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Ameliorative effects of African walnut on nicotine-induced reproductive toxicity in rat model
Address for correspondence: David Chibuike Ikwuka, Department of Physiology, University of Nigeria Enugu Campus, Enugu State, Nigeria. Phone: +2348037147603. E-mail: davidikwuka@gmail.com
Abstract
Objective:
Walnuts are widely consumed nut by men in Nigeria and it has been connected to improving male reproductive health. This study evaluated the effect of African walnut on sperm parameters and testicular architecture of nicotine (NIC)-induced reproductive toxicity in male Wistar rats.
Methods:
Wistar rats were randomly assigned into four groups, that is, GN0 (1 ml/day normal saline and normal rat chow), GN1 (1 ml/day NIC and normal rat chow), and GN1W6 and GN1W12 (1 ml/day of NIC daily fed with 6% and 12% walnut-rich feed), respectively. This continued for 28 days. The animals were euthanized and their sperm was collected and its parameters were analyzed. The testis was harvested and prepared for histological examination.
Results:
NIC significantly reduced sperm motility (P = 0.0006) and sperm count (P = 0.0001), induced mild apoptosis of Leydig cells and caused moderate spermatogenic arrest in GN1. However, walnut-supplemented diet significantly increased the NIC-induced reduction in sperm motility (P = 0.04) and sperm count (P = 0.0001) and its consumption was effective in attenuating testicular damage caused by NIC administration in GN1W6 and GN1W12.
Conclusion:
African walnut could exert therapeutic effect in the reduction of the adverse effect of NIC on the sperm motility, sperm count, and testicular architecture. It is worthwhile to consider it as a useful and affordable supplement to be added to the diet of males with infertility problems.
Keywords
Juglans
nicotine
oxidative stress
testis
tobacco
Introduction
Smoking is a critical public health problem that has adverse effects on human life,[1] and the interaction between lifestyle and reproductive health is a major source of concern in the world today.[2] Several studies from different parts of the world have identified that cigarette smoking has an adverse effect on the semen quality, especially in those who are heavy smokers or those who have been smoking for several years.[3-6]
Nicotine (NIC) is the most abundant alkaloid in tobacco leave and cigarette smoke, it sustains tobacco addiction and is suspected to be responsible for the deleterious effects cigarette smoking has on the reproductive system.[7] Studies have shown the adverse effect of NIC on semen parameters.[8-14] Interestingly, it has been observed that the concentration of NIC in seminal fluid is high in men passively exposed to cigarette smoke.[14,15] NIC increases oxidative stress that leads to the production of reactive oxygen species (ROS) which can predispose the patient to not only damage to the sperm chromatin compaction process but also cause an increase in sperm DNA fragmentation.[14]
Sperm motility and quality were shown to be increased following the addition of oral antioxidants to men diagnosed with oligoasthenoteratozoospermia.[16] African walnut is an edible nut that is widely consumed traditionally by men.[17] Studies have shown that African walnuts are rich in antioxidants such as saponins, phenols, flavonoids, and alkaloids.[18-20] It has been reported that walnuts possess the ameliorative effects against oxidative damage induced by hyperglycemia,[19] exhibited hepatoprotective activity against induced liver damage,[21] offer dose-dependent wound healing activity,[22] and reduce the oxidant and inflammatory load on brain cells.[23] Walnuts, when administered to men placed on a Western style diet, were also identified to, respectively, increase sperm motility, vitality, and normal morphology significantly.[24] Till date, there appears to be no study on the effects of walnut-enriched diet on sperm parameters conducted in an animal model induced with reproductive toxicity using NIC, hence this study.
Methods
Preparation of walnut meal
African walnut was obtained and a specimen of the walnut was identified and authenticated by taxonomists from the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, with herbarium voucher specimen number UNH711a. The nuts were gotten by breaking the pods. The nuts were washed and boiled at 100°C for 2 h. It was then allowed to cool. The shells were removed and the milky colored nuts were air dried. The dried nuts were made into powder with mechanical grinder. The powdered walnut granules were stored in an air-tight container. The powder was formulated into feed with grower’s mash in 6% and 12% weight of feed intake concentration. The procedure for its preparation is in accordance with Ghorbani et al.[25] The phytochemical qualitative analysis of the powdered walnuts was evaluated.
The mix ratio was calculated thus:
The percentage:(100-The percentage) =6%:(100–6%) = 6:94 =Walnut:feed.
=12%:(100–12%) = 12:88 = Walnut:feed.
Drug
NIC solution was prepared by mixing NIC hydrogentartrate (Sigma Chemical Company, St. Louis) at a concentration of 6.84 mg/ml (as the salt) into a 0.9 saline solution.[26]
Study design and animals
The study is an experimental study carried out from May to July 2018. Twenty-four sexually mature male Wistar rats (Rattus norvegicus) (200–220 g) obtained from the animal house of the Faculty of Basic Medical, University of Nigeria, Enugu, Nigeria. The animals were randomly assigned into four groups (n = 6/each) and were maintained in standard cages in a well-ventilated animal house of the Department of Physiology, University of Nigeria, Enugu Campus, Nigeria, with a 12:12 h light/dark cycle at room temperature. All efforts were used to reduce the number of rats used in the experiments.
Group 1 (GN0) received 1 ml/kg normal saline and normal rat chow daily, Group 2 (GN1) received 1 ml/kg NIC and normal rat chow daily, Group 3 (GN1W6) received 1 ml/kg NIC and 6% walnut-rich feed daily, and Group 4 (GN1W12) received 1 ml/kg NIC and 12% walnut-rich feed daily.
Ethical approval
The study was approved by College of Medicine Research Ethics Committee University of Nigeria Enugu Campus and it is coherent with the experimental guidelines of the U.S. National Institute of Health (NIH) and Institutional Animal Ethics Committee (IAEC) on the care and use of laboratory animals.
Sperm and tissue recovery
The total body weights of Wistar rats were taken before and after the experiment duration. Following the 4 weeks (28 days) experimental period, the rats were anesthetized with diethyl ether and sacrificed. Orchidectomy was performed by open castration method. A pre-scrotal incision was made and the testicles were milked out of the incision site and weighed with OHAUS electric weighing balance (OHAUS Corporation Parsippany, New Jersey, United States). The left testis was removed along with its epididymis. The caudal epididymis was removed and placed in 400 μl pre-warmed (to 37°C) human tubal fluid (with HEPES, Cat. # 2002, in vitro care, Frederick, MD), a capacitating medium used previously.[27,28] The samples were analyzed immediately after collection.
The testes were dewax in xylene for 30 min, xylene removed by rinsing in absolute alcohol, 90%, 70%, and 50% alcohol for 2 s each. It was wash in two changes of water, stained in hematoxylin for 20 min, washed in water, and differentiated in 1% acid. It was blue in tap water, washed in water, and counter stained in eosin for 5 min, washed in water, dried, and cleared in xylene before mounted in D.P.X and dry for micrograph and interpretation.
Sperm characteristics analysis
Sperm motility was immediately assessed. The sperm was squeezed on a pre-warmed slide, two drops of warm 2.9% sodium citrate were added, covered with a cover slip, examined, and scored under the microscope using ×40 objective lens with reduced light.[29] To determine sperm morphology, an portion of sperm was fixed, mounted on slides, and stained with Coomassie Brilliant Blue G-250 in 60% methanol-acetic acid for 10 min. Sperms were analyzed for morphological abnormalities according to the World Health Organization[30] standards. The prepared slides were afterward examined under the Nikon Eclipse E100 Biological microscope using oil immersion with ×100 objective lens. Sperm count was done with the aid of the improved Neubauer counting chamber (Hawksley Oxford Street, London) viewed under a light microscope (Nikon Eclipse E100). The counting was done in five Thoma chambers.[31]
Statistical Analysis of data
Data entry and analysis were performed using IBM SPSS version 22.0 (SPSS Inc., Chicago, IL., USA) for Windows. Data analysis was done using descriptive statistics of proportions (mean ± standard deviation). Independent samples t-test was used to analyze the difference among groups and P < 0.05 was considered to be statistically significant.
Results
In Table 1, the phytochemical screening of the mass of dried extracted walnuts meal indicated the presence of carbohydrates, reducing sugars, amino acids, oils, saponins, glycosides, alkaloids, and terpenoids. However, no tannins, flavonoids, phenols, nor resins were detected.
| Extract | Aqueous extract | Ethanolic extract |
|---|---|---|
| Carbohydrates | ++ | ++ |
| Reducing sugar | + | − |
| Amino acid | + | ++ |
| Oil | + | − |
| Saponins | +++ | − |
| Flavonoids | − | − |
| Glycosides | ++ | − |
| Alkaloids | ++ | + |
| Steroids | + | ++ |
| Terpenoids | + | + |
| Phenol | − | − |
(−): Absence, (+): Less presence, (++): Moderate presence, (+++): High presence
In Figure 1, the body mass of rats fed with 6% and 12% walnut-enriched diet did not differ significantly in comparison to rats fed the standard (control) diet over a 4 weeks period. The walnut-enriched diet had no effect on body weight. Body weight is a function of the number of weeks on the diet. Data expressed as mean (±SD) for GN0, GN1, GN1W6, and GN1W12. This result is parallel to the report of Nwaoguikpe et al.[18]
- Weight over time chart for GN0, GN1, GN1W6, and GN1W12 respectively
Table 2 shows a significant decrease (P = 0.0006) in sperm motility of GN1 (55.0%) following the administration of NIC as compared to GN0 (78.3%). There was, however, a significant increase (P = 0.04, P = 0.03) in sperm motility of GN1W6 and GN1W12 (70%, 68.3%) relative to GN1 (55.0%) following the addition of walnut at 6% and 12%, respectively, to the diet of the groups. Addition of walnut at 6% and 12% does not show to significantly increase (P = 0.2, P = 0.08) sperm motility in GN1W6 and GN1W12 (70%, 68.3%) when compared to GN0 (78.3%).
| Group parameters | GN0Mean±SD | GN1Mean±SD | GN1W6Mean±SD | GN1W12Mean±SD |
|---|---|---|---|---|
| Motility (%) | 78.3±7.6 | *55.0±8.7 | *70.0±13.2 | *68.3±10.4 |
| Count (Million/ml) | 78.7±7.8 | 49.0±1.7 | 73.3±6.7 | 81.3±4.5 |
| Morphology (%) | 90.0±2.0 | 89.3±1.5 | 91.0±2.6 | 89.3±1.5 |
Data presented in % ± SD, t-test statistics. *Represents significance difference as compared with control group (P<0.05)
The result data also revealed that there was a significant decrease (P < 0.0001) in sperm count of GN1 (49.0 Million/ml) relative to that of GN0 (78.7 Million/ml). The sperm count values for GN1W6 and GN1W12 (73.3 Million/ml and 81.3 Million/ml) are not significantly different (P = 0.2, P = 0.4) from GN0 (78.7 Million/ml). However, there was a progressive significant increase (P < 0.0001) in sperm count of GN1W6 and GN1W12 (73.3 Million/ml and 81.3 Million/ml) when compared to sperm count of GN1 (49.0 Million/ml).
Table 2 result further reveals that there was no significant difference (P = 0.51) in the mean percentages between GN0 (90.0%) and GN1 (89.0%), no significant difference (P = 0.47, 0.51) between the mean percentage of GN0 (90.0%) and GN1W6 and GN1W12 (91.0 and 89.0), and no significant difference (P = 0.19) between the mean percentage of GN1 (89.0%) as relative to GN1W6 and GN1W12 (91.0 and 89.0). This implies that sperm morphology was not significantly affected by the administration of NIC and the addition of walnut to diet.
Histological studies on testis
Figure 2 shows the histological analysis with ×400 which reveals that GN0 has a well enhanced spermatogenesis. In GN1, there was variation in the testicular architecture evidence by the mild apoptosis of interstitial cells of Leydig to moderate spermatogenic arrest due to the effect of NIC. In GN1W6, there was mild apoptosis of the interstitial cells of Leydig which is attenuated as compared to GN1, and GN1W12 showed a well-enhanced spermatogenesis as seen in GN0.
- Photomicrograph of sections of testis for GN0, GN1, GN1W6, and GN1W12 respectively WES: Well-enhanced spermatogenesis, SC: Sertoli cells, MSA: Moderate spermatogenic arrest, MAICL: Mild apoptosis of interstitial cells of Leydig
Discussion
In the present study, the effects of walnut-supplemented diet on sperm parameters conducted in male Wistar rats induced with reproductive toxicity using NIC were investigated. From our investigation, NIC proves to affect the male reproductive system and threatens male fertility.
Our finding on the negative effects of NIC on the male reproductive system is in line with the reports and findings of other researchers.[7,10,13,14,31,32] Condorelli et al.[14] posited that NIC increases oxidative stress leading to the production of ROS which can cause not only damage to the sperm chromatin compaction process but also increase sperm DNA fragmentation; it is of note that damage to sperm chromatin compaction process and increases sperm DNA fragmentation leads to defective sperm function which is identified as the most common cause of male fertility. Although physiologically controlled concentration level of ROS plays important roles in physiological processes such as capacitation, hyperactivation, acrosome reaction, and sperm-oocyte fusion to facilitate fertilization,[32] the excess production of ROS disrupts the delicate balance between ROS and antioxidants leading to lipid peroxidation, DNA damage, and sperm apoptosis.[33] ROS is produced endogenously in leucocytes, immature spermatozoa, and varicocele, and exogenously by radiation, toxins, smoking, and alcohol consumption.[33] However, in healthy men, there exists an effective scavenging system made up of various endogenous antioxidants which belong to both enzymatic and non-enzymatic groups that can remove the excess physiological ROS and prevent oxidative stress.[34] However, the effect of this scavenging system is attenuated with increased level of NIC in seminal fluid.[33]
In the present study, the supplementation of diet where walnuts composed of 6% and 12% of caloric intake for a 4-week period, compared to that in the study with humans,[24] caused no significant change in the body wight. However, the findings of this study are not consistent with the findings of Ting et al. and Rock et al.[35,36] who recorded that walnut-enriched diet promotes weight loss. This result may be probably due to the lower percentage of walnut added to the diet of the experimental animals.
There was significant improvement in the sperm motility and sperm count of rats supplemented with 6% and 12% walnut in diet but there was no clear observable increase in abnormal sperm morphology across the groups. The significant improvement in sperm motility is parallel to that reported in humans.[24] However, the report on human males also showed significant improvement in their sperm morphology following the addition of walnut to their diet.[24] The action of walnut in the improvement of sperm motility and count may be attributed to the antioxidants such as saponins, glycosides, and alkaloids present in walnut which is able to inhibit the production of malondealdehyde which is the end product of lipid peroxidation caused by the hydroxyl and superoxide radicals generated by NIC. Saponins in earlier studies have been associated with improvement in sperm parameters.[37]
Testicular histology damage by NIC corresponds with the report of Baker and Aitken[38] who posited that NIC linked oxidative stress is known to cause peroxidative damage in sperm plasma membranes. However, there was dose-dependent improvement in the testicular architecture of rats fed with 6% and 12% walnut-supplemented diet.
Finally, from our findings, it is worthy of note that NIC adversely affects sperm parameters and induces pathological changes in the interstitial cells of Leydig. However, walnut supplement improved sperm parameters such as motility and count and also attenuated the testicular damage caused by NIC.
Conclusion
Our study confirmed that NIC affects the male reproductive system through its adverse effects on the interstitial cells of Leydig, seminiferous tubules, and spermatogenesis. African walnut has demonstrated potential to attenuate the adverse effect of NIC on the sperm motility, sperm count testicular architecture. This effect which has been shown to be dose dependent did not have any significant effect on the morphology of sperm and weight of the rats. It is, therefore, worthwhile to consider walnut as a potential safe, useful, and affordable supplement to be added to the diet of males with infertility problems.
Acknowledgments
The authors would like to acknowledge Dr. M. Ezenwali and the technical staff of Brain Phosphorylation Laboratory, Enugu, for their assistance in conducting experimental sessions.
Authors’ Declaration Statements
Ethical approval
The study was approved by College of Medicine Research Ethics Committee University of Nigeria Enugu Campus and it is coherent with the experimental guidelines of the U.S. NIH and IAEC on the care and use of laboratory animals.
Data availability
The authors confirm that the data supporting the findings of the study are available within the article.
Competing interest
The authors declare that there are no commercial or financial relationships that could constitute as potential conflicts of interest in the conduct of the research.
Authors’ Contributions
The study arose from an original idea from DCI and BUA. All authors contributed to the study’s design. DCI wrote the first draft. EIU and CCN carried out sample analysis and data analysis. DCI, EN, EIU, and CCN are responsible for the preparation of the manuscript. BUA and EN revised and guided all the stages of the experiment and writing of the manuscript.
Funding statement: The authors declare that this study received no form of financial support or funding from any institution.
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