Influence of a 50 Hz extra low frequency electromagnetic field on spermatozoa motility and fertilization rates in rabbits

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1 Journal of Environmental Science and Health Part A (2009) 44, Copyright C Taylor & Francis Group, LLC ISSN: (Print); (Online) DOI: / Influence of a 50 Hz extra low frequency electromagnetic field on spermatozoa motility and fertilization rates in rabbits SHUBHADEEP ROYCHOUDHURY 1, JAROSLAV JEDLICKA 2, VLADIMIR PARKANYI 2, JAN RAFAY 2, LUBOMIR ONDRUSKA 2, PETER MASSANYI 1 and JOZEF BULLA 1 1 Slovak University of Agriculture, Nitra, Slovak Republic 2 Animal Production Research Center, Nitra, Slovak Republic Effects of a 50 Hz extra-low frequency electromagnetic field (ELF EMF) on in vitro rabbit spermatozoa motility were analyzed, as well as the effect on fertilization rates after insemination. Pooled semen samples and a control were exposed to 50 Hz ELF EMF. The difference of the samples of the test groups G1 and G2 with the control group CG (75.56%) for spermatozoa motility were found to be significant (P < 0.01). Differences were significant (P < 0.01) for curvilinear velocity (VCL) between the test group G3 ( µm/s) and the control group CG ( µm/s). Hormonally stimulated adult (9 12 months) females (n = 140) were inseminated with semen samples from G1, G2, G3 and CG ( spermatozoa/0.5 ml average insemination portion) immediately after ELF EMF exposure and fertilization (kindling) rates were calculated. For the G2 it was 54.28% data indicate 50 Hz ELF EMF induced alterations of spermatozoa motility and kindling rate in rabbits, therefore influencing fertility. Keywords: Spermatozoa motility, fertilization rate, CASA, rabbit, ELF EMF. Introduction The influence of radioactivity on the physiology and immunity of animals has long been an important topic of scientific investigations. [1] Studies of alterations in morphometric structures of small mammals in response to radioactive pollution have yielded contrasting results. [2 4] An increase of tension in physiological and immunological processes was reflected in the small mammal populations in response to chronic irradiation, which have a non-specific, stresscausing nature. [5] In the modern world, environmental protection is the concern of a variety of fields ranging from resource management to preservation to restoration of biotopes and biocenoses with respect to miscellaneous anthropic stresses including radiological protection. [6] Given the rapid expansion and possible large scale distribution of medical devices that utilize static magnetic fields health impact needs to be accurately assessed. [7] As societies develop, greater use of technologies leads to increasing exposure to static electric and magnetic fields. Possible health effects from static fields Address correspondence to Shubhadeep Roychoudhury, Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, Nitra, Slovak Republic. shubhadeep1@gmail.com Received February 16, have not been adequately assessed. To ensure the reliability of a future environmental radioprotection system, a solid scientific basis which reinforces the need to undertake relevant research in order to fil1 current deficiencies is required. [6] The widespread use of wireless telecommunications devices, particularly mobile phones, has resulted in an increased human exposure to radiofrequency (RF) fields. [8] Scientific controversy about long-term health effects of high-frequency EMFs lasting for at least 50 years exists, and national and international agencies have established safety guidelines for exposure to RF fields. The fact that never before has such a substantial proportion of the population been exposed to microwaves in RF field and at comparably high levels, has attracted the focus of the general public. Many more people are now concerned with the risks associated with RF exposure to the head from the small radiating antenna of mobile phones. [7 9] In a particularly interesting study, transgenic mice exposed to a digital telephone signal developed more than twice as many nonlymphoblastic lymphomas as the unexposed control group. [10] Although the significance of these results to human health is not known, they suggest a potential impact of the EMFs on biological processes. The issue of EMFs of all frequencies represents the area of largest concern with respect to environmental influences on human health. All populations are now exposed to varying degrees of EMF, and the levels will continue

2 1042 Roychoudhury et al. to increase as technology advances. Some animal studies suggest the possibility for ELF EMF exposures (50/60 Hz) to increase the risk of cancer by mechanisms yet to be elucidated. [11] Studies approaching reasonable latencies found an increased cancer risk associated with mobile phone use, with the highest overall risk for acoustic neuroma followed by uveal melanoma. Cancer risk was also found to be enhanced with increasing latency and duration of mobile phone use. [9] In vitro effects of an acute exposure to a sinusoidal ELF EMF (50Hz) on the ability of boar mature spermatozoa to acquire the fertilizing competence were also studied. [12] It was illustrated that reproductive capacity is more sensitive to the effects of radiation than life expectancy (mortality rate). [6] Theobjectiveofthisstudywastoevaluatethein vitro and in vivo effects of a 50 Hz ELF EMF on the motility of rabbit spermatozoa and subsequent fertilization rates after artificial insemination as ELF EMF is considered as one of the many environmental factors with potential to cause adverse health outcomes, especially concerning reproduction. Material and methods Animals Male rabbits (n = 15) belonging to the lines M91 and P91, Slovak Agricultural Research Centre (SARC), Nitra, Slovak Republic, selected on the basis of age normally associated with reproduction (12 14 months), were used in the experiment. Female rabbits (n = 140) after the second or third litter (SARC, Nitra) belonging to the same lines (n = 70 for line M91 and line P91 each) were selected for the experiment based on age (9 12 months). Animals were housed in a partially air-conditioned rabbit house (SARC, Nitra) under a photoperiod 16L:8D (minimum light intensity of 80 lux). They were placed in individual cages fed with a commercial diet and were provided water ad libitum. An air temperature of 17 ± 2 Candrelative humidity of 70 ± 5% were maintained in the rabbit house. This study was ethically approved by the Animal Ethics Committee of the Slovak Agricultural Research Centre, Hlohovská 2, Nitra, Slovak Republic. The animals were used in accordance with the guidelines for the use and care of laboratory animals (permission no. 2058/06 221/3b) as given by the State Veterinary and Food Administration of the Slovak Republic. Semen collection and ELF EMF exposure Two ejaculates per male were collected on a single day (early in the morning) with the help of an artificial vagina. Immediately after collection the individual doses of ejaculates exhibiting a white colour without presence of any Fig. 1. Schematic lay out of exposure device. gel were mixed together to acquire a pooled semen sample for insemination. The obtained pooled sample was diluted (dilution 1:10) with a Tris-citric acid-glucose diluent with addition of antibiotic (Minitüb-Verdünnungsmischung für Kaninchensperma mit Antibiotikum, Germany). After dilution, insemination doses were divided into four groups control group (CG), group 1 (G1), group 2 (G2) and group 3 (G3), stored in sterilized air-tight test tubes in thermostable environment (thermobox). For the experiment, a pulsed magnetic field was created (Belancik, Vrable, Slovak Republic) using an electromagnetic conductor assembled from an input electric cable, voltage regulator, input cables and an induction coil (Fig. 1). [13 15] The induction coil with an internal diameter of 55 cm (2R = 55 cm) produced an adjustable lowfrequency (50 Hz) magnetic impulse. [16] CG was not exposed to EMF. Experimental groups G1, G2 and G3 were exposed to EMFs of following intensities and exposure times at laboratory temperature (20 25 C). The three experimental pooled samples were placed at the centre of the induction coil of 3 individual exposure devices simultaneously for 20 minutes each. Their exposure schemestoemfswereasgivenbelow: G1 induction mt, (50 Hz) G2 induction mt, (50 Hz) G3 induction mt, (50 Hz) Thus ELF EMF exposed semen samples were immediately used for analysis of spermatozoa motility as well as artificial insemination of rabbits. Due to the lack of availability of scientific literature on ELF EMF exposure to mammalian spermatozoa, the minimum exposure duration of 20 minutes was selected based on data concerning the influence of a temporary magnetic field on chicken hatching where exposure times were 20, 30 and 40 minutes. [14,15] Spermatozoa motility: CASA analysis Each of the pooled samples (CG, G1, G2, G3) at a concentration ranging from cells/ml to cells/ml were evaluated using a Computer Assisted Semen Analyzer (CASA) system Sperm Vision (Minitüb, Tiefenbach, Germany) equipped with a microscope (Olympus BX 51, Japan) to assess the spermatozoa motility. [17,18] Each sample (10 µl drop) was placed into Makler Counting Chamber (depth 10 µm, Sefi Medical Instruments, Germany). The accuracy of CASA for evaluation of rabbit spermatozoa motility, as well as Makler chamber manual examination has already been described. [18,19] A heated stage (37 C) was used during the entire analysis

3 Electromagnetic fields and rabbit fertilization rates 1043 and multiple fields (up to 95 20x) in less than 2 seconds/field were analysed for each sample as per the specific CASA Sperm Vision software for rabbit spermatozoa motility analysis (Minitüb, Tiefenbach, Germany). Using the rabbit specific software settings the following parameters were evaluated total motile spermatozoa (MOT, %, motility > 5 µm/s), progressively motile spermatozoa (PRO, %, motility > 20 µm/s), average path distance (DAP, µm,the average distance ofthecell path using a smoothed line as a reference), curved line distance (DCL; µm, which is the actual distance that the spermatozoa moved during the analysis period), straight line distance (DSL,µm, the average distance measured in a straight line from the beginning to the end of the track), average path velocity (VAP, µm/s; theaverage velocity of the smoothedcell path), curvilinear velocity (VCL, µm/s; the actual velocity measured over the actual point to point track followed by the spermatozoa), straight line velocity (VSL, µm/s; the average velocity measured in a straight line from the beginning to the end of the track), straightness index (STR, the average value of the ratio VSL:VAP), linearity index (LIN, the average value of the ratio VSL:VCL,%), wobble (WOB = VAP:VCL, %, a measure of the oscillation of the actual trajectory about its spatial average path), amplitude of lateral head displacement (ALH, µm; the mean width of the head oscillation as the sperm cells swim) and beat cross frequency (BCF, Hz; the frequency of sperm head crossing the average path in either direction). Artificial insemination Artificial insemination (AI) was carried out for all hormonally treated females immediately after 20 minutes of ELF EMF exposure to the pooled samples except for the CG. Female rabbits were hormonally stimulated using an intramuscular application of 25 I.U. PMSG (Sergon, Bioveta, Czech Republic) 48 hours prior to assisted insemination. Fresh spermatozoa were inseminated into a total of 140 receptive females using uniform plastic curved pipettes (Mini Tüb, Germany) whereby the insemination doses (consisting of spermatozoa in 0.5 ml insemination portion on an average) were reposited into the reproductive tract of hormonally stimulated females. Concurrently with insemination, each female was given an intramuscular injection of synthetic GnRH (2.5 µg; Supergestran, Ferring- LéŁiva, Czech Republic) to induce ovulation. On the 25th day of pregnancy, palpation of inseminated females was performed in order to facilitate monitoring during pregnancy and expected fertility rate. The number of inseminations (NI) and number of females that gave birth (BIRTH), number of total kits born per litter and number of live kits born per litter were recorded to calculate the kindling rate (BIRTH/NI 100). [19] Statistical analyses Obtained data (expressed as means) was statistically analyzed with the help of the PC program Excel and a commercially available statistics package SAS 9.1 (SAS Institute Inc., USA) using t test and Scheffe s test. Statistical significance was indicated by P values of less than 0.01 and Results Spermatozoa motility Results of the spermatozoa motility analysis are presented in Figure 2. Analysis revealed the highest motility in the experimental sample G1 (93.81%), which received a onetime treatment of magnetic pole with the largest induction of mt (50 Hz) for 20 minutes. The lowest motility was showed by the sample G3 (74.67%), where a one-time lowest magnetic induction of mt (50 Hz) was applied. The test group G2 recorded a motility of 92.72% where spermatozoa were exposed to magnetic field of mt intensity. The difference of the test groups G1 and G2 with the control group CG (75.56%) were found to be significant (P < 0.01). Data for progressive motility of spermatozoa are presented in Figure 3. Significant differences were observed in the values of progressive motility of spermatozoa between the test group G1 (91.87%) and control group CG (70.99%) and also between test groups G2 and CG (89.09% vs %). Progressive motility was 71.77% in the test group G3. For both the parameters (motility and progressive motility), changes in magnetic field induction influenced the biological material (rabbit spermatozoa). Statistically significant differences ( P < 0.01) were also noted for Fig. 2. Rabbit spermatozoa motility in relation to a 50 Hz ELF EMF exposure in vitro.

4 1044 Roychoudhury et al. Table 1. Distance, velocity and other motility parameters of rabbit spermatozoa in relation to 50 Hz ELF EMF exposure in vitro. Group G1 G2 G3 CG Trait Mean SD Mean SD Mean SD Mean SD DAP DCL DSL VAP VCL a b VSL STR LIN WOB ALH BCF a, b: means at the same row with different letters are significantly different (P < 0.01) DAP average path distance, µm; DCL curved line distance, µm; DSL straight line distance, µm; VAP average path velocity, µm/s; VCL curvilinear velocity, µm/s; VSL straight line velocity, µm/s; STR straightness index; LIN linearity index,%; WOB wobble,%; ALH amplitude of lateral head displacement, µm; BCF beat cross frequency, Hz. the velocity parameter VCL (velocity curved line) between the test group G3 ( µm/s) and the control group CG ( µm/s; Table 1). For other velocity and distance parameters no significant differences were noticed. Fertilization rate Results of fertility analysis of female rabbits are presented in Table 2. The values for kindling rate obtained by palpation on day 25 and the highest kindling rate was obtained from experimental group G3 (74.28%). In this group, female rabbits were inseminated with spermatozoa treated with a single administration of magnetic field at an intensity of mt for 20 min. The lowest kindling rate was recorded in experimental group G1 (45.71%), in which sperm samples received a single administration of magnetic field at an intensity of mt for 20 min. In the control group, which remained free from the influence of magnetic field, the kindling rate was found to be 51.43%. Discussion As societies develop, greater use of certain technologies leads to increasing exposure to static electric and magnetic fields. RF fields are highly important and beneficial in many facets of everyday life, such as radio and TV transmission, telecommunications (e.g., mobile telephones), diagnosis and treatment of diseases and in the heating and sealing. [7] The issue of EMFs of all frequencies represents one of the areas of largest concern with respect to environmental influences on human health. Potential health effects of exposure to static and time varying EMFs need scientific clarification. [7] Studies concerning the effects of radioactive pollution on morphometric structures of small mammals have yielded contrasting results. [2 4] There is Table 2. Fertility rates of female rabbits inseminated artificially with a 50 Hz ELF EMF exposed spermatozoa. G1 G2 G3 CG Fig. 3. Progressive motility of rabbit spermatozoa in relation to a 50 Hz ELF EMF exposure in vitro. No. of females that gave birth (BIRTH) No. of total kits born/litter No. of live kits born/litter Kindling rate (%)

5 Electromagnetic fields and rabbit fertilization rates 1045 scientific controversy about long-term health effects of high frequency EMFs lasting for at least 50 years. [9] However, studies approaching reasonable latencies of RF fields found an increased cancer risk associated with mobile phone use with the highest overall risk for acoustic neuroma followed by uveal melanoma, which was related to increasing latency and duration of use. [9] Another study assessing association between RF exposure and human cancer reported positive associations suggesting an increased risk of some types of cancer in those individuals with greater exposure to RF. [20] Influence of magnetic resonance at 3.0 T on clonogenic ability, proliferation, and cell cycle in an embryonic human cell line has also been investigated. [21] Authoritative reviews completed during found no clear evidence of adverse health effects associated with RF fields. [8] However, following a recent review of nine epidemiological studies of mobile phones and cancer, it was concluded that the possibility of an enhanced cancer risk cannot be excluded. [9] These reviews support the need for further research to clarify the possible associations between RF fields and adverse health outcomes that have appeared in some reports. [8] Concerns still persist that exposure to pulsed and amplitude modulated RF fields may cause specific health effects. Investigators exposed mature boar spermatozoa to ELF EMF (50 Hz, 1 mt) during the 4hrs of incubation in vitro and evaluated for morphological (surface morphology and acrosome integrity) and functional parameters (cell viability, motility, induction of acrosomal reaction and the ability to fertilize oocytes in vitro). [12] In mouse epididymal extracts, kinematic parameters were evaluated to monitor maturation of spermatozoa movement in animals exposed to static magnetic fields using the Sperm-Class Analyzer computerized image analysis system. [22] For this purpose, animals were exposed to a field of 0.7 T generated by a permanent magnet over 10 or 35 days for either 1 or 24 hrs/day. Changes in motility were observed in all groups. The percentage of total motile and progressive motile spermatozoa increased during passage through the epididymis, with major changes between the caput and corpus epididymides, and the pattern of swimming changed clearly towards more rapid and straighter trajectories. The processes of initiation of spermatozoa motility and maturation of displacement patterns were not affected by magnetic field treatment. [22] The same authors are of the opinion that processes of initiation of spermatozoa motility and maturation of displacement patterns and eventually spermatozoa production in animals remain unaffected after static magnetic field (0.7 T over 10 or 35 days for either 1 or 24 hrs/day) exposure. [22] Another group of scientists illustrated the effects ELF EMFs generated by 170 kv (50 Hz) high power lines on the epididymal sperm characteristics, biochemical parameters, testosterone levels and histopathology of rat testis. [23] For indicators of dependence of catalytic activity on the peroid of exposure, authors observed no differences between control and treated animals due to glutathione and malondialdehyde levels. Another group of scientists could not find any exposure-related changes in exposed or shamexposed rats to a 60 Hz EMF (at field strengths of up to 500 µt from day 6 of gestation to day 21 of lactation) with respect to the anogenital distance, preputial separation, testis weight, testicular histology, sperm count, daily sperm cell production, spermatozoa motility, sperm cell morphology and reproductive capacity of Fl offspring. [24] Cytogenetic studies also confirmed no clastogenic effect of ELF EMF exposure (50 Hz, 20 mt for 2 h at 37 degreesc under 5% CO2 in air) on human sperm cell chromosomes in vitro. [25] Unlike spermatozoa of many lower species, mammalian spermatozoa do not possess the ability to fertilize an egg immediately upon ejaculation, although they are motile and appear to be morphologically mature. Under in vivo conditions, ejaculated spermatozoa requires a finite period of residence in the female reproductive tract to become fertilization-competent, which is termed as capacitation. [26,27] Historically, capacitation was originally defined as the time interval of sperm incubation (either in vivo or in vitro) that is required to bring about this final functional maturation of the spermatozoa. [28] The doses of ELF EMF exposure used in this experiment that caused high spermatozoa stimulation (54.50 mt and mt) can be best explained by the spermatozoa being activated to the state of capacitation and hyperactivation. This has been indicated by the CASA results in relation to the highest spermatozoa motility. Higher fertlity indicators (kindling rate of 74.28% in G3 vs % in CG) were also observed in the rabbits inseminated with spermatozoa receiving 21.33mT (50 Hz) induction. Capacitation of spermatozoa requires as long as 6 hours in the reproductive tract of females under in vivo conditions. [27] In contrast, results of the present study indicated capacitation of spermatozoa only after an ELF EMF exposure of 20 minutes. Electromagnetically influenced and hyperactivated spermatozoa were probably excited and eventually energetically exhausted (reduction of ATP) before they could participate in the fertilization of ovulated eggs under physiological conditions. Results from another study also suggested that ELF EMF exposure (waveform of 5 mt amplitude and frequency of 50 Hz) can improve human spermatozoa motility and this effect depends on the field characteristics. [29] ELF EMF-induced (60 Hz, 0.1 mt or 0.5 mt for 24 h/day; 8 weeks exposure) testicular germ cell apoptosis was reported in mice. [30] Early hyperactivation of spermatozoa had an impact on kindling rate as control group was compared with groups those received lower electromagnetic inductions (40.29 mt and mt). On the molecular and immunological level many membrane changes have been found in spermatozoa that prepare them for physiological changes such as hyperactivation, and morphological changes such as the acrosome reaction. [28] It was

6 1046 Roychoudhury et al. proposed that these events lead to more vigorous motility and to the release of various enzymes for the penetration of the egg. [28] The present in vitro study of a 50 Hz ELF EMF exposure indicates that magnetic field alters certain chatacteristic indicators of spermatozoa motility. Similarly, a significant reduction in spermatozoa count together with an increase in the rat serum levels of male luteinizing hormone after 18 weeks of consecutive exposure (50 Hz 25 mt), and significantly decreased testosterone levels only after 6 and 12 weeks of the exposure period suggested adverse effects on mammalian fertility and reproduction. [31] From the present study, we suggest that irradiated semen samples at certain inductions (21.33 mt) alter the kindling rate, thus influencing fertility. In light of the fact that evidence in the literature regarding similar investigations is scarce, we consider our results to be introductory and original, which will be compared with those of our future research projects in regards to species, time and other culture conditions. Acknowledgments The authors thank Prof. Mokhtar Ibrahim Yousef (University of Alexandria, Egypt) and Sara Patrawala (Northwestern Feinberg School of Medicine, Chicago, USA) for critical reading of the manuscript. This study was supported by Slovak Research and Development Agency grant no and the Ministry of Education of the Slovak Republic grant nos. VEGA 1/0834/08 and VEGA 1/0696/08. References [1] Moskalev, Y.I. Radiobiology of incorporated radionuclides. Nauka: Moscow, (In Russian.) [2] Ermakova, O.V. Changes of some demographic and morphophysiological indicators in the root vole, depending on the stage of populational cycle and radiational conditions. 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Effects of 50Hz extremely low frequency magnetic field on the morphology and function of boar spermatozoa capacitated in vitro. Theriogenology 2007, 67, [13] Veterany, L.; Jedlicka, J. The effect of an magnetic field on the chicken hatching. Acta fytotechnica et zootechnica 2001, Special Issue, [14] Toman, R.; Jedlicka, J.; Broucek, J. The influence of a temporary magnetic field on chicken hatching. J. Environ. Sci. Health, Part A, 2002, 37, [15] Jedlicka, J. Effect of electromagnetic field on the setting eggs of Shaver Starcross 288 during storage and incubation. In Proceedings of the International Scientific Conference: Risk Factors of Food Chain, Slovak University of Agriculture in Nitra, Slovak Republic; Massanyi, P., Toman, R., Lukac, N., Kramarova, M., Eds.; SAU: Nitra, Slavic Republic, [16] Horak, Z.; Krupka, F. Physics Volume I. State Publishing House of Technical Literature/ALFA: Prague, Czech Republic, (In Czech.) [17] Chen, Y.; Li, J.; Simkin, M.E.; Yang, X.; Foote, R.H. 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Effect of electromagnetic fields on the sperm characteristics and histopathology status of testis in rats. Vet. Med. 2007, 63, [24] Chung, M.K.; Lee, S.J.; Kim, Y.B.; Park, S.C.; Shin, D.H.; Kim, S.H.; Kim, J.C. Evaluation of spermatogenesis and fertility in F1 male rats after in utero and neonatal exposure to extremely low frequency electromagnetic fields. Asian J. Androl. 2005, 7, [25] Tateno, H.; Iijima, S.; Nakanishi, Y.; Kamiguchi, Y.; Asaka, A. No induction of chromosome aberrations in human spermatozoa exposed to extremely low frequency electromagnetic fields. Mutat. Res. 1998, 414, [26] Austin, C.R. The capacitation of the mammalian sperm. Nature 1952, 170, 326. [27] Chang, M.C. Development of fertilizing capacity of rabbit spermatozoa in the uterus. Nature 1955, 175,

7 Electromagnetic fields and rabbit fertilization rates 1047 [28] Chang, M.C. The meaning of sperm capacitation. J. Androl.1984, 5, [29] Iorio, R.; Scrimaglio, R.; Rantucci, E.; Delle Monache, S.; Di Gaetano, A.; Finetti, N.; Francavilla, F.; Santucci, R.; Tettamanti, E.; Colonna, R. A preliminary study of oscillating electromagnetic field effects on human spermatozoon motility. Bioelectromagnetics 2007, 28, [30] Lee, J.S.; Ahn, S.S.; Jung, K.C.; Kim, Y.W.; Lee, S.K. Effects of 60 Hz electromagnetic field exposure on testicular germ cell apoptosis in mice. Asian J. Androl. 2004, 6, [31] Al-Akhras, M.A.; Darmani, H.; Elbetieha, A. Influence of 50 Hz magnetic field on sex hormones and other fertility parameters of adult male rats. Bioelectromagnetics 2006, 27,

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