Acta Veterinaria (Beograd), Vol. 62, No. 5-6, 599-609, 2012.

DOI: 10.2298/AVB1206599K UDK 597.555.3:544.354-128.4:612.616.

THE EFFECT OF CATIONS ON SPERM MOTILITY PERFORMANCE AND FERTILIZING ABILITY

OF SILVER CARP Hypophtalmychtis molitrix

KHARA H*, BARADARAN SHAHROOZ N**, HADISEH DADRAS*, RAHBAR MINA***,

AHMADNEJAD MOHADESEH**** KHODADOOST A***

*Islamic Azad University – Lahijan Branch, Faculty of Natural Resource, Lahijan, Iran
**International Sturgeon Research Institute, Rasht, Iran
***Islamic Azad University, Young Researcher Club, Lahijan Branch, Iran
****Inland Water Aquaculture Research Center, Bandar Anzali, Iran

(Received 14th March 2012)

The objective of the study was to investigate the effect of saline

solution containing cations (Na+, K+, Ca+2, Mg+2) on sperm motility
performance (duration of sperm motility and percentage of motile
spermatozoa) and fertilizing capacity of sperm (fertilization rate,
hatching rate, larvae length during hatching, larvae length during active
feeding and survival rate) in silver carp. The results suggested that
solutions containing ions did not improve the duration of sperm motility.
The same was observed for the percentage of motile spermatozoa.
Fertilization rate influenced by solutions containing Ca+2, and other
ions could not affect this parameter. The results showed that hatching
rate was higher in solutions containing 99 mEq/L NaCl, 2 mEq/L MgCl2
and 2, 4 mEq/L CaCl2 respectively. Also, survival rate was higher in the
solution containing 2 mEq/L MgCl2 and 36 mg/dL KCl respectively. With
regard to the obtained results, it was concluded that using appropriate
activation medium can improve quality of fish sperm and subsequently
increases artificial reproduction performance.

Key words: cations, fertilization capacity. saline solution, seminal

plasma, silver carp, spermatozoa motility

INTRODUCTION

One of the most important factors responsible for the success of

reproduction is the quality of male gametes. This implies not only the quality of the
genetic material that is introduced into the egg during fertilization, but also the
ability of sperm to move toward the female gametes, i.e. sperm motility. Sperm
motility is a prerequisite factor determining sperm quality and fertilizing capacity
(Alavi et al., 2004). The quality of sperm is usually defined as motility, is a
prerequisite factor which determines the semen fertilizing ability (Lahnsteiner et
al., 1997). Several factors influence sperm motility, such as pH (Alavi and Cosson,
2005 a; b), cations (Cosson, 2004; Alavi et al., 2007), osmolality (Cosson, 2004;
Alavi and Cosson, 2006; Alavi et al., 2007) and dilution ratio (Alavi et al., 2004) in
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Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

either aqueous environment or diluent. Motility of freshwater fish spermatozoa is

triggered by a hypoosmotic medium (Cosson et al., 1999). The duration of sperm
motility is brief in most fish species (only lasts for 30 sec to few minutes) and varies
between species: it depends on many factors affecting biochemical,
physiological and metabolic characteristics of the broodstock and spermatozoa
(Alavi et al., 2008a). In industrial fish farming, one way to successful fertilization is
to prolonge sperm motility with a modified activation medium (Alavi et al., 2008a).
The determining parameters that influence sperm motility will provide us with
applied approaches to improve methods for artificial reproduction by developing
immobilizing or activating media for fertilization (Billard et al., 1995; Rodina et al.,
2004). To increase the efficiency in artificial fertilization trials, the composition of
diluents is very important and the components of activating solutions must be
adjusted according to species characteristics (Billard et al., 1995; Alavi et al.,
2005b). Sperm quality is a key factor that determines the ability of sperm to
successfully fertilise an egg. Also, fertilization success is depended on sperm
motility, sperm/egg ratio and egg quality (Billard et al., 1995). Previous studies
have confirmed sperm motility parameters, especially the percentage of motile
spermatozoa can be used to evaluate fertilization and hatching rate (Lahnsteiner
et al., 1998; Mansour et al., 2005). The role of ions on sperm motility and fertilizing
ability of spermatozoa has been reviewed by researchers (Stoss, 1983; Billard et
al., 1995; Cosson et al., 1991; Cosson, 2004; Linhart et al., 2008). The major ions
involved in improving motility characteristics include sodium (Na+), potassium
(K+), calcium (Ca+2) and magnesium (Mg+2). Silver carp, Hypophthalmichthys
molitrix belongs to cyprinids, which globally produced 3.78 million tonnes in the
world (FAO, 2008). Silver carp spawn in late spring and summer (April-
September) when water temperature is relatively high. Studies on silver carp
sperm are limited to cryopreservation. However, changes in silver carp sperm
quality and fertilizability during the spawning seasons were reported by Mofizur
Rahman et al., (2011). To date, there is no information available about the
influence of activation media on sperm motility of silver carp. Therefore, the
objectives of the present study were to investigate the effects of cations (Na+, K+,
Ca+2, Mg+2) on the motility performance of spermatozoa and their fertilizing ability
in silver carp. The ionic composition of seminal plasma was also measured during
the reproductive season.

MATERIAL AND METHODS

Brood fish, egg and sperm collection

The experiments were carried out in June 2010 at the Kasmahi Company,
Rasht, Iran. Broodstocks (15 mature males and 15 mature females) were captured
from the hatchery pools, during the spawning season. Fish were transferred to the
site of the experiment, and acclimated for 2 weeks in 4000 L tanks. Fish used in the
experiment ranged from 3.2 to 3.8 and 6.2 to 7.9 kg total weight and 63 to 82 and
71 to 86 cm total length for males and females, respectively. Silver carp males
were injected intramuscularly with Carp Pitatury Gland Hormone (cPG) at a dose
of 0.5 mg kg-1. In addition, females were injected intramuscularly with a double
Acta Veterinaria (Beograd), Vol. 62, No. 5-6, 599-609, 2012. 601
Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

injection of 2 mg kg-1 cPG. The first injection, 10% (0.2 mg/kg) cPG, was given 12h
before the second injection (1.8 mg/kg). Semen was collected after spermiation,
approximately 12 h after spawning induction. Semen of each male was collected
and sperm batches transported to the laboratory under cold conditions (4oC) until
used for analysis and fertilization. Care was taken to avoid contamination of the
semen with water, mucus, blood, faeces or urine. Stripping of females was carried
out 12 h after the second injection.

Sperm motility assessment

To examine the effects of cations on sperm motility performance, Na+ (NaCl)
(85, 92 and 99 mEq/L), K+ (KCl) (36, 42 and 48 mg/dL), Mg2+ (MgCl2) and Ca2+
(CaCl2) (2, 4 and 6 mEq/L) were used. All solutions were buffered with 30-40 mM
Tris–HCl, adjusted to pH 8.5 ± 0.2. Distilled water was used as the control. Sperm
motility was evaluated visually for the percentage of motile spermatozoa after
activation and total duration of motility (in seconds). To induce the initiation of
motility, sperm was triggered directly in activation solutions at a ratio of 1: 2000
and immediately recorded with a 3 CCD video camera (Panasonic 240 Japan)
mounted on a dark-field microscope (Leica USA). The duration of sperm motility
was measured immediately after initiation of sperm activation until 100%
spermatozoa were immotile. Percentage of motility was defined as the percentage
of progressively motile spermatozoa within each activated sample. Progressively
motile spermatozoa were defined as actively swimming in a forward motion. Only
forward moving sperm was judged as motile and sperm cells that vibrated in place
were not considered motile (Aas et al., 1991). Analysis of sperm motility was
carried out in triplicate for each sample at room temperature (20-22oC), using light
microscopy under 400 × magnification.

Evaluation fertilizing ability

Fresh eggs were obtained from females and pooled just prior to assay. To
control variation among the qualities of egg pools, the eggs were obtained from
same-age females cultivated under the same conditions. Fertilization was
performed in dry plastic dishes and 50 g of eggs (approximately 50000 eggs) was
placed into each dish. The fertilization solution (3 g of urea, 4 g of NaCl in 1 L
distilled water) was used according to the dry fertilization method. Batches of
eggs were inseminated with solutions containing 85, 92 and 99 mEq/L NaCl, 36,
42 and 48 mg/dL KCl, 30, 32, 35 2, 4 and 6 mEq/L CaCl2 and MgCl2 respectively.
Distilled water was used as the control. Following fertilization, the eggs were
stirred for 1 h and then rinsed with hatchery water and placed into the incubator.
Fertilization rate was determined as the percent of eyed eggs about 6 h after
fertilization. For incubation, the so-called "Vase" device was used. The success of
fertilization was evaluated by the percentage of eggs reaching the morula stage,
4h after fertilization. Hatching occurred between 1-2 days after fertilization.

Seminal plasma characteristics

Seminal plasma was separated from the semen by centrifugation
(Eppendorf AG, Hamburg, Germany). Plasma was centrifuged twice to avoid
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Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

possible contamination with spermatozoa. Plasma samples were frozen at -20oC

until analysis. The chemical composition of seminal plasma was measured by the
colorimetric method using an Autoanalyser Technican (Caretium-XI-921,
Germany) for Ca+2 and Mg+2 measurement and with a flamephotometer (Jenway
PFP, England, Standard kits from Parsazmoon, Tehran, Iran) for Na+ and K+ in the
Dr Fadaiees' Medicine Laboratory, Rasht, Iran.

Data analysis
The normal distribution of data was tested using the Shapiro-Wilk's test.
Data were analysed using one-way ANOVA (SPSS 16). Ducan test was used for
post hoc comparisons. Results are presented as mean ± SE. Differences with a
probability value of 0.05 (p<0.05) were considered significant.

RESULTS

Sperm motility performance

Sperm characteristics and ionic composition of seminal plasma of silver
carp are given in Table 1. Maximum (40.6 ± 8.5 s) duration of sperm motility was
observed in solutions containing 99 mEq/l NaCl (Fig. 1a). The higher percentage
of motile spermatozoa was obtained after triggering the motility in distilled water
(55±5%) compared to other concentrations of NaCl (Fig. 1b). A similar pattern
was detected in terms of percentage of motile spermatozoa when semen was
incubated with KCl solutions (Fig. 2b), but duration of sperm motility was higher in
the solution containing 36 mg/dL KCl (Fig. 2a). In the case of MgCl2 and CaCl2,
duration of sperm motility was not influenced by activation solutions and higher
values were observed in distilled water (Fig. 3 and 4a). A decreasing trend was
observed in the percentage of motile spermatozoa when the dose of the solutions
containing MgCl2 and CaCl2 gradually increased (Fig. 3 and 4b).

Figure 1. Effect of different concentrations of NaCl on (a) duration of sperm motility and (b)
percentage of motile spermatozoa in silver carp. Values with different alphabetic
letters are significantly different
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Khara Hossein et al.: The effect of cations on sperm motility performance
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Table 1. Sperm characteristics of silver carp

Variables Minimum Maximum Mean ± SE

Duration of sperm motility (sec) 45 27 36 ± 9
Percentage of motile spermatozoa (%) 94 97 95 ± 6
Sodium (mmol-1) 99 82 90 ± 4.90
Potassium (mmol-1) 48 43 45 ± 10.17
Magnesium (mmol-1) 14.6 10.1 7.3 ± 2.44
Calcium (mmol-1) 2.5 1.2 1.5 ± 0.47

Figure 2. Effect of different concentrations of KCl on (a) duration of sperm motility and (b)
percentage of motile spermatozoa in silver carp. Values with different alphabetic
letters are significantly different

Figure 3.Effect of different concentrations of MgCl2 on (a) duration of sperm motility and (b)
percentage of motile spermatozoa in silver carp. Values with different alphabetic
letters are significantly different
604 Acta Veterinaria (Beograd), Vol. 62, No. 5-6, 599-609, 2012.
Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

Figure 4. Effect of different concentrations of CaCl2 on (a) duration of sperm motility and (b)
percentage of motile spermatozoa in silver carp. Values with different alphabetic
letters are significantly different

Fertilization capacity
Effects of cations (Na+, K+, Ca2+ and Mg2+) on fertilizing capacity of silver
carp sperm are presented in Table 2-5, respectively. The solution containing
cations (Na+, K+, Ca2+ and Mg2+) did not affect fertilization rate and the highest
values were observed in distilled water (Table 2-5). The maximum hatching rate
was recorded when solutions containing 99 mEq/l NaCl, 2 mEq/L MgCl2 and
4 mEq/L CaCl2 were used respectively (Table 2, 4 and 5). Larvae length during
hatching and active feeding was not changed by activation mediums. Survival
rate was changed by solutions containing 99 mEq/l NaCl, 36 mg/dL KCl2 and
2 mEq/L MgCl2 and CaCl2, but their values were not significantly different (Table 2-
5).

Table 2. Effect of different concentrations of NaCl on fertilization capacity of silver

carp sperm

Na Larvae length Larvae length Survival

Fertilization Hatching
concentration during during active rate
rate rate
(mEq/L ) hatching feeding (%)
Distilled water 81.6±0.09a 43.2±7.5c 4.40±0.11 5.0±0.09 88.1±2.7
85 51.6±2.8b 60.0±5.0ab 4.07±0.12 0 0
92 20.0±5.0d 16.0±1.9 d 3.20±0.12 5.5±0.08 84±1.7
99 35.0±5.0c 70.0±5.0 a 4.00±0.15 4.8±0.13 92±0.61
Columns with different alphabetic letters are significantly different (p<0.05)
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Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

Table 3. Effect of different concentrations of KCl on fertilization capacity of silver carp

sperm

Larvae length Larvae length

K concentration Fertilization Hatching Survival
during during active
(mg/dL) rate rate rate (%)
hatching feeding
Distilled water 81.6±10.5a 43.2±7.5a 4.1±0.11 5.0±0.09 88.1±2.7
36 51.0±2.08b 41.6±2.8b 4.4±0.2 4.3±0.05 91.6±0.58
42 2.0±0.46d 1.3±0.31d 0 0 0
48 11.6±2.8c 6.6±1.8c 0 0 0
Columns with different alphabetic letters are significantly different (p<0.05)

Table 4. Effect of different concentrations of MgCl2 on fertilization capacity of silver

carp sperm

Mg Larvae length Larvae length

Fertilization Hatching Survival
concentration during during
rate rate rate (%)
(mEq/L) hatching active feeding
Distilled water 81.6±9.5a 43.20±7.5b 4.1±0.1 5.0±0.09 88.1±2.7
2 60.0±5b 80.00±5a 3.7±0.25 4.4±0.27 90.6±0.58
4 3.6±1.5c 2.00±0.65c 0 0 0
6 0.67±0.01d 0.33±0.05d 0 0 0

Table 5. Effect of different concentrations of CaCl2 on fertilization capacity of silver

carp sperm

Larvae length Larvae length

Ca concentration Fertilization Hatching Survival
during during active
(mg/dL ) rate rate rate (%)
hatching feeding
Distilled water 80.6±8.4a 43.2±7.2c 4.1±0.11 5.0±0.39 88.1±2.7
2 60.0±5b 70.0±5ab 4.0±0.06 5.1±0.08 91.1±0.16
4 50.0±5bc 75.0±5a 4.4±0.13 5.0±0.08 90.0±0.03
6 0d 0d 0 0 0
Columns with the different alphabetic letters are significantly different (p<0.05)

DISCUSSION

To our knowledge, this is the first observation about the effects of saline
solution containing cations (Na+, K+, Ca+2, Mg+2) on sperm motility performance
and fertilizing capacity of sperm in silver carp. The duration of sperm motility is
usually very short (a few minutes in freshwater species) and could be influenced
by external environmental factors such as pH, temperature, ions, and osmolality
606 Acta Veterinaria (Beograd), Vol. 62, No. 5-6, 599-609, 2012.
Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

(Cosson 2004; Alavi and Cosson, 2006). Therefore, determination of optimum

parameters for the activation medium is very important to increase the efficiency
of artificial reproduction (Alavi et al., 2004). It has been shown that the ionic
composition of the activating solution influences the initiation and duration of
sperm motility (Marian et al., 1993). Sperm motility is affected also by the ionic
composition of the diluent as previously reported (Billard and Cosson, 1992).
Research has documented the effect of ions on the initiation of motility and the
duration of sperm in an effort to determine the biosensitivity of sperm. The longer
period of sperm motility in saline solution is related to the delayed energy loss of
spermatozoa for movement (Alavi and Cosson, 2006). However, other factors
such as ATP content in the sperm, the number of mitochondria, osmolality and pH
are involved in this process. In contrast to other studies (Stoss, 1983; Billard et al.,
1995), the duration of sperm motility in H. molitrix was not prolonged in saline
solutions compared with freshwater. Morisawa et al., (1983) showed that K+
increases sperm motility (duration and percentage of motility) in some cyprinids.
Similar to other Cyprinidae, we found that Na+ and K+ were the most predominant
ions in seminal plasma. Literature reveals wide variations in the ionic composition
of seminal plasma. Na+ and K+ ions concentrations were reported to be in the 94-
107 and 39-78 in cyprinids (Kruger et al., 1984; Billard et al., 1995), respectively.
For example, K+ concentration differs among cyprinids. Values have ranged from
1.93 MmL–1 in Tinca tinca (Linhart et al., 2003) to 98 MmL–1 in Barbus barbus (Alavi
et al., 2008 b). K+ measured 28.8 MmL–1 in Barbus sharpeyi (Alavi et al., 2010).
This difference in seminal Na+ and K+ concentrations denotes species-specific
characteristics (Ciereszko et al., 2000). The differences between our results and
other studies could be related to several parameters, including spawning time of
the fish species (Suquet et al., 1994), contamination of semen by urine during
stripping (Suquet et al., 1994), phagocytosis of sperm in the testis during the
degeneration stage of spermatogenesis (Lahnsteiner et al., 1993), thinning and
hydration of semen during spermiation (Morisawa et al., 1979). Previous studies
have confirmed that sperm motility plays an important role to in the determination
of the fertilizing ability of sperm (Billard, 1992; Billard et al., 1995). Fertilizing ability
of spermatozoa increases by using appropriate activation mediums that increase
the duration of motility. In this study, the highest sperm motility was obtained in
distilled water compared to saline solutions; consequently the maximum
fertilization rate was observed after activation of sperm in distilled water. In our
experiment, the highest hatching rate was recorded in solutions containing
99 mEq/l NaCl, 2 mEq/L MgCl2 and 4 mEq/L CaCl2 respectively (Table 2, 4 and 5).
High sperm motility and fertilization rates were detected after activation of sperm
in 45 mM NaCl, 5 mM KCl, 30 mM Tris–HCl, pH 8.0 (Saad and Billard, 1987; Billard
et al., 1995; Perchec et al., 1995; Linhart et al., 2003; Linhart et al., 2008). Saad and
Billard (1987) reported fertilization rates lower than 5% and 25% after activation of
sperm in freshwater and carp activation solution (CAS, containing 45 mM NaCl, 5
mM KCl, 30 mM Tris–HCl adjusted to pH of 8.0). The spermatozoa incubated in K+
rich medium showed an interestingly high fertilizing ability after second motility
triggering in carp activation solution (CAS) (Linhart et al., 2008). Further studies,
reported that salmonid spermatozoa, when immersed in saline, ovarian fluid or
Acta Veterinaria (Beograd), Vol. 62, No. 5-6, 599-609, 2012. 607
Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

diluted seawater maintained motility and fertility for extended periods, i.e. hours or
days (Ellis and Jones, 1939). The differences observed between this study and
others might be related to the quality of eggs and the management conditions
used during artificial insemination.

ACKNOWLEDGEMENTS:
The present work was conducted at Lahijan Islamic Azad University, Iran. The authors wish to thank the
manager and staff of laboratory center for their valuable contribution.

Address for correspodence:

Khara Hossein
Islamic Azad University – Lahijan Branch
Faculty of Natural Resource
Department of Fishery and Aquaculture
Lahijan, Iran
P.O. Box: 1616
h_khara1974ªyahoo.com

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Khara Hossein et al.: The effect of cations on sperm motility performance
and fertilizing ability of silver carp Hypophtalmychtis molitrix

UTICAJ KATJONA NA POKRETLJIVOST SPERMATOZOIDA I NA PLODNOST

SREBRNOG [ARANA Hypophtalamychtis molitrix

KHARA H, BARADARAN SHAHROOZ N, HADISEH DADRAS,

RAHBAR MINA, AHMADNEJAD MOHADESEH i KHODADOOST A

SADR@AJ

Cilj ovog istra`ivanja je bio da se ispita uticaj rastvora koji sadr`e katjone
(Na+, K+, Ca+2, Mg+2) na pokretljivost spermatozoida (trajanje pokretljivosti
spermatozoida i procenat pokretljivih spermatozoa) i njihovu oplodnu sposob-
nost (stopa plodnosti, stopa izleganja, du`ina larve tokom izleganja, du`ina larve
tokom aktivne ishrane i stopa pre`ivljavanja) kod srebrnog {arana. Rastvori koji su
sadr`avali navedene jone nisu produ`ili trajanje poktretljivosti kao ni procenat
pokretnih spermatozoida. Procenat oplodnje nije bio izmenjen rastvorima koji su
sadr`avali Ca+2 i druge jone. Rezultati su ukazali da je stopa izleganja bila vi{a u
rastvorima koji su sadr`ali 99 mEq/L NaCl, 2 mEq/L MgCl2 i 2,4 mEq/L CaCl2.
Tako|e je stopa pre`ivljavanja bila ve}a u rastvoru koji je sadr`avao 2 mEq/L
MgCl2 i 36 mg/dL KCl. Na osnovu dobijenih rezultata se mo`e zaklju~iti, da
odgovaraju}i medijum za spermatozoide mo`e da pobolj{a kvalitet semena riba i
posledi~no pove}a uspeh ve{ta~ke oplodnje.