Animal Reproduction Science 241 (2022) 106991

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Motility and oxidative stress of common carp Cyprinus carpio

sperm during short-term storage
Anna Shaliutina-Kolešová a, b, *, Rui Nian c
a
University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture
and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25 Vodňany, Czech Republic
b
Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Průmyslová 595, 252 50 Vestec, Czech Republic
c
CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189
Songling Road, Qingdao 266101, China

A R T I C L E I N F O A B S T R A C T

Keywords: Short-term storage of semen is a simple and inexpensive procedure to deal with logistics of large-
Sperm storage scale hatchery operations but can lead to oxidative stress and a significant decrease in sperm
Carp motility and velocity. To better understand the mechanisms responsible for the association of
Reactive oxygen species
poor sperm quality with oxidative stress, in the present study we investigated the effect of
Sperm motility
refrigerated storage for 0, 24, 48, 72, and 144 h on sperm motility and curvilinear velocity and
Short-term
oxidant/antioxidant balance of common carp Cyprinus carpio. Percentage of motile sperm was
significantly (p < 0.05) reduced stored for 72 h storage compared to that of fresh sperm. Sperm
stored 144 h showed < 40% motility with an average velocity of 91.12 ± 10.4 µm s− 1. A time-
dependent increase in the level of the oxidative stress indices lipid peroxidation and carbonyl
derivatives of proteins was observed. Increase (p > 0.05) in total superoxide dismutase was
detected after 48 h, and glutathione reductase and glutathione peroxidase activity demonstrated a
significant increase after 72 h. These results provide an additional tool for the development and
improvement of short-term sperm preservation procedures commonly applied in aquaculture.

1. Introduction

In aquaculture, short-term liquid storage of semen is a common practice for routine management of reproduction. Sperm can be
maintained at 4 ◦ C for hours to weeks while preserving the sperm motility, fertilizing capacity, and metabolic activity (Shaliutina et al.,
2013). Short-term storage of sperm by refrigeration has become an important component of assisted reproduction technology that cuts
down on broodstock manipulation; reduces labour during spawning, the busiest season in commercial fish production; and allows
reproduction when the female maturation period is not synchronized with that of males (Kowalski et al., 2014). Procedures for
common carp Cyprinus carpio sperm storage have been developed, and modifications of the methods for carp-specific optimization
have been proposed (Bozkurt and Secer, 2005). It, however, short-term storage procedures that do not lead to impaired carp semen
quality have yet to be developed.

* Corresponding author at: University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian
Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Zátiší 728/II, 389 25
Vodňany, Czech Republic.
E-mail address: kolesova@frov.jcu.cz (A. Shaliutina-Kolešová).

https://doi.org/10.1016/j.anireprosci.2022.106991
Received 1 February 2022; Received in revised form 4 May 2022; Accepted 5 May 2022
Available online 10 May 2022
0378-4320/© 2022 Elsevier B.V. All rights reserved.
A. Shaliutina-Kolešová and R. Nian Animal Reproduction Science 241 (2022) 106991

The success of sperm storage depends on their quality, which is affected by fish age, spawning season, photoperiod, and collection
method as well as by storage conditions including temperature, oxygen availability, diluent composition, dilution rate, storage time,
and supplementation of extender with antibiotics or antioxidants (Penaranda et al., 2010; Contreras et al., 2017; Shaliutina-Kolešová
et al., 2019). Oxidative stress, in particular, can lead to cell aging and deterioration in sperm quality. Fish spermatozoa are especially
susceptible to peroxidative damage, since nearly all cytoplasm is depleted during the final stages of spermiogenesis, resulting in a
deficiency in enzymes that protect against damage induced by reactive oxygen species (ROS) (Lahnsteiner et al., 2010; Merino et al.,
2017; Sandoval-Vargas et al., 2020). This reduces sperm motility and viability, crucial factors in quality and fertilizing capacity
(Cosson, 2008). In Russian Acipenser gueldenstaedtii and Siberian sturgeon Acipenser baerii spermatozoa, the loss of motility and
movement velocity during short-term in vitro storage has been attributed to oxidative stress significantly impairing cell metabolism
(oxidative phosphorylation) (Shaliutina et al., 2013). In aquaculture, it will be useful to determine whether oxidative events occurring
during sperm storage affect carp sperm motility and if other mechanisms are involved.
Compounds that inhibit excessive production of free radicals or their reaction with biological structures are called antioxidants
(Niki, 2010). Aerobic organisms possess a defensive antioxidant system comprising molecular and enzyme defences (Wilhelm Filho,
2007; Potts et al. (2000)) suggested that antioxidant levels in sperm play an important role in its preservation. Further information is
needed.
The objective of the present study was to determine the effect of short-term storage on motility and velocity, oxidative stress
indices, and enzyme activity of spermatozoa of common carp Cyprinus carpio.

2. Materials and methods

2.1. Source of semen

The breeding and culture of common carp was carried out in Qingdao, Shandong Province, P.R. China. Ten sexually mature males
(4.0–4.3 kg) were held in three 3 m3 outdoor plastic tanks with flow-through pond water at 20–22 ◦ C. Spermiation was stimulated by
intramuscular injection of carp pituitary powder dissolved in 0.9% (w/v) NaCl solution at 1 mg kg− 1 body weight (Caille et al., 2006).
After 24 h, sperm were obtained via gentle abdominal massage and collected by micropipette, with care taken to avoid contamination
with urine, water, or faeces. Fresh sperm of each fish were divided into two parts to be used as control and for the short-term storage
procedure. Samples were stored for no longer than 30 min at 0–4 ◦ C in closed assay tubes until use.

2.2. Experimental protocol

The experiment was designed to follow standard protocols of short-term storage of carp spermatozoa under hatchery conditions. A
sperm sample from each male (n = 10) was diluted 5:1 (v:v) in immobilizing buffer (94 mM NaCl, 27 mM KCl, 50 mM glycine, 15 mM
Tris–HCl, 252 mOsm/kg, pH 7.5) (Rurangwa et al., 2002) and held in aerobic conditions at 4 ◦ C. Aliquots were removed at 24, 48, 72,
and 144 h post-collection for assessment of sperm function parameters including motility rate and velocity as well as oxidative stress
indices and antioxidant activity.
The concentration of sperm in each sample was measured by the hemocytometer method. Sperm concentration was and expressed
as 108 spermatozoa/mL of sperm.

2.3. Evaluation of spermatozoa motility and velocity

Percentage of motile spermatozoa (%) and sperm velocity (μm s− 1) were evaluated at 10 s post-activation with 10 mM Tris with
0.2% Pluronic F-127 (pH 8.0) medium (Linhart et al., 2000). Motility was assessed using a computer-assisted sperm motion analysis
system (CASAS-QH-III; Tsinghua Tongfang, Inc., Beijing, China). The software settings recommended by the manufacturer were
adjusted to obtain clear visualization of spermatozoa. Light microscopy at 200x magnification, 18 ◦ C ± 1 ◦ C, and a video digitization
rate of 24 frames s− 1 were used to analyse motility characteristics of > 100 spermatozoa. Assessment of sperm motility parameters was
conducted in triplicate for each sample.

2.4. Lipid peroxidation

Sperm samples were centrifuged at 5000g at 4 ◦ C for 10 min and the supernatant was discarded. The pellet was resuspended in 50
mM potassium phosphate (KPi) buffer, pH 7.0, containing 0.5 mM EDTA, to obtain a sperm concentration of 5 × 108 cells mL− 1, then
homogenized in an ice bath using a QSonica LLC ultrasonicator (Newtown, U.S.A). The frequency of the ultrasound in all the sonication
variants was 20 kHz. The homogenate was divided into two portions: one in which thiobarbituric acid reactive substances (TBARS) and
carbonyl derivatives of proteins (CP) was measured and a second to obtain the post-mitochondrial supernatant for antioxidant enzyme
activity assay. The TBARS method described by Lushchak et al. (2005) was used to evaluate sperm lipid peroxidation (LPO). Its
concentration was calculated by absorption at 535 nm and a molar extinction coefficient of 156 mM cm− 1. The TBARS content was
expressed as nanomoles per 108 cells. Carbonyl derivatives of proteins were detected by reaction with 2,4-dinitrophenylhydrazine
according to Lenz et al. (1989). The amount of CP was measured spectrophotometrically at 370 nm using a molar extinction coeffi­
cient of 22 mM cm− 1, expressed as nmol per 108 cells. Oxidative stress indices were obtained in triplicate for each sample.

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A. Shaliutina-Kolešová and R. Nian Animal Reproduction Science 241 (2022) 106991

2.5. Antioxidant parameters

The same protein extraction protocol as in lipid peroxidation was applied. The homogenate was centrifuged at 12 000g for 30 min
at 4 ◦ C to obtain the post-mitochondrial supernatant for antioxidant enzyme activity assay. Total superoxide dismutase (SOD) activity
was determined following Marklund and Marklund (1974) based on autoxidation of pyrogallol and assessed spectrophotometrically at
420 nm. Reduced glutathione level was assayed according to Sedlak and Lindsay (1968) with a modification. Glutathione peroxidase
(GPx) activity was based on the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase. The specific
activity was determined using the extinction coefficient of 6.22 × 103 M cm− 1 (Lawrence and Burk, 1976). Glutathione reductase (GR)
activity was determined spectrophotometrically, measuring NADPH oxidation at 340 nm (Carlberg and Mannervik, 1975). One unit of
SOD activity is defined as the quantity of the enzyme needed to induce 50% dismutation of the superoxide radical per min. One unit of
GPx or GR activity is defined as the quantity of the enzyme that consumes 1 µmol of substrate or generates 1 µmol of product min− 1.
Superoxide dismutase, GPx and GR activity was expressed in international units or milli-units, mU per 108 cells. All parameters of
antioxidant activity were made in triplicate for each sample.

2.6. Statistical analysis

Trials were conducted in triplicate. Normality and homogeneity of variance of data were first tested with the Kolmogorov test and
the Bartlett test, respectively. Data were expressed as mean ± standard deviation (n = 10) and compared using one-way analysis of
variance (ANOVA) at the significance level of p ˂ 0.05 followed by use of the Tukey’s HSD test. We used SPSS v.13.0 software (SPSS
Inc. Chicago, Illinois, USA).

3. Results

3.1. Sperm motility rate and velocity characteristics

Motility rate and sperm velocity in fresh and stored samples are presented in Figs. 1 and 2. The motility rate of fresh sperm at 10 s
post-activation ranged from 90% to 95%. There was no significant difference (p > 0.05) in motility rate of sperm in the control group
and that of stored for 24 and 48 h (Fig. 1). Semen storage for 72 h led to a significant reduction in the percentage of motile sperm (p <
0.05). After 144 h, motility had decreased to 37.21 ± 2.51%.
Estimates of sperm curvilinear velocity (VCL) were normally distributed, and ANOVA indicated lower velocity (p < 0.05) of the
stored sperm compared to fresh. Similar to sperm motility, no statistically significant differences were found in sperm VCL after 24 and
48 h of storage. Significant decrease of VCL was seen at 72 h (147.33 ± 9.17 µm s− 1) and 144 h (91.05 ± 10.46 µm s− 1).

3.2. Oxidative stress indices

Levels of TBARS and CP were considered indicators of the extent of lipid oxidation (LO) and protein oxidation, respectively.
The TBARS level was significantly higher than in fresh spermatozoa (p < 0.05) after 72 and 144 h storage (Table 1), whereas no
difference between fresh sperm and those stored for 48 h was observed. The maximum LO level was found in samples stored 144 h
(0.89 ± 0.04 nmol/108 cells). Carbonyl derivatives of proteins increased significantly in carp spermatozoa with increased storage
time. There was no significant difference (p ˃ 0.05) in CP of spermatozoa stored for 24 h and fresh control. The highest detected CP
level was 14.09 ± 1.01 nmol/108 cells in spermatozoa stored for 144 h, showing a trend similar to that of TBARS (Table 1).

Fig. 1. Effect of short-term storage on common carp Cyprinus carpio spermatozoa motility. Data are expressed as mean ± SD. Data in columns with
the same superscript do not significantly differ (p > 0.05; n = 10).

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A. Shaliutina-Kolešová and R. Nian Animal Reproduction Science 241 (2022) 106991

Fig. 2. Effect of short-term storage on common carp Cyprinus carpio spermatozoa velocity. Data are expressed as mean ± SD. Data in columns with
the same superscript do not significantly differ (p > 0.05; n = 10).

Table 1
Influence of short-term storage on thiobarbituric acid reactive substances (TBARS) and carbonyl derivatives
of proteins (CP) level in Cyprinus carpio spermatozoa. Data are presented as mean ± SD. Different super­
scripts indicate significant differences among samples (p < 0.05; n = 10).
Storage time, h TBARS, nmol/108cells CP, nmol/108cells
a
control 0.34 ± 0.03 5.24 ± 1.07a
24 0.39 ± 0.05a 5.97 ± 0.86a
48 0.44 ± 0.04a 7.84 ± 1.06b
72 0.62 ± 0.03b 9.16 ± 1.13b
144 0.89 ± 0.04c 14.09 ± 1.01c

3.3. Enzyme response

The antioxidant response was quantified as combined SOD, GR, and GPx activity. A gradual increase in total SOD activity was
observed with increasing time of storage with a significance difference from fresh (p < 0.05) beginning at 48 h (Table 2).
Glutathione reductase activity showed a trend similar to that of SOD (Table 2) with significant increase (p < 0.05) with storage
time. Maximum value of GR (13.61 ± 0.89 mU/108 cells) was observed in the sperm stored for 144 h, but the level did not significantly
(p ˃ 0.05) differ from that of samples held for 72 h. Significant differences (p < 0.05) in GPx activity were seen between the control
group and sperm stored for 72 h. In addition, the highest GPx activity was detected in samples stored 144 h.

4. Discussion

We demonstrated that refrigerated storage of common carp sperm allows sperm motility, velocity, and level of oxidative stress to be
maintained for several days and could be more effective than other non-freezing sperm preservation methods.
As a first step toward understanding how short-term storage might alter sperm physiology, motility, and velocity we found that
90–95% of fresh common carp spermatozoa could be activated after transfer to swimming medium. After the initial 48 h storage, no
significant reduction in parameters were detected 10 s post-activation. Sperm motility rates gradually declined over the storage period.
Our results agree with findings by Dietrich et al. (2021), who reported that procedures of short-term storage of common carp semen
prolonged sperm motility and viability. Bokor et al. (2018) concluded that common carp sperm shows tolerance to chilled storage for
two days, a period that could be extended to 16 days with the addition of 50 µg mL− 1 streptomycin and 50 IU bipenicillin. Native
Eurasian perch Perca fluviatilis sperm can be stored for 3 days, and samples placed in an immobilizing solution maintained motility for
48 days of storage (Sarosiek et al., 2013). Russian and Siberian sturgeon spermatozoa remained motile under refrigeration up to six
days, followed by a decline in motility attributed to oxidative stress that significantly impaired cell metabolism (Shaliutina et al.,

Table 2
Influence of short-term storage on superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) activity in Cyprinus
carpio spermatozoa. Data are presented as mean ± SD. Different superscripts indicate significant differences among samples (p < 0.05; n = 10).
Storage time, h SOD, mU/108 cells GR, mU/108 cells GPx, mU/108 cells

control 3.22 ± 0.92a 6.11 ± 1.21a 5.84 ± 0.87a

24 4.01 ± 0.96ab 6.97 ± 0.95a 6.77 ± 0.94a
48 5.84 ± 1.01bc 8.14 ± 1.10a 8.84 ± 1.01a
72 7.34 ± 0.98c 11.02 ± 1.08b 12.16 ± 0.93b
144 9.34 ± 1.07d 13.61 ± 0.89b 15.97 ± 0.99c

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A. Shaliutina-Kolešová and R. Nian Animal Reproduction Science 241 (2022) 106991

2013). Imbalance between ROS and the spermatozoon antioxidant system can cause metabolic and function disorders, reducing
spermatozoon motility and promoting peroxidation of the membrane phospholipids and oxidation of proteins (Li et al., 2009).
Oxidative stress occurs when ROS overcome the organism antioxidant defences, impacting sperm quality (Shaliutina-Kolešová
et al., 2013; Ulloa-Rodrígueza et al., 2018). In a healthy cell, it is estimated that approximately 2% of O2 is converted to ROS. In the
male reproductive system, spermatozoa produce high levels of ROS because of their aerobic metabolism. Especially in aquatic species,
spermatozoa are more vulnerable to attack by oxidative stress given their limited antioxidant defence and the high content of poly­
unsaturated fatty acid in the membranes (Shaliutina-Kolešová et al., 2013; Merino et al., 2020; Pintus and Ros-Santaella, 2021).
Polyunsaturated fatty acid has been reported to be a major contributor to the loss of cell function under oxidative stress, and its activity
in fish is usually inferred from TBARS (Shaliutina-Kolešová et al., 2014). In our study, the level of TBARS increased significantly after
72 h of storage. Aramli et al. (2013) reported an increased TBARS level associated with refrigerated storage of Persian sturgeon sperm
after 48 h. In the current work, we observed that the concentration of CP increased significantly in sperm after 48 h, which agrees with
data reported by Shaliutina et al. (2013) in Russian and Siberian sturgeon. Current evidence links the increased level of oxidative stress
to male infertility, reduced sperm motility, sperm DNA damage and increased risk of genetic diseases (Alahmar, 2019). Our results also
hypothesize that the loss of certain function parameters in common carp spermatozoa during refrigerated storage is caused by
oxidative stress, which can lead to a decrease in axon protein phosphorylation required for spermatozoon movement as well as to a
rapid decrease in ATP concentration and sperm motility and velocity.
It is widely accepted that ROS- and LPO-induced damage in living spermatozoa can be limited or even eliminated by the action of
enzymatic and non-enzymatic antioxidants (Partyka et al., 2012; Sandoval-Vargas et al., 2020). The antioxidant system, comprising
GSH, GPx, CAT, and SOD, has been described as a defence mechanism against LPO in semen of humans and numerous animal species
(Shaliutina-Kolešová et al., 2014; Alahmar, 2019). In the present research, antioxidant activity was evaluated as combined SOD, GR,
and GPx activity. Significant enhancement of SOD activity was seen after 48 h of storage. Similar observations were reported by Aramli
et al. (2013), who showed that total SOD activity increased significantly after 2 days storage in Persian sturgeon sperm. In contrast to
these results, Menegat et al. (2017) demonstrated that SOD activity did not change during storage at 17 ºC in homogenized samples of
liquid pig semen. We suggest that SOD activity can be used as a marker of sperm tolerance by protecting sperm cells from oxidative
stress induced by the preservation method. Activity of GR and GPx significantly increased with time of storage. This likely constitutes
an adaptive response to oxidative stress that serves to neutralize the impact of increased ROS generation; nevertheless, the level of
these antioxidants is not sufficiently high to completely prevent lipid peroxidation, especially during in vitro storage.

5. Conclusions

The data of the present study implied that diluted common carp sperm are sensitive to refrigerated storage. Decline in sperm
motility parameters is likely caused by oxidative stress and accumulation of LPO and CP in sperm cells resulting from cold storage of
sperm. We suggest that the addition of substances such as antioxidants or nanoparticles during short-term storage of fish spermatozoa
could prevent cell injury caused by oxidative stress. Studies are in development to identify the mechanisms involved in this substance
protection, including individual animal genetic variation.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgements

The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic projects CENAKVA
(LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370) and by CAS President’s International Fellowship for Post­
doctoral Researchers (2017PB0060), National Natural Science Foundation of China (21676286), and Primary Research Development
Plan of Shandong Province (2016GSF121006). The Lucidus Consultancy are gratefully acknowledged for English correction and
suggestions.

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