Protective Role of Garlic Extract Against Toxicity of Nickel Nitrate on Spermatocytes and Fertility Outcome
Sahar Hashim Al-Hindawi, Khalid Hamdan Gathwan, Zainab Abdul Jabaar Aldhaher, Rasha Mohammed Shaker
Department of Basic Science, College of Dentistry, University of Baghdad, Baghdad, Iraq.
Abstract | The current experiment was designed to detect the protective role of garlic extracts against the toxic effects of Nickel Nitrate (NiNO3) in white mice. Thirty male mice, were randomly distributed in five groups: (Group I) as control; other groups were orally administered different doses of NiNO3 daily with diet and water for 30 days through gavage (Group II: 7.1 mg/Kg), (Group III: 13.6 mg\ kg), (Group IV: 13.6 mg\ kg. + Garlic 120 mg\ kg), and (Group V: 13.6 mg\ kg + Garlic 180 mg\ kg). Spermatozoa of male mice were evaluated for count, motility, validity, sperm head abnormality, and live embryos in healthy female mice mated with males treated with NiNO3. Results reported in male mice show that NiNO3 substantially decreases sperm parameters (count, motility, and live spermatozoa) in groups II and III (low and high dose, respectively) compared to the control group. Garlic extract substantially enhanced sperm parameters in group V (with a high dose of garlic) related to group III (without garlic). Sperm count rose considerably in group V compared to group III. The mean sperm head abnormalities in group III were substantially higher than in groups I, II, IV, and V. Groups IV and V (with garlic extract) displayed no significant difference from group I, while mice subjected to NiNO3 showed more significant abnormalities in spermatozoa heads with increasing dosages. Female mice mated with males exposed to NiNO3 showed significantly decreased live embryos in group III compared to groups I, II, IV, and V. This study proved the protective role of garlic extract against the toxicity of NiNO3 on sperm mice, and confirmed the toxic properties of NiNO3 on sperm, leading to infertility in male mice.
Novelty Statement | This study provides novel evidence for the effects of NiNO3 on fertility and living embryos, revealing that NiNO3 causes infertility by affecting the quantity and quality of viable sperm during sperm formation and embryo development, which is treated by garlic extract.
Article History
Received: May 23, 2025
Revised: July 05, 2025
Accepted: July 23, 2025
Published: December 08, 2025
Authors’ Contributions
All authors contributed equally to planning and carrying out this work. All authors read the manuscript and confirmed its publication as the final version.
Keywords
Nickel, Nitrate, Garlic, Mice, Infertility, Healthy lifestyle
Copyright 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Corresponding author: Sahar H. Al-Hindawi
To cite this article: Al-Hindawi, S.H., Gathwan, K.H., Aldhaher, Z.A. and Shaker, R.M., 2025. Protective role of garlic extract against toxicity of nickel nitrate on spermatocytes and fertility outcome. Punjab Univ. J. Zool., 40(2): 225-232. https://dx.doi.org/10.17582/journal.pujz/2025/40.2.225.232
Introduction
The effects of harmful substances are currently a main focus of interest because they are everywhere (Mahdi et al., 2021). Nickel (Ni) is an essential trace element; modifications in the concentration of Ni have an influence on the construction and activity of hormones like prolactin, adrenaline, noradrenaline, and aldosterone. Ni toxicity influences membrane characterization variations and oxidation/reduction systems in cells. In animals, Ni toxicity causes cancer and leads to embryotoxicity, while the deficiency of Ni is linked to a reproductive rate, decreased development, and changes in lipid and glucose metabolism (Samal and Mishra, 2011). Ni overdose has been found to be a severe poison in the past few years (Rizwan et al., 2022). Nickel and nickel compounds are classified as carcinogens (Gathwan et al., 2013; Buxton et al., 2019).
Nickel nitrate (NiNO3) is classified as hazardous, but the data about NiNO3 acute toxicity are limited. This compound of nickel has bioavailability and bioaccessibility, releasing (Ni) ions into biological fluids (Henderson et al., 2012). Nickel nitrate has toxic action on rats’ kidney tissue, resulting in kidney cell atrophy (Varsha and Singh, 2021). Nickel oxide (NiO) was found to be a mutagenic agent, toxic, and essential trace element for all multicellular organs (Ali and Al-Mammar, 2024; Abbasa et al., 2024). Wyrobek et al. (1983) used sperm head morphology in mice as an index of mutagens or carcinogens. Nickel sulfate administered orally to adult male mice results in a strong reduction in body weight and sperm abnormalities associated with a decline in sperm mortality and sperm count (Pandey et al., 1999). Regularly, nitrite is employed as an addition to food conservation. Still, inordinate nitrite may be harmful and cause either acute or chronic poisoning (Sindelar et al., 2012). Time-dependent mouse infertility induced by nitrite exposure may be caused via apoptosis and oxidative stress (Wu et al., 2022).
Garlic (Allium sativum), a multifunctional crop, is appreciated for its therapeutic properties through its bioactive components of allicin, an extensively studied chemical, including phenolic compounds, organic sulfides, polysaccharides, and saponins (Jasim et al., 2023; Jain et al., 2025). Garlic components, with vital oil, matured garlic extract, matured black garlic, and garlic powder, have anti-inflammatory, antioxidant, antiviral, antibiotic, anticancer, anti-hyperlipidemic, and hypertensive properties (Jain et al., 2025). Garlic extraction protects testicles and boosts sperm (Shukry et al., 2021). Garlic, which directly affects estrogen and subsequently affects testosterone, may expected to stimulate sexual cells and sex hormones (Thuy et al., 2020; Hamed et al., 2021).
The study was intended to observe the protective effect of garlic extracts against the toxicity of NiNO3 on white mice. It also demonstrates the toxicity of nickel nitrate (NiNO3), which causes infertility in male mice, by evaluating sperm changes in morphology, count, motility, and validity.
Materials and Methods
Preparation of aqueous garlic extract
Fresh garlic was used, and the aqueous extract was obtained using a modified approach. After homogenizing thirty grams of garlic in one hundred milliliters of cold distilled water, the liquid was repeatedly filtered through cheesecloth and centrifuged at 200 g for ten minutes to produce the clear supernatant. Every mouse received a supernatant daily by oral gavage, based on weight (Martha et al., 1998).
Animals housing
The experiments began in March and ended in June 2024. The animals purchased from the National Centre for Drug Control and Research (NCDCR), Iraqi Ministry of Health, thirty (30) male mice weighing 32–36 g at the age of 8–10 weeks, with six female mice for mating. They were left for seven days to enable acclimation prior to beginning the therapy under regulated temperature settings of 22±3ºC and 12:12 light and dark cycle. Mice pellets and tap water were given to animals for eating and drinking.
Sample size
G*Power 3.1.9.7 (Franz Faul software) uses 8% study strength, 0.05 significance, and 0.5 Cohen’s D middle effect size. A total of 27 animals were studied. Cohen’s D effect sizes are: Small= 0.2, Medium= 0.5, Large= 0.8 (Cohen, 1988).
Experiment design
After one week of the adaptation period, A total of 30 healthy adult male mice were divided randomly into five main groups, with (6) mice in each group; the control group was fed only on a normal diet and water, and the other four groups were orally administered two doses of NiNO3 daily with diet and water for 30 days through gavage and two doses of garlic extract as follow (Pandey et al., 1999):
Sperm suspension preparation
After thirty days of exposure to NiNO3 and after twenty-four hours from the last day of treatment, four mice from each group were killed, and the remaining two live mice were left for fertility testing. Collect sperm samples by mixing the sperm from the vas deferens with 1 ml of standard water (0.9% NaCl) in a small dish. To stop sperm adhesiveness, the sperm suspension was kept at 37°C for ten minutes (Otitoloju et al., 2010).
Seminal fluid analysis
Motile spermatozoa were assessed using a Neubauer hemocytometer and a light microscope at 40× magnification with a drop of the sperm suspension solution. Sperm motility was evaluated as a percent of motile sperm of the whole sperm counted (Linder et al., 1986). The technique suggested by Evans and Maxwell helped to ascertain the approximate live-dead ratio of sperm cells (Evans and Maxwell, 1987).
Table 1: Effect of NiNO3 and garlic extract on spermatocyte parameters of male mice in groups of experiments.
|
Group/parameters |
Sperm cell count (x106/ mL) |
Sperm motility % |
Live spermatozoa % |
|
Group I (Control group) |
152.92 ± 3.1 |
89.20 ± 2.5 |
85.21 ± 2.8 |
|
Group II (NiNO3 7.1 mg\ kg) |
137.6 ± 3.4 |
76.8 ± 3.7 |
76.7 ± 2.8 |
|
Group III (NiNO3 13.6 mg\ kg) |
101.3± 4.6 |
70.3 ± 2.7 |
72.1 ± 2.9 |
|
Group IV NiNO3 13.6 mg\kg + garlic120 mg\ kg |
148.5±1.7 |
75.1 ± 2.1 |
79.6 ± 2.5 |
|
Group V NiNO3 13.6 mg\ kg + garlic180 mg\ kg |
153.3 ± 2.5 |
87.1 ± 2.1 |
86.4 ± 2.8 |
|
p-value |
0.021 |
0.032 |
0.038 |
The data expressed in mean ± standard deviation (Mean ± S.D); significant p-value < 0.05
The assessment of sperm abnormalities was conducted by preparing smears from the sperm solution. A drop of sperm sample was placed on a clean slide, let dry, and then fixed by ethanol for one hour. The sample was treated with Giemsa for 15–20 minutes, then washed and dried. After that, it was examined under a light microscope at 100× magnification to count the normal and abnormal sperm percentages (Otitoloju et al., 2010).
A fertility test was done by using the surviving mice from each group. After 24 hours after the final day of treatment, the treated males were paired with untreated healthy females overnight in a ratio of one male to three females. Every female was housed alone when the vaginal plug was noted. Pregnant mice were immolated at eighteen days of gestation, and embryos were separated from the uterus to determine the number of live and dead implants (Sasi et al., 2023).
Statistical analysis
The statistical analysis system (SAS Software, 2021) used a one-way analysis of variance (ANOVA) to evaluate the significance level between the control and treatment groups, with p < 0.05 indicating statistical significance. The data were expressed as mean ± standard deviation (mean ± SD).
Results
The observation reported that in NiNO3-treated mice, there was a reduction in food and water consumption, relative to the control group; moreover, the decrease was dose-dependent. The current work used ANOVA to demonstrate that NiNO3 at varying dosages (in groups II and III) substantially reduced sperm parameters, including sperm count, sperm motility (%), and live spermatozoa (%), in male mice compared to the control group (group I) (p < 0.05). The most pronounced toxicity of NiNO3 was seen in group III (NiNO3: 13.6 mg/Kg), whereas group II exhibited lower toxicity (NiNO3: 7.1 mg/Kg) in comparison to the control group, as shown in Table 1. However, the effect of garlic extract appeared in group V, with the sperm parameters [sperm count, sperm motility (%), live spermatozoa (%)] increased significantly (p < 0.05) related to the parameters in group III (with NiNO3 13.6 mg\ kg), but no vital difference in the groups IV (with NiNO3 13.6 mg\ kg + garlic120 mg\ kg) and V (with NiNO3 13.6 mg\ kg + garlic180 mg\ kg) related with a control group. About the differences between groups III (without garlic extract) and IV (with garlic extract), all parameters exhibited non-significant differences (p ˃ 0.05) except sperm count, which increased significantly (p < 0.05) compared to the count in group III.
In Table 2 the ANOVA test showed the sperm head abnormalities in group II mean value (9.9 ± 1.6) were non-significant (p ˃ 0.05) compared to group I mean value (6.80 ± 1.3), but in group III, the mean value (18.7 ± 1.9) elevated significantly (p < 0.05) related to group I, II, IV, and V (6.80 ± 1.3, 9.9 ± 1.6, 8.3 ± 1.7, and 7.6 ± 1.70) respectively. In group IV with garlic extract, 120 mg\ kg, the mean value (8.3 ± 1.7) and group V (7.6 ± 1.70) were non-significant (p < 0.05) with group I (6.80 ± 1.3), and the abnormalities of the head of spermatozoa of male mice exposed to NiNO3 were increased in abnormalities with multiplication of NiNO3 doses. The abnormalities were in some arbitrary types (amorphous, hammer-shaped, cylindrical, spinal, triangular, oval, curved, and double-head sperm) (Figure 1).
Table 2: The effect of NiNO3 and garlic extract on spermatocytes, with the abnormalities in the head in each group.
|
Group |
The mean of an abnormal head of sperm |
|
Group I |
6.80 ± 1.3 |
|
Group II |
|
|
Group III |
|
|
Group IV |
|
|
Group V |
7.6 ± 1.70 |
|
p-value |
0.018 |
The data are expressed in mean ± standard deviation (Mean ± S.D); significant p-value < 0.05.
The mean live embryos in female mice mated with males exposed to NiNO3 were revealed in Table 3, and there was a substantial (p < 0.05) decline in the number of live embryos in group III mean value (2.83 ± 2.1) treated with NiNO3 in high dose (13.6 mg\ kg) comparing with group I, II, IV, and V (10.30 ± 1.4, 7.37 ± 1.6, 8.42 ± 1.3 and 9.82 ± 2.6) respectively, and non-significant value (p > 0.05) in the number of live embryos in group V (13.6 mg\ kg + garlic180 mg\ kg ) compared to group I (10.30 ± 1.4) mean value. Table 3 and Figure 2 demonstrate the effect of NiNO3 and garlic extract on live embryos.
Table 3: The mean number of live embryos in female mice mated with male mice treated with NiNO3 and garlic extract.
|
Group |
The mean number of live implants |
|
Group I |
|
|
Group II |
7.37 ± 1.6 |
|
Group III |
2.83 ± 2.1 |
|
Group IV |
8.42 ± 1.3 |
|
Group V |
9.82 ± 2.6 |
|
p-value |
0.032 |
The data expressed in mean ± standard deviation (Mean ± S.D); significant p-value < 0.05
Discussion
The current study’s results improved the influence of Nickel (Ni) on spermatogenesis and fertility. This finding is in accordance with previous studies demonstrating that Nickel and Nickel’s compounds had a negative effect on spermatogenesis in male mice. Still, to our knowledge, no studies have considered the impact of nickel nitrate on mice fertility.
The experimental study conducted by Zeng et al. (2023) reported that nickel chloride (NiCl2) caused abnormalities in the testis and epididymis histopathological structure and depletions in sperm count with an increase in germ cell shedding in the lumen, indicating that NiCl2 is able to cause damage to the critical testicular structure and the blood-testis barrier (BTB) which is sensitive to reproductive toxicity of a variety of environmental contaminants. Another study by Pandey et al. (1999) on a different nickel compound showed that oral treatment with nickel sulfate (NiSO4) induces significant histological alterations in the testes and epididymis and reduces sperm in mice.
Prior studies demonstrated that nickel can build up in the testes, causing cell death, abnormal sperm formation, and spermatogenesis, which can harm reproduction in mice. It also affects (BTB), which provides a constant environment for spermatogenesis (Zeng et al., 2023). The results by Forgacs et al. (2012) found that nickel is toxic to the testes. However, the findings of Yang et al. (2021) demonstrated that various dosages of NiCl2 administered to mice may gather in the testes and disrupt the hypothalamic-pituitary-testicular axis, which plays a pivotal role in sperm production abnormalities in mice caused by NiCl2.
However, Zemanova et al. (2007) found that Ni affects the cyclic nucleotide-gated channels, which are vital to sperm functions. They noticed that a lack of Ni caused a big decrease in the motility and amount of sperm in the epididymis, epididymal transportation time of sperms, and testes’ sperm origination rate. Lack of Ni also reduced the weight of the seminal vesicles and prostate glands to a substantial extent.
About the effect of nitrate on fertility, research by Wu et al. (2022) reported that nitrate exposure may lead to infertility in mice, revealing further effects with repeated exposure. Previous studies by Amini et al. (2017, 2018), indicated the impact of nitrate on male mice, utilizing both direct and indirect assessments of sperm parameters, which suggested damaging effects displayed as decreased sperm motility, lowered sperm count, elevation of total abnormal sperm, and diminished testicular enzyme activity. Research on rabbits exposed a negative correlation between nitrate exposure and embryo count, testosterone levels, and sperm restrictions (Pant and Srivastava, 2002).
There is agreement with other studies regarding the embryos in female mice after mating with male mice treated with NiNO3. Greenlee et al. (2004) reported an elevated percentage of spontaneous abortion, embryonic demise, or heightened death in the embryos of female mice subjected to nitrate. Furthermore, additional research has shown links between nitrates and inborne with low weight, tiny for gestational age, premature delivery, stillbirth, and adverse reproductive outcomes (Ward et al., 2018). According to different research, around 30% of pregnancies terminate spontaneously; this might be because of poor semen quality, the causes of which are not well known (Weselak et al., 2008).
Concerning the findings of this study, verifying the constructive impact of garlic extract on sperm quality, motility, and viability, and against the toxicity of NiNO3, most studies agree with these results. A survey by Rabbani (2019) showed that garlic powder at a dosage of 150 mg/Kg mitigated the cytotoxic effects of NiCl2 on somatic and germinal cells, suggesting that it is associated with free radical scavenging activities. Garlic enhances male sexual function and positively influences the recovery of testicular functions, highlighting its beneficial effects on sperm concentration, motility, and viability (Bahrami et al., 2014; Qadariah et al., 2020).
Conversely, the current study’s findings contradict those of Qian et al. (1986), who stated that rats treated with garlic displayed a decline in sperm quality and functionality. The primary distinction and reasons for varying outcomes may be from the absence of constancy among study models and the disparate quantities of garlic administered to experimental subjects; nonetheless, providing more garlic supplementation over an extended duration to adult rats resulted in an increase in epididymis spermatozoa (Green et al., 1985).
Garlic includes phytoestrogens that directly influence estrogen, a precursor to testosterone development, suggesting that garlic may impact sexual cells and sex hormones (Hammami et al., 2013). Garlic complements enhance luteinizing hormone (LH) production from the pituitary gland, hence stimulating testosterone output from the testes (Oi et al., 2001). Cooked garlic is indicated to have superior corrective properties, while impacting the proliferation of sperms in the epididymis and testicles and enhancing spermatogenesis (Bahrami et al., 2014).
Garlic comprises components such as flavonoids, vitamins, fructose, and sulfur compounds, which may assist in removing free radicals. Sulfur compounds in garlic protect spermatogenesis, possess antioxidant characteristics, and may enhance fertility by diminishing lipid peroxidation (Moher et al., 2009). Garlic is recommended to help as a therapeutic for infertility. A study by Asadpour and others found that garlic has antioxidant properties because of vitamin E, which helps block oxygen peroxide (Asadpour et al., 2013). Nasr’s findings indicated that garlic’s antioxidant possessions may decrease the toxicity of destructive medications on the testes and enhance fertility and sperm production (Nasr, 2017).
To our knowledge, there is no research regarding the effects of NiNO3 on mice fertility and living embryos from mating between male mice treated with NiNO3 and healthy female mice. The findings of this study recorded that NiNO3 caused infertility, as fertility based on the quantity of viable sperm with good quality during sperm formation to produce healthy embryos, and consequently garlic extract (including its components) can be considered a respectable treatment for infertility cases resulting from pollutants such as nickel, nitrate, and other compounds, as it is an antioxidant and prevents toxicity by mechanism may be associated with the free radical removal activity.
Conclusions and Recommendations
This study proved the garlic extract’s protective effect, at high doses, against the toxicity of Nickel Nitrate on sperm mice. Furthermore, it confirmed the toxic effects of Nickel Nitrate on sperm count, morphology, fertility, and embryos, leading to the infertility of male mice.
Future research must include molecular or biochemical assays to understand mechanisms, such as antioxidant levels or DNA damage markers.
Declarations
Acknowledgment
Thanks to all participants in the present study.
Funding
This research was self-funded.
IRB approval
All animal procedures were conducted in accordance with the rules of the Animal Care and Use. Ethical approval for this study was arranged by the Institutional Review Board (IRB) of the College of Dentistry, University of Baghdad, under project No. 1008825.
Ethical statement
All experiments and procedures were approved by the Ethical Review Committee of Baghdad University, College of Dentistry, which permitted this experimental study under No. (1008).
Limitations of the study
More animals are needed to increase the confirmation of the results, and they need long-term evaluation.
Generative AI and AI-assisted technology statement
No Generative AI and AI-assisted technologies were used in the writing process .
Conflict of interest
The authors have declared no conflict of interest.
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