This web only provides the extract of this article. If you want to read the figures and tables, please reference the PDF full text on Blackwell Synergy. Thank you.
- Original Article -
Evaluation of spermatogenesis and fertility in F1 male rats
after in utero and neonatal exposure to extremely low frequency electromagnetic fields
M. K. Chung1, S. J. Lee1, Y. B.
Kim2, S. C. Park3, D. H.
Shin4, S. H. Kim4, J. C.
Division, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, Daejeon 305-600, Korea
2College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea
3College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea
4College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea
Aim: To determine whether in utero and neonatal exposure to a 60 Hz extremely low frequency electromagnetic field
(EMF) results in spermatotoxicity and reproductive dysfunction in the F1 offspring of rats.
Methods: Age-matched, pregnant Sprague-Dawley rats were exposed continuously (21 h/day) to a 60 Hz EMF at field strengths of 0 (sham
control), 5, 83.3 or 500 mT from day 6 of gestation through to day 21 of lactation. The experimentally generated
magnetic field was monitored continuously (uninterrupted monitoring over the period of the study) throughout the
study. Results: No exposure-related changes were found in exposed or sham-exposed animals with respect to the
anogenital distance, preputial separation, testis weight, testicular histology, sperm count, daily sperm production,
sperm motility, sperm morphology and reproductive capacity of F1 offspring.
Conclusion: Exposure of Sprague-Dawley rats to a 60 Hz EMF at field strengths of up to
500 mT from day 6 of gestation to day 21 of lactation did not
produce any detectable alterations in offspring spermatogenesis and fertility.
(Asian J Androl 2005 Jun; 7: 189-194)
Keywords: spermatogenesis; electromagnetic fields; prenatal exposure; postnatal exposure; rat
Correspondence to: Dr Jong-Choon Kim, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea.
Tel: +82-62-530-2827, Fax: +82-62-530-2809
Received 2004-04-05 Accepted 2004-10-08
Extremely low frequency (ELF, 50 and 60 Hz)
electromagnetic fields (EMF) are associated with the
production, transmission, and use of electricity; thus the
potential for human exposure is very high . Therefore,
the possible adverse effects of ELF EMF on reproduc
tive and developmental outcome have been extensively
studied in both experiments involving animals and
humans over the past several decades . However,
limited data have been published about these potential
adverse effects [3-11]. Moreover, there have been
conflicting findings regarding the alteration of
spermatogenic and reproductive functions. A number of studies
showed that exposure to ELF EMF did not induce any
adverse effects on spermatogenesis and reproductive
capacity in experimental animals and human [3-6]. In
contrast, some studies conducted by other investigators
showed clear damage to spermatogenesis [1, 7-11]. Therefore, more careful and detailed studies need to be
carried out to determine whether EMF exposure can
induce adverse effects on spermatogenesis and
In view of the fact that embryo-fetuses and young,
growing animals are more susceptible to the toxicity of
xenobiotics, exposure to any xenobiotic during
gestational and lactational stages of gonadal development may
lead to permanent damage to the gonads. The present
study was carried out on rats to investigate the potential
adverse effects of in utero and neonatal exposure to 60 Hz
EMF on the spermatogenesis and fertility of F1 male
2 Materials and methods
2.1 Animal husbandry and maintenance
Approximately 9-week-old, male and female
specific-pathogen-free Sprague-Dawley rats (Orient Co., Seoul,
Korea) were quarantined and acclimatized for 1 week
before the experiment. Two female rats were placed
into the cage of one male rat overnight. Vaginal smear
was performed the next morning and this was designated as day 0 of pregnancy if sperm were detected.
Forty pregnant females were then housed in a room maintained at a temperature of 23 ¡À 1¡æ, with good
ventilation, a relative humidity of 50 % ¡À 10 % and
artificial lighting from 08:00 am to 08:00 pm. Flow
cytometric analysis of the effects of 50 Hz magnetic fields
on mouse spermatogenesis. All animal rooms were
constructed of non-metallic materials. Mated females were
housed individually in clear polycarbonate cages with
polycarbonate lids, and the cages were placed on racks
designed and built specifically for EMF exposure. The
standard laboratory rodent diet (Jeil Feed Co., Daejeon, Korea)
and sterilized water were available ad
libitum. This experiment was conducted in facilities approved by the
Association for Assessment and Accreditation of
Laboratory Animal Care International and animals were
maintained in accordance with the Guide for the Care and
Use of Laboratory Animals .
2.2 EMF exposure facility
EMF generation equipment was designed and constructed by the Korea Electrotechnology Research
Institute as shown in Figure 1. The EMF was monitored
continuously throughout the study by observing the
current injected to the exposure system (this current is pro portional to the EMF). The generator housing capacity
was for approximately 50 rats. The field generator
consisted of four square coils and a shelf system with three
levels (top, middle and bottom) for the animals. Ten test
animals were placed on each level. The spatial variation
of the magnetic field (MF) was under 3 % within the
animal testing area (1 m ¡Á 1 m ¡Á 1 m). Also, the
temporal variation was negligible during the experimental
period because high-performance automatic voltage
regulation was adopted. The voltage fluctuation rate and
harmonic rate of automatic voltage regulation using a power
amplifier was nearly 0 %. The EMF exposure system is
described in detail in a previous study .
2.3 Experimental design and EMF exposure
The study was conducted using four groups of rats;
each group comprised 10 pregnant animals. Three groups
were exposed to EMF and the fourth group served as a
sham control. The sham-control female rats were handled
in an identical manner to the EMF-exposed female rats.
The field strength of 500 mT is the highest field strength
reasonably attainable within an experimental exposure
system and is approximately 2500 times higher than that
to which humans are routinely exposed in normal
residential environments. Most residential exposure is to
magnetic fields that are less than 0.2 mT in intensity,
although some areas in homes may exceed this intensity
. The field strength of 83.3 mT is the maximum
exposure level recommended by WHO/ICNIRP (World Health Organization/International Commission for
Non-Ionizing Radiation Protection)  and 5 mT is the
expected maximal exposure level under a 765 kV Korean
domestic electric cable. Based on these facts, the target
MF strengths were selected as 5, 83.3 and
500 mT. The dosimetry measurement was done on each day of the
experiment while the exposure of the rats to the EMF was taking place. Animals received sham exposure or
exposure to 60 Hz EMF for 21 hours per day (12:00
am-9:00 am) from day 6 of gestation to day 21 of lactation.
The study was blinded.
2.4 Examination of dams and F1 offspring
All dams were observed daily throughout gestation
and lactation for mortality, morbundity, general
appearance and behavior. Effects of EMF exposure on body
weight change and parturition were also monitored. On
day of delivery, the anogenital distance of male pups was
measured using a caliper to determine the distance be
tween the anus and the genital tubercle. On postnatal
day 4, eight pups were selected (four males and four
females, if possible) from each litter to produce
comparable litter sizes, whenever possible. At weaning, two
males and one female from each litter were randomly
selected. Clinical signs and the day of preputial
separation in male offspring were observed.
At 10 weeks old, one male rat from each litter was
selected and was killed by carbon dioxide overdose.
After external and internal macroscopic examinations, the
testes were removed and weighed. Sperm analysis was
conducted as has been previously described in other
studies [16, 17]. In order to ascertain sperm head count, the
left testis was homogenized and sonicated with 12 mL
distilled water. The number of spermatids in the sperm
suspension was counted under a light microscope with a
Neubauer hemacytometer (Paulmarine, Germany). Step
17-19 spermatids survive in this homogenization and can
be counted in a hemacytometer. In the rat, developing
spermatids spend about 6.3 days in these steps [18, 19].
Thus, daily sperm production was calculated by dividing
the number of spermatids determined, on a per-testis
basis, by 6.3. To assess motility, the left caudal
epididymis was minced in Hank's Balanced Salt Solution pH
7.2 (Sigma Chemical Co., St. Louis, MO, USA)
containing 10 mg.mL-1 bovine serum albumin and maintained at
37 ¡æ. Motility was observed using a microscope with a
microwarm plate. Sperm morphology was also examined under the light microscope using sperm smears
(sperm suspension containing 1 % eosin Y) collected from
the left caudal epididymis. A total of 2000 sperm from
each rat were examined for abnormalities in different
regions of spermatozoa. The right testis was fixed with
Bouin's solution for approximately 24 h, stored in 70 %
ethyl alcohol for several days, embedded in paraffin,
sectioned and stained with hematoxylin and eosin for
histopathological examination. Transversal testicular sections
were examined for testicular characteristics and the
When the offspring reached maturity (11 weeks old),
remaining one male and female from each litter were
mated for 2 weeks (one male to one female, with
brother-sister mating avoided) to evaluate their reproductive
capability. Successful mating was ascertained by the
presence of sperm in a vaginal smear and/or vaginal plug,
and the 24 hours immediately following this was
designated as day 0 of gestation. All females were subjected
to cesarean section on day 15 of gestation. Based on the
results, mating and fertility indices were calculated.
2.5 Statistical analyses
Statistical analyses were performed by comparing
the exposed groups with the sham control group using
SAS software (SAS Institute, NC, USA). Variables such
as the number of live young, anogenital distance, the day
of preputial separation, testes weight, sperm head count
and daily sperm production were subjected to one-way
analysis of variance (ANOVA) and the percentages of
motile sperm and sperm abnormalities were analyzed by
the Kruskal-Wallis nonparametric ANOVA . If
either of the tests showed a significant difference among
the groups, the data were analyzed by the multiple
comparison procedure of the Dunnett's post-hoc test
. The level of significance was taken as
P < 0.05.
As shown in Table 1, there were no statistically
significant differences in the number of live young at birth,
clinical signs, and body weight development of male
offspring between exposed and sham-exposed groups. The
anogenital distance of male offspring in the 5 mT group
was slightly shorter (about 7 %) than that in the sham
control group, but no significant difference was detected
between the groups. No exposure-related effects were
seen on the day of preputial separation; nor had the testis
weight of male offspring been affected. On testicular
histopathological examination, no exposure related
changes were observed in the number and morphology
of germ cells between the exposed and sham-exposed
groups. As shown in Table 2, copulation and fertility
indices in the exposed groups were similar to those in
the sham control group. The sperm head count and daily
sperm production of male offspring in the 500 mT group
were slightly less (about 8 %) than controls, respectively,
but the differences were not statistically significant when
compared with the sham control group. The results of
sperm motility and morphology in male offspring of the
exposure groups did not differ from the sham control
The present study was carried out in rats to
determine the potential adverse effects of in
utero and neonatal exposure to 60 Hz EMF on spermatogenesis and fertility of F1 male offspring. The results obtained in the
present study clearly showed that in utero and neonatal
exposure to 60 Hz at field strengths up to 500 mT did not
cause any adverse effects on spermatogenesis and
fertility in F1 male offspring.
The slight reduction of anogenital distance observed
in the 5 mT group was considered to be an accidental
finding, because this change was very slight and did not
show a dose-response relationship. This interpretation
was strengthened by the fact that there was no
significant differences between the exposed and sham control
groups with relation to preputial separation, and this is
one of the sensitive indicators of male reproductive
organ development. It was previously reported that
evaluation of testicular sperm head counts seems to be a good
indicator of spermatogenic damages and the number of
testicular sperm heads corresponds to the number of
elongate spermatids in the testis [16, 22]. In the present
study, however, the slight decrease in the number of
sperm heads observed in the 500 mT group is of
doubtful toxicological significance, because this change did
not exhibit a dose-response relationship and was
unaccompanied by correlated testicular weight changes and
histopathological findings. Histopathological
examinations revealed that there were no exposure related
adverse effects on the number and morphology of
sperma-togonia, spermatocytes, and spermatids were observed
at any doses tested. The results of sperm motility
observed in the three exposure groups also did not differ
from the sham control values. Although various sperm
abnormalities such as small head, amorphous head, two
heads/tails, excessive hook, blunt hook, folded tail, short
tail and no tail were observed in the both exposed and
sham-exposed groups, there was no obvious difference
in the incidence of abnormal sperm between the groups.
Conflicting observations have been reported
regarding the potential toxic effects of EMF on
spermatogenesis and reproduction in experimental animals and
humans. Multigeneration reproductive toxicity study
showed that continuous exposure of Spague-Dawley rats
to 60 Hz magnetic fields has no significant adverse
effects on adult reproductive capacity, developing fetus,
and neonatal development in rats . Lundsberg
et al.  reported that human sub-fertility was not associated
with occupational 0.3 mT exposure on
morphology, motility, and sperm concentration among males.
Kowalczuk et al.  also did not find dominant lethal
mutation in the male germ cells of mice when they ex
posed to power frequency magnetic fields at 10 mT for
the approximate period of spermatogenesis. Recently,
Heredia-Rojas et al.  reported that 60 Hz and 2 mT
magnetic field exposure did not affect meiotic
chromosomes and morphological characteristics of male germ
cells in mice. The lack of adverse effects of ELF EMF
exposure on spermatogenesis and fertility observed in
the present study is consistent with the results of
above-mentioned investigators [3-6]. On the contrary, De Vita
et al.  reported that exposure to 50 Hz and 1.7 mT
for 4 h caused a significant decrease in the number of
elongated spermatids on day 28 after treatment. Al-Akhras
et al.  reported that exposure of adult male rats to 50
Hz magnetic fields for 90 days had a significant effect
on the fertility of females impregnated by the exposed
males. Furuya et al.  suggested that long-term
exposure to ELF magnetic fields (1.0 mT) had a possible
effect on the proliferation and differentiation of
spermato-gonia. Recently, Ramadan et al.  also reported that
exposure of fractionated doses of magnetic fields (20
mT) caused a significant decrease in sperm count, motility, and daily sperm production in mice. Most
recently, Lee et al.  reported that continuous
exposure to EMF (60 Hz, 0.5 mT) for 8 weeks caused an
increased incidence of testicular germ cell death and this
finding resulted from an increased incidence of germ cell
apoptosis in mice. The apparent discrepancy among the
studies might be due to differences in animals used,
exposure period and intensity, environmental conditions,
etc. Meanwhile, exposure of mice to static magnetic
fields of 1.6 T, during a 30-day period, resulted in
eversible changes in spermatogenic epithelium and in a
considerable decrease in the number of mature germ cells
. According to the reports of Tablado et al.
[23, 24], however, the motility and morphological characteristics
of epididymal sperm were not affected after exposure to
0.7 T static magnetic fields in mice. These investigators
also demonstrated in subsequent study that in
utero exposure to 0.5-0.7 T static magnetic fields did not cause
any adverse effects on the development of testis and
epididymis in mice . In view of our findings and the
contradictory reports in the literature, it is necessary to
conduct much wider studies under different
experimental conditions, to help clarify the controversy
concerning the possible spermatotoxic risk associated with
magnetic field exposure.
Based on the results, it was concluded that in
utero and neonatal exposure of Sprague-Dawley rats to 60 Hz
EMF at field strengths up to 500 mT did not produce any
detectable alterations in the offspring spermatogenesis
The present study was supported by a grant from
the Korean Health 21 R&D Project, Ministry of Health
and Welfare, Republic of Korea.
1 World Health Organization (WHO). Environmental Health
Criteria 69: Magnetic fields. Geneva: 1987.
2 Brent RL. Reproductive and teratologic effects of
low-frequency electromagnetic fields: a review of
in vivo and in vitro studies using animal models. Teratology 1999; 59: 261-86.
3 Ryan BM, Symanski RR, Pomeranz LE, Johnson TR, Gauger
JR, McCormick DL. Multigeneration reproductive toxicity
assessment of 60-Hz magnetic fields using a continuous breeding
protocol in rats. Teratology 1999; 59: 156-62.
4 Lundsberg LS, Bracken MB, Belanger K. Occupationally
related magnetic field exposure and male subfertility. Fertil Steril
1995; 63: 384-91.
5 Kowalczuk CI, Robbins L, Thomas JM, Saunders RD.
Dominant lethal studies in male mice after exposure to a 50 Hz
magnetic field. Mutat Res 1995; 328: 229-37.
6 Heredia-Rojas JA, Caballero-Hernandez DE, Rodriguez-de la
Fuente AO, Ramos-Alfano G, Rodriguez-Flores LE. Lack of
alterations on meiotic chromosomes and morphological
characteristics of male germ cells in mice exposed to a 60 Hz and
2.0 mT magnetic field. Bioelectromagnetics 2004; 25: 63-8.
7 De Vita R, Cavallo D, Raganella L, Eleuteri P, Grollino MG,
Calugi A. Effects of 50 Hz magnetic fields on mouse
spermatogenesis monitored by flow cytometric analysis.
Bioelectromagnetics 1995; 16: 330-4.
8 Al-Akhras MA, Elbetieha A, Hasan MK, Al-Omari I, Darmani
H, Albiss B. Effects of extremely low frequency magnetic field
on fertility of adult male and female rats. Bioelectromagnetics
2001; 22: 340-4.
9 Furuya H, Aikawa H, Hagino T, Yoshida T, Sakabe K. Flow
cytometric analysis of the effects of 50 Hz magnetic fields on
mouse spermatogenesis. Nippon Eiseigaku Zasshi 1998; 53:
10 Ramadan LA, Abd-Allah AR, Aly HA, Saad-el-Din AA.
Testicular toxicity effects of magnetic field exposure and
prophylactic role of coenzyme Q10 and L-carnitine in mice. Pharmacol
Res 2002; 46: 363-70.
11 Lee JS, Ahn SS, Jung KC, Kim YW, Lee SK. Effects of 60 Hz
electromagnetic field exposure on testicular germ cell apoptosis
in mice. Asian J Androl 2004; 6: 29-34.
12 NRC (National Research Council). Guide for the Care and Use
of Laboratory Animals. Washington, USA: National Research
Council, National Academy Press; 1996.
13 Chung MK, Kim JC, Myung SH, Lee DI. Developmental
toxicity evaluation of ELF magnetic fields in Sprague-Dawley
rats. Bioelectromagnetics 2003; 24: 231-40.
14 Boorman GA, McCormick DL, Findlay JC, Hailey JR, Gauger
JR, Johnson TR, et al. Chronic toxicity/oncogenicity
evaluation of 60Hz (power frequency) magnetic fields in F344/N
rats. Toxicol Pathol 1999; 27: 267-78.
15 International commission on non-ionizing radiation
protection (ICNIRP). Guideline for limiting exposure to time
varying electric, magnetic and electromagnetic fields (up to 300
GHz). Health Physics 1998; 74: 494-522.
16 Kim JC, Kim KH, Chung MK. Testicular cytotoxicity of
DA-125, a new anthracycline anticancer agent, in rats. Reprod
Toxicol 1999; 13: 391-7.
17 Chung MK, Kim JC, Kim WB, Kang KK, Han SS. Fertility
study of the new pyrazolopyrimidinone derivative DA-8159
for erectile dysfunction in rats. Arzneimittelforschung 2004;
18 Robb GW, Amann RP, Killian GJ. Daily sperm production
and epididymal sperm reserves of pubertal and adult rats. J
Reprod Fertil 1978; 54: 103-7.
19 Takahashi O, Oishi S. Testicular toxicity of dietarily or
parenterally administered bisphenol A in rats and mice. Food Chem
Toxicol 2003; 41: 1035-44.
20 Kruskal WH, Wallis WA. Use of ranks in one criterion
variance analysis. J Aer Statist Assn 1952; 47: 583-621.
21 Dunnett CW. New tables for multiple comparisons with a
control. Biometrics 1964; 20: 482-91.
22 Meistrich ML. Evaluation of reproductive toxicity by
testicular sperm head counts. J Am Coll Toxicol 1989; 8: 551-66.
23 Tablado L, Perez-Sanchez F, Soler C. Is sperm motility
maturation affected by static magnetic field? Environ Health
Perspect 1996; 104: 1212-6.
24 Tablado L, Perez-Sanchez F, Nunez J, Nunez M, Soler C.
Effects of exposure to static magnetic fields on the
morphology and morphometry of mouse epididymal sperm.
Bioelectromagnetics 1998; 19: 377-83.
25 Tablado L, Soler C, Nunez M, Nunez J, Perez-Sanchez F.
Development of mouse testis and epididymis following
intrauterine exposure to a static magnetic field.
Bioelectromag-netics 2000; 21: 19-24.