Aquaculture 547 (2022) 737410

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Effect of short-term storage on sperm functional parameters in sex-reversed

female mandarin fish (Siniperca chuatsi)
Shuang Liu a, b, c, Yuqing Su a, b, c, Huadong Yi a, b, c, Xuange Liu a, b, c, Xiaoli Chen a, b, c, Han Lai a, b, c,
Sheng Bi a, b, c, Yong Zhang a, b, c, Xiaopin Zhao a, b, c, Guifeng Li a, b, c, *
a
School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China
b
Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University,
Guangzhou 510006, China
c
Guangdong Provincial Engineering Technology Research Center for Healthy Breeding of Important Economic Fish, Guangzhou 510006, China

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

Keywords: The sex-reversed female sperm is very important to produce all-females of mandarin fish (Siniperca chuatsi), and
Sperm storage the gametes collected in different locations usually needed short-distance transport. Short-term storage is a
Sperm function simple and inexpensive method to preserve sperm, however, the effect of short-term storage on sperm have yet to
Sex reversal
be studied in sex-reversed female mandarin fish. The purpose of this study was to evaluate the effects of short-
Siniperca chuatsi
term storage on sperm functional parameters in sex-reversed female mandarin fish. Samples of undiluted semen
(US), diluted semen 1:2 v/v (DS1), 1:3 v/v (DS2) and 1:4 v/v (DS3) in D-15 diluent were arranged in 50 mL
sterile cryogenic tubes and maintained in cold storage at 4 ◦ C in the dark for 5 days. The results showed that
sperm functional parameters were significantly affected by storage time, and the sperm motility could be better
preserved in diluted groups. Motility of DS1 and DS2 were not significantly different after storage for 1 or 3 days.
At day 3 of storage, spermatozoa showed a DNA fragmentation <18% in DS1 and DS2, plasma membrane
integrity >85% and mitochondrial membrane integrity >80% in all groups, and a fertility >73% in DS1. The
percentage of sperm functional parameters decreased significantly at 5 days for all samples of undiluted and
diluted semen. However, spermatozoa showed a motility >45%, plasma membrane integrity >75%, mito­
chondrial membrane integrity >80% and DNA fragmentation <35% in DS1. Fertility of DS1 still achieved 57.83
± 10.20% and the hatchability of DS1 and DS2 was not significantly different, showing values >70% at day 5.
Except for LIN, the values of VSL, VCL, VAP and ALH in DS1 was significantly higher than that of other groups
with storage time of 3–5 days. In conclusion, the results provided new data on sex-reversed female mandarin fish
sperm quality with respect to short-term storage evaluated by flow cytometry and confocal laser scanning mi­
croscopy, and demonstrated that semen of mandarin fish can be better stored for short term for 5 days in DS1.

1. Introduction for mandarin fish to produce genetically and phenotypically single sex
population (Liu et al., 2021). The first step to produce all-female of
Mandarin fish (Siniperca chuatsi) is one of the most economically and mandarin fish is artificially induced sex reversal (pseudo-males), thus
important commercial aquatic fish in the genus Siniperca for aquaculture the preservation of pseudo-male sperm is very important. The sex-
(Luo et al., 2011). The mandarin fish eat bait fish without eating fodder, reversed females lacked functional sperm duct (Geffen and Evans,
so it takes up a lot of land resources and bait fish for farming. Growth of 2000; Hliwa et al., 2014), therefore sperm was extracted directly from
mandarin fish is sexually dimorphic, with the growth rate of females the testes and cannot be discharged directly through the sperm duct
about 15% higher than that of males (He and He, 2005), therefore, (Robles et al., 2003), which was the difference from the normal-males.
producing all-female populations are conducive to saving resources and In breeding farm, sperm was directly extracted from the testes of sex-
raising productivity. Sex reversal has been used as a breeding strategy reversed females and the sperm in the testes cannot be used

* Corresponding author at: Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University, No. 132, East Outer Ring Road, Guangzhou
Higher Education Mega Center, Guangzhou 510006, China.
E-mail address: [email protected] (G. Li).

https://doi.org/10.1016/j.aquaculture.2021.737410
Received 1 March 2021; Received in revised form 26 August 2021; Accepted 27 August 2021
Available online 30 August 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
S. Liu et al. Aquaculture 547 (2022) 737410

completely, which will lead to waste of sperm. Moreover, gametes collection, fresh semen from sex-reversed females (n = 9) was mixed
collected in different locations usually needed short-distance transport. together and evaluated for fresh sperm quality parameters and fertil­
Therefore, we need to find a simple way to store semen in breeding farm. ization trial, then transferred in the container stored at a constant
Gamete preservation is a significant tool for fish reproduction and is temperature of 4 ◦ C.
of great importance for fish culture (Magnotti et al., 2016). Among the
techniques used to protect gametes, short-term storage is a simple and 2.2. Semen storage
inexpensive procedure that often needed a logistics procedure in a large
hatchery (Shaliutina et al., 2013). This technology is usually used for The mixed semen was divided into four groups: 1) undiluted semen
short-distance transportation of gametes collected in different locations, (US); 2) treatment 1 (DS1): diluted semen in D-15 diluent (0.8% NaCl,
avoiding sperm aging and experimental projects for genetic research 0.05% KCl and 1.5% glucose dissolved in 100 mL, PH 7.0) in a ratio of
(Bobe and Labbé, 2008; Cabrita et al., 2008). In addition, it is considered 1:2 semen: diluent (v/v); 3) treatment 2 (DS2): diluted semen in D-15
a useful strategy for protecting threatened or endangered fish species diluent in a ratio of 1:3 semen: diluent (v/v); 4) treatment 3 (DS3):
(Aguilar-Juárez et al., 2014). To our knowledge, short-term storage diluted semen in D-15 diluent in a ratio of 1:4 semen: diluent (v/v),
studies have already been developed for rainbow trout (Oncorhynchus which were arranged in 50 mL sterile cryogenic tubes and maintained in
mykiss; Aguilar-Juárez et al., 2014), Patagonian blenny (Eleginops cold storage at 4 ◦ C in the dark. At 0, 3 and 5 days of storage, Computer
maclovinus; Contreras et al., 2017), carp (Cyprinus carpio L.; Dietrich Assisted Sperm Analysis (CASA, Nanning Song Jing Tianlun Bio-
et al., 2021) and Catfish (Clarias macrocephalus; Vuthiphandchai et al., Technology Co. Ltd., Guangxi, China) was used to monitor sperm
2009). Short-term storage of semen is a feasible method to preserve motility parameters, furthermore, samples were extracted and adjusted
sperm in fish reproductive management, however, there is no report on to a final concentration of 3 × 106 spermatozoa mL− 1 to perform the
the spermatozoa of sex-reversed female mandarin fish during short-term evaluation of sperm function parameters by flow cytometry (Beckman
storage. CytoFLEX, Beckman Coulter, Inc., California, USA) and confocal laser
Sperm motility measurement is a key step to evaluate the sperm scanning microscopy (Leica TCS SP8 STED 3×, Leica Microsystems Ltd.,
quality and fertilizing ability. With the application of Computer Assisted Wetzlar, Germany).
Sperm Analysis (CASA) in sperm analysis, the motility of activated
sperm can be graded and the percentage of motility sperm at all levels 2.3. Sperm evaluations
can be counted. There are also many indicators for evaluating sperm
motility, including the percentage of motility, straight line velocity 2.3.1. Sperm concentration
(VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity Sperm concentration was determined using CASA, diluting 1 μL of
(LIN), amplitude of lateral head displacement (ALH; Kime et al., 2001; semen in 1200 μL of D-15 diluent. Each sample was analyzed in
Rurangwa et al., 2004). Generally, high fertilization rate and hatching triplicate.
rate of sperm are used as a sign of successful storage and the most direct
demand of production is to obtain high fertilization rate and hatching 2.3.2. Sperm motility parameters
rate of stored sperm. As a result of short-term storage, the quality of The sperm motility parameters were measured with the CASA, as
gametes decreased due to plasma membrane damage, mitochondrial described by Dietrich et al. (2005). The motility variables measured
membrane damage and DNA damage (Labbé, 2010; Merino et al., 2017). included the percentage of motility, straight line velocity (VSL), curvi­
All these factors can lead to changes in sperm function, resulting in linear velocity (VCL), average path velocity (VAP), linearity (LIN) and
reduced fertility and vitality (Lahnsteiner et al., 1997; Perez-Cerezales amplitude of lateral head displacement (ALH). The analysis in each trial
et al., 2010). However, the use of flow cytometry and laser confocal was replicated three times.
scanning microscopy to analyze the effects of short-term sperm storage
on plasma membrane damage, mitochondrial membrane damage and 2.3.3. Plasma membrane integrity
DNA damage of undiluted and diluted sperm of sex-reversed female The cytoplasm membrane integrity was assessed using the LIVE/
mandarin fish has not been reported. DEAD Sperm Viability Kit (Invitrogen Inc., Eugene, OR, USA; SYBR-14/
The aim of this study was to evaluate the effect of short-term storage PI dye). Semen was washed, centrifuged and resuspended at 4 × 106
at 4 ◦ C of diluted and undiluted semen of sex-reversed female mandarin spermatozoa/mL in ice-cold PBS. 5 μL of diluted SYBR-14 dye and 5 μL
fish for 5 days on the parameters of motility, plasma membrane damage, propidium iodide (PI) were added to a 1 mL sample of diluted semen.
mitochondrial membrane damage and DNA damage through CASA, flow After exposure of the semen to this solution for 5–10 min at 36 ◦ C, then
cytometry and confocal laser scanning microscopy. In addition, the observed by flow cytometry and confocal laser scanning microscopy.
fertilization capacity and hatchability of sex-reversed female mandarin The analysis in each trial was replicated three times.
fish were evaluated in this study.
2.3.4. Mitochondrial membrane potential (Δψ Mit)
2. Materials and methods The changes of mitochondrial membrane potential (ΔΨM) were
determined using a fluorescent cation dye, 5,5′ ,6,6′ -tetrachloro-1,1′ ,3,3′
2.1. Collection and maintenance of broodstock tetraethylbenzymidazolyl carbocyanine iodide, commonly known as JC-
1 (Figueroa et al., 2013). JC-1 is an ideal fluorescent probe which is
Mature sex-reversed females of mandarin fish (one-year old; n = 9, widely used in detecting mitochondrial membrane potential (ΔΨM).
average length of 30.25 ± 3.21 cm) and mature females of mandarin fish When the mitochondrial membrane potential is high, JC-1 aggregates in
(one-year old; n = 12, average length of 34.57 ± 5.89 cm) were collected the matrix of mitochondria to form a polymer (J-aggregates), and red
from aquatic breeding farm in Foshan, Guangdong Province in April. fluorescence is generated. When the mitochondrial membrane potential
Over the course of the experiments, the fish were maintained in tanks at is low, JC-1 is the monomer that cannot gather in the mitochondrial
water temperature of 25–27 ◦ C and natural photoperiod. All animal matrix and can produce green fluorescence. This test was performed in
experiments were approved by the Institutional Animal Care and Use accordance with the instructions of the manufacturer of Mitochondrial
Committee of Sun Yat-sen University and performed in accordance with membrane potential assay kit with JC-1 (JC-1, Beyotime, Shanghai,
the guidelines for experimental animals established by this committee. China). In short, 500 μL of sperm sample was centrifuged at 600 g for 4
Sex-reversed females of mandarin fish were anesthetized by MS-222 and min. The pellet was resuspended in 500 μL JC-1 solution (JC-1 (200×) in
dissected to obtain the individual gonads, and gently squeezing through ultrapure water) and incubated at 37 ◦ C in the dark for 20 min. The cell
a double-layer gauze to remove any testicular tissue. Immediately after suspension was centrifuged at 600 g for 4 min, supernatant was

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S. Liu et al. Aquaculture 547 (2022) 737410

discarded, and the spermatozoa were washed twice with JC-1 (1×). statistical tests was set at 5% (P < 0.05).
Finally, the sperm pellet resuspended in 500 μL PBS and analyzed
immediately by flow cytometry and confocal laser scanning microscopy. 3. Results
The analysis of each trial was repeated three times.
3.1. Sperm concentration and motility parameters
2.3.5. DNA fragmentation
The TUNEL (One step TUNEL Apoptosis Assay Kit, Beyotime, The sperm concentration of mandarin fish was 1.82 ± 0.25 × 1010
Shanghai, China) procedure was used to assess DNA fragmentation. The mL− 1. Sperm motility of diluted and undiluted semen decreased grad­
sperm suspension with a concentration of 4 × 106/mL was washed in ually as storage time increased (Fig. 1a). The initial motility of fresh
PBS at 600 g for 4 min. The pellet was fixed with 4% paraformaldehyde semen after activation showing average values of 90.48 ± 0.84%.
for 30 min at room temperature. The suspension was washed in PBS at Motility of undiluted semen decreased significantly during storage, that
400 g for 4 min, and the pellet was resuspended in 500 μL of 0.3% Triton was significantly different (P < 0.05) from that of diluted semen with
X-100 prepared in PBS for 5 min at 4 ◦ C, then washed in PBS at 600 g for storage time of 3–5 days. Motility of DS1 and DS2 were not significantly
4 min. The pellets were then resuspended in 50 μL of TUNEL reaction different (P < 0.05) after storage for 1 or 3 days, which were signifi­
mixture (TdT and fluorescein-dUTP) and incubated in the dark and a cantly higher than that of US and DS3 of 3–5 days. At 5 days of storage,
humidified atmosphere at 37 ◦ C for 60 min. Next, the sperm resus­ motility of DS1 (45.55 ± 2.05%) was significantly higher than those of
pended in PBS and washed at 600 g for 4 min. The pellet then was other groups.
resuspended in 400 μL PBS. After washing, the label at the damaged VSL, VCL, VAP and ALH values of sex-reversed female sperm
DNA sites was analyzed directly by flow cytometry and confocal laser decreased gradually with the storage period increased (Table 1). In
scanning microscopy. Sperm with TUNEL positive, or with fragmented addition to LIN, VSL, VCL, VAP and ALH values of stored semen declined
DNA, are stained green. The analysis in each trial was replicated three compared with that of fresh semen of sex-reversed females, and the
times. values of DS1 were significantly higher than those of the other groups
with a storage time of 3–5 days.
2.4. Fertilizing ability and hatchability
3.2. Plasma membrane integrity
The females were maintained in ambient water temperature of
25–27 ◦ C after the injection of LHRH-A2 (Ningbo second hormone fac­ The initial plasma membrane integrity rate of fresh semen showing
tory, Zhejiang, China) and DOM (Ningbo Sansheng Biotechnology Co., average values of 95.45 ± 2.06%. At day 1, plasma membrane integrity
Ltd., Zhejiang, China). After broodstock maturation, fresh and stored rates of undiluted and diluted semen were higher than 90% (Fig. 1b).
sperm (1,3 and 5 days) of sex-reversed females were artificially insem­ However, plasma membrane integrity rates of undiluted and diluted
inated in vitro to evaluate the fertilization rate and the hatching rate. semen were significantly affected (P < 0.05) with the storage time of
0.4% NaCl solution was used to activate gametes and then mixed them 3–5 days. At day 3 of storage, no significant differences were found
gently. After the gametes were activated, the eggs were rinsed twice between undiluted and diluted semen (P > 0.05), however, there were
with well‑oxygenated water and then incubated in 25 L bucket with significant differences in all groups at day 5, and DS1 (76.55 ± 6.22%)
well‑oxygenated water. Each trail was repeated three times. was significantly higher than the other three groups (Fig. 1b).

2.5. Flow cytometry 3.3. Mitochondrial membrane integrity

A flow cytometer (Beckman CytoFLEX, Beckman Coulter, Inc., Cal­ At day 0, average values (>90%) of mitochondrial membrane
ifornia, USA) was used to determine the following variables: plasma integrity were found in fresh sperm of Siniperca chuatsi. ΔψMit of un­
membrane integrity, mitochondrial membrane potential and DNA diluted and diluted semen gradually decreased as the storage time
fragmentation. At least 10,000 sperm were examined in each trial. The increased (Fig. 1c). At day 3, ΔψMit was significantly decreased in all
spermatozoon probe was gated using 90-degree and forward-angle light groups (US: 83.92 ± 3.21%; DS1: 86.30 ± 2.09%; DS2: 86.59 ± 0.82%
scatter to exclude debris and aggregates. The excitation wavelength was and DS3: 85.13 ± 2.47%). At day 5, ΔψMit in diluted semen showed no
488 nm and provided by an argon laser at 15 mW. FITC channels statistically significant differences, which significantly higher than that
detected green fluorescence (480–530 nm) and ECD/PE channels in undiluted semen (Fig. 1c).
detected red fluorescence (580–630 nm; Fig. 4).
3.4. DNA fragmentation
2.6. Confocal laser scanning microscopy
The DNA fragmentation rate of fresh semen was very low at day
Sperm samples were observed under a confocal laser scanning mi­ 0 (1.65 ± 0.32%; Fig. 1d). There were no significant differences between
croscopy (Leica TCS SP8 STED 3×, Leica Microsystems Ltd., Wetzlar, undiluted and diluted semen at day 1 (P > 0.05). Furthermore, the DNA
Germany). The probe emission was assessed in the wavelength range of fragmentation rate did not increase significantly with the storage time of
405–635 nm. The samples were excited by a multiline PMT laser (488 0–1 days. At 3–5 days of storage, DNA fragmentation were significantly
nm) and HyD laser (555 nm). increased in all groups (P < 0.05). DS1 (34.61 ± 2.70%) was signifi­
cantly lower than that of other three groups, and US showed the highest
2.7. Statistical analysis rate of DNA fragmentation (66.01 ± 4.96%) at day 5.

The dates (mean ± SD) were analyzed using the statistical software 3.5. Fertility and hatchability
GraphPad Prism®, version 8.0 (GraphPad Software, Inc., San Diego, CA,
USA). Percentage of sperm motility, fertilizing ability and hatchability At day 0, the value of fertility was observed in fresh sperm of Sini­
were arc-sine square root transformed to normalize variance prior to perca chuatsi, showing values 93.44 ± 1.49%. Fertility of US was
statistical analysis. Data were compared by means of one-way and two- decreased significantly (P < 0.05) as the storage time increased (Fig. 2a).
way repeated measurements ANOVA followed by Duncan's multiple However, no significant decline in fertility was observed in diluted
range test for post hoc comparisons of means. SPSS version 22.0 was semen with the storage time of 0–1 days. At 3–5 days of storage, fertility
used for the analyses in this study. The significance level for all was significantly decreased in all groups (P < 0.05), and DS1was

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S. Liu et al. Aquaculture 547 (2022) 737410

Fig. 1. Effect of short-term storage on sperm function of undiluted and diluted semen (1:2 v/v, 1:3 v/v and 1:4 v/v) of sex-reversed female Mandarin fish (Siniperca
chuatsi) for 5 days at 4 ◦ C. (a) Motility (%), (b) Plasma membrane integrity (%), (c) Mitochondrial membrane potential (ΔψMit; %) and (d) DNA fragmentation (%).
Data are presented as mean ± SD. a, b, c, dDifferent letters indicate statistically significant differences in the same group at different periods of storage time. *Asterisks
indicate statistically significant differences between groups in the same storage period (P < 0.05, n = 9).

significantly higher than that of other three groups. Fertility of DS1 still semen at low temperatures lead to a significant decline in sperm func­
achieved 57.83 ± 10.20%, while no fertility was found in US at day 5 tions, impairing motility, plasma membrane integrity (Contreras et al.,
(Fig. 2a). 2017; Merino et al., 2017), mitochondrial membrane potential (Merino
Hatchability of undiluted and diluted semen gradually decreased as et al., 2017; Trigo et al., 2015), DNA integrity (Shaliutina et al., 2013;
the storage time increased (Fig. 2b). The hatchability of DS2 showed no Contreras et al., 2017; Risopatron et al., 2018) and fertility (Geffen and
significant variation with the storage time of 0–3 days (P > 0.05), which Evans, 2000). The values of these parameters provide a well basis for
was higher than that of other three groups. The hatchability of DS1 and understanding the physiological parameters of sperm in the process of
DS2 had no significant difference (P > 0.05), showing values >70% at storage (Merino et al., 2011; Merino et al., 2012).
day 5. Successful storage is subject to species specificity and storage con­
ditions. Previous study found that the most effective extender for the
long-term cryopreservation of sex-reversed female mandarin fish sperm
3.6. Confocal microscopy was diluting in the D-15 extender (Liu et al., 2019). Our study found that
D-15 extender was also suitable for short-term storage of sex-reversed
Fig. 3 shows a confocal microscopy image of plasma membrane female sperm of mandarin fish. In fish, motility is the parameter
damage, mitochondrial membrane damage and DNA damage of sperm commonly used to evaluate sperm quality, as high motility is a prereq­
of sex-reversed female mandarin fish after short-term storage. uisite for fertilization and is closely related to the success of fertilization
(Rurangwa et al., 2004). Semen of Oncorhynchus kisutch diluted in
4. Discussion Storfish® maintained a higher motility than that of undiluted semen
during 3 days of storage at 4 ◦ C, and no significant differences were
Short-term storage of semen is a necessary key technique in aqua­ found between dilutions 1:2 and 1:3 (Merino et al., 2020), which is
culture to allow maximizing sperm quality, which is a crucial issue in the consistent with the results reported here for mandarin fish. Furthermore,
support of hatchery production in aquaculture (Fitzpatrick et al., 2005; the sperm of Refrigerated Atlantic cod and haddock diluted 1:3 with
Valdebenito et al., 2017). Successful short-term storage of semen has modified Mounib's extender maintained about 3% motility after for 40
been reported in some cultured teleost, such as walking catfish (Clarias and 38 days of storage, respectively, while undiluted sperm remained
macrocephalus; Vuthiphandchai et al., 2009), Patagonian blenny (Ele­ low motility for only 10 and 20 days (Ciereszko and Dabrowski, 1994). It
ginops maclovinus; Contreras et al., 2017), Siberian (A. baerii; Shaliutina is indicated that semen diluting in extender was avail for semen storage.
et al., 2013) and carp (Cyprinus carpio L.; Dietrich et al., 2021). Up to In our study, high motility was found in all groups at day 0, however, all
now, there is no date on short-term storage of semen from sex-reversed groups showed a significant decline at day 3 together with the decrease
females of mandarin fish currently. In this study, flow cytometry and in fertility, showing a positive correlation between the both parameters.
confocal laser scanning microscopy were used for the first time to The sperm motility parameters detected by CASA were consistent with
evaluate the effect of in vitro storage at 4 ◦ C of undiluted and diluted the result of sperm motility in sex-reversed females. The curvilinear
semen of mandarin fish on the sperm functional parameters. These re­ velocity (VCL) is considered as an indicator of sperm quality
sults are consistent with previous research that short-term storage of fish

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S. Liu et al. Aquaculture 547 (2022) 737410

Table 1 (Lahnsteiner et al., 1998), so VCL can be an important parameter for the
Sex-reversed female mandarin fish (Siniperca chuatsi) sperm motility variables at evaluation of sperm quality. In addition to the LIN, the parameters of
4 ◦ C for 5 days. VSL, VCL, VAP and ALH of sex-revered female sperm declined after
Storage Dilution Pseudo-male sperm parameters LIN (%) short-term storage. The value of LIN increased and was negatively
days rate
VSL VCL VAP ALH
correlated with the fertilization rate, which is consistent with the results
(4 ◦ C) of sex-reversal rainbow trout (Dietrich et al., 2014), which indicated
(μm/s) (μm/s) (μm/s) (μm)
that LIN can be used as a predictor of sperm quality of sex-reversal fish.
0 Control 26.56 48.83 34.53 14.31 55.33
± 0.61 ± 5.93 ± 4.19 ± 1.74 ± 8.50 In general, sperm motility decreased after storage in most fish, but the
A A A A A values of these parameters didn't decline all the time, and some of them
1 Undiluted 16.75 37.12 26.39 10.88 45.00 increased or remain the same (Dietrich et al., 2021; Babiak et al., 2006;
± 4.27 ± 7.08 ± 5.01 ± 2.08 ± 3.00 Dietrich et al., 2014, 2016). These changes in sperm motility parameters
Bb Bab Bab A A
1:2 20.67 43.37 30.67 12.70 47.67
may be related to different storage methods, or the positive correlation
± 2.99 ± 6.08 ± 4.30 ± 1.78 ± 0.58 between their values and sperm motility may be species specificity.
ABab Aa Aa AB A The plasma membrane is sensitive to cryopreservation (Anchordo­
1:3 24.83 41.01 29.00 12.01 61.33 guy et al., 1987; Muller et al., 2008). In this study, plasma membrane
± 1.78 ± 6.99 ± 4.95 ± 2.05 ± 6.51
integrity of DS1 was significantly higher than that of other three groups
Aa ABa Aa AB A
1:4 17.15 29.42 20.80 8.62 ± 58.33 at day 5, which was consistent with the result of sperm motility and
± 0.61 ± 2.89 ± 2.04 0.85 B ± 7.97 fertility. However, it is noteworthy that no motility was observed in the
Bb Bb Bb A undiluted semen at day 5 while the plasma membrane integrity of un­
3 Undiluted 11.23 20.47 14.80 6.00 ± 58.33 diluted semen remained high during storage period. We propose that
± 0.89 ± 6.61 1.94 B
short-term storage can avoid the formation of large crystals which may
± ±
B Cb 0.34C 15.28 A
1:2 17.19 32.37 20.12 9.25 ± 53.33 lead to the disruption of cytoplasm membranes. The change in mito­
± 0.65 ± 2.56 ± 0.91 1.47 ± 6.11 chondrial membrane integrity is a good indicator of functional
B Ba B BCE A normality. Although the lipophilic compound JC-1 has been used to
1:3 15.41 30.15 19.63 9.11 ± 51.33
evaluate the depolarization of the sperm mitochondrial membrane in
± 1.03 ± 3.18 ± 1.69 2.19 B ± 7.37
B BCab B A different species (Figueroa et al., 2013; Figueroa et al., 2015; Lie­
1:4 16.24 26.09 18.82 7.86 ± 62.00 bermann and Tucker, 2002; Viveiros and Godinho, 2009; Slabbert et al.,
± 1.95 ± 2.45 ± 1.86 1.07 B ± 2.00 2014), its application in the spermic mitochondria of mandarin fish has
B Bab B A
5 Undiluted 9.79 ± 16.63 11.67 3.94 ± 59.33
0.79 B ± 1.52 ± 1.93 B ±
Cb 1.53C 10.11 A
1:2 15.95 27.92 17.25 7.45 ± 57.00
± 0.57 ± 0.49 ± 2.16 1.26C ± 1.00
B Ba B A
1:3 13.62 22.54 15.47 8.16 ± 60.33
± 1.37 ± 2.15 ± 2.18 0.87 B ± 1.53
B Cab B A
1:4 12.38 19.85 14.70 6.41 ± 62.00
± 1.76 ± 0.98 ± 2.90 0.90 B ± 6.00
B Bab B A

All experimental data are expressed as mean ± SD. Different letters represent Fig. 3. Confocal microscopy of sex-reversed female mandarin fish (Siniperca
significant differences (P < 0.05), the difference of capital letters indicates that chuatsi) spermatozoa. (A) SYBR-14/PI staining: green spermatozoa with intact
the same group has significant difference in different storage time, and the cytoplasm membrane, red spermatozoa with disrupted cytoplasm membrane.
difference of lowercase letters indicates that the different groups have significant (B) JC-1 staining: red (arrow) spermatozoa with an intact mitochondrial
difference in the same storage time. membrane. (C) TUNEL staining, green spermatozoa with fragmented DNA. (For
interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

Fig. 2. Effect of short-term storage on Fertility and Hatchability of undiluted and diluted semen (1:2 v/v, 1:3 v/v and 1:4 v/v) of sex-reversed female Mandarin fish
(Siniperca chuatsi) for 5 days at 4 ◦ C. (a) Fertility (%), (b) Hatchability (%). Data are presented as mean ± SD. a, b, c, dDifferent letters indicate statistically significant
differences in the same group at different periods of storage time. *Asterisks indicate statistically significant differences between groups in the same storage period (P
< 0.05, n = 9).

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S. Liu et al. Aquaculture 547 (2022) 737410

Fig. 4. Three representative flow cytometry dot plots for IP/SYBR-14 (Aa, Ab, Ac), JC-1 (Ba, Bb, Bc) and TUNEL (Ca, Cb, Cc) staining. (Aa, Ba, Ca) Region R1:
spermatozoa set to FSC-A versus SSC-A, (Ab, Bb, Cb) Staining gated by region R1, the X-axis shows the intensity of fluorescence in the green spectrum (FITC-A
channel) on a linear scale, the Y-axis depicts the intensity of fluorescence in the red spectrum (ECD-A/PE-A channel) on a logarithmic scale. Cytoplasm membrane
integrity (Ab). Upper left (UL): cells with damaged membranes, lower right (LR): cells with intact cytoplasm membranes, upper right (UR): cells with partly damaged
cytoplasm membranes. Mitochondrial membrane integrity (Bb). Upper right (UR): cells with intact mitochondrial membranes; lower right (LR), cells with damaged
mitochondrial membranes. DNA fragmentation (Cb). Left, cells with intact DNA; Right, cells with fragmented DNA. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

not been reported. Mitochondria play a significant role in the activation sperm is an important basis for determining sperm quality. Damage to
mechanism of flagellar movement during sperm motility (Figueroa the DNA of sperm would affect embryonic development and lead to early
et al., 2013; Ulloa-Rodríguez et al., 2017). Mitochondria provide energy embryo loss (Kopeika et al., 2003). In this study, DNA fragmentation was
for the flagella, and many animals develop sperm with large number of consistent with the result of fertility and hatchability. US showed the
mitochondria, the function and number of which are positively corre­ highest rate of DNA fragmentation (66.01 ± 4.96%) at day 5. Fertility of
lated with sperm motility and fertilizing capacity (Ferramosca et al., DS1 (DNA fragmentation < 35%) still achieved 57.83 ± 10.20%, while
2013). Storage time had a significant effect on the ΔψMit of undiluted no fertility and hatchability were found in undiluted semen at day 5.
and diluted semen. These results are consistent with previous research This may allow diluents to have a good protective effect on sperm. At
showing that chilled storage of fish sperm leads to a decline of ΔψMit 3–5 days of storage, DNA fragmentation was significantly increased in
(Trigo et al., 2015; Merino et al., 2017). In this research, ΔψMit all groups, DS1 was significantly lower than that of other three groups,
decreased significantly in all groups at day 3. The results are consistent and the fertility was also significantly higher than that of other three
with previous studies (Figueroa et al., 2013; Figueroa et al., 2016), and groups, which suggest that low fertility may be strongly related to DNA
indicate that low motility observed after 3 days storage may be related damage. It is possible that DNA fragmentation may be due to oxidative
to the mitochondria damage. Moreover, mitochondria damage during stress caused by short storage in fish, and the oxygen consumed by
storage would lead to sperm motility reduction and thus to fertility sperm during storage is a certain, and a high concentration of semen
decreased definitely (Figueroa et al., 2015). reduces the oxygen dissolved in the system, which agree with those
DNA integrity plays a significant role in sperm quality and the previously studies (Lahnsteiner et al., 2011; Shaliutina et al., 2013;
development and growth of offspring, and nuclear DNA damage of Adiyodi and Adiyodi, 1983).The quality of diluted sperm post

6
S. Liu et al. Aquaculture 547 (2022) 737410

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