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C60 Fullerenes Suppress Reactive Oxygen Species Toxicity Damage in Boar Sperm

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Abstract We report the carboxylated C60 improved the survival and quality of boar sperm during liquid storage at 4 °C and thus propose the use of carboxylated C60 as a novel antioxidant semen extender supplement. Our results demonstrated that the sperm treated with 2 μg mL−1 carboxylated C60 had higher motility than the control group (58.6% and 35.4%, respectively; P ˂ 0.05). Moreover, after incubation with carboxylated C60 for 10 days, acrosome integrity and mitochondrial activity of sperm increased by 18.1% and 34%, respectively, compared with that in the control group. Similarly, the antioxidation abilities and adenosine triphosphate levels in boar sperm treated with carboxylated C60 significantly increased (P ˂ 0.05) compared with those in the control group. The presence of carboxylated C60 in semen extender increases sperm motility probably by suppressing reactive oxygen species (ROS) toxicity damage. Interestingly, carboxylated C60 could protect boar sperm from oxidative stress and energy deficiency by inhibiting the ROS-induced protein dephosphorylation via the cAMP-PKA signaling pathway. In addition, the safety of carboxylated C60 as an alternative antioxidant was also comprehensively evaluated by assessing the mean litter size and number of live offspring in the carboxylated C60 treatment group. Our findings confirm carboxylated C60 as a novel antioxidant agent and suggest its use as a semen extender supplement for assisted reproductive technology in domestic animals.
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C60 Fullerenes Suppress Reactive Oxygen Species
Toxicity Damage inBoar Sperm
XinhongLi1*, LiruiWang1,2, HuanLiu1, JieliFu1, LinqingZhen1, YuhuaLi1,
YaozhongZhang3, YafeiZhang4*
* Xinhong Li, lixinhong7172@sjtu.edu.cn; Yafei Zhang, yfzhang@sjtu.edu.cn
1 Shanghai Key Laboratory ofVeterinary Biotechnology, School ofAgriculture andBiology, Shanghai Jiao
Tong University, Shanghai200240, People’sRepublicofChina
2 Institute ofNano Biomedicine andEngineering, Shanghai Engineering Research Centre forIntelligent
Diagnosis andTreatment Instrument, Department ofInstrument Science andEngineering, School
ofElectronic Information andElectrical Engineering, Shanghai Jiao Tong University, Shanghai200240,
People’sRepublicofChina
3 Department ofElectrical andComputer Engineering, Michigan State University, EastLansing, USA
4 Key Laboratory ofThin Film andMicrofabrication (Ministry ofEducation), Department ofMicro/Nano
Electronics, School ofElectronics, Information andElectrical Engineering, Shanghai Jiao Tong University,
Shanghai200240, People’sRepublicofChina
HIGHLIGHTS
Carboxylated C60 may be considered a novel antioxidant agent to be used as a semen extender supplement for assisted reproductive
technology.
Carboxylated C60 could enhance the motility parameters and characteristics of boar spermatozoa during liquid storage at 4 °C and
protect boar sperm from oxidative stress by inhibiting the reactive-oxygen-species–induced protein dephosphorylation.
Novel insights into the molecular mechanisms contributing to the protective effects of carboxylated C60 are provided.
ABSTRACT We report the carboxylated C60 improved the survival and
quality of boar sperm during liquid storage at 4°C and thus propose the
use of carboxylated C60 as a novel antioxidant semen extender supple-
ment. Our results demonstrated that the sperm treated with 2μgmL−1
carboxylated C60 had higher motility than the control group (58.6% and
35.4%, respectively; P ˂ 0.05). Moreover, after incubation with carboxy-
lated C60 for 10days, acrosome integrity and mitochondrial activity of
sperm increased by 18.1% and 34%, respectively, compared with that
in the control group. Similarly, the antioxidation abilities and adenosine
triphosphate levels in boar sperm treated with carboxylated C60 signifi-
cantly increased (P ˂ 0.05) compared with those in the control group. The presence of carboxylated C60 in semen extender increases sperm
motility probably by suppressing reactive oxygen species (ROS) toxicity damage. Interestingly, carboxylated C60 could protect boar sperm
from oxidative stress and energy deficiency by inhibiting the ROS-induced protein dephosphorylation via the cAMP-PKA signaling pathway.
In addition, the safety of carboxylated C60 as an alternative antioxidant was also comprehensively evaluated by assessing the mean litter size
and number of live offspring in the carboxylated C60 treatment group. Our findings confirm carboxylated C60 as a novel antioxidant agent
and suggest its use as a semen extender supplement for assisted reproductive technology in domestic animals.
KEYWORDS Carboxylated C60; Semen preservation; Oxidative stress; Motility; Protein dephosphorylation
ISSN 2311-6706
e-ISSN 2150-5551
CN 31-2103/TB
ARTICLE
Cite as
Nano-Micro Lett.
(2019) 11:104
Received: 30 September 2019
Accepted: 29 October 2019
© The Author(s) 2019
https://doi.org/10.1007/s40820-019-0334-5
Nano-Micro Lett. (2019) 11:104 104 Page 2 of 17
https://doi.org/10.1007/s40820-019-0334-5
© The authors
1 Introduction
Fullerenes are typical zero-dimensional carbon nanoma-
terials and have been widely used for various purposes,
including electrocatalysts [1, 2], energy storage [3, 4], pho-
todetectors [5, 6], and solar cells [79]. Recently, carboxy-
lated C60 has been shown to be one of the most important
carbon nanoparticle derivatives and extensively studied in
biomedical applications [1012]. Notably, carboxylated
C60 is known to be a putative antioxidant [13, 14] with a
powerful bio-antioxidant ability, which can prevent tissue
dysfunction induced by oxidative stress [1517]. Previous
studies have demonstrated that in various invivo and invitro
pathological models the multidirectional positive biological
effects of carboxylated C60 are mediated by its antiradical
activity [18, 19]. Additionally, many beneficial effects of
carboxylated C60 have been observed even at extremely low
doses. Altogether, these data suggest that carboxylated C60
is a promising tool for the control of reactive oxygen species
(ROS)-dependent pathological damage, including brain and
immune system diseases. Despite substantial experimental
results highlighting the positive effect of carboxylated C60
on biological systems invivo and invitro, few attempts have
been made to clarify the molecular mechanism of its antioxi-
dant action. In particular, the effects of carboxylated C60 on
mammalian sperm motility and oxidative stress state remain
unexplored.
Although carboxylated C60 is a potential non-enzymatic
antioxidant, its practical production is still limited, espe-
cially in the biological field. In the modern pig husbandry,
artificial insemination (AI) is extensively used worldwide
[20]. Numerous studies have shown that the semen used
for AI must be diluted with suitable extenders and should
be preserved at 17°C or in liquid nitrogen. However, the
major drawback is the limited lifespan of the sperm, which
can only be preserved for 5days invitro [21]. Additionally,
although cryopreservation can prolong the semen storage
time invitro, only 1% of the artificial inseminations with
frozen-thawed boar sperm is successful worldwide [22].
Thus, to avoid boar sperm from being subjected to freezing
injury due to storage in liquid nitrogen and to prolong sperm
survival time invitro, many researchers are committed to
studying liquid storage at 4–5°C.
Due to a low cholesterol/phospholipid ratio, boar sper-
matozoa are particularly vulnerable to low temperatures,
which can lead to oxidative stress due to the inappropriate
formation of ROS [23]. It has previously been shown that
overproduction of ROS has an adverse impact on sperm
motility, viability, and morphology [24]. Therefore, regu-
lation of the generation of excessive ROS to eliminate the
accompanying ROS-mediated damage to the structural
integrity of the sperm plasma and acrosome, and mainte-
nance of the stability of the genetic material during exter-
nal storage of boar semen [25], are serious challenges that
urgently require solutions. In addition, because of the lim-
ited ability of sperm to establish a powerful defense sys-
tem of antioxidants, substantial efforts have been invested
to search for exogenous antioxidants to prevent oxidative
damage [26, 27]. To date, studies have suggested that the
addition of antioxidant agents to semen can protect sperm
from ROS attack and improve sperm quality parameters
[24, 28, 29]. However, the feasibility of the application
of carboxylated C60 in the reproductive field of animal
production is completely unknown, and in particular, little
is known about the protective mechanisms of antioxidants
with respect to ROS toxicity in sperm preserved at low
temperatures. Moreover, the effect of exposure of sperm
to carboxylated C60 on AI procedures has not been inves-
tigated. Therefore, it is extremely crucial to investigate
whether boar sperm exposed to antioxidant materials pre-
pared using carboxylated C60 can be used in current AI
procedures.
The present study aimed to explore the effect of carboxy-
lated C60 on spermatozoa, with a particular focus on the
sperm functionality, and whether carboxylated C60 could
exert significant antioxidant effects in boar spermatozoa
during invitro preservation at 4°C. Herein, we performed
a systematic assessment of the protective role of carboxy-
lated C60 against ROS oxidative damage in boar sperm pre-
served at 4°C. The tested viability and functional parame-
ters included boar sperm motility, membrane and acrosome
integrity, mitochondrial membrane potential, cAMP and
ATP levels, ROS production, antioxidation indexes, and the
protein phosphorylation level. Additionally, the safety of
carboxylated C60 as an alternative antioxidant was also eval-
uated. Our results suggested that carboxylated C60 exerted
Nano-Micro Lett. (2019) 11:104 Page 3 of 17 104
1 3
no obvious toxicity toward boar sperm cells. Given these
data, the good biocompatibility and antioxidative capacity
of carboxylated C60 demonstrated its great potential used
as a semen extender. Simultaneously, this study provides a
theoretical basis for broadening the use of carbon nanoma-
terials as antioxidants in the field of livestock and animal
breeding.
2 Experimental Section
2.1 Preparation ofHydrated C60 Fullerene
C60 samples with a purity of > 99.98% were obtained from
Shanghai Jiao Tong University. The carboxylated C60 was
prepared using the method described by Andrievsky, which
is based on the transfer of fullerene from an organic solu-
tion into the aqueous phase by ultrasonic treatment, as
described elsewhere [16]. The averaged FTLA concentra-
tion/size of carboxylated was determined as shown in Fig.
S1.
2.2 Animals
All the animal experiments strictly adhered to the stand-
ards of the institutional guidelines for ethics in animal
experimentation (Rule number 86/609/EEC-24/11/86).
All the experimental procedures were approved by the
Animal Ethics Committee of Shanghai Jiao Tong Univer-
sity. In our experiments, a total of 12 healthy and sexually
mature Duroc boars (2–3years old) with proven fertil-
ity were selected from Shanghai Sunsing Livestock Co.,
Ltd. (Shanghai, China). All the chemical products were
obtained from Sigma-Aldrich (St. Louis, MO, USA) unless
otherwise mentioned.
2.3 Semen Collection andProcessing
After collection from twelve boars, a computer-assisted
semen analysis (CASA) system (Hamilton Thorne
Research, Beverly, MA, USA) was used to evaluate
sperm motility. Only ejaculates with motility > 70%
were included in this study. Each ejaculate was diluted
in basal medium containing the following compounds:
27.5mg mL−1 d-fructose, 6.9 mg mL−1 trisodium
citrate dihydrate, 2.35mgmL−1 ethylenediaminetet-
raacetic disodium salt, 1.0mgmL−1 sodium hydro-
gen carbonate, 2.9mgmL−1 citric acid monohydrate,
5.65mgmL−1 tris (hydroxymethyl) aminomethane,
2mgmL−1 skim milk, and 0.2mgmL−1 amikacin sul-
fate. Skim milk (Foodhold USA LLC, Landover, MD,
USA) was prepared in advance by sonication on ice
for 40min using an ultrasonic cell crusher (Hielscher
Ultrasonics Gmbh, UP50H, Germany) (amplitude: 80%,
cycle: 0.5).
In our experiments, we designed four experimental
groups. Group I: carboxylated C60 was added to a final
concentration of 1, 2, 3, or 4μgmL−1 in the basal medium.
In this group, we analyzed sperm motility, acrosome integ-
rity, mitochondrial membrane potential, antioxidation abil-
ity, ATP level, and protein phosphorylation. Group II: dif-
ferent concentrations of H2O2 (0, 10, 100, 200, or 300μM)
were added to the basal medium that contained 2μgmL−1
C60. Group III: different concentrations of C60 (0, 1, 2, 3,
or 4μgmL−1) were added to the basal medium that con-
tained 100μM H2O2. Group IV: 100μM H2O2 was added
to the basal medium in the presence or absence of 1.0mM
dbcAMP, or 2μgmL−1 carboxylated C60 was added to
the basal medium in the presence or absence of 0.1mM
H-89. In groups II, III and IV, we assessed sperm protein
phosphorylation. All the semen samples reached a final
concentration of 1 × 108 cells mL−1 and were transported
to the laboratory at 37°C within 20min. Upon arrival
at the laboratory, the semen samples were immediately
stored at 4°C in an incubator sterilized with 75% alco-
hol. After 2–3h, the temperature of the semen samples
slowly decreased to 4°C. During the incubation process,
the samples were shaken gently three times a day to pre-
vent precipitation.
2.4 Sperm Quality Assessment
2.4.1 Sperm Motility
On each experimental day (days 3, 5, 10, and 15), 500μL
of each sample was taken from each bottle and incubated
in a water bath at 37°C for 30min. After incubation, 5μL
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© The authors
of every 500-μL semen sample was placed on pre-warmed
disposable counting chamber slides (Leja, Nieuw Vennep,
the Netherlands). The computer-assisted semen analysis
(CASA) system was used to measure sperm kinetic param-
eters [30]. The analysis was performed with images from
several fields containing at least 200 spermatozoa per sam-
ple, and the sample analysis was based on the examination
of 25 consecutive digitalized images.
2.4.2 Acrosome Integrity
Acrosome integrity was assessed after staining the sperm
with fluorescein isothiocyanate-conjugated peanut agglutinin
(PNA-FITC) as a marker of acrosomal status and propidium
iodide (PI) as an indicator of live or dead sperm. On each
experimental day (days 0, 3, 5, 10, and 15), 30μL of each
sample was taken from each bottle and collected by centrifu-
gation at 2500×g for 5min. After the removal of the super-
natant, the pellets were suspended in 500μL of phosphate-
buffered solution (PBS) (8g NaCl, 0.2g KH2PO4, 1.15g
Na2HPO4, and 0.2g KCl, diluted with double-distilled H2O
to 1L). The pellets were resuspended and incubated in the
dark at 37°C for 20min with 2μl PNA-FITC (100μgmL−1)
and 2 μL PI (1mgmL−1). After incubation, each sample was
centrifuged (2000×g, 5min) and washed once with PBS.
Then, 500μL of PBS was added to resuspend each sample
before flow cytometry analysis (Beckman Coulter Ltd., Brea,
CA, USA) [31]. The results were expressed as the percent-
age of viable spermatozoa with an intact acrosome (FITC
negative/PI negative).
2.4.3 Mitochondrial Membrane Potential
The specific probe 5,5,6,6-tetrachloro-1,1,3,3-
tetraethylbenzimidazolyl carbocyanine iodide (JC-1) (Beyo-
time Institute of Biotechnology, Shanghai, China) was used
to test the variation in mitochondrial membrane potential
ψm). The cells were stained with 0.5mL JC-1 working
solution (50µL JC-1 (200 ×), 8mL DDH2O, 2mL JC-1
staining solution (50 ×) and incubated in the dark at 37°C
for 30min. Following incubation, the samples were washed
three times with 1 × assay buffer (2mL JC-1 staining solu-
tion (50 ×, 8mL double-distilled H2O), and the observed
fluorescent signals were recorded using flow cytometry [32].
The FL-1 channel was used to detect JC-1 monomers and
shows the number of sperm cells at a high membrane poten-
tial. The FL-2 channel was used to detect JC-1 aggregates
and show the number of sperm cells at a low membrane
potential. FL2/FL1 served as the value of Δψm [33].
2.4.4 Total ROS (tROS) Assay
The tROS levels of each semen sample were evaluated
using the probe 2, 7-dichloride-hydrofluorescein diacetate
(DCFH-DA, Beyotime Institute of Biotechnology, Nanjing,
China). Intracellular DCFH-DA was oxidized by ROS to
produce dichlorofluorescein with strong fluorescence. On
each experimental day (days 0, 3, 5, 10, and 15), the semen
samples were washed three times with PBS, resuspended,
and incubated with 10μM DCFH-DA at 37°C in the dark
for 30min. The fluorescence intensity could be conveni-
ently monitored using a fluorescence spectrophotometer at
Ex/Em = 485/535nm [34].
2.4.5 Total Antioxidative Capacity (T‑AOC) Activity
Assay
T-AOC activity was quantified using a T-AOC assay kit
(Nanjing Jiancheng Bioengineering Institute, Jiangsu,
China). The sperm samples were washed three times
and resuspended in DDH2O. The suspension was lysed
ultrasonically (20kHz, 750W, operating at 40% for 3s,
off for 5s, 5 cycles) on ice and centrifuged (12,000×g,
10min)at 4°C. Supernatants were collected and mixed
with the reaction buffer. Finally, a spectrophotometer was
used to measure the T-AOC activity at 520nm [35]. The
T-AOC activity of each semen sample was converted into
units per mL of total protein in spermatozoa and expressed
as U mL−1.
2.4.6 Malondialdehyde (MDA) Content Assay
We used an MDA test kit (Nanjing Jiancheng Bioengineer-
ing Institute, Jiangsu, China) to measure the MDA content
according to the manufacturer’s protocol. Briefly, on each
experimental day (days 0, 3, 5, 10, and 15), the extracts of
sperm were prepared by sonication (20kHz, 750W, oper-
ating at 40%, on 3s, off 5s, 5 cycles) in ice-cold buffer
[50mM Tris–HCl (pH 7.5), 5mM EDTA, and 1mM DTT].
The lysed cells were centrifuged (12,000×g, 10min) to
Nano-Micro Lett. (2019) 11:104 Page 5 of 17 104
1 3
remove debris. Finally, the change in absorbance at 532nm
was measured using a spectrophotometer [35]. The sample
MDA levels were recorded as nmolmL−1.
2.5 Measurement ofATP Content andSafety
Evaluation ofCarboxylated C60
The ATP concentrations were measured using a biolumi-
nescence ATP assay kit. On each experimental day (days 0,
3, 5, 10, and 15), 1mL of each sample was collected from
each bottle and washed twice with PBS. The lysis buffer
was then added to the semen sample to extract the intracel-
lular ATP. The luciferase reagent was mixed with the ATP
extracts and serial dilutions of the ATP standard solution,
and a luminometer (Thermo Fisher Evolution 300) was used
to capture the fluorescent signals. The fluorescence values
were compared with those of the standard ATP dilutions
[36]. The safety of carboxylated C60 was evaluated by the
conventional artificial insemination and reproductive index
of sows.
2.6 SDS‑PAGE andImmunoblotting
One milliliter of each sample was collected from each bot-
tle and centrifuged at 12,500×g for 6min for four times;
the samples were resuspended in 100μL 5 × sample buffer
and boiled for 4min. The boiled samples were centrifuged
(13,500×g, 10min), and the supernatant was transferred to
a new centrifuge tube. Aliquots of 10% β-mercaptoethanol
were added to the samples, and the resulting sample mixture
was boiled again for 3min. The extracted sperm proteins
were quantified by spectrophotometry (NanoDrop 2000,
Thermo Scientific, USA) [27].
The extracted sperm proteins were resolved by SDS-
PAGE. The resolved proteins were transferred onto PVDF
membranes (Merck Millipore, Germany) at 90V for 2h on
ice. The membranes were blocked with 1% BSA or 5% skim
milk in T-TBS at room temperature for 1h. T-TBS (1 ×)
was then used to wash the membranes three times. After
washing, the membranes were immunoblotted with anti-
P-PKA antibody (Cell Signaling Technology, Cat# 9624,
clone 100G7E) or anti-phosphotyrosine antibody (Millipore,
Cat# 05-321, clone 4G10) followed by overnight incubation
at 4°C. The membranes were washed twice with T-TBS
(1 ×) and then incubated with the corresponding secondary
antibodies for 1h at room temperature. An enhanced chemi-
luminescence ECL-plus kit (GE Healthcare Worldwide,
USA) was used to develop the signals, which were detected
using a ChemiScope 3300mini-integrated chemilumines-
cence imaging system (CLINX, China). For all the experi-
ments, the molecular weights of the sperm proteins are indi-
cated in kDa [27].
2.7 Immunofluorescence
The semen samples were collected, centrifuged at 800×g for
10 min, and resuspended in 500μL of PBS. A total of 20μL
of each semen sample was placed on slides, smeared, and
air-dried for 1h. Then, 3.7% formaldehyde in PBS was used
to fix the sperm for 20min. After washing three times with
PBS, the sperm were permeabilized with 0.5% Triton X-100
in PBS for 10min. The semen samples were then washed
five times with PBS and blocked with 1% BSA in PBS for
2h at room temperature. After the slides were washed three
times, the sperm were incubated with the anti-P-PKA anti-
body or anti-phosphotyrosine antibody at 4°C overnight.
After incubation, the sperm were washed with PBS and
incubated with Alexa 555-conjugated anti-rabbit antibody
(Molecular Probes, Cat# A-21428) or Alexa 488-conjugated
anti-mouse antibody (Molecular Probes, Cat# A-11001) in
1% BSA in PBS for 2h at room temperature; these solutions
contained Alexa 488-conjugated PNA (Molecular Probes,
Cat# L-21409) (1:100) for staining acrosomes and DAPI
(1:100) for detection of the sperm nuclei. After 2h of incu-
bation, the sperm were washed four times and mounted on
slides with an antifade solution. Finally, a confocal fluo-
rescence microscope (Leica, TCS, SP5) equipped with a
400 × objective was used to examine the sperm samples [27].
2.8 Statistical Analysis
All data are indicated as the mean ± standard error of the
mean (SEM). The variances were first analyzed using a
homogeneity test. If the data met the assumption of homo-
scedasticity, the significance of differences in means was
determined by one-way analysis of variance (ANOVA) fol-
lowed by an LSD t test for multigroup comparisons. Oth-
erwise, significance was determined by the Tamhane’s T2
test. All the statistical analyses were performed using SPSS
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17.0 software. A P value < 0.05 was considered statistically
significant.
3 Results andDiscussion
3.1 The Effect ofCarboxylated C60 onBoar Sperm
Quality
The effects of different concentrations of carboxylated C60
supplementation on boar sperm motility during liquid stor-
age at 4°C are shown in Fig.1a. The results indicated that
supplementation with 1, 2, 3, or 4μgmL−1 carboxylated
C60 could significantly improve the sperm motility param-
eters when compared with those of the control. Moreover,
no significant difference was observed among the differ-
ent treatment groups (P > 0.05). Total motility (MOT) and
progressive motility (PRO) showed higher levels through-
out the entire storage time in the treatment groups than in
the control group (P < 0.05) (Fig.1a, b). Moreover, since
an intact acrosome in viable sperm is crucial to perform
the acrosome reaction, acrosome integrity is another key
performance indicator to assess sperm quality. As shown
in Fig.1c, the percentage of viable spermatozoa with an
intact acrosome significantly increased in the carboxylated
C60-treated group compared with that in the untreated group
(P < 0.05). Additionally, throughout the experimental peri-
ods, with regard to acrosome integrity, the 2μg/mL-carbox-
ylated C60-treated group performed better than the 3μg/mL-
or 4μg/mL-carboxylated C60-treated groups. After 15days
of preservation, the percentage of acrosome integrity in the
2μg/mL-carboxylated C60-treated group was higher than
in the 1, 3, and 4μg/mL-carboxylated C60-treated groups
(P < 0.05). Taken together, supplementation with 2μgmL−1
carboxylated C60 had the strongest protective effect on the
sperm acrosome.
3.2 The Effect ofcarboxylated C60 onBoar Sperm
Antioxidation Ability
Previous studies have demonstrated that sperm motility,
acrosome integrity, and lipid peroxidation are more sensi-
tive indicators of oxidative stress [37, 38], and ROS levels
can be managed by the addition of ROS-scavenging antioxi-
dants [39]. Therefore, the protection of spermatozoa against
oxidative stress by antioxidant supplementation is currently
pursued [40]. To verify the potential protective effects of
carboxylated C60 against oxidative damage, we analyzed the
effects of carboxylated C60 in the oxidative and antioxida-
tive states by measuring the tROS level, T-AOC activity,
and MDA content. As shown in Fig.2, when compared
with the control group, T-AOC activity in the carboxylated
C60-treated groups was significantly increased after 15days
of preservation (P < 0.01), while the MDA content and tROS
level were dramatically decreased (P < 0.01). Meanwhile,
the 2μg/mL-carboxylated C60-treated group had lower total
ROS and higher T-AOC than the 4μg/mL-carboxylated
C60-treated group (P < 0.05). Given the above findings, it is
reasonable to surmise that carboxylated C60 treatment could
maintain the antioxidative capacity of boar sperm and sup-
press the accumulation of ROS and MDA.
90
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Total motility MOT (%
)
0357
Periods of preservation (day)
10 15
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10
5
Progressive motility PRO (%
)
0357
Periods of preservation (day)
10 15
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20
Percentages of viable spermatozoa
with intact acrosome(%
)
035
Periods of preservation (day)
10 15
Control
1 µg mL
−1
2 µg mL
−1
3 µg mL
−1
4 µg mL
−1
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Fig. 1 Effects of carboxylated C60 on motility, acrosome integrity, and mitochondrial membrane potential of boar semen preserved at 4°C for
half a month. a Sperm motility parameters (MOT), b progressive motility parameters (PRO), c acrosome integrity. Each experiment was per-
formed at least six times and subjected to statistical analysis. Different lowercase letters within the same day demonstrate significant differences
(P < 0.05), whereas the same lowercase letters denote insignificant differences
Nano-Micro Lett. (2019) 11:104 Page 7 of 17 104
1 3
In the last decade, studies have indicated that carboxylated
C60, due to the physicochemical properties of its spherical
cage-like molecule built exclusively from carbon atoms,
is able to scavenge reactive oxygen species. Carboxylated
C60 can efficiently protect the central nervous system from
oxidative stress-induced damage [17]. Likewise, it can pro-
tect DNA from ionizing radiation-induced oxidative dam-
age invitro [16]. In particular, it is worth mentioning that
carboxylated C60 has been demonstrated to reduce diabetes-
induced oxidative stress and associated complications and
testicular dysfunction [15]. Consistent with these results,
we also determined that carboxylated C60 could protect boar
sperm against oxidative damage during liquid storage at 4°C.
This finding could explain why carboxylated C60 exhibited
antioxidant capacity at low concentrations and small doses,
further providing a theoretical basis for the present study.
Hence, the obtained data sufficiently expand our knowl-
edge concerning the potential involvement of fullerene nano-
structures in processes that occur in sperm at 4°C invitro.
Indeed, based on their antioxidant properties, fullerenes have
been shown to have protective effects in various cell types
[4143] and within multifarious disease contexts [4447].
It is worth mentioning that carboxylated C60 has recently
been demonstrated to be able to inactivate free radicals
(ROS) with values that substantially exceed the theoretically
expected level. Fullerene C60 has been observed to exert pro-
nounced testicular-protective efficacy in preventing oxidative
stress induced by doxorubicin [48]. This result is consistent
with previous studies that have reported that carboxylated
C60 prevents Fe2+/ascorbate-induced oxidative stress in goat
epididymal spermatozoa invitro [49]. Although much atten-
tion has been given to the effects of carboxylated C60 on the
**
**
**
**
** **
**
**
**
**
**
**
**
100
80
60
40
20
0
Total ROS (ε)
Control1
C60 (µg mL−1)
234
(c)
(b)(a)
8
6
4
2
0
T-AOC (U mL−1)
25
20
15
10
5
0
MDA (nmol mL−1)
Control1
C60 (µg mL−1)
234
Control 1
C
60
(µg mL−1)
234
Fig. 2 Dot plots of the tROS level, T-AOC activity, and MDA content, and ATP levels in the control and treatment groups after 15days of treat-
ment. a tROS level, b T-AOC activity, c MDA content (n = 6, *P < 0.05, **P < 0.01)
Nano-Micro Lett. (2019) 11:104 104 Page 8 of 17
https://doi.org/10.1007/s40820-019-0334-5
© The authors
reproductive health in experimental animals, it has not been
investigated whether carboxylated C60 has a protective effect
on sperm. Therefore, the present study remains the first to
reveal carboxylated C60 as a protective antioxidant against
ROS toxicity in boar sperm preserved at low temperatures.
3.3 The Effect ofCarboxylated C60 onBoar Sperm
ATP Levels
Mitochondrial membrane potential (Δψm) is used to char-
acterize the structural and functional integrity of mito-
chondria [50]. As indicated in Fig.3a, after 15days of
preservation, the Δψm values of the treatment groups were
higher than that of the control group (P < 0.01). Moreover,
with the increasing invitro storage time, the groups treated
with 1 and 2μgmL−1 carboxylated C60 showed higher
Δψm values than those of the 3 and 4μg/mL-carboxy-
lated C60 treatments (P < 0.05 and P < 0.01, respectively);
no significant difference was observed between the two
groups.
It is generally accepted that intracellular ATP, which
is an indicator of sperm motility, is required to maintain
sperm flagellar movement and signal transduction [36,
51]. Therefore, the ATP levels of boar sperm treated with
different concentrations of carboxylated C60 were investi-
gated. The results indicated that the untreated group had
the lowest ATP level among all the groups with increasing
invitro storage time (P < 0.01) (Fig.3b). This result was
in agreement with the sperm motility. Moreover, the ATP
level was higher in both 1 and 2μg/mL-treated groups than
that in untreated group, and no difference was observed
between these two treatment groups. Furthermore, the
safety of carboxylated C60 as an alternative antioxidant
was comprehensively evaluated using artificial insemina-
tion. Regardless of the mean litter size or the mean num-
ber of live offspring, the carboxylated C60 treatment group
showed an increasing trend, although there was no signifi-
cant difference with respect to the control group (Fig. S2).
3.4 The Effect ofCarboxylated C60 onProtein
Phosphorylation
In addition to the supply of ATP, protein phosphorylation
is another factor affecting sperm motility [52]. It has been
well documented that phosphorylation of PKA substrates
(P-PKAs) and protein tyrosine phosphorylation (PTP)
are closely related to the cAMP-PKA signaling pathway,
which is responsible for the regulation of sperm motil-
ity [53]. Although many previous studies have examined
protein phosphorylation in sperm, whether carboxylated
C60 impacts sperm protein phosphorylation modifications
is still not completely understood. Thus, we measured
protein phosphorylation in boar sperm and found that it
was affected by a series of carboxylated C60 concentra-
tions. The results showed that the level of P-PKAs was
noticeably higher in the treatment groups than that in the
control group (P < 0.05) (Fig.4a, b). In addition, com-
pared with the other treatment groups, supplementation
**
**
**
**
**
**
**
*
**
*
*
** ** *
**
2.5
2.0
1.5
1.0
0.5
0.0
FL2/FL1
Control1
C60 (µg mL−1)
2
(b)(a)
34
5.0
4.5
4.0
3.5
3.0
2.5
Control
ATP level (nmol/10
8 sperm)
1
C60 (µg mL
−1
)
234
Fig. 3 Dot plots of the mitochondrial membrane potential and ATP in the control and treatment groups after 15days of treatment. a Mitochon-
drial membrane potential, b ATP level (n = 6, *P < 0.05, **P < 0.01)
Nano-Micro Lett. (2019) 11:104 Page 9 of 17 104
1 3
with 2μgmL−1 carboxylated C60 had the most remarkable
impact on P-PKAs (P < 0.05) (Fig.4a, b). We also meas-
ured the effect of carboxylated C60 on PTP and obtained
approximately similar findings (Fig.4c, d). The changes in
PTP in the 1, 2, and 3μg/mL-carboxylated C60 treatment
groups were, as expected, greatly enhanced compared with
that in the control group and the 4μg/mL-carboxylated C60
treatment group (P < 0.05). Moreover, the most significant
increase in the level of PTP was observed in sperm incu-
bated with 1 or 2μgmL−1 carboxylated C60 (P < 0.05)
(Fig.4c, d). Consistently, these results demonstrated that
the optimal dose of carboxylated C60 was 2μgmL−1.
Additionally, immunolocalization of P-PKAs and tyrosine-
phosphorylated proteins in boar sperm also demonstrated
the promotion of protein phosphorylation by carboxylated
C60, in accordance with the western blot results (Fig.5).
In the present study, our results showed that the phos-
phorylation level decreased alongside decreased sperma-
tozoa motility, indicating that the phosphorylation levels
in sperm protein positively correlated with the motility
parameter [54].
In addition, to further explore the molecular mecha-
nism underlying the protective roles of carboxylated C60
from the perspective of protein phosphorylation, we ana-
lyzed the trend in P-PKAs between the control and 2μg/
mL-carboxylated C60 treatment groups after specific peri-
ods of preservation. As described in Fig.4e, f, the results
revealed a decrease in the level of P-PKAs in both groups
as the incubation progressed. Interestingly, a higher level
of P-PKAs was detected in the 2μg/mL-carboxylated C60
treatment group than in the control group after the same
period of preservation. Therefore, our results indicate that
1234
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250
KDa
150
100
75
50
37
25
20
15
10
β-tubulin
C60(μg ml−1)_
12345678 910
250
KDa
150
100
75
50
37
25
20
15
10
β-tubulin
Preservation days
C
60
(μg ml−1)_____22222
05710150 5710 15
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12 34
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(f)
(e)
(c)
(d)
(b)
(a)
45678910
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250
KDa
150
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50
37
25
20
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β-tubulin
C60(μg ml−1)_
**
*
*
3.0
2.7
2.4
2.1
1.8
1.5
1.2
0.9
0.6
0.3
0
Relative amount
12345
**
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
1.2
1.0
0.8
0.6
0.4
0.2
0
Relative amount
Relative amount
12 345
**
*
*
*
**
*
Fig. 4 Western blot analysis of P-PKAs and PTP under different concentrations of carboxylated C60. Western blot analysis was performed using
a, b an anti-phospho-PKA substrate antibody, c, d an anti-phosphotyrosine antibody, e, f an anti-phospho-PKA substrate antibody. The bands
used for histogram quantification are labeled #. β-Tubulin was used as an internal control. The experiment was performed at least three times; the
presented image is representative and repeatable. (n = 3,*P < 0.05)
Nano-Micro Lett. (2019) 11:104 104 Page 10 of 17
https://doi.org/10.1007/s40820-019-0334-5
© The authors
carboxylated C60 affected protein phosphorylation, at least in
part, by inhibiting protein dephosphorylation in boar sperm.
3.5 Carboxylated C60‑Induced Promotion ofProtein
Phosphorylation andAntioxidative Capability
It is noteworthy that sperm proteins are targets of redox-
dependent modifications, which seem to play a much greater
role, leading, depending on the levels of ROS, either to the
activation/inactivation of signaling pathways that are impor-
tant for sperm physiology or to the oxidative damage and
impairment of vital functions [55]. Moreover, it has been
previously shown that suitable ROS levels are essential for
the level of protein phosphorylation [56]. Low levels of
ROS induce protein phosphorylation [57]. In contrast, high
levels of ROS inhibit the synthesis of sAC, reduce intra-
cellular cAMP levels, and inhibit protein phosphorylation
[58]. To examine the effects of intracellular ROS on protein
phosphorylation in boar sperm, we used the oxidant H2O2
to simulate mitochondrial ROS. The results showed that
as the H2O2 concentration increased, the level of P-PKAs
decreased in a dose-dependent manner (Fig.6a, b). When
the H2O2 concentration was increased to 300μM, the level
of P-PKAs became very low. In contrast, the level of tyros-
ine phosphorylation decreased as the H2O2 concentration
increased up to 100μM and then remained stable at H2O2
concentrations > 100μM (Fig.6c, d). To further test whether
carboxylated C60 could eliminate excess mitochondrial ROS
to regulate protein phosphorylation, we performed another
experiment using H2O2. As described in Fig.6e–h, when
1, 2, 3, or 4μgmL−1 carboxylated C60 was added to the
basal medium in the presence of 100μM H2O2, the levels of
P-PKAs and PTP were all noticeably higher than in the con-
trol. Typically, 2μgmL−1 might be the optimum concentra-
tion of carboxylated C60 to restore protein phosphorylation
levels. These data suggested that the antioxidative ability
of carboxylated C60 prevented H2O2 from inhibiting protein
phosphorylation.
To further investigate whether the promotion of protein
phosphorylation by different concentrations of carboxy-
lated C60 occurred via the cAMP-PKA signaling pathway,
MergeDAPIPNACandidate
a1 a2 a3 a4
b1 b2 b3 b4
MergeCandidate DAPI
d1 d2 d3
c1 c2 c3
Fig. 5 Immunolocalization of phosphorylated PKA substrates (a1–4 are the control; b1–4 are in the presence of 2μg mL−1 carboxylated C60)
and tyrosine-phosphorylated (c1–3 are the control; d1–3 are in the presence of 2μgmL−1 carboxylated C60) proteins in boar sperm. Sperm were
incubated in the basal medium in the presence or absence of 2μg mL−1 carboxylated C60 with primary anti-P-PKAs and anti-phosphotyrosine
antibody. PNA (peanut agglutinin) was applied to stain the acrosome and DAPI to stain the nuclei of sperm. The merged images are also shown.
The experiment was performed at least three times, and the image presented is representative and repeatable. Sperm cells were visualized using a
confocal laser scanning microscope (× 400)
Nano-Micro Lett. (2019) 11:104 Page 11 of 17 104
1 3
the sperm were cultured with the cAMP analog dibutyryl-
cAMP (dbcAMP) in the presence or absence of 100μM
H2O2, or the PKA inhibitor H-89 in the presence or absence
of 2μgmL−1 carboxylated C60. The results showed that inhi-
bition of protein phosphorylation by 100μM H2O2 could
be reversed by the addition of 1.0mM dbcAMP (Fig.7).
In addition, protein phosphorylation was decreased by sup-
plementation with H-89 compared with that in the carboxy-
lated C60-treated group (Fig.7). The results indicated that
carboxylated C60 affected protein phosphorylation, at least in
part through the cAMP-PKA pathway in boar sperm.
To our knowledge, this is the first exploration of the
molecular mechanism underlying the protective action
of carboxylated C60 against ROS toxicity, which results
from the oxidative stress and energy deficiency, by inhib-
iting the protein dephosphorylation caused by ROS via
the cAMP-PKA signaling pathway. The data suggest that
carboxylated C60 nanoparticles may eliminate ROS from
sperm cells, resulting in reduced levels of intracellular
ROS, thus preventing mitochondrial damage and improv-
ing the motility parameters. Importantly, these discoveries
contribute to a more comprehensive view of the molecular
mechanisms underlying the protective effects of exogenous
antioxidants on sperm and indicate the practical feasibility
of using carboxylated C60 as a boar semen extender sup-
plement for assisted reproductive technology.
1234
22 22212345
010100 200 300
Relative amount
5
250
KDa
150
100
75
50
37
25
20
15
10
β-tubulin
C60(μg mL−1)
H2O2(μM)
(a)
(b)
*
**
*
1.2
1.0
0.8
0.6
0.4
0.2
0
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22 22212345
010 100 200 300
Relative amount
5
250
KDa
150
100
75
50
37
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20
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β-tubulin
C60(μg mL−1)
H2O2(μM)
(d)(c)
*
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1.0
0.8
0.6
0.4
0.2
0
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01 234
12345
100 100100 100 100
Relative amount
5
250
KDa
150
100
75
50
37
25
20
15
10
β-tubulin
C60(μg mL−1)
H2O2(μM)
(e)
(f)
****
1.8
1.6
1.4
1.2
1.0
0.8
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0.2
0
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01 234
12345
100 100100 100 100
Relative amount
5
250
KDa
150
100
75
50
37
25
20
15
10
β-tubulin
C60(μg ml−1)
H
2
O
2
(μM)
(g) (h)
**
**
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Fig. 6 C60-induced P-PKAs and PTP were affected by a series of H2O2 concentrations. a, b Western blot analysis was performed using an anti-
phospho-PKA substrate antibody. c, d Western blot analysis was performed using an anti-phosphotyrosine antibody. Spermatozoa were incu-
bated in basal medium containing 2μgmL−1 carboxylated C60 and a series of H2O2 concentrations (0, 10, 100, 200, and 300μM). β-Tubulin was
used as an internal control. e, f Western blot analysis was performed using anti-phospho-PKA substrate antibody. g, h Western blot analysis was
performed using anti-phosphotyrosine antibody. The experiment was performed at least three times, and the image presented is representative
and repeatable. (n = 3,*P < 0.05)
Nano-Micro Lett. (2019) 11:104 104 Page 12 of 17
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© The authors
3.6 The Interactions betweenSperm andCarboxylated
C60
To observe how carboxylated C60 interacted with sperm
cells, samples were examined by TEM (Fig.8). Carboxy-
lated C60 attached to sperm primarily as single nanoparticles
or aggregates on the sperm plasma membrane (Fig.8). We
re-examined the particle diameter in the TEM images and
found that primary particle diameters were similar to the size
of carboxylated C60 (75.5 ± 7.2nm)by NanoSight analysis
(Fig. S1). No nanoparticles were observed to penetrate mor-
phologically intact sperm in any case.
Interestingly, the present data suggest that the pro-
tective effects of carboxylated C60 on boar spermatozoa
can be attributed to its antioxidant activity, resulting
in an advanced fertility capacity of sperm cells during
1234
(c)(a)
(d)(b)
56
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KDa
12 3456KDa
150
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β-tubulin
250
150
100
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β-tubulin
Ca2+ (2 mM)
HCO3 (25 mM)
BSA (4 mg mL−1)
C60 (2 μg mL−1)
H2O2 (100 μM)
H-89 (0.1 mM)
dbcAMP (1.0 mM)
Ca2+ (2 mM)
HCO3 (25 mM)
BSA (4 mg mL−1)
C60 (2 μg mL−1)
H2O2 (100 μM)
H-89 (0.1 mM)
dbcAMP (1.0 mM)
Relative amount
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
*
**
*
*
123456
Relative amount
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
*
*
*
*
*
123456
Fig. 7 Effects of the cAMP-PKA pathway-related regulatory factors on P-PKAs and PTP with different concentrations of carboxylated C60. a
Western blot was performed using an anti-phospho-PKA substrate antibody. b Densitometric analysis of the western blot image shown in a. c
Western blot was performed using an anti-phosphotyrosine antibody. d Densitometric analysis of the western blot image shown in c. Spermato-
zoa were incubated in basal medium supplemented with 100μM H2O2 in the presence or absence of 1.0mM dbcAMP, or 2μgmL−1 carboxy-
lated C60 in the presence or absence of 0.1mM H-89. β-Tubulin was used as an internal control. The experiment was performed at least three
times, and the image presented is representative and repeatable. (n = 3,P < 0.05)
Nano-Micro Lett. (2019) 11:104 Page 13 of 17 104
1 3
preservation at 4°C. Numerous experimental studies
have been conducted to evaluate the application of various
antioxidants to protect spermatozoa against the oxidative
damage caused at low temperatures [27, 37]. Our findings
demonstrated that carboxylated C60 protected mitochon-
dria by scavenging ROS and increasing antioxidant abil-
ity, which might be related to the unique pseudo-aromatic
structure of carboxylated C60, resulting in delocalization of
π-electrons over its carbon core and readily reactivity with
oxygen free radicals. Over the last decade, the probable
function mechanism of carboxylated C60 has been exten-
sively studied. Carboxylated C60 is highly hydrophilic
and a highly stable donor–acceptor complex of C60 with
water molecules—C60@{H2O}n, n = 22–24 [59]. The cur-
rently recognized carboxylated C60 antioxidant mechanism
has the following aspects: carboxylated C60 as a novel
“structural” antioxidant that is characterized as a “radical
sponge” [60], the enzyme catalysis mechanism [61], the
stabilizing effects on alkaline phosphatase and peroxidase
invitro [13], and the binding of free radicals and removal
of hydroxyl radicals [16]. However, TEM analysis revealed
a modification-dependent attachment of carboxylated C60
to the cell membrane of spermatozoa (Fig.8), but provided
no evidence of the entrance of carboxylated C60 nanopar-
ticles into sperm cells. Therefore, the results obtained in
the present study suggest an alternative mechanism of the
antioxidative activity of carboxylated C60 that results in
ROS spillover in sperm cells, which could explain the
decrease in sperm ROS levels (Fig.9). We hypothesized
that the decrease in intracellular ROS levels in spermato-
zoa caused by carboxylated C60 is related to the surface
electron affinity. Additionally, antioxidant ability related to
Fig. 8 TEM images of spermatozoa samples treated with carboxylated C60 at 4°C. The spermatozoa were collected after 15days of treatment.
TEM images showing a the sperm head interacting with carboxylated C60 (2μgmL−1), b the sperm midpiece interacting with carboxylated C60
(2μg mL−1), c the sperm midpiece without carboxylated C60. d, e Cross-sectional TEM images showing the sperm midpiece interacting with
carboxylated C60 (2μg mL−1). f Cross-sectional TEM image showing the sperm midpiece without carboxylated C60. CM: cell membrane; M:
mitochondria
Nano-Micro Lett. (2019) 11:104 104 Page 14 of 17
https://doi.org/10.1007/s40820-019-0334-5
© The authors
an extended electron-conjugation system only determined
the high reactivity toward ROS.
4 Conclusions
To our knowledge, we report, for the first time, that carboxy-
lated C60 can be used as a safe antioxidant agent to serve
as semen extender supplement and that such a practice can
improve the survival and characteristics of boar sperm dur-
ing liquid storage. Our results suggested that carboxylated
C60 could effectively protect boar sperm against oxidative
injury. This approach can represent a good alternative to
the preservation methods used for boar semen and anti-ste-
rility drugs used for other reproductive diseases. Our results
provide reliable theoretical support for the futureapplication
of carboxylated C60 in breeding livestock boar. Furthermore,
the results present the first substantial evidence that carboxy-
lated C60 administration significantly reduces the oxidative
stress on boar spermatozoa stored at 4°C and the associated
complications, similar to a semen extender supplement.
Acknowledgements This work was supported by the National
Natural Science Foundation of China (No. 31772594), the Special
Fund for Agro-scientific Research in the Public Interest of China
(No. 200903056), and the Key Project of Shanghai Municipal Agri-
cultural Commission of China (2014-2-5).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you
ROS
ROS
ROS
ROS
ROS ROS
ROS
ROS
ROS
ROS
ROS
ROS
ROS
ROS
ROS
ROS
ROS
ROS
C
60
C60
ROS
ΔΨm
ATP
PY
PKA
cAMP
Cytoplasm
ROS
Motility
AMP/ATP
P-AMPK AMPK
Fig. 9 Putative mechanisms by which carboxylated C60 protects boar sperm from ROS-induced functional damage at 4°C. Carboxylated C60
may bind to ROS outside and reduce ROS levels inside sperm. Decreasing intracellular ROS levels may enhance the mitochondrial membrane
potential and cAMP levels and also reduce cellular protein dephosphorylation and enhance cellular ATP levels, subsequently increasing the
motility of spermatozoa at 4°C. Δψm:mitochondrial membrane potential; PKA: protein kinase A; PY: tyrosine phosphorylation
Nano-Micro Lett. (2019) 11:104 Page 15 of 17 104
1 3
give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons license, and indicate if
changes were made.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s4082 0-019-0334-5) contains
supplementary material, which is available to authorized users.
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... To our knowledge, to date, there are two scientific studies about the in vitro effect of C 60 HyFn on the quality of semen exposed to low temperature. Although, the positive effects of different doses of C 60 HyFn on human [17] and boar [18] semen were observed in those studies, there is no evidence related to its effect on ram semen incubated in low temperature. That's why this study was conducted to determine the effect of different doses of C 60 HyFn on ram semen during cool-storage at 5 °C by examining the changes in motility and kinematic parameters, membrane integrity, vitality, and morphology of spermatozoa. ...
... In the study conducted in goat epididymal spermatozoa, increments in glutathioneperoxidase, glutathione-reductase, and superoxide dismutase activities as well as decrement in LPO level in spermatozoa supplemented with 1, 10, or 100 µM fullerenol during incubation period at 32 °C for 3 h were found [31]. In addition, a current evidence has suggested that C 60 HyFn is able to protect boar spermatozoa against ROS injuries and energy insufficiencies by preventing protein dephosphorylation via the cAMP-PKA signalling pathway during cool-storage [18]. Although oxidative stress markers were not measured in this study, our unpublished data showed that each dose of C 60 HyFn between 100 nM and 40 µM provided significant reductions in LPO and significant increments in antioxidants such as glutathione, glutathione-peroxidase and catalase in frozen-thawed ram semen compared to the values in control. ...
... Although oxidative stress markers were not measured in this study, our unpublished data showed that each dose of C 60 HyFn between 100 nM and 40 µM provided significant reductions in LPO and significant increments in antioxidants such as glutathione, glutathione-peroxidase and catalase in frozen-thawed ram semen compared to the values in control. Findings of Murugan et al. [31], Li et al. [18] and our unpublished study clearly show that C 60 HyFn has potent antioxidant effect against oxidative stress caused by liquid-storage at low temperature and freeze-thawing process in mammalian semen. ...
... 4 C 60 fullerenes reduced reactive oxygen species (ROS) level in the preserved boar sperm samples and facilitated the survival of spermatozoa. 5 C 60 fullerenes showed beneficial effects in the number of experimental pathologies. Mice treated with hydrated C 60 fullerenes show decreased tumor growth. ...
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Several studies claimed C60 fullerenes as a prospective geroprotector drug due to their ability to capture free radicals effectively and caused a profound interest in C60 in life extension communities. Multiple additives are already sold for human consumption despite a small body of evidence supporting the beneficial effects of fullerenes on the lifespan. In order to test the effect of C60 fullerenes on lifespan and healthspan, we administered C60 fullerenes dissolved in virgin olive oil orally to 10-12 months old CBA/Ca mice of both genders for seven months and assessed their survival. To uncover C60 and virgin olive effects, we established two control groups: mice treated with virgin olive oil (vehicle) and mice treated with drinking water. To measure healthspan, we conducted daily monitoring of health condition and lethality and monthly bodyweight measurements. We also assessed physical activity, glucose metabolism, and hematological parameters every three months. We did not observe health deterioration in the animals treated with C60 compared with the control groups. Treatment of mice with C60 fullerenes resulted in an increased lifespan of males and females compared with the olive oil-treated animals. The lifespan of C60-treated mice was similar to the mice treated with water. These results suggest that the lifespan-extending effect in C60-treated mice appears due to the protective effect of fullerenes in opposition to the negative effect of olive oil in CBA/Ca mice.
... In previous studies, CUR administered to diabetic rats has been reported to decrease the level of MDA and increase the activity of GSH and antioxidant enzymes in kidney tissue (Kim et al., 2016;Selvi et al., 2014). It appears that C60 fullerene reduces oxidative stress in different tissues of diabetic rats (Bal et al., 2011;Li et al., 2019), however, no information has been found on its effects against oxidative stress in kidney tissue. Therefore, it can be concluded as a result of the present study that the combination of C60, C60 + CUR applied to diabetic rats is effective in reducing oxidative stress in kidney tissue (Table 1). ...
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... One hundred twenty collected semen samples were examined for bacterial communities and seven fertilizing criteria for semen quality, including semen volume, sperm concentration, morphology, motility, plasma membrane and acrosomal integrity, and mitochondrial activity. The semen volume and abnormal sperm percentage were detected according to the conventional methods in our lab (Li et al., 2019). Sperm concentration was evaluated by optical density using a calibrated spectrophotometer (Shanghai Spectrophotometer Co., Ltd., Shanghai, China). ...
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The presented review aims to summarize the knowledge regarding the reproductive and developmental toxicity of different types of carbon nanoparticles, such as graphene, graphene oxide, multi- and single-walled nanotubes, fullerenes, and nanodiamonds. Carbon nanoparticles have unique chemical and physical properties that make them an excellent material that can be applied in many fields of human activity, including industry, food processing, the pharmaceutical industry, or medicine. Although it has a high degree of biocompatibility, possible toxic effects on different tissue types must also be taken into account. Carbon nanoparticles are known to be toxic to the respiratory, cardiovascular, nervous, digestive system, etc., and, according to current studies, they also have a negative effect on reproduction and offspring development.
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This study was conducted to investigate the effect of different doses of hydrated C60 fullerene (C60HyFn) on freeze-thawing process-induced changes in lipid, vitamin and amino acid composition and also in motility, kinematic, sperm quality and oxidative stress parameters in ram semen. Semen was collected from seven rams twice a week for 3 weeks, so six repetitions were performed. The semen collected in each repetition was pooled. Each pooled sample was diluted with tris + egg yolk extender with (200 nM, 400 nM, 800 nM, 1 µM and 5 µM) and without (control) C60HyFn and they were frozen in mini straws. The doses of 800 nM, 1 µM and 5 µM had higher total, progressive motility, sperm membrane functionality rates, glutathione-peroxidase and catalase activities. All doses of C60HyFn significantly reduced dead and total abnormal sperm rates and malondialdehyde levels. Significant increases in vitamin A (400 and 800 nM doses), vitamin K1 (400 nM, 800 nM and 1 µM doses), total amino acid (all doses) levels, but significant decreases in vitamin D2 (800 nM, 1 and 5 µM doses), vitamin D3 (1 and 5 µM doses) and vitamin E (200 nM, 1 and 5 µM) levels were observed compared to control. In conclusion, the addition of C60HyFn to ram semen at 200 nM - 5 µM range, especially at a dose of 800 nM, provides a positive contribution to the protection of motility, vitamins A, K and total amino acid levels, and oxidant/antioxidant balance after freeze-thawing.
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Antiradical capacity of endohedrally and exohedrally functionalized C80-Ih fullerene is analyzed. Electrodonating and electroaccepting powers were calculated for 18 previously synthesized systems, additionally, adsorption energies of all systems were computed when one and two hydroxyl groups were added. Finally, the electrodonating and electroaccepting powers were also obtained for hydroxylated structures. The best acceptor systems were found as the worst donors, while the best donors were the worst acceptors. Endohedral functionalization does not improve acceptor capacity of C80 fullerene. Systems with the Sc3N cluster inside C80 fullerene have the best electrodonating character. In fact, Sc3[email protected]80-PCBM, whose exohedral functionalization is the same as that used in C60 to later be used as an acceptor molecule in photovoltaic cells, turns out to be the molecule with the lowest electroaccepting power but with the best electrodonating power. In general, closed-shell systems have smaller electrodonating and electroaccepting powers than open-shell systems.
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We study Ⅲ-nitride nanowire (NW) anti-reflection structures employed as photoelectrocatalyst for solar water splitting. We found that 1D vertical InGaN NW arrays tilted by 73° exhibit maximized photocurrent. Therefore, we grow 3D InGaN NW arrays on the facets of oblique pyramid-textured Si (311) (OPSi/InGaN) by plasma-assisted molecular beam epitaxy, exhibiting facets tilted by 73o. In addition, using finite difference time domain simulations we find the crucial impact of the asymmetry of the oblique pyramid InGaN NW arrays on the light trapping. Compared with InGaN NWs grown on a planar Si substrate (Si/InGaN), the OPSi/InGaN photoanode exhibits ~500% enhancement of the photocurrent due to various light trapping effects attributed to: 1D vertical NW arrays, their tilt, 3D arrangement and asymmetry. Decorated with NiOOH co-catalyst, the OPSi/InGaN photoanode exhibits photocurrent densities in the mA cm⁻² range. The present research provides a rational design for the fabrication of nanostructured photoelectrocatalyst with enhanced light absorption.
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Increasing evidence suggests that diabetes also targets lung tissues resulting in structural and physiological abnormalities. The present study evaluated the impact of pristine C60 fullerene (C60) against diabetes-induced lung damage for the first time. The objective was to evaluate the impacts of Curcumin (Cur), C60 and C60 fullerene+Curcumin (C60 + Cur) combination on oxidative stress (MDA, GSH, CAT, GST, Retinol, α-tocopherol), apoptosis (Caspase-3, Bcl-2), cholesterol and fatty acid profile (16:0,18:0,18:1,18:2, 22:4, 22:6) against changes in the lung tissue of diabetic rats. Streptozotocin (STZ) was used for inducing diabetes with Cur, C60 and C60 + Cur combination administered for eight weeks to treat diabetic and control rats. Increased oxidative stress, apoptosis and significant changes in cell structure were observed in the lung tissues of diabetic rats. The combination of Cur, C60 and C60 + Cur reduced oxidative stress in the lung tissue of diabetic rats while increasing the antioxidant defense capacity of the tissue, exhibiting tissue protective properties against apoptosis. The diabetic rats displayed favorable properties against lipotoxicity-induced tissue damage due to the increase in the fatty acid and cholesterol levels in lung tissue. It was observed that Cur, C60 and C60 + Cur combination displays protective effects against hyperglycemia induced oxidative damage to lung tissue. Oxidative stress, prevention of lipid and cholesterol accumulation, and weakening of lung apoptosis may be associated with these effects.
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Male infertility is a global problem in modern society of which capacitating defects are a major cause. Previous studies have demonstrated that Ca²⁺ ionophore A23187 can make mouse sperm capable of fertilizing in vitro, which may aid in clinical treatment of capacitating defects. However, the detailed role and mechanism of Ca²⁺ in the capacitating process are still unclear especially how A23187 quickly renders sperm immotile and inhibits cAMP/PKA-mediated phosphorylation. We report that A23187 induces a Ca²⁺ flux in the mitochondria enriched sperm tail and excess Ca²⁺ inhibits key metabolic enzymes involved in acetyl-CoA biosynthesis, TCA cycle and electron transport chain pathways resulting in reduced ATP and overall energy production, however this flux does not destroy the structure of the sperm tail. Due to the decrease in ATP production, which is the main phosphate group donator and the power of sperm, the sperm is rendered immobile and PKA-mediated phosphorylation is inhibited. Our study proposed a possible mechanism through which A23187 reduces sperm motility and PKA-mediated phosphorylation from ATP generation, thus providing basic data for exploring the functional roles of Ca²⁺ in sperm in the future.
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C60TEMPO10 catalytic system linked to a microspherical gold support through a covalent S-Au bond was developed. The C60TEMPO10@Au composite catalyst had a particle size of 0.5–0.8 μm and was covered with the fullerenes derivative of 2.3 nm diameter bearing ten nitroxyl groups; the organic film showed up to 50 nm thickness. The catalytic composite allowed for the oxidation under mild conditions of various primary and secondary alcohols to the corresponding aldehyde and ketone analogues with efficiencies as high as 79–98%, thus giving values typical for homogeneous catalysis, while retaining at the same time all the advantages of heterogeneous catalysis, e.g., easy separation by filtration from the reaction mixture. The catalytic activity of the resulting system was studied by means of high pressure liquid chromatography. A redox mechanism was proposed for the process. In the catalytic cycle of the oxidation process, the TEMPO moiety was continuously regenerated in situ with an applied primary oxidant, for example, O2/Fe³⁺ system. The new intermediate composite components and the final catalyst were characterized by various spectroscopic methods and thermogravimetry. Graphical abstractᅟ Electronic supplementary material The online version of this article (doi:10.1007/s11051-017-3857-z) contains supplementary material, which is available to authorized users.
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Mitochondrial oxidative phosphorylation (OXPHOS) is essential for ATP production to maintain sperm linear motility during migration from the uterus to the oviduct. However, ROS are generated as by-products of OXPHOS, causing stress and damaging the sperm quality. This study aimed to clarify the ROS targets in sperm mitochondria that decrease linear motility and to investigate whether mitochondria-target antioxidants (PQQ and CoQ10) affect mitochondrial activity and sperm motility. Sperm linear motility pattern, ATP production, and mitochondrial activity were decreased with increasing ROS levels during incubation in the low-glucose medium. However, sperm motility patterns and ROS levels were not significantly changed in the high-glucose medium. Moreover, the gene expression system (mt-DNA, mitochondrial transcription factor-A (TFAM) and RNA polymerase (POLRMT)) in sperm mitochondria was damaged during incubation in the low-glucose medium. Interestingly, PQQ treatment increased the mt-DNA stability and decreased the damage to TFAM and POLRMT, which resulted in high expression of mitochondrial genes. Furthermore, the antioxidants increased mitochondrial activity and maintained sperm linear motility under the low glucose condition. These results revealed that both ATP production and the mitochondrial transcription system are damaged with increasing ROS levels in sperm that show a linear motility pattern. Treatment with antioxidants, such as PQQ and CoQ10, is beneficial tool to maintain sperm linear motility.
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Protein post-translational modifications (PTMs) have been reported to be involved in various functions of sperm, yet the exact correlation between PTMs and sperm motility remains unclear. With the goal of contributing to this subject, motility variables were measured by computer-assisted sperm analysis system (CASA), and the amount of PTMs were evaluated using Western blot and immunofluorescence in fresh sperm and liquid stored sperm. Results of the present study indicate that the amount of the phosphorylated substrates of PKA (P-PKAs), protein tyrosine phosphorylation (PTP), global protein acetylation (Pan-Kac) and α-tubulin acetylation (Tub-Kac) was greater in sperm of fresh semen samples with relatively greater motility than in sperm of fresh semen samples with relatively lesser motility. Similarly, the amounts of phosphorylation and acetylation gradually decreased with the reduction in the motility of sperm in liquid stored semen samples. Interestingly, the P-PKAs (r = 0. 634, P < 0. 01) and Pan-Kac (r = 0. 380, P < 0. 05) were positively correlated with sperm motility in fresh semen, whereas only P-PKAs (r = 0.607, P < 0. 01) were positively correlated with sperm motility during liquid storage. Furthermore, it is noteworthy that the amounts of phosphorylation and acetylation were positively correlated with the acrosome integrity and mitochondrial membrane potential of fresh sperm and liquid stored sperm. This study is the first to explore the correlation between PTMs and sperm motility, and it may provide a new reproductive biomarker for evaluating semen quality and predicting sperm capacity for enhancing reproductive performance, which is meaningful for the pig breeding industry.
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Numerous studies have shown that Astragalus polysaccharide (APS) has strong antioxidant effects and high practical value for preserving semen at low temperatures in vitro. However, to date, little attention has been paid to the precise mechanism of APS in sperm preservation at 4 °C. Thus, to gain further insight into the protective effects of APS, the present study was performed to assess the changes in sperm quality parameters, antioxidant capacity, ATP content and protein phosphorylation levels. Here, we demonstrated that supplementation with APS could effectively preserve boar sperm quality parameters such as sperm motility, acrosome integrity and mitochondrial membrane potential. Moreover, we found that the positive effects of APS on boar sperm quality were mainly due to the elimination of excessive mitochondrial ROS, the improvement of antioxidant capacities and the enhancement of ATP levels. Interestingly, by conducting a series of studies on protein phosphorylation, we also discovered that APS could protect boar sperm from oxidative stress and energy deficiency through inhibiting the protein dephosphorylation caused by ROS via the cAMP-PKA signaling pathway. To our knowledge, this is the first exploration of the molecular mechanism underlying the protective roles of APS towards ROS toxicity from the perspective of energy metabolism and protein modification. This study comprehensively provides novel insights into the action mechanism of the protective effects of antioxidants on sperm stored at 4 °C and reveals the practical feasibility of using APS as a boar semen extender supplement for assisted reproductive technology. This article is protected by copyright. All rights reserved
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Fullerene C60 microbelts were fabricated using liquid-liquid interfacial precipitation method and converted into quasi 2D mesoporous carbon microbelts by heat-treatment at elevated temperatures of 900 and 2000 ○C. The carbon microbelts obtained by heat-treatment of fullerene C60 microbelts at 900 ○C showed excellent electrochemical supercapacitive performance exhibiting high specific capacitances ca. 360 F g-1 (at 5 mV s-1) and 290 F g-1 (at 1 A g-1) due to the enhanced surface area and robust mesoporous framework structure. Additionally, the heat-treated carbon microbelt showed good rate performance retaining 49% of capacitance at the high scan rate of 10 A g-1. The carbon belts exhibit super cyclic stability. Capacity loss was not observed even after 10,000 charge/discharge cycles. These results demonstrate that the quasi 2D mesoporous carbon microbelts derived from π-electron rich carbon source, fullerene C60 crystals could be used as a new candidate material for electrochemical supercapacitor applications.
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The buckminsterfullerene (C60) is considered as a relevant candidate for drug and gene delivery to the brain, once it has the ability to cross the blood-brain barrier. However, the biological implications of this nanomaterial are not fully understood, and its safety for intracerebral delivery is still debatable. In this study, we investigated if C60 particle size could alter its biological effects. For this, two aqueous C60 suspensions were used with maximum particle size up to 200nm and 450nm. The suspensions were injected in the hippocampus, the main brain structure involved in memory processing and spatial localization. In order to assess spatial learning, male Wistar rats were tested in Morris water maze, and the hippocampal BDNF protein levels and gene expression were analyzed. Animals treated with C60 up to 450nm demonstrated impaired spatial memory with a significant decrease in BDNF protein levels and gene expression. However, an enhanced antioxidant capacity was observed in both C60 treatments. A decrease in reactive oxygen species levels was observed in the treatments with suspensions containing particles measuring with up to 450nm. Thiobarbituric acid reactive substances, glutamate cysteine ligase, and glutathione levels showed no alterations among the different treatments. In conclusion, different particle sizes of the same nanomaterial can lead to different behavioral outcomes and biochemical parameters in brain tissue.
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Spermatogenesis is a complex and highly regulated process. The ability of spermatozoa to perform its function depends on multiple physiological and genetic factors that are not fully understood. Notably, due to lack of transcriptional and translational activity in spermatozoa, posttranslational modifications (PTMs) play key roles in determining their viability. PTMs not only confer structural changes in the proteome of the spermatozoa cells, but also increase the diversity of the proteome and introduce specific modifications that could be translated into functional changes in the affected spermatozoa. Multiple PTMs of active proteins have been identified in the developing spermatogonia. This review summarizes a diverse range of PTMs taking place in the developing spermatozoa, and analyzes their effects on male fertility and sperm viability. In particular, we discuss how SUMOylation, ubiquitination, phosphorylation, acetylation, glycosylation, and disulphide bond formation in proteins play a role in spermatogenesis, sperm maturation, movement of maturing spermatozoa to epididymis, capacitation, hyperactivation, spermatozoa motility, subversion of immune detection by spermatozoa, sperm to egg recognition and fusion, and the fertilization process. When possible, the specific proteins involved in these processes are highlighted. We point to existing knowledge gaps in the field of proteomics, and provide suggestions for future research on sperm viability and male fertility. We discuss briefly, as an example, the observations in water buffalo, Bubalus bubalis, which provides both meat and milk, and therefore is a reliable source for energy and protein needs of human populations. In conclusions, understanding the ways in which PTMs impact mammalian fertility and reproduction is important to make significant strides for diagnostic and therapeutic strategies in the near future.
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Primary memory impairments associated with increased level of amyloid-β (Aβ) in the brain have been shown to be linked, partially, with early pathological changes in the entorhinal cortex (EC) which spread on the whole limbic system. While the hippocampus is known to play a key role in learning and memory mechanisms, it is as yet unclear how its structures are involved in the EC pathology. In this study, changes in memory and neuronal morphology in male Wistar rats intrahippocampally injected with Aβ25-35 were correlated on days 14 and 45 after the injection to reveal specific cognitive-structural associations. The main focus was on the dentate gyrus (DG) and hippocampal areas of CA1 and CA3 because of their involvement in afferent flows from EC to the hippocampus through tri-synaptic (EC → DG → CA3 → CA1) and/or mono-synaptic (EC → CA1) pathways. Evident memory impairments were observed at both time points after Aβ25-35 injection. However, on day 14, populations of morphological intact neurons were decreased in CA3 and, drastically, in CA1, and the DG supramedial bundle was significantly damaged. On day 45, this bundle largely and CA1 neurons partially recovered, whereas CA3 neurons remained damaged. We suggest that Aβ25-35 primarily affects the tri-synaptic pathway, destroying the granular cells in the DG supramedial area and neurons in CA3 and, through the Schaffer collaterals, in CA1. Intrahippocampal pretreatment with hydrated fullerene C60 allows the neurons and their connections to survive the amyloidosis, thus supporting the memory mechanisms.
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Tuberculosis caused by Mycobacterium tuberculosis (MTB) is still a major threat to global public health. However, the existing methods for MTB detection are usually complicated and time consuming with unsatisfactory sensitivity and specificity. In this work, a relatively simple and ultrasensitive electrochemical aptasensor based on novel signal generation and amplification was constructed for the determination of MTB antigen MPT64. The coil-like fullerene (C60)-doped polyaniline (C60-PAn) nanohybrids with large surface area, abundant active groups and excellent electric performance were synthesized and used both as new redox nanoprobe and catalyst for the generation and amplification of electrochemical signal for the first time. Then gold nanoparticles decorated C60-PAn nanocomposites (GNPs-C60-PAn) were labeled with signal aptamer to form the tracer label. After the sandwich reaction of target MPT64 antigen between capture aptamer and the tracer label, a distinguishing detection signal of C60-PAn would be observed. Moreover, the detection signal could be enormously enhanced towards the efficient electrocatalytic oxidation of ascorbic acid based on C60-PAn, resulting in further improvement of the sensitivity. With the excellent redox and electrocatalytic activity of C60-PAn, a wide detection linear range from 0.02 to 1000 pg/mL was obtained with a detection limit of 20 fg/mL for MPT64. The proposed aptasensor showed high selectivity to target antigen compared with possible interfering substances. More importantly, it also exhibited excellent specificity and sensitivity for MPT64 detection in serum samples of tuberculosis patients, which provided a rapid and efficient detection method for MTB infection.
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High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, intact acrosome, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.