Acta Scientiae Veterinariae, 2019. 47: 1685.

RESEARCH ARTICLE ISSN 1679-9216

Pub. 1685

Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen

Juliano Pianowski Marques Silva1, André Felipe Berto de Almada1, Jonathan Soares de Lima1,
Carlos Renato de Freitas Guaitolini1, André Maciel Crespilho2, Camila de Paula Freitas Dell´Aqua3,
José Antônio Dell´Aqua Junior3, Danielle Andressa de Oliveira Sestari1 & Rosiara Rosaria Dias Maziero1

ABSTRACT

Background: Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases
can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood,
however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing
increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodex-
trins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from
the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram
naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%.
The aim of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in
the kinetics and viability of frozen and thawed ram spermatozoa.
Materials, Methods & Results: Five ejaculates, from five rams of Dorper breed were collected and divided into three groups:
control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×106 sperma-
tozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated
at 5°C for 4 h, exposed to LN2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics,
plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion (O2-) were evaluated.
Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP,
μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg (P < 0.05). In addition, 1 mg CLC
showed decreased levels of superoxide anion formation (O2-), a free radical detrimental to spermatozoa (P < 0.05). The
use of 2 mg of CLC reduce VAP (P < 0.05) and did not have any beneficial effect on the evaluated parameters.
Discussion: Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in
goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of
CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclo-
dextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H2O2. They obtained
superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group.
The superoxide anion (O2-) is a free radical formed from molecular oxygen by the addition of an electron. It is generated
spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases,
and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group.
Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a
concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid
sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed
decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

Keywords: ram, semen, cryopreserved, sperm parameters.

DOI: 10.22456/1679-9216.96576
Received: 21 June 2019 Accepted: 24 September 2019 Published: 10 October 2019
1
Paranaense University (UNIPAR), Umuarama, PR, Brazil. Santo Amaro University, São Paulo, SP, Brazil. School of Veterinary Medicine and Animal
2 3

Science, Unesp, Botucatu, SP. CORRESPONDENCE: R.R.D. Maziero [rosiaramaziero@prof.unipar.br]. Praça Mascarenhas de Morães n. 4282. CEP
87502-210 Umuarama, PR, Brazil.

1
J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

INTRODUCTION (CH3OH)1. Then, a 0.45 mL aliquot of the cholesterol

During cryopreservation process, the sperm and chloroform solution (C2432)1 was added to the
membrane undergoes a series of injuries provoked cyclodextrin solution and homogenized to obtain a
translucent solution.
by variations in temperature and the modification of
The solution was then transferred to a pre-
its physical state. These include osmotic and thermal
sterilized glass petri dish and the solvents removed
stress, the formation of intracellular ice crystals, and
by evaporation using hotplate at 40°C for a period of
excessive production of reactive oxygen species [7].
30 h, in a horizontal laminar flow hood. The crystals
These changes promote the destabilization of the sperm
resulting from the solution were removed from the plate
lipid membrane [23].
by scraping with a scalpel blade serving as a spatula
Studies report that young bulls have a higher
and stored in a sterile glass tube at room temperature.
intact plasma membrane index after thawing compared
The working solution of CLC was prepared
to adult or senior bulls. This may be associated with
by the addition of 50 mg of CLC in 1 mL of TRIS
the fact that young bulls produce spermatozoa rich in
medium (3.78 g TRIS (hydroxymethyl) aminomethane
fatty acids compared to older animals [1]. This high
(252859), 2.11 g citric acid (251275), 1.22 g fructose
concentration of fatty acids confers greater fluidity and
(1286504)1 at 37°C with subsequent homogenization
flexibility to the plasma membrane, which increases its
with the aid of a stirrer.
resistance to osmotic stress, as well as to the oxidative
This solution was packed in 1.5-mL micro
effects caused by cryopreservation [11]. centrifuge tubes and kept frozen at -20°C, then reheated
During the cryopreservation process, the semen at 37°C in a water bath and homogenized prior to use.
is initially refrigerated at room temperature, that is,
from 37ºC to 20ºC, which does not seem to cause great Animals and sample processing
damage to the spermatozoa. However, the initial stress Harvest and evaluation of fresh semen were
occurs when sperm cells pass from body temperature to carried out in the field, at a private property of the
5°C, characteristic of the transition phase, in which the municipality of Umuarama, located at latitude 23º 45’
plasma membrane changes from the crystalline liquid 59” S and at a longitude 53º 19’ 30” W, in the northwest
state to the gel state. This transition phase occurs both region of the state of Paraná, Brazil.
during the freezing process and thawed [11]. By increas- The diluted samples, packed in freezer vats
ing the cholesterol content of the membranes, there is an and refrigerated in a transport carton, were taken to
improvement in the sperm parameters after thawing [4]. the UNIPAR Animal Reproduction Laboratory, then
Considering reports of improvement in the frozen in a Styrofoam box containing liquid nitrogen
sperm parameters of frozen/thawed semen when using (LN2) before storing in cryogenic cylinders.
the cholesterol-loaded cyclodextrin (CLC), and knowing Sperm evaluations by flow cytometry and by
that sheep have naturally low amounts of fatty acids in the Computer Assisted Semen Analyses (CASA) were
sperm membrane, this study aimed to analyze the effects carried out at the Faculty of Veterinary Medicine and
of the addition of CLC in commercial diluent medium Animal Science (FMVZ) Unesp, in the Department
during the cryopreservation process of ram spermatozoa. of Animal Reproduction and Veterinary Radiology
located in the city of Botucatu, in the state of São Paulo,
MATERIALS AND METHODS
at latitude 22º 53’ 09” S and longitude 48º 26’ 42” West.
Five samples were taken, at weekly intervals,
Preparation of the cyclodextrin-loaded cholesterol solu- from five animals, totaling 25 collections of Dorper
tion (CLC) sheep sperm. The animals sampled were sexually
The preparation of cholesterol loaded methyl- mature at ages varying from 12 and 30 months. The
β-cyclodextrin1 was performed according to the meth- samples were collected with the use of an electroejacu-
odology of Purdy and Graham [21]. lator. After collection, the samples were subjectively
In a pre-sterilized glass tube, 200 mg of choles- evaluated by optical microscopy for total motility (MT,
terol (C8667)1 was dissolved in 1 mL of chloroform, %), vigor (1-5), sperm concentration, and morphology.
and in another tube, 1 g of Methyl-β-cyclodextrin Rams considered fit to sample presented a total
(C4555) 1 was dissolved in 2 mL of methanol motility ≥ 80%, vigor 3 (1-5), and a sperm concentra-

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J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

tion ≥ 1 billion sperm per ejaculate with a percentage Spermatic kinetics

of major and minor defects lower than 10%. For the evaluation of spermatic kinetics, five
For determination of the sperm concentration, fields were analyzed for each sample with the aid of
10 μL of the ejaculate was diluted in 2 mL of 10% CASA2.
formalin saline solution. Sperm count was performed The parameters evaluated were total sperm
using a hematometric method in a Neubauer chamber. motility (MT, %), progressive sperm motility (MP, %),
After the initial screening, each ejaculate was average path velocity (VAP, μm/s), linearity (LIN, %),
divided into 3 study groups: and percentage of spermatozoa with rapid spermatozoa
1) Control group: Botubov® commercial percentage (RAP, %). The CASA setup used in this
diluent3; experiment is described in Table 1.
2) Group 1: Botubov® commercial diluent For the rapid thermo-resistance test (TTR),
and 1 mg CLC for each 120 × 106 total spermatozoa; two straws from each group of studies were thawed
3) Group 2: Botubov® commercial diluent and conditioned in 1.5 mL microtubes. Samples were
and 2 mg CLC for each 120 × 106 total spermatozoa. kept in a water bath at 46°C for 30 min. After this time,
For each 120 × 106 total spermatozoa, 20 μL evaluations were performed by the CASA system.
and 40 μL of the prepared solution of cyclodextrin
were added in clusters 1 and 2, respectively. After the Table 1. Methodology of the computerized analysis (CASA) of the sper-
addition of CLC, the tubes were homogenized and in- matic kinetics of ram.
cubated at room temperature for 10 min, allowing for Analysis setup: OVINE
the incorporation of the cholesterol in the membrane Apply sort O
wall according to the protocol used by Watson [24]. Frames acquired 30
The sperm concentration in the diluted samples Frame rate 60 Hz
was adjusted to 100 million total spermatozoa per mL Minimum constrast 60
of diluent and filled in 0.5 mL French straw3 previ- Minimum cell size 60 pixels
Minimum static contrast 1
ously identified with animal number and collection
Straightness (STR), Threshold 70.0%
number. Thus, each vane contained a total of 50 × 106
Vapcutoff 30.0 μm/s
spermatozoa. Prog. Minvap 40.0 μm/s
The samples were freeze-dried and placed in VSL cutoff 20.0 μm/s
a Styrofoam box3 previously refrigerated at 5°C for 4 Cell size 5 pixels
h. Then, the freezing curve was performed in liquid Cell intensity 90
nitrogen vapor (LN2) by sealing the semen samples Static head size 0.10 to 3.40
in a conventional 40-liter polystyrene box at a fixed Static head intensity 0.30 to 1.50
distance of 5 cm from the LN2 level for 10 min. After Static elongation 8 to 95
this period, the vanes were immersed directly into the Slow cells motile NO
liquid nitrogen and raked for packaging in cryobiologi- Magnification 1.95
cal cylinders. Video frequency 60
Bright field NO
For the standardization of the post-thawing
LED illumination intensity 2240
analyses, the analyses were initiated after a minimum
IDENT illumination intensity 0
period of 3 days of storage [9]. Temperature 38ºC
Sperm analysis after thawing Chamber depth 10 μm
Chamber position 3.9 μm
For all analysis, the vanes were thawed in a Chamber position B 11.8 μm
water bath at 46°C for 20 s. For post-thaw evaluation, Chamber position C 16.3 μm
two straws of each ejaculate were used per treatment Chamber position D 26.6 μm
to remove the effect of the vane. Chamber type Makler
Post-thaw analysis of sperm kinetics was Field selection mode AUTO
performed by CASA2 and evaluation of plasma and IDENT fluorescente option OFF
acrosomal membrane integrity and intracellular level Integrating time 1 frames
of superoxide anion (O-2) by flow cytometry4. Remote image recall NO

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J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

Evaluation of spermatic characteristics by flow cytometry YP, 20 μM of MST, and 2 μM of DHE7 were incubated
For flow cytometry analysis, we used the BD for 20 min at 37°C.
LSR Fortessa equipment4, equipped with lasers: blue
Statistical analysis
(488 nm, 100 mW), red (640 nm, 40 mW), and violet
Statistical analysis were carried out using the
(405 nm, 100 mW). The data were evaluated using
the programs BD FACSDiva™ v6.1 and WinList 6.0 Statistical Analyses System (SAS)8. First, the Shapiro-
(Verify Software House). Wilk test (Proc-Univariate) was used for normal data
Samples were analyzed at a rate of acquisition of analysis and the Chi-Square test (Proc-GLM) was used
800 events/second, with 10,000 cells per sample. Cellular to analyze the homogeneity of the variations. The mean
debris and particles were excluded from acquisition and and standard deviation of the experimental groups and
analysis by adjustments in threshold and by labeling with their relationships with the study groups were made
Hoechst 333421 (100 μg/mL) excited by the violet laser. using analysis of variance (Proc-GLM), adopting a
significance level of 5% (P < 0.05)
Evaluation of the plasma and acrosomal membranes
RESULTS
The association of propidium iodide probes1
(P4170), FITC-PSA (L0770)1, and Hoechst 33342
Spermatic kinetics
(1533)1 was used. For each sample, 200 µL of diluted
semen in TALP-PVA1 at a concentration of 5 × 106 After the freezing/thawing process, the param-
sperm/mL were added to 5 µL H3342 (100 μg/mL), 5 eters of total motility (MT, %), progressive motility
µL of IP (50 mcg/mL), and 1 µL of FITC-PSA (100 (MP, %), average path velocity (VAP, μm/s), linearity
μg/mL), and then homogenized and incubated for 15 (LIN, %), rapid spermatozoa percentage (RAP, %),
min in the dark at 37°C. total motility post-test of thermoresistance (MT TTR,
%), and progressive motility post-test of thermoresis-
Evaluation of superoxide anion generation tance (MP TTR, %) were evaluated (Table 2).
For the evaluation of the production of super- According to the data presented in Table 2,
oxide (O2-), intracellular Yo-Pro® association7, with no difference was observed in the MT parameters be-
markings for cell plasma membrane destabilizing) tween the groups studied. However, the MP and RAP
and Dihydroethidium (DHE, D23107, generation of parameters showed that the data obtained by the 1 mg
superoxide anion intracytoplasmic)7 was used. Thus, CLC group were superior to the control and 2 mg CLC
in 500 μL of semen diluted in TALP-PVA1, 25 μM of groups (P < 0.05).

Table 2. Mean values and standard deviation of total motility (TM, %), progressive motility (PM, %), average path velocity (VAP, μm
/ s), linerality (LIN, %), percentage of rapid sperm (RAP, %), total motility post-test of thermoresistance (TM, TTR; %), progressive
motile post-test of thermoresistance (PM, TTR, %), frozen / thawed semen.
Parameter C Group1 Group 2
TM (%) 46.08 ± 21.4 58.96 ± 18.3 47.8 ± 23.5
PM (%) 21.64 ± 10.1b 28.96 ± 8.6a 23.84 ± 11.4b
LIN (%) 52.04 ± 8.3 51.08 ± 9.1 52.4 ± 5.8
VAP μm/s 58.68 ± 9a 59.88 ± 5.0a 52.96 ± 8b
RAP (%) 30.56 ± 17.5b 41.24 ± 18.8a 30.8 18.8b
TM TTR (%) 2.84 ± 5.3 7.84 ± 10.2 5.2 ± 9.3
PM TTR (%) 1.24 ± 3.5 4.08 ± 7.3 1.92 ± 3.7
C= control, Group 1= 1 mg CLC and Group 2= 2 mg CLC. Different lowercase letters on the same line indicate significant difference
(P < 0.05).

The VAP parameter was lower in the 2 mg Spermatic viability

CLC group compared to the control and 1 mg CLC In order to evaluate cell viability, the plasma
groups. However, linearity (LIN, %), MT post TTR and acrosomal membrane integrity parameters and in-
(%), and PM post TTR (%) did not show differences tracellular level of superoxide anion (O2-) were verified.
between the groups. The data obtained are presented in Table 3.

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J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

In the evaluation of the integrity of the plasma In the evaluation of the intracellular level of
and acrosomal membranes, it was verified that group 1 superoxide anion (O2-), we verified that group 1 was ef-
presented a higher percentage of intact cells, compared fective in decreasing the formation of this free radical,
to the control groups, and this result did not present compared to the control group, while group 2 provided
differences when compared to group 2 (P < 0.05). similar data to group 1 and the control.

Table 3. Mean and standard deviation of percentage of intact plasma and acrosomal membranes (MPAI, %), intracellular level of
superoxide anion (O2-) in the control groups, Group 1 and Group 2.
Parameter C 1 2
MPAI, (%) 54.94 ± 17.8b 56.04 ± 20.8a 55.86 ± 18.59ab
superoxide anion (O2-) 70.74 ± 14.9a 51.63 ± 17b 60.45 ± 2ab
C= control, Group 1= 1 mg CLC and Group 2= 2 mg CLC. Different lowercase letters on the same line indicate significant difference
(P < 0.05).

DISCUSSION However, when these authors used the slow freezing

Sperm cryopreservation plays an essential protocol for both bull and goat ejaculates, they did not
observe improvement in total motility parameters, in
role in the preservation of genetic material of endan-
accordance with the results of the present study.
gered species, as it allows for the storage of semen
Other authors did not observe improvement
for long periods of time. In addition, it facilitates
in the parameters of progressive motility when using
the management of animals, since they do not need
1 mg of CLC in goat semen and 2 mg in bull semen
to be transported over long distances, avoiding the
with the slow freezing protocol [16]. This differs from
stress caused by transportation, as well as avoiding
our work, as we found that 1 mg of CLC improved
the potential for contagion of sexually transmitted
the PM parameters, but not at the concentration of 2
diseases [10,13].
mg CLC.
However, despite these advantages, the cryo-
Different concentrations of CLC (0.5, 1, 1.5,
preservation process causes damage to sperm cells,
3, and 6 mg) were tested in the ejaculate of antelopes
in particular the plasma membrane. Studies show
(Nanger dama ruficollis). No differences in path veloc-
that, when added to the ejaculate alone, cyclodextrins ity values (VAP, μm/s) were observed after freezing/
stimulate the removal of cholesterol from the sperm thawing [25]. In the present study, we verified that
membrane. However, when they are preloaded with the concentration of 2 mg impaired sperm velocity,
cholesterol, they stimulate the incorporation therein showing lower VAP data when compared to the control
into the cell membranes. This occurs because the cy- group and group 1 (1 mg CLC). High concentrations
clodextrins have an internal hydrophobic core and a of cyclodextrin can cause an efflux of cholesterol from
high affinity for sterols [20]. the spermatozoa membrane, interfering with the value
Thus, several studies have reported improve- of VAP [8].
ments in sperm parameters in buffalo [22], bulls [5,26], In another work, the linearity (LIN, %) was
stallions [12,17] and sheep [11,15,18] after the process altered only with high concentrations of CLC (3 and
of sperm cryopreservation, with the addition of cyclo- 6 mg) [25]. In the present study, when we used a
dextrin to the ejaculate. maximum concentration of 2 mg of CLC, we did not
Authors tested fast freezing protocols using observe any interference of this parameter, similar to
direct freezing (vitrification) of the vane, with 2 h of that reported by these authors.
refrigeration at 5ºC and a concentration of 1 mg of The CLC used in stall refrigeration protocols
CLC in goat semen and 2 mg of CLC in bull semen. of stallions at 5°C at 24 and 48 h were able to inter-
They found that rapid freezing, associated with CLC, fere with the MT, MP, VAP, and RAP parameters. All
improved the total motile parameters, both in the semen parameters evaluated with refrigeration for 24 h, as
of the bulls and in the semen of the goats [15], contrary well as for 48 h, with the use of CLC, contributed to
to our study, in which we did not observe improvement an improvement in seminal quality. Concentrations
in this parameter, with the use of this concentration. of 1.5 and 2 mg of CLC were more effective than the

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J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

concentration of 1 mg. These authors observed that of cholesterol that high concentrations of cyclodex-
the concentration of 1 mg of CLC was more effective trin cause in spermatozoa [8]. In the present study,
than the control group (without addition of CLC) [12]. an improvement in plasma and acrosomal mem-
These results differ from our work, in which we used brane integrity was noted by adding 1 mg of CLC.
frozen samples of sheep semen and obtained better In the group receiving 2 mg of CLC, no difference
sperm parameters with the use of 1 mg of CLC com- was observed in relation to the control group, and
pared to 2 mg of CLC. in relation to group 1, group 2 did not present any
In a study using fresh sheep semen, authors significant differences.
induced oxidative stress by adding H 2O 2 to the The thermoresistance test (TTR) allows deter-
ejaculate before incubating for 12 h at 35ºC. These mination of the number of spermatozoa active after the
authors verified that cyclodextrin at 3 mg concentra- test and the fertilization rate, thus enabling the elimina-
tion was effective in protecting the sperm against the tion of inadequate semen samples, that is, those with a
deleterious effects of H2O2. They obtained superior final motility lower than 20% at the end of incubation
plasma membrane motility, viability, and integrity [14]. However, previous studies have stated that there
of the CLC-treated samples compared to the control is no correlation between this test and the pregnancy
group [19]. rate [2]. In the present study, we observed no difference
The superoxide anion (O2-) is a free radical in the TTR between the groups studied.
formed from molecular oxygen by the addition of an
CONCLUSION
electron. It is generated spontaneously, mainly in the
membrane of the mitochondria, by the respiratory chain In conclusion, the use of CLC at a concen-
and by flavoenzimes, lipoxygenases, and cicloxygen- tration of 1 mg was found to be effective for the
ases. It is a small reactive radical and does not have the improvement of parameters of sperm progressive
ability to penetrate the lipid membranes, acting only motility, rapid sperm percentage, and plasma and
in the compartment where it is produced [19]. In our acrosomal membrane integrity. In addition, the
study, we found a difference between the study group study group with 1 mg of CLC showed decreased
with 1 mg CLC and the control group. Thus, we suggest levels of superoxide anion formation, a free radical
that CLC may have a beneficial effect in stabilizing the detrimental to spermatozoa. The use of 2 mg of CLC
sperm plasma membrane. did not have any beneficial effect on the evaluated
In studies with sheep semen [3] tested the effect parameters, with a decrease in the sperm displace-
of different concentrations (0, 2, 4, and 6 mg) of CLC ment velocity.
added to vitamin E during cryopreservation and tested
MANUFACTURERS
their effects on plasma and acrosomal membrane. In the
Sigma Chemical Co. St. Louis, MO, USA.
1
tests performed, no differences in plasma membrane
Hamilton Thorne Research. Beverly, MA, USA.
2
integrity were observed between the control group and
Botupharma Animal Biotechnology. Botucatu, SP, Brazil.
3
the group in which 2 mg of CLC was added. However,
Becton Dickinson. Mountain View, CA, USA.
4
the highest concentrations (4 and 6 mg of CLC) were
IMV® Tecnologies. L’Aigle, Normandy, France.
5
harmful to the plasma membrane of spermatozoa.
Life Technologies. Carlsbad, CA, USA.
6
The percentage of sperm cells with the intact acro-
abcam. Cambridge, UK.
7
some did not differ between the control group and the
Institute Inc. Cary, NC, USA.
8
2 mg CLC group. Already in the study group with 4
mg of CLC, we found a decrease in the percentage of Acknowledgements. University Paranaense (UNIPAR) and
sperm cells with an intact acrosome. The group with Botupharma Biotecnologia Animal for financial support.
6 mg of CLC obtained worse results than the group Ethical approval. This study was approved by Ethics Commit-
that received 4 mg of CLC, in terms of percentage of tee on Animal Experimentation of the University of Umuarama,
intact acrosssoma. Paraná (UNIPAR): protocol nº 33704/2017, dated 08/07/2017.
Altering the integrity of the plasma mem- Declaration of interest. The authors report no conflicts of
brane and acrosome membrane, especially at high interest. The authors alone are responsible for the content and
concentrations of CLC, can be justified by the efflux writing of the paper.

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J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

REFERENCES

1 Argov-Argaman N., Mahgrefthe K., Zeron Y. & Roth Z. 2013. Variation in lipid profiles within semen compart-
ments - the bovine model of aging. Theriogenology. 80(7): 712-721.
2 Arruda R.P., Barnabe V.H., Alencar M.M. & Barnabe R.C. 1992. Avaliação de sêmen congelado de bovinos. Provas
lenta e rápida de termo-resistência: efeitos sobre a fertilidade. Brazilian Journal of Veterinary Research and Animal
Science. 29(1): 131-137.
3 Benhenia K., Rahab H., Smadi M.A., Benmakhlouf H., Lamara A., Idres T. & Iguer-Ouada M. 2018. Beneficial
and harmful effects of cyclodextrin-vitamin E complex on cryopreserved ram sperm. Animal Reproduction Science.
195: 266-273.
4 Blanch E., Tomás C., Graham J.K. & Mocé E. 2012. Response of boar sperm to the treatment with cholesterol-
loaded-cyclodextrins added prior to cryopreservation. Reproduction in Domestic Animals. 47(6): 959-964.
5 Boscarato A.G., Martins L.F., Pinho R.O., Silva Y.F.R.S., Papa F.O. & Macedo G.G. 2016. Efeito da adição de
ciclodextrina carregada com colesterol sobre a qualidade do sêmen congelado/descongelado de touros adultos da raça
Nelore. Revista Brasileira de Reprodução Animal. 40(3): 105-110.
6 Bucak M.N., Sarıözkan S., Tuncer P.B., Ulutaş P.A. & Akçadağ H.İ. 2009. Effect of antioxidants on microscopic
semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation. Small
Ruminant Research. 81(2-3): 90-95.
7 Chatterjee S. & Gagnon C. 2001. Production of reactive oxygen species by spermatozoa undergoing cooling, freez-
ing, and thawing. Molecular, Reproduction and Development: Incorporating Gamete Research. 59(4): 451-458.
8 Choi Y. H. & Toyoda Y. 1998. Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a
protein-free medium. Biology of Reproduction. 59(6): 1328-1333.
9 Crespilho A.M., Nichi M., Guasti P.N., Freitas-Dell’Aqua C.P., Sá Filho M.F., Maziero R.R. & Papa F.O. 2014.
Sperm fertility and viability following 48 h of refrigeration: evaluation of different extenders for the preservation of
bull semen in liquid state. Animal Reproduction Science. 146(3-4): 126-133.
10 Errandonea N., Fierro S., Viñoles C., Gil J., Banchero G. & Olivera-Muzante J. 2018. Short term protein sup-
plementation during a long interval prostaglandin-based protocol for timed AI in sheep. Theriogenology. 114: 34-39.
11 Graham J.K. 1996. Cryopreservation of stallion spermatozoa. Veterinary Clinics of North America: Equine Practice.
12(1): 131-147.
12 Hartwig F.P., Lisboa F.P., Monteiro G.A., Maziero R.R.D., Freitas-Dell’Aqua C.P., Alvarenga M.A. & Dell’Aqua
Jr. J.A. 2014. Use of cholesterol-loaded cyclodextrin: An alternative for bad cooler stallions. Theriogenology. 81(2):
340-346.
13 Lone S.A. 2018. Possible mechanisms of cholesterol-loaded cyclodextrin action on sperm during cryopreservation.
Animal Reproduction Science. 192: 1-5.
14 Luz S.L.N., Neves J.P. & Gonçalves P.B.D. 2000. Parâmetros utilizados na avaliação do sêmen congelado ovino para
inseminação laparoscópica. Brazilian Journal of Veterinary Research and Animal Science. 37(2): 141-145.
15 Mocé E., Purdy P.H. & Graham J.K. 2010. Treating ram sperm with cholesterol-loaded cyclodextrins improves
cryosurvival. Animal Reproduction Science. 118(2-4): 236-247.
16 Mocé E., Tomás C., Blanch E. & Graham J.K. 2014. Effect of cholesterol-loaded cyclodextrins on bull and goat
sperm processed with fast or slow cryopreservation protocols. Animal. 8(5): 771-776.
17 Moore A.I., Squires E.L. & Graham J.K. 2005. Adding cholesterol to the stallion sperm plasma membrane improves
cryosurvival. Cryobiology. 51(3): 241-249.
18 Motamedi-Mojdehi R., Roostaei-Ali Mehr M. & Rajabi-Toustani R. 2014. Effect of Different Levels of Glycerol
and Cholesterol Loaded Cyclodextrin on Cryosurvival of Ram Spermatozoa. Reproduction in Domestic Animals. 49(1):
65-70.
19 Naseer Z., Ahmad E., Aksoy M., Küçük N., Serin I., Ceylan A. & Kum C. 2015. Protective effect of cholesterol-
loaded cyclodextrin pretreatment against hydrogen peroxide induced oxidative damage in ram sperm. Cryobiology.
71(1): 18-23.
20 Navratil A.M., Bliss S.P., Berghorn K.A., Haughian J.M., Farmerie T.A., Graham J.K. & Roberson M.S. 2003.
Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdo-
mains is necessary for GnRH signaling to ERK. Journal of Biological Chemistry. 278(34): 31593-31602.

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J.P.M. Silva, A.F.B. Almada, J.S. Lima, et al. 2019. Action of Cholesterol-Loaded Cyclodextrin on Viability of Ram Semen.
Acta Scientiae Veterinariae. 47: 1685.

21 Purdy P.H. & Graham J.K. 2004. Effect of cholesterol-loaded cyclodextrin on the cryosurvival of bull sperm. Cryo-
biology. 48(1): 36-45.
22 Rajoriya J.S., Prasad J.K., Ramteke S.S., Perumal P., Ghosh S.K., Singh M. & Srivastava N. 2016. Enriching
membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa. Animal Reproduction Science. 164:
72-81.
23 Trevizan J.T., Carreira J.T., Carvalho I.R., Kipper B.H., Nagata W.B., Perri S. H.V. & de Koivisto M.B. 2018.
Does lipid peroxidation and oxidative DNA damage differ in cryopreserved semen samples from young, adult and aged
Nellore bulls? Animal Reproduction Science. 195: 8-15.
24 Watson P.F., Morris G.J. & Clarke A. 1981. The effects of cold shock on sperm cell membranes. In: Effects of low
temperatures on biological membranes. New York: Academic Press, pp.189-218.
25 Wojtusik J., Pennington P., Songsasen N., Padilla L.R., Citino S.B. & Pukazhenthi B.S. 2016. Pretreatment of
Addra gazelle (Nanger dama ruficollis) spermatozoa with cholesterol-loaded cyclodextrins improves cryosurvival.
Cryobiology. 73(3): 388-395.
26 Yadav H.P., Kumar A., Shah N., Chauhan D.S., Saxena A., Yadav S. & Swain D.K. 2017. Effect of cholesterol
loaded cyclodextrin supplementation on tyrosine phosphorylation and apoptosis like changes in frozen thawed Hariana
bull spermatozoa. Theriogenology. 96: 164-171.

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