SCUC_SI_AAVS_12_s1

INTRODUCTION

In both industrialized and developing nations, there has been a notable decrease in fertility rates during the last few decades. Approximately 70 million people worldwide suffer from the prevalent issue of infertility (Aşır et al., 2024), 15% of all reproductive-aged couples, and 50% of infertility cases are of male origin (Saleem et al., 2020). The most commonly identified risk factors for male infertility include aging, stress, smoking, chemotherapy, occupational toxins, environmental pollution, nutrition, and genetics (Aşır et al., 2024). These issues, either separately or in combination, may have an impact on sperm production and contribute to overall male infertility and subfertility (Jung and Schuppe, 2007). Furthermore, around 30% of cases of male infertility are caused by abnormalities in the sperm (Cavallini, 2006). Male infertility is primarily associated with hormonal changes brought on by dietary variables (Lotti and Maggi, 2018). These jeopardy reasons are linked to augmented reactive oxygen species (ROS) production and levels of oxidative stress which can oxidize sperm protein, DNA, and lipids, influence the enzymatic function and lead to significant changes resulting in infertility, damaging the sperm quality, and cell death (Redza-Dutordoir and Averill-Bates, 2016). The first cells in which ROS generation was recognized were spermatozoa (Maghsoumi-Norouzabad et al., 2021).

The widely held investigations that is now accessible focuses on selective serotonin reuptake inhibitors (SSRIs), which have been proven to have detrimental effects on reproduction. Examples of SSRIs include citalopram, escitalopram, paroxetine, fluoxetine, fluvoxamine, and sertraline (Kumar et al., 2024). Compared to other antidepressants in its class, citalopram is a selective SSRI antidepressant with a more focused and selective pharmacological profile (Bezchlibnyk - Butler et al., 2000). Treatments with citalopram decreased libido and arousal and anorgasmia in thirty percent of patients (Lorenz et al., 2016). Male rats given citalopram both acutely and chronically have less ejaculation and mounting (de Jong et al., 2006). In a recent study, citalopram evoked testicular toxicity and spermatogenesis impairment in mice (Moradi et al., 2024).

Phytotherapy is based on the idea that plants possess natural properties that support the body and reduce ailments. For this reason, plants have been utilized as energizing supplements, vitamin supplements, and potential enhancers of male sexual performance (Derbak et al., 2023). Panax ginseng Meyer (ginseng) of the Araliaceae family has been regarded as one of the best natural herbs for enhancing health. Ginsenosides, a broad class of steroidal saponins, are the main active ingredients in ginseng. They have the capacity to target a variety of tissues and elicit a range of pharmacological effects (Li et al., 2014).Additionally, ginseng reversed testicular dysfunction in aged rats (Kopalli et al., 2015).

Due to its pleiotropic role-which includes autocrine, paracrine, and endocrine actions on a variety of target organs and systems-vitamin D has been a widely studied substance. It is a fat-soluble vitamin that helps to support bone mineralization and preserve calcium and phosphorus homeostasis (Dusilová-Sulková, 2009). Vitamin D is well-thought-out as an imperative micronutrient with various biological properties (Maghsoumi-Norouzabad et al., 2021). Reduced sperm functioning may be the cause of male infertility; low vitamin D levels can impact the number and growth of motile sperm (Aşır et al., 2024).

This study aimed at investigation of the possible mitigating effects of ginseng and vitamin D against citalopram-induced testicular toxicity and damage in male rats.

MATERIALs AND METHODS

Animal Ethical Statement

Animal handling, medications, and scarification were carried out following the guidelines for the care of experimental animals and approved by the Research Ethical Committee, Fac. of Vet. Med., Zagazig Univ., Zagazig, Egypt according to the Animal Research Ethics Committee Guide for care and use of laboratory animals (Approval number: ZU-IACUC/2/F/118/2024).

Drugs

Citalopram tablets [citalopram hydrochloride (C20H22BrFN2O), 20 mg] were gained from Eva Pharmaceutical Com., Egypt.
Ginseng tablets (Panax Ginseng Extract Tablets - 200mg), natural herbal supplements, were purchased from Ema pharm Co., Egypt.
Vitamin D tablets (concentrations of 125 mg (5000 IU)] were bought from Eva Pharmaceutical Company, Egypt.

Experimental Animals

Clinically healthy adult male albino rats aged 7 to 9 months and weighing between 150-200 grams were obtained from the Animal House of Fac. of Vet. Med., Zagazig Univ., Egypt. The animals were maintained in metal cages with typical laboratory settings, which included room temperature of around 25ºC and aeration. Throughout the experiment, the animals were provided a regular diet consisting of pellets and unlimited access to filtered water ad libitum.

Experimental Design

The forty-two rats were divided randomly and blindly into 7 groups, each of 6 rats. Group 1 received 0.2 ml/ day of saline solution orally for 60 days and served as a negative control. Groups 2-4 received citalopram (1.8 mg/kg b.wt), ginseng (18 mg/kg b.wt) and vitamin D (0.01mg/kg b.wt). Groups 5-7 were given citalopram plus ginseng (G5), vitamin D (G6), ginseng or vitamin D (G7).

All medications were administered orally one time daily for 60 days after being dissolved in regular saline at 0.2 ml containing the above calculated doses according to (Paget and Barnes, 1964). Drug solutions were freshly prepared before administration.

Sampling

On the day 60, rats were euthanized and the blood samples were collected from vena cava, and permitted to be clot at room temp for 10-20 minutes. After that, samples were centrifuged for 15 minutes at 3000 rpm to separate the serum, which was then kept at -20°C (Hashem et al., 2018) until analysis of hormonal parameters.

All of the animals’ testicular and epididymal tissues were removed, and they were then cleaned in saline. The epididymis was cleaned and minced in order to determine the morphology and total number of epididymal sperm. Testes was washed in ice cold saline and stored in freezer (−20°C) for antioxidants measurements. Testicular and seminal vesicles tissues were fixed in 10% formalin for histological studies.

Antioxidants Analysis

The testicular samples stored at −20°C were thawed and homogenized in 3 milliliters of phosphate buffer (pH 7.4). The homogenate was centrifuged for 30 minutes at 4°C at 100,800 g. To ascertain the testicular tissue’s antioxidant level, supernatant was collected.

The activity of antioxidants like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were estimated in the testicular tissue of experimental animals according to the methods described by (Kakkar et al., 1984), (Aebi, 1984) and (Paglia and Valentine, 1967) respectively using Bio Rad SmartSpecTM Plus Spectrophotometer. Lipid peroxidation represented by malondialdehyde (MDA) was assessed in the testicular tissue according to the method of (Mihara and Uchiyama, 1978).

Hormonal Analysis

Serum concentrations of testosterone (TS) (Diametra Co, Italy, with 0.07 ng/ml sensitivity), luteinizinghormone (LH) (Bioassay Technology Laboratory, China, with 0.051 µIU/ml sensitivity) and follicle -stimulating hormone (FSH) (Bioassay Technology Laboratory, China, with 0.12 µIU /ml sensitivity) were measured through enzyme-linked immunosorbent assay using commercial enzyme immunoassay kits (BiocheckInc, USA), according manufacturer’s instructions provided with the kit.

Semen Analysis

Epididymal sperm count: The epididymis and vas deferens were minced in 1ml of Phosphate Buffer Saline (PBS) (pH 7.2) to obtain a suspension. The sperm count was counted using the standard hemocytometric method using the improved Neubauer hemocytometer (Deep 1/10 mm, LABART, Germany) as described by (Marianti et al., 2020). Briefly, an aliquot from the suspension (up to 0.5) was taken in leukocyte pipette and diluted with PBS up to the mark 11. Prior to the count, the chamber was washed and cleaned. A cover slip was placed on the counting chamber. The sperm suspension was well-mixed and charged in to Neubauer counting chamber and examined under a light microscope with x10 objective lens. The total sperm count in eight squares (except the central erythrocyte area) of 1 mm2each was determined and multiplied by 5x 104to calculate the number of spermatozoa per milliliter.

Evaluation of sperms motility: On a slide that had been preheated beforehand, sperm from the epididymis filtrate were wet mounted. The percentage of spermatozoa that moved gradually in one direction on a slide per field was observed using a light microscope to count the motile and non-motile sperms. Spermatozoa that exhibited round, pendulous, or retrograde movements were not counted; only those traveling in a single direction were counted (Zemjanis, 1970).

Assessment of sperm morphology: Fresh sperm smears were prepared for morphometric analysis by placing 5 μl of the fresh semen on the clear end of a frosted slide by dragging the drop across the slide. The smears were air-dried before staining. Three semen smears were prepared and stained with Eosin nigrosine stain. The assessment of sperm morphology was qualitative and quantitative and included microscopic evaluation for atypical spermatozoa morphology involving any changes involving the head, neck, mid-piece and tail and the diagnosis of abnormal spermatozoa (Horri et al., 2021).

Histopathological Analysis

The formalin preserved specimens collected from rat’s testicular and SV tissues were processed in an automated tissue processor. The samples were then cleared in multiple changes of xylene after being dehydrated using a gradient of ethanol concentrations (70–100%). After that, samples were imbedded, blocked out, and impregnated with melted paraffin wax. Hematoxylin and eosin was used to stain 4-to 5-micrometer paraffin sections (Suvarna et al., 2018). A veterinary pathologist looked at the pathologic structural abnormalities under a light microscope.

Statistical Analysis

The standard error of the mean (mean ± SEM) was used to express the results. Version 22 of the SPSS program was used to conduct the statistical analysis (IBM corporation, SPSS Inc., 2010). One-way analysis of variance (ANOVA) followed by post hoc Tukey’s test was used to assess the statistical significance of data between different groups. If P<0.05, it was deemed significant. There were no outliers observed in the current study. In addition, no data normalization procedures were applied.

RESULTS AND DISCUSSION

The obtained results were exposed to one-way analysis of variance followed by post hoc Tukey’s test to assess the statistical significance of data between different groups.

Antioxidant and Stress Biomarkers

Citalopram treatment resulted in significant (p < 0.0001) decrease in the activity of antioxidant enzymes (SOD, CAT and GPx) in the testicular tissues, in addition significant rise (p<0.0001) of MDA levels within testes when compared to control animals (G1). No significant changes (p > 0.05) in the activity of antioxidant enzymes and MDA levels with exception of significant increase of SOD (p < 0.05) were observed in treated groups with ginseng (G3) and vitamin D (G4) alone in comparison with control group. Co-administrations of citalopram with ginseng (G5), vitamin D (G6), ginseng +vitamin D (G7) ensued a significant (p < 0.001) upsurge and marked enhancement in testicular SOD, CAT and GPx activity, with significant (p < 0.001) reduction in MDA levels when compared with G2 (Figure 1).

Hormonal Analysis

Repeated oral treatment of citalopram (1.8 mg/kg/day) for 60 days resulted in significant (p<0.001) rises in the serum levels of LH, and FSH with non- significant (p > 0.05) difference in TS level when compared to the untreated control (G1). Treatments of rats with ginseng (18 mg/kg b.wt) or vitamin D (0.01mg/kg b.wt) alone caused non-significant (p > 0.05) alterations in serum TS, LH, and FSH levels when compared to the untreated control values, while serum TS was significantly (p < 0.05) decreased in vitamin D- treated group. Repeated oral treatments of rats with citalopram in combination with ginseng (G5),vitamin D (G6), or both (G7) for 60 days resulted in significant declines in the circulating serum TS (p<0.01), LH (p<0.001), and FSH (p<0.001) levels when matched with the positive control (citalopram group) neighboring to the normal values of negative control (Figure 2).

Semen Analysis

When compared to the control group, the citalopram-treated group showed a significant reduction (p<0.001) in sperm count and motility and a significant rise (p<0.0001) in the percentage of sperm abnormalities (Table 1). However, co-administration of citalopram with ginseng, vitamin D, and ginseng plus vitamin D to normal rats caused significant (p< 0.0001) improvement in count of sperms, motility and sperm morphology, compared with the positive control (G2). This improvement was towards the normal of G1. Treated rats with either ginseng or vitamin D alone showed nearly similar sperm parameters when compared with the negative control values (Table 1).

Histopathological Findings

Upon histological analysis of the testicular tissues of the control group (G1), seminiferous tubules (ST) were found to be seemingly normal. The lumen of the tubules was filled with spermatids, regular spermatogonia, and a sequence of spermatocytes. The interstitial tissue exhibited a variable number of Leydig cells. The seminal vesicles (SV) denoted

Table 1: Semen analysis in the control and treated groups of adult male rats after 60 days of experiment. Mean ± SEM (n= 6).

Parameters/ Groups	Sperm concentration (x106/ml)	Sperm motility (%)	% Abnormal sperms
G1 (-ve control)	107.67±2.60 ab	86.33±3.66 ab	15.67±0.51 b
G2 (Citalopram)	53.00±22.11 b	56.67±2.32 c	28.69±2.92 a
G3 (Ginseng)	156.00±8.66 a	93.33±2.85 a	11.97±0.77 bc
G4 (Vit D)	136.00±14.73 a	93.30±3.07 a	10.89±0.37 bc
G5 (Citalopram+ ginseng)	128.33±21.40 a	80.33±2.66 b	13.47±0.45 bc
G6 (Citalopram+ Vit D)	108.67±6.01 ab	81.32±3.00 b	14.15±0.14 bc
G7 (Citalopram+ ginseng +Vit D)	116.00±7.21 a	83.33±2.44 b	13.02±0.18 bc

Means in the same column not followed by the same letter differ significantly (p<0.05).

a normal structural appearance with seminal acinar structures and papillary fronds lined by simple secretory columnar epithelium and eosinophilic intra-luminal secretory materials. No pathological changes could be detected in any stage of examined testicular or SV tissues (Figure 3). Citalopram-treated group (G2) revealed moderate destruction of the ST affecting about 30 to 45% of the tubular structures including some of them in the central tubules and others at the periphery. The affected ST showed marked interstitial edema, vascular dilatation, edema, and degenerative and necrotic changes in the germinal epithelium, spermatocyte, and spermatozoa. Some of the ST showed degeneration of the primary and secondary spermatozoa with vacuolation of the spermatogonia. The interstitial tissue in some tubules appeared thickened by Leydig cells proliferation and edema. Other ST appeared atrophied with irregular contours. The SV thickened interstitial tissue. Other parts of the SV showed cystically dilated papillae and acini with large amounts of secretory materials, atrophy of the lining epithelium, and interstitial edema. The proportion of the normal to the cystically dilated parts is one-fifth (1:5) (Figure 4). Ginseng-treated group (G3) showed normal structure compared to the control group with keeping features of the regarding the germinal epithelium, spermatocyte, spermatids, and spermatozoa. The SV revealed normal structures including the acini and the papillary fronds with normal secretory materials and lining epithelium. Mild interstitial edema and fibroblastic proliferation could be detected in some lobules. A few lobules showed cystic degeneration (Figure 5). Vitamin D-treated group (G4) revealed highly active ST with the presence of dividing spermatocytes and well-formed spermatocytes and spermatozoa. However, focal thickening of the interstitial fibrous tissue and cystic dilatation of some follicles could also be detected (Figure 6). Citalopram plus ginseng-treated group (G5) revealed about 25-30% of the ST with characteristic cytotoxic effect most probably citalopram-induced as represented by marked necrosis and/or apoptosis of the affected ST cells. The interstitial tissue showed edema and atrophy of some tubules. The remaining testicular tissue showed actively normal tubular structure with apparently healthy ST with normal histo-morphological structures. The SV showed a focal cystic change in the acinar and papillary structures with partial atrophied epithelium calls. There was also interstitial edema and vascular dilatation. The remaining SV, follicles, and papillary structures were apparently normal (Figure 7). In citalopram plus vitamin D-treated group (G6), about 5-10 % of testicular ST showed degenerative and early necrotic changes in the cellular structures together with focal atrophy and disorganization of the tubules. Focal interstitial edema and vascular dilatation were also detected. The interstitial septa were mildly thickened due to edema, fibroblastic and Leydig cell proliferation. The SV showed mild to moderate interstitial edema and fibrosis besides cystic dilatation of some follicles. The remaining part showed a normal configuration of the papillary and follicular structures with apparently normal columnar epithelium cells and eosinophilic secretory materials (Figure 8). Citalopram combined with ginseng and vitamin D (G7) showing that about 5-10% of the testicular ST were affected as represented by mild to moderate degenerative, apoptotic and necrotic changes in tubular linings, focal interstitial edema with atrophy of some tubules could also be detected. The remaining testicular tissue showed normal ST structures with active spermiogenesis and spermatogenesis. The Leydig cells appear with normal structure and normal population. However, a slight rise in the number of Leydig cells could be observed in some cases. Apparently normal seminal vesicular structures with keeping features of the folliculo-papillary pattern and active secretory function could be recorded. Mild interstitial fibrosis was seen in a few cases. The lining epithelium of the follicle and papillae appeared normal (Figure 9).

One hypothesis for the etiology of male infertility is oxidative stress (Kumar et al., 2013). Studies have publicized an augmentation in the levels of ROS in 40 to 80% of infertile men (Hamada et al., 2012). An imbalance between oxidants and antioxidants, favoring oxidants, can lead to oxidative stress, which can deteriorate testicular structure and spermatogenesis processes, ultimately resulting in infertility (Guz et al., 2013). Cells have antioxidant enzymes such as glutathione reductase (GR), SOD, CAT, GPx, and GPx to guard against the potentially detrimental effects of ROS. These enzymes mitigate the destructive effects of ROS by inactivating them. Antioxidant enzymes therefore serve as the cell’s defense mechanism (Nita and Grzybowski, 2016).

The results of current study showed significant decrease in SOD, CAT and GPx activities with increased levels of MDA in testicular tissue of citalopram-treated rats. This is considered as indicator of deterioration in the antioxidant /oxidant balance in the citalopram-administered group. The present findings are consistent with previous studies (Hashim, 2020). Marked alterations noticed in antioxidant activities in the testis of citalopram-treated rats might have been initiating and propagating the oxidative damage (Kumar et al., 2024). Spermatogenesis is hampered by lipid peroxidation (LPO), which breaks down the lipid matrix’s structure in the membranes of germ cells and spermatozoa. Testicular MDA activity is increased, which is indicative of damaged cell membranes caused by high LPO levels (Herbet et al., 2015). In conventionality with prior investigations (Atli et al., 2017), administration of sertraline initiated a state of oxidative stress in the rat testicular tissue, revealed by augmented lipid peroxidation and cooperated antioxidant defense system. Recent evidence stated that fluoxetine as a one of SSRI increased MDA level and decreased the antioxidant enzyme activity (SOD, CAT and GSH) (El Sayed et al., 2021). Furthermore, citalopram administration significantly increased oxidative stress with significantly increased MDA levels (Moradi et al., 2023). Administration of ginseng and vitamin D in combination with citalopram caused an improvement in the antioxidants and MDA levels. This improvement in the antioxidant enzymes and amelioration in MDA levels improved infertility in aged rats treated with ginseng (Won et al., 2014) or vitamin D (Maghsoumi-Norouzabad et al., 2021).

Endocrine activity, which is mediated by the hypothalamic-pituitary-testes axis, controls spermatogenesis (Ciarrocca et al., 2013). FSH and LH are secreted by the anterior pituitary, and testosterone is secreted by the testes to carry out crucial functions in spermatogenesis. LH promotes the production of testosterone from Leydig cells in the testes, while FSH promotes the production of androgen-binding proteins from Sertoli cells during the process of sperm maturation. Testosterone is essential for the maintenance of semen production and the spermatogenesis process (Wdowiak et al., 2014).

Citalopram treatment for 60 days increased the sera LH, and FSH levels with non-significant (p > 0.05) difference in serum TS level. The hormonal results of the present study were in agreement with other reported investigations (Erdemir et al., 2014). There were preceding reports showed some differences in the evaluations of hormonal parameters. Sertraline administration resulted in a significant increase in serum TS (Atli et al., 2017), increased FSH and lower TS levels with no significant differences in the LH levels (Hamdi, 2019). Moreover, a repeated oral treatment citalopram (10 mg/kg) twice daily for 21 consecutive days significantly increase serum TS concentration (Przegaliński et al., 1987). When rats were administered citalopram orally once daily at varied concentrations for four weeks in a row, the study did not uncover a significant variation in their testosterone levels (Khan et al., 2023). In addition, adult male rats given 200 mg/kg of fluoxetine for 60 days demonstrated a noteworthy drop in TS and FSH levels (Bataineh and Daradka, 2007). Testicular failure is known to be indicated by elevated LH and FSH levels with normal TS levels (Ilgin et al., 2017).

Oral treatment of rats with citalopram in combination with ginseng, vitamin D, or both resulted in significant declines in the circulating serum TS (p<0.01), LH (p<0.001), and FSH (p<0.001) levels when matched with citalopram group. These results indicated that an improvement in the hormones levels particularly LH and FSH and the potential benefits of the used ginseng or vitamin D against testicular damage in rats induced by citalopram. Ginseng therapy is crucial for preserving essential sex hormone receptor levels, which in turn promotes healthy androgen production and regular spermatogenesis (Lee et al., 2019). Vitamin D treatment decreased the adverse effects of citalopram causing infertility. An investigation showed that the TS and LH concentrations were significantly decreased during vitamin D treatment (Behmanesh et al., 2019).

With reference to semen analysis in the existing study, citalopram treatment for 60 days lowered sperm concentration, motility, and normal sperm morphology with increases in the percentage of sperm abnormalities. Reduction in sperm count and motility may be attributed to oxidative stress made by high production of free radicals as a result of testicular damage-induced by citalopram. Sperm cell membranes are rich in polyunsaturated fatty acids, making them the target of the ROS produced. (Dandekar et al., 2002). The consumption of mitochondrial oxygen and the production of ROS are recognized to elevate during the fertilization and spermatogenesis process (Guerriero et al., 2014). An excess of ROS can result in infertility due to a reduction in sperm count and motility (Kumar et al., 2013). Abnormal sperm morphology is produced during the process of sperm membrane remodeling in spermatogenesis, along with the generation of ROS. (Guerriero et al., 2014). Serum hormone levels, specifically FSH and LH, were negatively correlated with sperm concentration, motility, and morphology (Keskin et al., 2015). Other papers that demonstrate a decrease in normal sperm morphology, concentration, and motility after citalopram exposure corroborated our findings (Elnazer and Baldwin, 2014). The increases in morphologically abnormal sperm percentage (28.69±2.92%) in this study are considered as an important indicator of disorders that occur during sperm production and maturation that lead to infertility. Male infertility is generally regarded to be present when sperm abnormalities are found to be more than 10%. (Saba et al., 2009). Morphological abnormalities in the sperm alone are thought to influence the sperm’s fertilization potential (Agarwal et al., 2014). Similarly, the sperm concentration and normal sperm morphology were significantly decreased in rats received oral doses of citalopram hydrobromide at 5, 10, and 20 mg/kg/day for 28 days (Ilgin et al., 2017). The percentage of sperm count and sperm motility were significantly decreased while the sperm abnormalities was increased with increased dose of antidepressant drug fluoxetine treatment (2.6, 7.8 and 13.0 mg/kg b.wt.) for 5 consecutive days (Alzahrani, 2012). Likely, Atli et al. (2017) showed that sertraline decreased the sperm concentration, motility, and increased abnormalities of sperm morphology, induced histopathological changes in testicular tissue.

The quality of the semen was enhanced by ginseng and/or vitamin D treatment. Our results align with previous study, since multiple preclinical studies have demonstrated that ginseng, as a cyclic adenosine monophosphate-responsive element modulator, enhances spermatogenesis and improves testicular issues, sperm quality, and sperm motility (Lee et al., 2020). Ginseng has been shown to increase sperm count and quality in both healthy persons and treatment-related infertile patients (Leung and Wong, 2013). As well as other studies recorded the benefits effect of vitamin D on spermatogenesis and sperm parameters (BaSalamah et al., 2018). Comprehensive studies confirmed that supplementing with fluoxetine and vitamin D significantly decreased testicular damage and restored all pertinent parameters (El Sayed et al., 2021).

Histopathological examinations are often used as important biomarkers in toxicological researches. Histopathological changes in the testes can accompany decreases in sperm quantity and quality (Ben Slima et al., 2017). In the current study, citalopram induced injurious effects on the testicular tissue including marked interstitial edema, vascular dilatation, edema, and degenerative and necrotic changes in the germinal epithelium, spermatocyte, and spermatozoa in ST matched to the control group. The seminal vesicle showed cystically dilated papillae and acini with large amounts of secretory materials, atrophy of the lining epithelium, and interstitial edema. The histopathological observations confirmed that citalopram caused testicular damage. These findings are in harmony to previous reporters (Elsedawi et al., 2021; Moradi et al., 2024). Such findings observed in the current study were in harmony with others in which SSRIs use caused significant damage to the testicular histology (Soliman et al., 2017). Other researchers (Ilgin et al., 2017) revealed sever testicular damage including germinal cell degeneration, atrophy of STs, extracellular vacuolization, and reduction of cells in the spermatogenesis series, especially at high doses of citalopram. Furthermore, fluoxetine treatment resulted in marked alterations in the testis, indicated by disorganized and shrunken STs, thickening of the tunica propria, and vacuolization of germ cells (Kumar et al., 2024), cells with darkly stained nuclei (denoting apoptosis) and separated epithelium from the underlying basement membrane, degenerated germ cells were detached and found in the lumen of the tubules (Elsedawi et al., 2021), In addition to deformation of the seminiferous tubules, deteriorated spermatogenic cells with extensive cytoplasmic vacuoles, and sloughing of germ cells. (Hajizadeh et al., 2016; Sayed et al., 2021). The aforementioned findings noticed in the existing work in which citalopram usage initiated major damage to the testicular histology which was upturned in the treated rats (G5-7). In comparison to the citalopram group, the histopathological lesions in the testis and SV were at a lower level in the citalopram- treated rats with ginseng (G5), vitamin D (G6), or both (G7). The affections of STs were mostly better in rats of groups 6 and 7 (about 5-10%) thatgroup5 (about 25-30%). The existing investigations verified that vitamin D or with ginseng can provide promising protection against citalopram -induced testicular damage. These results may be attributed to ginsenosides, and saponins which are the foremost active components of ginseng (Li et al., 2014), or due to vitamin D metabolizing and signaling molecules have all been identified in male and female reproductive organs from different species (Carlberg and Campbell, 2013; Mohamed et al., 2021). These findings go in agreement with previous studies (Kopalli et al., 2015; Sayed et al., 2021; Moradi et al., 2024). Earlier studies showed that ginseng protected against testicular damage induced by doxorubicin in rats (Chen et al., 2013), while vitamin D ameliorated testis injury caused by imidacloprid (Keles et al., 2022), or lead (BaSalamah et al., 2018).

Treatments with ginseng and vitamin D alone produced no histopathological changes in the testicular and SV architecture, STs, and spermatozoa (spermatogenesis) when compared to the control group. Similar results were reported in former models for ginseng (Shojaeepour et al., 2022) or vitamin D (Marahovskiy et al., 2022).

CONCLUSIONs AND RECOMMENDATIONS

The results of the current investigation indicated that ginseng and vitamin D could improve the citalopram-induced damage of testicular tissue and impaired sexual function. Such effects might be attributed to their pronounced antioxidant activities. Therefore, ginseng and vitamin D might act as potential candidates to improve citalopram-induced testicular toxicity and damage in male rats.

ACKNOWLEDGMENTS

Researchers would like to thank Dr. El-Sayed R. El-Attar, Professor of Pathology, Fac. of Vet. Med., Zagzig Univ., for histopathological examination.

NOVELTY STATEMENT

This study indicated the importance of ginseng and vitamin D to improve citalopram-induced male infertility in rats

AUTHORS’ CONTRIBUTIONS

All authors directly contributed to the manuscript’s composing, revising, and approving its final version. Gamal E. Shams, Gihan G. El-Sayed, Reda M. Abd El-Aziz developed the study design and controlled its progress. Aya Salah Eldin Mohamed, and Reda M. Abd El-Aziz performed the animal treatment, examination, and follow-up. Aya Salah Eldin Mohamed, Gamal E. Shams, and Gihan G. El-Sayed analyzed the estimated parameters obtained throughout the study. Aya Salah Eldin Mohamed performed the statistical processing of the experimental data.

Funding

None.

Conflict of Interest

None of the authors have any conflict of interest to declare.

REFERENCES

Aebi, H. Catalase in vitro. Methods in Enzimology, 1984; 105: 121–126. https://doi.org/10.1016/S0076-6879(84)05016-3

Agarwal, A., Tvrda, E., Sharma, R. Relationship amongst teratozoospermia, seminal oxidative stress and male infertility. Reproductive Biology and Endocrinology, 2014; 12: 1-8. https://doi.org/10.1186/1477-7827-12-45

Alzahrani, H. A. Sister chromatid exchanges and sperm abnormalities produced by antidepressant drug fluoxetine in mouse treated in vivo. European Review in Medical and Pharmacological Sciences, 2012; 16(15): 2154-2161.

Aşır, F., Duran, S., Afşin, M., Duran, E., Korak, T., Şahin, F. Investigation of Vitamin D Levels in Men with Suspected Infertility. Life (Basel), 2024; 14: 2. https://doi.org/10.3390/life14020273

Atli, O., Baysal, M., Aydogan-Kilic, G., Kilic, V., Ucarcan, S., Karaduman, B., Ilgin, S. Sertraline-induced reproductive toxicity in male rats: evaluation of possible underlying mechanisms. Asian Journal of Andrology, 2017; 19(6): 672-679. https://doi.org/10.4103/1008-682X.192637

BaSalamah, M. A., Abdelghany, A. H., El-Boshy, M., Ahmad, J., Idris, S., Refaat, B. Vitamin D alleviates lead induced renal and testicular injuries by immunomodulatory and antioxidant mechanisms in rats. Scientific Reports, 2018; 8(1): 4853. https://doi.org/10.1038/s41598-018-23258-w

Bataineh, H. N., Daradka, T. Effects of long-term use of fluoxetine on fertility parameters in adult male rats. Neurology and Endocrinology Letters, 2007; 28(3): 321-325.

Behmanesh, N., Abedelahi, A., Charoudeh, H. N., Alihemmati, A. Effects of vitamin D supplementation on follicular development, gonadotropins and sex hormone concentrations, and insulin resistance in induced polycystic ovary syndrome. Turkish Journal of Obstetrics in Gynecology, 2019; 16(3): 143-150. https://doi.org/10.4274/tjod.galenos.2019.46244

Ben Slima, A., Chtourou, Y., Barkallah, M., Fetoui, H., Boudawara, T., Gdoura, R. Endocrine disrupting potential and reproductive dysfunction in male mice exposed to deltamethrin. Human Experimental Toxicology, 2017; 36(3): 218-226. https://doi.org/10.1177/0960327116646617

Bezchlibnyk-Butler, K., Aleksic, I., Kennedy, S. H. Citalopram--a review of pharmacological and clinical effects. Journal of Psychiatry Neuroscience, 2000; 25(3): 241-254.

Carlberg, C., Campbell, M. J. Vitamin D receptor signaling mechanisms: integrated actions of a well-defined transcription factor. Steroids, 2013; 78(2): 127-136. https://doi.org/10.1016/j.steroids.2012.10.019

Cavallini, G. Male idiopathic oligoasthenoteratozoospermia. Asian Journal of Andrology, 2006; 8(2): 143-157. https://doi.org/10.1111/j.1745-7262.2006.00123.x

Chen, X., Gong, L., Xu, J. Antioxidative activity and protective effect of probiotics against high-fat diet-induced sperm damage in rats. Animal, 2013; 7(2): 287-292.

Ciarrocca, M., Capozzella, A., Tomei, F., Tomei, G., Caciari, T. Exposure to cadmium in male urban and rural workers and effects on FSH, LH and testosterone. Chemosphere, 2013; 90(7): 2077-2084.

Dandekar, S., Nadkarni, G., Kulkarni, V., Punekar, S. Lipid peroxidation and antioxidant enzymes in male infertility. Journal of Postgraduate Medicine, 2002; 48(3): 186-189.

de Jong, T. R., Veening, J. G., Waldinger, M. D., Cools, A. R., Olivier, B. Serotonin and the neurobiology of the ejaculatory threshold. Neuroscience & Biobehavioral Reviews, 2006; 30(7): 893-907. https://doi.org/10.1016/j.neubiorev.2006.01.001

Derbak, H., Imre, K., Benabdelhak, A. C., Moussaoui, M., Kribeche, A., Kebbi, R., Ayad, A. Effect of Peganum harmala Total Alkaloid Extract on Sexual Behavior and Sperm Parameters in Male Mice. Veterinary Sciences, 2023; 10: 8. https://doi.org/10.3390/vetsci10080498

Dusilová-Sulková, S. Vitamin D metabolism and vitamin D traditional and nontraditional, target organs: implications for kidney patients. Journal of Renal Care, 2009; 35: 39-44. https://doi.org/10.1111/j.1755-6686.2009.00066.x

El Sayed, Y., Ali, N., Elshazly, A., Salim, R., Baioumy, B., El Noury, H. The Role of Vitamin D on Fluoxetine Induced Testicular Toxicity in Adult Male Rats. Mansoura Journal of Forensic Medicine and Clinical Toxicology, 2021; 29(2): 41-56.

Elnazer, H. Y., Baldwin, D. S. Treatment with citalopram, but not with agomelatine, adversely affects sperm parameters: a case report and translational review. Acta Neuropsychiatr, 2014; 26(2): 125-129. https://doi.org/10.1017/neu.2013.60

Elsedawi, B. F., Hussein, Y., Sabry, M. A., Aziz, J. A. Effect of fluoxetine on the testes of adult albino rats and the possible protective role of curcumin. Anatomical Science International, 2021; 96: 187-196. https://doi.org/10.1007/s12565-020-00573-9

Erdemir, F., Atilgan, D., Firat, F., Markoc, F., Parlaktas, B. S., Sogut, E. The effect of sertraline, paroxetine, fluoxetine and escitalopram on testicular tissue and oxidative stress parameters in rats. International Brazilian Journal of Urology, 2014; 40: 100-108

Guerriero, G., Trocchia, S., Abdel-Gawad, F., Ciarcia, G. Roles of reactive oxygen species in the spermatogenesis regulation, Frontiers in Endocrinology, 2014; 5: 56. https://doi.org/10.3389/fendo.2014.00056

Guz, J., Gackowski, D., Foksinski, M., Rozalski, R., Zarakowska, E., Siomek, A., Szpila, A., Kotzbach, M., Kotzbach, R., Olinski, R. Comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. PLoS One, 2013; 8(7): e68490. https://doi.org/10.1371/journal.pone.0068490

Hajizadeh, Z., Soleimani Mehranjani, M., Najafi, G., Shariatzadeh, S., Shalizar-Jalali, A. Black Grape Seed Extract Modulates Fluoxetine-Induced Oxidative Stress and Cytotoxicity in the Mouse Testis. Jundishapur Journal of Natural Pharmaceutical Products, 2016; 11(2): e27512. https://doi.org/10.17795/jjnpp-27512

Hamada, A., Esteves, S. C., Nizza, M., Agarwal, A. Unexplained male infertility: diagnosis and management. International Brazilian Journal of Urology, 2012; 38: 576-594. https://doi.org/10.1590/S1677-55382012000500002

Hamdi, H. The preventive role of wheat germ oil against sertraline-induced testicular damage in male albino rats. Andrologia, 2019; 51(10): e13369. https://doi.org/10.1111/and.13369

Hashem, M. A., El-Deen, N. A. N., Ghareeb, O. A. Biochemical effects of ginger and/or green tea extracts in high fat diet-induced obese rats. Slovenian Veterinary Research, 2018; 55: 241–249. https://doi.org/10.26873/SVR-651-2018

Hashim, W. Physiological Disturbances Caused by Citalopram in Rats. Journal of Research on the Lepidoptera, 2020; 51: 408-416. https://doi.org/10.36872/LEPI/V51I2/301107

Herbet, M., Gawrońska-Grzywacz, M., Jagiełło-Wójtowicz, E. Evaluation of selected biochemical parameters of oxidative stress in rats pretreated with rosuvastatin and fluoxetine. Acta Poloniae Pharmaceutica, 2015; 72(2): 261-265.

Horri, E., Esmaeilnejad Moghadam, A., Talebpour Amiri, F., Ebrahimzadeh, M. A. Protective effect of Feijoa sellowianan fruit on testicular toxicity-induced by cadmium chloride. Andrologia, 2021; 53(2): e13926. https://doi.org/10.1111/and.13926

Ilgin, S., Kilic, G., Baysal, M., Kilic, V., Korkut, B., Ucarcan, S., Atli, O. Citalopram Induces Reproductive Toxicity in Male Rats. Birth Defects Research, 2017; 109(7): 475-485. https://doi.org/10.1002/bdr2.1010

Jung, A., Schuppe, H. C. Influence of genital heat stress on semen quality in humans. Andrologia, 2007; 39(6): 203-215. https://doi.org/10.1111/j.1439-0272.2007.00794.x

Kakkar, P., Das, B., Viswanathan, P. N. A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biochemical Biophysics, 1984; 21(2): 130-132.

Keles, H., Yalcin, A., Aydin, H. Protective effect of vitamin D on imidacloprid-induced testicular injury in rats. Archives of Medical Sciencs, 2022; 18(6): 1659-1665.

Keskin, M. Z., Budak, S., Zeyrek, T., Çelik, O., Mertoglu, O., Yoldas, M., Ilbey, Y. Ö. The relationship between serum hormone levels (follicle-stimulating hormone, luteinizing hormone, total testosterone) and semen parameters. Archivio Italiano di Urologia e Andrologia, 2015; 87(3): 194-197. https://doi.org/10.4081/aiua.2015.3.194

Khan, W., Ahmad, F., Bhutta, M. S. A., Inam U., Jadoon, M., Imran, T. Effect of citalopram on sex hormones and testicular histomorphology in male albino rats: An experimental study. International Journal of Health Sciences, 2023; 7(S1): 1083-1101. https://doi.org/10.53730/ijhs.v7nS1.14319

Kopalli, S. R., Hwang, S.-Y., Won, Y.J., Kim, S.-W., Cha, K.-M., Han, C.-K., Hong, J.-Y., Kim, S.-K. Korean red ginseng extract rejuvenates testicular ineffectiveness and sperm maturation process in aged rats by regulating redox proteins and oxidative defense mechanisms. Experimental Gerontology, 2015; 69: 94-102. https://doi.org/10.1016/j.exger.2015.05.004

Kumar, S., Behari, J., Sisodia, R. Influence of electromagnetic fields on reproductive system of male rats. International Journal of Radiation Biology, 2013; 89(3): 147-154. https://doi.org/10.3109/09553002.2013.741282

Kumar, S., Tripathi, A., Singh, P. Antidepressant-Induced Testicular Alterations in the Normal and Depressed Mice. JSM Sexual Medicine, 2024; 8: 1-11. https://doi.org/10.47739/2578-3718.sexualmedicine.1126

Lee, H. W., Kil, K. J., Lee, M. S. Ginseng for Improving Semen Quality Parameters: A Systematic Review. World Journal of Mens Health, 2020; 38(3): 377-384. https://doi.org/10.5534/wjmh.190125

Leung, K. W., Wong, A. S. Ginseng and male reproductive function. Spermatogenesis, 2013; 3(3): e26391. https://doi.org/10.4161/spmg.26391

Li, H., Liu, S.-Y., Wang, B. Progress of the regulation effect of ginsenosides on HPA axis. Yao xue xue bao= Acta Pharmaceutica Sinica, 2014; 49(5): 569-575.

Lorenz, T., Rullo, J., Faubion, S. Antidepressant-Induced Female Sexual Dysfunction. Mayo Clinic Proceeding, 2016; 91(9): 1280-1286. https://doi.org/10.1016/j.mayocp.2016.04.033

Lotti, F., Maggi, M. Sexual dysfunction and male infertility. Nature Reviews Urology, 2018; 15(5): 287-307. https://doi.org/10.1038/nrurol.2018.20

Maghsoumi-Norouzabad, L., Zare Javid, A., Mansoori, A., Dadfar, M., Serajian, A. Evaluation of the effect of vitamin D supplementation on spermatogram, seminal and serum levels of oxidative stress indices in asthenospermia infertile men: a study protocol for a triple-blind, randomized controlled trial. Nutrition Journal, 2021; 20(1): 49. https://doi.org/10.1186/s12937-021-00711-7

Marahovskiy, I., Laryanovska, Y., Korenieva, Y., Smolienko, N., Chystiakova, E., Belkina, І., Velychko, N., Misiura, K., Bondarenko, V. Influence of vitamin D on the histostructure of the testis and morphometric indications of spermatogenesis of intact rats. Reports of Morphology, 2022; 28: 25-31. https://doi.org/10.31393/morphology-journal-2022-28(2)-04

Marianti, A., Isnaeni, W., Setiati, N., Sumadi, S. Effects of Chitosan on Sperm Quality of Lead Acetate-Induced Rats. Journal of Physics: Conference Series, 2020; 1567: 032061. https://doi.org/10.1088/1742-6596/1567/3/032061

Mihara, M., Uchiyama, M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry, 1978; 86(1): 271-278. https://doi.org/10.1016/0003-2697(78)90342-1

Mohamed, D. I., Abou-Bakr, D. A., Ezzat, S. F., El-Kareem, H. F. A., Nahas, H. H. A., Saad, H. A., Mehana, A. E., Saied, E. M. Vitamin D3 Prevents the Deleterious Effects of Testicular Torsion on Testis by Targeting miRNA-145 and ADAM17: In Silico and In Vivo Study. Pharmaceuticals, 2021; 14(12): 1222. https://doi.org/10.3390/ph14121222

Moradi, M., Hashemian, M. A., Fathi, M., Peysokhan, M., Hashemian, A. H., Moradi, B., Jalili, C., Faramarzi, A. Utility of vitamin C in ameliorating citalopram-induced testicular toxicity via modulating nitro-oxidative stress and apoptosis in mice. Journal of Biochemical and Molecular Toxicology, 2024; 38(1): e23543. https://doi.org/10.1002/jbt.23543

Nita, M., Grzybowski, A. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxidative Medicine and Cellular Longevity, 2016; 2016. https://doi.org/10.1155/2016/3164734

Paget, G. E., Barnes, J. M. 1964: CHAPTER 6 - Toxicity Tests. In D. R. Laurence & A. L. Bacharach (Eds.), Evaluation of Drug Activities (pp. 135-166). Academic Press. https://doi.org/10.1016/B978-1-4832-2845-7.50012-8

Paglia, D. E., Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical Medicine, 1967; 70(1): 158-169.

Przegaliński, E., Warchoł-Kania, A., Budziszewska, B., Jaworska, L. Effect of repeated administration of antidepressant drugs on the serum and brain concentration of testosterone and its metabolites. Polish Journal of Pharmacology and Pharmacy, 1987; 39(6): 683-689.

Redza-Dutordoir, M., Averill-Bates, D. A. Activation of apoptosis signalling pathways by reactive oxygen species. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2016; 1863(12): 2977-2992. https://doi.org/10.1016/j.bbamcr.2016.09.012

Saba, A. B., Oridupa, O. A., Oyeyemi, M. O., Osanyigbe, O. D. Spermatozoa morphology and characteristics of male wistar rats administered with ethanolic extract of Lagenaria Breviflora Roberts. African Journal of Biotechnology, 2009; 8: 7.

Saleem, U., Zubair, S., Riaz, A., Anwar, F., Ahmad, B. Effect of venlafaxine, pramipexole, and valsartan on spermatogenesis in male rats. ACS Omega, 2020; 5(32): 20481-20490. https://doi.org/10.1021/acsomega.0c02587

Sayed, Y., Ali, N., Elshazly, A., Salim, R., Badr Eldeen, B., El Noury, H. The Role of Vitamin D on Fluoxetine Induced Testicular Toxicity in Adult Male Rats. Mansoura Journal of Forensic Medicine and Clinical Toxicology, 2021; 29: 41-56.

Shojaeepour, S., Sharififar, F., Haghpanah, T., Iranpour, M., Imani, M., Dabiri, S. Panax ginseng ameliorate toxic effects of cadmium on germ cell apoptosis, sperm quality, and oxidative stress in male Wistar rats. Toxin Reviews, 2022; 41(2): 389-401. https://doi.org/10.1080/15569543.2021.1884095

Soliman, M., Mahmoud, B., Kefafy, M., Yassien, R., El-Roghy, E. Effect of antidepressant drug (fluoxetine) on the testes of adult male albino rats and the possible protective role of omega-3. Menoufia Medical Journal, 2017; 30: 1135. https://doi.org/10.4103/mmj.mmj_521_17

Suvarna, K. S., Layton, C., Bancroft, J. D. 2018: Bancroft’s theory and practice of histological techniques E-Book. Elsevier health sciences.

Wdowiak, A., Raczkiewicz, D., Stasiak, M. Levels of FSH, LH and testosterone, and sperm DNA fragmentation. Neuroendocrinology Letters, 2014; 35: 1.

Won, Y.-J., Kim, B.-k., Shin, Y.-K., Jung, S.-H., Yoo, S.-K., Hwang, S.-Y., Sung, J.-H., Kim, S.-K. Pectinase-treated Panax ginseng extract (GINST) rescues testicular dysfunction in aged rats via redox-modulating proteins. Experimental Gerontology, 2014; 53: 57-66. https://doi.org/10.1016/j.exger.2014.02.012

Zemjanis, R. 1970: Diagnostic and therapeutic techniques in animal reproduction. Williams & Wilkins; Second Edition.