ARTIFICIAL SEMINAL PLASMA IMPROVES MOTILITY AND FERTILISATION

CAPACITY OF COMMON CARP CYPRINUS CARPIO L. SPERM DURING ONE

HOUR OF STORAGE
, Daniel Żarskia, Katarzyna Palińska-Żarskab, Mariola Słowińskaa,
Beata Irena Cejkoa,⁎
Radosław Kajetan Kowalskia
a Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food
Research, Polish Academy of Science, Olsztyn, Poland
b Department of Ichthyology, Faculty of Environmental Sciences, University of Warmia
and Mazury, Olsztyn, Poland

ABSTRACT

The quality of fish sperm is characterized by relatively high individual variation.

Moreover, under artificial conditions, the collected sperm loses its motility and viability
very rapidly, which may decrease fertilisation success. Therefore, techniques able to
secure and maintain a high biological value of collected fish sperm are needed in
hatchery practice. Herein, we used previously composed artificial seminal plasma
(ASP) containing 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris, 110 mM NaCl and 40 mM
KCl (pH 7.5 and 310 mOsm kg−1) to dilute common carp sperm samples for 1 h storage
prior to fertilisation. Sperm was collected from mature males and, after checking its
motility (MOT) using the CASA system, was divided into high (range 55–65%) and low
(range 5–10%) quality. Samples of both sperm qualities (high/low) were diluted tenfold
(1: 9; sperm: extender) in ASP and stored for 24 h at 10 °C. Motility (MOT, PRG) and
velocity (VCL, VSL) of low- and high-quality sperm diluted in ASP increased after 1 h of
storage compared to undiluted sperm. Moreover, the fertilisation capacity of low-
quality sperm (previously diluted and stored for 1 h in ASP) increased three times
compared to undiluted sperm of the same quality. Prolonging the time of sperm storage
in ASP to 24 h results in the further increase of MOT, PRG and VCL in the low quality
samples and PRG in the high quality samples. The results of the presented study
confirm the possibility of common carp sperm revitalisation using ASP. The utilisation of
such sperm in hatchery practice is possible since the fertilisation capacity of sperm,
previously diluted and stored for 1 h in ASP, was over 90% regardless of the initial
sperm quality (high/low).

INTRODUCTION

Under controlled conditions, sperm of common carp Cyprinus carpio

L. are collected manually (massage of abdominal parts of males) and subsequently used
in the fertilisation of eggs (Billard et al., 1995). This technique is commonly used in hatchery
practice although the quality of the sperm obtained may differ significantly between
individuals, which may affect the reproduction success (Kaspar et al., 2008). One factor
which reduces the quality of common carp sperm may be urine contamination (during
collection), which causes premature activation of sperm (by the low osmotic pressure of
the urine), which as a con- sequence loses its fertilisation ability (Perchec et al., 1995).
Moreover, the sperm quality variation might results from different maturity stage of the male
(Cejko et al., 2018a). It is also true that in some Cyprinide species fresh sperm (undiluted)
loses its fertilisation ability within a few hours after collection and its motility usually does
not exceed 20% over 24 h of storage (Dietrich et al., 2015). All those issues have negative
effects on the fertilisation success in the hatcheries practice.
It was confirmed that under controlled conditions low quality common carp sperm may be
revitalized after a few hours of sperm in- cubation in 200 mM KCl buffer (Redondo-Müller et
al., 1991). This indicates that obtaining potentially non-motile sperm and reversing its quality
in vitro requires the use of an appropriate extender or artificial seminal plasma (ASP) for any
given species. The function of ASP is not only related to the increasing osmolality, in case of
urine contamina- tion, but also to limiting the negative impact of temperature changes and
the toxic action of the products of cell metabolism, as well as se- curing the appropriate gas
exchange during storage (Ulloa-Rodríguez et al., 2018). One of the main problem related to
the toxic effects of cell metabolism products during sperm storage is their lipids peroxidation
level due to ROS propagation during the cellular respiration process (Shaliutina-Kolešová et
al., 2013). On the other hand, CO2 accumula- tion might also reduce their motility (Ingermann
et al., 2002). Dilution of sperm samples might simply reduce the possibilities of ROS and
CO2 sperm poisoning by reducing their total concentration in the aliquots. It was
confirmed that in extender containing 130 mM NaCl, 13.5 mM KCL, 13.5 mM CaCl 2,
30 mM Tris (pH 8.5 and osmolality 235 mOsm kg −1), the sperm motility of common carp
dropped sig- nificantly from 81 to 29% over 24 h (Ravinder et al., 1997). On the contrary,
sperm motility stored in Tyrode's medium (TLP:100 mM NaCl, 3.1 mM KCl, 2 mM CaCl2,
0.4 mM MgCl2, 25 mM NaHCO3, 0.3 mM NaH2PO4; pH 8.6 and osmolality 236 mOsm kg−1)
and modified Krebs- Ringer's (BWW:95 mM NaCl, 48 mM KCl, 1.7 mM CaCl 2, 25 mM
NaHCO3; pH 8.8 and 326 mOsm kg−1) extenders remained stable (88%) over 24 h of
storage (Ravinder et al., 1997). Recently, Cejko et al. (2018b) showed that diluting
common carp sperm in ASP containing 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris, 110 mM
NaCl and 40 mM KCl prolongs their motility during storage for longer than 24 h as
compared to undiluted sperm. It was shown that after 72 h of storage in samples diluted in
ASP the sperm motility was more than twice as high (79.0%) in comparison to undiluted
(fresh) sperm (29.0%). This indicates a high efficiency of ASP in common carp sperm
storage under in vitro condi- tions.
Under controlled conditions, collecting a sufficient number of ma- ture gametes
determines the success of reproduction. Due to its high variabilities, this task requires
hatcheries to secure more males than minimally needed for reproduction. Therefore , a
technique able to se- cure and maintain a high biological value of collected common carp
sperm might help increase the efficiency of reproduction in hatchery practice.
The aim of the presented study was to evaluate the effect of ASP on sperm motility
parameters in samples of different sperm quality (high/ low) stored for 24 h under in vitro
conditions. The high- and low-quality samples were also used for fertilisation to check the
effectiveness of previous dilution and storage of sperm (for 1 h) in ASP on their ferti- lisation
capacity.
MATERIALS AND METHODS

Sperm collection and CASA analysis

Sperm was collected (using a syringe) from mature common carp males (n = 8; average
body weight = 7 kg) obtained from ground ponds during the spawning season (mid-May).
Before collection, males were transported to the hatchery and placed in a 20 °C pool with
closed water circulation. Next, males were subjected to anaesthesia using 2-
phenoxyethanol (Merc, Darmstadt, Germany) administered at a dose of
0.5 ml l−1. Special care was taken to avoid contamination of the sam- ples with urine, faeces
or blood during collection. Sperm motility was performed using the Computer-Assisted Sperm
Analysis (CASA) system. As an activation buffer (AB) 10 mM Tris buffer containing 100 mM
NaCl at pH 9.0 and osmolality 200 mOsm kg−1 was used (Cejko et al., 2013). To prevent
sticking of the sperm samples to the microscope slide, 0.5% Bovine Serum Albumin (BSA;
Sigma-Aldrich; St. Louis, MO) was added to the AB. To activate movement, 1 μl of sperm
prediluted 10 times with ASP was mixed with 100 μl of AB and placed into the well of a multi-
test glass slide (Tekdon, Inc., Myakka City, USA) and covered with a cover- slip. Next,
recordings were made approximately 6 s after the activation of sperm motility. The percentage
of motile sperm (MOT, %), pro- gressive motile sperm (PRG, %), curvilinear velocity (VCL, μm
s−1), straight-linear velocity (VSL, μ ms−1), beat cross frequency (BCF, Hz) and amplitude of
lateral head displacement (ALH, μm) were de- termined. Recordings were made using a Basler
202 K (Basler, Ah- rensburg, Germany) digital camcorder integrated with the Olympus BX51
(Olympus, Tokyo, Japan) microscope (lens Plan FL N 20×/0.5 NH ph 1). The recording speed
was 47 frames per second. The first 200 frames of each recording were then analysed using
the CRISMAS pro- gram (ImageHouse, Ltd., Birmingham, UK). Sperm motility was mea- sured
twice (duplicate measurements) for each sperm sample. During analysis, sperm was stored
in a thin layer (Eppendorf tube) under anaerobic conditions ( ± 10 °C) using a
thermomixer (ThermoMixer C, Eppendorf, Hamburg, Germany).

Sperm sample preparation

After measuring the sperm motility using the CASA system, the undiluted, i.e., fresh,
sperm was divided into high (n = 4) and low (n = 4) quality. The high-quality sperm was
characterized by sperm motility in the range 55–65% whereas low-quality sperm was char-
acterized by sperm motility in the range 5–10%. Next, 200 μl of sperm samples were
diluted tenfold (1: 9; sperm: extender) in ASP containing 2 mM CaCl2, 1 mM Mg2SO4, 40
mM KCl, 100 mM NaCl, 20 mM Tris at pH 7.5 and 310 mOsm kg−1 (Cejko et al., 2018b).
The samples of high and low quality sperm diluted in ASP were stored ( ± 10 °C) in a thin
layer (a layer of sperm no > 1 cm of high) for 24 h. Undiluted samples equal to the diluted
volume (2 ml) were also preserved in the same condition.
After 1 h of preservation, the sperm motility in the samples of high- and low-quality sperm
was analysed using the CASA system. Sperm motility was also checked 24 h after ASP
dilution.
Fertilisation trial

One female with a body weight of 10 kg was selected for re- production. Before the
breeding, female was kept in pool with closed circulation of water and temp. 20 °C. For the
egg fertilisation, samples of high- and low-quality sperm undiluted and diluted in ASP were
used. Before fertilisation, the sperm concentration was calculated using a Bürker chamber
and the appropriate amount of sperm was added to 100 eggs to obtain the concentration
of 200,000 sperm per egg in all the fertilisation batches. As an activator in the fertilisation
trial, 5 ml of AB buffer was used for each fertilisation sample. The sperm concentration in
AB was equal to 4 × 106 ml−1. Next, samples of fertilised eggs were left for 5 min. The
eggs were then rinsed with hatchery water and in- cubated in Petri dishes at a temperature
of 21 ± 1 °C. After 24 h of incubation, the fertilisation rate was calculated.

Statistical analysis

The effect of the sperm quality (high/low) and time of sperm sto- rage (0, 1 and 24 h) in
undiluted and diluted trials on the sperm motility parameters and fertilisation success was
analysed using a two-way ANOVA and the results were presented as mean ± SEM.
Differences among means were determined by LSD tests. The differences were
considered significant at P ≤ .05. All data representing percentages were transformed
using the arc-sin transformation prior to statistical analysis. Statistica software (Version
9.0, 2007, StatSoft, Inc., Tulsa, OK, USA) was used for statistical calculations.

RESULTS

Quality of undiluted sperm

Immediately after collection, significant variability in the quality of the undiluted (fresh)
sperm was noted (Fig. 1A–F). The values of all the CASA parameters differed significantly
between the high and low- quality undiluted sperm (P < .05). There was no statistically
sig- nificant difference in the mean value of the sperm concentrations be- tween the high
(24.36 × 109 ml−1) and low (22.62 × 109 ml−1) quality samples. During the 1 h storage of
undiluted sperm, we found no statistically important differences in the motility parameters,
whereas after 24 h of storage all the undiluted samples were immotile (data not shown).
Effect of sperm dilution with ASP on CASA parameters

One-hour storage of high-quality sperm in ASP resulted in a sig- nificant increase of the
MOT, PRG, VCL and VSL values compared to the high-quality undiluted sperm (Fig. 1A–D;
P < .05). On the other hand, there were no differences in the BCF and ALH values of high-
quality sperm between undiluted sperm and sperm diluted in ASP for 1 h (Fig. 1E, F; P >
.05). Within 24 h of storage in ASP, further increase of the high-quality sperm was observed
only in the PRG values compared to the values of this parameter determined after 1 h of
storage.
In the low-quality sperm stored in ASP, all the CASA parameters significantly increased
during 1 h in comparison to low-quality un- diluted sperm (Fig. 1A–F; P < .05). A significant
increase of the MOT, PRG and VCL values was also observed after 24 h of low- quality
sperm storage in ASP compared to the values of these parameters determined the day
before (Fig. 1A–C; P < .05). There were no differences in the high- and low-quality sperm
 between 1 and 24 h of storage in ASP for the VSL, BCF and ALH parameters determined
using the CASA system.

Fig. 1. Sperm motility (A), progressive motile sperm (B), curvilinear velocity (C), straight-
linear velocity (D), amplitude of lateral head displacement (E) and beat cross frequency
(F) of high (n = 4) and low (n = 4) quality of undiluted and diluted in ASP sperm stored
for 1 and 24 h under in vitro conditions. Undiluted (fresh) sperm samples were not
preserved in ASP. Boxes marked with various letters indicate significant differences
between groups (P < .05).

Effect of sperm storage in ASP on fertilisation success

The significant decrease in the fertilisation capacity of low-quality compared to high-

quality undiluted sperm was noted (Fig. 2). On the other hand, there were no differences
in the fertilisation rate of high- quality sperm when undiluted and diluted in ASP.
Conversely, the fertilisation capacity of low-quality undiluted sperm increased three times
after 1 h of sample storage in ASP (Fig. 2).
Fig. 2. Fertilisation rate (%) of high (n = 4) and low (n = 4) quality undiluted and diluted
in ASP sperm stored for 1 h under in vitro conditions. Undiluted (fresh) sperm samples
were not preserved in ASP. Boxes marked with various letters indicate significant
differences between groups (P < .05).

DISCUSSION
The results of the presented study indicate for the first time the possibility of common carp
sperm revitalisation after 1 h of incubation in ASP containing 2 mM CaCl 2, 1 mM Mg2SO4,
40 mM KCl, 100 mM NaCl, 20 mM Tris at pH 7.5 and 310 mOsm kg −1, and its usefulness in
the fertilisation procedure. Already within one hour a significant in- crease in the most
important CASA parameters (MOT, PRG, VCL and VSL) was observed in both high- and
low-quality sperm in comparison to the values of these parameters determined in undiluted
(fresh) sperm. Moreover, the fertilisation capacity of sperm diluted in ASP and stored for 1 h
was over 90% regardless of the quality of the sperm (high/low) used for reproduction. This
indicates that in hatchery practice ASP could be used as a beneficial tool for common carp
sperm revival in the case of obtaining reduced quality sperm.
In our study, the quality of the fresh sperm collected from common carp under hatchery
conditions was characterized by great variability and the sperm motility differed significantly
between individuals (5–65%). However, just 1 h after sperm dilution in ASP the most im-
portant CASA parameters such as MOT, PRG, VCL and VSL significantly increased in both
low- and high-quality sperm in comparison to the values of these parameters determined
in undiluted (fresh) sperm. The low-quality samples could have originated from the different
maturity levels of the males used (Cejko et al., 2018a). This could indicate that, similar to
rainbow trout (Oncorhynchus mykiss) sperm (Kobayashi et al., 2004), the final stages of
common carp sperm maturation might be induced artificially by dilution in the appropriate
ASP medium.
The presence of high and low quality sperm in common carp might also be related to
urine contamination (Perchec et al., 1995). However, this contamination is unlikely to be
the source of low quality sperm samples in our study, as we identified a high sperm
concentration in samples of low-quality and, moreover, the lowest sperm concentration
(equal to 20.0 × 109 ml−1) was found in a sample of high-quality. However, we did not
measure osmolality in the samples to examine possible seminal plasma dilution. Therefore,
we cannot exclude the possibility of individual males exhibiting differences in sperm con -
centration, and urine contamination might be one of the reasons pro- ducing low quality
sperm in the present study.
This effect is related to the ASP used in common carp sperm pre- servation, since its
composition was close to the seminal plasma of common carp as determined by Morisawa
et al. (1983). It seems that some of the main inorganic components of the seminal plasma
of common carp are cations such as K+ (82 mM) and Na+ (75 mM), which play a crucial role
in maintaining osmotic balance during sperm storage and are important components of
numerous enzymes. It was confirmed that in salmonids and sturgeons, ions such as K+,
rather than osmol- ality, are an essential factor in sperm motility inhibition (Morisawa and
Suzuki, 1980). In contrast, cyprinid sperm motility is inhibited by the high osmolality of the
seminal plasma, with potassium ions not essen- tial to this process (Perchec Poupard et al.,
1997; Cosson, 2004). However, requirements for external K+ (50 mM) suggest that its
ab- sence might produce K+ efflux from the cell, which will therefore be- came non-functional
as a possible consequence of changes in the plasma membrane potential (Redondo-Müller et
al., 1991). Interestingly, common carp sperm may be re-activated a second time after the first
activation, although sperm storage in K + -rich incubation medium is necessary. It was
confirmed that sperm exposure to 200 mM potassium ions allows ATP reloading within the
flagellum, which may help to increase the motility and fertilisation capacity of sperm (Linhart et
al., 2008). As the authors pointed out, a resting period in an immobilizing solution is species
specific and requires room temperature (20 °C), which allows the mitochondrial metabolism
to operate to reload the ATP level. It was also found that the specific ratio of KCl to NaCl ions
(40: 110) in ASP had a beneficial effect on common carp sperm motility during 72 h of storage
(Cejko et al., 2018b). The above data indicate that, for effective common carp sperm storage
under in vitro conditions, a potassium concentration around 40 mM is required in ASP.
Moreover, such concentration is not toxic for common carp eggs and will not lead to a
reduction of the fertilisation rate (Saad and Billard, 1987).
It was confirmed that the motility of cyprinids sperm indicated their fertilisation capacity
(Lahnsteiner et al., 1996). In our study, we ob- served that, indeed, the increase in sperm
motility improves their fer- tility potential. This indicated that ASP used for common carp
sperm storage had a beneficial effect on sperm motility by rebuilding the en- ergy reserves
(ATP) necessary to raise its potential to move. On the other hand, the right pH (7.5) and
osmolality (310 mOsm kg−1) of ASP prevent damage of sperm motility, potentially caused
by a reduction in intracellular pH and osmotic pressure. From a practical point of view, the
use of sperm with similar quality (without great variability) is beneficial due to the
fertilisation of a large number of eggs. This reduces the risk of obtaining low fertilisation
rate. Despite unfertilised eggs representing the lost potential, they are also a source of
bacterial and fungal infections which may contaminate all the incubated eggs, and
consequently dramatically reduce the overall effect of reproduction.
It should also be indicated that prolonging the time of sperm storage in ASP to 24 h
resulted in further improvements of their motility po- tential. We observed increasement of
the PRG values in the high and low quality sperm and additionally MOT and VSL values
in the low quality samples. This finding is important from a practical point of view, since the
preparation of sperm samples one day before female spawning might have a beneficial
effect through optimising the hatchery fertilisation procedure.

CONCLUSIONS
To conclude, the revitalisation of common carp sperm is possible in a very short time (one
hour) using ASP containing 2 mM CaCl 2, 1 mM Mg2SO4, 20 mM Tris, 110 mM NaCl and 40
mM KCl at pH 7.5 and os- molality of 310 mOsm kg−1. The application of such solution
improves sperm motility and velocity during 1 h, regardless of the quality (high/ low) of the
fresh sperm collected under controlled conditions. It was also noted that the fertilisation
capacity of the revived sperm was at a high level since the fertilisation rate using sperm
previously diluted in ASP of different quality (high/low) was > 90%. Therefore, the appli-
cation of ASP in hatchery practice may increase the fertilisation rate regardless of the
sperm quality (high/low) collected, although the sperm needs to be stored for one hour in
ASP before fertilisation.