Sperm selection techniques in cattle: Microfilter device versus conventional methods

Abstract

Microfluidics and microfilter devices have been developed to mimic the characteristics of the female reproductive tract, minimizing the risk of sperm damage. This study aimed to compare the use of a microfilter device versus conventional methods for sperm selection used in in vitro fertilization (IVF). For selecting spermatozoa, the pooled samples were processed in a microfilter device, swim‐up and mini‐Percoll gradient. Kinematic and morphometric parameters, vitality and DNA damage were analysed before and after sperm selection. After selection, 10,000 motile spermatozoa per oocyte were used in IVF drops. Embryos were assessed at three (cleavage rate) and seven (blastocyst rate) days post‐IVF. Results of sperm kinematic parameters including average path velocity, velocity straight line, curvilinear velocity, linearity, lateral head displacement with the microfilter device were superior to density gradient (p < 0.05), but similar to swim‐up method. Likewise, sperm DNA damage was significantly reduced using the microfilter device and swim‐up method. Regarding the total sperm recovery rate post selection, results with the microfilter device (17.64%) and mini‐Percoll gradient (18.27%) were higher than with swim‐up method (6.52%). However, the cleavage and blastocyst rates were the lowest using the microfilter device. In conclusion, sperm selection using the microfilter device and swim‐up method can improve kinematic parameters, although the mini Percoll gradient was the most efficient method for embryo production.

Full text

ORIGINAL ARTICLE
Sperm selection techniques in cattle: Microfilter device versus
conventional methods
Jhorjhi Vega-Hidalgo | Misael Rodriguez | Deysi Dipaz-Berrocal |
Josselin Rivas | Carmen Huayhua | Edwin Mellisho
Centro de Investigaci
on en Tecnología de
Embriones (CIETE), Programa de
Mejoramiento Animal, Universidad Nacional
Agraria La Molina, Lima, Peru
Correspondence
Edwin Mellisho, Centro de Investigaci
on en
Tecnología de Embriones (CIETE), Programa de
Mejoramiento Animal
Universidad Nacional Agraria, La Molina
Avenue, 15024, Lima, Peru.
Email: emellisho@lamolina.edu.pe
Funding information
Fondo Nacional de Desarrollo Científico,
Tecnol
ogico y de Innovaci
on Tecnol
ogica,
Grant/Award Number: 178-2015
Abstract
Microfluidics and microfilter devices have been developed to mimic the characteris-
tics of the female reproductive tract, minimizing the risk of sperm damage. This study
aimed to compare the use of a microfilter device versus conventional methods for
sperm selection used in in vitro fertilization (IVF). For selecting spermatozoa, the
pooled samples were processed in a microfilter device, swim-up and mini-Percoll gra-
dient. Kinematic and morphometric parameters, vitality and DNA damage were ana-
lysed before and after sperm selection. After selection, 10,000 motile spermatozoa
per oocyte were used in IVF drops. Embryos were assessed at three (cleavage rate)
and seven (blastocyst rate) days post-IVF. Results of sperm kinematic parameters
including average path velocity, velocity straight line, curvilinear velocity, linearity,
lateral head displacement with the microfilter device were superior to density gradi-
ent (p< 0.05), but similar to swim-up method. Likewise, sperm DNA damage was sig-
nificantly reduced using the microfilter device and swim-up method. Regarding the
total sperm recovery rate post selection, results with the microfilter device (17.64%)
and mini-Percoll gradient (18.27%) were higher than with swim-up method (6.52%).
However, the cleavage and blastocyst rates were the lowest using the microfilter
device. In conclusion, sperm selection using the microfilter device and swim-up
method can improve kinematic parameters, although the mini Percoll gradient was
the most efficient method for embryo production.
KEYWORDS
blastocyst rate, kinematics, microfilter device, sperm selection
1|INTRODUCTION
Sperm selection techniques may affect the quality of spermatozoa
used in in vitro embryo production systems (Arias et al., 2017). For
years, assisted reproductive technologies have used traditional tech-
niques such as swim-up and density gradient (Parrish et al., 1995;
Parrish, 2014) which provide highly motile and functional sperm popu-
lation. Nevertheless, as a mechanical process, centrifugation may have
adverse effects on sperm DNA integrity (Samuel et al., 2018), there-
fore this could affect the fertilization and embryonic development.
In the last decade, several methods have been developed that imi-
tate the natural process of sperm selection. Microfluidics and microfil-
ter devices mimic the characteristics of the female oviducts and
uterus, minimizing the risk of sperm damage, based on a mechanism
of biokinetic sperm classification (Asghar, et al., 2014; Smith &
Takayama, 2017; Parrella et al., 2019).
In humans, using microfluidic technology, researchers have
selected spermatozoa with significantly improved motility and mor-
phology (Schuster et al., 2003). Furthermore, this technology has out-
performed conventional centrifugation in selecting spermatozoa with
Received: 20 March 2022 Revised: 18 August 2022 Accepted: 26 August 2022
DOI: 10.1111/and.14585
Andrologia. 2022;e14585. wileyonlinelibrary.com/journal/and © 2022 Wiley-VCH GMbH. 1of8
https://doi.org/10.1111/and.14585

high DNA integrity, (Shirota et al., 2016; Parrella et al., 2019). Like-
wise, the blastocyst rate of fertilized embryos with sperm selection
using glass wool filtration was significantly higher than with Percoll
technique (Lee et al., 2009). Furthermore, recent reports (Quin
et al., 2018; Ozcan et al., 2021) showed that microfluidic sorting of
unprocessed semen allows selection of highly motile spermatozoa
with very low levels of DNA damage. In bovines, it is well known that
the sperm selection technique is correlated to the variability in sperm
quality and blastocyst productivity. Thus, this study aimed to compare
the performance of a microfilter device versus conventional methods
to select spermatozoa to be used in in vitro fertilization (IVF).
2|MATERIAL AND METHODS
The approval of the ethics committee was not required by the Gradu-
ate School of the Universidad Nacional Agraria La Molina, as the study
involved the use of commercial frozen semen and ovaries collected in
a local slaughterhouse. The chemicals were purchased from Sigma-
Aldrich (St. Louis, MO, USA), and the in vitro embryo culture media
from Vitrogen (YVF Biotech LTDA EPP, Sao Paulo, Brazil).
2.1 |Experimental design
For this work we selected commercial semen from two bulls that have
shown a high production of blastocysts and less variability between
batches in previous in vitro procedures. Three straws from the same bull
per replicate were thawed. The pooled sample was divided to be used in
three different sperm selection procedures. Sperm quality analysis
(kinematics and morphometric parameters, vitality and DNA fragmenta-
tion) was performed before and after the sperm selection procedures. A
total number of 10,000 motile spermatozoa per oocyte was used in IVF.
The experiment was replicated 11 times. Embryonic development evalua-
tion was performed at three times point post-IVF: cleavage (day 3), moru-
lae (day 5) and blastocyst rate (day 7) (see Figure 1).
2.2 |Sperm selection techniques
Semen straws were thawed in a water bath at 37C for 30 s. Sperm
selection was carried out using microfilter devices, mini-Percoll gradi-
ent and swim-up method.
2.2.1 | Microfilters
Semen (300 μl) with sperm washing medium (550 μl) was deposited in
the inlet duct of the microfilter device (ZyM
ot Multi 850 μl, DxNow,
USA) using a 1-ml syringe. Sperm washing medium (850 μl) was
loaded into the outlet duct of the microfilter device with 8 μm porous
membrane. After loading, the device was placed in an incubator at
38C for 30 min. The processed sperm sample (300 μl) was collected
from the outlet duct for sperm quality analysis and to be used for IVF.
2.2.2 | Mini-Percoll gradient
A 45%/90% discontinuous Percoll density gradient (Parrish et al., 1995)
was prepared by combining the sperm washing medium (sperm TALP
FIGURE 1 Schematic illustration of
experimental design. Sperm quality
analysis was performed after thawing
semen and after spermatic selection.
Embryonic development evaluation was
performed at days 3, 5 and 7 post-IVF
2of8 VEGA-HIDALGO ET AL.

supplemented with 100 μM sodium pyruvate and 6 mg of bovine serum
albumin free fatty acid) with Percoll
®
. Semen (300 μl) was slowly pipetted
downthesideofthemicrotubetocreateathirdlayerontopofthe45%
gradientandthencentrifugedat600g for 6 min. The pellet was
washed in 400 μl of sperm washing medium and centrifuged again at
600 g for 3 min. Finally, a 100 μl pellet was recovered for sperm quality
analysis and to be used for IVF.
2.2.3 | Swim-up
Semen (300 μl) was layered carefully under 1 ml of equilibrated sperm
washing medium in a 1.5 ml microtube. After loading the tube at an
angle of 45, it was placed in an incubator at 38C with 5% CO
2
for 1 h.
After incubation, 800 μl of the supernatant was placed into an empty
1.5 ml microtube and centrifuged at 600 g for 3 min. Finally, a 100 μl
pellet was recovered for sperm quality analysis and to be used for IVF.
2.3 |Sperm quality
Kinematic parameters were assessed using samples of 7 μl which were
placed in a Sperm Tracker Chamber (Proiser R+D, Spain) and analysed
in a computer-assisted sperm analysis (CASA) (ISAS
®
V, Integrated
Semen Analysis System, Proiser R+D, Spain). The video camera
employed was a Proiser 782 m attached to a microscope UB203
(UOP/Proiser) equipped with a 10negative phase contrast objective
and an integrated heated stage that was maintained at a constant tem-
perature of 37C. Samples were captured at 25 fps following the set-up
configuration of the manufacturer for bull semen. The CASA settings
used were a particle area between 15 and 70 μm
2
and connectivity of
12. An average sperm population per sample was analysed according to
the sperm selection procedure: Microfilter device (896 spz), mini-Percoll
gradient (1478 spz) and swim-up method (906 spz). The kinematic
parameters measured were: curvilinear velocity (VCL), linear velocity
(VSL), average path velocity (VAP), linearity (LIN), straightness (STR),
wobble (WOB), lateral head displacement (ALH) and beat cross- fre-
quency (BCF). The parameters defining progressive motility were STR
≥70% and VAP ≥25 μm/s, as previously described by Barquero
et al. (2021).
Morphometric parameters were assessed using the Diff Quik Kit
(Medion Diagnostics, Germany) as previously described by Barquero
et al. (2021). For this analysis, 6557 sperm (200 cells/sample) images
were captured at 400magnification. The sperm head measurements
were calculated automatically by ISAS1 (CASA Integrated Semen
Analysis System, Proiser R+D, Spain) including the size (length [L,μm],
width [W,μm], area [A,μm
2
] and perimeter [P,μm]) and shape vari-
ables (ellipticity [L/W], elongation [(LW)/(L+W)], roughness [4πA/
P2], regularity [πLW/4A] and acrosome percentage).
Sperm vitality was assessed using acridine orange and propidium
iodide (AO/PI) from Sigma-Aldrich. Each aliquot was incubated with
AO/PI for 10 min at 37C. Samples were placed on a glass slide and
observed at 100objective using a fluorescence microscope
(Axioscope, Carl Zeiss, USA). For analysis, 200 cells/sample were ana-
lysed to determine the sperm vitality rate.
Total sperm recovery was determined in samples (10 μl) diluted in 90 μl
of distilled water, and counting was performed through a Neubauer cham-
berat40in a microscope. The sperm recovery rate was calculated as (final
concentration final volume)/(initial concentration initial volume) 100.
DNA fragmentation was measured using the sperm chromatin dis-
persion test (Halomax
®
, Halotech DNA SL, Spain) following the manu-
facturer's instructions. Spermatozoa were visualized in a fluorescence
microscope (Axioscope, A2, Carl Zeiss, USA), and about 200 cells per
sample were counted. Spermatozoa with unfragmented DNA showed
a large halo of dispersed chromatin, while spermatozoa with fragmen-
ted DNA showed a small or null halo.
2.4 |Embryo development
Ovaries were obtained from local abattoirs following the standard proce-
dure described by Rodríguez et al. (2008). COCs in groups (10–12) were
in vitro matured (IVM) in a drop (70 μl) of IVM medium (Vitrogen, Brazil)
for 20–22 h. Immediately after sperm selection, a total number of 10,000
motile spermatozoa per oocyte was used in IVF and incubated with COCs
at 38Cin5%CO
2
in an air atmosphere. The insemination volume on IVF
drop varied according to sperm selection technique: 13.47 ± 1.78 μl(micro-
filter device), 3.59±1.12μl (mini-Percoll gradient) and 13.88 ± 2.55 μl
(swim-up). Fertilization was carried out in an IVF medium (Vitrogen, Brazil).
After 18–22 h of IVF, presumptive zygotes were mechanically denuded by
pipetting and then in vitro cultured (IVC) in a drop (70 μl) of IVC medium
(Vitrogen, Brazil) at 38Cin5%CO
2
in an air atmosphere for 7 days post-
IVF. At days 3 and 5 post-IVF, 50% of IVC medium was changed. Embryo
development evaluation (cleavage, and blastocyst rate) was carried out on
days 3 and 7 post-IVF, under a stereoscopic microscope using morphologi-
cal criteria described in the International Embryo Transfer Society (IETS)
manual (Stringfellow & Givens, 2010).
2.5 |Statistical analysis
Analysis of variance and Duncan test were used to compare the effect
of three sperm selection techniques on sperm quality (kinematics and
morphometric parameters, vitality and DNA fragmentation) and embryo
development (cleavage and blastocyst rate). Statistical analysis was per-
formed using the IBM SPSS Statistics version 20 (IBM, Armonk, NY,
USA), and significance was determined at the p< 0.05 level.
3|RESULTS
3.1 |Effect of sperm selection technique on sperm
quality
The results generally showed that the separation method does affect
(p< 0.05) the kinematics parameters (Table 1). The most relevant
VEGA-HIDALGO ET AL.3of8

parameters—VCL, VSL, VAP, LIN, STR and ALH—were higher
(p< 0.05) when using the microfilter device and swim-up method with
respect to the mini-Percoll gradient (Table 1). Upon analysing other
motility parameters, higher (p< 0.05) total motility and progressive
motility were observed in the mini-Percoll gradient technique com-
pared with the others. Also, the microfilter and mini-Percoll technique
had an effect (p< 0.05) on size of sperm selected (width and elonga-
tion) (Table 2). The results of DNA fragmentation of spermatozoa
show that spermatozoa selected through the microfilter device and
swim-up techniques had less DNA fragmentation (p< 0.05). Likewise,
the total sperm recovery was higher using the microfilter device and
mini Percoll gradient (Table 3).
TABLE 1 Sperm kinematics values (mean ± SD) for sperm selected using microfilter device versus conventional methods
Parameter
Pre-selection Post-selection
p-ValueInitial Microfilter device
Conventional methods
Swim-up Density gradient
n(sperm) 40,134 9851 9967 16,255
Repeat 11 11 11 11
VCL (μm/s) 101.49 ± 9.30 124.17 ± 33.96
a
128.13 ± 19.17
a
98.45 ± 30.17
b
0.042
VSL (μm/s) 37.94 ± 8.21 64.43 ± 16.67
a
73.60 ± 12.64
a
45.01 ± 14.23
b
0.000
VAP (μm/s) 59.59 ± 8.53 79.83 ± 17.34
a
88.42 ± 11.33
a
59.91 ± 17.61
b
0.001
ALH (μm) 4.28 ± 0.29 5.23 ± 0.82
a
5.06 ± 0.27
a
4.00 ± 0.99
b
0.001
BCF (Hz) 9.64 ± 0.51 10.02 ± 1.28 11.04 ± 1.15 9.81 ± 1.24 0.057
LIN 37.30 ± 6.48 49.77 ± 7.15
ab
55.33 ± 7.33
a
46.17 ± 7.38
b
0.021
STR (%) 63.27 ± 4.74 79.63 ± 6.37
ab
82.88 ± 5.18
a
75.08 ± 6.75
b
0.020
TM (%) 83.49 ± 9.52 73.69 ± 10.36
b
63.56 ± 8.59
c
88.83 ± 6.59 0.000
PM (%) 31.64 ± 4.25 53.89 ± 9.68
a
43.39 ± 10.25
b
57.09 ± 12.19
a
0.015
Note: Values with different superscripts between columns are significantly different at p< 0.05. Data in the initial column (pre-selection) were not included
in the statistical analyses.
Abbreviations: ALH, lateral head displacement; BCF, beat cross-frequency; LIN, linearity; PM, progressive motility; SD, standard deviation; STR,
straightness; TM, total motility; VAP, average path velocity; VCL, curvilinear velocity; VSL, linear velocity; WOB, wobble.
TABLE 2 Morphometric parameter measurements (mean ± SD) for sperm selected using microfilter device versus conventional methods
Parameter
Pre-selection Post-selection
P-valueInitial Microfilter device
Conventional methods
Swim-up Density gradient
n(spermatozoa) 1273 1322 1321 1274
Repeat 11 11 11 11
Sperm head measurements—size
Length 6.21 ± 0.13 6.23 ± 0.20 6.22 ± 0.16 6.21 ± 0.08 0.941
Width 3.47 ± 0.17 3.54 ± 0.20
b
3.50 ± 0.22
ab
3.33 ± 0.16
a
0.045
Area 18.14 ± 0.72 18.67 ± 1.05 18.24 ± 1.13 19.06 ± 0.23 0.118
Perimeter 19.30 ± 0.52 19.62 ± 0.87 19.39 ± 0.42 19.65 ± 0.06 0.497
Sperm head measurements—shape
Ellipticity 1.79 ± 0.10 1.78 ± 0.11 1.79 ± 0.11 1.71 ± 0.04 0.116
Elongation 0.28 ± 0.02 0.28 ± 0.03
b
0.28 ± 0.03
b
0.26 ± 0.01
a
0.045
Roughness 0.61 ± 0.01 0.61 ± 0.03 0.60 ± 0.04 0.62 ± 0.03 0.564
Regularity 0.93 ± 0.01 0.93 ± 0.01 0.94 ± 0.02 0.94 ± 0.01 0.325
Acrosome, % 32.7 ± 3.72 33.60 ± 3.05 32.74 ± 4.04 32.71 ± 0.81 0.735
Note: Values with different superscripts between columns are significantly different at p< 0.05. Data in the initial column (pre-selection) were not included
in the statistical analyses.
Abbreviation: SD, standard deviation.
4of8 VEGA-HIDALGO ET AL.

3.2 |Effect of sperm selection techniques on
embryonic development
Values of cleavage and blastocyst rate in the mini-Percoll gradient
method were higher (p< 0.05) than those using the microfilter device
and swim-up method (Table 4).
4|DISCUSSION
In the laboratory, sperm selection techniques help us to separate the
motile from the immotile and dead sperm fraction and to eliminate
the seminal plasma, diluents and cryoprotectant. Traditional sperm
selection techniques were simple washing (Edwards et al., 1969),
swim-up and Percoll gradient (Parrish et al., 1995), which have been
used in human and bovine IVF.
The present study is the first report comparing the use of micro-
filter devices for sperm separation versus conventional methods used
in bovine IVF. There are several sperm selection techniques available
that use different principles to remove dead and abnormal spermato-
zoa, seminal plasma, cryoprotective agents and other factors. Our
results indicate that the use of the microfilter device and swim-up
method for sperm selection allows us to obtain the best kinematics
parameters (Table 1). Several publications (Asghar et al., 2014; Parrella
et al., 2019; Gode et al., 2019) consider the “Zymot or Fertile plus
devices”as microfluidics even though the specifications indicate that
the collection chamber is separate by an 8-μm porous microfilter.
Microfluidic technology operates on the principle of fluid dynamics in
a space-constricted environment and offers an alternative for sperm
sorting (Lopez-Garcia et al., 2008), emulating sperm migration in the
reproductive tract. This sperm selection method is highly related to
the motility of the isolated sample, as sperm stream has a rheotactic
behaviour in smaller flow rates. Nagata et al. (2018) reported the
selection of 100% normal spermatozoa with high progressive motility
using microfluidics. Similar results were observed by Gode et al.
(2019) and Parrella et al. (2019), when comparing microfilters versus
density gradient centrifugation, as progressive motility was greater
than 96% versus 64 to 91%, respectively. However, Hamacher et al.
(2020) showed that the quality of microfluidics processed bull sperma-
tozoa did not differ significantly from that processed by conventional
methods. Furthermore, Lee et al. (2009) reported that the most effi-
cient technique for removing spermatozoa with damaged membranes
is using glass wool filtration.
The scientific publications indicate that sperm DNA damage is
associated with a low performance in fertilization, low embryo quality,
less blastocyst formation, implantation and causes spontaneous mis-
carriage (Seli et al., 2004). In our study, sperm selection procedures
did not affect sperm DNA fragmentation (p> 0.05). However, in
humans, an extremely low sperm DNA fragmentation (0 to 1.6% ver-
sus control 7 to 26%) is observed with the use of microfluidics/
microfilter device (Quinn et al., 2018; Nagata et al., 2018; Parrella
et al., 2019). Since conventional techniques for sperm selection use
centrifugation as a common denominator it is possible that they pro-
duce a higher generation of reactive oxygen species and DNA
TABLE 3 Total sperm recovery, vitality and DNA damage (mean ± SD) of sperm selected with microfilter device versus conventional methods
Parameter
Pre-selection Post-selection
P-valueInitial Microfilter device
Conventional methods
Swim-up Density gradient
Total sperm recovery (10
6
) 26.25 4.63 ± 0.42
a
1.71 ± 0.12
b
4.79 ± 1.02
a
0.000
Sperm recovery rate, % 100 17.64 ± 1.60
a
6.52 ± 0.48
b
18.27 ± 3.89
a
0.000
Vitality, % 64.78 ± 6.17 77.80 ± 6.37
a
67.04 ± 8.11
b
74.67 ± 5.37
a
0.002
DNA damage % 13.13 ± 3.34 11.30 ± 3.15
b
12.50 ± 2.62
b
16.56 ± 1.92
a
0.020
Note: Values with different superscripts between columns are significantly different at p< 0.05. Data in the initial column (pre-selection) were not included
in the statistical analyses.
Abbreviation: SD, standard deviation.
TABLE 4 Embryonic development of
oocytes fertilized in vitro with
spermatozoa selected using microfilter
device versus conventional methods
Embryo development Microfilter device
Conventional methods
p-ValueSwim-up Density gradient
n(oocytes) 324 321 327
Repeat 11 11 11
Volume AI per drop, μl 13.47 ± 1.78
b
13.88 ± 2.55
b
3.59 ± 1.12
a
0.000
Cleavage rate (D 3) 67.10 ± 10.07
b
75.21 ± 7.64
b
83.75 ± 5.10
a
0.000
Blastocyst rate (D 7) 14.93 ± 5.41
c
21.89 ± 4.26
b
28.07 ± 5.40
a
0.000
Note: Values with different superscripts between columns are significantly different at p< 0.05.
Abbreviation: SD, standard deviation.
VEGA-HIDALGO ET AL.5of8

fragmentation. Besides, the presence of colloidal particles can inter-
fere with the sperm velocity in the Percoll gradient technique being
responsible for the reduction of VCL, VSL, VAP and STR (Celeghini
et al., 2008).
Another characteristic in which the microfilter device stands out
in our study is in the total sperm recovery (17.64%) compared to mini-
Percoll gradient (18.27%) and swim-up technique (6.52%) method
(p< 0.05). A greater recovery of spermatozoids would allow greater
efficiency in the production of embryos in vitro (Table 3). Meanwhile,
Quinn et al. (2018) showed that the total recovered spermatozoa
decreased after processing with microfluidics compared with density
gradient centrifugation. Also, Cesari et al. (2006) showed that the total
sperm recovered was similar using the Percoll gradient (38.9%) and
swim-up techniques (30.2%).
Currently, spermatozoa morphometry analysis is extraordinarily
complex because it lacks of universal parameters for normal sperma-
tozoa morphology (Walters et al., 2004). It is reported that sample
processing techniques, such as fixation, staining and drying, can sig-
nificantly change the dimensions of the spermatozoa (Kondracki
et al., 2017). Some reports indicate that high fertility in bulls is
related to the size and shape of the sperm head (Valverde
et al., 2016). Moreover, these head size variations of spermatozoa
can be affected by the species (Bos taurus vs. Bos indicus) (Yániz
et al., 2016), sperm subpopulation (Valverde et al., 2016), without
changing their ability to fertilize. In our study, sperm head measure-
ments showed no significant differences (p> 0.05) amongst sperm
selection techniques, which differs from the results reported by Gar-
cía-Herreros, & Leal (2014).
In vitro embryo production is a gold standard test for the assess-
ment of sperm fertilization potential. In this study, the blastocyst rate
using the mini-Percoll gradient (28.1%) method was higher (p< 0.05)
than those obtained through the swim-up method (21.9%) and using
the microfilter device (15.3%). However, adding different volumes of
sperm suspension to the IVF drop (See Table 4) could be a problem in
this experimental design. These results are similar to those obtained
by Yetkinel et al. (2019) in humans and Parrish et al. (1995), Cesari
et al. (2006) and Muiño et al. (2009) in cattle. However, Sepúlveda
et al. (2018) observed no major differences on in vitro embryonic
development using sperm selected from Percoll and Isolate
®
gradients.
The loss of decapacitation factors from the sperm surface is
important in the success of fertilization (Fraser and Adeoya-
Osiguwa, 2005). In this study, the low rate of blastocysts produced
in vitro using selected spermatozoa by swim up method and using
the microfilter device, could be explained considering the partial
conservation of decapacitating factors found in processing by
these methods. Jeyendran et al. (2019) showed that the swim-up
technique cannot remove ROS and decapacitation factors. In con-
trast, density-gradient centrifugation techniques have been used
extensively to separate motile spermatozoa from immotile sperma-
tozoa and other cells, and to eliminate decapacitation factors,
prostaglandin and reactive oxygen species (ROS) (Henkel and
Schill, 2003).
In conclusion, these results indicate that sperm selection using
the microfilter and swim-up techniques can improve kinematic param-
eters and reduce DNA fragmentation, although, mini-Percoll gradient
was the most efficient method according to embryo production
parameters evaluation.
AUTHOR CONTRIBUTIONS
Edwin Mellisho and Jhorjhi Vega-Hidalgo designed the experi-
ment. Jhorjhi Vega-Hidalgo, Josselin Rivas, Misael Rodriguez and
Carmen Huayhua performed the experiment. Jhorjhi Vega-
Hidalgo, Misael Rodriguez, Deysi Dipaz-Berrocal and Edwin Mel-
lisho analysed the data and drafted the manuscript. Edwin Mel-
lisho, Jhorjhi Vega-Hidalgo, Josselin Rivas, Deysi Dipaz-Berrocal,
Carmen Huayhua and Misael Rodriguez revised and edited the
manuscript.
ACKNOWLEDGEMENTS
This research was supported by FONDECYT, Peru Grant FONDECYT
178-2015.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on
request from the corresponding author. The data are not publicly
available due to privacy or ethical restrictions.
ORCID
Jhorjhi Vega-Hidalgo https://orcid.org/0000-0002-8950-0700
Misael Rodriguez https://orcid.org/0000-0002-9342-7067
Deysi Dipaz-Berrocal https://orcid.org/0000-0003-3992-6735
Josselin Rivas https://orcid.org/0000-0003-3321-362X
Carmen Huayhua https://orcid.org/0000-0002-4170-0412
Edwin Mellisho https://orcid.org/0000-0001-7171-1991
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SUPPORTING INFORMATION
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ing Information section at the end of this article.
How to cite this article: Vega-Hidalgo, J., Rodriguez, M.,
Dipaz-Berrocal, D., Rivas, J., Huayhua, C., & Mellisho, E.
(2022). Sperm selection techniques in cattle: Microfilter device
versus conventional methods. Andrologia, e14585. https://doi.
org/10.1111/and.14585
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