1. Introduction
The use of mobile phones has become abundant in recent years throughout the world. According to Agarwal et al , 4.8 billion people are said to be using mobile phones worldwide and the usage is continuously increasing from younger to older generations. Mobile phones are operating between 800 to 2 200 MHz frequency bands and emit radiofrequency (RF) electro-magnetic radiation (EMR). The organic effect of RF-EMR release from mobile phones is still a debatable issue[1 2 3 4 5 6 7
Sexual abnormalities increase with etiological factors and ageing, including degenerative diseases and industrialized life cycle with stress. Reactive oxygen species are highly reactive oxidizing agents, belonging to the class of free radicals. Pomegranate fruit is nutrient dense antioxidant rich fruit which has been revered as a symbol of health and fertility. Rich numbers of phenolic compounds are present in pomegranate juice, including punicalagin, isomers, gallic acid, and anthocyanins[8 9
2. Materials and methods
2.1. Animals and groupings
Thirty healthy adult male Sprague Dawley rats (10-14 weeks old, weighing 150-180 g) were procured from Biogen Laboratory Animal Facility, Bangalore, India. The Sprague Dawley rats were housed in polypropylene cages. The experimental rats were maintained under well-ventilated standard laboratory, where the room temperature of (24±2) °C, with 45%-50% constant humidity and kept in a “12 hours light/12-hour dark” cycle throughout the study. The rats had free access to pellets and water ad libitum . Before beginning of the experiment, the rats were allowed to adapt lab condition (12 days). After acclimatization, 30 male Sprague Dawley rats were divided into 5 groups (n =6/group) such as group I : the control group, group II : the mobile RF-EMR group, group III : the mobile RF-EMR + pomegranate juice group, group IV : the mobile RF-EMR recovery group, and group V : the pomegranate juice group. Group I was neither exposed to RF-EMR nor given pomegranate juice, group II, III and IV were exposed to mobile emitted RF-EMR (800 MHz to 2 400 MHz) for 60 min/day for 90 days[10 8
2.2. Mobile RF-EMR exposure technique
The 4G mobile phone (Vivo 1803 mobile phone) with a specific absorption rate of 0.53 Watt/kg was hung down from the center of the rat roof cage at a distance 5 cm between cage floor and mobile phone, and during the exposure period food and water was available ad libitum . Radiation that was emitted during the exposure was quantified by radiation frequency meter (Meco-GRF-EMR) which was kept at the periphery. The control rats were placed in a separate room where there was no exposure of mobile RF-EMR[10
2.3. Plant material and authentication
Pomegranate fruit (bhagwa variety) was collected from local market in Kolar. The pomegranate fruit material (botanical name: Punica granatum L.; voucher number: 0320) was authenticated by Dr. Madhava Chetty K, Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupathi-517502, Andhra Pradesh, India.
2.4. Pomegranate juice preparation
Fresh and healthy pomegranate fruit was used in this study. Pomegranate fruit was washed under tap water and cut into four pieces and manually peeled in aseptic conditions. Pomegranate fruit juice was obtained by using mixer blender (Bajaj GX 11 750W), filtrated with a funnel and juice was immediately poured into dark-bottle wrapped with aluminum foil[11 8
At the end of intervention, the rats were euthanized with ketamine (50 mg/kg body weight, i.p.) . The epididymides were harvested via center incisions in the scrotal sac. Immediately, cauda epididymides were dissected and placed in a clean small petri dish.
2.5. Sperm counts
A 1 cm incision was done at the epididymal cauda by using sterilized blade and all the semen was squeezed by using forceps into petri dish, containing 5 mL of phosphate buffered saline. The glass petri dishes were closed and incubated at 34 °C for 8-10 min to allow for sperm swim-up. The epididymal sperm was transferred to “Neubauer Hemocytometer”. The total sperm count was counted by using a light microscope (Labomed LX 200 LED) at 40× magnification[12 6 /mL[13
2.6. Sperm motility
Caudal epididymis was mixed with few drops of normal saline. One drop sample was placed on a clean slide and covered with a cover slip. The 200 sperms were examined at 40× magnification. The motile and non-motile sperm number was counted in 10 random fields and motility was expressed as a ratio of number of motile sperms to the total number of sperms[12
2.7. Sperm viability
One drop of semen sample was placed on a clean glass slide and one drop of 0.5% eosin solution was added. After 2 min, the slides were observed under a light microscope (40× objective). Percentage of viable sperm (color less) and non-viable sperm (colored) was expressed. The relative sperm viability was calculated, total “viable sperms” (unstained) by the total “viable sperms” (stained and unstained) × 100[14
2.8. Sperm morphology
One drop of sperm suspension was placed on a glass slide, a thin smear was done and few drops of 95% ethanol and smear were allowed to air dry overnight. The slides were stained with Papanicolaou staining technique. The slide was observed under microscope at 400× magnification, to detect the sperm morphological abnormalities which were as follows: detached head, pyriform head, coiled tail, and multiple abnormalities. For each slide, the % of abnormal sperms was scored from at least 10 fields and 200 sperms[15 16
2.9. Sperm progressivity
The sperm progressivity was determined by subjecting grading system (grades): 4 or A: Excellent forward directional movement; 3 or B: Good forward direction movement; 2 or C: Fair forward directional movement; 1 or D: Poor forward directional movement, as described by the World Health Organization (2005)[17
2.10. Statistical analysis
The statistical analysis was carried out by using the SPSS (version 20; SPSS Inc) software program. Normal distribution of the data was presented as mean±standard deviation (mean±SD). One-way analysis of variance and Bonferroni's post-hoc test were used to determine the significance among the multiple comparisons of 4G mobile RF-EMR and pomegranate juice treatment groups. P<0.05 was considered statistically significant.
2.11. Ethics statement
The study was approved by Institutional Animal Ethical Committee (IAEC /PHARMA/SDUMC/2018/12a), SDUAHER Tamaka, Kolar, India. The experiments were performed at Central Animal House Laboratory, Sri Devaraj Urs Medical College, Tamaka, Kolar, India.
3. Results
3.1. Effect of mobile emitted RF-EMR and ameliorative effect of pomegranate juice on sperm count
The sperm count was significantly decreased in the mobile RF-EMR group compared to the control and pomegranate juice groups (P both<0.05). However, the sperm count was significantly increased in the mobile RF-EMR + pomegranate juice group compared to the mobile RF-EMR group (P<0.05). The sperm count was not significantly increased in the mobile RF-EMR recovery group compared to the mobile RF-EMR group (P >0.05). The sperm count was increased in the pomegranate juice group compared to the control group; however, it was not statistically significant (P >0.05) (Table 1 ).
Table 1: Effects of 4G mobile emitted RF-EMR and ameliorative effect of pomegranate juice on sperm parameters.
3.2. Effect of mobile emitted RF-EMR and ameliorative effect of pomegranate juice on sperm motility
The sperm motility was significantly decreased in the mobile RF-EMR group compared to the control and pomegranate juice groups (P both<0.05). However, the sperm motility was significantly increased in the mobile RF-EMR + pomegranate juice group compared to the mobile RF-EMR group (P <0.05). The sperm motility was significantly increased in the mobile RF-EMR recovery compared to the mobile RF-EMR group (P <0.05); however, it did not reach to the control and RF-EMR + pomegranate juice group levels. The sperm motility was not significantly increased in the pomegranate juice group compared to the control group (P >0.05) (Table 1 ).
3.3. Effect of mobile emitted RF-EMR and ameliorative effect of pomegranate juice on sperm viability
The sperm viability was significantly decreased in the mobile RF-EMR group compared to the control and pomegranate juice groups (P both <0.05). However, the sperm viability was significantly increased in the mobile RF-EMR + pomegranate juice group compared to the mobile RF-EMR group rats (P <0.05). The sperm viability was significantly decreased in the mobile RF-EMR recovery group compared to the mobile RF-EMR group (P <0.05). The sperm viability was not significantly different between the pomegranate juice group and the control group (P>0.05) (Table 1 ).
3.4. Effect of mobile emitted RF-EMR and ameliorative effect of pomegranate juice on sperm progressivity
The sperm progressivity was remarkably declined in the mobile RF-EMR group (grade C) compared to the control group (grade A) and the pomegranate juice group (grade A). However, the sperm progressivity was increased in the mobile RF-EMR + pomegranate juice group (grade B) compared to the mobile RF-EMR group (grade C). The sperm progressivity was not increased in the mobile RF-EMR recovery group (grade C) compared to mobile RF-EMR group (grade C) (Table 1 ).
3.5. Effect of mobile emitted RF-EMR and ameliorative effect of pomegranate juice on abnormality sperm morphology
The abnormality of sperm morphology (detached head, pyriform head, coiled tail, and bent tail) was significantly increased in the mobile RF-EMR group compared to the control and pomegranate juice groups (P both<0.05). However, the abnormality of sperm morphology was significantly decreased in the mobile RF-EMR + pomegranate juice group compared to the mobile RF-EMR group (P <0.05). The abnormality of sperm morphology was comparable between the mobile RF-EMR recovery group and the mobile RF-EMR group. The abnormality of sperm morphology was decreased in the pomegranate juice group compared to the control group; however, it was not statistically significant (P >0.05) (Table 2 ).
Table 2: Effects of 4G mobile emitted RF-EMR and ameliorative effect of pomegranate juice on sperm morphology (percentage of abnormality).
4. Discussion
The mobile phones have become a part of daily life. In view of COVID-19 pandemic, the usage of smart phones was increased in adults including adolescents[18 19 20
Our current study results showed that the chronic exposure [90 days (1 hour/day)] to RF-EMR emitted by 4G mobile leads to decrease of the sperm count, viability, motility, and progressivity in rat semen. In addition, RF-EMR exposure also increased the abnormality of sperm morphology in rat semen. It indicates the chronic exposure of RF-EMR emitted by 4G mobile causes male infertility in rats. Jong et al reported that the 30-day exposure (18 hours/day) to the 4G mobile emitted RF-EMR leads to decrease of spermatogonia, germ cells and leydig cell count in rats[21 et al reported that seven hours/day for 30 days exposure to the 2.45 GHz Wi-Fi EMR (high frequency radiation) induced a decrease in sperm parameters and increase of apoptosis positive cells and increased the caspase 3 activity in seminiferous tubules in rats[22 et al reported that long-term exposure (3 hours/day for 365 days) of 900 MHz RF radiation altered male reproductive parameters in rats[23
In contrast, studies reported that RF-EMR exposure did not decrease the sperm count in the experimental group compared to the control animals[24 25 et al stated that the 1 hour/day for 14 days exposure to the RF-EMR increases the oxidative stress in testis of rats[26
The direct biological effects of mobile RF-EMR are divided into thermal effects by the RF-EMR energy absorption, stimulation function by the induced electric current and athermic action by the long term RF-EMR exposure. No clinical studies have evaluated the effect of mobile RF-EMR and co-administration of pomegranate juice on the sperm parameters, but some epidemiologic studies have shown the mobile phone use has negative effects of sperm parameters[27 Punica granatum has protective effects due to its ingredients like phenolic acids[28 29 30 31 32 Punica granatum fruits[33
Plant products such as leaves, seeds and fruits have natural antioxidants. Pomegranate juice has an ancient history of being used in several diseased conditions[34 35 2 , to chelate metal ions and to exert a synergistic effect of other anti-oxidant metabolites. Our results in conjunction with the others mentioned above, suggest that flavonoids could constitute one of the active components of pomegranate[36 37
In addition, the abnormality of sperm morphology in rat semen was also decreased in the RF-EMR exposed rats supplemented with pomegranate juice compared to RF-EMR group rats. Turk et al[8 et al observed that 52 days of pomegranate juice supplementation increased the structural integrity of testis and testosterone levels in old rats compared to age-matched rats[38 et al stated that pomegranate juice supplementation increases the sperm count, progressive sperm motility, testosterone levels and epithelial diameters of the seminiferous tubule of the exposed male mice[39
The limitation in the present study was that we did not analyze the oxidative status of testis and also the testosterone levels in rat, which can give us a better understanding of the role of pomegranate juice on reduced sperm parameters induced by the mobile emitted RF-EMR.
In conclusion, the study results indicate that the mobile RF-EMR exposure reduces the sperm parameters and increases the sperm abnormality. However, the pomegranate juice supplementation reverses the 4G mobile RF-EMR induced damage on sperm quality in rats. It indicates that the pomegranate juice can be used as a nutritional supplement to improve the sperm quality.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Authors’ contributions
The design of the study was done by Anjaneyababu Naik Banavath. The literature search was done and the experiment was conducted by Anjaneyababu Naik Banavath. The data analysis as well as “drafting of the manuscript” was done by Anjaneyababu Naik Banavath. The editing and reviewing of the draft manuscript as well as revising the manuscript for intellectual content were done by Anjaneyababu Naik Banavath and Sridevi Nangali Srinivasa.
Acknowledgements
The authors sincerely acknowledge Dr. Sarala N Professor, Department of Pharmacology, Sri Deva raj Urs Medical College, Kolar, Karnataka for permitting to use the animal house facility. We acknowledge, Dr. Venkateswarlu Raavi, Assistant Professor, Department of Cell Biology and Molecular Genetics and K. Suresh, Tutor/Ph.D. Scholar Department of Physiology, SDUAHER and Dr. K. Sreedhar Naik, Research Associate, Indian Institute of Science Education and Research, Tirupati, for his valuable suggestions and reviewing the manuscript. We also acknowledge Mr. Ravi Shankar, statistician for his suggestions for the present study.
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