Dosadašnja istraživanja o učinku radiofrekvencijskog elektromagnetskog zračenja (RF-EMZ) na muški reproduktivni sustav osobito u ljudi temelje se na modernoj tehnologiji koja je u svakodnevnoj upotrebi. Biološki učinci zračenja na organizam/stanice su različiti, a ovise o frekvenciji zračenja te dugotrajnosti izlaganja. Potencijalno negativni utjecaji EMZ-a većih frekvencija od 2500 MHz do sada nisu istraživana na uzorcima sperme ljudi i životinja. Nadalje, u dostupnoj literaturi nema podataka o učinku RF-EMZ-a na pokazatelje antioksidacijske zaštite i vitalnosti spermija te strukturnu stabilnosti kromatina spermija in vitro ozračenoga sjemena rasplodnih nerasta. Jedino je istraživan učinak RF-EMZ-a vrlo male frekvencije 50 MHz na pokazatelje gibljivosti i brzine spermija nerasta.
Cilj ovog istraživanja bio je istražiti učinak elektromagnetskog zračenja na pokazatelje vitalnosti, gibljivosti i brzine spermija, pokazatelje antioksidacijske zaštite te strukturnu stabilnosti kromatina spermija in vitro ozračenoga sjemena rasplodnih nerasta. Također, cilj je bio utvrditi ovisi li učinak kontinuiranog neionizirajućeg elektromagnetskog zračenja o frekvenciji zračenja na prethodno spomenute pokazatelje.
Uzimanje uzoraka, ozračivanje te analize sjemena provele su se u tri razdoblja radi lakše provedbe istraživanja. U svakom od tri razdoblja, uzorkovani su uzorci sperme 12 rasplodnih nerasta pasmine pietrain i landras, starosti od 1,5 do 3,5 godina. Svježe razrijeđeno sjeme svakog nerasta razdijelilo se u kontrolni uzorak (n = 12) te pokusni uzorak (n = 12) koji je bio ozračen. Pokusni su uzorci bili ozračeni s tri različita tretmana izlaganja kontinuiranom neionizirajućem elektromagnetskom zračenju različite frekvencije. U prvom razdoblju uzorci svježeg razrijeđenog sjemena su bili ozračeni s frekvencijom od 700 MHz, u drugom s 2500 MHz, a u trećem s 3500 MHz pri jakosti elektromagnetskog polja od 10 V/m u trajanju od 2 sata pomoću gigahercne transverzalne elektromagnetske komore. Sveukupno je uzorkovano 36 uzoraka sperme za sva tri pokusna razdoblja što u konačnici predstavlja broj od 72 uzorka gdje 36 čine kontrolne a 36 pokusne uzorke. U uzorcima ozračenog i neozračenog sjemena određivani su pokazatelji gibljivost i brzine spermija pomoću kompjuterske analize spermija, udio oštećenja stanične i akrosomske membrane, oštećenje DNK-a te oštećenje i depolarizacija mitohondrija pomoću specifičnih metoda bojenja. U staničnim lizatima spermija na automatskom biokemijskom analizatoru određene su aktivnosti glutation reduktaze, glutation peroksidaze, ukupne superoksid dismutaze, koncentracija ukupnog antioksidacijskog statusa te malondialdehida.
U ukupno svim ozračenim uzorcima sperme utvrđeno je statistički značajno manji udio gibljivih, progresivno gibljivih i brzih spermija te spermija pravolinijskog indeksa, indeksa linearnosti i indeksa oscilacije u odnosu na kontrolnu skupinu uzoraka. Najveći pad u udjelu progresivno gibljivih spermija, u odnosu na kontrolnu skupinu, zabilježen je u uzorcima ozračenima sa 700 MHz te s 2500 MHz dok je nešto manji pad zabilježen u uzorcima ozračenima s 3500 MHz. U uzorcima sperme ozračenima s 2500 MHz zabilježen je značajan pad u udjelu progresivno gibljivih spermija, u udjelu spermija pravolinijskog indeksa, u udjelu indeksa linearnosti, u vrijednostima pravolinijske brzine spermija i frekvenciji prelaska pravolinijske putanje u odnosu na neozračene uzorke. Značajan pad u udjelu progresivno gibljivih i brzih spermija, u udjelu indeksa oscilacije, u vrijednostima pravolinijske brzine spermija zabilježen je u uzorcima ozračenima sa 700 MHz u odnosu na neozračene uzorke. Statistički značajno veći udio spermija s oštećenjem DNK-a utvrđen je u ukupno svim ozračenim uzorcima, u odnosu na kontrolne skupine uzoraka. Najveći porast u udjelu spermija s oštećenjem DNK-a zabilježen je u uzorcima ozračenima s 2500 MHz, a nešto je manji porast zabilježen u uzorcima ozračenima s 3500 MHz u odnosu na neozračene uzorke. Vrijednosti pokazatelja antioksidacijskog statusa spermija nisu se statistički značajno razlikovale u ozračenim i neozračenim uzorcima sperme nerasta.
|Abstract (english)|| |
With the development of modern technology, there is an increasing exposure of organisms to artificially created electromagnetic fields. The presence of different frequencies from non-ionizing radiation field of anthropogenic origin (radio communication systems, mobile phones, microwave ovens, sources from the field of wireless communication system in the environment) is known as electromagnetic smog or electrosmog. Also, with the development of modern technology, public interest in the potential negative effects of electromagnetic radiation (EMR) has increased as the number of users increases daily, especially those of younger age who are most exposed.
Public concern about the impact of EMR on health is controversial. One of perhaps the greatest threats to humanity today is electromagnetic pollution in the environment due to its cumulative effect over a long period of time. In addition, the negative effects of electromagnetic pollution are often not recognized but are associated with other factors such as stress, hereditary traits or diet.
Biological effects can be direct that occur when radiation acts on mitochondrial DNA, indirect that occur due to the effect of radiation on water within the cell itself and somatic that occur on an irradiated individual while genetic effects can occur and be transmitted genetically to offspring.
The majority of research has focused on the negative impact of radiofrequency electromagnetic radiation (RF-EMR) on the male reproductive system, especially humans, while data on the effect of RF-EMR on the reproductive system of male domestic animals are scarce. Currently known data indicate that due to exposure to EMR, there may be a decrease in sperm motility, DNA damage and the creation of excessive amounts of reactive oxygen species or oxidative stress. However, the potentially negative effects of EMRs of higher frequencies (higher than 2500 MHz) have not been investigated on human and animal ejaculates so far. Also, so far there is no data on the effect of RF-EMR on antioxidant protection indicators and vitality of sperm and the structural stability of sperm chromatin in vitro irradiated semen of breeding boars. Only the effect of RF-EMR of very low frequency, 50 MHz, on sperm motility and velocity parameters of boars was investigated.
The aim of this study was to investigate the effect of electromagnetic radiation on sperm vitality, motility and velocity indicators, antioxidant protection indicators and structural stability of sperm chromatin in vitro irradiated semen of breeding boars. Also, the aim was to determine whether the effect of continuous non-ionizing electromagnetic radiation depends on the frequency of radiation on the previously mentioned indicators.
The hypothesis of the research is that there is a negative effect of electromagnetic radiation on the quality of boar semen and on the indicators of antioxidant protection and on the structural stability of sperm chromatin. Also, it is assumed that lower frequencies of radio frequency electromagnetic radiation can also have a negative effect on the aforementioned indicators.
The research was performed on ejaculate samples of 8 breeding boars of the pietren breed and 4 breeding boars of the German Landrace breed, aged from 1,5 to 3,5 years, which were obtained by manual fixation of the penis. To make the research easier to conduct, sampling and irradiation of sperm semen were carried out in three periods, each period lasting one week. In each of the three periods, ejaculates of 12 breeding boars were sampled. After sampling of the ejaculates, their volume, concentration, motility, proportions of vital and abnormal spermatozoa were determined. The concentration of spermatozoa was determined by electronic counter; their motility was assessed natively under binocular microscope with built-in sperm heater plate, and for determining morphologically normal and pathologic forms of spermatozoa a commercial staining set Spermac was used. Freshly diluted semen of each boar were divided into a control sample (n = 12) and a test sample (n = 12) that was irradiated.
The test samples were irradiated with three different treatments of exposure to continuous non-ionizing electromagnetic radiation of different frequencies. In the first period, test samples of fresh diluted semen were irradiated with a frequency of 700 MHz, in the second with 2500 MHz and in the third with 3500 MHz at an electromagnetic field strength of 10 V / m for 2 hours using gigahertz transverse electromagnetic (HCTEM) chamber made at the Department of Communication and Space Technologies, Faculty of Electrical Engineering and Computing, University of Zagreb. In addition to the HCTEM irradiation chamber, an HP 8657A signal generator and an RFGA0101-05 linear amplifier were used to obtain higher electromagnetic field strengths. In the samples of irradiated and non-irradiated semen, the indicators of motility (sperm motility, progressive motility) and sperm velocity indicators were determined (curvilinear velocity - VCL, straight-line velocity - VSL, average path velocity -VAP, amplitude of lateral head displacement - ALH, linearity - LIN, wobble - WOB, straightness - STR, beat cross frequency - BCF) using computer assisted sperm analyzer.
Furthermore, in the samples of irradiated and non-irradiated semen, the proportion of sperm with cell and acrosomal membrane damage, the proportion of sperm with damage and depolarization of mitochondria using PI⁄SYBR-14 LIVE⁄DEAD with JC-1 and the proportion of sperm with DNA damage were determined using Halomax DNA Fragmetation Test. Part of the experimental and control samples after irradiation were centrifuged at 21 ° C for 15 minutes at 2400 g. The supernatants from both groups were taken by pipette, and the remaining spermatozoa were washed three times with saline and after each washing they were centrifuged for 5 minutes at 500 g and soon after that preserved to - 80 ° C in the freezer until analyzed. After thawing the sperm samples, cell lysates were prepared. The activities of glutathione reductase, glutathione peroxidase, total superoxide dismutase and the concentration of total antioxidant status, as well as the concentration of malondialdehyde by the Grotto method were determined in the cell supernatants lysates by commercial kits of the company "Randox Laboratories" on an automatic biochemical analyzer.
In all irradiated samples, a statistically significantly lower share of motile, progressively motile, rapid sperm count, straightness, linearity and wobble index, was found in relation to the control group of ejaculate. Furthermore, in all irradiated ejaculates, a statistically significantly higher proportion of medium-fast and immobile sperm was recorded compared to the control group of ejaculates. Likewise, all irradiated ejaculates had, compared to the control group of ejaculates, statistically significantly lower values of straight-line and average path velocity.
The largest decrease in the proportion of progressive sperm motility, compared to the control group, was recorded in ejaculates irradiated with 700 MHz (p<0,001) and 2500 MHz (p<0,001) while a slightly smaller decrease was recorded in ejaculates irradiated with 3500 MHz (p<0,05). In ejaculates irradiated with 2500 MHz, a significant decrease was recorded in the share of progressive sperm motility, in the share of the straightness, in the share of the linearity index, in the values of the straight-line velocity and the beat cross frequency.
A significant decrease in the share of progressively motile and rapid sperm, in the share of wobble index, in the values of straight-line velocity was recorded in ejaculates irradiated with 700 MHz in relation to non-irradiated ejaculates. Furthermore, in ejaculates irradiated
with 700 MHz, a simultaneous increase in the proportion of medium-fast sperm and slow sperm was recorded (p<0,05).
The largest increase in the proportion of sperm with DNA damage was recorded in ejaculates irradiated with 2500 MHz (p <0,01) and a slightly smaller increase was recorded in ejaculates irradiated with 3500 MHz (p<0,05) compared to non-irradiated ejaculates. A negative correlation was found between DNA damage with almost all investigated indicators of sperm velocity (VAP, VCL, ALH, BCF, STR and LIN). A significantly higher proportion of sperm with damaged membrane and good mitochondrial potential was recorded in ejaculates irradiated with 3500 MHz.
Although there was an increase in the sum of the proportion of sperm with damaged membrane and / or low mitochondrial potential in all irradiated ejaculates, depending on and independent of the radiation frequency, the difference was not statistically significant. The values of sperm antioxidant status indicators did not differ statistically significantly in irradiated and non-irradiated boar ejaculates.
According to the results of this study, it can be concluded that RF-EMR has a negative effect on sperm motility, velocity indicators and on the structural stability of sperm chromatin in vitro irradiated semen of breeding boars. The negative effect of RF-EMR on indicators of sperm motility and velocity sperm of breeding boar ejaculates was manifested in a significant decline: the share of motile, progressively motile and rapid sperm, average path velocity and straight-line velocity values and linearity and wobble values. The effect of RF-EMR on the aforementioned indicators significantly depends on the radiation frequency. The most harmful effect on sperm motility, velocity indicators and on the structural stability of sperm chromatin was found in ejaculates irradiated with 2500 MHz.
The established negative correlation between DNA damage of irradiated boar sperm with almost all investigated indicators of sperm velocity indicates that irradiated sperm with DNA damage had a lower velocity.
The negative effect of RF-EMR with a frequency of 2500 MHz on the indicators of motility and sperm velocity of irradiated ejaculates was recorded in a significant decline: the proportion of progressively motile sperm, the proportion of straightness index, linearity index, values of straight-line velocity and beat cross frequency index.
The effect of RF-EMR on irradiated ejaculates, depending on and independent of the radiation frequency, is manifested by an increase in the proportion of sperm with damaged membrane and / or low mitochondrial potential compared to non-irradiated ejaculates.
RF-EMR has no significant effect on the indicators of antioxidant protection of sperm in vitro irradiated semen of breeding boars.