The plant material used in this study comprised Manihot esculenta (M. esculenta) leaves, which were procured from a local vegetable market in the commune of Abomey-Calavi and authenticated at the National Herbarium of the University of Abomey-Calavi (Herbier national, UAC, Benin).

Sixty (60) adult male Wistar rats (Rattus norvegicus), weighing 150 - 200 g, were obtained from the Department of Animal Production and Health at the Institute of Applied Biomedical Sciences (ISBA), University of Abomey-Calavi (UAC, Benin), for the experiments. The animals were housed under standard laboratory conditions in well-ventilated cages with a 12-hour light/dark cycle. The rats were allowed to acclimatize to the laboratory environment for 3 weeks before the start of the experiments. During this period, they were provided with a standard rodent diet and water ad libitum. All procedures involving animals were conducted in compliance with the ethical guidelines for the care and use of laboratory animals.

Ethanolic extraction was performed following the protocol of the Laboratory of Pharmacognosy and Essential Oils at the Faculty of Science and Technology (FAST, UAC, Benin). Young leaves were dried at room temperature (16˚C) in the open air, protected from light and heat, for approximately two to three weeks before being ground into powder. The ethanolic extract was prepared by macerating the powdered material in 96% ethanol for 72 hours. The mixture was stirred twice daily, and the resulting macerate was filtered using Whatman paper and evaporated under reduced pressure at 40˚C until a dry extract of constant weight was obtained. The extraction yield (r) was calculated using the formula:

$\text{r}=\frac{\text{Mass of extract}}{\text{Mass of powder before extraction}}\times 100$.

Phytochemical screening was conducted based on differential reactions (coloration and precipitation) of major chemical groups present in plants, following the method described by Houghton and Raman [14] , as adapted to the Laboratory of Pharmacognosy and Essential Oils’ conditions. The presence of the following compounds was assessed: alkaloids, tannins, flavonoids, anthocyanins, saponosides, triterpenoids, steroids, cardenolides, mucilage, coumarins, quinone derivatives, cyanogenic derivatives, reducing compounds, and anthraquinone derivatives.

Male Wistar rats were divided into two groups of 30 rats each (Treated and Control groups) and were allowed three weeks to acclimate to the animal facility conditions at the Non-Communicable Diseases and Cancer Research Unit. This size was calculated based on group comparison of one-way ANOVA test using Degree of Freedom as reported by Charan and Kantharia [15] . The extracts were dosed based on the body weight of each rat at 200 mg per kg and administered to the treated group orally in 1 mL of distilled water daily according to the method described by Abu et al. [16] .

Throughout the study, all male rats were provided with a free diet of pellets and water ad libitum. The effects of the extract were assessed at 6 and 12 weeks, focusing on body weight, testicular weight, serum testosterone levels, testicular histology, and sperm parameters in both the Treated and Control groups.

Weekly body weight measurements were conducted using an electronic scale. At the start of the study (W0), the rats were weighed, and their body weight was monitored weekly up to the twelfth week (W12) of the experiment. After six weeks of extract administration, half of the representative rats were sacrificed, while the remaining half were sacrificed at the end of week 12. Testes were collected at the beginning (W0), after six weeks (W6), and at the end of the study (W12) through laparotomy in both control and treated groups.

Testicular weight was measured using Archimedes’ principle, based on the volume (mL) of physiological saline solution (0.9% NaCl) displaced by each testis. The displaced volume was converted to mass (mg) to accurately estimate testicular weight. These measurements of body and testicular weight were further utilized to calculate the gonadosomatic index (GSI), defined as the ratio of the testicular weight to the total body weight, expressed as a percentage.

Blood samples were collected from rats through retro-orbital bleeding following an overnight fast. Plasma obtained from fluoride tubes was used for glucose testing, while serum was used for the measurement of other parameters. The following biomarkers were analyzed using an automated biochemical analyzer and reagent kits from ELITech (ELITech Group, France): fasting blood glucose, triglycerides, total cholesterol, and high-density lipoprotein cholesterol, considering their impact on spermatogenesis [17] . Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured to assess liver function, while creatinine and urea were determined to monitor the effect of treatments on kidney function. Testosterone levels were measured using an ELISA method with a kit supplied by Bio-Techne (Bio-Techne, Abingdon, UK).

Male Wistar rats were anesthetized, and an abdominal incision was performed to expose the reproductive organs. The caudal epididymis was carefully excised using sterile scissors and placed in a Petri dish containing 10 mL of physiological saline solution (0.9% NaCl). The method of Akpovi et al., with slight modification, was used to determine the number of sperm in the epididymis [18] . The tissue was finely minced to release spermatozoa, and the suspension was incubated under orbital agitation at room temperature (approximately 24˚C) for 30 minutes to ensure optimal dispersion. Following incubation, the suspension was transferred to a sterile test tube and centrifuged at 250 g for 15 minutes. The spermatozoa formed a pellet at the bottom of the tube, while the supernatant was carefully discarded. The pellet was resuspended in 1 mL of 0.9% NaCl solution, creating the final suspension used for counting.

Spermatozoa counting was performed using a Malassez hemocytometer. A 10 µL aliquot of the suspension was placed into the hemocytometer chamber, covered with coverslip to ensure no air bubbles were present. After allowing the suspension to stabilize for 3 to 5 minutes, spermatozoa were observed under a light microscope at 40× magnification. The sperm concentration was calculated using standard World Health Organization protocols and expressed as 10⁶ sperm/mm³ [19] .

Rats were sacrificed, and their testicles were immediately removed and fixed in a 10% formalin solution. After rinsing with water, the testicles were dehydrated through a graded series of ethanol concentrations (70%, 95%, and 100%) and embedded in paraffin wax. The paraffin-embedded testicles were carefully positioned in cassettes to ensure proper orientation. Tissue sections were prepared by slicing the paraffin blocks into 6-µm-thick sections. The histological sections were subsequently stained using hematoxylin and eosin (HE) for microscopic examination.

Data were recorded using Microsoft Excel 2016, and statistical analyses were conducted using SigmaPlot version 14. Results are expressed as the mean ± standard error of the mean (SEM). Statistical tests included one-way analysis of variance (ANOVA) for parametric data and the Kruskal-Wallis ANOVA for non-parametric data. Post-hoc multiple comparison analyses, including Dunnett’s test, were applied to compare the exposed groups with the control. A p-value of less than 0.05 was considered statistically significant.

The ethanolic extract of M. esculenta leaves was obtained as a fractionable dark-green solid, weighing 29 g from an initial mass of 351 g prior to extraction. The extraction yield was calculated to be 8.26%.

The phytochemical screening of M. esculenta leaves showed the presence of alkaloids, tannins (catechin and gallic tannins), flavonoids, leuco-anthocyanins, quinone derivatives, saponosides, mucilage, reducing compounds, C-glycosides, and coumarins. In contrast, free and combined anthraquinones, O-glycosides, reduced O-glycosides, terpenoids, steroids, cyanogenic derivatives, and cardenolides were absent ( Table 1 ).

The table summarizes the presence (+) or absence (−) of various phytochemical compounds identified in ethanolic leaf extracts of Manihot esculenta.

The average body weights of the rats in the treated group receiving M. esculenta extracts compared to the control group over time were monitored ( Figure 1 ). Results showed no significant differences in the body weight of the treated rats compared to the control group throughout the study from week zero (W0) to week 12 (W12) (p > 0.5) ( Figure 1 ).

Consistent with the body and testicular weights, the mean gonadosomatic index showed no significant differences in treated group compared to control at 6 weeks (p = 0.87) and 12 weeks (p = 0.53) ( Figure 2 ).

Table 2 provides an overview of the biochemical parameters measured before and throughout the treatment period. Before the initiation of treatment, there were no significant differences observed between the groups across all measured biochemical parameters, indicating baseline equivalence. However, after 12 weeks of treatment with M. esculenta extracts, significant increases were recorded in glucose levels (p = 0.015) and LDL cholesterol (p = 0.001) in the treated group compared to the control group ( Table 2 ).

Other biochemical parameters, including urea, creatinine, ALAT, ASAT, total cholesterol, HDL cholesterol, triglycerides, and total proteins, remained unchanged throughout the 12-week treatment period, showing no statistically significant differences between the groups ( Table 2 ).

Biochemical parameters are measured in control and treated rats at Week 6 and Week 12 of treatment. Values are expressed as mean ± standard deviation (SD). The number of rats per group (n) is indicated.

Serum testosterone levels were comparable in both treated and control groups before treatment ( Table 3 ). However, a significant decrease was observed in treated group after 6 weeks (p < 0.04) and 12 weeks (p < 0.005) of treatment compared to the control group ( Table 3 ).

Values are presented as mean ± standard error of the mean (SEM) of serum testosterone levels expressed in ng/mL. The number of rats tested in each group (n) at the respective time points is indicated. Comparisons were made between the control group, which received distilled water, and the treated group, which received 200 mg/kg of M. esculenta extract. Significant reductions in testosterone levels were observed in the treated group at Week 6 (p = 0.040) and Week 12 (p = 0.005). No significant difference was noted at baseline (Week 0, p = 0.630).

The epididymal spermatozoa level in the treated group was compared to the control group over the course of the study ( Figure 3 ). Results showed a significant reduction in sperm count in treated groups at week 6 of treatment (p < 0.005) compared to the control group ( Figure 3 ). The decrease became even more pronounced after 12 weeks (p < 0.001) ( Figure 3 ). This progressive decline suggests a significant adverse effect of M. esculenta extracts on spermatogenesis, likely impacting sperm production and/or maturation in the epididymis.

Representative pictures of histological sections of testis of control and treated rats at baseline (A), after 6 weeks (B), and after 12 weeks (C) are shown in Figure 4 .

At baseline ( Figure 4(A) ), the seminiferous tubules of control rats displayed normal spermatogenesis, characterized by the presence of germinal cells at various stages of development, including spermatogonia, spermatocytes I and II, spermatids, and spermatozoa. The seminiferous epithelium was densely populated with spermatozoa, filling the lumen of the tubules.

At 6 weeks ( Figure 4(B) ), the treated group showed evidence of complete spermatogenesis. However, there was a notable reduction in the concentration of spermatozoa within the seminiferous tubule lumen compared to the control group. Additionally, partial deformation of some seminiferous tubules was observed in the treated rats ( Figure 4(B) ).

By 12 weeks ( Figure 4(C) ), the treated group exhibited a significant decline in the concentration of spermatozoa within the seminiferous tubule lumen. This reduction was more pronounced compared to both the control group and the 6-week treated group, indicating progressive disruption of spermatogenesis over the treatment period ( Figure 4(C) ).