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Curcuma longa, Methamphetamine, Histomorphometric parameters, Reticular fibers, Seminiferous tubules, Antioxidants, Male infertility
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Elum Fischer C, Otu Otu Ee, Abang Agbor C et al. Testicular Histomorphometric Parameters, Reticular Fibres Expression and antioxidant markers Assessment on Testicular Tissue of Methamphetamine-Exposed Wistar Rats Treated with Curcuma longa [version 1; peer review: 1 approved with reservations]. F1000Research 2026, 15:57 (https://doi.org/10.12688/f1000research.175565.1)
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Research Article
Testicular Histomorphometric Parameters, Reticular Fibres Expression and antioxidant markers Assessment on Testicular Tissue of Methamphetamine-Exposed Wistar Rats Treated with Curcuma longa
[version 1; peer review: 1 approved with reservations]
PUBLISHED 14 Jan 2026
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Abstract
Corresponding author:
Ekementeabasi Aniebo Umoh
Competing interests:
No competing interests were disclosed.
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The author(s) declared that no grants were involved in supporting this work.
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© 2026 Elum Fischer C et al.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Elum Fischer C, Otu Otu Ee, Abang Agbor C et al. Testicular Histomorphometric Parameters, Reticular Fibres Expression and antioxidant markers Assessment on Testicular Tissue of Methamphetamine-Exposed Wistar Rats Treated with Curcuma longa [version 1; peer review: 1 approved with reservations]. F1000Research 2026, 15:57 (https://doi.org/10.12688/f1000research.175565.1)
First published: 14 Jan 2026, 15:57 (https://doi.org/10.12688/f1000research.175565.1)
Latest published: 14 Jan 2026, 15:57 (https://doi.org/10.12688/f1000research.175565.1)
1. Introduction
The escalating global prevalence of substance dependence and abuse, particularly among adolescents and young adults at reproductive age, poses a significant threat to male fertility.1,2 Methamphetamine (METH), a potent psychostimulant with addictive and neurotoxic properties, ranks among the most abused drugs worldwide, contributing to rising rates of male infertility.3,4 In Nigeria, its abuse under local monikers like mkpuru mmiri reflects a growing public health crisis, with detrimental socioeconomic and reproductive consequences.5 Studies indicate that chronic methamphetamine use disrupts male reproductive function through oxidative stress, hormonal imbalances, and structural damage to testicular tissue, leading to reduced sperm count, motility, and viability.6,7 These alterations, including apoptosis in seminiferous tubules and impaired spermatogenesis, underscore the urgent need for interventions to mitigate drug-induced infertility.8
The resurgence of herbal medicine as a viable alternative to conventional treatments aligns with the World Health Organization’s advocacy for integrating plant-based therapies into primary healthcare.9,10 In recent times, studies have found herbal remedies useful in ameliorating infertility caused by exposure to cadmium.11,12 Antimalarial treatment using herbal remedies such as Mangifera indica has also proven a better remedy as aftermath of reproductive disorder wasn’t associated with malaria treatment using this herb.13 Even in other aspect of physiological systems, herbs have shown notable result in improving health. Ameliorating ulcer and liver disorders has been associated to ginger extract14,15 and locally prepared Tom brown weaning meal.16 Medicinal plants like Curcuma longa (turmeric), renowned for their antioxidant and anti-inflammatory properties, offer promising avenues for addressing oxidative stress-related pathologies, including reproductive dysfunction.17 Turmeric’s active compound, curcumin, demonstrates potent antioxidant activity and has been shown to enhance sperm function in vitro, improving motility and fertilization capacity.18 Despite its safety profile and therapeutic potential, its application in mitigating drug-induced testicular damage remains underexplored, particularly in the context of methamphetamine exposure.
Methamphetamine-induced oxidative stress disrupts the redox balance in gonadal tissues, overwhelming endogenous antioxidants like superoxide dismutase (SOD) and glutathione peroxidase (GPx), and triggering lipid peroxidation (LPO) and cellular damage.19,20 This oxidative milieu compromises sperm quality and testicular architecture, with histopathological changes such as reticular fiber proliferation around seminiferous tubules further impairing spermatogenesis.21 Histo-morphometric analysis of testicular tissue, including parameters like tubular diameter and epithelial height, alongside reticular fiber density, provides critical insights into structural and functional alterations caused by toxicants.22 Curcuma longa has been shown to counteract the neurotoxic effects of METH and other drug stimulants on different parts of the brain, by aiding autophagy in the cells, modulation of inflammatory cytokines, maintenance of ion homeostasis, epigenetic regulation, enhancement of antioxidant capacity, as well as the activation of the cAMP response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) signaling pathways.23,24 Given that oxidative stress and inflammation are common underlying mechanisms of METH-induced toxicity in both brain and reproductive tissues,25,26 and considering the protective effects of Curcuma longa in the brain, it is plausible that Curcuma longa may also mitigate METH-induced oxidative stress and damage in testicular tissues, warranting further investigation in this area. Again, Curcumin, a polyphenol compound which is sourced from Curcuma longa has been found to be a good remedy for treating testicular cancer.27 Hence, there is possibility of Curcuma ameliorating any detrimental effect that may emanate from the use of Methamphetamine on the testis. Hence, the purpose of this research.
In this study, we investigate the protective effects of Curcuma longa on histo-morphometric parameters, reticular fiber expression and level of antioxidant markers in the testicular tissue of methamphetamine-exposed Wistar rats treated with graded doses of turmeric. By evaluating its capacity to counteract oxidative damage and restore testicular architecture, this research aims to advance the development of accessible herbal interventions for drug-induced male infertility.
2. Materials and methods
2.1 Preparation of extract
Fresh rhizomes of Curcuma longa were purchased from Watt Market, Calabar, Cross River State, Nigeria. The plant was authenticated by Mr. Effa Effa A, a taxonomist at the Department of Botany, University of Calabar, and assigned a voucher number (Bot/Herb/UCC/318). The rhizomes were washed, air-dried at room temperature, and pulverized into fine powder using a motorized blender. A cold maceration method was employed for extraction: 1500 g of powdered material was soaked in 4.5 L of absolute ethanol for 48 hours with periodic agitation. The mixture was filtered first with muslin cloth and then through Whatman No. 1 filter paper. The filtrate was concentrated using a water bath (35–40°C) to obtain a crude extract (127.9 g), which was stored at 4°C until use.
2.2 Procurement of crystal methamphetamine
The crystal methamphetamine used was procured with the necessary permits and approvals from the Nigeria Drug Law Enforcement Agency (NDLEA) Calabar command Cross River State, ensuring compliance with national laws and regulations governing the use of controlled substances in scientific research.
2.3 Experimental animals
Twenty-five (25) adult male Wistar rats (150–200 g) were procured from the Animal Facility of the College of Medical Sciences, University of Calabar. Animal handling was done according to ARRIVE guidelines. The rats were housed in ventilated plastic cages under standard laboratory conditions (25°C, 50–60% humidity, 12/12-hour light-dark cycle) with ad libitum access to feed and water. Bedding material (sawdust) was replaced daily to maintain hygiene. Ethical approval (320ANA2924) was obtained from the Faculty Animal Research Ethics Committee (FAREC-FBMS), University of Calabar, in compliance with institutional and national guidelines.
2.4 Acute toxicity study
The median lethal dose (LD50) of methamphetamine was determined using Lorke’s three-phase method. In the initial phase, doses of 0.05, 0.15, 0.25, and 0.50 mg/kg were administered to four groups (n = 3 rats/group). Subsequent phases tested higher doses (0.75–50.00 mg/kg). No mortality or acute toxicity (e.g., hyperactivity, defecation) was observed. The LD50 was calculated as the geometric mean of the highest non-lethal dose (10.00 mg/kg) and the lowest lethal dose (20.00 mg/kg).
2.5 Experimental design
After a 14-day acclimatization period, rats were randomly divided into five groups (n = 5/group) through balloting:
− Group 1 (Normal Control): Received distilled water.
− Group 2 (Methamphetamine-Only): Administered methamphetamine (5 mg/kg b.w. orally).
− Groups 3–5 (Meth + C. longa): Received methamphetamine (5 mg/kg b.w.) alongside graded doses of C. longa extract (100, 200, and 400 mg/kg b.w., respectively).
Treatments were administered daily for 28 days orally using orogastric tubes.
2.6 Termination and sample collection
After fasting overnight, rats were humanely euthanized under deep anesthesia induced by intraperitoneal administration of ketamine (dose: 200 mg/kg body weight). Death was confirmed by cervical dislocation and absence of vital signs (respiratory arrest, loss of corneal reflex, and absence of heartbeat). Blood was collected via cardiac puncture and centrifuged (3000 rpm, 10 min) to separate serum, which was stored at −20°C. Testes were excised, weighed, and fixed in 10% buffered formalin for histological processing.
2.7 Histomorphometric analysis
Testicular tissues were dehydrated through a graded ethanol series, embedded in paraffin, and sectioned (5 μm) using a rotary microtome. Sections were stained with hematoxylin and eosin (H&E) using a standard protocol. Photomicrographs with calibrated scales were captured using a light microscope equipped with a digital camera. Images were analyzed using ImageJ software (NIH, USA) to measure seminiferous tubule diameter and germinal epithelial height.
2.8 Reticular fiber staining
Reticular fibers were visualized using Gomori’s methenamine silver stain (Sigma-Aldrich) according to the manufacturer’s instructions. Deparaffinized sections were oxidized in 0.5% periodic acid (15 min), incubated in methenamine silver solution (60°C, 1 h), toned with gold chloride (0.2% solution, 1 min), and counterstained with nuclear fast red (5 min). Reticular fibers appeared black against a pink background. Sections were examined under a light microscope and photomicrographs were captured using a digital camera.
2.9 Oxidative stress assay
Superoxide Dismutase (SOD) activity was determined by its ability to inhibit the auto-oxidation of epinephrine, measured by the increase in absorbance at 480 nm.28 The reaction mixture contained 2.95 ml of 0.05 M sodium carbonate buffer (pH 10.2), 0.02 ml of the sample, and 0.03 ml of epinephrine, with hydrochloric acid used to initiate the reaction. Enzyme activity was calculated by measuring the change in absorbance at 480 nm for 5 minutes. Catalase activity was determined by measuring the decrease in absorbance at 620 nm due to H2O2 decomposition.29 The reaction mixture included 1.0 ml of 0.01 M phosphate buffer (pH 7.0), 0.1 ml of the sample, and 0.4 ml of 2 M H2O2, and was stopped with dichromate-acetic acid reagent. Lipid peroxidation was assessed via malondialdehyde (MDA) levels using the method of Buege and Aust (1978), with absorbance read at 532 nm. Glutathione peroxidase (GPx) activity was measured by adding the sample to a reaction mixture containing Tris buffer, EDTA, sodium azide, GSH, and H2O2, and expressed as μg of GPx consumed/min/mg protein.30
3. Results
3.1 Reticular fibre and basement membrane examination
Methenamine Silver (Gomori PAM) staining of testicular tissue in the Control Group (Group 1) revealed moderate reticular fibre expression with intact basement membranes and minimal formation around seminiferous tubules (Figure 1), indicative of normal testicular architecture. In contrast, the Methamphetamine-Only Group (Group 2) exhibited markedly increased reticular fibre density, characterized by thickened fibres encircling the basement membrane of seminiferous tubules (Figure 2), suggesting methamphetamine-induced structural alterations. The Low Dose Group (Group 3), treated with methamphetamine and 100 mg/kg b.w. C. longa, demonstrated moderate reticular fibre expression with preserved basement membrane integrity (Figure 3), indicating partial attenuation of drug-induced changes. Further reduction in fibre density was observed in the Medium Dose Group (Group 4), where reticular fibre formation around tubules decreased significantly compared to Group 2 (Figure 4). Notably, the High Dose Group (Group 5) showed the most pronounced protective effect, with sparse reticular fibre expression and intact basement membranes resembling near-normal morphology (Figure 5).
Figure 1. Testis control (Silver Nitrate stain X 400).
Photomicrograph testes showing moderate expression of reticular fibers (RF). Intact basement membrane (BM) and decreased formation of reticular fiber around the seminiferous tubules (ST).
Figure 2. Testis methamphetamine (Silver Nitrate stain X 400).
Photomicrograph of testes showing increased expression of reticular fibers (RF). Basement membrane (BM) of seminiferous tubules (SM) is surrounded by thickened reticular fiber (RF).
Figure 3. Testis low dose (Silver Nitrate stain X 400).
Photomicrograph of testes showing moderate expression of reticular fibers (RF). Basement membrane (BM) surrounding seminiferous tubules (ST) not intact.
Figure 4. Testis medium dose (Silver Nitrate stain X 400).
Photomicrograph of the testis, showing moderate expression of reticular fibers.
Figure 5. Testis high dose (Silver Nitrate stain X 400).
Photomicrograph of the testis, showing less expression of reticular fibers. Intact basement membrane and decreased formation of reticular fibers around the seminiferous tubules.
3.2 Seminiferous tubular diameter
The results revealed a significant increase (p < 0.05) in seminiferous tubular diameter in the Methamphetamine-Only Group (315.00 ± 10.18 μm) compared to the Control Group (283.39 ± 5.63 μm). Tubular diameters in the treatment groups exhibited a dose-dependent reduction, with the Low Dose (294.24 ± 9.27 μm) and Medium Dose (270.52 ± 12.30 μm) groups showing a decrease, while the High Dose Group (247.35 ± 4.40 μm) demonstrated a significant decrease (p < 0.05) relative to both the Methamphetamine-Only and Control groups ( Table 1, Figure 6).
Table 1. Histomorphometric analysis of the seminiferous tubules of the testis.
| Groups | Seminiferous tubular diameter (μm) | Seminiferous epithelial height (μm) |
|---|---|---|
| Control group (n = 50) | 283.39 ± 5.63 | 95.17 ± 4.56 |
| Methamphetamine group (n = 50) | 315.00 ± 10.18a | 98.46 ± 7.24 |
| Low dose group (n = 50) | 294.24 ± 9.27 | 97.21 ± 3.94 |
| Medium dose group (n = 50) | 270.52 ± 12.30b | 97.05 ± 5.37 |
| High dose group (n = 50) | 247.35 ± 4.40a,b,c | 81.63 ± 2.87 |
| p-value | <0.001 * | 0.099 |
Figure 6. Comparison of diameter of the seminiferous tubules of the testes in the different groups.
Values are expressed as mean ± SEM, n = 50 for each group.
a = significantly different from control group.
b = significantly different from Methamphetamine group.
c = significantly different from low dose group.
d = significantly different from medium dose group.
3.3 Germinal epithelial height
From the result, the seminiferous epithelial height (μm) for the various groups were; 95.17 ± 4.56 for Control group, 98.46 ± 7.24 for Methamphetamine group, 97.21 ± 3.94 for Low dose group, 97.05 ± 5.37 for Medium dose group, and 81.63 ± 2.87 for High dose group. There was no significant (p > 0.05) differences in the seminiferous epithelial height among the various groups ( Table 1, Figure 7).
Figure 7. Comparison of the epithelial height of the seminiferous tubules of the testes in the different groups.
Values are expressed as mean ± SEM, n = 50 for each group.
3.4 Effect of different doses of Curcuma longa extracts on oxidative stress markers
Results obtained on the level of oxidative stress markers showed a significant (p > 0.05) increase in the activities of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and glutathione peroxidase (GPx) in the methamphetamine group, compared to the normal control. On administration of the low, medium & high doses of Curcuma longa extract, the antioxidant enzymes concentrations were observed to have reduced significantly (p < 0.05) when compared to the Methamphetamine group (see Table 2).
Table 2. Testicular tissue antioxidant level.
| Groups | SOD (μmol/mL) | CAT (μmol/mL) | MDA (μmol/mL) | GPx (μmol/mL) |
|---|---|---|---|---|
| Control group (n = 5) | 26.74 ± 2.40 | 49.64 ± 0.12 | 5.01 ± 0.07 | 4.13 ± 0.04 |
| Methamphetamine group (n = 5) | 55.25 ± 7.01a | 89.22 ± 4.06 | 13.74 ± 1.49a | 5.42 ± 0.31 |
| Low dose group (n = 5) | 41.14 ± 1.49a | 69.52 ± 16.41 | 10.49 ± 1.28a | 4.25 ± 0.81 |
| Medium dose group (n = 5) | 38.72 ± 4.50b | 75.36 ± 18.02 | 8.16 ± 2.03b | 4.45 ± 0.44 |
| High dose group (n = 5) | 31.94 ± 0.33b | 55.56 ± 3.68 | 8.23 ± 0.15b | 4.16 ± 0.04 |
| p-value | 0.024 * | 0.153 | 0.032 * | 0.317 |
4. Discussion
This study investigated the protective effects of Curcuma longa on histomorphometric parameters and reticular fiber expression in the testicular tissue of methamphetamine-exposed Wistar rats. The findings revealed that methamphetamine exposure significantly increased reticular fiber density around seminiferous tubules and altered tubular diameter, while C. longa administration attenuated these changes in a dose-dependent manner. The thickening and proliferation of reticular fibers observed in the methamphetamine-only group align with previous reports linking psychostimulants to extracellular matrix remodeling and basement membrane disruption.31 Such structural alterations likely impair spermatogenesis by destabilizing the microenvironment required for germ cell differentiation and migration.32 The reduction in reticular fiber density in C. longa-treated groups, particularly at high doses (400 mg/kg b.w.), underscores the plant’s capacity to mitigate methamphetamine-induced fibrosis. This protective effect may be attributed to curcumin, the primary bioactive compound in turmeric, which exhibits potent antioxidant and anti-inflammatory properties.33 By scavenging reactive oxygen species (ROS) and inhibiting lipid peroxidation, curcumin likely preserves basement membrane integrity and prevents collagen deposition, thereby restoring testicular architecture. Notably, methamphetamine exposure caused a significant increase in seminiferous tubular diameter, consistent with apoptosis-related pathological changes such as membrane blebbing and vesicle expansion.34 The dose-dependent reduction in tubular diameter observed in C. longa-treated groups suggests its anti-apoptotic efficacy. Curcumin’s ability to modulate pro-apoptotic pathways (e.g., caspase-3 inhibition) and enhance endogenous antioxidant defenses (e.g., SOD, GPx) may counteract methamphetamine-induced germ cell loss.35 The antioxidant effect of C. longa was further proven by the significant reduction in tissue antioxidant markers in the curcumin-treated group compared to the methamphetamine group, which caused an increase in antioxidant markers, further supporting the antioxidant effects of the plant as noted in other studies.33 However, the absence of significant differences in germinal epithelial height across groups may reflect the study’s shorter duration (28 days) compared to long-term interventions in other studies.36 The ameliorative effects of C. longa align with its historical use in traditional medicine for enhancing male fertility. Its rich phytochemical profile, including curcuminoids, flavonoids, and phenolics, synergistically combats oxidative stress and inflammation, key drivers of drug-induced testicular damage.37 These findings reinforce the potential of herbal therapies as adjunct treatments for substance abuse-related infertility.
5. Limitation of study
Study was limited to assessing only histomorphometric parameters and recticular fiber density. The bioinformatics study of various extract and its subsequent effect on other reproductive parameters will be a novel approach to future research studies.
6. Conclusion
This study demonstrates that methamphetamine exposure induces detrimental structural changes in testicular tissue, including reticular fiber proliferation and seminiferous tubular dilation. Curcuma longa extract, particularly at higher doses, effectively mitigates these alterations, likely through its antioxidant and anti-apoptotic mechanisms. These results highlight turmeric’s potential as a therapeutic agent for addressing methamphetamine-associated male infertility. Further clinical studies are warranted to validate its efficacy in humans.
Ethics approval and consent to participate
Ethical approval was obtained from the Faculty Animal Research Ethics Committee (FAREC-FBMS), University of Calabar, Nigeria (Certificate No. 320ANA2924), in compliance with national and institutional guidelines for the humane use of laboratory animals. The ethics approval granted by the Faculty Animal Research Ethics Committee (FAREC-FBMS), University of Calabar, Nigeria included specific restrictions on data sharing, which are detailed in the Data and Material Availability Statement. All the work in the experiment was done according to ARRIVE guidelines.
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Elum Fischer C, Otu Otu Ee, Abang Agbor C et al. Testicular Histomorphometric Parameters, Reticular Fibres Expression and antioxidant markers Assessment on Testicular Tissue of Methamphetamine-Exposed Wistar Rats Treated with Curcuma longa [version 1; peer review: 1 approved with reservations]. F1000Research 2026, 15:57 (https://doi.org/10.12688/f1000research.175565.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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iqbal t. Reviewer Report For: Testicular Histomorphometric Parameters, Reticular Fibres Expression and antioxidant markers Assessment on Testicular Tissue of Methamphetamine-Exposed Wistar Rats Treated with Curcuma longa [version 1; peer review: 1 approved with reservations]. F1000Research 2026, 15:57 (https://doi.org/10.5256/f1000research.193561.r451222)
The direct URL for this report is:
https://f1000research.com/articles/15-57/v1#referee-response-451222
https://f1000research.com/articles/15-57/v1#referee-response-451222
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 20 Jan 2026
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This study investigates Curcuma longa's protective effects against methamphetamine-induced testicular damage in rats. Results show dose-dependent amelioration of histomorphometric alterations (tubular diameter, reticular fiber density) and oxidative stress markers, supporting turmeric's therapeutic potential for drug-associated male infertility.
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iqbal t. Reviewer Report For: Testicular Histomorphometric Parameters, Reticular Fibres Expression and antioxidant markers Assessment on Testicular Tissue of Methamphetamine-Exposed Wistar Rats Treated with Curcuma longa [version 1; peer review: 1 approved with reservations]. F1000Research 2026, 15:57 (https://doi.org/10.5256/f1000research.193561.r451222)
The direct URL for this report is:
https://f1000research.com/articles/15-57/v1#referee-response-451222
https://f1000research.com/articles/15-57/v1#referee-response-451222
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Author Response 22 Jan 2026Ekementeabasi Umoh, Department of Physiology, Kampala International University - Western Campus, Bushenyi, Uganda22 Jan 2026Author Response(Replicability (Partly): While methods are detailed, legal restrictions on data sharing hinder reproducibility. Authors should provide a detailed, anonymized data availability plan or synthetic datasets.)
Reviewers comments are explicit. ... Continue reading
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Author Response 22 Jan 2026Ekementeabasi Umoh, Department of Physiology, Kampala International University - Western Campus, Bushenyi, Uganda22 Jan 2026Author Response(Replicability (Partly): While methods are detailed, legal restrictions on data sharing hinder reproducibility. Authors should provide a detailed, anonymized data availability plan or synthetic datasets.)
Reviewers comments are explicit. ... Continue reading
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