Gonadal health benefits of black cumin (Nigella sativa) seed oil on chronic lead-exposed male Wistar rats

Lead (Pb) is widely recognized as one of the most perilous toxic heavy metals that possess the potential to inflict severe harm on nearly all essential organ systems, encompassing the neurological, renal, and reproductive systems [1]. As per the World Health Organization (WHO) estimates, Pb exposure results in roughly 1 million fatalities and 21.7 million disability-adjusted life years (DALYs) worldwide each year. Moreover, it is linked to idiopathic intellectual disability (accounting for 30% of cases), cardiovascular disease (approximately 4.6%), and chronic kidney diseases (around 3%)[2]. Numerous industrial chemicals are well-documented for their detrimental effects on human reproductive health [3], with a particular focus on the adverse outcomes associated with the workplace as well as atmospheric contact with heavy metals such as Pb [4, 5]. Exposure to inorganic Pb in male health is linked to decreased semen quality, impacting spermatogenesis and fertility. Elevated Pb levels in seminal plasma can also reduce sperm count [6]. Moreover, Pb exposure can adversely affect semen quality parameters, such as sperm density, motility, and morphology [7]. Epidemiological studies have indicated that male employees having blood Pb concentrations between 10 and over 40 µg/dl may face an elevated risk of experiencing infertility [8].

It is important to note that Pb poisoning can affect not only humans but also domestic animals, ranging from livestock to pet animals [1]. Animals can get intoxicated from Pb-contaminated feed and water, primarily from soil, industrial pollution, agricultural practices, and feed processing. Notably, Pb in poultry feed often comes from bone, blood meals, and tannery waste, leading to poisoning, reduced performance, and animal fatalities [9]. Acute Pb poisoning in animals can result in a 100% fatality rate, while chronic exposure affects the male reproductive system by causing testicular damage, atrophy of Leydig cells, and semen abnormalities like azoospermia, asthenozoospermia, teratozoospermia, and morphologically abnormal sperm via leading to reduced fructose and succinic dehydrogenase levels [10]. For instance, Pb exposure leads to reduced sperm counts within the epididymis along with ceased sperm production [11]. Additionally, microscopic examinations have revealed noteworthy alterations in the morphological structure of testicular tissues and reduced germ cell populations [12]. Moreover, mice exposed to Pb exhibited degeneration of the seminiferous tubule [13].

Herbal medications, which have been used for therapeutic purposes since antiquity and are often free of side effects, are becoming increasingly popular over synthetic treatments due to their natural origin and the fact that nature supplies the primary resources for human health [14]. The alcoholic extract of Nigella sativa has been found to improve reproductive health, including body weight gain, reproductive parameters (e.g. seminiferous tubules, spermatogonia, spermatocytes, spermatids, Sertoli and Leydig cells, Leydig cell diameter, and epididymal caudal region epithelial cell height), and testosterone along with follicle-stimulating hormone (FSH) levels [15]. In rams, black cumin seed extract increased ejaculation volume, sperm activity, and motility, improving accessory gland production [16], while in male rats, this improved histomorphological functions of testicles while decreasing sperm abnormalities [17]. However, the toxic effect of Pb on reproduction has already been revealed but mitigation of toxic effects by natural herbs like Nigella sativa seed oil (NSO) is still unclear in Wistar male rats. Therefore, the present study aimed to reveal the effects of NSO on the testicular morphology, hormonal assay, and histological changes in chronic Pb-exposed male Wister rats.

Ethical Approval

The Animal Welfare and Experimentation Ethics Committee at Bangladesh Agricultural University, Mymensingh, granted approval for the animal care, management, and experimental procedures under reference number AWEEC/BAU/2023 (42).

Experimental site and layout

The research was carried out at the Department of Physiology, Bangladesh Agricultural University, Mymensingh, over 16 weeks. Forty male albino Wistar rats weighing between 270-280 g were sourced from the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR’B) in Dhaka. The animals were kept in a meticulously controlled environment, ensuring precise regulation of factors such as temperature, humidity, ventilation, and lighting. Rats were randomly divided into four groups of 10 rats each. Group A served as a control group that received standard rat chow only. Group B rats were administered 75 mg Pb acetate/kg body weight (BW) in the drinking water whereas group C rats were given 1 mL NSO/kg feed. Lastly, Group D rats were subjected to combined treatment, receiving daily doses of 75 mg Pb acetate/kg BW and 1 mL NSO/kg feed. All the experimental treatments were given for 16 weeks. Pb acetate and NSO were purchased from “Sweety Scientific Traders Ltd”, Mymensingh, Bangladesh, and the doses of NSO and Pb was selected according to results obtained from our earlier research [18, 19].

Blood collection and serum preparation

After the 16-week experiment, the rats were subjected to an overnight fast. Subsequently, each rat was placed individually in a sealed chamber containing cotton soaked with diethyl ether. Once unconscious, the rats were removed from the chamber, and blood samples were collected directly from the heart using a sterile syringe. Approximately 3-3.5 ml of blood had been withdrawn and the tubes containing the blood were gently slanted and left at normal temperature for 60 minutes. Afterward, the blood clot had been carefully removed from the inner tube wall, and then given some time for settling, following which the serum was obtained. This serum was then subjected to centrifugation at 3000 rpm for 15 minutes to get clean serum, which was subsequently kept at -20°C till required.

Hormonal assay

At the Institute of Nuclear Medicine & Allied Sciences (INMAS; Mymensingh Medical College, Mymensingh, Bangladesh), serum levels of T₄ and testosterone were measured using a radioimmunoassay kit (Berthold, Bad Wildbad, Germany) in accordance with the standard protocol [20], with an assay sensitivity of 0.10 nmol (0.0288 ng/ml). For testosterone, the inter-assay coefficient of variation was 12.2%.

Assessment of sperm physiological characteristics along with testicular parameters

After sacrifice, the testis was checked for weight by using an electronic balance. The testis diameter was measured by using slide calipers. The epididymis was taken out to test sperm motility and concentration. In brief, a small amount of the diluted mixture was put on a previously warmed slide, and an outer cover slip was placed on it. The percentage (%) of sperm motility had been established. Both the epididymal sperm collection and sperm computation had been completed [21]. To do this, the rat's cauda epididymis was removed in a Petri dish and then minced with the help of scissors. The macerated epididymis was put into a test tube containing 4 milliliters of prewarmed (37 °C) normal saline. After that, 5-10 minutes were given so that the sperm could come out. Finally, the sperm was estimated using Neubauer's chamber and a microscope's high-power objective.

Histopathological analysis

Testicles were obtained from each group of rats, and thorough removal of blood was achieved through perfusion with phosphate-buffered saline. These collected testicles were then placed in 10% neutral buffered formalin and preserved for a duration of 15 days. After this preservation period, the properly fixed tissues were subjected to processing, sectioning, and staining (hematoxylin and eosin) in collaboration with the Department of Pathology at Bangladesh Agricultural University, Mymensingh. This followed the established protocol as outlined by [22]. The stained slides were examined using an Olympus Photomicroscope (Model CX43) at the Department of Physiology, BAU, Mymensingh, and photographs of these slides were taken.

Johnsen scores were used to assess spermatogenesis in seminiferous tubules [23]. At a magnification of 40X, twenty cross-sectional segments of seminiferous tubules were examined in every sample and ranked from 1 to 10. 10: Indicates the presence of perfect tubules and complete sperm production; 9: Multiple sperm cells present but with disorganized sperm production; 8: Few sperm cells present; 7: No sperm cells present but numerous immature sperm cells (spermatids) present; 6: Few immature sperm cells present; 5: Neither sperm cells nor immature sperm cells present but numerous early-stage sperm cells (spermatocytes) present; 4: Few early-stage sperm cells present; 3: Only early-stage sperm cells (spermatogonia) present; 2: No germ cells (cells that give rise to sperm) present; 1: Neither germ cells nor Sertoli cells (cells that support sperm development) present.

Statistical analysis

Initially, laboratory data were recorded using Microsoft Excel 2016 and then imported into GraphPad Prism 5 for a thorough analysis. To evaluate mean values, standard error of the mean (SEM), and determine statistical significance (p-value), we conducted a Bonferroni multiple comparison test across various parameters. Given the utilization of multiple comparisons, we calculated and adjusted the P-values to significant levels of 0.001, 0.01, and 0.05. For the comparative analysis of hormonal, sperm and testicular parameters in rats corresponding to fed supplementation, an Analysis of Variance (ANOVA) was employed.

Effect of NSO and Pb on testosterone and thyroxin concentration

The results of the hormonal assay, specifically measuring testosterone levels, revealed a noteworthy impact of Pb treatment. The serum testosterone level was markedly reduced to 1.15 ± 0.25 ng/mL, a significant decrease (p ≤ 0.001) when compared to the control group of rats, where it averaged 2.59 ± 0.17 ng/mL (Figure 1A). Interestingly, the serum testosterone level demonstrated resilience following supplementation with NSO, with levels reaching 3.10 ± 0.10 ng/mL, and in the NSO singly supplemented group, where levels reached 3.85 ± 0.05 ng/mL (Figure 1B).

Furthermore, the influence of Pb treatment extended beyond testosterone, impacting serum T₄ levels. Notably, T₄ levels significantly declined after Pb exposure but exhibited a notable increase following NSO supplementation.

Effect of NSO and Pb on sperm motility and concentration

The findings indicate that sperm motility in the control group was 82.50 ± 2.50%, but this motility significantly decreased to 34.00 ± 1.00% after exposure to Pb. However, when NSO supplementation was administered alongside Pb exposure, there was a notable improvement in sperm motility, which increased to 64.00 ± 4.00% (Figure 2A). Similarly, sperm concentration experienced a substantial decline after Pb exposure but exhibited a significant increase when NSO supplementation was introduced (Figure 2B).

Effect of NSO and Pb on testis weight and diameter

Testis weight was measured during the necropsy. The study findings indicated that in the control group, the mean weight of both the left and right testis was recorded as 1.56 ± 0.02 g. However, in rats treated with Pb, there was a significant decrease in testicular weight. Conversely, Pb-treated rats supplemented with NSO exhibited a notable increase in testicular weight compared to those treated with Pb alone (Figure 3A).

Testis diameter measurements were obtained during necropsy, revealing distinct findings among the experimental groups. In the control group, the mean diameter of both left and right testis was 2.28 ± 0.04 cm. However, significant shrinkage in testicular diameter was observed in Pb-exposed rats. Notably, the Pb-treated rats that received supplementation with NSO exhibited a notably greater testicular diameter than rats that were administered Pb only (Figure 3B).

Effect of NSO and Pb on testicular histopathology

Histological examination of testicular tissue in the control group revealed a normal tissue structure. In contrast, the Pb-treated group exhibited significant degeneration of seminiferous tubules. However, both the group treated with NSO alone and the group treated with both Pb and NSO displayed normal histological characteristics in their testicular tissues (Figure 4).

The mean Johnsen score (MJS) of the untreated mice was 9.40 ± 0.19, whereas mice that received Pb had a significant reduction in MJS of 7.6 ± 0.28. The group supplemented with NSO along with Pb had increased the MJS to 9.53 ± 0.19, which clearly indicates the restoring effect of the male reproductive system by NSO on Pb intoxication (Figure 5).

In the era of the industrial revolution and rapid expansion of agricultural sectors, environmental pollutants comprising toxic heavy elements, including Pb, are indeed entering the food chain resulting in increased exposure to Pb for both humans and animals [24]. Pb toxicity has been linked to male reproductive health reducing sperm quality and fertility in men, potentially leading to reproductive failure [25]. Therefore, this research investigates the detrimental consequences of Pb exposure in experimental animals and assesses whether Nigella sativa, as a natural product, may offer protective effects against Pb-induced damage to male fertility.

The findings indicate a notable decrease in serum testosterone (Figure 1A) and T₄ levels (Figure 1B) following Pb exposure, which were subsequently restored to normal levels with NSO supplementation. These results suggest that Pb toxicity likely disrupts the functioning of the pituitary and hypothalamus, leading to impaired luteinizing hormone secretion causing direct damage to the seminiferous tubules in the testes, resulting in reduced testosterone secretion from Leydig cells [26, 27]. Furthermore, research has indicated that Pb exposure in albino rats can impact the steroidogenic activity of the testes, along with the alterations in testosterone and gonadotropin serum levels [28]. Thyroid function often becomes compromised in individuals exposed to Pb [29]. Specifically, a decline in thyroid hormone levels has predominantly been identified through research involving employees at greater exposure limits, typically with mean blood Pb levels exceeding 60 μg/dL. Conversely, at lesser Pb contact limits, the findings showed varying outcomes, with some researches indicating a rise, others showing a decline, and some revealing no significant changes in hormone concentration [30].  Blood Pb levels are shown to be negatively related to total thyroxine levels, which suggests that Pb may lower the number of proteins bound to thyroxine and enhance its clearance from the blood [31]. In our current research context, rats treated with NSO exhibited a marked elevation in both testosterone and thyroxin concentrations (Figure 1). These findings align with the outcomes of previous studies conducted by [32] and [33]. A notable rise in testosterone and thyroxin levels in mice fed with NSO by their antioxidant properties that inhibit ROS generation and enhance the activity of antioxidant enzymes such as superoxide dismutase and glutathione [34]. Following treatment with an alcoholic extract of black cumin seed, there are notable increases in body weight gain, reproductive characteristics (such as the thickness and diameters of seminiferous tubules, the number of spermatogonia, primary and secondary spermatocytes, spermatids, spermatozoa, the amount of Leydig cells as well as Sertoli, the diameter of Leydig cells, and the height of epithelial cells that completely filled the epididymal caudal), as well as hormones (such as testosterone and FSH) [35].

In this study, we observed reductions in motility (Figure 2A) and concentrations (Figure 2B), testis weight (Figure 3A), and diameter (Figure 3B) after Pb exposure. Sperm motility, structural integrity, and viability are all significantly impacted by this membrane fluidity  [36, 37]. There are several mechanisms through which ROS production can impact sperm motility. One such mechanism by Pb is the potential reduction in phosphorylation of axonemal proteins essential for sperm movement and concentrations [38]. Importantly, this reduced motility may occur without a concurrent drop in mitochondrial membrane potential. Similar outcomes were also observed in investigations examining the impacts of Pb on the weight of testes [39]. Many variables, including an early onset of edema, inflammation, cellular infiltration, and other aspects, can explain this seeming contradiction. Another possibility is the development of Leydig cell hyperplasia [40]. This study observed an increase in the weight and diameter of the testis and the motility and concentrations of sperm in the group treated with both NSO and Pb. Interestingly, NSO enhanced the rate of reproduction in hyperlipidemic rats [41]. Furthermore, a blend comprising equal proportions of Eruca sativa, Nigella sativa, and Raphanus sativus meals has shown promising effects on semen’s physical properties while reducing free radicals in seminal plasma [42]. In addition, when NSO is orally administered daily for up to 60 days, it notably improves the weight, circumference, volume, length, width, and thickness of testicles in comparison to the control. All of the results align with similar observations made in rat studies [32, 43].

This study’s findings indicated that 16 weeks of daily oral consumption of Pb had noticeable effects on the testicular structure (Figure 4), suggesting adverse impacts on the process of spermatogenesis. Recent research has also reported comparable or even greater severe alterations in male gonadal tissue in rats and mice exposed to Pb [44, 45]. According to [46], any harm to Sertoli cells can quickly disrupt their paracrine control over other dependent spermatogenic cells, potentially resulting in subsequent cell necrosis, rapid exfoliation into the lumen, and thinning of the seminiferous tubule walls. However, it is important to note that the obvious detrimental effects of Pb on germ cells cannot be dismissed [47]. In the current study, co-treating Pb-exposed rats with NSO effectively protected them from histological changes in the testicles (Figure 4). NSO has demonstrated remarkable potential in preserving testicular health against damage caused by various agents that generate free radicals. For instance, NSO supplementation has been shown to restore spermatogenesis in rats following testicular injury induced by chronic toluene exposure [48]. NSO has also provided protection to experimental mice from testicular tissue damage induced by methotrexate [33] and significantly reversed deviations in semen quality and reproductive characteristics caused by heat stress [49]. The adverse impacts of Pb acetate upon antioxidant defenses, testosterone levels, and spermatogenesis; however, oral NSO treatment mitigated these negative effects [50].

NSO consumption has protective effects on serum testosterone and thyroxine levels and testicular and sperm physiological parameters in chronic Pb-exposed rats, suggesting it may be a useful dietary supplement against Pb-induced reproductive toxicity. Further research is needed to understand protective the cellular level mechanism of NSO against Pb exposure. This could expand knowledge of NSO advantages and may help to its use as a dietary intervention to reduce heavy metal exposure's health risks.

The authors would like to express their gratitude to the Ministry of Science and Technology and the Government of the People's Republic of Bangladesh for the study's funding grant (Project ID: SRG-221134). The authors also acknowledge the Bangladesh Agricultural University Researcher System (BAURES) as well as everyone engaged with the research project for being willing to get involved.

AM planned and executed the experiment, monitored it, analyzed the results, and evaluated the final version of the report. In addition to executing the experiment, MA, SR, and AUM analyzed the data and wrote the initial draft of the paper. MAM and EHC provided significant revisions to the article.

There is no conflict of interest among the authors.