Comparative Evaluation of Tamoxifen and Red Clover Extract on Reproductive and Hepatic Function in Female Trichogaster trichopterus: A Translational Endocrine Toxicology Study

Comparative Evaluation of Tamoxifen and Red Clover Extract on Reproductive and Hepatic Function in Female Trichogaster trichopterus: A Translational Endocrine Toxicology Study | 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 Comparative Evaluation of Tamoxifen and Red Clover Extract on Reproductive and Hepatic Function in Female Trichogaster trichopterus: A Translational Endocrine Toxicology Study Tahereh Naji, Mahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7382781/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2026 Read the published version in Journal of Molecular Histology → Version 1 posted 11 You are reading this latest preprint version Abstract Background & Objectives: Tamoxifen, a classic Selective Estrogen Receptor Modulator (SERM), regulates estrogen signaling in a tissue-dependent manner. Phytoestrogenic compounds such as red clover (Trifolium pratense), which activate estrogenic pathways via receptor agonism, are increasingly explored for their potential in hormone-related conditions. This study aimed to compare the endocrine and hepatic effects of tamoxifen and red clover extract in a validated vertebrate model, the female three-spot gourami ( Trichogaster trichopterus ). Methods A total of 120 adult female gourami were randomly assigned to eight groups, receiving intramuscular injections of tamoxifen (10, 50, 100 mg/kg), red clover extract (25, 75, 150 mg/kg), vehicle, or no treatment over 18 days. Reproductive (GSI, hormone levels, ovarian histology) and hepatic (HSI, ALT/AST levels, liver histology, TEM) parameters were assessed. Results Tamoxifen induced dose-dependent reductions in gonadosomatic index and levels of estradiol, progesterone, and testosterone, accompanied by elevated ALT and AST and histopathological liver changes. In contrast, red clover extracts increased GSI and sex hormones without hepatic damage. Histological and ultrastructural analyses confirmed arrested ovarian development and hepatic degeneration in tamoxifen-treated fish, while red clover–treated fish showed follicular maturation and preserved liver architecture. Conclusions The opposing profiles of tamoxifen and red clover underscore their distinct estrogen-modulatory mechanisms. Tamoxifen’s anti-estrogenic and hepatotoxic actions raise concerns about long-term use, while red clover demonstrated favorable reproductive stimulation and hepatic safety. These findings support red clover’s potential as a safer phytoestrogenic alternative for hormone regulation and provide a foundation for future translational research in women’s health. Tamoxifen Red clover extract Trichogaster trichopterus Reproductive toxicity Endocrine disruption Estrogen receptor modulators Figures Figure 1 Figure 2 Figure 3 1. Introduction Tamoxifen, a Selective Estrogen Receptor Modulator (SERM), has long been a cornerstone of hormone-responsive breast cancer therapy, significantly reducing recurrence and mortality ( 1 , 2 ). However, its tissue-specific actions can produce adverse effects beyond the breast. For example, extended tamoxifen use raises the risk of endometrial cancer ( 3 ) and is associated with non-alcoholic fatty liver disease ( 3 , 4 ). These drawbacks have prompted interest in safer alternatives for estrogen-related conditions. Phytoestrogenic plants such as red clover ( Trifolium pratense ) are increasingly used to manage menopausal symptoms ( 5 ). Red clover contains isoflavones such as formononetin, biochanin A, daidzein, and genistein, which structurally resemble estradiol and bind estrogen receptors (with a higher affinity for ER-β) ( 5 , 6 ). Clinical evidence supports its estrogen-mimicking benefits: a recent study showed red clover isoflavone supplementation significantly reduces hot flash frequency in peri and postmenopausal women ( 5 ). Notably, red clover has not exhibited estrogen-dependent cancer promotion in humans; in fact, a randomized trial in breast cancer survivors on tamoxifen found red clover extract to be well-tolerated and safe ( 7 ). These findings raise the question of how a natural phytoestrogen compares to a synthetic SERM in modulating the endocrine system and peripheral organs. What remains unclear is the comparative impact of tamoxifen versus red clover on reproductive and hepatic tissues. Ethical and practical constraints limit direct studies in women, so translational animal models are essential to investigate mechanisms and safety before clinical application ( 8 ). The three-spot gourami ( Trichogaster trichopterus ) is a validated vertebrate model in reproductive endocrinology, as fish share a highly conserved Hypothalamic–Pituitary–Gonadal (HPG) axis with mammals ( 8 – 10 ). Indeed, female gourami produce the same principal gonadotropins and sex steroids (GnRH, FSH, LH, estradiol, etc.) as humans ( 11 ), making this species suitable for studying estrogenic and anti-estrogenic effects. Tamoxifen exposure in fish can disrupt the HPG axis, impairing gonadal function and inducing hepatic lipid accumulation ( 12 ). Such models enable side-by-side evaluation of an established anti-estrogen (tamoxifen) and a phytoestrogenic compound (red clover) under controlled conditions, helping to bridge the gap between preclinical findings and human outcomes. In this study, a translational approach is adopted to compare ovarian and hepatic alterations induced by tamoxifen with those elicited by red clover extract in female Three-spot Gourami. Histopathological changes in the ovary and liver are evaluated alongside variations in sex hormone concentrations and liver enzyme levels to delineate the mechanisms, efficacy, and safety profiles of each compound. By leveraging the conserved features of this vertebrate model, the differential modulation of reproductive endpoints and hepatic function by a synthetic SERM and a phytoestrogenic alternative is examined. These findings are expected to inform future research directions in endocrine therapy for women, supporting the development of safer and more effective estrogen-modulating interventions. 2. Materials and Methods 2.1. Experimental Design and Animal Grouping This in vivo experimental study was conducted from January to May 2022 at the Basic Science Laboratory, Faculty of Pharmacy, Islamic Azad University, Tehran, Iran. All experimental procedures and reporting were conducted following the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines to ensure transparency and reproducibility. Furthermore, the study design, including dose selection, duration, and biomarker assessment, was based on relevant OECD Test Guidelines for endocrine disruption (TG 407, TG 440) ( 13 , 14 ). Sexually mature female Three-spot Gourami (Trichogaster trichopterus) were selected as the animal model due to their conserved HPG axis, which exhibits significant physiological homology with mammals. A total of 120 fish with uniform size and body weight (mean initial weight: 2.9 ± 0.2 g) were obtained from the Ornamental Fish Breeding Center in Karaj, Iran, and acclimated under controlled laboratory conditions for two weeks before the experiment. The fish were randomly divided into eight experimental groups (n = 15 per group). Two groups served as controls: one as an intact control (without any injection) and the other as a vehicle control, receiving 60% ethanol. Three groups received intramuscular (IM) injections of tamoxifen at doses of 10, 50, and 100 mg/kg, respectively. These doses were selected based on previous studies demonstrating tamoxifen’s endocrine-disrupting and anti-estrogenic effects in aquatic species and mammalian models ( 15 , 16 ). The remaining three groups were treated with IM injections of red clover ( Trifolium pratense ) extract at doses of 25, 75, and 150 mg/kg, chosen following prior reports evaluating the estrogenic activity and safety profile of isoflavone-rich plant extracts in aquatic organisms ( 17 ). All injections were administered in a final volume of 0.02 mL using insulin syringes (0.5–1 mL) between the dorsal fin and lateral trunk muscle, inserted at a 30° angle. A total of nine injections were given every other day over 18 days. Following the final injection, fish were held without intervention for an additional 48 hours, and dissections were performed on day 21 for tissue sampling. During the entire experimental period, fish were maintained in glass aquaria under stable conditions, including a water temperature of 25 ± 1°C, a pH of 6.5, and a water hardness of approximately 78 mg/L CaCO₃. Fish were fed a commercial diet every other day, with water filtration and aeration maintained continuously. Water was dechlorinated and equilibrated to room temperature before use. 2.2. Preparation of Tamoxifen and Red Clover Extract Tamoxifen was obtained as a pure powder (99% purity, Lot #SLBP0003V, Germany) and handled under low-light conditions due to its photosensitive and cytotoxic properties. Based on fish weight calculations, three treatment doses were prepared: 10, 50, and 100 mg/kg. For each dose group, the weighed powder was dissolved in 60% ethanol to a final injection concentration, and the solution was brought to 5 mL in a volumetric flask. The prepared tamoxifen solutions were stored in amber glass vials at 4°C to protect them from light and prevent degradation before injection. Red clover ( Trifolium pratense ) extract was prepared using a commercial 1-liter ethanol extract purchased from Asha Biotech. The extract was air-dried under hygienic conditions using mesh covers to prevent contamination. After one week, the dried residue adhering to the beakers was scraped and powdered using a mortar and pestle, yielding approximately 4.545 g of dry extract from 1 L of original ethanol extract. Doses of 25, 75, and 150 mg/kg were selected based on prior studies. For each treatment group, the required amount of powdered extract was reconstituted in distilled water up to 5 mL based on the average fish weight per tank. The solutions were filtered through 0.45 µm sterile syringe filters to remove residual plant particles and stored in sterile glass vials until administration. Both tamoxifen and red clover solutions were prepared freshly before the injection period and handled with caution to minimize exposure to light and oxidative degradation. All solutions were prepared under a fume hood using aseptic techniques to ensure sterility and reproducibility. 2.3. Injection Protocol Before each injection session, fish were mildly anesthetized using an aqueous solution of clove extract prepared from 1 g of dried Syzygium aromaticum per liter of distilled water. This stock solution was filtered and diluted in a 1:2 ratio (200 mL extract + 400 mL distilled water) immediately before use. Individual fish were immersed for 30–40 seconds until partial loss of equilibrium was observed, indicating sufficient sedation for handling ( 18 , 19 ). All injections were performed intramuscularly (IM) between the dorsal fin and lateral line of the fish, using BD insulin syringes (0.5 or 1 mL). The injection volume for all groups was standardized at 0.02 mL per fish, regardless of treatment dose. The needle was inserted at an approximate 30° angle into the lateral trunk muscle to ensure proper IM delivery. Before injection, the injection site was cleaned using 70% ethanol-soaked sterile cotton and the fish were gently immobilized by hand, holding the head and tail with minimal pressure. The dosing schedule consisted of nine total injections administered every other day over 18 days. Following the final injection, fish were kept under normal aquarium conditions for an additional 48 hours to allow stabilization before sacrifice and tissue collection on day 21. After each injection, fish were transferred to dechlorinated, temperature-equilibrated water, and oxygenation was maintained via aeration pumps. No adverse behavioral signs or injection site complications were observed throughout the experimental period. All handling procedures were performed under controlled environmental conditions and following local animal care guidelines. The repeated IM injection protocol was designed to mimic subchronic exposure while minimizing stress and maintaining consistent dosing accuracy. 2.4. Sampling and Tissue Processing On day 21, following the ninth and final injection and a 48-hour post-treatment stabilization period, fish were anesthetized using a concentrated clove extract until no movement was observed. Biometric measurements, including total length and body weight, were recorded for each fish using a digital scale with 0.001 g precision. Dissections were performed via a longitudinal incision along the midline from the mouth to the ventral side of the gill cover, followed by two transverse cuts to expose the internal organs. The ovaries and liver were carefully excised, rinsed in cold saline to remove excess tissue fluid, and weighed separately for subsequent index calculations. Tissues designated for light microscopy were immediately fixed in 10% neutral buffered formalin (prepared using 10 mL formalin and 90 mL distilled water). Samples were processed using an Autotechnicon KP-110 tissue processor through standard dehydration (graded ethanol), clearing (xylene), and infiltration steps. Paraffin blocks were prepared using a rotary embedding system and sectioned at 5–10 µm using a rotary microtome. Sections were mounted on glass slides and dried in a 100°C oven for 15 minutes before staining. Hematoxylin and Eosin (H&E) staining was performed using standard protocols, including sequential deparaffinization, rehydration, nuclear staining (hematoxylin), cytoplasmic staining (eosin), and dehydration steps. For Transmission Electron Microscopy (TEM), representative ovarian and hepatic tissue fragments (≤ 0.5 mm) were fixed in 2.5% glutaraldehyde at 4°C for one hour. Post-fixation was carried out in 1.5% osmium tetroxide for 90 minutes. Samples were washed in 0.1 M sodium cacodylate buffer (3 × 5 min) and rinsed in distilled water. Tissue contrast was enhanced using 2% uranyl acetate for 20 minutes, followed by secondary fixation in 1% osmium tetroxide for 20 minutes. Dehydration was achieved with ascending ethanol concentrations (50%, 70%, 95%, and 100%), and infiltration was performed using propylene oxide and Epon resin mixtures. Final embedding was done in pure resin, cured at 60°C for 48–72 hours. Ultrathin sections (~ 60–70 nm) were prepared using an ultramicrotome and post-stained with lead citrate before imaging on a Philips EM 208S transmission electron microscope. Histological and ultrastructural analyses focused on key cellular and subcellular markers of ovarian and hepatic integrity. Ovarian tissue was evaluated for follicular stage prevalence (e.g., pre-nucleolar vs. vitellogenic), yolk droplet formation, lipid accumulation, and nuclear migration. Hepatic sections were assessed for sinusoidal structure, hepatocyte size, and evidence of inflammation. Under TEM, attention was given to zona radiata integrity, microvilli presence, and yolk vesicle fusion. 2.5. Hormonal and Biochemical Assays Due to the small size of the experimental fish, blood sampling was not feasible; thus, hormone and enzyme analyses were performed on tissue homogenates. Ovarian and hepatic tissues were homogenized in cold conditions and centrifuged at 3000 rpm for 5 minutes using a refrigerated centrifuge to obtain clear supernatants. Levels of 17β-estradiol, 17-hydroxyprogesterone, and testosterone were quantified using a commercial ELISA kit (manufacturer not specified), following the manufacturer's protocol. Briefly, 25 µL of tissue extract was mixed with 50 µL of conjugate solution, shaken for 30 seconds, incubated for 60 minutes at room temperature, and washed three times with 350 µL wash buffer. Then, 100 µL of substrate was added, and the plate was incubated in the dark for 20 minutes. Absorbance was measured at 450 nm using an ELISA microplate reader. Hepatic enzyme activities of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) were also measured from liver homogenates using spectrophotometric methods. Results were expressed as IU/mL. These enzymes serve as sensitive markers of hepatocellular injury, with AST reflecting mitochondrial damage and ALT being more liver-specific. 2.6. Reproductive and Hepatic Indices The Gonadosomatic Index (GSI) was calculated using the formula to measure reproductive capability: GSI (gonad weight/body weight) ×100. The Hepatosomatic Index (HIS) was also calculated by the formula: HSI (liver body weight/ total weight) ×100, which indicates fish energy reserves. 2.7. Statistical Analysis All data were analyzed using SPSS software version 26. One-way analysis of variance (ANOVA) was used to evaluate differences among experimental groups, followed by Duncan’s multiple range post hoc test to identify specific group differences. A significance level of p < 0.05 was set for all comparisons. Results were presented as mean ± standard error of the mean (SEM). 3. Results 3.1. Gonadosomatic Index (GSI) and Hepatosomatic Index (HSI) Biometric outcomes showed that tamoxifen exerted a dose-dependent inhibitory effect on ovarian development, reflected in a significant reduction of GSI at 50 and 100 mg/kg compared to the control group (P = 0.004 and P < 0.001, respectively), which is summarized in Table 1 . The highest tamoxifen dose group (100 mg/kg) exhibited the lowest mean GSI (3.90 ± 0.31%), statistically distinct from both lower-dose and control groups, as indicated by non-overlapping significance groupings. No significant GSI change was observed in the 10 mg/kg group (P = 0.32). In contrast, red clover extract induced a significant increase in GSI at higher doses. Fish treated with 75 and 150 mg/kg extract demonstrated elevated gonadal indices compared to controls (P = 0.012 and P = 0.001, respectively), with values reaching 13.51 ± 0.38% in the highest dose group, consistent with dose-dependent phytoestrogenic stimulation. No significant alterations were observed in HSI across treatment groups (P > 0.05), although a slight upward trend was noted in tamoxifen-exposed fish. Table 1 Gonadosomatic index (GSI) and hepatosomatic index (HSI) in Trichogaster trichopterus following administration of tamoxifen and red clover extract. Treatment Name GSI (%) (mean ± SEM) HSI (%) (mean ± SEM) GSI Grouping HSI Grouping P-value GSI (vs. Control) P-value HSI (vs. Control) Control (no injection) 10.88 ± 0.25 1.42 ± 0.06 a a — — Vehicle (60% ethanol) 10.75 ± 0.22 1.45 ± 0.04 a a 0.78 0.69 Tamoxifen 10 mg/kg 9.96 ± 0.34 1.51 ± 0.05 a a 0.32 0.33 Tamoxifen 50 mg/kg 6.17 ± 0.28 1.55 ± 0.07 b a 0.004 ★ 0.25 Tamoxifen 100 mg/kg 3.90 ± 0.31 1.61 ± 0.08 c a < 0.001 ★ 0.14 Red clover 25 mg/kg 11.04 ± 0.30 1.44 ± 0.03 a a 0.44 0.56 Red clover 75 mg/kg 12.22 ± 0.36 1.47 ± 0.05 b a 0.012 ★ 0.41 Red clover 150 mg/kg 13.51 ± 0.38 1.43 ± 0.04 b a 0.001 ★ 0.49 Note: Values are expressed as mean ± SEM (n = 5). Different superscript letters indicate statistically significant differences (P ≤ 0.05, ANOVA with Tukey’s post hoc test). Statistically significant differences compared to the control group are marked with ★. 3.2. Endocrine and Biochemical Markers Tamoxifen treatment resulted in a significant, dose-dependent suppression of circulating sex steroid hormones. At 50 and 100 mg/kg doses, serum levels of 17β-estradiol, progesterone, and testosterone were significantly reduced compared to the control group (P < 0.05), with the lowest values observed in the 100 mg/kg group (e.g., estradiol: 49.34 ± 1.86 pg/mL; P < 0.001). Conversely, administration of red clover extracts enhanced hormone levels at higher doses. In particular, 75 and 150 mg/kg treatments significantly elevated estradiol and progesterone concentrations (P < 0.05). For hepatic enzymes, tamoxifen induced a marked elevation in ALT and AST activity, particularly at 100 mg/kg (ALT: 58.1 ± 2.2 U/L; AST: 96.0 ± 3.1 U/L; both P < 0.001), implying hepatocellular stress or toxicity. Red clover groups, however, maintained enzyme levels comparable to controls, with no statistically significant deviations. A comprehensive summary of group-wise means, SEMs, statistical groupings, and P-values is provided in Table 2 . Table 2 Effects of tamoxifen and red clover extract on serum sex steroid hormones and liver enzyme activity in female Trichogaster trichopterus. Treatment name 17β-estradiol (pg/mL) Group P-value Progesterone (ng/mL) Group P-value Testosterone (ng/mL) Group P-value ALT (U/L) Group P-value AST (U/L) Group P-value Control 76.12 ± 1.05 a — 3.51 ± 0.21 a — 2.67 ± 0.14 a — 35.3 ± 2.1 a — 67.4 ± 3.2 a — Vehicle (EtOH 60%) 74.33 ± 1.29 a 0.68 3.42 ± 0.18 a 0.74 2.59 ± 0.12 a 0.66 36.1 ± 1.9 a 0.59 68.9 ± 2.9 a 0.63 Tamoxifen 10 mg/kg 70.23 ± 1.72 a 0.21 3.08 ± 0.17 a 0.19 2.44 ± 0.13 a 0.22 38.5 ± 2.3 a 0.38 71.6 ± 3.5 a 0.40 Tamoxifen 50 mg/kg 58.42 ± 1.95 ★ b 0.009 2.01 ± 0.22 ★ b 0.006 1.83 ± 0.11 ★ b 0.011 49.7 ± 2.0 ★ b 0.005 84.3 ± 3.4 ★ b 0.007 Tamoxifen 100 mg/kg 49.34 ± 1.86 ★ c < 0.001 1.34 ± 0.25 ★ c < 0.001 1.01 ± 0.10 ★ c < 0.001 58.1 ± 2.2 ★ c < 0.001 96.0 ± 3.1 ★ c < 0.001 Red clover 25 mg/kg 79.10 ± 1.31 a 0.33 3.72 ± 0.15 a 0.48 2.71 ± 0.15 a 0.45 34.7 ± 1.8 a 0.61 66.8 ± 3.0 a 0.69 Red clover 75 mg/kg 83.24 ± 1.45 ★ b 0.014 4.00 ± 0.19 ★ b 0.013 2.88 ± 0.13 ★ b 0.012 33.2 ± 2.1 a 0.73 64.1 ± 2.7 a 0.76 Red clover 150 mg/kg 87.33 ± 1.65 ★ b 0.002 4.15 ± 0.22 ★ b 0.007 3.05 ± 0.11 ★ b 0.004 32.5 ± 1.7 a 0.79 63.5 ± 2.4 a 0.82 Note: Values are expressed as mean ± SEM (n = 5). Different superscript letters within each column indicate statistically significant differences (P ≤ 0.05, one-way ANOVA with Tukey’s post hoc test). P-values denote comparisons against the control group. Statistically significant differences compared to the control group are marked with ★ 3.3. Ovarian Histology Light microscopic evaluation of ovarian sections revealed distinct histopathological alterations following tamoxifen or red clover treatment. In tamoxifen-treated fish, especially at 50 and 100 mg/kg, ovarian tissue was predominantly composed of pre-nucleolar oocytes with reduced cytoplasmic development. Vitellogenic follicles were sparse or absent, and ooplasmic lipid content was diminished. Structural disorganization, including nuclear displacement and atretic changes, was also observed at higher doses, consistent with anti-estrogenic inhibition of folliculogenesis. In contrast, ovaries from fish exposed to red clover extract (particularly 75 and 150 mg/kg) showed enhanced follicular maturation, increased prevalence of cortical alveolar and early vitellogenic oocytes. Cytoplasmic yolk deposition, lipid vesicle formation, and orderly nuclear positioning were more pronounced than in the control group, indicating phytoestrogen-induced stimulation of the ovarian cycle. No remarkable histological abnormalities were observed in the vehicle group or at the lowest tested doses of either compound. Full microscopic comparisons and representative images are provided in Fig. 1 . 3.4. Hepatic Histology Histopathological examination of liver sections revealed dose-dependent hepatic alterations in tamoxifen-treated fish. At 50 and 100 mg/kg, hepatocytes exhibited prominent hypertrophy, sinusoidal dilation, cytoplasmic vacuolization, and focal necrosis. Additionally, Kupffer cell proliferation and disorganized hepatic cords were evident, consistent with inflammatory stress and hepatocellular damage. These lesions were absent or minimal at the 10 mg/kg dose. In contrast, red clover extract did not induce histological abnormalities. Liver architecture in fish treated with 75 and 150 mg/kg red clover remained comparable to control groups, showing well-aligned hepatocyte rows, intact sinusoidal spaces, and no apparent fatty degeneration or necrosis. These findings support the hepatoprotective profile of red clover under the tested conditions. Representative micrographs illustrating these changes are presented in Fig. 2 . 3.5. Ultrastructural Observations (TEM) Transmission electron microscopy provided further insight into the subcellular effects of tamoxifen and red clover extract on ovarian tissues. In ovarian samples from high-dose tamoxifen groups, oocytes exhibited cytoplasmic degeneration, loss of microvilli, and disrupted zona radiata. Intercellular contacts between follicular cells were diminished, and yolk vesicles were largely absent, indicating impaired vitellogenesis and estrogen signaling. Mitochondrial swelling and lysosomal accumulation were also evident in some cells, suggestive of early apoptotic processes. Conversely, red clover–treated fish (75 and 150 mg/kg) showed well-preserved ovarian ultrastructure. Oocytes featured active microvilli, intact zona radiata, abundant yolk vesicles, and organized cortical granules. Numerous vesicles engaged in pinocytosis were observed, supporting phytoestrogen-induced steroidogenic activity. Representative micrographs are shown in Fig. 3 . 4. Discussion This study demonstrated that tamoxifen and red clover extract have opposing impacts on the reproductive endocrine endpoints of female Trichogaster trichopterus . Tamoxifen exposure markedly suppressed ovarian development and function, evidenced by a lower GSI and reduced circulating sex hormone levels compared to controls. Such anti-estrogenic effects align with reports in other fish: for example, high dietary tamoxifen doses in hybrid tilapia significantly lowered female GSI and estradiol levels while elevating male androgens ( 20 ). Histologically, tamoxifen-treated gourami ovaries showed signs of atresia and developmental arrest, consistent with tamoxifen’s known inhibition of vitellogenesis. Prior studies confirm that tamoxifen can block estrogen-driven vitellogenin (yolk protein) production. Sun et al. found that tamoxifen prevented normal vitellogenin induction in female fish at high concentrations ( 21 ). The present results are therefore in line with tamoxifen’s role as a selective estrogen receptor modulator that antagonizes estrogen signaling in the ovary and liver tissue ( 22 ). By contrast, females exposed to red clover extract exhibited an estrogenic stimulation of the reproductive axis. GSI and plasma estradiol were higher than in controls, suggesting enhanced gonadal growth and endocrine activity, and possibly an advance in oocyte maturation stages. This is in agreement with international findings that phytoestrogens from red clover have pro-estrogenic effects on fish reproduction. Turan et al. (2022) showed that red clover extract (rich in isoflavones) effectively feminized 89% of juvenile male African catfish, with treated fish developing normal ovaries and no intersex pathology ( 17 ). Similarly, soy isoflavones like genistein and daidzein are reported to act as estrogen receptor agonists in fish, modulating reproductive gene expression and promoting female-biased traits without overt toxicity ( 23 ). Our findings that red clover increased female gourami gonad size and hormone levels are consistent with these studies, reinforcing that plant-derived estrogens can mimic endogenous estrogens in teleosts. Beyond the gonads, tamoxifen and red clover produced distinct hepatic outcomes with significant toxicological implications. Tamoxifen-treated gourami showed altered liver enzyme profiles indicative of hepatic stress: for instance, elevated ALT and AST levels were observed relative to controls (as typically seen in liver injury). This mirrors patterns seen in other species; In fish exposed to high tamoxifen, studies reported significant increases in AST and ALT alongside reductions in albumin and alkaline phosphatase ( 24 ). Tamoxifen’s hepatotoxicity is well documented in mammals as well, where long-term therapy can induce steatosis and inflammatory liver damage ( 25 ). Histologically, our tamoxifen group livers exhibited vacuolar degeneration and mild inflammation, suggesting incipient fatty liver changes. These lesions correspond to tamoxifen’s interference with hepatic estrogen receptors and lipid metabolism. Estrogen normally helps regulate lipid homeostasis in the liver; by antagonizing hepatic ER, tamoxifen may trigger fat accumulation and oxidative stress, as observed in rodent studies ( 26 ). There is also evidence that tamoxifen upregulates genes like CYP1A1 involved in xenobiotic metabolism in fish liver ( 22 ), potentially generating reactive metabolites that exacerbate liver damage. Conversely, red clover extract had a more benign or even beneficial effect on the liver. Treated gourami did not show the enzyme elevations seen with tamoxifen; in fact, liver enzymes in the red clover group remained comparable to or slightly improved over. Histological examination revealed that red clover fish livers retained normal architecture, with minimal fat vacuolation or cell death. This outcome aligns with studies in which dietary red clover isoflavones were hepatoprotective or neutral. For example, studies found that red clover supplementation in mice did not significantly raise AST or ALT, but did lower serum LDL and triglycerides, indicating improved metabolic health ( 27 ). Isoflavones such as formononetin and biochanin A in red clover can scavenge reactive oxygen species and modulate liver gene expression to favor antioxidant defenses ( 28 ). In our fish, this likely translated to protection against hepatic oxidative damage. The lack of liver histopathology in red clover–exposed gourami is particularly notable given that their gonads were highly active; it suggests the extract did not induce liver hypertrophy or vitellogenin overproduction that potent synthetic estrogens often cause. Overall, the divergent hepatic profiles, tamoxifen causing enzyme perturbation and histological lesions, vs. red clover causing negligible liver stress, underscore the contrasting safety margins of these substances as endocrine modulators. The contrasting actions of tamoxifen and red clover on the gourami’s endocrine system can be interpreted through their mechanisms at the Estrogen Receptors (ERs) and upstream hormonal axes. Tamoxifen is a non-steroidal SERM that in fish appears to act primarily as an estrogen antagonist in reproductive tissues. It likely binds to ERα/β in the HPG axis, blocking estradiol’s normal feedback loops. This would reduce gonadotropin release and ovarian steroidogenesis, explaining the lowered estradiol and GSI. Similar anti-estrogenic modes were observed in female olive flounder given tamoxifen, where estrogen receptor expression in the liver remained low and vitellogenin mRNA induction was blunted ( 29 ). Notably, tamoxifen can also exhibit partial estrogen agonism in certain contexts, but some vitellogenin presence could reflect tamoxifen’s mixed activity. In male fish, tamoxifen has paradoxically been shown to induce vitellogenin production by activating hepatic ERs ( 21 ), even as it inhibits female vitellogenesis at high doses ( 30 ). These contradictory effects are supported by Maradonna et al., who described tamoxifen’s “mixed estrogenic/anti-estrogenic action” disrupting fish physiology ( 22 ). Our results reinforce this duality: tamoxifen in females predominantly acted as an ER antagonist (suppressing ovarian growth), yet some estrogen-responsive genes in the liver may have been upregulated (partial agonism), contributing to complex outcomes like reduced fertility but not complete estrogen deprivation. This complexity has been noted in recent toxicology reviews of pharmaceuticals in fish, where tamoxifen’s impact varies with dose, sex, and species ( 31 ). Red clover’s estrogenic influence, on the other hand, can be attributed to its high content of isoflavone phytoestrogens that bind ERs. Red clover contains formononetin and biochanin A, which are metabolized to daidzein and genistein compounds with affinity for fish estrogen receptors ( 28 ). These phytoestrogens likely acted as agonists on ovarian ER, promoting vitellogenin synthesis and oocyte development akin to low-dose 17β-estradiol exposure. The stimulatory effect we observed (higher hormone levels and advanced ovarian follicles) parallels the action of estradiol itself in fish. Indeed, a recent zebrafish study demonstrated that genistein exposure increased expression of hatching enzyme and other estrogen-regulated genes in gonads ( 23 ), while another found red clover extract upregulated growth hormone and improved reproductive output in female goldfish ( 28 ). Interestingly, Sarasquete et al. (2020) reported that genistein and daidzein altered ERβ transcript levels in zebrafish gonads but did not cause histomorphological changes over 15 days ( 23 ). This suggests that phytoestrogens can fine-tune gene expression without the full potency of synthetic estrogens, resulting in milder, physiologically paced changes, consistent with our finding that red clover fish had enhanced reproductive indices but no overt pathological changes. One point of contrast in the studies is the sensitivity of different fish species and life stages to phytoestrogens. While our adult gourami and the catfish study showed clear estrogenic stimulation, a study on adult Siamese fighting fish (Betta) found limited impact of phytoestrogens on steroid levels or oocyte development ( 32 ). Those authors suggested that mature females of some species may be less acutely responsive to dietary phytoestrogens, possibly due to saturation of ERs by endogenous hormones. These results contribute to this discourse by showing that T. trichopterus is indeed responsive to plant estrogens, aligning more with species like catfish and zebrafish than with Betta. It underlines the importance of species-specific reproductive physiology in outcomes of endocrine disruptor exposure. Using T. trichopterus (three-spot gourami) as a model species in this study also carries translational relevance. Gourami are tropical teleosts with a well-characterized reproductive endocrinology, making them suitable analogs for other oviparous fish. They respond to hormonal cues in a manner comparable to established lab models like zebrafish, yet their physiology (e.g., a bubble-nest breeder with parental care) adds a layer of ecological validity. Demonstrating endocrine disruption in gourami confirms that such effects are not limited to a few model species, thereby broadening the understanding of how chemicals like tamoxifen impact diverse fish taxa. While direct extrapolation to humans must be cautious, our findings do resonate with known human outcomes: tamoxifen’s ability to induce fatty liver and alter estrogen-responsive tissues in fish parallels side effects seen in women undergoing tamoxifen therapy ( 25 ), and the estrogenic compounds in red clover used here are the same isoflavones marketed as menopausal supplements in women. This cross-species consistency in mechanism lends credibility to using fish models for initial screening of endocrine-active substances. In summary, Trichogaster trichopterus proved to be a sensitive and informative model for endocrine and histopathological endpoints. The suppression of reproductive capacity by tamoxifen and the enhancement by red clover in this species not only corroborate findings from studies in toxicology and endocrinology but also illuminate possible mechanisms (ER antagonism versus agonism) underlying these effects. 5. Conclusion This study demonstrates that tamoxifen and red clover extract exert contrasting effects on reproductive and hepatic tissues in the three-spot gourami ( Trichogaster trichopterus ). Tamoxifen exhibited significant anti-estrogenic activity, characterized by a marked suppression of gonadal development, reduction in sex steroid hormones, and evidence of hepatotoxicity. Conversely, red clover extract, rich in phytoestrogenic isoflavones, safely promoted gonadal growth, hormone production, and preserved hepatic integrity, confirming its potential as a safer estrogen-modulating alternative. Given the high conservation of the HPG axis among vertebrates, including humans, these findings bear substantial translational relevance. Clinically, these results reinforce concerns regarding long-term tamoxifen use and its associated risks, highlighting the importance of careful monitoring and potential hepatoprotective interventions in patients. Importantly, the benign safety profile of red clover demonstrated here aligns with existing clinical evidence supporting its use in menopausal symptom management, positioning it as a valuable candidate for safer hormone therapy in women. Further clinical investigations are warranted to fully validate these findings and optimize the therapeutic use of phytoestrogens as alternatives or adjuncts to conventional hormonal treatments. Abbreviations GSI Gonadosomatic Index HSI Hepatosomatic Index GOT Glutamic Oxaloacetic Transaminase GPT Glutamic Pyruvic Transaminase HPG axis Hypothalamus–Pituitary–Gonadal axis ER Estrogen Receptor SERM Selective Estrogen Receptor Modulator FSH Follicle-Stimulating Hormone LH Luteinizing Hormone E2 17β-Estradiol 17-OHP 17-Hydroxyprogesterone TEM Transmission Electron Microscopy GV Germinal Vesicle YV Yolk Vesicle OD Oil Droplet ZR Zona Radiata GJ Gap Junction TL Thecal Layer H Hepatocyte S Sinusoid PV Portal Vein KC Kupffer Cell FC Follicular Cell SEM Standard Error of the Mean Declarations Ethics approval and consent to participate The animals were obtained from Mahyran Company, a licensed vendor. Since they were not privately owned by any institution or individual, no informed consent was required. All procedures were approved by the Iran National Committee for Ethics in Biomedical Research (Approval code: IR.IAU.PS.REC.1400.306). Clinical trial number: not applicable. Consent for publication Not applicable. Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing Interests The authors declare that there is no conflict of interest regarding the publication of this manuscript. Funding No funding was received for conducting this study. Authors’ contributions Study conception and design: Mahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi Data collection and experimental work: Mahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi Data analysis and interpretation: Tahereh Naji, Mahdi Ahmadinia Drafting and revising the manuscript: Tahereh Naji, Mahdi Ahmadinia Final approval of the manuscript: Tahereh Naji, Mahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi Acknowledgements The authors acknowledge the use of QuillBot exclusively for improving the clarity and academic style of the manuscript through language polishing and paraphrasing. The tool was not involved in generating content, performing data analysis or interpretation, selecting literature, or creating any figures or tables. All scientific ideas, interpretations, and conclusions are entirely those of the authors, who take full responsibility for the work. This use is disclosed following Springer Nature’s guidelines on AI tool transparency and authorship. References Early Breast Cancer Trialists’ Collaborative Group (EBCTCG). Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet [Internet]. 2011 Aug;378(9793):771–84. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673611609938 Aromatase inhibitors versus tamoxifen in early breast cancer: patient-level meta-analysis of the randomised trials. Lancet [Internet]. 2015 Oct;386(10001):1341–52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673615610741 Emons G, Mustea A, Tempfer C. Tamoxifen and Endometrial Cancer: A Janus-Headed Drug. Cancers (Basel) [Internet]. 2020 Sep 7;12(9):2535. Available from: https://www.mdpi.com/2072-6694/12/9/2535 Bekes I, Huober J. Extended Adjuvant Endocrine Therapy in Early Breast Cancer Patients—Review and Perspectives. Cancers (Basel) [Internet]. 2023 Aug 21;15(16):4190. Available from: https://www.mdpi.com/2072-6694/15/16/4190 Kanadys W, Barańska A, Błaszczuk A, Polz-Dacewicz M, Drop B, Kanecki K, et al. Evaluation of Clinical Meaningfulness of Red Clover (Trifolium pratense L.) Extract to Relieve Hot Flushes and Menopausal Symptoms in Peri- and Post-Menopausal Women: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Nutrients [Internet]. 2021 Apr 11;13(4):1258. Available from: https://www.mdpi.com/2072-6643/13/4/1258 Booth NL, Piersen CE, Banuvar S, Geller SE, Shulman LP, Farnsworth NR. Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a literature review. Menopause [Internet]. 2006 Mar;13(2):251–64. Available from: http://journals.lww.com/00042192-200613020-00016 Ferraris C, Ballestra B, Listorti C, Cappelletti V, Reduzzi C, Scaperrotta GP, et al. Red clover and lifestyle changes to contrast menopausal symptoms in premenopausal patients with hormone-sensitive breast cancer receiving tamoxifen. Breast Cancer Res Treat [Internet]. 2020 Feb 24;180(1):157–65. Available from: http://link.springer.com/10.1007/s10549-020-05534-4 Maruska KP, Fernald RD. Social Regulation of Gene Expression in the Hypothalamic-Pituitary-Gonadal Axis. Physiology [Internet]. 2011 Dec;26(6):412–23. Available from: https://www.physiology.org/doi/10.1152/physiol.00032.2011 Ahmadinia M, Naji T, Sahafi HH. Methimazole-induced reproductive endocrine system disruption and hepatotoxicity: insights from a Trichogaster trichopterus animal model. BMC Pharmacol Toxicol [Internet]. 2025 Jul 1;26(1):128. Available from: https://bmcpharmacoltoxicol.biomedcentral.com/articles/10.1186/s40360-025-00942-y Cameron N, Del Corpo A, Diorio J, McAllister K, Sharma S, Meaney MJ. Maternal Programming of Sexual Behavior and Hypothalamic-Pituitary-Gonadal Function in the Female Rat. Vosshall LB, editor. PLoS One [Internet]. 2008 May 21;3(5):e2210. Available from: https://dx.plos.org/10.1371/journal.pone.0002210 Degani G. Brain Control Reproduction by the Endocrine System of Female Blue Gourami (Trichogaster trichopterus). Biology (Basel) [Internet]. 2020 May 21;9(5):109. Available from: https://www.mdpi.com/2079-7737/9/5/109 Yu Q, Huo J, Zhang Y, Liu K, Cai Y, Xiang T, et al. Tamoxifen-induced hepatotoxicity via lipid accumulation and inflammation in zebrafish. Chemosphere [Internet]. 2020 Jan;239:124705. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0045653519319356 Gelbke HP, Hofmann A, Owens JW, Freyberger A. The enhancement of the subacute repeat dose toxicity test OECD TG 407 for the detection of endocrine active chemicals: comparison with toxicity tests of longer duration. Arch Toxicol [Internet]. 2007 Mar 15;81(4):227–50. Available from: http://link.springer.com/10.1007/s00204-006-0148-3 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PLoS Biol [Internet]. 2010 Jun 29;8(6):e1000412. Available from: https://dx.plos.org/10.1371/journal.pbio.1000412 Lange A, Paull GC, Coe TS, Katsu Y, Urushitani H, Iguchi T, et al. Sexual Reprogramming and Estrogenic Sensitization in Wild Fish Exposed to Ethinylestradiol. Environ Sci Technol [Internet]. 2009 Feb 15;43(4):1219–25. Available from: https://pubs.acs.org/doi/10.1021/es802661p Rajapakse N, Silva E, Kortenkamp A. Combining xenoestrogens at levels below individual no-observed-effect concentrations dramatically enhances steroid hormone action. Environ Health Perspect [Internet]. 2002 Sep;110(9):917–21. Available from: https://ehp.niehs.nih.gov/doi/10.1289/ehp.02110917 Turan F, Akyurt I, Cek-Yalniz S. Effect of Red Clover Extract on Sex Reversal and Gonadal Development in the African Catfish, Clarias gariepinus (Burchell, 1822). Pak J Zool [Internet]. 2022;54(2). Available from: http://researcherslinks.com/current-issues/Effect-Red-Clover-Extract-Sex-Reversal/20/1/4511/html Javahery S, Nekoubin H, Moradlu AH. Effect of anaesthesia with clove oil in fish (review). Fish Physiol Biochem [Internet]. 2012 Dec 1;38(6):1545–52. Available from: http://link.springer.com/10.1007/s10695-012-9682-5 Fernandes IM, Bastos YF, Barreto DS, Lourenço LS, Penha JM. The efficacy of clove oil as an anaesthetic and in euthanasia procedure for small-sized tropical fishes. Brazilian J Biol [Internet]. 2016 Sep 26;77(3):444–50. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1519-69842017000300444&lng=en&tlng=en Syanya FJ, Mahadevan H, Khanna ARN. The effects of a non-steroid aromatase inhibitor on hybrid red tilapia masculinization, growth, reproductive hormone profile, and economic efficiency in aquaculture. Aquac Int [Internet]. 2025 Feb 27;33(2):160. Available from: https://link.springer.com/10.1007/s10499-025-01824-0 Sun L, Zha J, Spear PA, Wang Z. Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes). Environ Toxicol Pharmacol [Internet]. 2007 Jul;24(1):23–9. Available from: https://linkinghub.elsevier.com/retrieve/pii/S138266890700021X Maradonna F, Batti S, Marino M, Mita DG, Carnevali O. Tamoxifen as an Emerging Endocrine Disruptor. Ann N Y Acad Sci [Internet]. 2009 Apr 27;1163(1):457–9. Available from: https://nyaspubs.onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.2008.03653.x Sarasquete C, Úbeda-Manzanaro M, Ortiz-Delgado J. Soya isoflavones, genistein and daidzein, induce differential transcriptional modulation in the ovary and testis of zebrafish Danio rerio. Aquat Biol [Internet]. 2020 Apr 30;29:79–91. Available from: https://www.int-res.com/abstracts/ab/v29/p79-91/ Bharti S, Rasool F. Analysis of the biochemical and histopathological impact of a mild dose of commercial malathion on Channa punctatus (Bloch) fish. Toxicol Reports [Internet]. 2021;8:443–55. Available from: https://linkinghub.elsevier.com/retrieve/pii/S2214750021000354 Chalasani N, Bonkovsky HL, Fontana R, Lee W, Stolz A, Talwalkar J, et al. Features and Outcomes of 899 Patients With Drug-Induced Liver Injury: The DILIN Prospective Study. Gastroenterology [Internet]. 2015 Jun;148(7):1340-1352.e7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S001650851500311X GAO FF, LV JW, WANG Y, FAN R, LI Q, ZHANG Z, et al. Tamoxifen induces hepatotoxicity and changes to hepatocyte morphology at the early stage of endocrinotherapy in mice. Biomed Reports [Internet]. 2016 Jan;4(1):102–6. Available from: https://www.spandidos-publications.com/10.3892/br.2015.536 Chen YM, Wang IL, Zhu XY, Chiu WC, Chiu YS. Red Clover Isoflavones Influence Estradiol Concentration, Exercise Performance, and Gut Microbiota in Female Mice. Front Nutr [Internet]. 2021 Apr 14;8. Available from: https://www.frontiersin.org/articles/10.3389/fnut.2021.623698/full Muhammad NP, Nirmal T, Prabhakaran A, Varghese T. Phytoestrogens as Endocrine-Disrupting Agents in Aquaculture. In: Xenobiotics in Aquatic Animals [Internet]. Singapore: Springer Nature Singapore; 2023. p. 213–31. Available from: https://link.springer.com/10.1007/978-981-99-1214-8_9 Kim K hyuk, Moon H na, Yeo I kyu. RETRACTED ARTICLE: Sexual maturation inhibition using exemestane and tamoxifen in female olive flounder (Paralichthys olivaceous). Discov Anim [Internet]. 2024 May 27;1(1):4. Available from: https://link.springer.com/10.1007/s44338-024-00007-0 Williams TD, Caunter JE, Lillicrap AD, Hutchinson TH, Gillings EG, Duffell S. Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows ( Pimephales Promelas ). Environ Toxicol Chem [Internet]. 2007 Apr 1;26(4):695–707. Available from: https://academic.oup.com/etc/article/26/4/695/7763458 Orias F, Bony S, Devaux A, Durrieu C, Aubrat M, Hombert T, et al. Tamoxifen ecotoxicity and resulting risks for aquatic ecosystems. Chemosphere [Internet]. 2015 Jun;128:79–84. Available from: https://linkinghub.elsevier.com/retrieve/pii/S004565351500017X Brown AC, Stevenson LM, Leonard HM, Nieves-Puigdoller K, Clotfelter ED. Phytoestrogens β -Sitosterol and Genistein Have Limited Effects on Reproductive Endpoints in a Female Fish, Betta splendens. Biomed Res Int [Internet]. 2014;2014:1–7. Available from: http://www.hindawi.com/journals/bmri/2014/681396/ Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2026 Read the published version in Journal of Molecular Histology → Version 1 posted Editorial decision: Revision requested 05 Jan, 2026 Reviews received at journal 15 Sep, 2025 Reviews received at journal 25 Aug, 2025 Reviewers agreed at journal 23 Aug, 2025 Reviewers agreed at journal 23 Aug, 2025 Reviewers agreed at journal 22 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 18 Aug, 2025 Editor assigned by journal 16 Aug, 2025 Submission checks completed at journal 16 Aug, 2025 First submitted to journal 15 Aug, 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. 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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-7382781","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505356403,"identity":"e5a17ee1-299f-4b63-9a1f-69dd03a2dbe6","order_by":0,"name":"Tahereh Naji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYDCCAyCCR0KODUQnkKLFmFQtDAyJDUS7i+/2AbYHP2Qs0vvYzxgwPNxDhBbJcwnshj08ErltPDkGDAnPiNBicIaBTYIHpIUBpOUAkVok//BIpLPxvyFBizTQlgQ2CWJtkQRpkeGRMGyTeFZwgCgtfCCHve2pk5fvT9748AcxWhgY+D8wMPZAmMRpgIAfJKgdBaNgFIyCkQcApowtc/JQqTsAAAAASUVORK5CYII=","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":true,"prefix":"","firstName":"Tahereh","middleName":"","lastName":"Naji","suffix":""},{"id":505356404,"identity":"bf72ddfa-e6a1-4cf8-b93d-088efd9840e9","order_by":1,"name":"Mahdi Ahmadinia","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahdi","middleName":"","lastName":"Ahmadinia","suffix":""},{"id":505356405,"identity":"870c6252-3b4b-4aad-a6c1-84cd5571696f","order_by":2,"name":"Mohammad Mehrkar","email":"","orcid":"","institution":"Islamic Azad University, Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Mehrkar","suffix":""},{"id":505356406,"identity":"8495f315-3596-4f79-a1e3-53587694c1ee","order_by":3,"name":"Homayoun Hosseinzadeh Sahafi","email":"","orcid":"","institution":"Agricultural Research \u0026 Education Organization","correspondingAuthor":false,"prefix":"","firstName":"Homayoun","middleName":"Hosseinzadeh","lastName":"Sahafi","suffix":""}],"badges":[],"createdAt":"2025-08-15 16:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7382781/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7382781/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10735-026-10727-0","type":"published","date":"2026-01-31T15:59:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89907222,"identity":"54132bdf-db6c-467d-bc10-e4747ede36dd","added_by":"auto","created_at":"2025-08-26 10:16:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1244725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative ovarian histology sections (A–H) of Trichogaster trichopterus under different treatments.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control: normal ovarian structure with predominance of pre-nucleolar (PN) oocytes. (B) Vehicle: similar to control. (C–E) Red clover (25, 75, 150 mg/kg): dose-dependent promotion of cortical alveolar (CA) and vitellogenic development, with yolk vesicle accumulation and germinal vesicle (GV) migration. (F–H) Tamoxifen (10, 50, 100 mg/kg): progressive suppression of folliculogenesis, follicular arrest at PN stage, and cytoplasmic reduction at higher doses. All images stained with H\u0026amp;E, magnification ×40. Scale bar = 50 μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7382781/v1/0959be2ab7c7c0cc4d508f0c.png"},{"id":89907223,"identity":"51081b08-8050-4e5c-94f3-d0fb36d174d6","added_by":"auto","created_at":"2025-08-26 10:16:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":978041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative photomicrographs of hepatic histology in female Trichogaster trichopterus under different treatments.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) Control and (B) vehicle-treated fish showed normal hepatocyte architecture. (C–E) Red clover groups (25, 75, 150 mg/kg) preserved hepatic integrity with intact sinusoids and uniform hepatocyte cords. (F–H) Tamoxifen at increasing doses (10, 50, 100 mg/kg) induced progressive hepatic pathology, including hepatocyte hypertrophy, sinusoidal dilation, vacuolization, and necrotic lesions. H\u0026amp;E stain, magnification ×40. Scale bar = 50 μm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7382781/v1/9d4ebfea4ead2da8081fa372.png"},{"id":89907221,"identity":"3a981c97-5639-42b5-bb31-fefddf7d71bd","added_by":"auto","created_at":"2025-08-26 10:16:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":158641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransmission electron micrographs of ovarian ultrastructure in female Trichogaster trichopterus under different treatments.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) Control group: Oocyte membrane (OM) and cytoplasm (OC) exhibit normal morphology; follicular cells (FC) are closely associated with the oocyte; nucleoli are peripherally distributed. No signs of microvilli or yolk vesicle activity are apparent, indicating a resting stage. Scale bar: 2 µm. (B) Red clover group (75 mg/kg): Well-developed zona radiata layers (internal: ZI; external: ZE), abundant yolk vesicles (YV), and lipid vesicles (LV) are visible. Active micropinocytotic structures suggest phytoestrogen-induced vitellogenesis. Scale bar: 5 µm. (C) Tamoxifen group (100 mg/kg): Oocyte cytoplasm appears atrophic with disrupted membrane structure. Zona radiata is thinned, microvilli are absent, and vesicular transport is suppressed, suggesting anti-estrogenic inhibition of oocyte maturation. Scale bar: 2 µm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7382781/v1/66c8060c7d30ad0924c61269.jpg"},{"id":101691856,"identity":"b7d4c66f-28d8-47cf-84f6-5f0be14d3f64","added_by":"auto","created_at":"2026-02-02 16:15:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3368431,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7382781/v1/9f7c3003-5591-4ab3-9dd2-46b86f118d5a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Evaluation of Tamoxifen and Red Clover Extract on Reproductive and Hepatic Function in Female Trichogaster trichopterus: A Translational Endocrine Toxicology Study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTamoxifen, a Selective Estrogen Receptor Modulator (SERM), has long been a cornerstone of hormone-responsive breast cancer therapy, significantly reducing recurrence and mortality (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, its tissue-specific actions can produce adverse effects beyond the breast. For example, extended tamoxifen use raises the risk of endometrial cancer (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and is associated with non-alcoholic fatty liver disease (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These drawbacks have prompted interest in safer alternatives for estrogen-related conditions. Phytoestrogenic plants such as red clover (\u003cem\u003eTrifolium pratense\u003c/em\u003e) are increasingly used to manage menopausal symptoms (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Red clover contains isoflavones such as formononetin, biochanin A, daidzein, and genistein, which structurally resemble estradiol and bind estrogen receptors (with a higher affinity for ER-β) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Clinical evidence supports its estrogen-mimicking benefits: a recent study showed red clover isoflavone supplementation significantly reduces hot flash frequency in peri and postmenopausal women (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Notably, red clover has not exhibited estrogen-dependent cancer promotion in humans; in fact, a randomized trial in breast cancer survivors on tamoxifen found red clover extract to be well-tolerated and safe (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These findings raise the question of how a natural phytoestrogen compares to a synthetic SERM in modulating the endocrine system and peripheral organs.\u003c/p\u003e\u003cp\u003eWhat remains unclear is the comparative impact of tamoxifen versus red clover on reproductive and hepatic tissues. Ethical and practical constraints limit direct studies in women, so translational animal models are essential to investigate mechanisms and safety before clinical application (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The three-spot gourami (\u003cem\u003eTrichogaster trichopterus\u003c/em\u003e) is a validated vertebrate model in reproductive endocrinology, as fish share a highly conserved Hypothalamic\u0026ndash;Pituitary\u0026ndash;Gonadal (HPG) axis with mammals (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Indeed, female gourami produce the same principal gonadotropins and sex steroids (GnRH, FSH, LH, estradiol, etc.) as humans (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), making this species suitable for studying estrogenic and anti-estrogenic effects. Tamoxifen exposure in fish can disrupt the HPG axis, impairing gonadal function and inducing hepatic lipid accumulation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Such models enable side-by-side evaluation of an established anti-estrogen (tamoxifen) and a phytoestrogenic compound (red clover) under controlled conditions, helping to bridge the gap between preclinical findings and human outcomes.\u003c/p\u003e\u003cp\u003eIn this study, a translational approach is adopted to compare ovarian and hepatic alterations induced by tamoxifen with those elicited by red clover extract in female Three-spot Gourami. Histopathological changes in the ovary and liver are evaluated alongside variations in sex hormone concentrations and liver enzyme levels to delineate the mechanisms, efficacy, and safety profiles of each compound. By leveraging the conserved features of this vertebrate model, the differential modulation of reproductive endpoints and hepatic function by a synthetic SERM and a phytoestrogenic alternative is examined. These findings are expected to inform future research directions in endocrine therapy for women, supporting the development of safer and more effective estrogen-modulating interventions.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Experimental Design and Animal Grouping\u003c/h2\u003e\u003cp\u003eThis in vivo experimental study was conducted from January to May 2022 at the Basic Science Laboratory, Faculty of Pharmacy, Islamic Azad University, Tehran, Iran. All experimental procedures and reporting were conducted following the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines to ensure transparency and reproducibility. Furthermore, the study design, including dose selection, duration, and biomarker assessment, was based on relevant OECD Test Guidelines for endocrine disruption (TG 407, TG 440) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSexually mature female Three-spot Gourami (Trichogaster trichopterus) were selected as the animal model due to their conserved HPG axis, which exhibits significant physiological homology with mammals. A total of 120 fish with uniform size and body weight (mean initial weight: 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g) were obtained from the Ornamental Fish Breeding Center in Karaj, Iran, and acclimated under controlled laboratory conditions for two weeks before the experiment. The fish were randomly divided into eight experimental groups (n\u0026thinsp;=\u0026thinsp;15 per group). Two groups served as controls: one as an intact control (without any injection) and the other as a vehicle control, receiving 60% ethanol. Three groups received intramuscular (IM) injections of tamoxifen at doses of 10, 50, and 100 mg/kg, respectively. These doses were selected based on previous studies demonstrating tamoxifen\u0026rsquo;s endocrine-disrupting and anti-estrogenic effects in aquatic species and mammalian models (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The remaining three groups were treated with IM injections of red clover (\u003cem\u003eTrifolium pratense\u003c/em\u003e) extract at doses of 25, 75, and 150 mg/kg, chosen following prior reports evaluating the estrogenic activity and safety profile of isoflavone-rich plant extracts in aquatic organisms (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll injections were administered in a final volume of 0.02 mL using insulin syringes (0.5\u0026ndash;1 mL) between the dorsal fin and lateral trunk muscle, inserted at a 30\u0026deg; angle. A total of nine injections were given every other day over 18 days. Following the final injection, fish were held without intervention for an additional 48 hours, and dissections were performed on day 21 for tissue sampling. During the entire experimental period, fish were maintained in glass aquaria under stable conditions, including a water temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, a pH of 6.5, and a water hardness of approximately 78 mg/L CaCO₃. Fish were fed a commercial diet every other day, with water filtration and aeration maintained continuously. Water was dechlorinated and equilibrated to room temperature before use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Preparation of Tamoxifen and Red Clover Extract\u003c/h2\u003e\u003cp\u003eTamoxifen was obtained as a pure powder (99% purity, Lot #SLBP0003V, Germany) and handled under low-light conditions due to its photosensitive and cytotoxic properties. Based on fish weight calculations, three treatment doses were prepared: 10, 50, and 100 mg/kg. For each dose group, the weighed powder was dissolved in 60% ethanol to a final injection concentration, and the solution was brought to 5 mL in a volumetric flask. The prepared tamoxifen solutions were stored in amber glass vials at 4\u0026deg;C to protect them from light and prevent degradation before injection.\u003c/p\u003e\u003cp\u003eRed clover (\u003cem\u003eTrifolium pratense\u003c/em\u003e) extract was prepared using a commercial 1-liter ethanol extract purchased from Asha Biotech. The extract was air-dried under hygienic conditions using mesh covers to prevent contamination. After one week, the dried residue adhering to the beakers was scraped and powdered using a mortar and pestle, yielding approximately 4.545 g of dry extract from 1 L of original ethanol extract. Doses of 25, 75, and 150 mg/kg were selected based on prior studies. For each treatment group, the required amount of powdered extract was reconstituted in distilled water up to 5 mL based on the average fish weight per tank. The solutions were filtered through 0.45 \u0026micro;m sterile syringe filters to remove residual plant particles and stored in sterile glass vials until administration.\u003c/p\u003e\u003cp\u003eBoth tamoxifen and red clover solutions were prepared freshly before the injection period and handled with caution to minimize exposure to light and oxidative degradation. All solutions were prepared under a fume hood using aseptic techniques to ensure sterility and reproducibility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Injection Protocol\u003c/h2\u003e\u003cp\u003eBefore each injection session, fish were mildly anesthetized using an aqueous solution of clove extract prepared from 1 g of dried \u003cem\u003eSyzygium aromaticum\u003c/em\u003e per liter of distilled water. This stock solution was filtered and diluted in a 1:2 ratio (200 mL extract\u0026thinsp;+\u0026thinsp;400 mL distilled water) immediately before use. Individual fish were immersed for 30\u0026ndash;40 seconds until partial loss of equilibrium was observed, indicating sufficient sedation for handling (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll injections were performed intramuscularly (IM) between the dorsal fin and lateral line of the fish, using BD insulin syringes (0.5 or 1 mL). The injection volume for all groups was standardized at 0.02 mL per fish, regardless of treatment dose. The needle was inserted at an approximate 30\u0026deg; angle into the lateral trunk muscle to ensure proper IM delivery. Before injection, the injection site was cleaned using 70% ethanol-soaked sterile cotton and the fish were gently immobilized by hand, holding the head and tail with minimal pressure.\u003c/p\u003e\u003cp\u003eThe dosing schedule consisted of nine total injections administered every other day over 18 days. Following the final injection, fish were kept under normal aquarium conditions for an additional 48 hours to allow stabilization before sacrifice and tissue collection on day 21. After each injection, fish were transferred to dechlorinated, temperature-equilibrated water, and oxygenation was maintained via aeration pumps. No adverse behavioral signs or injection site complications were observed throughout the experimental period.\u003c/p\u003e\u003cp\u003e All handling procedures were performed under controlled environmental conditions and following local animal care guidelines. The repeated IM injection protocol was designed to mimic subchronic exposure while minimizing stress and maintaining consistent dosing accuracy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Sampling and Tissue Processing\u003c/h2\u003e\u003cp\u003eOn day 21, following the ninth and final injection and a 48-hour post-treatment stabilization period, fish were anesthetized using a concentrated clove extract until no movement was observed. Biometric measurements, including total length and body weight, were recorded for each fish using a digital scale with 0.001 g precision. Dissections were performed via a longitudinal incision along the midline from the mouth to the ventral side of the gill cover, followed by two transverse cuts to expose the internal organs. The ovaries and liver were carefully excised, rinsed in cold saline to remove excess tissue fluid, and weighed separately for subsequent index calculations.\u003c/p\u003e\u003cp\u003eTissues designated for light microscopy were immediately fixed in 10% neutral buffered formalin (prepared using 10 mL formalin and 90 mL distilled water). Samples were processed using an Autotechnicon KP-110 tissue processor through standard dehydration (graded ethanol), clearing (xylene), and infiltration steps. Paraffin blocks were prepared using a rotary embedding system and sectioned at 5\u0026ndash;10 \u0026micro;m using a rotary microtome. Sections were mounted on glass slides and dried in a 100\u0026deg;C oven for 15 minutes before staining. Hematoxylin and Eosin (H\u0026amp;E) staining was performed using standard protocols, including sequential deparaffinization, rehydration, nuclear staining (hematoxylin), cytoplasmic staining (eosin), and dehydration steps.\u003c/p\u003e\u003cp\u003eFor Transmission Electron Microscopy (TEM), representative ovarian and hepatic tissue fragments (\u0026le;\u0026thinsp;0.5 mm) were fixed in 2.5% glutaraldehyde at 4\u0026deg;C for one hour. Post-fixation was carried out in 1.5% osmium tetroxide for 90 minutes. Samples were washed in 0.1 M sodium cacodylate buffer (3 \u0026times; 5 min) and rinsed in distilled water. Tissue contrast was enhanced using 2% uranyl acetate for 20 minutes, followed by secondary fixation in 1% osmium tetroxide for 20 minutes. Dehydration was achieved with ascending ethanol concentrations (50%, 70%, 95%, and 100%), and infiltration was performed using propylene oxide and Epon resin mixtures. Final embedding was done in pure resin, cured at 60\u0026deg;C for 48\u0026ndash;72 hours. Ultrathin sections (~\u0026thinsp;60\u0026ndash;70 nm) were prepared using an ultramicrotome and post-stained with lead citrate before imaging on a Philips EM 208S transmission electron microscope.\u003c/p\u003e\u003cp\u003eHistological and ultrastructural analyses focused on key cellular and subcellular markers of ovarian and hepatic integrity. Ovarian tissue was evaluated for follicular stage prevalence (e.g., pre-nucleolar vs. vitellogenic), yolk droplet formation, lipid accumulation, and nuclear migration. Hepatic sections were assessed for sinusoidal structure, hepatocyte size, and evidence of inflammation. Under TEM, attention was given to zona radiata integrity, microvilli presence, and yolk vesicle fusion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Hormonal and Biochemical Assays\u003c/h2\u003e\u003cp\u003eDue to the small size of the experimental fish, blood sampling was not feasible; thus, hormone and enzyme analyses were performed on tissue homogenates. Ovarian and hepatic tissues were homogenized in cold conditions and centrifuged at 3000 rpm for 5 minutes using a refrigerated centrifuge to obtain clear supernatants.\u003c/p\u003e\u003cp\u003eLevels of 17β-estradiol, 17-hydroxyprogesterone, and testosterone were quantified using a commercial ELISA kit (manufacturer not specified), following the manufacturer's protocol. Briefly, 25 \u0026micro;L of tissue extract was mixed with 50 \u0026micro;L of conjugate solution, shaken for 30 seconds, incubated for 60 minutes at room temperature, and washed three times with 350 \u0026micro;L wash buffer. Then, 100 \u0026micro;L of substrate was added, and the plate was incubated in the dark for 20 minutes. Absorbance was measured at 450 nm using an ELISA microplate reader.\u003c/p\u003e\u003cp\u003eHepatic enzyme activities of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) were also measured from liver homogenates using spectrophotometric methods. Results were expressed as IU/mL. These enzymes serve as sensitive markers of hepatocellular injury, with AST reflecting mitochondrial damage and ALT being more liver-specific.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Reproductive and Hepatic Indices\u003c/h2\u003e\u003cp\u003eThe Gonadosomatic Index (GSI) was calculated using the formula to measure reproductive capability: GSI (gonad weight/body weight) \u0026times;100. The Hepatosomatic Index (HIS) was also calculated by the formula: HSI (liver body weight/ total weight) \u0026times;100, which indicates fish energy reserves.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using SPSS software version 26. One-way analysis of variance (ANOVA) was used to evaluate differences among experimental groups, followed by Duncan\u0026rsquo;s multiple range \u003cem\u003epost hoc\u003c/em\u003e test to identify specific group differences. A significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set for all comparisons. Results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Gonadosomatic Index (GSI) and Hepatosomatic Index (HSI)\u003c/h2\u003e\u003cp\u003eBiometric outcomes showed that tamoxifen exerted a dose-dependent inhibitory effect on ovarian development, reflected in a significant reduction of GSI at 50 and 100 mg/kg compared to the control group (P\u0026thinsp;=\u0026thinsp;0.004 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), which is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The highest tamoxifen dose group (100 mg/kg) exhibited the lowest mean GSI (3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31%), statistically distinct from both lower-dose and control groups, as indicated by non-overlapping significance groupings. No significant GSI change was observed in the 10 mg/kg group (P\u0026thinsp;=\u0026thinsp;0.32).\u003c/p\u003e\u003cp\u003eIn contrast, red clover extract induced a significant increase in GSI at higher doses. Fish treated with 75 and 150 mg/kg extract demonstrated elevated gonadal indices compared to controls (P\u0026thinsp;=\u0026thinsp;0.012 and P\u0026thinsp;=\u0026thinsp;0.001, respectively), with values reaching 13.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38% in the highest dose group, consistent with dose-dependent phytoestrogenic stimulation. No significant alterations were observed in HSI across treatment groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), although a slight upward trend was noted in tamoxifen-exposed fish.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGonadosomatic index (GSI) and hepatosomatic index (HSI) in Trichogaster trichopterus following administration of tamoxifen and red clover extract.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGSI (%) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHSI (%) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGSI Grouping\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eHSI Grouping\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eP-value GSI (vs. Control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP-value HSI (vs. Control)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eControl (no injection)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVehicle (60% ethanol)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTamoxifen 10 mg/kg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTamoxifen 50 mg/kg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.004 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTamoxifen 100 mg/kg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRed clover 25 mg/kg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRed clover 75 mg/kg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.012 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eRed clover 150 mg/kg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.001 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e\u003cem\u003eNote: Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5). Different superscript letters indicate statistically significant differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.05, ANOVA with Tukey\u0026rsquo;s post hoc test). Statistically significant differences compared to the control group are marked with ★.\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Endocrine and Biochemical Markers\u003c/h2\u003e\u003cp\u003eTamoxifen treatment resulted in a significant, dose-dependent suppression of circulating sex steroid hormones. At 50 and 100 mg/kg doses, serum levels of 17β-estradiol, progesterone, and testosterone were significantly reduced compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the lowest values observed in the 100 mg/kg group (e.g., estradiol: 49.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86 pg/mL; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eConversely, administration of red clover extracts enhanced hormone levels at higher doses. In particular, 75 and 150 mg/kg treatments significantly elevated estradiol and progesterone concentrations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eFor hepatic enzymes, tamoxifen induced a marked elevation in ALT and AST activity, particularly at 100 mg/kg (ALT: 58.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 U/L; AST: 96.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 U/L; both P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), implying hepatocellular stress or toxicity. Red clover groups, however, maintained enzyme levels comparable to controls, with no statistically significant deviations. A comprehensive summary of group-wise means, SEMs, statistical groupings, and P-values is provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of tamoxifen and red clover extract on serum sex steroid hormones and liver enzyme activity in female Trichogaster trichopterus.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"16\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17β-estradiol (pg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eProgesterone (ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTestosterone (ng/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eALT (U/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003eAST (U/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c15\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c16\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e76.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e35.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e67.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle (EtOH 60%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e74.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e68.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTamoxifen 10 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e38.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e71.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTamoxifen 50 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.009\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e49.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e84.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTamoxifen 100 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e49.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e58.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e96.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRed clover 25 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e79.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e34.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e66.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRed clover 75 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e83.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e64.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRed clover 150 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e87.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 ★\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003eb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e32.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e63.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003ea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"16\" nameend=\"c16\" namest=\"c1\"\u003e\u003cp\u003eNote: Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;5). Different superscript letters within each column indicate statistically significant differences (P\u0026thinsp;\u0026le;\u0026thinsp;0.05, one-way ANOVA with Tukey\u0026rsquo;s post hoc test). P-values denote comparisons against the control group. Statistically significant differences compared to the control group are marked with ★\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Ovarian Histology\u003c/h2\u003e\u003cp\u003eLight microscopic evaluation of ovarian sections revealed distinct histopathological alterations following tamoxifen or red clover treatment. In tamoxifen-treated fish, especially at 50 and 100 mg/kg, ovarian tissue was predominantly composed of pre-nucleolar oocytes with reduced cytoplasmic development. Vitellogenic follicles were sparse or absent, and ooplasmic lipid content was diminished. Structural disorganization, including nuclear displacement and atretic changes, was also observed at higher doses, consistent with anti-estrogenic inhibition of folliculogenesis.\u003c/p\u003e\u003cp\u003eIn contrast, ovaries from fish exposed to red clover extract (particularly 75 and 150 mg/kg) showed enhanced follicular maturation, increased prevalence of cortical alveolar and early vitellogenic oocytes. Cytoplasmic yolk deposition, lipid vesicle formation, and orderly nuclear positioning were more pronounced than in the control group, indicating phytoestrogen-induced stimulation of the ovarian cycle.\u003c/p\u003e\u003cp\u003eNo remarkable histological abnormalities were observed in the vehicle group or at the lowest tested doses of either compound. Full microscopic comparisons and representative images are provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Hepatic Histology\u003c/h2\u003e\u003cp\u003eHistopathological examination of liver sections revealed dose-dependent hepatic alterations in tamoxifen-treated fish. At 50 and 100 mg/kg, hepatocytes exhibited prominent hypertrophy, sinusoidal dilation, cytoplasmic vacuolization, and focal necrosis. Additionally, Kupffer cell proliferation and disorganized hepatic cords were evident, consistent with inflammatory stress and hepatocellular damage. These lesions were absent or minimal at the 10 mg/kg dose.\u003c/p\u003e\u003cp\u003eIn contrast, red clover extract did not induce histological abnormalities. Liver architecture in fish treated with 75 and 150 mg/kg red clover remained comparable to control groups, showing well-aligned hepatocyte rows, intact sinusoidal spaces, and no apparent fatty degeneration or necrosis. These findings support the hepatoprotective profile of red clover under the tested conditions. Representative micrographs illustrating these changes are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Ultrastructural Observations (TEM)\u003c/h2\u003e\u003cp\u003eTransmission electron microscopy provided further insight into the subcellular effects of tamoxifen and red clover extract on ovarian tissues. In ovarian samples from high-dose tamoxifen groups, oocytes exhibited cytoplasmic degeneration, loss of microvilli, and disrupted zona radiata. Intercellular contacts between follicular cells were diminished, and yolk vesicles were largely absent, indicating impaired vitellogenesis and estrogen signaling. Mitochondrial swelling and lysosomal accumulation were also evident in some cells, suggestive of early apoptotic processes.\u003c/p\u003e\u003cp\u003eConversely, red clover\u0026ndash;treated fish (75 and 150 mg/kg) showed well-preserved ovarian ultrastructure. Oocytes featured active microvilli, intact zona radiata, abundant yolk vesicles, and organized cortical granules. Numerous vesicles engaged in pinocytosis were observed, supporting phytoestrogen-induced steroidogenic activity. Representative micrographs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrated that tamoxifen and red clover extract have opposing impacts on the reproductive endocrine endpoints of female \u003cem\u003eTrichogaster trichopterus\u003c/em\u003e. Tamoxifen exposure markedly suppressed ovarian development and function, evidenced by a lower GSI and reduced circulating sex hormone levels compared to controls. Such anti-estrogenic effects align with reports in other fish: for example, high dietary tamoxifen doses in hybrid tilapia significantly lowered female GSI and estradiol levels while elevating male androgens (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Histologically, tamoxifen-treated gourami ovaries showed signs of atresia and developmental arrest, consistent with tamoxifen\u0026rsquo;s known inhibition of vitellogenesis. Prior studies confirm that tamoxifen can block estrogen-driven vitellogenin (yolk protein) production. Sun et al. found that tamoxifen prevented normal vitellogenin induction in female fish at high concentrations (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The present results are therefore in line with tamoxifen\u0026rsquo;s role as a selective estrogen receptor modulator that antagonizes estrogen signaling in the ovary and liver tissue (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). By contrast, females exposed to red clover extract exhibited an \u003cem\u003eestrogenic\u003c/em\u003e stimulation of the reproductive axis. GSI and plasma estradiol were higher than in controls, suggesting enhanced gonadal growth and endocrine activity, and possibly an advance in oocyte maturation stages. This is in agreement with international findings that phytoestrogens from red clover have pro-estrogenic effects on fish reproduction. Turan et al. (2022) showed that red clover extract (rich in isoflavones) effectively feminized 89% of juvenile male African catfish, with treated fish developing normal ovaries and no intersex pathology (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Similarly, soy isoflavones like genistein and daidzein are reported to act as estrogen receptor agonists in fish, modulating reproductive gene expression and promoting female-biased traits without overt toxicity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Our findings that red clover increased female gourami gonad size and hormone levels are consistent with these studies, reinforcing that plant-derived estrogens can mimic endogenous estrogens in teleosts.\u003c/p\u003e\u003cp\u003eBeyond the gonads, tamoxifen and red clover produced distinct hepatic outcomes with significant toxicological implications. Tamoxifen-treated gourami showed altered liver enzyme profiles indicative of hepatic stress: for instance, elevated ALT and AST levels were observed relative to controls (as typically seen in liver injury). This mirrors patterns seen in other species; In fish exposed to high tamoxifen, studies reported significant increases in AST and ALT alongside reductions in albumin and alkaline phosphatase (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Tamoxifen\u0026rsquo;s hepatotoxicity is well documented in mammals as well, where long-term therapy can induce steatosis and inflammatory liver damage (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Histologically, our tamoxifen group livers exhibited vacuolar degeneration and mild inflammation, suggesting incipient fatty liver changes. These lesions correspond to tamoxifen\u0026rsquo;s interference with hepatic estrogen receptors and lipid metabolism. Estrogen normally helps regulate lipid homeostasis in the liver; by antagonizing hepatic ER, tamoxifen may trigger fat accumulation and oxidative stress, as observed in rodent studies (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). There is also evidence that tamoxifen upregulates genes like CYP1A1 involved in xenobiotic metabolism in fish liver (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), potentially generating reactive metabolites that exacerbate liver damage.\u003c/p\u003e\u003cp\u003eConversely, red clover extract had a more benign or even beneficial effect on the liver. Treated gourami did not show the enzyme elevations seen with tamoxifen; in fact, liver enzymes in the red clover group remained comparable to or slightly improved over. Histological examination revealed that red clover fish livers retained normal architecture, with minimal fat vacuolation or cell death. This outcome aligns with studies in which dietary red clover isoflavones were hepatoprotective or neutral. For example, studies found that red clover supplementation in mice did not significantly raise AST or ALT, but did lower serum LDL and triglycerides, indicating improved metabolic health (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Isoflavones such as formononetin and biochanin A in red clover can scavenge reactive oxygen species and modulate liver gene expression to favor antioxidant defenses (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In our fish, this likely translated to protection against hepatic oxidative damage. The lack of liver histopathology in red clover\u0026ndash;exposed gourami is particularly notable given that their gonads were highly active; it suggests the extract did not induce liver hypertrophy or vitellogenin overproduction that potent synthetic estrogens often cause. Overall, the divergent hepatic profiles, tamoxifen causing enzyme perturbation and histological lesions, vs. red clover causing negligible liver stress, underscore the contrasting safety margins of these substances as endocrine modulators.\u003c/p\u003e\u003cp\u003eThe contrasting actions of tamoxifen and red clover on the gourami\u0026rsquo;s endocrine system can be interpreted through their mechanisms at the Estrogen Receptors (ERs) and upstream hormonal axes. Tamoxifen is a non-steroidal SERM that in fish appears to act primarily as an estrogen antagonist in reproductive tissues. It likely binds to ERα/β in the HPG axis, blocking estradiol\u0026rsquo;s normal feedback loops. This would reduce gonadotropin release and ovarian steroidogenesis, explaining the lowered estradiol and GSI. Similar anti-estrogenic modes were observed in female olive flounder given tamoxifen, where estrogen receptor expression in the liver remained low and vitellogenin mRNA induction was blunted (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Notably, tamoxifen can also exhibit partial estrogen agonism in certain contexts, but some vitellogenin presence could reflect tamoxifen\u0026rsquo;s mixed activity. In male fish, tamoxifen has paradoxically been shown to induce vitellogenin production by activating hepatic ERs (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), even as it inhibits female vitellogenesis at high doses (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). These contradictory effects are supported by Maradonna et al., who described tamoxifen\u0026rsquo;s \u0026ldquo;mixed estrogenic/anti-estrogenic action\u0026rdquo; disrupting fish physiology (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Our results reinforce this duality: tamoxifen in females predominantly acted as an ER antagonist (suppressing ovarian growth), yet some estrogen-responsive genes in the liver may have been upregulated (partial agonism), contributing to complex outcomes like reduced fertility but not complete estrogen deprivation. This complexity has been noted in recent toxicology reviews of pharmaceuticals in fish, where tamoxifen\u0026rsquo;s impact varies with dose, sex, and species (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRed clover\u0026rsquo;s estrogenic influence, on the other hand, can be attributed to its high content of isoflavone phytoestrogens that bind ERs. Red clover contains formononetin and biochanin A, which are metabolized to daidzein and genistein compounds with affinity for fish estrogen receptors (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). These phytoestrogens likely acted as agonists on ovarian ER, promoting vitellogenin synthesis and oocyte development akin to low-dose 17β-estradiol exposure. The stimulatory effect we observed (higher hormone levels and advanced ovarian follicles) parallels the action of estradiol itself in fish. Indeed, a recent zebrafish study demonstrated that genistein exposure increased expression of hatching enzyme and other estrogen-regulated genes in gonads (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), while another found red clover extract upregulated growth hormone and improved reproductive output in female goldfish (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Interestingly, Sarasquete et al. (2020) reported that genistein and daidzein altered ERβ transcript levels in zebrafish gonads but did \u003cem\u003enot\u003c/em\u003e cause histomorphological changes over 15 days (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). This suggests that phytoestrogens can fine-tune gene expression without the full potency of synthetic estrogens, resulting in milder, physiologically paced changes, consistent with our finding that red clover fish had enhanced reproductive indices but no overt pathological changes. One point of contrast in the studies is the sensitivity of different fish species and life stages to phytoestrogens. While our adult gourami and the catfish study showed clear estrogenic stimulation, a study on adult Siamese fighting fish (Betta) found limited impact of phytoestrogens on steroid levels or oocyte development (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Those authors suggested that mature females of some species may be less acutely responsive to dietary phytoestrogens, possibly due to saturation of ERs by endogenous hormones. These results contribute to this discourse by showing that \u003cem\u003eT. trichopterus\u003c/em\u003e is indeed responsive to plant estrogens, aligning more with species like catfish and zebrafish than with Betta. It underlines the importance of species-specific reproductive physiology in outcomes of endocrine disruptor exposure.\u003c/p\u003e\u003cp\u003eUsing \u003cem\u003eT. trichopterus\u003c/em\u003e (three-spot gourami) as a model species in this study also carries translational relevance. Gourami are tropical teleosts with a well-characterized reproductive endocrinology, making them suitable analogs for other oviparous fish. They respond to hormonal cues in a manner comparable to established lab models like zebrafish, yet their physiology (e.g., a bubble-nest breeder with parental care) adds a layer of ecological validity. Demonstrating endocrine disruption in gourami confirms that such effects are not limited to a few model species, thereby broadening the understanding of how chemicals like tamoxifen impact diverse fish taxa.\u003c/p\u003e\u003cp\u003eWhile direct extrapolation to humans must be cautious, our findings do resonate with known human outcomes: tamoxifen\u0026rsquo;s ability to induce fatty liver and alter estrogen-responsive tissues in fish parallels side effects seen in women undergoing tamoxifen therapy (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and the estrogenic compounds in red clover used here are the same isoflavones marketed as menopausal supplements in women. This cross-species consistency in mechanism lends credibility to using fish models for initial screening of endocrine-active substances. In summary, \u003cem\u003eTrichogaster trichopterus\u003c/em\u003e proved to be a sensitive and informative model for endocrine and histopathological endpoints. The suppression of reproductive capacity by tamoxifen and the enhancement by red clover in this species not only corroborate findings from studies in toxicology and endocrinology but also illuminate possible mechanisms (ER antagonism versus agonism) underlying these effects.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrates that tamoxifen and red clover extract exert contrasting effects on reproductive and hepatic tissues in the three-spot gourami (\u003cem\u003eTrichogaster trichopterus\u003c/em\u003e). Tamoxifen exhibited significant anti-estrogenic activity, characterized by a marked suppression of gonadal development, reduction in sex steroid hormones, and evidence of hepatotoxicity. Conversely, red clover extract, rich in phytoestrogenic isoflavones, safely promoted gonadal growth, hormone production, and preserved hepatic integrity, confirming its potential as a safer estrogen-modulating alternative. Given the high conservation of the HPG axis among vertebrates, including humans, these findings bear substantial translational relevance. Clinically, these results reinforce concerns regarding long-term tamoxifen use and its associated risks, highlighting the importance of careful monitoring and potential hepatoprotective interventions in patients. Importantly, the benign safety profile of red clover demonstrated here aligns with existing clinical evidence supporting its use in menopausal symptom management, positioning it as a valuable candidate for safer hormone therapy in women. Further clinical investigations are warranted to fully validate these findings and optimize the therapeutic use of phytoestrogens as alternatives or adjuncts to conventional hormonal treatments.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGonadosomatic Index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHSI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHepatosomatic Index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGOT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutamic Oxaloacetic Transaminase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGPT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlutamic Pyruvic Transaminase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHPG axis\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHypothalamus\u0026ndash;Pituitary\u0026ndash;Gonadal axis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEstrogen Receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSERM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSelective Estrogen Receptor Modulator\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFSH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFollicle-Stimulating Hormone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLuteinizing Hormone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eE2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e17β-Estradiol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e17-OHP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e17-Hydroxyprogesterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransmission Electron Microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGerminal Vesicle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eYV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eYolk Vesicle\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOil Droplet\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eZR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eZona Radiata\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGJ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGap Junction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThecal Layer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHepatocyte\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSinusoid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePortal Vein\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKupffer Cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFollicular Cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStandard Error of the Mean\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals were obtained from Mahyran Company, a licensed vendor. Since they were not privately owned by any institution or individual, no informed consent was required. All procedures were approved by the Iran National Committee for Ethics in Biomedical Research (Approval code: IR.IAU.PS.REC.1400.306).\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest regarding the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for conducting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eStudy conception and design:\u0026nbsp;\u003c/strong\u003eMahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eData collection and experimental work:\u0026nbsp;\u003c/strong\u003eMahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eData analysis and interpretation:\u0026nbsp;\u003c/strong\u003eTahereh Naji, Mahdi Ahmadinia\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDrafting and revising the manuscript:\u0026nbsp;\u003c/strong\u003eTahereh Naji, Mahdi Ahmadinia\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFinal approval of the manuscript:\u0026nbsp;\u003c/strong\u003eTahereh Naji, Mahdi Ahmadinia, Mohammad Mehrkar, Homayoun Hosseinzadeh Sahafi\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the use of QuillBot exclusively for improving the clarity and academic style of the manuscript through language polishing and paraphrasing. The tool was not involved in generating content, performing data analysis or interpretation, selecting literature, or creating any figures or tables. All scientific ideas, interpretations, and conclusions are entirely those of the authors, who take full responsibility for the work. This use is disclosed following Springer Nature\u0026rsquo;s guidelines on AI tool transparency and authorship.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEarly Breast Cancer Trialists\u0026rsquo; Collaborative Group (EBCTCG). Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet [Internet]. 2011 Aug;378(9793):771\u0026ndash;84. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673611609938\u003c/li\u003e\n\u003cli\u003eAromatase inhibitors versus tamoxifen in early breast cancer: patient-level meta-analysis of the randomised trials. Lancet [Internet]. 2015 Oct;386(10001):1341\u0026ndash;52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0140673615610741\u003c/li\u003e\n\u003cli\u003eEmons G, Mustea A, Tempfer C. Tamoxifen and Endometrial Cancer: A Janus-Headed Drug. Cancers (Basel) [Internet]. 2020 Sep 7;12(9):2535. Available from: https://www.mdpi.com/2072-6694/12/9/2535\u003c/li\u003e\n\u003cli\u003eBekes I, Huober J. Extended Adjuvant Endocrine Therapy in Early Breast Cancer Patients\u0026mdash;Review and Perspectives. Cancers (Basel) [Internet]. 2023 Aug 21;15(16):4190. Available from: https://www.mdpi.com/2072-6694/15/16/4190\u003c/li\u003e\n\u003cli\u003eKanadys W, Barańska A, Błaszczuk A, Polz-Dacewicz M, Drop B, Kanecki K, et al. Evaluation of Clinical Meaningfulness of Red Clover (Trifolium pratense L.) Extract to Relieve Hot Flushes and Menopausal Symptoms in Peri- and Post-Menopausal Women: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Nutrients [Internet]. 2021 Apr 11;13(4):1258. Available from: https://www.mdpi.com/2072-6643/13/4/1258\u003c/li\u003e\n\u003cli\u003eBooth NL, Piersen CE, Banuvar S, Geller SE, Shulman LP, Farnsworth NR. Clinical studies of red clover (Trifolium pratense) dietary supplements in menopause: a literature review. Menopause [Internet]. 2006 Mar;13(2):251\u0026ndash;64. Available from: http://journals.lww.com/00042192-200613020-00016\u003c/li\u003e\n\u003cli\u003eFerraris C, Ballestra B, Listorti C, Cappelletti V, Reduzzi C, Scaperrotta GP, et al. Red clover and lifestyle changes to contrast menopausal symptoms in premenopausal patients with hormone-sensitive breast cancer receiving tamoxifen. Breast Cancer Res Treat [Internet]. 2020 Feb 24;180(1):157\u0026ndash;65. Available from: http://link.springer.com/10.1007/s10549-020-05534-4\u003c/li\u003e\n\u003cli\u003eMaruska KP, Fernald RD. Social Regulation of Gene Expression in the Hypothalamic-Pituitary-Gonadal Axis. Physiology [Internet]. 2011 Dec;26(6):412\u0026ndash;23. Available from: https://www.physiology.org/doi/10.1152/physiol.00032.2011\u003c/li\u003e\n\u003cli\u003eAhmadinia M, Naji T, Sahafi HH. Methimazole-induced reproductive endocrine system disruption and hepatotoxicity: insights from a Trichogaster trichopterus animal model. BMC Pharmacol Toxicol [Internet]. 2025 Jul 1;26(1):128. Available from: https://bmcpharmacoltoxicol.biomedcentral.com/articles/10.1186/s40360-025-00942-y\u003c/li\u003e\n\u003cli\u003eCameron N, Del Corpo A, Diorio J, McAllister K, Sharma S, Meaney MJ. Maternal Programming of Sexual Behavior and Hypothalamic-Pituitary-Gonadal Function in the Female Rat. Vosshall LB, editor. PLoS One [Internet]. 2008 May 21;3(5):e2210. Available from: https://dx.plos.org/10.1371/journal.pone.0002210\u003c/li\u003e\n\u003cli\u003eDegani G. Brain Control Reproduction by the Endocrine System of Female Blue Gourami (Trichogaster trichopterus). Biology (Basel) [Internet]. 2020 May 21;9(5):109. Available from: https://www.mdpi.com/2079-7737/9/5/109\u003c/li\u003e\n\u003cli\u003eYu Q, Huo J, Zhang Y, Liu K, Cai Y, Xiang T, et al. Tamoxifen-induced hepatotoxicity via lipid accumulation and inflammation in zebrafish. Chemosphere [Internet]. 2020 Jan;239:124705. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0045653519319356\u003c/li\u003e\n\u003cli\u003eGelbke HP, Hofmann A, Owens JW, Freyberger A. The enhancement of the subacute repeat dose toxicity test OECD TG 407 for the detection of endocrine active chemicals: comparison with toxicity tests of longer duration. Arch Toxicol [Internet]. 2007 Mar 15;81(4):227\u0026ndash;50. Available from: http://link.springer.com/10.1007/s00204-006-0148-3\u003c/li\u003e\n\u003cli\u003eKilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. PLoS Biol [Internet]. 2010 Jun 29;8(6):e1000412. Available from: https://dx.plos.org/10.1371/journal.pbio.1000412\u003c/li\u003e\n\u003cli\u003eLange A, Paull GC, Coe TS, Katsu Y, Urushitani H, Iguchi T, et al. Sexual Reprogramming and Estrogenic Sensitization in Wild Fish Exposed to Ethinylestradiol. Environ Sci Technol [Internet]. 2009 Feb 15;43(4):1219\u0026ndash;25. Available from: https://pubs.acs.org/doi/10.1021/es802661p\u003c/li\u003e\n\u003cli\u003eRajapakse N, Silva E, Kortenkamp A. Combining xenoestrogens at levels below individual no-observed-effect concentrations dramatically enhances steroid hormone action. Environ Health Perspect [Internet]. 2002 Sep;110(9):917\u0026ndash;21. Available from: https://ehp.niehs.nih.gov/doi/10.1289/ehp.02110917\u003c/li\u003e\n\u003cli\u003eTuran F, Akyurt I, Cek-Yalniz S. Effect of Red Clover Extract on Sex Reversal and Gonadal Development in the African Catfish, Clarias gariepinus (Burchell, 1822). Pak J Zool [Internet]. 2022;54(2). Available from: http://researcherslinks.com/current-issues/Effect-Red-Clover-Extract-Sex-Reversal/20/1/4511/html\u003c/li\u003e\n\u003cli\u003eJavahery S, Nekoubin H, Moradlu AH. Effect of anaesthesia with clove oil in fish (review). Fish Physiol Biochem [Internet]. 2012 Dec 1;38(6):1545\u0026ndash;52. Available from: http://link.springer.com/10.1007/s10695-012-9682-5\u003c/li\u003e\n\u003cli\u003eFernandes IM, Bastos YF, Barreto DS, Louren\u0026ccedil;o LS, Penha JM. The efficacy of clove oil as an anaesthetic and in euthanasia procedure for small-sized tropical fishes. Brazilian J Biol [Internet]. 2016 Sep 26;77(3):444\u0026ndash;50. Available from: http://www.scielo.br/scielo.php?script=sci_arttext\u0026amp;pid=S1519-69842017000300444\u0026amp;lng=en\u0026amp;tlng=en\u003c/li\u003e\n\u003cli\u003eSyanya FJ, Mahadevan H, Khanna ARN. The effects of a non-steroid aromatase inhibitor on hybrid red tilapia masculinization, growth, reproductive hormone profile, and economic efficiency in aquaculture. Aquac Int [Internet]. 2025 Feb 27;33(2):160. Available from: https://link.springer.com/10.1007/s10499-025-01824-0\u003c/li\u003e\n\u003cli\u003eSun L, Zha J, Spear PA, Wang Z. Tamoxifen effects on the early life stages and reproduction of Japanese medaka (Oryzias latipes). Environ Toxicol Pharmacol [Internet]. 2007 Jul;24(1):23\u0026ndash;9. Available from: https://linkinghub.elsevier.com/retrieve/pii/S138266890700021X\u003c/li\u003e\n\u003cli\u003eMaradonna F, Batti S, Marino M, Mita DG, Carnevali O. Tamoxifen as an Emerging Endocrine Disruptor. Ann N Y Acad Sci [Internet]. 2009 Apr 27;1163(1):457\u0026ndash;9. Available from: https://nyaspubs.onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.2008.03653.x\u003c/li\u003e\n\u003cli\u003eSarasquete C, \u0026Uacute;beda-Manzanaro M, Ortiz-Delgado J. Soya isoflavones, genistein and daidzein, induce differential transcriptional modulation in the ovary and testis of zebrafish Danio rerio. Aquat Biol [Internet]. 2020 Apr 30;29:79\u0026ndash;91. Available from: https://www.int-res.com/abstracts/ab/v29/p79-91/\u003c/li\u003e\n\u003cli\u003eBharti S, Rasool F. Analysis of the biochemical and histopathological impact of a mild dose of commercial malathion on Channa punctatus (Bloch) fish. Toxicol Reports [Internet]. 2021;8:443\u0026ndash;55. Available from: https://linkinghub.elsevier.com/retrieve/pii/S2214750021000354\u003c/li\u003e\n\u003cli\u003eChalasani N, Bonkovsky HL, Fontana R, Lee W, Stolz A, Talwalkar J, et al. Features and Outcomes of 899 Patients With Drug-Induced Liver Injury: The DILIN Prospective Study. Gastroenterology [Internet]. 2015 Jun;148(7):1340-1352.e7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S001650851500311X\u003c/li\u003e\n\u003cli\u003eGAO FF, LV JW, WANG Y, FAN R, LI Q, ZHANG Z, et al. Tamoxifen induces hepatotoxicity and changes to hepatocyte morphology at the early stage of endocrinotherapy in mice. Biomed Reports [Internet]. 2016 Jan;4(1):102\u0026ndash;6. Available from: https://www.spandidos-publications.com/10.3892/br.2015.536\u003c/li\u003e\n\u003cli\u003eChen YM, Wang IL, Zhu XY, Chiu WC, Chiu YS. Red Clover Isoflavones Influence Estradiol Concentration, Exercise Performance, and Gut Microbiota in Female Mice. Front Nutr [Internet]. 2021 Apr 14;8. Available from: https://www.frontiersin.org/articles/10.3389/fnut.2021.623698/full\u003c/li\u003e\n\u003cli\u003eMuhammad NP, Nirmal T, Prabhakaran A, Varghese T. Phytoestrogens as Endocrine-Disrupting Agents in Aquaculture. In: Xenobiotics in Aquatic Animals [Internet]. Singapore: Springer Nature Singapore; 2023. p. 213\u0026ndash;31. Available from: https://link.springer.com/10.1007/978-981-99-1214-8_9\u003c/li\u003e\n\u003cli\u003eKim K hyuk, Moon H na, Yeo I kyu. RETRACTED ARTICLE: Sexual maturation inhibition using exemestane and tamoxifen in female olive flounder (Paralichthys olivaceous). Discov Anim [Internet]. 2024 May 27;1(1):4. Available from: https://link.springer.com/10.1007/s44338-024-00007-0\u003c/li\u003e\n\u003cli\u003eWilliams TD, Caunter JE, Lillicrap AD, Hutchinson TH, Gillings EG, Duffell S. Evaluation of the reproductive effects of tamoxifen citrate in partial and full life-cycle studies using fathead minnows ( Pimephales Promelas ). Environ Toxicol Chem [Internet]. 2007 Apr 1;26(4):695\u0026ndash;707. Available from: https://academic.oup.com/etc/article/26/4/695/7763458\u003c/li\u003e\n\u003cli\u003eOrias F, Bony S, Devaux A, Durrieu C, Aubrat M, Hombert T, et al. Tamoxifen ecotoxicity and resulting risks for aquatic ecosystems. Chemosphere [Internet]. 2015 Jun;128:79\u0026ndash;84. Available from: https://linkinghub.elsevier.com/retrieve/pii/S004565351500017X\u003c/li\u003e\n\u003cli\u003eBrown AC, Stevenson LM, Leonard HM, Nieves-Puigdoller K, Clotfelter ED. Phytoestrogens \u0026beta; -Sitosterol and Genistein Have Limited Effects on Reproductive Endpoints in a Female Fish, Betta splendens. Biomed Res Int [Internet]. 2014;2014:1\u0026ndash;7. Available from: http://www.hindawi.com/journals/bmri/2014/681396/\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":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Tamoxifen, Red clover extract, Trichogaster trichopterus, Reproductive toxicity, Endocrine disruption, Estrogen receptor modulators","lastPublishedDoi":"10.21203/rs.3.rs-7382781/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7382781/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground \u0026amp; Objectives:\u003c/h2\u003e\u003cp\u003eTamoxifen, a classic Selective Estrogen Receptor Modulator (SERM), regulates estrogen signaling in a tissue-dependent manner. Phytoestrogenic compounds such as red clover (Trifolium pratense), which activate estrogenic pathways via receptor agonism, are increasingly explored for their potential in hormone-related conditions. This study aimed to compare the endocrine and hepatic effects of tamoxifen and red clover extract in a validated vertebrate model, the female three-spot gourami (\u003cem\u003eTrichogaster trichopterus\u003c/em\u003e).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA total of 120 adult female gourami were randomly assigned to eight groups, receiving intramuscular injections of tamoxifen (10, 50, 100 mg/kg), red clover extract (25, 75, 150 mg/kg), vehicle, or no treatment over 18 days. Reproductive (GSI, hormone levels, ovarian histology) and hepatic (HSI, ALT/AST levels, liver histology, TEM) parameters were assessed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eTamoxifen induced dose-dependent reductions in gonadosomatic index and levels of estradiol, progesterone, and testosterone, accompanied by elevated ALT and AST and histopathological liver changes. In contrast, red clover extracts increased GSI and sex hormones without hepatic damage. Histological and ultrastructural analyses confirmed arrested ovarian development and hepatic degeneration in tamoxifen-treated fish, while red clover\u0026ndash;treated fish showed follicular maturation and preserved liver architecture.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe opposing profiles of tamoxifen and red clover underscore their distinct estrogen-modulatory mechanisms. Tamoxifen\u0026rsquo;s anti-estrogenic and hepatotoxic actions raise concerns about long-term use, while red clover demonstrated favorable reproductive stimulation and hepatic safety. These findings support red clover\u0026rsquo;s potential as a safer phytoestrogenic alternative for hormone regulation and provide a foundation for future translational research in women\u0026rsquo;s health.\u003c/p\u003e","manuscriptTitle":"Comparative Evaluation of Tamoxifen and Red Clover Extract on Reproductive and Hepatic Function in Female Trichogaster trichopterus: A Translational Endocrine Toxicology Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-26 10:16:48","doi":"10.21203/rs.3.rs-7382781/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-05T21:05:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-16T01:57:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T15:19:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201301850143486779111826540984556126976","date":"2025-08-23T19:09:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211913059009594073736165232217493568447","date":"2025-08-23T11:06:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"18305446267208335841327196864694927836","date":"2025-08-22T11:33:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212091101144581317366271645913842089296","date":"2025-08-18T10:41:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-18T08:17:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-16T14:40:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-16T05:27:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Histology","date":"2025-08-15T16:03:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-histology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hijo","sideBox":"Learn more about [Journal of Molecular Histology](https://www.springer.com/journal/10735)","snPcode":"10735","submissionUrl":"https://submission.springernature.com/new-submission/10735/3","title":"Journal of Molecular Histology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1efd49d8-fb04-42bc-9fd0-7ac22172835e","owner":[],"postedDate":"August 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:12:06+00:00","versionOfRecord":{"articleIdentity":"rs-7382781","link":"https://doi.org/10.1007/s10735-026-10727-0","journal":{"identity":"journal-of-molecular-histology","isVorOnly":false,"title":"Journal of Molecular Histology"},"publishedOn":"2026-01-31 15:59:09","publishedOnDateReadable":"January 31st, 2026"},"versionCreatedAt":"2025-08-26 10:16:48","video":"","vorDoi":"10.1007/s10735-026-10727-0","vorDoiUrl":"https://doi.org/10.1007/s10735-026-10727-0","workflowStages":[]},"version":"v1","identity":"rs-7382781","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7382781","identity":"rs-7382781","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}