The project was presented and validated by the scientific committee of the Department of Animal Biology, University of Dschang, which follows the internationally accepted standard ethical guidelines for laboratory animal use and care.

Commercial IMI (Colibri®: 30% IMI and 70% inactive ingredients) was purchased from the Dschang market (Sun Valley Hall Limited-Hong Kong; Bach N˚ SVH161104) and, the working solution prepared in distilled water. Methanol and Anhydrous sodium sulfate were purchased from Sigma Chemical Co., Germany. Assay kits for testosterone, LH and FSH (Accubind, Monobind. Lake Forest, USA) were used according to the manufacturer’s instructions. All other chemicals and reagents were of analytical grade and purchased from local suppliers.

The stem barks of L. acida were received from the Alango Foundation (Dschang, Cameroon) and shade-dried. The dried material was reduced into powder for extract’s preparation. Aqueous and methanol extracts were prepared as previously described [19] . Briefly, the aqueous extract was prepared by decoction of 250 g of plant powder in 1.5 L of boiled distilled water for 10 minutes. The solution was then filtered at room temperature and the filtrate was oven-dried to obtain a brown powder. The methanol extract was prepared by maceration of 250 g of the powder of L. acida in 1 L of methanol for 72 h at room temperature. The product was then filtered and the filtrate was evaporated under reduced pressure and oven-dried to obtain a brown powder representing the methanol extract.

In our previous study, the aqueous and methanol extracts of L. acida at 340 mg/kg and 170 mg/kg, respectively, were the most active plant doses in alleviating sperm, hormonal, oxidative and testis histological impairments induced by IMI and were chosen for this study [19] . Moreover, the same doses of IMI (1/20 of the LD₅₀: 22.5 mg/kg bw), clomiphene citrate and vitamin E were used as described in our previous study. The working solution of IMI was freshly prepared by mixing 1.5 mL of the commercial IMI to 18.5 mL of distilled water to obtain 20 mL of IMI solution at 2.25 mg/mL. For aqueous extract solution, 680 mg of the extract powder were dissolved in 14 mL of distilled water. The volume of the homogenized mixture was then adjusted to 20 mL to obtain a concentration of 34 mg/mL. The preparation of the methanol solution followed the same procedure using 340 mg of methanol extract powder. All working solutions were freshly prepared and administered at a volume of 10 mL/kg.

Adult male (30) and female (60) Wistar rats weighing 150 - 200 g were obtained from the animal house of the Department of Animal Biology, Faculty of Science of University of Dschang, Cameroon. They were acclimated in plastic cages (4/cage) at room temperature with a natural light/dark cycle for 2 weeks before experiments. Animals had free access to standard rat diet and tap water throughout the experiment.

The induction of the reproductive toxicity using IMI was done as previously described [19] . 25 male rats were daily administered 22.5 mg/kg of IMI for two consecutive weeks. The weight of animals was monitored every 3 days and IMI solution volume was adjusted accordingly. 5 other rats constituted the control group and received distilled water (10 mL/kg) throughout the experiment.

After two weeks of repeated oral exposure to IMI, 25 sexually experienced male rats were randomly divided into 5 groups of 5 animals each. Group 1 was the negative control animals and received distilled water (IMI + DW). Groups 2 and 3 were the positive control animals and were treated with clomiphene citrate (IMI + CC) and vitamin E (IMI + Vit E), respectively. Groups 4 and 5 were respectively treated with aqueous (IMI + AE) and methanol (IMI + ME) extract of L. acida. Control animals without IMI exposure received distilled water (Group 6) throughout the experiment. All treatments were orally given for 8 consecutive weeks, the time necessary for spermatogenesis achievement in rats.

The fertility test was conducted as described by [3] with minor adjustments. The test was performed during the last 10 days of treatments (days 47 to 56). For this purpose, each male was paired with 2 adult females of proven fertility. Vaginal smears were daily examined under a light microscope (OLYMPUS, ×400) for the presence of spermatozoa. Sperm positive females were considered inseminated, caged separately and followed-up till delivery. Quantic gestation [(number of sperm positive females/number of pregnant rats) × 100], fertility index [(number of pregnant rats/number of females mated) × 100] and mean litter size were determined for each treatment group

At the end of the treatment (day 57), male rats were sacrificed under diazepam/ketamine anesthesia. Blood sample was collected from the abdominal artery and centrifuged for 15 min at 3000 rpm. The supernatant plasma was pipetted and kept in sealed tubes at −20˚C for the measurement of sexual hormone levels. Testes, vas deferens, epididymis, seminal vesicles and prostate were excised, freed from surrounding tissues and weighted using a sensitive laboratory scale. The left cauda epididymis served for sperm parameters analysis. Testes were kept at −20˚C for the measurement of oxidative stress markers.

The plasma testosterone, FSH and LH were measured through ELISA technic according to the standard guidelines instructed by the kit supplier (Accubind, Monobind. Lake Forest, USA).

After sacrifice, the fresh left cauda epididymis of each rat was crushed in 10ml of saline solution (0.9%) at 34˚C to evaluate sperm motility, count, viability and morphology (normality). The procedure followed the steps described by [20] and [3] . 10 μl of a diluted solution were inserted in A (Ratio: 1/1) and B (Ratio: 1/2) chambers of the Mallassez hemocytometer. Motile and non-motile spermatozoa were then counted in 10 random squares (5 in either chamber) using a light microscope (OLYMPUS, 40×). This permitted to determine sperm motility as the as the percentage of motile spermatozoa over the total number of spermatozoa counted.

The epididymis solution was kept for 24 h to allow sperm cells migration from the tissue to the physiological solution before sperm count estimation. After dilution (1/11 ratio), 10 μl of the solution were placed in each chamber of the Mallassez hemocytometer. The density was expressed as the total number of spermatozoa counted in 20 random squares using light microscope (OLYMPUS, 40×).

For the measurement of sperm viability and morphology, sperm smear was prepared using a drop of the solution obtained from the cauda epididymis and eosin-nigrosine staining. The preparation was dried at room temperature and a minimum of 200 sperm cells were examined in 20 randomly selected fields using light microscope. Sperms with stained cytoplasm were considered nonviable and the total viability was calculated as the percentage of viable sperm cells over the total number. In the same fields, sperms with morphological abnormalities were counted and the total normality was estimated as the percentage of total cells without morphological abnormalities over the total number of sperm cells observed.

Testis homogenate was prepared at 10% in ice-cold 10 mM Tris buffer (pH 7.4) and centrifuged for 10min at 3000 rpm at 4˚C. The supernatant was collected separately and stored at −20˚C for oxidative stress markers estimation. Total proteins were quantified using bovine serum albumin as standard and lipid peroxidation (LPO) was measured in terms of malondialdehyde (MDA) production using the thiobarbituric acid method [21] . Super oxide dismutase (SOD) and catalase (CAT) activities were measured following the procedure described by [22] . Potassium iodate method was employed for total peroxidase activities measurement [23] .

Results are presented as mean ± SEM. After testing conformity to normality of data, comparisons were analyzed by a one-way analysis of variance (ANOVA) followed by a Tukey-HSD post-hoc test to determine statistical difference. Values were considered statistically significant if p < 0.05. STATISTICA/PC program (Version 8.0.) was used for data analysis.

After two weeks of repeated oral exposure to IMI, a significant decrease (p ≤ 0.0001) in body weight was recorded in all exposed rats compared to the control (Table 1). During the treatment phase, there was a marked recovery in body weight. The increase was balanced in all treated groups except for control which recorded the lowest increase (Table 1).

The effects of treatments on testis, seminal vesicles and prostate weights are

Values are presented as mean ± Standard Error of the Mean. Number of animals per group = 5. Control group received only distilled water throughout the experiment. IMI + DW = Imidacloprid + Distilled Water (10 ml/kg). IMI + CC = Imidacloprid + Clomiphene Citrate (0.25 mg/kg). IMI + Vit E = Imidacloprid + Vitamin E (200 mg/kg). IMI + AE = Imidacloprid + Aqueous Extract at 340 mg/kg. IMI + ME = Imidacloprid + Methanol Extract at 170 mg/kg. ###; P < 0.001: Significantly different compared to Control group.

presented in Table 2. After 8 weeks of continuous gavage, no major difference was observed between the absolute and relative weights of IMI + DW and Control groups. However, a significant increase (p < 0.05) in the relative weight of testis was noted in animals treated with the methanol extract at 170 mg/kg.

Similar to effects on sexual glands, no noticeable difference was observed between treatment groups (Table 3).

The administration of IMI induced a significant decrease (P < 0.05 - 0.001) in testosterone, LH and FSH as compared with the untreated control group. Similar to clomiphene citrate and vitamin E, aqueous and methanol extract of L. acida significantly increased testosterone (P < 0.05 - 0.001), and LH (P < 0.001) levels compared with the untreated control group. Moreover, a significant (P < 0.05) improvement of FSH concentration was noticed in animals receiving the aqueous extract. Comparatively, the aqueous extract of L. acida appeared to be more active than the methanol extract (Table 4).

As described in Table 5 there was a moderate change in testis total proteins, but no significant difference was recorded across the groups. In contrast, IMI exposition led to a significant increase in testis MDA content (P ≤ 0.001), SOD (P ≤ 0.05) coupled with a significant increase in Catalase (P ≤ 0.01) and total peroxidases (P ≤ 0.001) activities. Similar to vitamin E, treatment with plant extracts, especially the aqueous extract, significantly decreased (P ≤ 0.05 - 0.001) these oxidative stress markers compared to IMI + DW group.

Values are presented as mean ± Standard Error of the Mean. Number of animals per group = 5. Abs weight = Absolute weight. Rel weight = relative weight. bw= Body Weight. Control group received only distilled water throughout the experiment. IMI + DW = Imidacloprid + Distilled Water (10 ml/kg). IMI + CC= Imidacloprid + Clomiphene Citrate (0.25 mg/kg). IMI + Vit E = Imidacloprid + Vitamin E (200 mg/kg). IMI + AE = Imidacloprid + Aqueous Extract at 340 mg/kg. IMI + ME = Imidacloprid + Methanol Extract at 170 mg/kg. *: P < 0.05 = Significantly different compared to IMI + DW group

Values are presented as mean ± Standard Error of the Mean. Number of animals per group = 5. Abs weight = Absolute weight. Rel weight = relative weight. bw = Body Weight. Control group received only distilled water throughout the experiment. IMI + DW = Imidacloprid + Distilled Water (10 ml/kg). IMI + CC = Imidacloprid + Clomiphene Citrate (0.25 mg/kg). IMI + Vit E = Imidacloprid + Vitamin E (200 mg/kg). IMI + AE = Imidacloprid + Aqueous Extract at 340 mg/kg. IMI + ME = Imidacloprid + Methanol Extract at 170 mg/kg.

Values are presented as mean ± Standard Error of the Mean. Number of animals per group = 5. LH = Luteinizing Hormone. FSH = Follicle Stimulating Hormone. Control group received only distilled water throughout the experiment. IMI + DW = Imidacloprid + Distilled Water (10 ml/kg). IMI + CC = Imidacloprid + Clomiphene Citrate (0.25 mg/kg). IMI + Vit E = Imidacloprid + Vitamin E (200 mg/kg). IMI + AE = Imidacloprid + Aqueous Extract at 340 mg/kg. IMI + ME = Imidacloprid + Methanol Extract at 170 mg/kg. ###; P < 0.001: Significantly different compared to Control group. *: P < 0.05; ***: P < 0.001: Significantly different compared to IMI + DW group.

Values are presented as mean ± Standard Error of the Mean. Number of animals per group = 5. MDA = Malondialdehyde. SOD = Superoxide Dismutase. Control group received only distilled water throughout the experiment. IMI + DW = Imidacloprid + Distilled Water (10 ml/kg). IMI + CC = Imidacloprid + Clomiphene Citrate (0.25 mg/kg). IMI + Vit E = Imidacloprid + Vitamin E (200 mg/kg). IMI + AE = Imidacloprid + Aqueous Extract at 340 mg/kg. IMI + ME = Imidacloprid + Methanol Extract at 170 mg/kg. ##; P ≤ 0.01; ###; P ≤ 0.001: Significantly different compared to Control group. *: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001: Significantly different compared to IMI + DW group.

The effects of L. acida on sperm parameters are summarized in Table 6. Animals exposed to IMI and treated with distilled water for 8 weeks showed a significant drop in sperm count (P ≤ 0.01), sperm motility (P ≤ 0.01), sperm viability (P ≤ 0.001) and sperm normality (P ≤ 0.001) compared with control group. Plant extracts significantly improved (P ≤ 0.05 - P ≤ 0.001) the sperm quality and quantity as compared with IMI + DW. The aqueous extract was more active than the methanol extract.

Results of the fertility test performed in two weeks IMI-exposed male rats followed by 8 weeks of treatment are presented in Table 7. A drop by 40% and 60% in quantic gestation and fertility index, respectively, was recorded in females paired with males of IMI-DW group in comparison to control. Moreover, IMI caused a significant decrease (P ≤ 0.01) in litter size in the females of this group. Interestingly, plant extract treatments reversed these fertility impairments. IMI + CC, IMI + Vit E and aqueous extract groups significantly (P < 0.01) improved the litter size.