Cryopreservation of scaly carp (Cyprinus carpio) sperm:effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching successof embryos

Ilker Yavas • Yusuf Bozkurt • Cengiz Yıldız

Received: 29 January 2013 / Accepted: 28 August 2013� Springer Science+Business Media Dordrecht 2013

Abstract The aim of the present study was to determine the effect of various cryoprotec-

tants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyp-

rinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a

practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura’s

extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl2, 0.08 g/l MgCl2 and 0.2 g/l

NaHCO3. The extender contained three different cryoprotectants (DMSO, DMA and egg

yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid

nitrogen vapor (-120 �C) using an insulated box with an adjustable tray for 10 min and then

plunged into liquid nitrogen (-196 �C) tank. The thawing process was performed in a water

bath at 40 �C for 10 s. The results indicated that type of cryoprotectants and their concen-

trations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality,

while they are very important in order to obtain high fertilization rates. The highest fertil-

ization rate was determined as 96.4 ± 0.15 % with 15 % egg yolk, while the highest hatching

rate was determined as 99.3 ± 0.80 with 15 % DMA. In conclusion, the applied cryopres-

ervation method for scaly carp sperm is suitable to fertilize high amounts of eggs.

Keywords Scaly carp � Cyprinus carpio � Cryoprotectant � Cryopreservation �Motility � Fertilization � Hatching

Introduction

Cryopreservation of fish semen provides many benefits such as genetic manipulation,

selective breeding and maintaining continuous and stable supply of gametes for hatchery

I. Yavas � C. YıldızDepartment of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Universityof Mustafa Kemal, Antakya, Hatay, Turkey

Y. Bozkurt (&)Department of Aquaculture, Faculty of Marine Science and Technology, University of Mustafa Kemal,Iskenderun, Hatay, Turkeye-mail: yfbozkurt@hotmail.com

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Aquacult IntDOI 10.1007/s10499-013-9698-6

seed production or laboratory experimentation (Suquet et al. 2000). In addition, cryo-

preservation biotechnology may also be used for gene banking of valuable species and for

synchronization of artificial reproduction in the field of aquaculture. However, there is no

one protocol that seems to work with all species of fish.

Cryopreservation techniques involve addition of cryoprotectants, freezing and thawing

of sperm samples that may result in some damage to the spermatozoa and may decrease

egg fertilization rate. Therefore, before cryopreservation of spermatozoa, a thorough

evaluation of different extender solutions, cryoprotectants, cooling and thawing rates is

essential to develop optimum cryopreservation protocols for various species (Routray et al.

2008).

The cryopreservation procedure needs a suitable extender that containing optimum

amount of cryoprotectant reducing the cell damage associated with dehydration, cellular

injuries and ice crystal formation (Leung 1991). Although cryoprotectants help to the

prevention of cryoinjuries during freezing and thawing, they may become toxic to the cells

when exposure time and concentration are increased (Jamieson 1991; Christensen and

Tiersch 1996). Therefore, the optimum cryoprotectant concentration may vary according to

the cryoprotectant type, fish species, equilibration period and the other criteria used for the

evaluation of post-thaw sperm quality.

Although there are many reports for sperm cryopreservation of the Cyprinidae, detailed

investigations on cryobiological parameters are inadequate and cryopreservation protocols

derived mainly from empirical data (Billard et al. 1995; Yavas and Bozkurt 2011). For this

reason, standardization and simplification of cryopreservation procedure for scaly carp

sperm are needed for commercial applications.

From this point of view, the main aim of the present study was to develop an appropriate

protocol for scaly carp sperm cryopreservation and also minimize deleterious effect of the

cryopreservation process using Kurokura’s solution containing DMSO, DMA and egg yolk

at different concentrations to determine post-thaw motility, motility period and fertilizing

ability of cryopreserved scaly carp sperm.

Materials and methods

Broodstock management and collection of gametes

The experiment was carried out during spawning season of scaly carp (Cyprinus carpio).

The broodstock were kept in earthen ponds under natural photoperiod regime. In these

ponds, water temperature varied between 22 and 24 �C during spawning season. The

broodstock was collected from wintering ponds by seining and transported into the

hatchery 48 h prior to gamete collection. In the hatchery, male and female broodfish were

kept separately and were not fed in shadowed tanks (V = 1,000 l) that supplied with

continuously (2.5 l/min) well-aerated water at 23 �C.

Before each injection and gamete collection, the fish were anaesthetized in a solution

containing 5 ppm quinaldine (Reanal Ltd, Budapest, Hungary). Following, the broodfish

removed from the water, and their genital apertures were wiped dry. Before stripping, one

dose of carp pituitary extract (CPE) (2 mg/kg) was injected intramuscularly to the males

12 h before semen collection. Sperm was stripped from ten males by abdominal massage

directly into 20-ml glass tubes. Care was taken to avoid contamination with urine, mucus,

feces or water. Sperm samples were not pooled, and the beakers were covered with

parafilm and stored on ice in aerobic conditions. Ovulation was also induced by

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intramuscular double injection of 3.5 mg kg/kg CPE. The first injection of CPE (0.35 mg/kg)

was given 10 h before the second (3.15 mg/kg) (Bozkurt et al. 2012).

Sperm quality and semen dilution

Sperm motility was determined under light microscope (Olympus, Japan) at 4009 mag-

nification. Samples were activated by mixing 1 ll of sperm with 20 ll activation solution

(0.3 % NaCl) on a glass slide. The percentage of motility was defined as the percentage of

spermatozoa moving in a forward motion every 20 % motile increment (i.e., 0, 20, 40, 60,

80, and 100 %) (Vuthiphandchai and Zohar 1999). Sperm cells that vibrated in place were

not considered to be motile. Sperm motility was determined with three replications of

samples. For cryopreservation experiments, samples showing motility below 80 % were

discarded. Sperm motility period was determined using a sensitive chronometer (sensi-

tivity: 1/100 s) by recording the time following addition of the activation solution to the

sperm samples.

Spermatozoa density was determined according to the haemocytometer method. Sperm

was diluted at ratio of 1:1,000 with Hayem’s solution (5 g Na2SO4, 1 g NaCl, 0.5 g HgCl2,

200 ml bicine), and density was determined using a 100 lm deep Thoma haemocytometer

(TH-100, Hecht-Assistent, Sondheim, Germany) at 4009 magnification with Olympus

BX50 phase contrast microscope (Olympus, Japan) and expressed as spermatozoa 9109/ml

(three replicates). Counting chambers were always kept in a moist atmosphere for at least

10 min before cell counting. Sperm pH was measured using indicator papers (Merck, 5.5-9)

within 30 min of sampling.

Collected sperm from 10 males showing [80 motility was pooled into equal aliquots

and chosen for cryopreservation experiments. Semen and extenders were kept at 4 �C prior

to dilution. Pooled semen was diluted at 1:3 ratio with an extender containing 3.6 g/l NaCl,

10 g/l KCl, 0.22 g/l CaCl2, 0.08 g/l MgCl2 ve 0.2 g/l NaHCO3 (Kurokura et al. 1984). The

extender contained three different cryoprotectants that are DMSO, DMA and egg yolk at

three different rates such as 5, 10 and 15 %.

General procedure for sperm freezing and thawing

Diluted semen is around (1.0–2.5) 9 109 cells/ml spermatozoa density in the extender. This

dilution was enough to avoid damage due to sperm compression during freezing and

thawing (Lahnsteiner et al. 2000). The diluted samples were drawn into 0.25-ml plastic

straws (IMV, France) and were sealed with polyvinyl alcohol (PVA). Before freezing, the

semen samples were equilibrated at 4 �C for 10 min to reach the appropriate temperature.

The tray floating on the surface of liquid nitrogen in Styrofoam box was adjusted according

to the desired freezing level. Finally, the straws were frozen in liquid nitrogen vapor

(-140 �C) 4 cm above of liquid nitrogen surface for 10 min. Following, the straws were

kept in liquid nitrogen (-196 �C) container until thawing. For the aim of thawing, the

straws were removed from the liquid nitrogen tank and submerged into a water bath at 40 �C

for 10 s. Afterward, post-thaw sperm motility and periods were immediately evaluated.

Fertilization and hatching experiments

For the aim of fertilization, pooled eggs from mature females were used to determine

fertilization rates. Cryogenic straws randomly selected from the liquid nitrogen tank were

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used to fertilize eggs. Egg samples (about 1,000 eggs) were inseminated in dry Petri dishes

with fresh sperm or frozen sperm immediately after thawing at a spermatozoa–egg ratio of

1 9 105:1. Eggs were inseminated by the dry fertilization technique using a solution of 3 g

urea and 4 g NaCl in 1 l distilled water. The sperm and eggs were slightly stirred for

30 min and washed with hatchery water (23 �C, 9 mg/l O2). Following, the eggs were

transferred into clean Petri dishes and were left untouched for 30 min. Afterward, the eggs

were rinsed twice with 5 % tannic solution and transferred into Zuger glasses supplied with

flow-through hatchery water (23 �C) where they were kept until hatching (3–4 days).

Living and dead eggs were counted in each incubator during incubation, and the dead eggs

were removed. The fertilization and hatching rates were calculated.

Statistical analysis

Results are presented as mean ± SE. Data for percentage of sperm motility and fertil-

ization were transformed by angular transformation prior to statistical analysis by SPSS

10.0 software. Differences between parameters were analyzed by repeated analysis of

variance (ANOVA). Significant means were subjected to a multiple comparison test

(Duncan) for post hoc comparison at a level ofa = 0.05. All analyses were carried out

using SPSS 10 for Windows statistical software package.

Results

Fresh sperm quality parameters

In scaly carp, fresh semen volumes were rather variable and ranged from 7 to 19 ml and

mean volume was 14.2 ± 3.46 ml. Motility values were ranged from 85 to 100 %. The

mean motility value of fresh sperm samples was 95.3 ± 4.27 %. Mean spermatozoa

movement duration (s), sperm density (9109/ml) and pH values were determined as

120.7 ± 4.8 s, 25.6 9 109/ml and 7.3 ± 4.15, respectively.

Cryopreservation and fertilization experiments

Mean post-thaw motility of scaly carp sperm was 88.4 ± 1.5 %, while the mean highest

post-thaw motility and motility periods were determined in sperm cryopreserved with

15 % egg yolk as 93.2 ± 1.27 % and 26 ± 1.2 s., respectively (Table 1). The overall

mean fertilization rate was determined as 91.0 ± 1.1, while the mean highest fertilization

rate was 96.4 ± 0.15 % when sperm cryopreserved with 15 % egg yolk. On the other

hand, the mean highest hatching rate (99.3 ± 0.8 %) was determined when sperm cryo-

preserved with 15 % DMA (Table 2).

The fertilization and hatching rates were similar for fresh and post-thaw sperm frozen

with 10 and 15 % DMA and also 5 and 15 % egg yolk. In addition, post-thaw sperm

motility showed high positive correlations between fertilization (r2 = 0.906) and hatching

rates (r2 = 0.910). Similarly, high positive correlation was determined between fertiliza-

tion and hatching rates (r2 = 0.843). The overall fertilization rate of the frozen-thawed

sperm was similar to that of fresh sperm (98.7 ± 1.28 %). According to the results, post-

thaw motility, fertilization and hatching rates of cryopreserved scaly carp sperm were

determined statistically different between experimental groups (P \ 0.05). Interaction

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between the extenders and cryoprotectants was not significant with regard to fertilization

rates (F = 2.06, P [ 0.05).

Discussion

Sperm cryopreservation has been implemented as an important tool for the management of

captive breeding program, genetic conservation of endangered species and improvement in

selective breeding in aquaculture. During this process, spermatozoa have been subjected to

drastic physical and chemical changes such as ice crystal formation, physical stress and

destabilization of the plasmatic membrane (Labbe et al. 1997). Research on sperm cryo-

preservation in scaly carp has not yet addressed to the use of extenders with simplified

ingredients of ions in combination with various cryoprotectants. Therefore, standardization

and simplification of cryopreservation procedure for scaly carp sperm are needed for

commercial application.

During cryopreservation process, one of the important issues is the choice of cryo-

protectant that its role is to prevent cell damage during freezing and thawing stages.

Table 1 Effect of different cry-oprotectants on post-thaw motil-ity and periods of frozen scalycarp sperm (mean ± SE, n = 3)

Different superscripts indicatesignificant difference withincolumns (p \ 0.05)

Cryoprotectants Cryoprotectantconcentrations(%)

Post-thawmotility (%)

Post-thawmotilityperiods (s)

DMSO 5 80.2 ± 0.47a 23 ± 0.9b,c

10 90.6 ± 1.24c 19 ± 1.4a,b

15 84.5 ± 2.46a,b 14 ± 0.8a

DMA 5 82.7 ± 0.25a 19 ± 1.4a,b

10 89.5 ± 1.42b,c 25 ± 1.8c

15 92.3 ± 1.20c 23 ± 1.2b,c

Egg yolk 5 90.6 ± 2.07c 17 ± 2.5a

10 92.5 ± 0.36c 24 ± 1.7b,c

15 93.2 ± 1.27c 26 ± 1.2c

Table 2 Effect of different cry-oprotectants on fertilization ofeggs and hatching success ofembryos in scaly carp(mean ± SE, n = 3)

Different superscripts indicatesignificant difference withincolumns (p \ 0.05)

Cryoprotectants Cryoprotectantconcentrations(%)

Fertilizationrates (%)

Hatching rates(%)

DMSO 5 86.4 ± 1.48a 84.6 ± 0.91a

10 90.5 ± 2.17a,b,c 92.3 ± 2.14b,c

15 89.2 ± 1.29a,b 94.6 ± 3.25c,d

DMA 5 87.5 ± 0.18a,b 87.3 ± 2.17a,b

10 91.6 ± 0.42a,b,c,d 98.0 ± 1.82c,d

15 90.2 ± 1.14a,b,c 99.3 ± 0.80d

Egg yolk 5 92.3 ± 0.46b,c,d 95.0 ± 7.52c,d

10 95.6 ± 2.27c,d,e 99.0 ± 0.54d

15 96.4 ± 0.15d,e 99.0 ± 0.53d

Control (freshsperm)

– 98.7 ± 1.28e 99.2 ± 0.84d

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Cryoprotectants can suppress most of cryoinjuries when used at higher concentrations, but

at the same time, it can become toxic to the cells (Tekin et al. 2007). Therefore, suitable

cryoprotectant concentration was needed for the development of cryopreservation protocol.

Comparison of different cryoprotectants and freeze–thaw protocols is difficult when

each treatment tested for the ability of sperm to fertilize eggs. The protective effect of

different cryoprotectants varies in different fish species. Several cryoprotectants have been

used for fish sperm cryopreservation such as dimethyl sulphoxide (DMSO), methanol,

ethylene glycol, glycerol and propylene glycol (Routray et al. 2007; Bozkurt et al. 2011). It

is generally known that DMSO is the most widely used cryoprotectant for the cryopres-

ervation of fish sperm and provides better protection at concentrations between 5 and

25 %. On the other hand, in the present study, it was determined that 10 and 15 %

concentrations of DMA and 5, 10 and 15 % concentrations of egg yolk were more suitable

than DMSO for the cryopreservation of scaly carp sperm.

Motility is an important characteristic to determine fresh and cryopreserved sperm

quality. Compared with the current results, Fresneda et al. (2004) determined higher sperm

motility and motility duration using DMSO and methanol as cryoprotectant (80 and 78 %,

respectively) in cachama blanca (Piaractus brachypomus). In addition, Cabrita et al.

(2001) reported sperm motility close to 45 % in rainbow trout when sperm was cryopre-

served with 7 % DMSO and thawed at 25 �C for 30 s. In the present study, the freezing

and thawing process did not significantly influence the sperm motility and ranged from 75

to 97 % by using DMSO, DMA and egg yolk.

In the present study, an ionic-based extender was used to cryopreserve scaly carp sperm

and also DMSO, DMA and egg yolks were tested as cryoprotectant. As a result, the

extender and cryoprotectants yielded comparatively high fertilization rates. The best fer-

tilization rate determined with egg yolk as 96.4 ± 0.15 %. The high fertilization results

can be linked to adequate penetration of used cryoprotectants into the cell membranes. Egg

yolk has been reported to cover the cell membrane wall and thereby reducing lysis during

the freezing process (Scott and Baynes 1980). Although the specific action of egg yolk is

unknown, it has been theorized that a low-density lipoprotein fraction loosely interacts

with the sperm plasma membrane (Quinn et al. 1980). However, the beneficial effects of

egg yolk supplementation appear to be species specific as increased fertilization rates have

been observed in Atlantic salmon (Alderson and Macneil 1984) and rainbow trout (Tekin

et al. 2003).

In the present study, the applied sperm/egg ratio was 1 9 105:1 for fresh and as well as

frozen/thawed sperm which probably resulted in excessive sperm concentrations in all

batches. Excessive sperm concentrations do not only mask poor sperm quality, but also

other suboptimal conditions like reduced egg quality (Lubzens et al. 1997) and pollution

effects (Rurangwa et al. 1998). Nevertheless, according to Lubzens et al. (1997), the

concentration of frozen/thawed sperm to be used to achieve optimal fertilization and

hatching success is approximately 100 times higher than for fresh semen. This may be due

to differences in extender compositions, cryoprotectant types, equilibration periods, egg

quality or applied protocols. In the present study, high positive correlation was determined

between post-thaw sperm motility and fertilization. This was consistent to the results that

obtained from turbot (Dreanno et al. 1999), common carp (Linhart et al. 2000) and African

catfish (Rurangwa et al. 2001).

According to the results of the present study, the cryopreservation protocol developed in

the present study is rather effective and scaly carp sperm can be successfully cryopre-

served. It seems that cryopreservation of scaly carp sperm with Kurokura extender con-

taining egg yolk, DMA and DMSO as cryoprotectant is rather effective on post-thaw sperm

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quality. In addition, based on the results determined in the present study, it is possible to

suggest that sperm cryopreserved with ionic extender containing egg yolk, DMA and

DMSO and also packed in 0.25-ml straws are suitable to retain sperm quality in scaly carp

having optimal sperm motility, motility duration as well as high fertility close to the values

obtained with fresh sperm.

In conclusion, the results obtained in the present study contribute significantly

improving of the sperm cryopreservation protocol development in scaly carp at large scale.

In addition, the results indicated that type of cryoprotectants and their concentrations are

very important in order to obtain high fertilization rates. On the other hand, further efforts

are necessary to fertilize larger amount of eggs with thawed sperm in order to apply this

protocol for commercial aquaculture.

Acknowledgments This research was funded by a grant from Scientific Research Institute of MustafaKemal University (MKU-08-E-0206). The authors would like to thank the staff of the State HydraulicWorks (SHW) Fish Production Station in Adana (Turkey) for their technical assistance. Also, the valuablecomments and suggestions from anonymous reviewers are deeply thanked.

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