amp;E staining). The images were captured at 40x and 20um scale bar (A) Control (B) Cyclophosphamide group (CP), (C) Sodium selenite group (NaSe), (D) Selenium Nanoparticles group (SeNP), (E)Cyclophosphamide + selenium nanoparticle (CP +SeNP) group.\u003c/p\u003e","description":"","filename":"Fig9.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/c1c9c457783cf9c43ea10d68.jpg"},{"id":99172453,"identity":"d79157d1-7c58-4ac9-a7c1-3dd80bbb189d","added_by":"auto","created_at":"2025-12-29 16:09:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6970874,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/f1dec755-e56b-4fc3-b8ba-3a971b5c816c.pdf"},{"id":84212818,"identity":"4ef4cea4-d406-4cb8-89d2-31ba428bdc0c","added_by":"auto","created_at":"2025-06-09 10:21:11","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":501852,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6759007/v1/7613251540dc89a989ab7d23.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Green Synthesized Selenium Nanoparticles Mitigate Cyclophosphamide-Induced Reproductive Toxicity in Male Wistar Rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCyclophosphamide (CP), a widely used antineoplastic and immunosuppressive agent, is particularly detrimental to the reproductive system. The primary mechanism of CP involves disrupting cell division, growth, and function by cross-linking DNA strands. This mechanism is responsible for both the therapeutic effects and toxic properties of CP, particularly in rapidly proliferating tissues like the reproductive system. Studies have demonstrated that CP treatment causes biochemical and structural disruptions in the testes and epididymis of male rats [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Additionally, CP exposure often leads to oligospermia and azoospermia, as evidenced by experimental animal studies. Testicular injury induced by CP is associated with aberrant gonadotropin secretion and reduced testosterone levels in male patients. Given the significant impact of chemotherapy-induced infertility on quality of life, it is crucial to develop strategies for protecting germ cells during and after chemotherapy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNanotechnology has garnered significant attention globally due to its biocompatible properties and potential to enhance the safety and efficacy of treatments. Among various nanoparticles, selenium nanoparticles (SeNPs) have emerged as a promising candidate, owing to their potent antioxidant properties. Studies have demonstrated that nanoparticulate selenium formulations offer superior bioavailability and targeted delivery compared to conventional selenium supplements, thereby enhancing their therapeutic potential [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The protective effects of SeNPs against various forms of toxicity, including reproductive toxicity, have been increasingly explored in preclinical studies. These nanoparticles have shown efficacy in neutralizing reactive oxygen species (ROS), reducing lipid peroxidation, and enhancing the function of the body's natural antioxidant enzymes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Given their antioxidant properties, SeNPs present a promising intervention to counteract the oxidative damage induced by cyclophosphamide in male reproductive organs.\u003c/p\u003e \u003cp\u003eGreen synthesis offers several benefits over chemical method, including being non-toxic [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], pollution-free [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], economical and more sustainable [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The phytoconstituents present in the plant extract imparts a wide range of therapeutic effects due to their diverse biological activities It also provide characteristic size and shape to the nanoparticles. The nanoparticles synthesised from plant extract are often have superior biocompatibility in \u003cem\u003eIn-vitro\u003c/em\u003e and \u003cem\u003eIn-vivo\u003c/em\u003e experiments. In this study we have synthesized selenium nanoparticles by \u003cem\u003eAzadirachta indica\u003c/em\u003e leaves extract, an ecofriendly green approach. After the synthesis and characterization of nanoparticles the nanoparticles were investigated in mitigating cyclophosphamide (CP)-induced reproductive toxicity in male rats. The dose of CP, 15mg/kg weekly [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]and SeNP, 0.2mg/kg/day [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] was decided based on previous research. Various parameters, including sperm count, motility, morphology, and antioxidant enzyme activities, hormonal parameters were evaluated to elucidate the protective mechanisms of SeNPs and explore their potential as a therapeutic strategy to safeguard male fertility during chemotherapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemical Reagents\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSodium selenite (214885-5G), cyclophosphamide (PHR1404-1G), were procured from Sigma-Aldrich chemicals., St. Louis (USA). The serum total protein kit and the serum albumin kit were procured from Accurex Biomedical Private Limited, Thane, Maharashtra. All ELISA kits such as FSH (Cat: ELK1315), Rat testosterone (Cat: ELK8314), Rat LH (Luteinising hormone) Cat: ELK2367), General Estradiol Cat: ELK1208, Rat T3 (triiodothyronine) Cat: ELK1339, Rat T4 Cat: ELK1204, TNF-α (Cat: ELK1396), and caspase 3 (Cat: ELK1396), were obtained from ELK Biotechnology Wuhan. The ELISA kit Rat IL-6 (Cat. #IT17833) was purchased from G-bioscience, while all other chemicals were purchased from Hi media and Merck.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Green Synthesis of selenium nanoparticles\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Plant source\u003c/h2\u003e \u003cp\u003eThe fresh green leaves of \u003cem\u003eAzadirachta indica\u003c/em\u003e were collected from botanical garden at NIPER Raebareli – transit campus Lucknow. The identification of collected leaves is done at the research laboratory in NIPER facility.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Preparation of aqueous extract\u003c/h2\u003e \u003cp\u003eAfter collection the leaves were cleaned with water and dried. The dried leaves were then ground and extraction was done with water for 1 hour. The extract was concentrated and filtered through a crude cellulose filter paper and then Whatman #1 filter paper. The resulting filtrate was stored at 2–4 ͦ C until required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Synthesis of selenium nanoparticle\u003c/h2\u003e \u003cp\u003eSelenium nanoparticles (SeNPs) were synthesized using a green synthesis method, as described by [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], with slight modifications. For SeNP synthesis, sodium selenite was added dropwise to the neem extract under continuous stirring on a magnetic stirrer. A red colloidal solution of SeNPs formed instantaneously. After completion of the reaction, the solution was centrifuged at 9000 rpm for 30 minutes. The supernatant was discarded, and the pellet was resuspended in milli-Q water and washed 3–5 times. The pellet was stored in a -20°C deep freezer and lyophilized for further use.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of selenium nanoparticles\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Dynamic light scattering (DLS)\u003c/h2\u003e \u003cp\u003eThe particle size, zeta potential and polydispersity index (PDI) of the biomolecule-functionalized SeNPs were ascertained by dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments Ltd., UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Infrared spectroscopy using Fourier transform (FT-IR)\u003c/h2\u003e \u003cp\u003eThe FTIR spectrophotometer (ECO-ATR, ALPHA, Bruker, Germany) was used to examine the FTIR spectral analysis of biosynthesized SeNPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 X-ray diffraction (XRD) analysis\u003c/h2\u003e \u003cp\u003eAt 40 keV, the crystalline characterization was carried out at IIT-Roorkee, India (Grazing Incidence-X-Ray Diffractometer (GI-XRD)), in the range of 20~ # 2q # 80. PowderX software was used to design the crystals' lattice constant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Microscopic analysis via SEM and TEM\u003c/h2\u003e \u003cp\u003eA Jeol JSM-IT200 scanning electron microscope (Japan) was used to examine the surface morphology of biosynthesized SeNPs. The sample preparation was done on clean electric stub, the stub was covered with a drop of the sample, which was then let to dry. The dried sample was prepared for imaging by mounting it using double adhesive tape on an aluminum stub. The microscope had an 8 mm working distance and an accelerating voltage of 10 kV. Energy-dispersive X-ray spectroscopy (EDX) was used to analyze the elemental content present in the nanoparticles.\u003c/p\u003e \u003cp\u003eThe transmission electron microscopy (TEM) was performed at Jamia Hamdard. It was used to analyze the size and shape of the SeNPs. To prepare the samples for examination, a single drop of aqueous biosynthesized SeNPs was placed on a copper-coated grid. A Jeol JEM-2100 electron microscope (Japan) was used to record a TEM micrograph.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Animals and experimental study design\u003c/h2\u003e \u003cp\u003eThe experiment was performed on male Wistar rats having body weight of 120–140 grams. The rats were procured from CSIR-IITR, Lucknow. Prior to the experimentation the protocol was approved by the IAEC (Institutional Animal Ethics Committee) members of NIPER-Raebareli (Approval No. NIPER/RBL/IAEC/96/AUG 2022). The rats were kept under standard conditions and feed with standard pellet diet and had unrestricted access to water. Housing condition of animals included a temperature of 20 ± 2°C, relative humidity of 50 ± 10%, and a 12-hour light/dark cycle.\u003c/p\u003e \u003cp\u003eAfter one-week of acclimatization period, the animals were randomly assigned to five groups, each comprising six rats. The groups were designated as follows:\u003c/p\u003e \u003cp\u003eGroup 1 (Control): normal saline.\u003c/p\u003e \u003cp\u003eGroup 2 (CP): cyclophosphamide only (15 mg/kg/Week).\u003c/p\u003e \u003cp\u003eGroup 3 (NaSe): sodium selenite (0.2 mg/kg/day).\u003c/p\u003e \u003cp\u003eGroup 4 (SeNPs): selenium nanoparticles (0.2 mg/kg/day).\u003c/p\u003e \u003cp\u003eGroup 5 (CP + SeNPs): cyclophosphamide (15 mg/kg/week) and SeNP (0.2 mg/kg/day).\u003c/p\u003e \u003cp\u003eAll study treatments were administered orally to the animals over a 28-day period. The care and use of the animals were conducted in accordance with CPCSEA guidelines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue Processing\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUpon completion of the study, rats were anesthetized with urethane (5mg/kg) via intraperitoneal injection. Blood samples were then collected by retro-orbital sinus puncture. The blood samples were divided into two groups: one was transferred into non-EDTA microcentrifuge tubes for serum collection, while the other was transferred into EDTA-coated microcentrifuge tubes for whole blood collection.\u003c/p\u003e \u003cp\u003eFor serum separation, the non-EDTA blood samples were allowed to clot for 30 minutes at room temperature, followed by centrifugation at 10,000xg for 10 minutes at 4°C. The resulting serum was stored at -80°C for subsequent serum analysis and hormonal determination. The anticoagulated blood samples (EDTA-coated tubes) were stored at 2–4°C for hematological estimation.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFollowing blood collection, the animals were humanely euthanized by cervical dislocation. The testes and epididymides were carefully excised, washed with ice-cold phosphate-buffered saline (PBS), and divided into two portions. One portion was fixed in 10% formalin for histopathological examination. The other portion was stored at -80°C for biochemical assays.\u003c/p\u003e \u003cp\u003eTissue homogenates were prepared by homogenizing the testicular tissue in a 10-fold volume of ice-cold PBS buffer (0.01 M, pH 7.4) using a tissue homogenizer. The homogenates were then centrifuged at 10,000xg for 10 minutes at 4°C. The resulting supernatants were collected as tissue homogenates and stored at -80°C for further analysis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sperm collection and sperm parameters\u003c/h2\u003e \u003cp\u003eOne epididymis was placed in a Petri dish containing Hanks' Balanced Salt Solution (HBSS) medium. The caudal portion of the epididymis was gently incised at a few sites using a scalpel, taking care to minimize exposure to air. The tissue was then dispersed in the medium for 5–15 minutes, allowing the sperm to be released. Following this incubation period, the tissue was removed, and the sperm were collected for further analysis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sperm count\u003c/h2\u003e \u003cp\u003eThe sperm-containing HBSS medium (1 ml) was centrifuged at 1000 rpm for 3 minutes. The resulting supernatant was collected and used for sperm counting. Sperm count was estimated using a hemocytometer, and the results were expressed as millions of sperm per milliliter (ml)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAbwAAABBCAYAAACqyxGsAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAA0sSURBVHhe7Z1PjtRGG4cbjhEhhBik3IAFYrIMHIAFc4FIcACymQVI2RCxYpFIWbEjRwgs2CSnQAxCiHvk8+Pxb/L2+1W53e0ed/f490g10/X/favKVa4qu3zt34aFMcYYc8W53v03xhhjrjQe8IwxxswCD3jGGGNmgQc8Y4wxs8ADnjHGmFngAc8YY8ws8IBnjDFmFnjAM8YYMws84BljjJkFHvCMMcbMAg94xhhjZoEHPGOMMbPAA54xxphZ4AHP7JRff/11cefOnc62W5ADeXYF+V+7dq01nz9/7lz3nz///LOVec5Qd5TDtshlOuY6cf38hwc8cwEXlTrcknn69GkXcjtwIf7888+dbbc8fPhwcXZ21tmmh/zfvHmz4GtdR0dHi1evXnU++8/jx49bueeIbpJ+/PHHxd27d9vrpDTwxZuZbErXVSzTsdfJZdTPuvrsCx7wzBJv375tLw51/vzH/vfff7f2Ifzzzz+DZkpciC9fvuxsu+Wvv/5qB5pd8e7du8V3333X/v706dPit99+a3+b/Ya6YjD6/fff2/bz5MmTtl1nCMe1RRiuJxnciMtAUWPd62To9TeGmj6YsVym/B7wzBKlixXu37+/uHXrVmfr55dfful+mSEc0vKlWWZs3XG96WaSWf422PX1N/Zm7VLlb0ZkY/6PZmbHrVr7vwR+Ms3dZ+f677/NHd+SHzR3vUtuMU3iEqcE7vhjYnqgfJT3gwcPluyg+Mpf+WS7UHilnf0Bd5mmo+pcz+NGPTMxXpSR39GvlKfSbe6o299C8kp3TEbumD55lVYum2wvoTITSivqVqOmG5CO4iM7YaI76UOu+6gXJrY34kZ/iGFjGfXJJqQr/rpmJGcGd8KXUF6SlTRj2GjnP2FL+stPBnJaEMMoHUFY3DAxnUzWh/Cl8pPJfUn0y3UrI6JblJfwMZ9VrA5hZoku3txIgUamBkoDLzVC2ZWOUAMVhMMtEztxpRXThWwnjuwxvmQt2fUbSA836YyddAR+gnxkV14lPQD3vvLqK2vCq7z4rzyifupkNpU3pjW0rARu+JXSko74RX1FTTeIcVQ+Mf+cJvli72tvWW+IYYkve59sJfAvlU8E/1o6ypt8c5kC/tHO75L+EP1KafFbspIf/gq/Tv0p7WjUFvvqAWJY0s7yxfxq8pbqcxVe0jRroQ15LX2y1Nk05MUff/zR2jO3b9+m1Xe2Rbu5PwTtqTUNe/Hs2bPWbZ2n1GJ8yVqyf/36tf0t8Edm4CES9tZAemtjXg8RsN+gvH766afWLbJueZVgj4elM5aK2DuBqB9pAktBm8i7aVkB/oQTMa0h9VbSTbIrPvVBmkPoa29Z774ygpJsNfBXWY0ll+kYclqr2uO69UdYyhvTDEqda389IEMzWF20W/KplW+fvLk+h+ABz6xFqdP74YcfVj7hSMdBp0IncqjEi1tGF22NTctL6AInb8pPHUCJGzdudL/O2UTeKanpRpmtc3NTYmh7q5XROuW+Db58+dL+v+z6Gdse+2BQyvKX6qEkQ41ty+sBz6zFzZs328ZGQ47QMdSgwXOHR2dCJ3IofPv2bUmvfJHRCWo2UGOT8soww6DsuOs+OTnpzXOsvFNT023VjKqPddpbXxmtU+5jIF0GhCmujW20x6HU6gEZhtbvtuX1gGfWQksL8V0blv7ik1W6O6fzeP36dds4cwPfxh0z+ejOmE6D5TyWpcY80qz04PT09EIv6R2fpEPvVXfkQ8qrD/RSXJZ+WAqKfPjwoft1vqSp5Z1N5Z2Smm6PHj1qO7m+eqzVPe5D21tfGa0q922BXMfHx63MDLBD6Wv78frLA8XY9jgU8q7VAzLk+uW3ZI3y824jbE3eZvQ1ZonmbqzdDJbBnon+2lAWzR3xUrwYtrkI2v+EUTi5RxROYbVBLTtoAxuDP0Z+Of4qu4juWa/mIr3ww2jTvZZWJMaL6RI++qFDhDyi7rEuyLfmB0PlXdceifITLoeN8uW4fbpRRnKXieXWV/cxjuTBr6RHrYz6ZNuEmHc2uVywy4942Q59+is8MpfigtwwsVxzGcUyUPqQ9cnXCkR/hVcaudxj2pI5lnkMq7yyrEO4xp8mgjHmwOBOmBmdHi64yqArd/WaoRizCV7SNMYYMws84BlzgDDjYR9k7J7lISBdeXCEfR1jNsVLmsYYY2bBpDM8HlOV4Ymc+OSNMcYYc5lMNuDx6O/Lly/bdzIwvJ/x/v37ztcIHjG+6ktUxhizCyYb8HhP5N69e53t/MXSeNyMOWfb78MYY4w5Z7IB7+joqH3BMr4IGV+0ZGOamQ0zQS17ZuKSKDMhQVyWR2M8pSd37JDtNZQWJm+UR784GyPN6CZdchjsGKUh8NMLpNEdYrmUTH7B1BhjTIKHVqYgv2gYiS836sVPXirEXcQ4ejERFJfwIqanlxRL9tLLkoCf5CCvmDa/FU8vf8aXHvGPdmSRPcoltxw+240xxmyHyWZ4Oj37SXemGrMSPbQST+jW0Ucs7THbAc2wNJthBgTM8konZsf09KJqyV46mJS8moHpQo54krfkUBqEQZ+hJ9+vexK5McaY7THpU5qgA1kZKDgwtfZeza5Ofu87yfsyTxpfhZc0jTFmHJM+pRlh4GMm1TfAMMiJPKgwUMZ9vG3Rd5L3lCeNZ5gd5gE/Gn3DzRhjTJlJn9KMD28waOQnN/fh5HfyYlCLsvIbeSVHfH8wn9zNEuWYE/y1xMmAPsWs7cWLF4vr16/b2NjY7I35+PFj10NtmWZ2MAn55G6MHgyBZpa09FBHPCkb8kMv8eEWuZF+yW2VPZPzymGiX37wRQ+yYNAHU5Mr6qsw/Mee9TfGGDOOvTlajJnNXE5+N8YYMz2TP7RijDHG7IK9GPCY3bFvNoeT340xxuwGfy3BGGPMLPCSpjHGmFngAc8YY8ws8IBnzArYY9713jIyXOVTddCvdurSlGy7rjept6na21T57BMe8Mzeow4DkzvF2KHEAwG2BYcd8EDVLkEGDjhgu/3o6Gjx6tWrzufwUafLp8Lu3r27VMexbrM5hLrepN6mam/70K53Ag+tGHMI0FwxzYXauZzDy/rxEINtw4EBOhhgF5R0vkqoXjH5wAUOdqD8I7gp/FhoN7Fut1nXyLdJvU3V3nbdrneBZ3jmYODA8eYindWHg6/i8mVkE/044q8ZqNrfzFTGcFkfXL7q9XaoeMAzB8X79+/bpZjakpaWwbQ3QYcY7aBlNNLADztke0Zpl/xxl1n1ceJMjBvl5DcDPPC/lK/SZhkwl4nkxSBTXiqslVGUFxM775I+MWzUvU82wc1LM8tob2ao174vqET0aS7OqpV8NTmQGd2kJ0buOutWbiLrWENhMCq/IfUGMW5J55oM0R0j/aWn/JVvtmdwL/krfcy6bXpv6WZ6xuw9Wu7SkpbOMWVZJi5pNp3M0lJNPM80nl+q+CW7fgPp4ablKeykI/AT5CO78iJ8DfyUl85hjbKTZ8w7QniVCf9jPvxWOkoj61QqI4UVhFMeJX1i2Kh7n2wl8I/yCdxqcZWfyk1EOWJ9q42QnuqP37EcsBNWstTkguiX666v3iDKQxzSEn0y1Oon6qmwJbt+g/KRjNhVLriLUnkS9hD5Tytj9hx1oMBvXaxckOo8gIsxdmJcpNGe/de1xw6WDoTf2cTONcaNEBf/SB4c+jpOyZH9Suliz51dlCuXkUCeWO4xXp/uNdnWpaSLQA7yeP78+VL+MrU6kNyQ/YaWS0muWHd99UZcDSwlhsoAsX5W6bLKrjrrq1fI8Q4JL2mag4TvKTYX3l7s5yFHcy0tmSGfrhr7QWEt65E/S0taFiPd2vLVUFgmI02WGPuo6V6TbZvoM1zff//9WnWQPy69CWPqru8boEMZWj+bsmmb3nc84JmDRft57MFMybdv39oOQeROjs497nnU2MYHhRn46YyaO+7FycnJRb61jxgPgY6UGwnSZdDqo0/3mmzbgLTo7CXfunWwThmXGFN3fR+ZHsI69bMOsV1v2qb3HQ945mDhK+9v377tbP/B7GbMR3hLKD04PT29eLpvzMeJh3xQuA90U1w+q/XgwYP296NHj9oOq0/fWhnhTqeXO+TSDK1P95ps2wBZjo+PWzkZVIfUQe3j0poJk2YevPoYU3fEzfXD7yH5I+fQ+hlCqV0PKc+DpblLMGavYb+ApirTdBadzzn4a38BtBeBYf8j7oE0ncWFH26r7CK6s8cRQR75YeJeh9xiWpkYN6ZNnOiHHhHywU3+zd1+51PeX4tp95VRjCMdamVT071PtqHE/LLJ5VmTA0inJovKGLesX4yT8xPyx6h8laYM6WSyvEp/iAyyY0plRLicTraL6B7bx9g2va/4awnGzABmMvHufU6guz8ubcBLmsYYY2aBBzxjrjjMcM7OztoHRzbd6zlUpLs/Lm3AS5rGGGNmgWd4xhhjZoEHPGOMMbPAA54xxphZ4AHPGGPMLPCAZ4wxZgYsFv8D67SDB0EoZb0AAAAASUVORK5CYII=\" width=\"444\" height=\"65\"\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Sperm motility\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSperm motility was assessed by placing a 10 µl aliquot of the sperm suspension on a microscope slide, which was then covered with a coverslip. For each animal, at least five microscopic fields were examined, with a minimum of 200 sperm evaluated per field. The percentage of motile sperm was calculated by determining the proportion of total sperm that exhibited movement [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Sperm morphology\u003c/h2\u003e \u003cp\u003eSperm morphology was evaluated by preparing thin smears of semen on clean, grease-free microscope slides. The slides were air-dried overnight and then stained with a 1% eosin-Y dye solution. Using a light microscope equipped with a 40× objective lens, the stained sperm cells were examined for any morphological abnormalities. Sperm with irregularities, such as amorphous or irregularly shaped heads, missing or malformed acrosomes, double heads, coiled tails, or other deviations from normal morphology, were identified and counted [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.8 JC-1 microplate assay\u003c/h2\u003e \u003cp\u003eThe JC-1 microplate assay is a widely used method for assessing mitochondrial membrane potential (ΔΨm\\Delta \\Psi_mΔΨm​), a critical indicator of mitochondrial health and function. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, which is reflected in a shift in its fluorescence emission. For the fluorescence microplate analysis, a 200 µL aliquot of the sample (25 x 10^6 cells/mL) were placed undiluted into every well (doublet) in a 96-well plate. Fluorescence was measured using a microplate reader equipped with a 485 nm excitation filter, with the gain set to 100% for all assays. Initially, a 595 nm emission filter was used to detect total orange fluorescence, which corresponds to JC-1 aggregates. Subsequently, a 535 nm emission filter was employed to detect all green fluorescence, which corresponds to JC-1 monomers. Each well's total fluorescence was corrected by subtracting the blank fluorescence from HBSS containing 2 pM JC-1 dye only. The mean of total and blank corrected fluorescence values were then calculated for both treatment and control groups [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Hematological parameter\u003c/h2\u003e \u003cp\u003eBlood samples were isolated from the retro-orbital plexus to assess hematological parameters. Key metrics, including RBC count, WBC count, hemoglobin concentration (HBT), hematocrit percentage (HCT), granulocyte, lymphocyte, and monocyte counts, were measured using a Mindray BC-5130 automated hematoanalyzer following transfer of the blood into EDTA-treated microcentrifuge tubes[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Serum biochemical analysis\u003c/h2\u003e \u003cp\u003eWe assessed serum total protein content, albumin levels, and the albumin/globulin (A/G) ratio to evaluate the overall health and physiological status of the organism. The A/G ratio serves as a crucial marker for immune function and inflammation, whereas total protein and albumin levels can indicate liver dysfunction or malnutrition. Serum biochemical indices were measured using an Accurex colorimetric assay kit (Accurex Biomedical Pvt. Ltd., Mumbai, India), following the manufacturer's protocols (Accurex Biomedical Pvt. Ltd., Thane, India). Specifically, the biuret method was employed to determine serum total protein levels, and the Bromocresol Green (BCG) method was used to estimate serum albumin levels [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.11 TBARS Assay\u003c/h2\u003e \u003cp\u003eThe estimation of lipid peroxidation was performed by measuring malondialdehyde (MDA), a well-known lipid peroxidation marker. Tissue homogenate was treated with acetic acid, sodium dodecylsulphate (SDS), followed by the addition of thiobarbituric acid. The mixture was then heated at 95°C for 1hr and centrifuged at 12,000 g for 10 minutes. The resulting pink supernatant was measured at an absorbance of 532 nm. MDA concentration was determined using a standard curve generated with tetraethoxypropane (TEP) and expressed as nmol/mg of protein [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Nitrate assay\u003c/h2\u003e \u003cp\u003eUsing the Griess reagent, the nitric oxide (NO) level was determined. The diazotization process mediated by Gries reagent, which spectrophotometrically identifies nitrite generated by spontaneous oxidation of NO under physiological circumstances, is the principle at play here. Here, sulfanilic acid reacts with nitrite in an acidic solution to quantitatively produce a diazonium salt. Following this, the diazonium salt is combined with N-(1-naphthyl) ethylenediamine to create an azo dye, which can be measured spectrophotometrically at the absorbance at 548 nm [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.13 Glutathione assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe concentration of glutathione (GSH) in the testis was determined using a colorimetric method. Tissue homogenates were treated with an equal volume of 5% sulphosalicylic acid, vortexed, and incubated in an ice bath for 30 minutes to precipitate proteins. The resulting supernatant was then used to measure GSH levels using Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) solution. The results were expressed as µM GSH/mg protein [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Catalase assay\u003c/h2\u003e \u003cp\u003eTo determine the catalase activity in the testis, a modified version of the method described by Sinha et al. was employed. The assay involved adding 250 µl of the sample to 250 µl of hydrogen peroxide, followed by incubation at 37°C for 1 minute. The reaction was terminated by adding 1 ml of a solution containing potassium dichromate and acetic acid in a 1:3 ratio, and the mixture was then placed in a boiling water bath at 95°C for 10 minutes. After cooling, 300 µl of each solution was loaded into well plates, and the absorbance was measured at 570 nm[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Superoxide Dismutase (SOD)\u003c/h2\u003e \u003cp\u003eTo determine the antioxidant enzyme SOD, The Sodium pyrophosphate was added to the supernatant, and then phenazine methosulphate, nitro blue tetrazolium, nicotinamide adenine dinucleotide, and distilled water was added in a test tube. The mixture was incubated at 30°C for 90 s. The reaction was ceased by adding glacial acetic acid within 90 s and the violet color complex formed was determined at the absorbance of 560nm [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Measurement of serum proteins\u003c/h2\u003e \u003cp\u003eThe blood serum was used to estimate the level of serum total protein, albumin, globulin and albumin/globulin ratio by using Elisa kits procured from ELK biotechnology. Assay was performed as per manufacturer’s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Measurement of serum hormonal levels\u003c/h2\u003e \u003cp\u003eThe serum was used for estimation of level of Follicular stimulating hormone (FSH), Testosterone, Luteinizing hormone (LH), Estradiol (E2), T3 (triiodothyronine) and T4 (Thyroxine). These hormones level was measured by ELISA which were procured from ELK biotechnology. Assay was performed according to manufacturer’s protocol[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e2.18 Measurement of inflammatory biomarkers\u003c/h2\u003e \u003cp\u003eTestis homogenate was used to measure inflammatory biomarkers. Testis inflammatory mediators TNF-α and IL-6 were measured by ELISA. ELISA kits were procured from ELK biotechnology. The assays were performed as per manufacturer’s instruction[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e2.19 Estimation of apoptotic biomarker Caspase-3\u003c/h2\u003e \u003cp\u003eTestis homogenate was used to measure apoptosis biomarker. Testis apoptosis biomarker Caspase-3 was measured by ELISA. ELISA kits were procured from ELK biotechnology. The assay was performed as per manufacturer’s instruction[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e2.20 Histology of testis and epididymis of rat\u003c/h2\u003e \u003cp\u003eTestis and epididymis collected in the formalin were processed in ascending concentrations of ethanol such as 70%, 80%, 90%, and 3 times in 100%, two times changed with xylene, and three times changed with paraffin (60C) for 40 min per each solution. Finally, the samples were sectioned at a thickness of 5 µm and these sections were mounted on the coated slides. After that deparaffinization is done in xylene and rehydration is done in graded ethanol ranging from 100–50%, followed with hematoxylin and Eosin (H\u0026amp;E) staining. Further, gross cellular damage was observed by using light microscope[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e2.21 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data was presented as the Mean ± Standard Error (SE). Graph Pad Prism 8 software was utilized to analyze the statistical differences using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. The level of statistical significance was set at *p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of Selenium Nanoparticles (SeNPs)\u003c/b\u003e \u003c/p\u003e\u003cp\u003e \u003cb\u003eZeta Potential and Size\u003c/b\u003e \u003c/p\u003e\u003cp\u003eUsing dynamic light scattering (DLS), the average particle size, polydispersity index (PDI), and zeta potential of the selenium nanoparticles were ascertained. The results revealed the formation of selenium nanoparticles with an average size of 100.2 nm, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a) and the polydispersity index was found to be 0.024, signifying a homogeneous size population of nanoparticles in the aqueous medium. The zeta potential of the synthesized nanoparticles was − 4.75 mV, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b).\u003c/p\u003e\u003cp\u003e \u003cb\u003eFourier Transform Infrared (FTIR) and XRD Analysis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe FTIR analysis was conducted to identify the functional groups present on the selenium nanoparticles. The spectra covered the range from 400 cm⁻¹. The FTIR data of selenium nanoparticles shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c) shows the peak at 3300 cm-1 = Hydroxyl group of phenol, 1655cm-1 = Amide group, 588 cm-1 = Se-O interaction.\u003c/p\u003e\u003cp\u003eThe X-ray diffraction (XRD) pattern of the synthesized selenium nanoparticles is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d). The analysis revealed prominent diffraction peaks at 2θ values of 23.62°, 29.81°, 41.42°, 43.74°, 45.45°, 51.80°, 55.71°, 61.32°, and 65.28°. The most intense peak was observed at 29.81°, indicating a high degree of crystallinity.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSEM, EDX, and TEM Analysis of Selenium Nanoparticles\u003c/b\u003e \u003c/p\u003e\u003cp\u003eEnergy-dispersive X-ray (EDX) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) confirmed the presence of selenium, sodium, carbon, and oxygen. The elemental composition of Se was found to be 0.057 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e±\u003c/span\u003e 0.03. The size and shape of the selenium nanoparticles were analyzed using Scanning electron microscopy (SEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), revealing an average size of 190.2 nm. Transmission electron microscopy (TEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg) analysis showed that the nanoparticles were not aggregated, and their surface morphology was consistent with the size found using the zeta-sizer.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etoxicity assessment of Cyclophosphamide\u003c/b\u003e\u003c/p\u003e\u003cp\u003e \u003cb\u003eEffect of treatment on body and organ (testis) weight\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe body weight of rats in each group was monitored throughout the study, and organ weight (testis) was measured at the end of the study. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a) \u0026amp; (b). Cyclophosphamide (CP) treatment (Group 2) caused a slight decrease in body weight compared to the control group (Group 1). In contrast, rats treated with selenium nanoparticles (SeNPs, Group 4) exhibited a gradual and consistent increase in body weight throughout the study, suggesting the non-toxic nature of SeNPs. In Group 5, where animals received both CP and SeNPs, the body weight also increased steadily, although the increment was slightly lower than in Group 4. However, compared to the CP-only group, the SeNP treatment significantly mitigated CP's toxic effects, resulting in a notable improvement in body weight. Similarly, Group 3 (sodium selenite) showed a trend towards increased body weight compared to the CP group, but the protective effect was less pronounced than that observed with SeNPs.\u003c/p\u003e\u003cp\u003eCyclophosphamide treatment also caused a significant reduction in testis weight compared to the control group (***P \u0026lt; 0.004 vs. Control), indicating CP-induced testicular toxicity. This reduction was significantly reversed by SeNPs treatment in Group 5 (#P \u0026lt; 0.011, ###P \u0026lt; 0.0005 vs. CP), suggesting their protective effect. While sodium selenite in Group 3 showed a mild improvement in testis weight compared to the CP group, it was less effective than SeNPs. Overall, the findings demonstrate that SeNPs not only counteract CP-induced testicular toxicity but also improve body weight, highlighting their potential as a protective agent against CP-induced systemic toxicity.\u003c/p\u003e\u003cp\u003eThe data was expressed as mean ± SEM (n = 5). The statistical analysis included Tukey's multiple comparison tests after a one-way ANOVA. The significance level was set at **p \u0026lt; 0.01, ***p \u0026lt; 0.001 in comparison to the control. And \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 vs CP.\u003c/p\u003e\u003cp\u003e \u003cb\u003eCyclophosphamide's Influence on Sperm Characteristics, and Mitochondrial Function\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe effects of green synthesized selenium nanoparticles (SeNPs) on cyclophosphamide (CP)-induced reproductive toxicity were evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The CP-treated group (Group 2) exhibited a significant reduction in sperm count compared to the control group (Group 1, p \u0026lt; 0.0002). Notably, treatment with SeNPs (Group 5) significantly improved sperm count compared to the CP group (p \u0026lt; 0.0005), indicating a reversal of CP-induced toxicity.\u003c/p\u003e\u003cp\u003eSperm motility was significantly impaired in the CP group compared to the control group (p \u0026lt; 0.0002). SeNP treatment (Group 5) restored sperm motility to near-control levels (p \u0026lt; 0.0001 vs. CP), demonstrating its protective potential.\u003c/p\u003e\u003cp\u003eThe JC-1 assay was carried out to assess mitochondrial membrane potential (MMP) of the sperm as MMP is \u003cb\u003ea\u003c/b\u003e critical parameter for assessing sperm function and quality. As we know Mitochondria in sperm provide the energy (ATP) necessary for motility and capacitation. Sperm with high mitochondrial membrane potential are considered more viable, motile, and healthy. Whereas decreased membrane potential​ may indicate poor sperm quality. In our study, we noticed a significant reduction in mitochondrial membrane potential in the sperm of CP-treated rats (Group 2) compared to the control group. Treatment with SeNPs (Group 5) markedly restored mitochondrial membrane potential compared to the CP group.\u003c/p\u003e\u003cp\u003eSperm morphology analysis showed that CP treatment led to a higher incidence of abnormalities, including hooked heads, coiled tails, and detached hooks. These abnormalities were significantly reduced in the SeNP-treated group, with morphology comparable to the control group.\u003c/p\u003e\u003cp\u003eThese findings indicate that SeNPs effectively mitigate CP-induced reproductive toxicity, as evidenced by improved sperm parameters, restored mitochondrial function, and reduced sperm morphological abnormalities. Statistical significance was noted as ***p \u0026lt; 0.0004 vs. control and ###p \u0026lt; 0.001 vs. CP.\u003c/p\u003e\u003cp\u003e \u003cb\u003eEffect of cyclophosphamide on hematological parameters of rat blood\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe effects of cyclophosphamide (CP) on haematological parameters were assessed by measuring white blood cells (WBC), red blood cells (RBC), eosinophils (Eos), neutrophils (neu), monocytes (Mon), lymphocytes (Lym), hemoglobin (HGB), and hematocrit percentage (HCT%) and protective effect of SeNP were evaluated (Fig .4).\u003c/p\u003e\u003cp\u003eCyclophosphamide treatment (Group 2) caused a significant reduction in all measured parameters compared to the control group (Group 1), reflecting the hematotoxic effects of CP. Specifically, WBC counts were significantly decreased (**p \u0026lt; 0.014 vs. Control), as were RBC counts (***p \u0026lt; 0.00051 vs. Control), eosinophils (**p \u0026lt; 0.0021 vs. Control), neutrophils (***p \u0026lt; 0.0002 vs. Control), and monocytes (*p \u0026lt; 0.03 vs. Control). Lymphocyte counts (**p \u0026lt; 0.0053 vs. Control), HGB levels (**p \u0026lt; 0.0027 vs. Control), and HCT% (***p \u0026lt; 0.0001 vs. Control) were also markedly reduced in the CP group compared to the control group.\u003c/p\u003e\u003cp\u003eTreatment with SeNPs (Group 5) significantly reversed these CP-induced alterations. WBC counts in the SeNP-treated group showed a marked recovery (#p \u0026lt; 0.02 vs. CP), indicating restoration of immune function. RBC counts were significantly improved compared to the CP group (#p \u0026lt; 0.01, ###p \u0026lt; 0.0008 vs. CP), suggesting enhanced erythropoiesis. Eosinophils, neutrophils, and monocyte counts demonstrated substantial recovery with SeNP treatment (#p \u0026lt; 0.031 for eosinophils, #p \u0026lt; 0.03 for neutrophils, and #p \u0026lt; 0.03 for monocytes vs. CP), reflecting improved immune and inflammatory responses.\u003c/p\u003e\u003cp\u003eLymphocyte counts in the SeNP group were significantly higher compared to the CP group (##p \u0026lt; 0.0023, ##p \u0026lt; 0.0093 vs. CP), highlighting the mitigation of CP-induced lymphotoxicity. HGB levels (#p \u0026lt; 0.0130 vs. CP) and HCT% (###p \u0026lt; 0.0003, ###p \u0026lt; 0.0007 vs. CP) were also significantly restored in the SeNP-treated group, indicating recovery of oxygen-carrying capacity and blood volume integrity.\u003c/p\u003e\u003cp\u003eThese findings demonstrate that SeNPs effectively counteracted the hematotoxic effects of CP by improving all evaluated hematological parameters. Statistical significance was observed for all comparisons, indicating the robust protective potential of SeNPs.\u003c/p\u003e\u003cp\u003e \u003cb\u003eEstimation of serum total protein content, albumin and albumin/globulin (A/G) ratio\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe effects of cyclophosphamide (CP) on serum total protein, albumin, and albumin/globulin (A/G) ratio were evaluated to assess the impact of treatments on protein metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCyclophosphamide treatment (Group 2) significantly reduced serum total protein, albumin levels, and the albumin-to-globulin (A/G) ratio compared to the control group (Group 1). Specifically, total protein levels were markedly decreased in the CP group (***p \u0026lt; 0.0005 vs. Control), reflecting impaired protein synthesis. Similarly, albumin levels (**p \u0026lt; 0.009 vs. Control) and the A/G ratio (**p \u0026lt; 0.0067 vs. Control) were significantly lower in the CP group, indicating disruptions in hepatic function and protein homeostasis.\u003c/p\u003e\u003cp\u003eTreatment with selenium nanoparticles (Group 5) significantly restored these parameters. Total protein levels were significantly improved in the SeNP group compared to the CP group (##p \u0026lt; 0.0099, ##p \u0026lt; 0.028 vs. CP), highlighting a recovery in overall protein synthesis. Albumin levels also showed a significant recovery (#p \u0026lt; 0.0342 vs. CP), suggesting an improvement in liver function. Furthermore, the A/G ratio was significantly enhanced in the SeNP-treated group compared to the CP group (#p \u0026lt; 0.01, ##p \u0026lt; 0.02 vs. CP), reflecting a restored balance between albumin and globulin levels.\u003c/p\u003e\u003cp\u003eThese results indicate that SeNP treatment effectively deciphers the adverse effects of CP on serum protein metabolism, with statistically significant improvements in total protein, albumin levels, and A/G ratio.\u003c/p\u003e\u003cp\u003e \u003cb\u003eEstimation of oxidant (MDA, nitrite level) and Antioxidant parameters (GSH, Catalase, SOD, Gpx activity)\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe impact of cyclophosphamide (CP), sodium selenite (NaSe), selenium nanoparticles (SeNPs), and CP combined with SeNPs (CP + SeNP) on oxidative stress was evaluated through the measurement of lipid peroxidation (TBARS), nitrate levels, and antioxidant parameters including catalase, superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCyclophosphamide treatment (Group 2) significantly elevated TBARS and nitrate levels, indicating increased oxidative stress, while reducing the activities of catalase, SOD, GPx, and GSH levels compared to the control group (Group 1). Specifically, TBARS levels were significantly increased in the CP group (*p \u0026lt; 0.0308, **p \u0026lt; 0.0065 vs. Control), while nitrate levels were markedly higher (***p \u0026lt; 0.0003 vs. Control), reflecting an enhanced production of reactive oxygen and nitrogen species. Antioxidant parameters, including catalase (***p \u0026lt; 0.001 vs. Control), SOD (***p \u0026lt; 0.0005 vs. Control), GSH (*p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. Control), and GPx (**p \u0026lt; 0.001 vs. Control), were significantly diminished, underscoring a compromised antioxidant defense system.\u003c/p\u003e\u003cp\u003eTreatment with selenium nanoparticles (Group 5) significantly ameliorated oxidative stress by reducing TBARS levels (#p \u0026lt; 0.02, #p \u0026lt; 0.01 vs. CP) and nitrate levels (###p \u0026lt; 0.0008, ###p \u0026lt; 0.006 vs. CP). Antioxidant defense markers showed significant recovery in the SeNP-treated group compared to the CP group. Catalase activity was significantly restored (###p \u0026lt; 0.0006, ###p \u0026lt; 0.0003 vs. CP), as were SOD (##p \u0026lt; 0.002, ##p \u0026lt; 0.007 vs. CP), GSH (##p \u0026lt; 0.007, ##p \u0026lt; 0.006 vs. CP), and GPx activity (#p \u0026lt; 0.01, ##p \u0026lt; 0.0090 vs. CP). These improvements highlight the capacity of SeNPs to counteract CP-induced oxidative damage and enhance endogenous antioxidant defenses.\u003c/p\u003e\u003cp\u003eOverall, the results demonstrate that SeNP treatment significantly alleviates CP-induced oxidative stress, restoring a balance between pro-oxidant and antioxidant systems, with statistically significant improvements across all parameters.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMeasurement of serum hormonal levels\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe serum hormonal levels of follicle-stimulating hormone (FSH), testosterone, luteinizing hormone (LH), estradiol, triiodothyronine (T3), and thyroxine (T4) were evaluated in different treatment groups. The results are expressed as mean ± SEM (n = 5) and analysed using one-way ANOVA followed by Tukey's multiple comparison tests, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e (a)–(f).\u003c/p\u003e\u003cp\u003eFSH levels in the CP group (Group 2) showed a slight increase compared to the control (Group 1); however, this difference was not statistically significant across the groups. Testosterone levels were significantly decreased in the CP-treated group compared to the control (**p \u0026lt; 0.005), indicating CP-induced testicular toxicity. Treatment with SeNPs (Group 4) and the CP + SeNP combination (Group 5) significantly restored testosterone levels compared to the CP group (#p \u0026lt; 0.012, ##p \u0026lt; 0.0385). Sodium selenite (Group 3) also showed an improvement, although the effect was less pronounced than SeNPs.\u003c/p\u003e\u003cp\u003eLH levels followed a similar trend, with significant reduction in the CP group compared to the control (**p \u0026lt; 0.0038, *p \u0026lt; 0.0244). Both SeNP (Group 4) and CP + SeNP (Group 5) treatments significantly increased LH levels compared to the CP group (#p \u0026lt; 0.0457), highlighting the protective potential of SeNPs.\u003c/p\u003e\u003cp\u003eEstradiol levels were significantly increased in the CP group compared to the control (**p \u0026lt; 0.0022), reflecting hormonal imbalance caused by CP. This elevation was effectively normalized in Group 5, while sodium selenite group doesn’t have any significant changes. The estradiol level was slightly higher in SeNP group compared to cyclophosphamide (#p \u0026lt; 0.0104 vs. CP) indication beneficial effect of low dose of selenium on estradiol hormone.\u003c/p\u003e\u003cp\u003eTriiodothyronine (T3) levels were severely reduced in the CP group compared to the control (***p \u0026lt; 0.0001, **p \u0026lt; 0.0022). Treatment with SeNPs and CP + SeNPs significantly restored T3 levels compared to the CP group (###p \u0026lt; 0.0009, ###p \u0026lt; 0.0006), with SeNPs showing the most pronounced effect. Similarly, thyroxine (T4) levels were significantly decreased in the CP group compared to the control (****p \u0026lt; 0.0001), and treatment with SeNPs significantly improved T4 levels (#p \u0026lt; 0.0415, ###p \u0026lt; 0.0004 vs. CP).\u003c/p\u003e\u003cp\u003eOverall, SeNPs effectively mitigated CP-induced alterations in serum hormonal levels, highlighting their potential to restore hormonal homeostasis disrupted by CP toxicity.\u003c/p\u003e\u003cp\u003e \u003cb\u003eMeasurement of inflammatory and Apoptosis markers\u003c/b\u003e \u003c/p\u003e\u003cp\u003eThe levels of inflammatory markers, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), as well as the apoptosis marker caspase-3, were assessed across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c).\u003c/p\u003e\u003cp\u003eTNF-α levels were significantly elevated in the CP-treated group (Group 2) compared to the control (**p \u0026lt; 0.0024), indicating CP-induced inflammation. In contrast, treatment with SeNPs (Group 5) significantly reduced TNF-α levels compared to the CP group (##p \u0026lt; 0.051, ##p \u0026lt; 0.0099), demonstrating a potent anti-inflammatory effect. Similarly, IL-6 levels were significantly increased in the CP group compared to the control (**p \u0026lt; 0.006), reflecting a pro-inflammatory response. Treatment with SeNPs (Group 5) effectively reduced IL-6 levels compared to the CP group (#p \u0026lt; 0.02), highlighting the anti-inflammatory potential of SeNPs.\u003c/p\u003e\u003cp\u003eCaspase-3 activity, a marker of apoptosis, was markedly elevated in the CP group compared to the control (***p \u0026lt; 0.0001). Treatment with SeNPs (Group 5) significantly reduced caspase-3 activity compared to the CP group (###p \u0026lt; 0.0002), demonstrating its role in mitigating CP-induced apoptotic damage. Notably, Group 3 (Sodium Selenite) and Group 4 (SeNPs only) showed lower caspase-3 levels compared to the CP group (#p \u0026lt; 0.02, ##p \u0026lt; 0.01). These findings collectively suggest that SeNPs effectively attenuate CP-induced inflammation and apoptosis, thereby reducing oxidative damage and tissue injury.\u003c/p\u003e\u003cp\u003e \u003cb\u003eHistopathological investigation of rat testis and epididymis\u003c/b\u003e \u003c/p\u003e\u003cp\u003eHematoxylin and Eosin (H\u0026amp;E) staining was performed to evaluate histological alterations in the epididymis and testis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (a) and (b). Sections were examined under a light microscope to identify structural and cellular changes.\u003c/p\u003e\u003cp\u003eIn the seminiferous tubules of the testis, the cyclophosphamide (CP)-treated group (Group 2) displayed significant histopathological abnormalities, including severe degeneration of spermatocytes and Sertoli cells, perivascular cell degradation, disturbed Leydig cells, and a marked loss of mature spermatids, compared to the control group (Group 1). In contrast, the selenium nanoparticles (SeNPs) group (Group 4) and the CP + SeNPs-treated group (Group 5) exhibited normal seminiferous structure, indicating retained testicular physiology and reduced CP-induced damage.\u003c/p\u003e\u003cp\u003eThe histology of the cauda epididymis in the control group showed well-organized luminal epithelial cell layers with abundant sperm. In contrast, the CP-treated group exhibited a significant reduction in sperm count and disrupted epithelial cell integrity. Treatment with SeNPs and the combination of CP + SeNPs effectively preserved the normal architecture of principal cells and luminal structure, demonstrating the protective effects of SeNPs against CP-induced reproductive toxicity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCyclophosphamide (CP), a widely used alkylating agent in chemotherapy, is known to cause severe reproductive toxicity in males, posing challenges to long-term reproductive health. The mechanisms of CP-induced toxicity include germ cell damage, oxidative stress, hormonal disruptions, and depletion of spermatogonial stem cells, collectively impairing spermatogenesis and testosterone production. These findings highlight the urgent need for protective strategies against CP-induced reproductive dysfunction. In this study, green selenium nanoparticles (SeNPs) were synthesized and extensively characterized to evaluate their protective role against CP-induced reproductive toxicity.\u003c/p\u003e\u003cp\u003eThe synthesized SeNPs demonstrated desirable physicochemical properties, including a particle size of 100.2 nm and a zeta potential of -4.75 mV, as confirmed by dynamic light scattering (DLS). Spectroscopic and microscopic techniques further validated the nanoparticles' composition and morphology. FTIR analysis revealed characteristic peaks of SeNPs, while XRD data confirmed their crystalline nature. TEM and SEM images showed spherical, monodisperse particles, and EDX analysis identified selenium as a major component. These findings confirm the suitability of SeNPs for biological applications, enabling their evaluation in vivo.\u003c/p\u003e\u003cp\u003eIn CP-treated rats, significant reductions in body weight gain and testis weight were observed, indicating systemic and organ-specific toxicity. SeNP treatment mitigated these effects, suggesting a protective role in preserving testicular integrity. Sperm morphology assessment revealed severe abnormalities, including hooked and damaged sperm in the CP group, while SeNP treatment restored normal morphology. Similarly, mitochondrial potential, measured using the JC-1 assay, was significantly improved in SeNP-treated groups, further supporting the nanoparticles' protective effects.\u003c/p\u003e\u003cp\u003eCP exposure adversely affected blood parameters, with reductions in hematocrit and hemoglobin levels. Serum biochemical analysis revealed a decrease in total protein, albumin, and the albumin-to-globulin (A/G) ratio, indicating systemic inflammation and metabolic dysregulation. SeNP administration reversed these alterations, restoring blood parameters closer to control levels.\u003c/p\u003e\u003cp\u003eOxidative stress, a key contributor to CP toxicity, was evident from elevated malondialdehyde (MDA) and nitrate levels in CP-treated rats. This was accompanied by a reduction in antioxidant defenses, as indicated by decreased levels of superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPX). Treatment with SeNPs significantly reduced oxidative stress markers and restored antioxidant enzyme levels, demonstrating their potential as a potent antioxidant therapy.\u003c/p\u003e\u003cp\u003eHormonal disruptions were also prominent in CP-treated rats, with significant reductions in thyroid hormones (T3, T4) and thyroid-stimulating hormone (TSH), alongside minor, non-significant increases in follicle-stimulating hormone (FSH). These alterations can severely impair spermatogenesis and overall reproductive health. SeNP treatment partially reversed these hormonal imbalances, underscoring their role in preserving endocrine function.\u003c/p\u003e\u003cp\u003eInflammatory and apoptotic markers, including TNF-α, IL-6, and caspase-3, were markedly elevated in CP-treated groups, indicating inflammation and testicular cell death. Notably, SeNP treatment significantly reduced these markers, demonstrating their anti-inflammatory and anti-apoptotic potential.\u003c/p\u003e\u003cp\u003eHistopathological analysis corroborated the biochemical findings, revealing profound testicular damage in CP-treated rats, including distorted seminiferous tubules, germ cell exfoliation, and disrupted germinal epithelium. Conversely, SeNP-treated groups exhibited near-normal testicular morphology, with preserved seminiferous tubules and reduced interstitial space.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates the protective effects of green SeNPs against CP-induced reproductive toxicity through their antioxidant, anti-inflammatory, and anti-apoptotic properties. By mitigating oxidative stress, preserving hormonal balance, and reducing inflammatory responses, SeNPs offer a promising therapeutic strategy to safeguard male reproductive health during chemotherapy. Further studies are warranted to elucidate their precise molecular mechanisms and evaluate their clinical potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare, no competing interests related to this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBA and RM conducted the experiments. BA drafted the initial manuscript. RM prepared the final manuscript.SN conceptualized the study, reviewed, and corrected the final manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors acknowledges financial support provided by the Ministry of chemical and fertilizers, Department of Pharmaceuticals (DoP), Government of India and NIPER-Raebareli for other assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFraiser, L.H., S. 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Shalet, \u003cem\u003eGonadal damage from chemotherapy and radiotherapy.\u003c/em\u003e Endocrinol Metab Clin North Am, 1998. \u003cstrong\u003e27\u003c/strong\u003e(4): p. 927-43.DOI: 10.1016/s0889-8529(05)70048-7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cyclophosphamide (CP), cyclophosphamide toxicity, testicular toxicity, Selenium nanoparticles (SeNPs), serum hormonal level, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-6759007/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6759007/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCyclophosphamide (CP), a widely used alkylating chemotherapeutic and immunosuppressive agent, is associated with significant reproductive toxicity in male patients, primarily through oxidative stress and inflammatory damage to testicular tissue. This study examines the protective effects of green-synthesized selenium nanoparticles (SeNPs) from \u003cem\u003eAzadirachta indica\u003c/em\u003e leaf extract against CP-induced reproductive toxicity in male Wistar rats. SeNPs were characterized using FTIR, SEM, EDX, TEM, XRD, and dynamic light scattering (DLS); XRD confirmed crystalline SeNPs, with elemental analysis (EDX) revealing 0.57% selenium content. TEM and SEM imaging indicated average particle sizes of 72.32 ± 5.00 nm and 190.2 ± 2.0 nm, respectively. CP administration (15 mg/kg/week, i.p.) induced significant reductions in enzymatic antioxidants and serum hormone levels, alongside abnormal spermatogenesis and histopathology. SeNPs (0.2 mg/kg/day, oral) restored antioxidant enzyme activity, normalized testosterone and gonadotropin levels, improved sperm quality, and ameliorated testicular histoarchitecture. Moreover, SeNPs reduced pro-inflammatory markers, suggesting an anti-inflammatory mechanism. These findings highlight the potential of SeNPs as a pharmacological intervention to mitigate CP-induced reproductive toxicity, with implications for preserving fertility during chemotherapy.\u003c/p\u003e","manuscriptTitle":"Green Synthesized Selenium Nanoparticles Mitigate Cyclophosphamide-Induced Reproductive Toxicity in Male Wistar Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 10:21:06","doi":"10.21203/rs.3.rs-6759007/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-07T13:37:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-30T13:43:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199297805662156242027403580768858129590","date":"2025-06-30T09:55:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119340898674662644801343311679700937513","date":"2025-06-29T17:50:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-03T14:42:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-30T06:21:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-30T06:17:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2025-05-27T11:34:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"384c2e35-5d4e-4f2d-904c-9df0d369fb38","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T16:04:36+00:00","versionOfRecord":{"articleIdentity":"rs-6759007","link":"https://doi.org/10.1007/s00210-025-04929-8","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2025-12-26 15:57:49","publishedOnDateReadable":"December 26th, 2025"},"versionCreatedAt":"2025-06-09 10:21:06","video":"","vorDoi":"10.1007/s00210-025-04929-8","vorDoiUrl":"https://doi.org/10.1007/s00210-025-04929-8","workflowStages":[]},"version":"v1","identity":"rs-6759007","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6759007","identity":"rs-6759007","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}