Fish Physiol Biochem
https://doi.org/10.1007/s10695-020-00860-2
Effect of chilled storage on sperm quality of basa catfish
(Pangasius bocourti)
Sen Yang & Wenhua Huang & Huichong Chen &
Muzhen Huang & Yongzhong Liufu & Zining Meng
Received: 5 February 2020 / Accepted: 13 August 2020
# Springer Nature B.V. 2020
Abstract Basa catfish (Pangasius bocourti) is an indig-
enous species of the Lower Mekong River with increas-
ing aquaculture value in Southeast Asia. Short-term
semen storage has proven to be a valuable tool for
assisted reproduction in fish, but little information is
available on basa catfish. The present study was aimed
at evaluating the effect of short-term semen storage on
the sperm quality of basa catfish. Semen samples were
kept at 4 °C for 7 days, either undiluted or diluted with
Ca-F HBSS at different ratios (1:1, 1:3, and 1:6;
semen:medium). Results showed that sperm quality
was significantly affected by the time of chilled storage,
characterized by a decline in sperm motility, viability,
mitochondrial membrane potential (MMP), ATP con-
tent, and increased level of lipid peroxidation
Sen Yang and Wenhua Huang contributed equally to this work.
S. Yang
College of Animal Science and Technology, Zhongkai University
of Agriculture and Engineering, Guangzhou 510000, China
W. Huang : Z. Meng (*)
State Key Laboratory of Biocontrol, Institute of Aquatic Economic
Animals and Guangdong Province Key Laboratory for Aquatic
Economic Animals, School of Life Sciences, Sun Yat-Sen
University, Guangzhou 510275, China
e-mail: mengzn@mail.sysu.edu.cn
H. Chen : M. Huang : Y. Liufu
Guangdong Provincial Fishery Germplasm Conservation Center,
Guangzhou 511453, China
Z. Meng
Southern Laboratory of Ocean Science and Engineering,
Zhuhai 519000, China
throughout the storage period. Compared with undiluted
semen, diluted in Ca-F HBSS allowed better preserva-
tion of sperm quality during 7 days of chilled storage;
dilution ratio at 1:1 was more effective than higher ratios
(1:3 and 1:6) for prolonging sperm storability. In addi-
tion, sperm motility, viability, and ATP content de-
creased more rapidly than MMP, suggesting these indi-
cators are more sensitive in detecting sperm damage of
basa catfish during short-term chilled storage. These
results obtained here will contribute to a better under-
standing of reproductive management in this species.
Keywords Basa catfish . Pangasius bocourti . Sperm
quality . Chilled storage
Introduction
The basa catfish (Pangasius bocourti) is an indigenous
species in the Lower Mekong River that belongs to the
Pangasiidae family (Cacot et al. 2003). Owing to the
desirable traits such as fast growth, high yield, and lack
of horizontal bones, it has been widely cultured as a new
economic fish in freshwater systems of the Mekong
Delta during the past decades, especially in Vietnam
and Thailand (Jiwyam 2010). Moreover, the white flesh,
low-fat content, and good taste also make it an attracting
fish food source (Thammapat et al. 2010). It is estimated
that the Pangasius fish production has reached 800,000
tons per year in 2013, supplying more than 100 coun-
tries worldwide with an export value at USD 1.5 billion
(Trifković 2014; Van Doan et al., 2016). Although

artificial propagation has been successfully achieved in
basa catfish since 1995 (Le Thanh et al. 1999), adequate
seed supply is gradually becoming one of the bottle-
necks for the rapid expansion culturing of this fish, due
to the shortage of broodstocks or asynchrony in gamete
production under captivity (Cacot et al. 2003; Kainin
et al. 2014). Consequently, gamete conservation in this
species is of obvious benefit as it would maximize the
reproductive potential of the breeding fish and increase
flexibility for the organization of hatchery practice.
Currently, many assisted reproductive technologies
are used in fish breeding and fish farming with the aim
of improving reproductive efficiency. Among the tech-
niques used for gamete conservation, short-term storage
(refrigerated) and cryopreservation of semen have prov-
en to be two valuable tools for routine management in
aquaculture (Contreras et al. 2019; Magnotti et al.,
2018). In a previous research conducted by Kainin
et al. (2014), a sperm cryopreservation protocol of basa
catfish has been developed and Ca-F HBSS was shown
to be the most suitable extender; the results of which
could be used to facilitate breeding programs in
P. bocourti hatcheries. In recent years, the storage of
refrigerated semen has an increasing number of appli-
cations in aquaculture practice (Contreras et al. 2019),
but there are few reports on basa catfish. In fact, appro-
priate semen short-term storage can be a powerful sup-
plement to the technology of cryopreservation from a
practical point of view, as it is a simple and cost-
effective procedure with no need of complicated instru-
ments or specific training (Trigo et al. 2015; Ubilla et al.,
2015), which is particularly important under circum-
stances lacking instruments or species-specific protocol
for cryopreservation.
Short-term storage of semen can be achieved with
samples preserved either undiluted or diluted at low
temperatures, allowing maintaining viable spermatozoa
for hours, days, and even weeks (Contreras et al. 2019).
Different factors, oxidative stress in particular, however,
would inevitably lead to cellular aging and cause dete-
rioration of sperm quality within the storage period; the
duration that sperm maintains its quality may vary
greatly, as has been assessed in various fish species
(Aramli et al., 2013; Gao et al. 2019; Sun et al. 2010;
Trigo et al. 2015); thus, a better understanding of this
process would help to optimize gamete management
during artificial reproduction.
With undiluted semen as control, the aim of this
study was to (1) fully evaluate the effect of chilled
Fish Physiol Biochem
storage (4 °C) on sperm quality and (2) determine the
proper dilution ratio for semen storage in basa catfish,
using sperm functional parameters including motility,
viability, MMP, ATP content, and lipid peroxidation.
Materials and methods
Broodstock management and semen collection
Sexually mature broodstock basa catfish (3 to 5 years
old, 4 to 7 kg) were provided by the Guangdong Pro-
vincial Fishery Germplasm Conservation Center in
Guangzhou, China (N 22° 53′ 43″, E 113° 24′ 56″).
During the experimental period, the fish were kept at
indoor flow-through cement tanks (40 m3) at 26–28 °C
under a natural photoperiod. The raising density was
about 3 kg/m3 and fish were fed once a day with a
commercial food (30% protein, Yuehai Feeds Group,
Guangdong, China) at 1% body weight, but feeding was
stopped 12 h prior to semen collection. The broodfish
were removed from the water and anesthetized by eu-
genol at a dose of 40 mg/L before sampling.
Semen was collected into 10-ml tubes by gentle
abdominal massage of mature males; contaminated
samples with urine, blood, or freshwater were discarded.
Samples were then stored at 4 °C for no more than
30 min before the following processing.
All animal handling and procedures in this study
were approved by the Animal Ethics Committee of
Sun Yat-Sen University.
Experimental design
Considering the variability in sperm concentration
among males (3.67–5.28 × 1010 cells/mL), samples with
initial total motility > 85% were selected and sperm
concentration was adjusted to 3.5 × 1010 cells/ml by
sperm-free seminal plasma for the experiment (n = 6);
seminal plasma was obtained by the centrifugation of
semen samples from another two individuals at 8000×g
for 10 min (4 °C). Then, samples were diluted at ratios
of 1:1, 1:3, and 1:6 (semen:medium, v/v) with Ca-F
HBSS (Kainin et al. 2014), and undiluted semen was
served as the control. The stored samples (final volume
700 μL) were maintained in separate 1.5-ml Eppendorf
tubes at 4 °C, and the sampling times were 12 h, 1, 3, 5,
and 7 days after chilled storage. At each time point,
aliquots of each sample were removed to assess the
Fish Physiol Biochem
sperm quality. To prevent sperm sedimentation, all sam-
ples were mixed daily by turning the tubes upside down.
Assessment of sperm quality
Sperm motility and concentration
Sperm motility was evaluated with an Integrated System
for Semen Analysis system (ISAS 2.0, Valencia, Spain)
according to the procedure described by Yang et al.
(2017). Briefly, 1 μL of semen sample was diluted in
200 μL activating solution (distilled water plus 0.1% w/
v bovine serum albumin). Then, 10 μL of the mixture
was quickly deposited in the chamber under × 10
negative-phase objective for sampling. At least 500
spermatozoa from 3 to 5 fields were captured for each
sample. Sperm swimming with a curvilinear velocity
(VCL) > 10 μm/s was considered as motile. After eval-
uation, the following motility parameters were chosen
for further analysis in this study: total motility (TM, %),
progressive motility (PM, %), and VCL (μm/s). PM was
defined as sperm moved with straightness > 80%.
Sperm concentration was measured by the concen-
tration module of ISAS software after dilution of semen
by 1:1000 with Ca-F HBSS.
Sperm viability
Sperm viability was assessed by staining with the com-
bination of SYBR-14/propidium iodide (PI) probes
(Live/Dead Sperm Viability Kit, Thermo Fisher Scien-
tific, Waltham, USA). Briefly, 200 μL sperm suspen-
sion (106 cells/mL) was mixed with 1 μL SYBR-14
(25 μM) and 1 μL PI (2.4 mM), incubated at 4 °C in
the dark for 10 min and then analyzed by flow
cytometer. Live sperm (SYBR-14 labeled, green) and
dead sperm (PI labeled, red) were counted, and viability
was determined as the proportion of PI-negative cells.
Mitochondrial membrane potential
To evaluate the mitochondrial function, a commonly
used dye, JC-1 (JC-1 Assay Kit, Beyotime, Shanghai,
China), was adopted to sort cells with relatively higher
mitochondrial membrane potential (MMP). In brief,
about 107 sperm from each sample was stained in
500 μL JC-1 working solution and incubated at room
temperature for 20 min; after which, samples were
washed twice with the washing buffer and resuspended
for analysis by flow cytometer. Cells stained with red
(JC-1 aggregates) were recorded as high MMP, and
those showing green fluorescence (JC-1 monomer)
was recorded as cell population with low MMP.
ATP content
The level of ATP was determined with the ATP Biolu-
minescence Assay Kit (Beyotime, Shanghai, China)
following the manufacturer’s instruction. Briefly, 109
sperm was fully lysed in 500 μL lysis buffer; the super-
natant was collected by centrifugation at 12, 000g for
5 min at 4 °C. Afterwards, 20 μL supernatant was mixed
with 100 μL ATP detection working solution in a 96-
well plate, and the luciferase activity was then evaluated
by a multifunction microplate reader (Thermo Fisher,
USA). The ATP content was calculated from a concur-
rent standard curve and expressed as nmol/109 cells.
Quantification of lipid peroxidation
As malondialdehyde (MDA) was a typical metabolite of
lipid peroxidation and could be used to reflect the level
of lipid peroxidation, here, we measured MDA content
in sperm using a Lipid Peroxidation MDA Assay Kit
(Beyotime, China) in accordance with the manufac-
turer’s protocol. Diluted sperm was lysed and centri-
fuged at 12,000×g for 10 min at 4 °C to collect the
supernatant. Subsequently, 100 μL supernatant was
mixed with 200 μL reagent provided by the kit and
heated at 100 °C for 15 min. The absorbance of the
mixture was read at 532 nm using a microplate reader.
MDA content was calculated from a standard curve and
presented as nmol/108 cells.
Flow cytofluorimetric analysis
A CytoFlex flow cytometer (Beckman Coulter, USA)
was used to assess the proportion of viable sperm and
sperm with relatively higher MMP. Samples with
probes were kept in the dark at 4 °C prior to analysis.
Cells were excited by a 488-nm laser (50-mW laser
output) and the emitted green and red fluorescence
was captured through the 525/40-nm and 610/20-nm
band pass filter, respectively. A minimum of 10,000
sperm-specific events was examined for each sample
at a flow rate of 300 cells/s. Cell gating and data analysis
were performed by the software CytExpert (Beckman
Coulter, USA).

Statistical analysis
Results are presented as mean ± SD (standard devia-
tion). Percentage data was normalized by arcsine
square-root transformation prior to analysis. Two-way
ANOVA was applied to examine the effect of storage
time and dilution treatment, followed by Tukey’s test for
post hoc comparisons of means. The significance level
was set at P < 0.05. All the analyses were conducted in
the software program SPSS version 20.0 (SPSS, Chica-
go, IL, USA).
Results
Sperm motility
With the increasing of chilled storage time, all the
motility parameters (TM, PM, and VCL) decreased in
different treatments (Table 1). TM, PM, and VCL de-
tected in undiluted semen after 12-h chilled storage were
92.7 ± 2.3%, 42.2 ± 8.5%, and 87.1 ± 5.7 μm/s, respec-
tively, which were not significantly different with semen
diluted at different ratios. At each time point in day 1 to
day 7, TM in semen diluted at the ratio of 1:1 was
relatively higher than that in other treatments. A 3-day
storage led to dramatic declines of sperm motility in
undiluted semen (TM: 58.6 to 12.2%; PM: 24.0 to
0.8%; VCL: 61.3 to 27.7 μm/s). Compared with the
undiluted sample, diluted semen exhibited a lesser de-
crease in sperm motility from day 1 to day 7. At the end
of the experiment (day 7), most of the spermatozoa in
undiluted sample remained immotile (TM, 6.8 ± 1.2%;
PM, 0.3 ± 0.1%; VCL, 25.5 ± 3.0 μm/s), whereas corre-
sponding parameters in semen diluted at 1:1, 1:3, or 1:6
still showed relatively higher values (P ≤ 0.05).
Sperm viability
At the beginning of the experiment (12 h), sperm via-
bility in diluted and undiluted semen was not signifi-
cantly different, with values > 90% (Fig. 1). At day 1,
the viability results decreased with varying degrees but
little difference was detected between treatments
(P > 0.05). However, semen diluted at 1:1 showed
higher viability than other samples at day 3 and day 5.
After 7 days of storage, all the treatments retained sim-
ilar viability with values ranging from 7.4 ± 2.8 to 15.6
± 4.4% (P > 0.05).
Fish Physiol Biochem
Mitochondrial membrane potential
During the first 3-day storage period, a high MMP rate
was observed in diluted and undiluted samples, showing
mean values > 90% with little difference over time or
between treatments (P > 0.05; Fig. 2). From day 3 to day
5, a significant reduction in MMP was detected but still
maintained over 75% in all treatments. At day 7 of
storage, it decreased remarkably regardless of the treat-
ment; the corresponding MMP for the undiluted sample
and semen diluted at 1:1, 1:3, and 1:6 was 21.1 ± 3.4%,
8.9 ± 1.7%, 13.1 ± 2.3%, and 5.6 ± 1.1%, respectively
(Fig. 2).
Evaluation of ATP content
The content of ATP at 12 h in undiluted and diluted
semen 1:1, 1:3, and 1:6 was 99.9 ± 12.1, 266.5 ± 19.2,
303.2 ± 21.3, and 369.3 ± 13.1 nmol/109 cells, respec-
tively. A decline in ATP content was observed over time
at different rates, depending on the treatment (Fig. 3). At
12 h and day 1, the ATP content of diluted semen was
significantly higher than that of undiluted one, but no
significant difference was observed among samples di-
luted at ratios from 1:1 to 1:6 (Fig. 3). At day 3 and day
5, much higher ATP content was recorded in semen
diluted at 1:1 when compared with others (P < 0.05),
while no difference was observed among all treatments
on day 7.
Lipid peroxidation
With regard to lipid peroxidation, the level of which
elevated with the storage time as reflected by the MDA
content (Fig. 4). In particular, from day 5 to day 7, the
level of lipid peroxidation increased to more than three-
fold in all the studied treatments. At 12 h of storage, the
lowest MDA content was found in undiluted semen
(2.7 ± 1.1 nmol/108 cells). In contrast, from day 1 to
day 7, diluted semen exhibited lower MDA content in
comparison with the undiluted sample.
Discussion
Compared with oocytes, it is relatively easier to preserve
semen in fish, either by cryopreservation or chilled
storage. As reviewed by Contreras et al. (2019), suc-
cessful semen cool storage has been reported in many
Fish Physiol Biochem

Table 1 Changes of sperm kinematic parameters in basa catfish (P. bocourti) after chilled storage of undiluted and diluted semen. TM, total
motility; PM, progressive motility; VCL, curvilinear velocity

Time Undiluted Diluted

1:1 1:3 1:6

TM (%)
12 h 92.7 ± 2.3a,1 92.3 ± 3.8a,1 91.6 ± 1.5a,1 93.3 ± 2.1a,1
1 day 58.6 ± 1.5b,1 80.3 ± 3.7b,2 64.2 ± 6.7b,1 59.4 ± 4.6b,1
c,1 c,2 c,2
3 day 12.2 ± 2.4 54.7 ± 3.4 47.0 ± 3.3 36.3 ± 5.3c,3
c,1 d,2 d,23
5 day 7.3 ± 2.9 44.8 ± 4.5 34.1 ± 5.2 32.7 ± 6.9c,3
c,1 e,2 e,2
7 day 6.8 ± 1.2 21.3 ± 2.9 20.8 ± 5.5 16.0 ± 4.7d,2
PM (%)
12 h 42.2 ± 8.5a,1 39.8 ± 5.3a,1 40.7 ± 4.5a,1 40.0 ± 5.6a,1
b,1 b,1 b,1
1 day 24.0 ± 5.1 24.1 ± 2.7 25.4 ± 3.6 25.2 ± 3.7b,1
c,1 c,2 c,2
3 day 0.8 ± 0.4 10.7 ± 3.0 9.8 ± 1.7 7.4 ± 0.6c,2
c,1 d,2 d,2
5 day 0.3 ± 0.1 2.9 ± 0.5 2.6 ± 0.9 1.3 ± 0.3c,1
c,1 d,2 d,2
7 day 0.3 ± 0.1 2.6 ± 0.4 1.7 ± 0.8 1.6 ± 0.7c,2
VCL (μm/s)
12 h 87.1 ± 5.7a,1 90.3 ± 3.0a,1 85.1 ± 6.7a,1 87.1 ± 4.6a,1
b,1 b,1 b,1
1 day 61.3 ± 4.2 63.7 ± 1.6 63.2 ± 4.5 66.5 ± 6.9b,1
c,1 c,2 c,2
3 day 27.7 ± 2.5 48.2 ± 1.7 45.3 ± 2.1 40.8 ± 1.4c,3
c,1 d,2 d,12
5 day 26.9 ± 3.5 35.7 ± 1.0 32.9 ± 4.3 28.3 ± 1.1d,1
7 day 25.5 ± 3.0c,1 34.4 ± 0.9d,2 31.8 ± 2.5d,12 30.1 ± 4.7d,12
a–e
Different superscripts within the same columns indicate significant differences (P < 0.05) in the same treatment along time
1, 2, 3
Different superscripts within the same rows indicate significant differences (P < 0.05) among treatments at a given time point

different fish species. Regarding basa catfish, the effect
of 7-day storage on sperm motility was fully evaluated
in the present study, a series of physiological and bio-
chemical changes in spermatozoa were observed. In
fact, it is not uncommon detecting sperm quality degra-
dation during chilled storage of fish semen; sperm me-
tabolism itself and stressors such as cooling, storage
duration, oxygen consumption, and possible bacterial
growth would contribute to the accumulation of reactive
oxygen species (Contreras et al. 2019), causing a
compromised plasma membrane integrity, motility and
MMP (Contreras et al. 2017; Trigo et al. 2015), increas-
ing the level of lipid peroxidation, and DNA fragmen-
tation (Aramli 2014; Shaliutina et al. 2013).
Among the different semen quality biomarkers, mo-
tility is nowadays the most commonly used parameter in
fish (Gallego and Asturiano, 2019), which is highly
correlated with fertilization success in several fish spe-
cies (Gallego et al. 2018). In this study, a significant
decline in sperm motility over time was observed, with a

Fig. 1 Effect of chilled storage

on sperm viability in diluted and
undiluted semen of basa catfish
(P. bocourti). a–eDifferent
superscripts indicate significant
differences in the same treatment
at different periods of storage
time. Asterisks (*) indicate
statistically significant differences
among treatments at the same
time point (P < 0.05)

Fig. 2 Effect of chilled storage on sperm mitochondrial mem-
brane potential (MMP) in diluted and undiluted semen of basa
catfish (P. bocourti). a, b, c Different superscripts indicate
higher level of motility parameters in diluted samples
from day 1 to day 7. The loss of motility could be
explained by the depletion of intracellular ATP over
the storage time, as has been demonstrated in beluga
(Huso huso) (Aramli 2014), Persian sturgeon
(Acipenser persicus) (Aramli et al. 2013), Atlantic salm-
on (Salmo salar L.) (Dziewulska et al., 2010), and
Patagonian blenny (Eleginops maclovinus) (Contreras
et al. 2017). Indeed, the intracellular ATP content ex-
hibited a similar decreasing trend with motility in the
present study (Table 1 and Fig. 3). As for undiluted
semen, the anoxia atmosphere may accelerate the con-
sumption of ATP for maintenance of vital functions
during storage, thus resulting in a drastic reduction in
motility (Table 1). At 12 h of storage, it is noteworthy
that ATP content in undiluted semen was significantly
lower than diluted ones (Fig. 3), although no significant
difference was found in motility parameters at the same
time, implying that dilution after semen stripping should
be conducted as soon as possible to avoid extra ATP
consumption for the purpose of short-term storage.
The plasma membrane and mitochondrial integrity is
of fundamental importance for sperm functionality and
Fig. 3 Changes of ATP content
in undiluted and diluted semen of
basa catfish (P. bocourti) during
chilled storage. Different
superscripts indicate significant
differences (P < 0.05) in the same
treatment at different periods of
storage time (a, b, c, d) and among
treatments at the same time point
(1, 2, 3)
Fish Physiol Biochem
significant differences in the same treatment at different periods
of storage time. Asterisks (*) indicate statistically significant dif-
ferences among treatments at the same time point (P < 0.05)
fertilizing capacity (Dzyuba and Cosson 2014; Figueroa
et al. 2013). As expected, sperm viability obtained in
this study showed a significant reduction in all the
treatments from day 1–7, matching the deterioration of
motility parameters at the same storage time. Compared
with motility, viability, or ATP content, mitochondrial
integrity seemed to be less sensitive in reflecting sperm
functional impairment of basa catfish, as little difference
was observed in MMP either over time or between
treatments in the first 3 days of storage (P > 0.05); a
significant decrease occurred until day 5. Similarly, a
previous study on striped bass (Morone saxatilis) has
revealed that sperm ATP was affected more significant-
ly than MMP during a 24-h storage (Guthrie et al. 2011).
In contrast to the present study, changes in mitochon-
drial membrane potential have proven to be a good
indicator of sperm function in rainbow trout (Oncorhyn-
chus mykiss) (Figueroa et al. 2013), Atlantic salmon
(S. salar) (Figueroa et al. 2015), and Patagonian blenny
(E. maclovinus) (Contreras et al. 2017). In this sense, it
is necessary to integrate different parameters to evaluate
the sperm quality, given that one indicator alone may
not determine so accurately.
Fish Physiol Biochem
Fig. 4 Changes of sperm lipid peroxidation (MDA concentration)
in undiluted and diluted semen of basa catfish (P. bocourti) during
chilled storage. a, b, cDifferent superscripts indicate significant
In this study, the MDA content was increasing with
the storage period in all the samples, indicating an
elevated level of lipid peroxidation. This is consistent
with the results obtained from beluga (H. huso) (Aramli
2014), Russian sturgeon (A. gueldenstaedtii), and Sibe-
rian sturgeon (A. baerii) (Shaliutina et al. 2013), show-
ing that short-term storage induces a significantly in-
creasing level of sperm lipid peroxidation. Because of
the relatively higher amounts of unsaturated fatty acids
than other species, aquatic species’ sperm is particularly
susceptible to oxidative damage (Trenzado et al. 2006).
According to Li et al. (2009), disequilibrium between
oxidative stress and the spermatozoa antioxidant was the
main contributor for the increasing lipid peroxidation.
As a consequence, the sperm plasma membrane may
lose the fluidity and integrity due to the attack of reac-
tive oxygen species, thereby subsequently reducing its
fertilizing ability (Hagedorn et al. 2012). It has been
reported in paddlefish (Polyodon spathula) that melato-
nin supplement minimized the oxidative damage to
sperm during in vitro storage (Gao et al. 2019); hence,
the addition of antioxidants could be tested as an option
for further improvement of our results obtained in basa
catfish.
Generally, our data demonstrated that chilled storage
of semen could be an effective strategy for gamete
management considering of its simplicity and low cost,
compared with that of freezing. According to the results,
chilled storage of basa catfish semen in diluted form had
a clear improvement in sperm storability and seemed to
be more beneficial to sustain the sperm function; a rapid
loss of sperm quality was detected in undiluted semen
after only 1 day of incubation (Table 1, Fig. 1 and Fig.
2). Although dilution may not always essential for short-
term semen storage, it has been reported in some teleosts
differences in the same treatment at different periods of storage
time. Asterisks (*) indicate statistically significant differences
among treatments at the same time point (P < 0.05)
that sperm stored undiluted tend to yield poor quality.
For instance, extender-preserved semen of walking cat-
fish (Clarias macrocephalus) could keep motile for up
to 10 days while the undiluted sample lost its motility
within 3 days (Vuthiphandchai et al. 2009). In an en-
demic trout (O. mykiss nelsoni), semen can be success-
fully preserved at 4 °C and maintain high sperm motility
(≥ 80%) for 7 days under diluted conditions (Aguilar-
Juárez et al. 2014). Semen samples from pufferfish
(Takifugu niphobles) diluted in a seminal-like solution
remained quality parameters as the same in fresh sperm
for even 1 week, whereas undiluted decreased signifi-
cantly after 1 day of storage (Gallego et al. 2013).
Dilution also improved sperm quality during storage in
rainbow trout (O. mykiss) (Trigo et al. 2015), Patagonian
blenny (E. maclovinus) (Contreras et al. 2017), and
European eel (Anguilla anguilla) (Peñaranda et al.
2010).
A suitable diluent medium (or extender) could help to
improve sperm storability by providing an adequate
aerobic atmosphere, maintaining a constant pH and
osmolality, avoiding the agglutination and dehydration
of the cells, etc. The tested dilution ratios in this study
were 1:1, 1:3, and 1:6, although a degradation of sperm
quality was detected in all treatments with the duration
of storage time; better results were obtained at a low
dilution ratio of 1:1 than higher ratios (1:3 and 1:6),
which yielded significantly higher ATP content at day 3
and day 5. For short-term semen storage, the optimal
dilution ratio normally ranged from 1:1 to 1:10 in var-
ious fish species (reviewed by Contreras et al., 2019).
The negative effect of higher semen dilution ratio may
possibly contribute to the over dilution of antioxidant
compounds that naturally exist in seminal plasma (e.g.,
enzymes, vitamins, glucose, and ions), which have been

suggested as the major defense against oxidative stress
in fish, since spermatozoa are characterized by a low
content of cytoplasm (Lahnsteiner et al., 2010). On the
other hand, high sperm density under a low dilution ratio
may induce agglutination and hinder gaseous exchange
between sperm and the surrounding atmosphere, thus
leading to hypoxic conditions. Therefore, it is recom-
mended that the optimal dilution ratio should be
established with a species-specific balance between suf-
ficient gas exchange and the protective effect of seminal
plasma.
Conclusions
Overall, our data provides valuable information on basa
catfish sperm quality regarding short-term storage.
Based on the parameters analyzed in this study, semen
quality is affected significantly by chilled storage, with a
decreased sperm motility, viability, MMP, ATP content,
and increased lipid peroxidation throughout the storage
period. Compared with undiluted semen, dilution with
Ca-F HBSS maintains better sperm quality and the
dilution ratio at 1:1 (semen: medium) is more suitable
to improve the sperm storability of basa catfish. Further
studies should be performed on testing the supplement
of antioxidants to see if oxidative damage could be
reduced during semen in vitro storage. Moreover, fertil-
ization trials need to be involved to examine the effec-
tiveness of the protocol in practice.
Funding information This work was supported by funds from
the Science and Technology Planning Project of Guangzhou
(grant number 201804020013), the Agriculture Research System
of China (grant number ARS-47), the Program of the China-
ASEAN Maritime Cooperation Fund of the Chinese government
(grant number 42000-41170002), and the National Natural Sci-
ence Foundation of China (grant number 31872572).
Compliance with ethical standards All animal handling and
procedures in this study were approved by the Animal Ethics
Committee of Sun Yat-Sen University.
Conflict of interest The authors declare that they have no con-
flict of interest.
Ethical approval All applicable international, national, and/or
institutional guidelines for the care and use of animals were
followed by the authors.
Fish Physiol Biochem
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