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Citation: Žaja, I.Ž.; Vince, S.; Butkovi´c,
I.; Senaši, K.; Milas, N.P.; Malari´c, K.;
Lojki´c, M.; Folnoži´c, I.; Tur, S.M.;
Kreszinger, M.; et al. The Distribution
of Boars Spermatozoa in
Morphometrically Distinct
Subpopulations after In Vitro
Exposure to Radiofrequency
Electromagnetic Radiation at 2500
MHz and Their Motility. Animals 2024,
14, 828. https://doi.org/10.3390/
ani14060828
Academic Editor: Anna Wysoki´nska
Received: 5 February 2024
Revised: 23 February 2024
Accepted: 25 February 2024
Published: 7 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
animals
Article
The Distribution of Boars Spermatozoa in Morphometrically
Distinct Subpopulations after In Vitro Exposure to
Radiofrequency Electromagnetic Radiation at 2500 MHz and
Their Motility
Ivona Žura Žaja 1, Silvijo Vince 2, Ivan Butkovi´c 2,*, Kim Senaši 3, Nina Poljiˇcak Milas 4, Krešimir Malari´c 5,
Martina Lojki´c 2, Ivan Folnoži´c 2, Suzana Milinkovi´c Tur 1, Mario Kreszinger 6,*, Marko Samardžija 2,
Snježana ˇ
Cipˇci´c 7, Nikolino Žura 7, Mario Ostovi´c 8and Marinko Vili´c 1
1Department of Physiology and Radiobiology, Faculty of Veterinary Medicine, University of Zagreb,
10 000 Zagreb, Croatia; izzaja@vef.unizg.hr (I.Ž.Ž.); tur@vef.unizg.hr (S.M.T.); mvilic@vef.unizg.hr (M.V.)
2Clinic of Obstetrics and Reproduction, Faculty of Veterinary Medicine, University of Zagreb,
10 000 Zagreb, Croatia; svince@vef.hr (S.V.); mlojkic@vef.unizg.hr (M.L.); folnozic@vef.unizg.hr (I.F.);
smarko@vef.unizg.hr (M.S.)
3Faculty of Veterinary Medicine, University of Zagreb, 10 000 Zagreb, Croatia; kim.senasi996@gmail.com
4Department of Pathological Physiology, Faculty of Veterinary Medicine, University of Zagreb,
10 000 Zagreb, Croatia; nmilas@vef.unizg.hr
5Department of Communication and Space Technologies, Faculty of Electrical Engineering and Computing,
University of Zagreb, 10 000 Zagreb, Croatia; kresimir.malaric@fer.hr
6Clinic of Surgery, Orthopedics and Ophthalmology, Faculty of Veterinary Medicine, University of Zagreb,
10 000 Zagreb, Croatia
7University of Applied Health Sciences, 10 000 Zagreb, Croatia; bielienoci@gmail.com (S. ˇ
C.);
nikolino.zura@zvu.hr (N.Ž.)
8Department of Animal Hygiene, Behaviour and Welfare, Faculty of Veterinary Medicine,
University of Zagreb, 10 000 Zagreb, Croatia; mostovic@vef.unizg.hr
*Correspondence: ibutkovic@vef.unizg.hr (I.B.); kreszinger@vef.unizg.hr (M.K.);
Tel.: +385-12390168 (I.B.); +385-12390128 (M.K.)
Simple Summary: The global use of anthropogenic radiofrequency electromagnetic radiation (RF-
EMR) in wireless technologies is increasing exponentially and presents a potential risk to animals,
especially domestic animals and pets. Additionally, the semen of boar is, in the process of collection,
manipulation and storage for the artificial insemination (AI) of sows, surrounded by and exposed
to these different sources of wireless technologies devices. A frequency of 2.5 GHz (this frequency
band is used in 5G technology) is of particular interest because many studies have used the frequency
bands of 4G technology. For the efficiency of pig production and breeding, it is extremely important
to determine the effects of such radiation on semen quality and sow fertilization success. Therefore,
we aimed to investigate the effect of RF-EMR at 2500 MHz on
in vitro
exposed breeding boar semen
spermatozoa motility and the proportions of spermatozoa subpopulations according to morpho-
metric parameters. The progressive spermatozoa motility and the proportion of the spermatozoa
subpopulation with a higher fertilizing potential were significantly reduced in the experimental
group. These results indicate the importance of further research on the effects of RF-EMR on different
animal species, especially in those undergoing AI procedures, which are important both in terms of
the quality of semen and fertilization and production and breeding goals.
Abstract: Anthropogenic radiofrequency electromagnetic radiation (RF-EMR) from wireless tech-
nologies has increased dramatically. The boar semen used for artificial insemination is essential
in sustaining the pig industry, and additionally it is also exposed to the effects of the RF-EMR of
wireless technologies. Furthermore, there are no data on the effects of RF-EMR on semen quality, and
this is the first analysis of sperm’s morphometric parameters for assessing the effect of RF-EMR on
the spermatozoa subpopulations of boars. This study investigated the effect of RF-EMR on
in vitro
exposed breeding boar semen spermatozoa motility and the proportions of spermatozoa subpopula-
tions according to their morphometric head and tail parameters. The semen samples of
12 boars
were
Animals 2024,14, 828. https://doi.org/10.3390/ani14060828 https://www.mdpi.com/journal/animals
Animals 2024,14, 828 2 of 13
divided into control and experimental groups. The samples in the experimental group were exposed
in a gigahertz transverse electromagnetic chamber at a frequency of 2500 MHz (the frequency band
used in 5G technology) and an electric field strength of 10 Vm
−1
for two hours. After exposure,
the spermatozoa motility was evaluated for both groups. A morphometric analysis of the semen
smears was performed using SFORM software (Version 1.0; VAMS, Zagreb, Croatia). The progressive
spermatozoa motility was significantly reduced in the experimental group (74.7% vs. 85.7%). PC
analysis and cluster analysis revealed two spermatozoa subpopulations: S1, spermatozoa with a
more regular head shape and a smaller midpiece outline, and S2, spermatozoa with a more elongated
head shape and a larger midpiece outline. The experimental semen samples had a greater proportion
of the S1 spermatozoa subpopulation (68.2% vs. 64.4%). The effect of RF-EMR at
2500 MHz
on the
in vitro
exposed boar semen resulted in decreased progressive spermatozoa motility and a lower
proportion of the spermatozoa subpopulation with a higher fertilizing potential.
Keywords: boars; radiofrequency electromagnetic radiation; exposure; morphometric analysis;
spermatozoa subpopulations
1. Introduction
An enormous surge in wireless communication, with the consequent increase in hu-
man and animal exposure to radiofrequency electromagnetic radiation (RF-EMR), has been
evident in recent decades. Based on many studies, there is sufficient evidence that RF-EMR
of anthropogenic origin has increased many times over in nature and that this radiation
affects the environment. For example, anthropogenic radiofrequency electromagnetic ra-
diation from wireless technologies has increased the natural levels of the around 1 GHz
frequency band in nature by about 10
18
times [
1
]. Therefore, RF-EMR at today’s intensity
is known as “electro-pollution”. Wi-Fi-based technology and receivers, such as laptops,
tablets and mobile phones with their base stations, as well as Bluetooth devices, are now
routinely used [
2
–
4
]. Although such technology has significantly improved our quality
of life, it cannot be ruled out that it is also the cause of many ailments and diseases. The
harmful effect of RF-EMR can be manifested in most organ systems; however, one of the
most sensitive organ systems is the male reproductive system [
3
,
4
]. It is well known that
RF-EMR reduces the quality of semen and has genotoxic effects on humans and animals
both
in vitro
and
in vivo
[
5
–
7
]. The exposure of men to RF-EMR through various devices,
such as mobile phones, wireless internet and laptops, causes abnormal spermatozoa mor-
phology, a decrease in spermatozoa count due to apoptosis, reduced spermatozoa motility
and viability, increased testosterone levels, decreased luteinizing hormone levels and in-
creased spermatozoa DNA fragmentation [
8
–
14
]. These effects are correlated with the
time of exposure [
15
]. A decrease in semen quality in humans (a decrease in progressive
spermatozoa motility and an increase in spermatozoa DNA fragmentation) is also evident
when the semen is exposed ex vivo to a laptop connected to a wireless network, i.e., a
Wi-Fi frequency of 2.4 GHz for 4 h [
16
]. Moreover, an increased percentage of damaged
epididymal spermatozoa heads was found in rats exposed to 24 h RF-EMR at a frequency
of 2.4 GHz for a year [
17
]. The biological targets of RF-EMR are cell structures such as
the plasma membrane (causing cell membrane permeability, including changes in calcium
levels, ionic distribution and ion permeability), mitochondria and DNA [
5
,
18
]. Sperma-
tozoa motility and morphology are important characteristics to assess in determining
semen quality [
19
]. The introduction of computer-assisted sperm analysis (CASA) has
advanced quality assessments of human and animal semen and the diagnosis of fertility,
enabling the assessment of parameters such as motility and morphology [
19
]. The current
computer-assisted sperm morphometric analysis (ASTMA) can be used to more accurately
analyze individual spermatozoa morphometrics [20,21]. By applying ASTMA technology
and multivariate procedures such as cluster analysis, it was observed that boar semen
samples contained spermatozoa subpopulations of different morphometric characteristics
Animals 2024,14, 828 3 of 13
that are not detectable by conventional subjective methods [
22
,
23
]. Morphometric results
can vary depending on internal factors such as individual variability, species, breed, sexual
maturity and age [
23
–
25
], and external factors including environmental factors, sample
preparation and the morphometric analysis of semen [20,26].
To date, there are no data on the effects of RF-EMR on the reproductive system of
domestic animals. Moreover, it cannot be ignored that the environment surrounding
boars at farms is subject to constant increases in radiofrequency electromagnetic fields
from different sources, including mobile phone base stations and wireless communication
devices. Therefore, for the efficiency of pig production and breeding, it is extremely
important to determine the effects of such radiation on the reproductive system of boars.
The aim of this study was to investigate the effect of RF-EMR at 2500 MHz (the frequency
band used in 5G technology), since it is very close to 2.45 GHz, the frequency of wireless
communication devices (Wi-Fi and Bluetooth), on
in vitro
exposed breeding boar semen
spermatozoa motility and proportions of spermatozoa subpopulations using principal
component (PC) and cluster analyses of morphometric head and tail parameters.
2. Materials and Methods
2.1. Animals, Housing and Feeding
The study included a total of 12 boars of the Pietrain (8) and German Landrace (4)
breeds, aged 1.5–3.5 years, from which semen was routinely taken twice a week, on a certain
day of the week for each boar for the artificial insemination of sows. The boars are owned
by the Centre for Artificial Insemination, Stoˇcar d.o.o., Varaždin, Croatia. The boars were
housed individually in 12 m
2
(4
×
3 m) pens, with straw bedded floors and natural lighting,
and were fed twice a day, around 6:00 a.m. and 2:00 p.m., with a mixture produced by
Stoˇcar d.o.o., Varaždin, Croatia. The daily requirements of the boar are approximately 2 kg
of mixture, with the following composition: crude protein (17.15%), crude fat (3.13%), crude
cellulose (6.29%), ash (5.30%), metabolic energy (12.49 MJ/kg), lysine (1.24%), methionine
(0.47%), tryptophan (0.18%), methionine + cystine (0.76%), zinc (126 mg/kg), magnesium
(56 mg/kg), digestible protein (38.28%), copper (31.50 mg/kg), selenium (0.42 mg/kg),
calcium (0.83%), total phosphorus (0.53%), usable phosphorus (0.22%), sodium (0.23%),
vitamin D3 (1750.01 IU/kg), vitamin A (9100 IU/kg) and vitamin E (70 IU/kg).
2.2. Collection and Evaluation of Semen from the Boars
Collection of semen samples was performed in the morning hours (around 7:00 a.m.).
The procedure for obtaining the ejaculate was carried out by the method of manual fixation
of the penis. Standard evaluation of boar ejaculate was performed at the Centre for Artificial
Insemination, Stoˇcar d.o.o., Varaždin, Croatia. The semen was collected in wide-mouthed
glass containers. The semen volume was determined using a measuring cup, and the
concentration of sperm in the ejaculate was determined using an Accucell photometer
type 60CI0394 (IMV technologies, Normandy, France). Mass motility was determined
in native semen using an Olympus BX50F (Olympus, Tokyo, Japan) microscope with a
built-in spermotherm. After determining sperm concentration and mass motility, a certain
amount of Cronos diluent (Medi-Nova, Reggio Emilia, Italy) was added to the semen and
it was transferred to 80 mL plastic bottles. The samples of semen were transported in
specialized containers with thermometer (to protect against the harmful effects of light and
temperature) for 45 min from the collection place to the laboratory.
2.3. Exposure of Samples to RF-EMR in Laboratory Conditions
Upon arrival at the clinic, samples of each boar (n= 12) were divided into two Petri
dishes (control and experimental sample (12 each for a total of 24 samples). The experi-
mental samples were exposed in a gigahertz transverse electromagnetic (GTEM) chamber
to RF-EMR at a frequency of 2500 MHz and an electric field strength of 10 Vm
−1
for two
hours. GTEM was located at the Clinic for Reproduction and Obstetrics at the Faculty of
Veterinary Medicine University of Zagreb (45
◦
48
′
25.91
′′
North and 16
◦
0
′
20.49
′′
East). A
Animals 2024,14, 828 4 of 13
GTEM-chamber was made at the Department of Communication and Space Technologies,
Department of Radiocommunications and High-Frequency Electronics, Faculty of Electrical
Engineering and Computing, University of Zagreb, Zagreb, Croatia. The chamber con-
tained a digital thermometer to measure temperature to determine if there was a thermal
effect of the radiation (Figure 1). The average temperature inside the chamber during
semen exposure was 19.1
◦
C (range 18.7–19.5
◦
C). Control samples (unexposed samples)
were placed in a metal container (imitation for GTEM-chamber) and kept under the same
conditions (temperature and time) as the experimental groups but without exposure to
RF-EMR. Experimental samples (exposed) and control samples (unexposed) were assessed
after exposure/sham-exposure of 2 h. In addition to the chamber, an HP 8657A signal
generator and an RFGA0101-05 linear amplifier were used to achieve electromagnetic field
strength. The amplifier was connected to a computer and the desired frequency was set
using the SynthNV program (Windfreak Technologies, LLC., New Port Richey, FL, USA).
The GTEM-chamber is a transmission line that is based on TEM-chambers where the letter
G indicates that the GTEM chamber works in the GHz range up to 18 GHz. The GTEM-
chamber is adjustable in the pyramidal part of the TEM-chamber with an impedance of
50
Ω
. After exposure, the control and experimental semen samples were evaluated for
spermatozoa motility.
Animals 2024, 14, x FOR PEER REVIEW 4 of 14
2.3. Exposure of Samples to RF-EMR in Laboratory Conditions
Upon arrival at the clinic, samples of each boar (n = 12) were divided into two Petri
dishes (control and experimental sample (12 each for a total of 24 samples). The experi-
mental samples were exposed in a gigaher transverse electromagnetic (GTEM) chamber
to RF-EMR at a frequency of 2500 MHz and an electric field strength of 10 Vm
−1
for two
hours. GTEM was located at the Clinic for Reproduction and Obstetrics at the Faculty of
Veterinary Medicine University of Zagreb (45°48′25.91″ North and 16°0′20.49″ East). A
GTEM-chamber was made at the Department of Communication and Space Technologies,
Department of Radiocommunications and High-Frequency Electronics, Faculty of Electri-
cal Engineering and Computing, University of Zagreb, Zagreb, Croatia. The chamber con-
tained a digital thermometer to measure temperature to determine if there was a thermal
effect of the radiation (Figure 1). The average temperature inside the chamber during se-
men exposure was 19.1 °C (range 18.7–19.5 °C). Control samples (unexposed samples)
were placed in a metal container (imitation for GTEM-chamber) and kept under the same
conditions (temperature and time) as the experimental groups but without exposure to
RF-EMR. Experimental samples (exposed) and control samples (unexposed) were as-
sessed after exposure/sham-exposure of 2 h. In addition to the chamber, an HP 8657A
signal generator and an RFGA0101-05 linear amplifier were used to achieve electromag-
netic field strength. The amplifier was connected to a computer and the desired frequency
was set using the SynthNV program (Windfreak Technologies, LLC., New Port Richey,
FL, USA). The GTEM-chamber is a transmission line that is based on TEM-chambers
where the leer G indicates that the GTEM chamber works in the GHz range up to 18
GHz. The GTEM-chamber is adjustable in the pyramidal part of the TEM-chamber with
an impedance of 50 Ω. After exposure, the control and experimental semen samples were
evaluated for spermatozoa motility.
Figure 1. Gigaher transverse electromagnetic (GTEM) chamber. View of the GTEM chamber in
which the boar semen samples were placed together with a digital thermometer.
2.4. Computer-Assisted Sperm Analysis
Spermatozoa motility was determined using a computer-assisted sperm analysis
(CASA) device (Integrated Visual Optical System, Version 12; Hamilton Thorne Research,
Beverly, MA, USA) located at the Clinic of Obstetrics and Reproduction, Faculty of Veter-
inary Medicine, University of Zagreb, Zagreb, Croatia. After the exposure procedure of
the experimental samples, diluted experimental and control semen samples (5 µL each)
were applied to a 20 µm deep Leja-chamber (Leja Products B.V., Nieuw Vennep, The Neth-
erlands) and placed on a heated spermotherm (Minitub, Tiefenbach, Germany). After the
cessation of passive spermatozoa movement, imaging was performed on all eight fields of
the chamber. The program was set to analyze 45 frames obtained per field at a frame rate
of 60 Hz. The analysis determined spermatozoa motility (%) and progressive motility (%).
Figure 1. Gigahertz transverse electromagnetic (GTEM) chamber. View of the GTEM chamber in
which the boar semen samples were placed together with a digital thermometer.
2.4. Computer-Assisted Sperm Analysis
Spermatozoa motility was determined using a computer-assisted sperm analysis
(CASA) device (Integrated Visual Optical System, Version 12; Hamilton Thorne Research,
Beverly, MA, USA) located at the Clinic of Obstetrics and Reproduction, Faculty of Veteri-
nary Medicine, University of Zagreb, Zagreb, Croatia. After the exposure procedure of the
experimental samples, diluted experimental and control semen samples (5
µ
L each) were ap-
plied to a 20
µ
m deep Leja-chamber (Leja Products B.V., Nieuw Vennep,
The Netherlands
)
and placed on a heated spermotherm (Minitub, Tiefenbach, Germany). After the cessation
of passive spermatozoa movement, imaging was performed on all eight fields of the cham-
ber. The program was set to analyze 45 frames obtained per field at a frame rate of 60 Hz.
The analysis determined spermatozoa motility (%) and progressive motility (%).
2.5. Preparation and Staining of Semen Smears
The control and experimental semen samples were then used to make a smear on a
glass slide. The semen smears were then stained with the Spermac set of reagents (Minitube,
Tiefenbach, Germany) for diagnostic staining of spermatozoa, which is generally used to
visualize the head, acrosome, equatorial region, central part and tail of spermatozoa. The
Spermac method in brief: a thin smear was made on a clean slide, then placed in fixative for
5 min. After fixation, the slides were placed on a heating plate at 37
◦
C for 15 min. Then the
slides were washed with distilled water and staining followed by first immersing the smear
in 50 mL of red liquid (Spermac “A”) for 1 min, followed by rinsing again with distilled
Animals 2024,14, 828 5 of 13
water; afterwards, the smear was immersed in 50 mL of pale green liquid (Spermac “B”)
for 1 min, after which the smear was rinsed with distilled water and the last smear was
immersed in 50 mL of dark green liquid (Spermac “C”) for 1 min, with a final rinse. After
staining, the final drying of the preparation followed on a heating plate at a temperature
of 37 ◦C.
2.6. Morphometric Analysis of Spermatozoa
In total, 24 stained semen smears were analyzed, with approximately 70 spermatozoa
measured for each smear (n= 1691). The spermatozoa morphometric analysis was per-
formed using the SFORM program for image processing and analysis (VAMSTEC, Zagreb,
Croatia). Only spermatozoa heads that did not overlap with those of other spermatozoa and
non-banded tails were measured and analyzed. The borders of the head, midpiece and tail
of the spermatozoa were marked automatically using the marking option of SFORM (first
the head, then the midpiece and finally the tail) with manual correction using a computer
mouse and then the calculated data were printed in the program table [
27
]. The area (
µ
m
2
),
outline (
µ
m), minimum radius (
µ
m), maximum radius (
µ
m), length (
µ
m) and width (
µ
m)
were the primary morphometric parameters (size parameters) calculated for the spermato-
zoa head and midpiece, while for spermatozoa tail, only length was calculated. Different
ratios of morphometric parameters were also calculated, such as the total length of the
spermatozoa, which is the sum of the head length and the tail length, then head length/total
length, head length/tail length, tail length/total length, head outline/total length, head
area/total length and head length
×
head width/total length. Some primary morphomet-
ric parameters (head size morphometric parameters) were used to calculate head shape
morphometric parameters using the following formulas: ellipticity (length/width), ru-
gosity (4
π×
area/outline2), elongation
(length −width)/(length + width)
and regularity
(π×length ×width/4 ×area).
2.7. Statistical Data Processing
A statistical data analysis was performed using the SAS 9.4 software package (Statisti-
cal Analysis Software 2002–2012 by the SAS Institute Inc., Cary, NC, USA). A descriptive
data analysis was performed using MEANS and FREQ procedures. The dependent param-
eters between groups were analyzed via a multivariate analysis of variance (MANOVA)
based on Wilks’ lambda criterion using the GLM procedure. The results are expressed
as least squares means (LSM) and 95% confidence intervals. To compare mean values,
the TukeyKramer method of multiple comparisons was used at the level of statistical
significance p< 0.05.
Multivariate clustering analyses (CLUSTERS) of data were performed through several
steps to obtain spermatozoa subpopulations based on the data of the main morphome-
tric parameters of the spermatozoa head and tail. The first step was the analysis of the
main components (PROC FACTOR) to obtain the characteristic values (eigenvalues) of
the morphometric parameters using the Kaiser criterion (
λ≥
1) to determine the number
of main components. The number of clusters in the K-means cluster analysis was deter-
mined using the HPCLUS procedure, which selects the best k value (number of clusters
or subpopulations) using the aligned box criterion value (Figure 2). The third step was to
group the data using non-hierarchical analysis (K-means method and Euclidean distance)
of the most important parameters for each component from the previous analysis using the
FASTCLUS procedure. In order to better interpret the data of the obtained spermatozoa
subpopulations, stepwise discrimination analysis (PROC STEPDISC) and testing of atypical
values (PROC FASTCLUS) were performed. Testing for differences in the distribution of
spermatozoa subpopulations between the control and experimental semen sample groups
was done using Chi-square and Mantel-Haenszel Chi-square tests (PROC FREQ).
Animals 2024,14, 828 6 of 13
Animals 2024, 14, x FOR PEER REVIEW 6 of 14
the data using non-hierarchical analysis (K-means method and Euclidean distance) of the
most important parameters for each component from the previous analysis using the
FASTCLUS procedure. In order to beer interpret the data of the obtained spermatozoa
subpopulations, stepwise discrimination analysis (PROC STEPDISC) and testing of atyp-
ical values (PROC FASTCLUS) were performed. Testing for differences in the distribution
of spermatozoa subpopulations between the control and experimental semen sample
groups was done using Chi-square and Mantel-Haenszel Chi-square tests (PROC FREQ).
Figure 2. Two subpopulations of boar spermatozoa. The figure shows that two optimal subpopula-
tions were obtained using the values of the equalized box criterion.
3. Results
3.1. Overall Semen Variables
Standard evaluation of ejaculate revealed that the semen samples correspond to the
criteria valid for boars (minimum motility > 60%) and in this respect the conditions were
met since average spermatozoa motility was about 80%. The mean volume (±SD) of the
boar semen sample was 354.5 ± 126.3 mL. Sperm concentration in mL of semen (±SD) was
358.7 ± 169.4 million. The total sperm concentration (±SD) was 113.5 ± 34.6 billion.
3.2. Individual Morphometric Parameters of the Spermatozoa Head and Tail
Morphometric analysis was performed on a total of 1691 spermatozoa, of which 839
spermatozoa (49.6%) in the control sample and 852 (50.4%) in the experimental group.
Statistical analysis of individual spermatozoa morphometric parameters between the ex-
perimental group exposed to RF-EMR at a frequency of 2500 MHz and the control group
(Tables 1–3) revealed no statistically significant differences. The only morphometric pa-
rameter whose value was close to statistical significance was the midpiece convex (p =
0.06). Sperm motility determined by CASA between the experimental and control groups
was not statistically significantly different, although motility was reduced in the experi-
mental group (74.7%) as compared to the control group (85.7%). Progressive sperm motil-
ity was statistically significantly reduced (p < 0.001) in the experimental group (35.0%)
compared to the control group (60.1%).
Figure 2. Two subpopulations of boar spermatozoa. The figure shows that two optimal subpopula-
tions were obtained using the values of the equalized box criterion.
3. Results
3.1. Overall Semen Variables
Standard evaluation of ejaculate revealed that the semen samples correspond to the
criteria valid for boars (minimum motility > 60%) and in this respect the conditions were
met since average spermatozoa motility was about 80%. The mean volume (
±
SD) of the
boar semen sample was 354.5
±
126.3 mL. Sperm concentration in mL of semen (
±
SD) was
358.7 ±169.4 million. The total sperm concentration (±SD) was 113.5 ±34.6 billion.
3.2. Individual Morphometric Parameters of the Spermatozoa Head and Tail
Morphometric analysis was performed on a total of 1691 spermatozoa, of which
839 spermatozoa
(49.6%) in the control sample and 852 (50.4%) in the experimental group.
Statistical analysis of individual spermatozoa morphometric parameters between the ex-
perimental group exposed to RF-EMR at a frequency of 2500 MHz and the control group
(Tables 1–3) revealed no statistically significant differences. The only morphometric param-
eter whose value was close to statistical significance was the midpiece convex (p= 0.06).
Sperm motility determined by CASA between the experimental and control groups was
not statistically significantly different, although motility was reduced in the experimental
group (74.7%) as compared to the control group (85.7%). Progressive sperm motility was
statistically significantly reduced (p< 0.001) in the experimental group (35.0%) compared
to the control group (60.1%).
Table 1. Boar spermatozoa morphometric head parameters in the control and experimental semen
sample groups.
Spermatozoa Morphometric Parameters Control Group Experimental Group
Mean 95% Confidence Interval Mean 95% Confidence Interval pValue
Morphometric
parameters of
the head
Area (µm2)46.79 46.47–47.11 46.75 46.43–47.07 0.84
Outline (µm) 27.31 27.21–27.41 27.26 27.16–27.36 0.49
Minimal radius (µm) 2.48 2.47–2.49 2.48 2.47–2.49 0.81
Maximal radius (µm) 5.34 5.32–5.37 5.34 5.32–5.37 0.93
Convex (µm) 47.39 47.06–47.72 47.43 47.11–47.76 0.85
Length (µm) 10.31 10.26–10.36 10.3 10.26–10.35 0.86
Bredth (µm) 5.45 5.42–5.47 5.45 5.42–5.47 0.89
Ellipticity 1.9 1.89–1.91 1.89 1.89–1.91 0.78
Rugosity 0.79 0.785–0.789 0.79 0.787–0.791 0.27
Elongation 0.31 0.305–0.311 0.31 0.30–0.31 0.8
Regularity 0.94 0.941–0.944 0.94 0.94–0.95 0.26
Ellipticity = length/bredth; Rugosity = (4
π×
area/outline2); Elongation = [(lenght
−
bredth)/(length + bredth)];
Regularity = (π×length ×bredth/4 ×area).
Animals 2024,14, 828 7 of 13
Table 2. Boar spermatozoa morphometric midpiece and tail parameters in the control and experimen-
tal semen sample groups.
Spermatozoa Morphometric Parameters Control Group Experimental Group
Mean
95% Confidence Interval Mean 95% Confidence Interval pValue
Parameters of
morphometric
characteristics of
the midpiece and
the tail
Midpiece area (µm2)19.42 19.29–19.55 19.48 19.35–19.60 0.52
Midpiece outline (µm) 30.44 30.32–30.56 30.35 30.23–30.46 0.27
Midpiece min. radius (µm) 0.39 0.37–0.39 0.39 0.38–0.40 0.66
Midpiece max. radius (µm) 6.78 6.75–6.81 6.76 6.73–6.78 0.25
Midpiece convex (µm) 24.58 24.32–24.84 24.92 24.67–25.18 0.06
Midpiece length (µm) 13.25 13.19–13.30 13.2 13.15–13.26 0.27
Midpiece width (µm) 2.05 2.03–2.08 2.08 2.05–2.11 0.21
Tail length (µm) 34.05 33.84–34.26 33.92 33.71–34.12 0.37
Table 3. Boar spermatozoa morphometric head and tail parameters ratios in the control and experi-
mental semen sample groups.
Spermatozoa Morphometric Parameters Control Group Experimental Group
Mean
95% Confidence Interval Mean 95% Confidence Interval pValue
Different ratios of
morphometric
parameters
Total length * 44.36 44.13–44.59 44.22 43.99–44.45 0.4
Head length/Total length 0.23 0.232–0.234 0.23 0.23–0.24 0.52
Head length/Tail length 0.31 0.30–0.31 0.31 0.30–0.31 0.53
Tail length/Total length 0.77 0.766–0.768 0.77 0.765–0.767 0.52
Head outline/Total length 0.62 0.615–0.621 0.62 0.616–0.621 0.78
Head area/Total length 1.06 1.05–1.06 1.06 1.05–1.07 0.75
Head length and width/
Total length * 1.27 1.26–1.28 1.27 1.27–1.28 0.53
* Total length = head length + tail length.
3.3. Spermatozoa Subpopulations Based on Morphometric Parameters of Spermatozoa Head
and Tail
Analysing the main components before grouping, four components (factor 1, 2, 3 and
4) with a characteristic value (
λ≥
1) were retained. All four components in total explained
84.6% of the variance of the morphometric parameters of the spermatozoa head, midpiece
and tail (Table 4).
Table 4. Eigenvalues of boar spermatozoa morphometric head and tail parameters in the analysis
of the main components. Four components (factor 1, 2, 3, 4) with a characteristic root
λ≥
1 were
retained—Kaiser’s criterion.
Spermatozoa Indicators Factor 1 Factor 2 Factor 3 Factor 4
Head length 0.93 *
Head width 0.97 *
Head area 0.62
Head outline 0.83
Ellipticity 0.72
Rugosity −0.69
Elongation 0.72
Regularity 0.89 *
Midpiece length 0.70
Midpiece width 0.39
Midpiece area 0.65
Midpiece outline 0.72 *
Tail length 0.39
Characteristic root (λ)
and explained variance (%) 4.78 (36.8) 3.66 (28.2) 1.53 (11.8) 1.01 (7.8)
* The most important parameters for each factor.
Animals 2024,14, 828 8 of 13
Using Table 4, the most important parameters were selected from each component
(head length, head width, midpiece outline and regularity of the spermatozoa head). The
final number of subpopulations was obtained using the value of the equalised box criterion.
This analysis determined that the two subpopulations are the most optimal because the
value of the equalised box criterion was the highest (Figure 2).
The grouping analysis revealed well-defined differences between head length, width
and regularity and the midpiece outline in the two spermatozoa subpopulations (S1
subpopulation—smaller length and larger head width, more regular head shape and
smaller midpiece outline, and S2 subpopulation—longer length and smaller head width,
more elongated head shape and larger midpiece outline) (Table 5).
Table 5. Subpopulations of boar spermatozoa (S1 and S2) obtained using the analysis of grouping of
spermatozoa morphometric head and midpiece parameters.
Spermatozoa Subpopulation
Spermatozoa Morphometric Head and Midpiece Parameters
Mean ±SD S1 S2
n(%) 942 (55.71) 749 (44.29)
Head length (µm) 10.16 ±0.65 10.77 ±0.68
Head width (µm) 5.44 ±0.38 5.45 ±0.35
Midpiece outline (µm) 29.67 ±1.17 32.66 ±1.09
Regularity 0.943 ±0.02 0.941 ±0.02
S1—Spermatozoa of smaller length and larger head width, more regular head shape and smaller midpiece
outline; S2—Spermatozoa of longer length and smaller head width, more elongated head shape and larger
midpiece outline.
Statistical analysis of the obtained subpopulations between the control and experi-
mental groups of semen samples revealed that the experimental semen samples group
had a higher percentage of spermatozoa of the S1 subpopulation (68.2% vs. 64.4%) and
a lower percentage of the S2 subpopulation (31.8% vs. 35.6%) which is close to statistical
significance (p= 0.09) (Figure 3).
Animals 2024, 14, x FOR PEER REVIEW 9 of 14
S1—Spermatozoa of smaller length and larger head width, more regular head shape and smaller
midpiece outline; S2—Spermatozoa of longer length and smaller head width, more elongated head
shape and larger midpiece outline.
Statistical analysis of the obtained subpopulations between the control and experi-
mental groups of semen samples revealed that the experimental semen samples group
had a higher percentage of spermatozoa of the S1 subpopulation (68.2% vs. 64.4%) and a
lower percentage of the S2 subpopulation (31.8% vs. 35.6%) which is close to statistical
significance (p = 0.09) (Figure 3).
Figure 3. Proportion of spermatozoa subpopulations in control and experimental semen samples
groups.
4. Discussion
This study showed that the exposure of semen of breeding boars in vitro to RF-EMR
at a frequency of 2500 MHz and an electric field strength of 10 V/m for a duration of 2 h
did not cause changes of spermatozoa individual morphometric parameters and sperm
motility, but it decreased progressive sperm motility.
Although it is known that RF-EMR has a harmful effect on the male reproductive
system, by reducing the number of Leydig cells, motility, and number of spermatozoa,
and altering spermatozoa morphology in humans and animals [9,15,28–32], the results
presented here do not support this. This could be due to the application of different study
designs (protocols) during the experiment, different species and ages of animals, and dif-
ferent analysis methods. Furthermore, to the extent of our knowledge, the effect of RF-
EMR on spermatozoa morphometric parameters has not previously been investigated in
humans or other species. In addition, spermatozoa morphometry in boars is performed
using different software and on semen smears stained with different methods.
Wysokińska et al. [33] performed spermatozoa morphometric analysis on spermatozoa
samples collected from 35 boars of the Polish Landrace breed at the age of 7 to 8 months
stained with the Bydgoszka method using a computer image analysis package (Screen
Measurement v. 4.1, Laboratory Imaging S.r.o. LIM, Prague, Czech Republic). Their re-
search showed a lower mean length, width, area and outline of the head and a higher
mean value for the total length and tail length compared to the results for the control
group in the present study. This study used the computer program SFORM (VAMSTEC,
Zagreb, Croatia) for the morphometric analysis, and there is no information about its use
in the morphometric analysis of boar spermatozoa in the literature, and therefore, it is
possible that the difference in the stated values was result of the differences in the pro-
grams for morphometric analysis or differences in staining methods, age and breed of
Figure 3. Proportion of spermatozoa subpopulations in control and experimental semen
samples groups.
4. Discussion
This study showed that the exposure of semen of breeding boars
in vitro
to RF-EMR
at a frequency of 2500 MHz and an electric field strength of 10 V/m for a duration of 2 h
did not cause changes of spermatozoa individual morphometric parameters and sperm
motility, but it decreased progressive sperm motility.
Animals 2024,14, 828 9 of 13
Although it is known that RF-EMR has a harmful effect on the male reproductive
system, by reducing the number of Leydig cells, motility, and number of spermatozoa,
and altering spermatozoa morphology in humans and animals [
9
,
15
,
28
–
32
], the results
presented here do not support this. This could be due to the application of different study
designs (protocols) during the experiment, different species and ages of animals, and
different analysis methods. Furthermore, to the extent of our knowledge, the effect of
RF-EMR on spermatozoa morphometric parameters has not previously been investigated
in humans or other species. In addition, spermatozoa morphometry in boars is performed
using different software and on semen smears stained with different methods. Wysoki´nska
et al. [
33
] performed spermatozoa morphometric analysis on spermatozoa samples collected
from 35 boars of the Polish Landrace breed at the age of 7 to 8 months stained with the
Bydgoszka method using a computer image analysis package (Screen Measurement v.
4.1, Laboratory Imaging S.r.o. LIM, Prague, Czech Republic). Their research showed a
lower mean length, width, area and outline of the head and a higher mean value for the
total length and tail length compared to the results for the control group in the present
study. This study used the computer program SFORM (VAMSTEC, Zagreb, Croatia) for
the morphometric analysis, and there is no information about its use in the morphometric
analysis of boar spermatozoa in the literature, and therefore, it is possible that the difference
in the stated values was result of the differences in the programs for morphometric analysis
or differences in staining methods, age and breed of boars. Górski et al. [
34
] showed a
lower mean value for the length, width, area and outline of the spermatozoa head and
a higher mean value for the spermatozoa total length and the tail length as compared to
the control group of this study. Those authors performed a spermatozoa morphometric
analysis on semen samples collected from 12 boars of the Duroc breed, stained using the
method according to Kondracki et al. [
24
], and using the computerised image analysis
system Screen Measurement v. 4.1. for morphometric measurements. One reason for the
differences between the results of our control group and that study could be due to the
different staining methods [26], animal breed [23] or the analysis method used.
Other aspects of spermatozoa physiology and morphology may also need to be con-
sidered, as they may affect their ability to actively move through the female reproductive
system. In many species, the first barrier is the cervical mucus, which allows only progres-
sively motile spermatozoa with normal morphology to pass into the uterus and through
which they progressively move (with the help of myometrial contractions) to the fallopian
tube, where fertilization occurs [
35
]. Therefore, spermatozoa motility is crucial. This study
showed a significant reduction in progressive spermatozoa motility after exposure of the
semen of the boars
in vitro
to RF-EMR at a frequency of 2500 MHz. Mailankot et al. [
10
]
reported similar results, showing that exposing rats to RF-EMR 1 h a day for 28 days at
a frequency of 900 and 1800 MHz originating from mobile devices caused a drop in the
number of motile spermatozoa, and reduced progressive sperm motility. Oni et al. [
36
] and
Gorpinchenko et al. [
37
] investigated the effect of electromagnetic radiation at the frequency
of mobile telecommunications (900/1800 MHz) and laptops (2.45 GHz) on
in vitro
samples
of human semen, and found that exposure to these frequencies also reduced the number
of motile and progressively motile spermatozoa. On the other hand, some studies have
reported no changes in spermatozoa motility after rats had been long-term exposed to
RF-EMR frequency of 2.4 GHz [
17
]. A possible mechanism that leads to a decrease in sper-
matozoa motility after exposure of semen to RF-EMR is a lowered mitochondrial potential
or oxidative stress, and consequently impaired spermatozoa vitality [
28
,
38
]. Namely, some
studies on the impact of RF-EMR on spermatozoa, though not on boars, have indicated
that RF-EMR at the frequency of mobile telephony can cause the formation of reactive
oxygen species (ROS) and thus oxidative stress. It is also known that in aerobic organ-
isms, a balance between antioxidant processes and reactive compounds formed requires
an oxidative-reduction balance, because otherwise, during oxidative stress, an excess of
ROS leads to the damage of numerous molecules. Kumar et al. [
39
] and Meena et al. [
40
]
showed that exposing rats to RF-EMR frequencies of 2.45 GHz and 10 GHz for 2 h a day
Animals 2024,14, 828 10 of 13
for 45 days led to cell damage mediated by oxidative stress, i.e., caused an increase in the
concentration of ROS, an increase in the percentage of spermatozoa apoptosis in testicles,
and DNA damage.
This study has shown that RF-EMR at a frequency of 2500 MHz could have a negative
effect on the success of egg fertilisation. We base the above assumption not only on the
obtained results that RF-EMR reduces the number of progressively motile spermatozoa,
but also on the obtained percentages of the spermatozoa subpopulation after exposure to
RF-EMR. The obtained proportions of spermatozoa subpopulations using PC and cluster
analysis according to morphometric head and tail parameters showed that the experimental
group of semen had a higher percentage of the less desirable subpopulation, characterised
by spermatozoa of smaller length and larger head width, more regular head shape and
smaller midpiece outline as compared to the more desirable spermatozoa characterised by
longer length and larger head width, more elongated shape of the head and larger midpiece
outline. It has been empirically proven in different species that spermatozoa function is
related to its morphometry, which includes the area of the head, midpiece and tail [
41
,
42
].
Ramon et al. [
43
] stated that deer ejaculate containing a high percentage of spermatozoa
with fast and linear motility have small and elongated heads and achieve higher fertility.
An elongated spermatozoa head can have an important function in that such sperm will
be hydrodynamically more efficient due to less resistance in forward movement, which
can affect the fertilising ability of sperm [
20
,
44
]. However, Barquero et al. [
23
] reported
that boars with a larger litter size had significantly less elongated spermatozoa, and the
mortality of piglets was greater in these males. Evolutionary biology is still debating which
of the two spermatozoa components, head characteristics or midpiece traits of the sperma-
tozoa, is more important in spermatozoa competition during the egg fertilisation process.
Namely, the increase in the midpiece of the spermatozoa increases its energy due to the
increased area housing the mitochondria [
45
], as more energy is needed for faster sperm.
FIRMAN and SIMMONS [
46
] reported that midpiece size is a predictor of swimming speed
of Mus musculus domesticus spermatozoa. It is known that RF-EMR can cause cell/sperm
apoptosis, and mitochondria are the main initiators of apoptosis [
47
]. In addition, RF-EMR
promotes increased mitochondrial ROS production and expression of mitochondrial apop-
totic markers [
48
] with decreased mitochondrial membrane potential [
47
,
49
]. Although
the exact mechanism of apoptotic changes in spermatozoa, and in somatic cells, is still
unknown, one of the signs of apoptosis was described as typical cell shrinkage [
50
]. The
decrease in the mitochondria outline with reduced progressive motility of spermatozoa of
the experimental group in this study could indicate the initiation of spermatozoa apoptosis.
Furthermore, exposure of cells to RF-EMR if the intensity of the fields increases beyond the
threshold, causes electroporation, during which water pores are created in the membrane,
disrupting the ion balance and leading to water ingress in the cell [
18
], which is likely to
cause a change in spermatozoa shape in a higher proportion in the experimental group in
this study. If this was the case, then the increased ROS production generated in these highly
vulnerable cells could reasonably be expected to impose an oxidative stress environment
upon the aforementioned the sperm population. Given that there is no literature on the
effects of RF-EMR on the proportion of boars or other species spermatozoa subpopulations
obtained based on morphometric parameters, the results of the present study cannot be
compared. There are scarce studies on the proportions of boar spermatozoa subpopulations
using PC and cluster analysis according to morphometric parameters, therefore it is not
possible to compare the obtained results. However, Barquero et al. [
23
] investigated the
spermatozoa morphometry of boars without exposure to RF-EMR, and found four subpop-
ulations of spermatozoa using PC and cluster analysis according only to morphometric
head parameters: subpopulation 1 with lower values for ellipticity and the widest heads,
sub-population 2 with the highest values for head area and perimeter, subpopulation
3 with
shorter length, with a smaller area and head perimeter values, and subpopulation
4 with the highest values for head length, ellipticity, and elongation. We obtained two
subpopulations related to not only morphometric head but also tail parameters, giving a
Animals 2024,14, 828 11 of 13
more complete picture of spermatozoa morphology, which is related to their function, i.e.,
fertilisation ability.
5. Conclusions
The effect of RF-EMR at 2500 MHz on
in vitro
exposed breeding boar semen for two
hours was seen in the decreased progressive spermatozoa motility and proportion of
the spermatozoa subpopulation with a more elongated head shape and larger midpiece
outline. Further research on the effects of RF-EMR on different animal species and breeds,
especially domestic animals, is important both for the quality of semen and fertilization
and for production and breeding goals. The observed results could also be crucial for
comparison with human reproductive medicine and potential adverse effects during the
specific technological process of semen processing on breeding pigs farms.
Author Contributions: Conceptualization, I.Ž.Ž., S.V., I.B., K.M. and M.V.; methodology, M.L., I.B.;
software, S.V., N.P.M., M.K. and I.Ž.Ž.; validation, M.L.; investigation, I.Ž.Ž., S.V., I.B., K.S., M.L., I.F.,
S. ˇ
C., N.Ž. and M.V.; writing—original draft preparation, I.Ž.Ž., I.B., M.K. and K.S.; writing—review
and editing, I.Ž.Ž., M.V., M.S., S.M.T., M.O. and N.P.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: This research was approved by the Ethics Committee in
Veterinary Medicine, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia (record
No.: 640-01/21-02/03; file No.: 251-61-01/139-21-39), approval date: 19 February 2020.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on reasonable request
from the corresponding author. The data are not publicly available due to planned research in
the future.
Acknowledgments: The authors would like to thank Sead Džubur, Vams Tec d.o.o., Zagreb, Croatia
for his help and assistance with SFORM Software.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1.
Bandara, P.; Carpenter, D.O. Planetary Electromagnetic Pollution: It Is Time to Assess Its Impact. Lancet Planet. Health 2018,
2, e512–e514. [CrossRef]
2. Kesari, K.K.; Agarwal, A.; Henkel, R. Radiations and Male Fertility. Reprod. Biol. Endocrinol. 2018,16, 118. [CrossRef]
3.
Yadav, H.; Rai, U.; Singh, R. Radiofrequency Radiation: A Possible Threat to Male Fertility. Reprod. Toxicol. 2021,100, 90–100.
[CrossRef]
4.
Prli´c, I.; Šiško, J.; Varnai, V.M.; Paveli´c, L.; Macan, J.; Kobeš´cak, S.; Hajdinjak, M.; Jurdana, M.; Cerovac, Z.; Zauner, B.; et al.
Wi-Fi Technology and Human Health Impact: A Brief Review of Current Knowledge. Arch. Ind. Hyg. Toxicol. 2022,73, 94–106.
[CrossRef]
5.
La Vignera, S.; Condorelli, R.A.; Vicari, E.; D’Agata, R.; Calogero, A.E. Effects of the Exposure to Mobile Phones on Male
Reproduction: A Review of the Literature. J. Androl. 2012,33, 350–356. [CrossRef]
6.
Liu, K.; Li, Y.; Zhang, G.; Liu, J.; Cao, J.; Ao, L.; Zhang, S. Association between Mobile Phone Use and Semen Quality: A Systemic
Review and Meta-Analysis. Andrology 2014,2, 491–501. [CrossRef] [PubMed]
7.
Kaur, P.; Rai, U.; Singh, R. Genotoxic Risks to Male Reproductive Health from Radiofrequency Radiation. Cells 2023,12, 594.
[CrossRef] [PubMed]
8.
Erogul, O.; Oztas, E.; Yildirim, I.; Kir, T.; Aydur, E.; Komesli, G.; Irkilata, H.C.; Irmak, M.K.; Peker, A.F. Effects of Electromagnetic
Radiation from a Cellular Phone on Human Sperm Motility: An In Vitro Study. Arch. Med. Res. 2006,37, 840–843. [CrossRef]
9.
Agarwal, A.; Singh, A.; Hamada, A.; Kesari, K. Cell Phones and Male Infertility: A Review of Recent Innovations in Technology
and Consequences. Int. Braz. J. Urol. 2011,37, 432–454. [CrossRef] [PubMed]
10.
Mailankot, M.; Kunnath, A.P.; Jayalekshmi, H.; Koduru, B.; Valsalan, R. Radio Frequency Electromagnetic Radiation (RF-EMR)
from GSM (0.9/1.8 GHZ) Mobile Phones Induces Oxidative Stress and Reduces Sperm Motility in Rats. Clinics 2009,64, 561–565.
[CrossRef] [PubMed]
11.
Kesari, K.K.; Behari, J. Effects of Microwave at 2.45 GHz Radiations on Reproductive System of Male Rats. Toxicol. Environ. Chem.
2010,92, 1135–1147. [CrossRef]
Animals 2024,14, 828 12 of 13
12.
Gutschi, T.; Mohamad Al-Ali, B.; Shamloul, R.; Pummer, K.; Trummer, H. Impact of Cell Phone Use on Men’s Semen Parameters.
Andrologia 2011,43, 312–316. [CrossRef]
13.
Rago, R.; Salacone, P.; Caponecchia, L.; Sebastianelli, A.; Marcucci, I.; Calogero, A.E.; Condorelli, R.; Vicari, E.; Morgia, G.; Favilla,
V.; et al. The Semen Quality of the Mobile Phone Users. J. Endocrinol. Investig. 2013,36, 970–974. [CrossRef]
14.
Yildirim, M.E.; Kaynar, M.; Badem, H.; Cavis, M.; Karatas, O.F.; Cimentepe, E. What Is Harmful for Male Fertility: Cell Phone or
the Wireless Internet? Kaohsiung J. Med. Sci. 2015,31, 480–484. [CrossRef]
15.
Agarwal, A.; Deepinder, F.; Sharma, R.K.; Ranga, G.; Li, J. Effect of Cell Phone Usage on Semen Analysis in Men Attending
Infertility Clinic: An Observational Study. Fertil. Steril. 2008,89, 124–128. [CrossRef]
16.
Avendaño, C.; Mata, A.; Sanchez Sarmiento, C.A.; Doncel, G.F. Use of Laptop Computers Connected to Internet through Wi-Fi
Decreases Human Sperm Motility and Increases Sperm DNA Fragmentation. Fertil. Steril. 2012,97, 39–45.e2. [CrossRef]
17.
Dasdag, S.; Ta¸s, M.; Akdag, M.Z.; Yegin, K. Effect of Long-Term Exposure of 2.4 GHz Radiofrequency Radiation Emitted from
Wi-Fi Equipment on Testes Functions. Electromagn. Biol. Med. 2015,34, 37–42. [CrossRef] [PubMed]
18.
Koohestanidehaghi, Y.; Khalili, M.A.; Fesahat, F.; Seify, M.; Mangoli, E.; Kalantar, S.M.; Annarita Nottola, S.; Macchiarelli, G.;
Grazia Palmerini, M. Detrimental Effects of Radiofrequency Electromagnetic Waves Emitted by Mobile Phones on Morphokinetics,
Oxidative Stress, and Apoptosis in Mouse Preimplantation Embryos. Environ. Pollut. 2023,336, 122411. [CrossRef] [PubMed]
19.
Verstegen, J.; Iguer-Ouada, M.; Onclin, K. Computer Assisted Semen Analyzers in Andrology Research and Veterinary Practice.
Theriogenology 2002,57, 149–179. [CrossRef] [PubMed]
20.
Yániz, J.L.; Soler, C.; Santolaria, P. Computer Assisted Sperm Morphometry in Mammals: A Review. Anim. Reprod. Sci. 2015,
156, 1–12. [CrossRef]
21.
Wysoki´nska, A.; Wójcik, E.; Chłopik, A. Evaluation of the Morphometry of Sperm from the Epididymides of Dogs Using Different
Staining Methods. Animals 2021,11, 227. [CrossRef] [PubMed]
22.
Peña, F.J.; Saravia, F.; García-Herreros, M.; Núñezmartínez, I.; Tapia, J.A.; Johannisson, A.; Wallgren, M.; Rodríguez-Martínez,
H. Identification of Sperm Morphometric Subpopulations in Two Different Portions of the Boar Ejaculate and Its Relation to
Postthaw Quality. J. Androl. 2005,26, 716–723. [CrossRef] [PubMed]
23.
Barquero, V.; Roldan, E.R.S.; Soler, C.; Yániz, J.L.; Camacho, M.; Valverde, A. Predictive Capacity of Boar Sperm Morphometry
and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination. Animals 2021,11, 920. [CrossRef]
[PubMed]
24.
Kondracki, S.; Bonaszewska, D.; Mielnicka, C. The Effect of Age on the Morphometric Sperm Traits of Domestic Pigs (Sus Scrofa
Domestica). Cell. Mol. Biol. Lett. 2005,10, 3–13. [PubMed]
25.
Banaszewska, D.; Andraszek, K. Assessment of the Morphometry of Heads of Normal Sperm and Sperm with the Dag Defect in
the Semen of Duroc Boars. J. Vet. Res. 2021,65, 239–244. [CrossRef] [PubMed]
26.
Szablicka, D.; Wysoki´nska, A.; Pawlak, A.; Roman, K. Morphometry of Boar Spermatozoa in Semen Stored at 17
◦
C—The
Influence of the Staining Technique. Animals 2022,12, 1888. [CrossRef] [PubMed]
27.
Žaja, I.Ž.; Vince, S.; Milinkovi´c-Tur, S.; Milas, N.P.; Samardžija, M.; Valpoti´c, H.; Berta, V.; Vili´c, M.; Raki´c, K. Exogenous
Melatonin Influences Distribution of French Alpine Buck Spermatozoa in Morphometrically Distinct Subpopulations during the
Non-Breeding Season. Anim. Reprod. Sci. 2018,192, 154–163. [CrossRef]
28.
De Iuliis, G.N.; Newey, R.J.; King, B.V.; Aitken, R.J. Mobile Phone Radiation Induces Reactive Oxygen Species Production and
DNA Damage in Human Spermatozoa In Vitro. PLoS ONE 2009,4, e6446. [CrossRef]
29.
Saygin, M.; Caliskan, S.; Karahan, N.; Koyu, A.; Gumral, N.; Uguz, A. Testicular Apoptosis and Histopathological Changes
Induced by a 2.45 GHz Electromagnetic Field. Toxicol. Ind. Health 2011,27, 455–463. [CrossRef]
30.
Gye, M.C.; Park, C.J. Effect of Electromagnetic Field Exposure on the Reproductive System. Clin. Exp. Reprod. Med. 2012,39, 1–9.
[CrossRef]
31.
Al-Bayyari, N. The Effect of Cell Phone Usage on Semen Quality and Fertility among Jordanian Males. Middle East Fertil. Soc. J.
2017,22, 178–182. [CrossRef]
32.
Kıvrak, E.G.; Yurt, K.K.; Kaplan, A.A.; Alkan, I.; Altun, G. Effects of Electromagnetic Fields Exposure on the Antioxidant Defense
System. J. Microsc. Ultrastruct. 2017,5, 167–176. [CrossRef]
33.
Wysoki´nska, A.; Kondracki, S.; Banaszewska, D. Morphometrical Characteristics of Spermatozoa in Polish Landrace Boars with
Regard to the Number of Spermatozoa in an Ejaculate. Reprod. Biol. 2009,9, 271–282. [CrossRef] [PubMed]
34.
Górski, K.; Kondracki, S.; Wysoki´nska, A. Ejaculate Traits and Sperm Morphology Depending on Ejaculate Volume in Duroc
Boars. J. Vet. Res. 2017,61, 121–125. [CrossRef] [PubMed]
35.
Druart, X. Sperm Interaction with the Female Reproductive Tract. Reprod. Domest. Anim. 2012,47, 348–352. [CrossRef] [PubMed]
36.
Oni, O.; Amuda, D. Effects of radiofrequency radiation from wifi devices on human ejaculated semen. Int. J. Res. Rev. Appl. Sci.
2011,9, 292–294.
37.
Gorpinchenko, I.; Nikitin, O.; Banyra, O.; Shulyak, A. The Influence of Direct Mobile Phone Radiation on Sperm Quality. Cent.
Eur. J. Urol. 2014,67, 65–71. [CrossRef]
38.
Ghanbari, M.; Mortazavi, S.B.; Khavanin, A.; Khazaei, M. The Effects of Cell Phone Waves (900 MHz-GSM Band) on Sperm
Parameters and Total Antioxidant Capacity in Rats. Int. J. Fertil. Steril. 2013,7, 21–28.
39.
Kumar, S.; Kesari, K.K.; Behari, J. Influence of Microwave Exposure on Fertility of Male Rats. Fertil. Steril. 2011,95, 1500–1502.
[CrossRef]
Animals 2024,14, 828 13 of 13
40.
Meena, R.; Kumari, K.; Kumar, J.; Rajamani, P.; Verma, H.N.; Kesari, K.K. Therapeutic Approaches of Melatonin in Microwave
Radiations-Induced Oxidative Stress-Mediated Toxicity on Male Fertility Pattern of Wistar Rats. Electromagn. Biol. Med. 2014,
33, 81–91. [CrossRef]
41.
Immler, S.; Pryke, S.R.; Birkhead, T.R.; Griffith, S.C. Pronounced Within-Individual Plasticity in Sperm Morphometry Across
Social Environments. Evolution 2010,64, 1634–1643. [CrossRef] [PubMed]
42.
Shahani, S.K.; Revell, S.G.; Argo, C.G.; Murray, R.D. Mid-Piece Length of Spermatozoa in Different Cattle Breeds and Its
Relationship to Fertility. Pak. J. Biol. Sci. PJBS 2010,13, 802–808. [CrossRef] [PubMed]
43.
Ramón, M.; Soler, A.J.; Ortiz, J.A.; García-Alvarez, O.; Maroto-Morales, A.; Roldan, E.R.S.; Garde, J.J. Sperm Population Structure
and Male Fertility: An Intraspecific Study of Sperm Design and Velocity in Red Deer. Biol. Reprod. 2013,89, 110. [CrossRef]
[PubMed]
44.
Malo, A.F.; Garde, J.J.; Soler, A.J.; García, A.J.; Gomendio, M.; Roldan, E.R.S. Male Fertility in Natural Populations of Red Deer Is
Determined by Sperm Velocity and the Proportion of Normal Spermatozoa1. Biol. Reprod. 2005,72, 822–829. [CrossRef] [PubMed]
45.
Gomendio, M.; Roldan, E.R.S. Implications of Diversity in Sperm Size and Function for Sperm Competition and Fertility. Int. J.
Dev. Biol. 2008,52, 439–447. [CrossRef] [PubMed]
46.
Firman, R.C.; Simmons, L.W. Sperm Midpiece Length Predicts Sperm Swimming Velocity in House Mice. Biol. Lett. 2010,
6, 513–516. [CrossRef] [PubMed]
47.
Costa, J.; Braga, P.C.; Rebelo, I.; Oliveira, P.F.; Alves, M.G. Mitochondria Quality Control and Male Fertility. Biology 2023,12, 827.
[CrossRef] [PubMed]
48.
Liu, Q.; Si, T.; Xu, X.; Liang, F.; Wang, L.; Pan, S. Electromagnetic Radiation at 900 MHz Induces Sperm Apoptosis through Bcl-2,
Bax and Caspase-3 Signaling Pathways in Rats. Reprod. Health 2015,12, 65. [CrossRef]
49.
Houston, B.J.; Nixon, B.; King, B.V.; Iuliis, G.N.D.; Aitken, R.J. The Effects of Radiofrequency Electromagnetic Radiation on Sperm
Function. Reproduction 2016,152, R263–R276. [CrossRef]
50.
Talarczyk-Desole, J.; Kotwicka, M.; Jendraszak, M.; Pawelczyk, L.; Murawski, M.; J˛edrzejczak, P. Sperm Midpiece Apoptotic
Markers: Impact on Fertilizing Potential in in Vitro Fertilization and Intracytoplasmic Sperm Injection. Hum. Cell 2016,29, 67–75.
[CrossRef]
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