Effect of Supplementation of Zinc on Count, Motility and in vitro Fertilization Capacity of Spermatozoa of Magnetic Field Exposed Rats
The aim of this study was to investigate the preventive effect of zinc on count, motility and fertilization capacity of rat spermatozoa that exposed to 1.5 Tesla magnetic fields. Thirty two adult male rats were subdivided randomly to 4 groups: group 1, serve as untreated controls; group 2, was exposed to the magnetic field for 30 min but received no additional treatment; groups 3 and 4, were exposed to a magnetic field for 30 min and received 200 and 500 ppm zinc sulfate oral daily, respectively. After 50 days all rats were killed and their epidydimises were removed. Then after incubation of sperm in the incubator within 37°C and 5% CO2 for 1 h, the sperms count and motility were examined by an inverted microscope. For in vitro fertilization at first the sperm suspension of different groups of rats added to the freshly ovulated ova than combined sperm-oocyte suspension was incubated for 4-6 h. Sperm counts in 1 g of the epididymis were 2998.7±322.70 in group 1 and in groups 2, 3 and 4, 1022.9±128.66, 1978.4±457.79 and 2126.1±308.90, respectively. Therefore, group 2 has a significant lower sperm count in comparison with other groups (p<0.05). Sperm with progressive motility was 52.25±3.88 in group 1, 22.35±1.82 in the group 2, 49±26±1.66 in group 3 and 46.11±4.05 in group 4. Therefore, group 2 has a significant lower sperm motility in comparison with other groups (p<0.05). The same results were obtained in the case of pregnancy. The sperm count, motility and fertilization capacity may be preserving by zinc supplementation. Therefore, zinc might have the potential for usage for MRI patients as well as workers.
Infertility explains as inability of a couple to become pregnant after unprotected
one year sexual intercourse. In the United States about 10% of couples are affected
by infertility (Philippov et al., 1998). Both
genders can affect by infertility. According to the American Society for Reproductive
Medicine, around 33% of the time the diagnosis is due to female infertility,
33% of the time it is linked to male infertility and the rest 33% is due to
a combination of factors from both partners (Philippov et
al., 1998). A variety of stress factors such as microorganisms, hyperthermia,
exposure to heavy metals (Ozawa et al., 2002)
stressful stimuli such as prolonged immobilization (Almeida
et al., 2000) and forced swimming (Mingoti et
al., 2003; Saki et al., 2009) inhibit
male reproductive functions and spermatogenesis. Magnetic Resonance Imaging
(MRI) for medical diagnosis is one of the most important sources of static magnetic
fields. Earlier study showed that a 30 min exposure to 1.5-T of static magnetic
fields appears to have effect on spermatogenesis in mice (Chakeres
and Vocht, 2005; Narra et al., 1996; Monfared
et al., 2009). On the other hand, the gonads are the fastest growing
tissues in the body, with zinc metalloenzymes essentially involved in nucleic
acid and protein synthesis (Bedwal and Bahuguna, 1994).
Zinc participates in mechanisms of the major metabolic pathways involving protein
synthesis and turnover and it also has an important function in gene expression
and embryogenesis (Aggett and Comerford, 1995). Zinc
plays an important role in the physiology of spermatozoa, in sperm degeneration
and in sperm membrane stabilization (Lewis-Jones et al.,
1996). The present study was designed to investigate the protective effects
of zinc supplementation on count, motility and in vitro fertilization
capacity of spermatozoa of 1.5 Tesla magnetic field exposed rats.
MATERIALS AND METHODS
Animals: This experimental study was performed in the physiology research center of Ahwaz Joundishapour University of Medical Sciences from March 2008 to August 2009. Total 32 adult male Wister rats with 3 months of age and weighing 210±10.6 g were used in this study. The animals were obtained from Laboratory Animals Care and Breeding Center of Ahwaz Joundishapour University of Medical Sciences, Ahwaz, Iran. The fertilizing ability of male mice was proven at the beginning of the experiment. All rats were randomly divided into four equal groups (n = 8); (1) control and (2) experimental groups. All animals were housed individually per cage under a 12 h light/dark cycle, 20±2°C temperature and 60-65% humidity-controlled room with food and water ad libitum. All procedures were approved by international guidelines and by the Institute Research Ethics and Animal Care and Use Committee of Ahwaz Joundishapour University of Medical Sciences. Every effort was made to minimize the number of animals used and their suffering.
Experimental setup: Thirty two adult male rats were subdivided randomly to 4 groups: group 1, serve as untreated controls; group 2 was exposed to an 1.5 Tesla magnetic field but received no additional treatment; groups 3 and 4, were exposed to 1.5 Tesla magnetic field but received 200 and 500 ppm zinc sulfate orally daily, respectively. After 50 days all rats were killed and their epidydimises were excised and weighed.
Static Magnetic Field (SMF) irradiation and grouping: Irradiation was
carried out a 1.5 T, superconductive coil medical MRI unit of Ahwaz Joundishapour
University of Medical Sciences. Rats in experimental groups were exposed in
a perforated in 35x35 cm of the chamber to SMF in the core of MRI gantry at
room temperature (24±1°C) for 30 min (Narra et
al., 1996; Monfared et al., 2009).
Sperm motility analysis: Sperm motility of two study groups was determined
using a Makler chamber. All counts were performed at 37°C in T6 media. The
sperm motility was assessed and classified as progressive, no progressive. Initial
sperm motility was manually assessed by a single individual in duplicate for
each sample by evaluating 100 sperms. Total motility was defined as any movement
of the sperm head and progressive motility was defined as the count of those
spermatozoa that moved in a forward direction (Movassaghi
et al., 2009).
Measurements of sperm count: Sperms were collected from the epididymis
of each rat by flushing with the same volume (about 8 mL) of T6 medium. Collected
samples were centrifuged at 100 g for 2 min and the precipitate portion was
resuspended in 10 mL of fresh T6 medium. A fraction of suspension (100 μL)
was mixed with an equal volume of 1% Trypan blue in the same medium and numbers
of sperms were counted in four chambers of hemocytomere slide (Alvares
and Story, 1984). The sperm number was expressed per milliliter of suspension.
Oocyte collection: Adult female Wistar rats that were between
10 to 12 weeks old were administered intraperitoneally with 10 IU Pregnant Mare
Gonadotropine (PMG) serum for superovulation; this was followed 46-48 h later
by the intraperitoneal administration of 10 IU Human Chorionic Gonadotropine
(HCG). Mice were killed 12-14 h after HCG injection by cervical dislocation
method. After disinfection with 70% alcohol and opening the abdomen wall, the
Y shaped uterus, ovaries and oviducts were identified. The oviducts were excised
as follows: clamping cornuas, dissecting the peritoneum and fat between ovary
and tube and then cutting the fallopian tube from the proximal end and cumulus-
oocyte complexes were collected in T6 medium. The granulosa cells of oocytes
were removed by pipetting in T6 medium containing 80 IU mL-1 hyaluronidase
and mature oocytes obtained and randomly divided into two groups (Malkov
et al., 1998).
In vitro fertilization: In vitro fertilization was carried
out in drops of T6 medium added to 5 mg mL-1 BSA under mineral oil.
A preincubated capacitated sperm suspension of different groups as mentioned
above was gently added to the freshly ovulated ova which divided in four groups
to give a final motile sperm concentration on 100000 ML-1. The combined
sperm-oocyte suspension was incubated for 4-6 h. The ova was then washed through
several changes of a medium and finally incubated in drops of T6+5 mg mL-1
BSA under mineral oil. Fertilization was assessed by recording the number
of 2 cell embryos 24-26 h after completion of fertilization in vitro
(Saki et al., 2006).
Statistical analysis: Data are reported as the Mean±SD and percentage. The statistical significance of difference between the control and experimental groups was determined by the unpaired t-test. Differences between the means were considered to be significant when p<0.05 was achieved.
In every group of this study 8 adult male rats with 10 to 12 weeks old were
used. Sperm count was 2998.7±322.70 in group 1 and in groups 2-4, the
1022.9±128.66, 1978.4±457.79 and 2126.1±308.90 values were
observed, respectively (Table 1). Therefore, group 2 has a
significant lower sperm count in comparison with other groups (p<0.05).
count in 1 g of the epididymis, different type of motility and fertilization
capacity of sperm in groups of study|
Table 1 shows that sperm with progressive motility was 52.25±3.88 in group 1, 22.35±1.82 in the group 2, 49±26±1.66 in group 3 and 46.11±4.05 in group 4. Therefore, group 2 has a significant lower sperm motility in comparison with other groups (p<0.05). In this regards no difference between other groups was encountered (p>0.05). In the term of non-progressive motility the data showed the values of 27.12±2.11 in group 1, 28.42±5.9 for group 2, 26.12±1.66 for group 3, 24.12 ±2.43 in group 4. Therefore, there was no significant difference between all groups (p>0.05).
The fertilization capacity of sperm observed values of 20.3% in group 2, 49% in group 3, 55.4% in group 4 comparing to 66.9% in group 1 (Table 1). The fertilization capacity of sperm of group 2 was significantly lower than other groups (p>0.05).
The present study was carried out in rats to determine the effect of administration
of zinc on count, motility and fertilization capacity of spermatozoa of 1.5
Tesla magnetic field exposed rats. The results obtained in the present study
clearly showed that a 1.5 Tesla magnetic field may reduce the number of sperm
of rats. This finding is in agreement with the two earlier studies that reported
the exposure to the 1.5 Tesla magnetic field caused a statistically significant
reduction in testicular sperm on the 16th and 29th days after exposure (Narra
et al., 1996; Monfared et al., 2009).
This finding may be resulted from the increased incidence of testicular germ
cell death. It previously reported that continuous exposure to 0.5 mili-Tesla
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 (Lee et al., 2004). In this study, we observed
that exposure to 1.5 Tesla magnetic fields for 30 min a day caused a significant
decrease in motility of sperm on day 50 after treatment. Previously, reported
by Tablado et al. (1998) that sperm motility
was not affected by 0.7 Tesla magnetic fields. The inconsistency between the
above report and present study might be due to variation of field strengths.
The reduced fertilization capacity of 1.5 Tesla magnetic field exposed rats
is probably due to a decrease in the number and motility of sperm.
Bernabò et al. (2007) conducted a study
to evaluate the effect of an acute exposure to a sinusoidal magnetic field (50
Hz, 1 mili-Tesla) on the ability of boar mature spermatozoa to acquire the fertilizing
competence in vitro. As a consequence, they showed after 1 h of incubation
magnetic field exposed cells displayed a reduced motility, a modest reactivity
when co-incubated with solubilized zonae pellucidae and a reduction in oocyte
penetrating ability. The present study observed that exposure to 1.5 Tesla magnetic
fields for 30 min a day caused a significant decrease in motility of sperm on
day 50 after treatment.
The protective effects of zinc supplementation on count, motility and fertilization
capacity of spermatozoa of 1.5 Tesla magnetic field exposed rats investigated
in the present study. Previous study has been shown that zinc is important for
normal testicular development, maintenance of the germinal epithelium and motility
(Favier, 1992) and also zinc administration minimized
oxidative damage and reversed the impairment of spermatogenesis and testosterone
production induced by cadmium in the rat testis (Amara et
As our knowledge this present study was evaluated the effect of administration of zinc on count, motility and fertilization capacity of spermatozoa of 1.5 Tesla magnetic fields exposed rats for first time. We observed that zinc treatment of 1.5 Tesla magnetic fields exposed rats significantly enhanced the sperm count, motility and fertilization capacity so zinc might have the potential for usage for Magnetic Resonance Imaging (MRI) patients as well as workers.
This project was financially supported by the research deputy of Ahwaz Jondishapour University of Medical Sciences (AJUMS). We would like to express our great appreciation for their support.
1: Aggett, P.J. and J.G. Comerford, 1995. Zinc and human health. Nutr. Rev., 53: S16-22.
2: Almeida, A.S., W.G. Kempinas and T.L.L. Carvalho, 2000. Sexual behavior and fertility of male rats submitted to prolonged immobilization-induced stress. Braz. J. Med. Biol. Res., 33: 1105-1109.
3: Alvares, J.S. and B.T. Story, 1984. Assessment of cell damage caused by spontaneous lipid peroxidation in rabbit spermatozoa. Biol. Reprod., 30: 323-331.
4: Amara, S., H. Abdelmelek, C. Garrel, P. Guiraud and T. Douki et al., 2008. Preventive effect of zinc against cadmium-induced oxidative stress in the rat testis. J. Reprod. Dev., 54: 129-134.
5: Bedwal, R.S. and A. Bahuguna, 1994. Zinc, copper and selenium in reproduction. Experientia, 50: 626-640.
CrossRef | Direct Link |
6: Bernabo, N., E. Tettamanti, M.G. Pistilli, D. Nardinocchi, P. Berardinelli, M. Mattioli and B. Barboni, 2007. Effects of 50 Hz extremely low frequency magnetic field on the morphology and function of boar spermatozoa capacitated in vitro. Theriogenology, 67: 801-815.
7: Chakeres, D.W. and F.D. Vocht, 2005. Static magnetic field effects on human subjects related to magnetic resonance imaging systems. Prog. Biophys. Mol. Biol., 67: 255-265.
8: Favier, A.E., 1992. The role of zinc in reproduction: Hormonal mechanisms. Biol Trace Elem. Res., 32: 363-382.
9: Lee, J.S., S.S. Ahn, K.C. Jung, Y.W. Kirn and S.K. Lee, 2004. Effects of 60 Hz electromagnetic field exposure on testicular germ cell apoptosis in mice. Asian J. Androl., 6: 29-34.
PubMed | Direct Link |
10: Lewis-Jones, D.I., I.A. Aird, M.M. Biljan and C.R. Kingsland, 1996. Effects of sperm activity on zinc and fructose concentrations in seminal plasma. Hum. Reprod., 11: 2465-2467.
11: Mingoti, G.Z., R.N. Pereita and C.M.R. Monteiro, 2003. Fertility of male adult rats submitted to forced swimming stress. Brazil. J. Med. Biol. Res., 36: 677-681.
12: Monfared, A.S., S.G. Jorsaraei and R. Abdi, 2009. Protective effects of vitamins C and E on spermatogenesis of 1.5 Tesla magnetic field exposed rats. J. Magn Reson Imaging., 30: 1047-1051.
13: Movassaghi, S., G. Saki, F. Javadnia, M. Panahi, M. Mahmoudi and F. Rhim, 2009. Effects of methyl-beta-cyclodextrin and cholesterol on cryosurvival of spermatozoa from C57BL/6 mouse. Pak. J. Biol. Sci., 12: 19-25.
CrossRef | PubMed | Direct Link |
14: Malkov, M., Y. Fisher and J. Don, 1998. Developmental schedule of the postnatal rat testis determined by flowcytometry. Biol. Reprod., 54: 84-92.
15: Narra, V.R., R.W. Howell, S.M. Goddu and D.V. Rao, 1996. Effect of 1.5-Tesla static magnetic field on spermatogenesis and embryogenesis in mice. Invest. Radiol., 31: 586-590.
16: Ozawa, N., N. Goda, N. Makino, T. Yamaguchi, Y. Yoshimura and M. Suematsu, 2002. Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress. J. Clin. Invest., 109: 457-467.
CrossRef | PubMed | Direct Link |
17: Philippov, O.S., A.A. Radionchenko, V.P. Bolotova, N.I. Voronovskaya and T.V. Potemkina, 1998. Estimation of the prevalence and causes of infertility in Western Sibria. Bull. Word Health Organ., 76: 183-187.
18: Saki, G., F. Rahim and K. Alizadeh, 2009. Effect of forced swimming stress on count, motility and fertilization capacity of the sperm in adult rats. J. Hum. Reprod. Sci., 2: 72-75.
19: Saki, G., Z. Safikhani, A. Sobhani and M. Salehnia, 2006. The in vitro fertilization rate of mouse ova in the absence or presence of recombinant human leukemia inhibitory factor. Pak. Med. Sci., 22: 438-441.
Direct Link |
20: Tablado, L., F. Perez-Sanchez, J. Nunez, M. Nunez and C. Soler, 1998. Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm. Bioelectromagnetics, 19: 377-383.